Annals of sbv vol 5 iss 1 jan jun 2016 by Dept of Medical Informatics

ISSN 2395 - 1982

SRI BALAJI VIDYAPEETH ACADEMY OF HEALTH PROFESSIONS EDUCATION AND ACADEMIC DEVELOPMENT

ANNALS OF SBV Volume 5 - Issue 1 JAn - jun 2016

Theme

Advances in Inter-Disciplinary Research in Health Sciences

A Publication of

SRI BALAJI VIDYAPEETH

Annals of SBV Editorial Advisors Rajaram Pagadala K R. Sethuraman Editor-in-Chief N. Ananthakrishnan Core Committee M. Ravishankar

V.N. Mahalakshmi Karthiga Jayakumar

Seetesh Ghose

K.A. Narayan

R. Pajanivel Issue Editor Adithan C

R. Jagan Mohan

Assistant Editors Pooja Pratheesh Sam Vijay Kumar J Benet Bosco Dhas D Managing Editor A.N. Uma Statistical Advisor G. Ezhumalai Published, Produced and Distributed by

Sri Balaji Vidyapeeth

Editorial correspondence to Editorial, Technical and Production Consultant

Annals of SBV Sri Balaji Vidyapeeth

(Deemed University, Accredited by NAAC with 'A' Grade) Mahatma Gandhi Medical College & Research Institute Campus Pillaiyarkupam, Puduchery - 605 403 INDIA E.mail:annals@sbvu.ac.in | Phone : +91 413 2615449 to 58 | Fax : +91 413 2615457 Visit Annals of SBV Online at http://www.annals.sbvu.ac.in

Annals of SBV

From the Editor’s Desk

INDEX From the Editor's desk - Adithan C

1. Mutiple facets of a progressive research facility - Central Inter-Disciplinary Research Facility (CIDRF)

- Balanehru Subramanian., Adithan C. 6

2. Bringing pharmacogenomics and personalized medicine into clinical practice. - Adithan C.

3. Genome editing tools and its potential applications in translational medicine- A brief overview. - Agieshkumar B, Elanthiraiyan S

4. microRNA, A clinical diagnostic and prognostic biomarker - Anitha TS

5. Therapeutic epigenetics- a boon to the future - Benet Bosco Dhas D.

6. “Memory” in the mammalian brain - Jaichandar Subramanian

7. Music therapy in neonatology: what is known and what is unknown - Parin N Parmar, Sumathy S

8. Osteoprotective effect of few indian herbs: an update - Veni S, Srinivasan N

9. Glioblastoma: evolving niches and challenges - Pooja Pratheesh

10. Glioblastoma multiforme metabolism: adding fuel to the flame - Preethi S

11. Cancer stem cells – a brief overview - Sam Vijay Kumar J

12. Mannose binding lectin- genetic variations, deficiency and disease associations - Farzana Begum Liakath

13. Adipose tissue hypoxia in obesity - Akshayavardhani A.

14. Post-mortem examination : combining conventional autopsy techniques with virtual autopsyConcerted efforts by the departments of Forensic medicine and Toxicology and Radiology - Dipayan Deb Barman, Vijaya Kumar Nair G.

Advances in Inter-Disciplinary Research in Health Sciences Interdisciplinary research in health science is the collaboration between the predominant health sciences disciplines and the “bench” science. It is directly associated with translational research that harnesses knowledge from basic sciences to initiate a “bench-to-bedside” channel starting from laboratory experiments through clinical trials to point-of-care for patient benefit. Interdisciplinary research in health sciences is the need of the hour for improving the management of complex and chronic diseases Advances in the understanding of cellular functions in normal and disease states, sequencing of human genome, fast growing technological advances in modern science, emerging importance of traditional medicines and introduction of biologics in clinical practice ushers hope of mitigating some chronic health problems. This hope can turn into a reality if more interdisciplinary approaches are launched in health science research. Keeping this in mind the theme of the present issue of “Annals of SBV” is chosen as “Advances in inter-disciplinary research in health sciences”. In this issue we received review articles from Central Inter-Disciplinary Research Facility, (CIDRF), Picower Institute for Learning and Memory (USA), Centre for Music Therapy Education and Research (CMTER) and Sri Sathya Sai Medical College and Research Institute (SSSMCRI). The articles range from basic research on memory to clinical research on personalized medicine. The first article describes the genesis, facility and the future plan of CIDRF. Small Animal Research Facility which will soon be established at CIDRF is described. Introduction of personalized genetic testing at SBVU is highlighted. The next three articles discuss genome editing tools, such as micro RNA’s potential as diagnostic and prognostic markers and therapeutic epigenetics which in recent times have been at the forefront of translational research. Understanding the mechanism of memory storage is an important aspect of modern neuroscience. An article from Massachusetts Institute of Technology, USA reviews the recent findings in the nature of memory traces in mammalian brain. Music therapy is a novel therapeutic approach and its possible use in neonatology is discussed in the next article. Advances in traditional medicine research and its application has gained momentum in recent times and there is a need to explore the medicinal potential of Indian herbs. An article describing available Indian medicinal plants used in bone metabolic disorders is an apt entry in this publication. The next 3 articles discuss the molecular and metabolic basis of glioblastoma multiforme and the importance of characterizing cancer stem cells. The subsequent articles describe the influence of mannose-binding lectins on the immunity and the therapeutic potential of MBL replacement therapy. An interesting article on molecular mechanism underlying adipose tissue remodeling in obesity may help to evolve preventive and therapeutic strategies towards obesity related complications. The last article brings forth the new role of medical imaging in virtual autopsy which in addition with conventional autopsy technique holds potential for augmenting forensic medicine investigations in future. It is hoped that the content of this issue will stimulate interdisciplinary health research. Dr.C.Adithan, Issue Editor, Director, Central Inter-Disciplinary Research Facility Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus, Pillaiyarkuppam, Puducherry-607403, India

Ann. SBV, Jan-Jun 2016;5(1)

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Multiple Facets of A Progressive Research Facility – Central Inter-Disciplinary Research Facility (CIDRF)

Multiple Facets of A Progressive Research Facility – Central Inter-Disciplinary Research Facility (CIDRF) Balanehru Subramanian, Deputy Director Adithan C, Director Central Inter-Disciplinary Research Facility Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India Email: director@cidrf.res.in

CIDRF was established as an inventive and independent research institute under the Sri Balaji Educational and Charitable Public Trust of Puducherry. It was inaugurated on October 29, 2012 by Dr. S.R. Rao, Advisor, Dept. of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi. CIDRF has a state-of-the-art biomedical research facility. It has modern and modular infrastructure; unique and first of its kind in Pondicherry and this part of Tamilnadu. The uniqueness lies in the conception and organization of laboratories, all of which are clean rooms with totally controlled and regulated airflow; temperature and pressure. Of these laboratories, the culture laboratories (two in number with Class II and Class III bio-safety cabinets) meant to culture stem cells and other cell lines is Class 1000 while the other laboratories are Class 10,000 and the corridor, as well as common areas are Class 1lakh in international standards of air purity. The Servostabilized inverter backed up power is made available around the clock. Smoke and fire detectors and other safety measures are in place. A walk in cold room maintained at 4oC exclusively for large scale media storage and column separation etc., biology laboratory with class 10,000 area is kept away from other labs to avoid contamination. There is a media kitchen and store with specially ventilated cupboards for organic solvents and acids separately, minus 80 and minus 20oC storage cabinets etc. There is an emergency shower for face, eye and body wash in case of accidents. There are very strict and specific protocols to be followed by workers and even visitors in CIDRF. Page 6

CIDRF has a defined clear vision and mission:

Vision: • Leadership in Translational Personalized Healthcare Research

Mission: • • • • •

Expertise in cutting edge research Increased impact on patient care Expanded scope of clinical practice Institutional knowledge coordination Knowledge-base in healthcare research

CIDRF bridges and coordinates the research interests of medical, dental, nursing and scientific faculty under Sri Balaji Vidyapeeth and beyond. Research projects that originate with pre-, para- and clinical researchers are jointly developed by CIDRF in a consortium style of functioning sharing mutual Ann. SBV, Jan-Jun 2016;5(1)

strengths and resources available at Mahatma Gandhi Medical College & Research Institute, Shri Sathya Sai Medical College and Research Institute, Indira Gandhi Institute of Dental Sciences, Kasturiba Gandhi Nursing College and external collaborators.

stronger and larger network of “Centers of Biomedical Excellence” in India.

CIDRF unites the forces of government, academia and entrepreneurial entities to promote and support biomedical research and healthcare. The corporal outcomes will include new medicines, novel diagnostics and new technologies that advance biomedical research and patient care resulting in

Research Activities

Ann. SBV, Jan-Jun 2016;5(1)

PRIDE is the acronym for the thrust areas of research of CIDRF

• Since its inception, CIDRF’s research activities involved securing 6 extramural research grants from Government of India and Government of Puducherry. Page 7

Multiple Facets of A Progressive Research Facility – Central Inter-Disciplinary Research Facility (CIDRF)

Annals of SBV

• Nine intramural research grants from and Sri Balaji Vidyapeeth Research Fund. • This is in addition to the 10 inter-disciplinary clinical research projects established in collaboration with clinicians of MGMCRI, SSSMCRI and IGIDS. • Nine Research collaborations with both national/ international, academic/industry have been established so far. • In-house research activities till date has resulted in 21 publications and filing of three provisional patents with Government of India. • Doctoral degree program affiliated to Sri Balaji Vidyapeeth has enrolled seven Ph.D. internal/ external candidates. • An undergraduate medical student research preceptorship program was established in 2014. • Research mentorship to both medical/dental PG students and faculty is active. • CIDRF has so far conducted 5 conference/seminar/ workshop as part of its knowledge dissemination program.

Innovative Programs 1. With the aim of training interested postgraduates to gain expertise in fundamentals of Page 8

Pharmacogenomics and Personalized Medicine, CIDRF is starting a one year PG Diploma program under the umbrella of CIDRFInnovative PG Diploma programs. 2. With the aim of training interested postgraduates to be sound in fundamentals of modern Bio-Medical / Life Science research, CIDRF proposes following programs under the umbrella of Central Inter Disciplinary Research Facility’s Innovative Certificate and Fellowship Program in Bio-Medical Research Techniques (CIDRF-IceFeP BMRT) :

quality of medical/surgical education, and increase employment opportunities. These courses will have 3 months and 6 months duration including a period for internship exposure to all the techniques in the new facility.

a) Certificate Program in Bio-Medical Research Techniques (Cert.in BMRT) – Three Months (One trimester) b) Fellowship Program in Bio-Medical Research Techniques (FBMRT) - One Year (Four trimesters)

The new facility will house laboratories dedicated to: • Small animal housing and testing area • Tissue Bank • Cell Culture Research • Molecular Biology and Genetics • Toxicology, Histology/Pathology • Biomaterials & Bioengineering

CIDRF-DBT Center for Animal Research, Training and Services (CAReTS, pronounced as carrets) CIDRF has teamed up with DBT, Government of India to establish a new state of art small animal research facility to serve the community. This new research facility designed with modern infrastructure and latest tools would result in the establishment of a GLP compliant pathogen free animal housing and a state of art small animal research facility first of its kind, in this part of the country. This will be engaged in preclinical research, services dedicated for safety assessment, toxicology and development of medical therapeutics and devices. This new facility will serve the R & D needs of researchers in both public and private sectors in and around Pondicherry as well as Central and Southern Tamilnadu. Pondicherry has more than thirty biotechnology and pharmaceutical companies and twelve medical and life sciences research institutions. However, the availability and accessibility of Specific Pathogen Free animals for quality and advanced animal research in preclinical studies and therapeutics has been a great limitation. This gap will now be filled for the benefit of users. This state of art specific pathogen free animal facility is therefore a unique research initiative to serve this part of the country.

Yet another unique objective of the new facility would be to make available to needy scientists, knock-out animals and indigenous disease models that can be valuable for understanding the etiology of disease or for testing potential therapies specific to Indian population.

The new small animal facility will be custom designed and built featuring latest laboratory concepts and systems equipped with advanced equipment; allowing regional researchers to gain global recognition.

Personalized Genetic Testing Service Pharmacogenomics and personalized medicine promises to markedly improve the individualized drug therapy. Realizing its importance and clinical utility, CIDRF, for the first time in Pondicherry, is introducing genetic testing of CYP2D6, CYP2C9, CYP2C19, VKORC1 and P2Y12 polymorphic genes for patient care services. The results of these tests will help to tailor the posology of anti-platelet drugs, anti-epileptics, anticoagulants, anticancer drugs, beta blockers and other commonly used drugs. This technological advancement of CIDRF will shortly be introduced at MGMCRI and to other needy hospitals.

Milestones for next five years • Develop a nutraceutical product based on the research outcomes of DBT funded project entitled “Prophylactic potential of the extract of the bark of Terminalia arjuna (Roxb.ex.Dc.) Wight & Arn. in preventing osteoporosis”. • Establish and extend PCR based testing, on demand, for H. pylori treatment • Add Pharmacogenetics of cardio-protective drugs and anti-psychotic drugs to the newly established personalized genetic testing services. • Establish research programs on MRSA and Salivary biomarkers. • Secure Early Career Research Awards from National and International agencies for Scientists of CIDRF. In order to achieve our research objectives, the available research facility will be extended to accommodate a state-of-art • • • • • • • •

Pharmacogenomics Lab. Proteomics Lab Regenerative Medicine Lab. Bio-informatics Lab Bio-analytical Lab Microbiome Lab. Natural Products Lab and Molecular virology Lab

With a defined roadmap, we hope the facets of CIDRF in interdisciplinary research, human resource development, new facility and laboratory space development, innovative academic activities and unique patient care services are well set to be the progressive inter-disciplinary research facility in Puducherry.

The proposed small animal facility also proposes to provide medium to high skill development courses for qualified low-skilled technical manpower, improved Ann. SBV, Jan-Jun 2016;5(1)

Ann. SBV, Jan-Jun 2016;5(1)

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Bringing Pharmacogenomics and Personalized Medicine into Clinical Practice

Personalized Medicine Bringing Pharmacogenomics and Personalized Medicine into Clinical Practice Adithan C, Director Central Inter-Disciplinary Research Facility and Professor of Pharmacology, Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India Email: director@cidrf.res.in

It has been observed that genetics can account for 20– 95% of variability in drug disposition and effects3. The environmental factors, concurrent drug intake, disease process etc. can further contribute to the existing variability. Therefore, it is preferable that personalised medicine be used for a more successful pharmacotherapy. Personalised medicine is concerned with providing medical care tailored to the genomic profile, family medical history and other environmental factors related to the patient. At present, drug therapy is done by trial and error method and ‘one size fits all’ concept. Personalized

medicine involves conducting genetic testing in order to select the right drug, in the right dosage. Factors such as liver and kidney function, concomitant drug intake etc. that could influence the pharmacokinetics and the pharmacodynamics of the drug are also taken into consideration.

Pharmacogenomics of Drug Metabolising Enzymes Cytochrome P450 enzymes present in the liver and other parts of the body play a major role in the metabolism of drugs. The important drug metabolising enzymes are shown in Table 1.

Table 1. Important polymorphic genes encoding Phase 1 and 2 drug metabolizing enzymes 4, 5 Abstract 

harmacogenomics and personalised medicine are emerging as important tools in P individualised drug therapy. Many of the genes which encode drug metabolising enzymes, transporters and receptors are polymorphic. Individuals with polymorphic genes are likely to experience therapeutic failure or drug toxicity. This is clinically important for drugs with low safety margin such as oral anticoagulants, anti-epileptics and anticancer drugs. Prior genetic testing may help to predict responders and non-responders to the above mentioned drugs, besides avoiding drug toxicity. CIDRF is planning to introduce CYP2C9, CYP2C19 and CYP2D6 genetic testing shortly, which shall help to integrate pharmacogenomics with clinical practice. Key Words: Pharmacogenomics, Personalized medicine, Genetic testing, Drug toxicity.

Introduction

Pharmacogenomics

The present scenario in drug therapy is not satisfactory. In spite of advances in the understanding of pathophysiology of diseases and introduction of newer drugs, the optimal therapy for major diseases is still elusive. For example, 30 % of schizophrenic patients do not respond to drug therapy, 27 % of hypertensive patients are poorly controlled and many anticancer drugs fail to bring remission. Further, adverse drug reactions (ADR) remain a problem. A USA study reported an annual mortality of about 1 lakh patients due to ADR1. Most of these problems are due to variability in drug responses seen in patients. Hence, there is a need to improve the outcome of pharmacotherapy. One of the ways to achieve this is by selecting drugs and posology based on individual genetic profile of patients. Thus, pharmacogenomics and personalized medicine is a new, promising and emerging area of medical science.

Pharmacogenomics is the study of the effect of genetic variability of an individual in drug response. It is known that DNA sequence of all human beings is 99.9% identical and we differ by 0.1%. Since human genome has about 3 billion bases, the 0.1% difference (genetic variations) amounts to around 3 million spelling differences in the human genome.

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Majority of genetic variations are due to single nucleotide polymorphism2. The genetic polymorphism of an individual can influence the susceptibility to disease, and the absorption, distribution, metabolism and excretion of drugs. Besides, the function of receptors and response to drugs also gets modified. The presence of genetic polymorphism may result in poor drug response or drug toxicity.

Gene

Mutant Alleles

Drug Metabolising Enzymes

Substrates (Drugs)

Phase 1 enzymes CYP2C9

*2, *3, *4, *5, *6

CYP2C9

Warfarin, losartan phenytoin, tolbutamide

CYP2C19

*2, *3, *4, *5, *6, *7, *8

CYP2C19

Proguanil, imipramine, ritonavir, nelfinavir, cyclophosphamide

CYP2D6

*1XN, *2XN, *3,*4,*5, *6 *9,*10,*17

CYP2D6

Clonidine, codeine, promethazine, propranolol, clozapine, fluoxetine, haloperidol, amitriptyline

CYP2E1

*2,*1B

CYP2E1

Acetaminophen, halothane, enflurane, fluoxetine, chlorzoxazone

CYP1A2

*1C, *1F, *1K, *3,*4,*6

CYP1A2

Clozapine, fluvoxamine, haloperidol, imipramine, tacrine, verapamil

Phase 2 enzymes NAT2

*5, *6,*7, *10,*14

NAT2

Isoniazid, hydralazine,

GST

P1, M1 null, T1 null

GST

D-penicillamine

TPMT

*2,*3A,*3C

TPMT

Azathioprine, 6-mercaptopurine

UGT1A1

*28

UGT1A1

Irinotecan

DPD

*2A

DPD

Fluorouracil

Red: Absent; Blue: Reduced; Green: Increased activity Ann. SBV, Jan-Jun 2016;5(1)

Ann. SBV, Jan-Jun 2016;5(1)

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Bringing Pharmacogenomics and Personalized Medicine into Clinical Practice

Annals of SBV

The syntheses of these enzymes are encoded by specific genes. Any genetic variability in these genes will result in either deficiency or increased quantity of enzymes which can predispose an individual to drug toxicity or therapeutic failure. The various molecular mechanisms that can alter drug metabolism are as follows: 1. Deletion of gene. This results in complete cessation of production of the concerned enzyme. As a result, the metabolism of the drug by that particular enzyme does not take place (e.g., CYP2C19*2, *3) 2. Alteration of function of a single gene. This can result in three possible scenarios. a. Production of an unstable enzyme – In this case, the normal drug metabolism is compromised (e.g., CYP2D6*10). b. Normal enzyme – Normal metabolism of drug (e.g., CYP2C9*1, CYP2D6*1) c. Altered substrate specificity – Other metabolites may be formed. 3. Duplication or multiduplicated genes This results in higher enzyme levels leading to an increased metabolism of drugs (e.g., CYP2D6*1 X N) In the body, drug metabolism takes place in two phases – phase 1 and phase I2. In phase 1, the drugs undergo oxidation, reduction or hydrolysis. In phase 2, drugs

undergo glucuronidation, sulfatation or acetylation which makes the drug more water soluble and easily excretable. CYP3A4/A5, CYP2D6, CYP2C9, CYP2C19, CYP2E1 are the important phase I enzymes. Among them CYP3A4/A5 accounts for metabolism of more than 50% of currently used drugs. However, no significant polymorphism has been reported in the genes encoding them. CYP2C9, CYP2C19 and CYP2D6 are the important genes which are polymorphically expressed. Important variant alleles of phase 1 and phase 2 enzymes and their drug substrates are given in Table 1. The frequency of variation in alleles of drug metabolising enzymes can differ in a population depending on their ethnicity. It was found to be different among Caucasians, Orientals, Africans and Indians. Even among Indians, the frequency of variant alleles was found to be different between South Indians, Western Indians and North Indians. A detailed review on this topic is published elsewhere6.

Pharmacogenomics of Drug Transporters Drug transporters located in the cell membranes are the major determinants of pharmacokinetic profile of drugs. Two major superfamilies, namely, ATP-Binding Cascade (ABC) and Solute Carrier (SLC) were found to have important genetic variation in the coding regions7. Among them, P-glycoprotein (ABCB1) and Organic Cation Transporters 1 and 2 (OCT1 and OCT2) are functionally well characterized. In ABCB1 P-glycoprotein, a synonymous polymorphism (3435C>T) was extensively studied and found to be associated with various drug responses. Some of the drug substrates for P-glycoprotein are mentioned in Table 2.

Table 2. Important drug substrates of P-glycoprotein

Drug Category

Substrates for P-glycoprotein

Cardiac drugs

Digoxin, quinidine

Anti-cancer agents HIV protease inhibitors Immunosuppressants Antibiotics

Lipid lowering agents Page 12

Actinomycin D, vincristine, paclitaxel . Ritonavir, indinavir

Cyclosporine A, tacrolimus Erythromycin,levofloxacin Lovastatin, atorvastatin

Ann. SBV, Jan-Jun 2016;5(1)

Pharmacogenomics of Drug Receptors Drugs produce their effect by binding to receptors, present either in the membrane or inside the cell. The genes encoding these receptors too show polymorphism, which may modify the receptor activity and drug response8. For example – asthmatic patients with Gly16 polymorphism of beta 2 receptor gene are likely to be about 5 fold less responsive to salbutamol9. Beta 1 receptor blockers such as metoprolol may have more profound action in individuals having Gly49 polymorphism of beta 1 receptors10.

Clinical Applications A number of papers have been published supporting the role of pharmacogenomics in clinical practice. A few examples of works done by the author at JIPMER, Pondicherry and by other workers elsewhere are given below Anticonvulsant drug: Influence of CYP2C9 polymorphism on phenytoin dosage requirement and toxicity were studied in the Indian population. It showed that epileptic patients with homomutant genotype of CYP2C9 may require about 110 mg/day of phenytoin when compared to its normal dose of 300 mg/day. Phenytoin induced neurological toxicity was found to be 4 to 10 times higher if patients have either of CYP2C9*2 or *3 variant allele.11 Oral anticoagulants: Warfarin related bleeding complications were about 3 to 4 times more in patients who were carriers of at least 1 mutant allele (*2 or *3) of CYP2C9. The optimum dosage of warfarin or acenocoumarol can be predicted by genetic testing of CYP2C9 and VKORCI. The genetic factors contribute about 45 to 55% of oral anticoagulant response in the Tamilian population12. Antiplatelet drug: Clopidogrel‘s action was significantly influenced by CYP2C19, CYP3A5, MDR1 and P2Y12 genetic polymorphism. Poor metabolisers having CYP2C19 polymorphic genes fail to respond satisfactorily to clopidogrel even after doubling the dose13. Antidiabetic drugs: The beneficial effect of glibenclamide and metformin were partially determined by CYP2C9 and OCT1 polymorphic genes respectively 14, 15 . Ann. SBV, Jan-Jun 2016;5(1)

Anticancer drug: Tamoxifen is used as an adjuvant therapy in carcinoma of breast. It is a prodrug which is metabolised by CYP2D6 enzyme into an active endoxifen metabolite. It was reported that in patients having carcinoma of breast with CYP2D6 variant alleles and treated with tamoxifen, disease recurrence was increased and survival period was reduced16. Antipsychotic drugs: Tardive dyskinesia is a known complication of antipsychotic drugs occurring in 10 to 30 % of cases. Most severe form of tardive dyskinesia was reported in individuals with Ser9Gly DRD3 gene polymorphism and CYP1A2*1F polymorphism17.

International Status Worldwide the potential value of pharmacogenomics testing is being realised. New and rapid methods for pharmacogenetic testing are also being developed. A technology called SmartAmp claims to provide the result within 45-60 minutes without the need for DNA extraction and PCR amplification18. However, there are just a few controlled clinical trials to establish the cost-effectiveness for recommending routine genetic testing. Precision medicine (personalized medicine) received a big boost in 2015 by President Obama’s initiative to establish a voluntary national research cohort of at least 1 million USA population. Under this initiative, the participants are required to contribute their core data including genetic profile, metabolites profile, microbiome profile in and on the body, life style data etc. which shall ultimately promote precision medicine. FDA (USA) based on the available pharmacogenomics data has recommended label changes for abacavir, azathioprine, carbamazepine, citalopram, clopidogrel, warfarin and a few other drugs. An article published in Cleveland Clinical Journal of Medicine recommends pharmacogenetic testing for abacavir, carbamazepine, allopurinol (HLA testing), clopidogrel (CYP2C19), tamoxifen (CYP2D6), azathioprine, 6-mercaptoprine (TPMT) and few more drugs19, 20. Now, many biotechnology companies have started commercial genetic testing for important drugs. But the issue of reimbursement by insurance companies is still unresolved.

National Status Most of the genetic studies done in India have focussed on disease susceptibility to cancer, diabetes and other conditions. Relatively fewer studies have been carried out with anti-epileptic, cardiovascular, and anti-cancer drugs which were briefly discussed earlier. Page 13

Annals of SBV

In private sector, there are more than a dozen companies offering genetic testing. Among them, Acton Biotech (India) Pvt. Ltd, Ayugen Biosciences, Datar genetics are more focussed on pharmacogenetic tests. Government funding agencies such as Department of Biotechnology and Indian Council of Medical Research have identified pharmacogenomics as one of their priority areas and have established Task Forces to promote pharmacogenomics research. The Indian government is planning to link Pharmacovigilance Program of India (PvPI) with pharmacogenomics research. With this, PvPI will include pharmacogenomics as a part of its scientific component to address concerns of vulnerable populations susceptible to adverse drug events induced by a certain drug triggered due to genetic factors.

Proposed Role of CIDRF CIDRF is planning to start collaborative research work in cancer and cardiovascular diseases. It also plans to start hospital services for genotyping

important genes such as CYP2C9, CYP2C19, CYP2D6, P2Y12 and VKROC1. The results of the above genetic testing may be useful for tailoring the dosages of anti-epileptics (phenytoin), antiplatelet drugs (clopidogrel), oral anticoagulant (warfarin and acenocoumarol), antimalarial drugs (proguanil), anti-depressant drugs (fluoxetine, imipramine), antipsychotic drugs (clozapine, haloperidol), antihypertensives (beta blockers, losartan), antidiabetic drugs (glibenclamide and metformin), antiretroviral drugs (ritonavir, nelfinavir), NSAIDs and anticancer drugs (cyclophosphamide, tamoxifen). The cost of these genetic tests may vary from Rs. 2000 - 15,000 depending upon the number of genes to be tested. Similar to the blood group testing, these results are valid throughout life. We believe that integration of selected pharmacogenetic testing into clinical practice will benefit patients receiving drugs with low safety margin.

Genome Editing Tools and its Potential Applications in Translational Medicine - A Brief Overview Agieshkumar B, Senior Scientist Elanthiraiyan S, Project Trainee Central Inter-Disciplinary Research Facility Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India Email: agiesh.b@gmail.com

Abstract 

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Lazarou J, Pomeranz BH, Corey PN. Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies. JAMA. 1998; 279:1200-5. Stoneking M. Single nucleotide polymorphisms. From the evolutionary past. Nature. 2001;409:821-2. Kalow W, Tang BK, Endrenyi L. Hypothesis: comparisons of inter- and intra-individual variations can substitute for twin studies in drug research. Pharmacogenetics. 1998;8:283-9. http://www.cypalleles.ki.se/ (accessed on 15 May 2016). Jancova P, Anzenbacher P, Anzenbacherova E. Phase II drug metabolizing enzymes. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2010;154:103-16. Umamaheswaran G, Kumar DK, Adithan C. Distribution of genetic polymorphisms of genes encoding drug metabolizing enzymes & drug transporters - a review with Indian perspective. Indian J Med Res. 2014;139:27-65. Yee SW, Chen L, Giacomini KM. Pharmacogenomics of membrane transporters: past,present and future. Pharmacogenomics. 2010 ;11:475-9. Johnson JA, Lima JJ. Drug receptor/effector polymorphisms and pharmacogenetics: current status and challenges. Pharmacogenetics. 2003;13:525-34. Martinez FD, Graves PE, Baldini M, Solomon S, Erickson R. Association between genetic polymorphisms of the beta2-adrenoceptor and response to albuterol in children with and without a history of wheezing. J Clin Invest. 1997;100:3184-8. Levin MC, Marullo S, Muntaner O, Andersson B, Magnusson Y. The myocardium-protective Gly-49 variant of the beta 1-adrenergic receptor exhibits constitutive activity and increased desensitization and down-regulation. J Biol Chem. 2002 ;277:30429-35. Kesavan R, Narayan SK, Adithan C. Influence of CYP2C9 and CYP2C19 genetic polymorphisms on phenytoin-induced neurological toxicity in Indian epileptic patients. Eur J Clin Pharmacol. 2010;66:689-96. Krishna Kumar D, Shewade DG, Loriot MA, Beaune P, Balachander J, et al. Effect of CYP2C9, VKORC1, CYP4F2 and GGCX genetic variants on warfarin maintenance dose and explicating a new pharmacogenetic algorithm in South Indian population. Eur J Clin Pharmacol. 2014 ;70:47-56. Subraja K, Dkhar SA, Priyadharsini R, Ravindra BK, Shewade DG, et al. Genetic polymorphisms of CYP2C19 influences the response to clopidogrel in ischemic heart disease patients in the South Indian Tamilian population. Eur J Clin Pharmacol. 2013;69:415-22. Vilvanathan S, Gurusamy U, Mukta V, Das AK, Chandrasekaran A. Allele and genotype frequency of a genetic variant in ataxia telangiectasia mutated gene affecting glycemic response to metformin in South Indian population. Indian J Endocrinol Metab. 2014 ;18:850-4. Surendiran A, Pradhan SC, Agrawal A, Subrahmanyam DK, Rajan S, et al. Influence of CYP2C9 gene polymorphisms on response to glibenclamide in type 2 diabetes mellitus patients. Eur J Clin Pharmacol. 2011;67:797-801. Jung JA, Lim HS. Association between CYP2D6 genotypes and the clinical outcomes of adjuvant tamoxifen for breast cancer: a meta-analysis. Pharmacogenomics. 2014 ;15:49-60. Zhang JP, Malhotra AK. Pharmacogenetics and antipsychotics: therapeutic efficacy and side effects prediction. Expert Opin Drug Metab Toxicol. 2011 ;7:9-37. Aw W, Lezhava A, Andoh A, Tanaka H, Hayashizaki Y, et al. The SmartAmp method: rapid detection of SNPs in thiopurine S-methyltransferase and ABC transporters ABCC4 and ABCG2. Curr Drug Metab. 2012 ;13:968-77. Wang B, Canestaro WJ, Choudhry NK. Clinical evidence supporting pharmacogenomic biomarker testing provided in US Food and Drug Administration drug labels. JAMA Intern Med. 2014;174:1938-44. Kitzmiller JP, Groen DK, Phelps MA, Sadee W. Pharmacogenomic testing: relevance in medical practice: why drugs work in some patients but not in others. Cleve Clin J Med. 2011;78 :243-57.

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Ann. SBV, Jan-Jun 2016;5(1)

Recent understandings in the genetic basis of diseases emphasize the need for potential therapeutic strategies. Latest molecular techniques such as RNA interference, gene therapy and gene editing that can modify nucleic acids within disease affected tissues can have great potential for treating genetic disorders. In this context, the present review summarizes the tools available for human gene editing and advances in research with reference to CRISPR/Cas9 based human genome editing. This review also discusses various ethical concerns associated with human gene editing. Key Words: Genome editing, CRISPR, translational medicine

Introduction Genome-editing is a technique that is applied to target a particular deleterious and disease causing genes in certain genetic disorders1. Genome editing of somatic cells alters the non-functional cells into functional cells, is at various clinical stages and is considered a promising area of therapeutic development. On the other hand if the targeted genes are altered at the germinal level the disease causing genes can be entirely rectified and cannot be taken over to the next generation2,3. There are several ways by which replacement of the faulty gene with functional gene or repair of the faulty gene are reported in case of gene therapy. Homologous recombination is the generally applied method for introducing any change to specific sequence in the genome called genome targeting4. Later RNA interference (RNAi) has been used to knockdown the function of genes implicated in cancer, age-related macular degeneration etc., which was reviewed in detail5,6. Despite promise and recent success, the techniques suffers from certain limitations like, RNAi often cannot fully repress the gene expression and is therefore unlikely to provide Ann. SBV, Jan-Jun 2016;5(1)

a benefit for diseases in which complete removal of gene function is necessary for therapy. This limits their utility for a large number of diseases. Similarly the techniques described thus far also possess significant off target effect that may switch out genes that are not targeted and leads to undesired effects. However recently developed genomic editing technologies have the potential to be powerful tools for gene therapy because of their ability to inactivate genes, correct mutated sequences, or insert intact genes with reduced off target activities and thus establishing the desired mutations in more cells in any given experiment. A brief overview of recently developed tools in gene editing, potential clinical applications and its ethical issues are given in the below section.

Overview of Genome Editing Technologies The four well described methods of Genome editing technologies with clinical applications are mega nucleases (MNs), zinc finger nucleases (ZFN’s) transcription activator–like effector nucleases Page 15

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Figure 1. Mechanism of genome editing

Figure 2. Mode of gene correction by gene editing tools

Table 2. Examples showing the application of gene editing tools in correcting genetic disorders

S. no

(TALEN’s) and the clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease Cas979 These four major tools are currently being explored for the possibility of achieving therapeutic genome editing in diseased cells and tissues, resulting in the removal or correction of deleterious mutations or the insertion of protective mutations10. All these genome editing methods works on a common phenomenon in

Tool

Interaction

Off-Target effects

FokI nuclease domain

Off-Target effects have been reported

FokI nuclease domain

Off-target effects are not well reported

Cas9

Potential offtarget effects have been reported

ZFN (Zinc Finger Nuclease)

Proteins-DNA

Multiplexing

Very low

Efficiency & Cost effectiveness Expensive & less efficient

TALEN (Transcription activator-like effector nuclease)

Proteins-DNA

Used Occasionally

Moderately expensive & efficient

High

Cost effective & highly efficient

CRISPR/Cas9 (Clustered regularlyinterspaced short palindromic repeats)

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RNA-DNA

Ann. SBV, Jan-Jun 2016;5(1)

Mode of Genome editing tool HDR- Edition of mutation in mouse zygote HDR-Repair of CFTR in intestinal stem cell NHEJ and HDR mediated gene correction

Cataract

CRISPR

Cystic fibrosis

CRISPR

Duchenne muscular dystrophy (DMD)

CRISPR and TALEN

HIV

ZFN and CRISPR

NHEJ-mediated repair

HBV

TALEN and CRISPR

NHEJ-mediated depletion of viral DNA

Hemophilia B

ZFN

Table 1. Comparison of commonly used Gene editing tools in Translational medicine

DNA cleavage

Disease

Genome editing tool

Hereditary tyrosinemia Severe Combined Immunodeficiency (SCID)

CRISPR

HDR-mediated insertion of correct gene sequence HDR-mediated correction of mutation in liver

Mice model

References [12]

Primary adult [14] stem cells Cells from DMD patients,Mice [15, 16] germline DNA HIV Patient cells, Pluripotent [11, 17-19] stem cells (iPSCs) Mouse model, Cultured cells

[20-21]

mouse model

[12]

Mouse model

[13]

ZFN

HDR-mediated insertion of correct gene sequence

Hematopoietic [22] stem cells (HSCs)

NHEJ-mediated genome editing

Induced pluripotent stem cells

[23,24]

NHEJ-mediated genome editing

Human Embryo

[25]

β-thalassemia

TALEN and CRISPR

β-thalassemia

CRISPR

which body cells are able to repair any double stranded breaks made to its DNA by endonucleases either by non homologous end joining (NHEJ) or homology-directed repair (HDR). Mostly, the former one is not perfect thereby resulting in deletion or addition of several bases thus mutating the target sequence whereas the latter one uses a homologous sequence that adds specific changes directly on the target site. Thus all the tools mentioned here make use of nucleases to make sitespecific double-stranded breaks (DSB’s) in the target genome and the basic working mechanism of TALEN’s and ZFN augmenting an error in the genome with a functional copy through homologous recombination is shown in Fig.1. The two different modes of repair (NHEJ and HDR) adapted by the genome editing tools described in this review is depicted in Fig 2. Ann. SBV, Jan-Jun 2016;5(1)

Models used

The genome editing tools are broadly classified into two categories based on their mode of DNA recognition. For instance ZFN, TALEN, and meganucleases achieve specific DNA binding via protein-DNA interactions whereas Cas9 system is targeted to specific DNA sequences with the help of a short RNA guide molecule. All the four nucleases have been reported to yield efficient gene editing in different model organisms and mammalian cells and efforts are now underway to develop these tools as therapeutics11-13. Though all the tools work in an efficient manner, a potential advantage of Cas9 is its ability to introduce multiple double strand breaks in the same cell via expression of distinct guide RNAs that drew attention among researchers and currently considered as a best tool for gene editing. Table 1 shows a comparison of ZFN, TALEN and Cas9 Page 17

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in terms of its efficiency and specificity. Table 2 shows some of the diseases in which the tools are being applied to correct the disorder.

CRISPR-Cas9 System CRISPR stands for Clustered Regularly-Interspaced Short Palindromic Repeats discovered in some bacterial DNA26. The repeats are used by archaea bacteria for adaptive immunity from invading viruses. Cas9 is a specific endonuclease that can cleave DNA. This CRISPR-Cas9 system is considered a highly specific and convenient tool for gene editing (including gene deletion/gene insertion or gene silencing). The technology is also considered cheap and easy for genetic manipulation. A brief account of the evolution of this technology is given below: A research team led by Emmanuelle Charpentier and Jennifer Doudna utlilised the CRISPR system from Streptococcus pyrogenes for genome editing for the first time in 201227. They generated a single guide RNA (sgRNA) by fusing the crRNA to the tracrRNA, which recruits the Cas9 nuclease to specific genomic locations. The creation of site-specific double-strand breaks by the CRISPR/Cas9 complex then triggers genome editing through 2 different mechanisms. First, in the absence of a homologous DNA template double stranded breaks can be repaired by non-homologous end joining (NHEJ), which is an error-prone process that causes small insertions or deletions. Second, in the presence of a synthetic repair template DSBs can be repaired by homology-directed repair (HDR), which enables the introduction of any desired base-pair changes as described earlier (Fig 1 &2). The simplicity of this technique drew attention among molecular biologists to use the tool extensively for gene editing and other functional genomic studies though it has not been adequately tested in human. The therapeutic possibility of CRIPSR was first opened by Feng Zhang group, where an engineered novel form of CRISPRCas9 was used to edit the human genome9. Based on the speed, efficiency and specificity of CRISPR-Cas9, it is now widely used in the field of cancer biology and considered to be a potential tool for clearing stemness among cancer cell though several of these studies are still in animal models and has not tried in human. Despite of all these salient features of CRISPR-Cas9 system, it reached the limelight among genetic scientists when the first report came out which

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described its use in editing “human embryo”25. The group studied the ability of the CRISPR/Cas9 system to edit the gene called HBB, which encodes the human β-globin protein. Mutations in the gene are responsible for β-thalassaemia, a blood disorder and the team successfully used the system as therapeutic agent to repair the mutant gene from the germ line of the human embryo. Though the system could successfully edit the genome, the study reported off-target mutations introduced by the CRISPR/Cas9 system on other parts of the genome pose a serious threat in using the system as such for clinical applications.

Ethical Concerns and Limitations of the Technology The simplicity of creating CRISPR paves way to scientists across the globe to carry out genome editing experiments for therapeutic applications, but working on human embryos or germinal cells poses a serious threat as if any unintended off-target effects introduced by the system during editing could have unpredictable effects to the future generations. Apart from promising clinical applications by treating genetic diseases, the technology may be used for treating unwanted characteristics, for example a report on successful change of the coat color of the rat, open door for inducing pigmentation change in human through genome editing in embryo. This may lead to genetic enhancement of a specific appearance and loss of human diversity and eugenics. Thus Genome editing of the human embryo could hinder the ongoing research that involve gene editing of somatic cells that hold promise for therapeutic development. An important limitation of the technology is the cost of the gene-editing tools. The cost of the technique is too high for the developing countries to afford and thus may not be implemented effectively all over the world.

expression of the gene, thus delivery methods should be properly optimized so that there is no off target activity or immunological responses. Nevertheless, despite all the challenges and the serious ethical issues

associated, with improvement in the designing of the tool and careful ethical considerations, CRISPR/Cas9 technology holds immense promises for bringing gene therapy into clinics.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Krishan K, Kanchan T. Human genome editing and ethical considerations. Sci Eng Ethics. 2016; 22:597-9. Billings PR, Hubbard R, Newman SA. Human germline gene modification: a dissent. Lancet 1999;353:1873−5. Lanphier E, Urnov F, Haecker SE, Werner M, Smolenski J. Don’t edit the human germ line. Nature. 2015; 519: 410-11. Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol. 2001; 21:289–97. Vaishnaw AK, Gollob J, Gamba VC, Hutabarat R, Sah D, et al. A status report on RNAi therapeutics. Silence. 2010;1:14. Kay MA. State-of-the-art gene-based therapies: the road ahead. Nature Rev Genet. 2011;12:316–28. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nature Rev Genet. 2010;11:636–46. Scharenberg AM, Duchateau P, Smith J. Genome engineering with TAL-effector nucleases and alternative modular nuclease technologies. Curr Gene Ther. 2013;13:291–303. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR Cas9 for genome engineering. Cell. 2014;157:1262–78. Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: Prospects and challenges. Nat Med. 2015; 21: 121-31. Tebas P, Stein D, Tang WW, Frank I, Wang SQ, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014;370:901–10. Li H,Haurigot V, Doyon Y, Li T, Wong SY, et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature.2011;475:217–22. Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 2014;32:551–3. Schwank G, Koo BK, Sasselli V, Dekkers JF, Heo I, et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell. 2013;13:653–8 Ousterout DG, Pinera PP, Thakore PI, Kabadi AM, Brown MT. et al.Reading frame correction by targeted genome editing restores dystrophin expression in cells from Duchenne Muscular Dystrophy patients. Mol Ther.2013;21:1718–30. Long C, McAnally R, Shelton JM, Mireault A, Duby R, et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014;345:1184–8. Holt N, Wang J, Kim K, Friedmann G, Wang X et al. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat. Biotechnol. 2010;28: 839–47. Perez EE, Wang J, Miller J, Jouvenot Y, Kim AK, et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 2008;26: 808–16. Ye L, Wang J, Beyer AI, Teque F, Cradick TJ, et al. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Delta32 mutation confers resistance to HIV infection. Proc Natl Acad Sci USA. 2014;111:9591–6 Lin SR, Yang HC, Kuo YT, Liu CJ, Yang TY, et al. The CRISPR/Cas9 System facilitates clearance of the intrahepatic HBV templates In Vivo. Mol Ther Nucleic Acids. 2014;3:e186. Bloom K, Ely A, Mussolino C, Cathomen T, Arbuthnot P. Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activatorlike effector nucleases. Mol Ther. 2013; 21:1889-97. Genovese P, Schiroli G, Escobar G, Tomaso T, Firrito C, et al. Targeted genome editing in human repopulating haematopoietic stem cells. Nature.2014; 510:235–40. Ma N, Liao B, Zhang H, Wang L, Shan Y et al. Transcription activator-like effector nuclease (TALEN)-mediated gene correction in integration-free β-thalassemia induced pluripotent stem cells. J Bio Chem. 2013; 288:34671-79. Xie F, Ye L, Chang JC, Beyer A, Wang J, et al. Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggy Bac. Genome Res. 2014; 24:1526–33. Liang P, Xu Y, Zhang X, Ding C, Huang R, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6:363-72. Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature 2012; 482: 331–8. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337:816–21.

Future Implications and Conclusions Though CRISPR-Cas9 genome editing tools holds promise to personalized medicine, translating genome editing tools to the clinic involves major challenges with respect to efficacy and safety. The specificity of the tool should be improved so that the system will not introduce any off-target effect in the genome. Similarly the mode of delivery of the editing tools into the desired cells decides the transient or permanent

Ann. SBV, Jan-Jun 2016;5(1)

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microRNA, A Clinical Diagnostic and Prognostic Biomarker

miRNA Biogenesis microRNA, A Clinical Diagnostic and Prognostic Biomarker Anitha TS, Scientist Central Inter-Disciplinary Research Facility Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India Email:tsanitha6@gmail.com

Abstract 

microRNAs, small non-coding RNAs, have recently been emerged as powerful regulators in a variety of cellular processes especially important roles in disease and tissue remodeling. Apart from involvement in a variety of biological processes, microRNAs were early recognized for their potential use as biomarkers in disease diagnostics and prognosis. Currently, there are a number of microRNAs helping clinicians to determine the origins of cancer in disseminated tumors. The development of microRNA therapeutics has proved more challenging mainly due to delivery issues. This review focuses on the potential role of microRNA as clinical diagnostic and prognostic biomarkers. In addition, it highlights the microRNA profiling techniques, thereby leading to the advance opportunities to safely pursue microRNA as therapeutic modalities. Key Words: miRNAs, biomarkers, diagnostic markers, prognostic markers, profiling

Introduction

miRNA Basics

microRNAs (miRNAs) are evolutionarily conserved small non-coding RNA molecules that regulate gene expression, and their recent discovery is revolutionizing both basic biomedical research and drug discovery. The human genome is believed to encode ~1,000 miRNAs. A repository of miRNAs from many organisms has been listed in the miRBase Sequence Database1, that contains sequences and annotation2. More than 25,000 miRNAs have been described in man, worms, Drosophila, and also in the small plant Arabidopsis thaliana3. This review aims to describe the basics of miRNA and their role as biomarkers in clinical diagnostics and prognostic values. Moreover, this review also discusses the promising methods for measuring miRNA expression profiles in various biological samples such as cells, tissues and body fluids.

Gary Ruvkun and Victor Ambros in 1990, first discovered miRNAs in Caenorhabditis elegans and their target gene4,5. Together, these two seminal discoveries identified a novel mechanism for post-transcriptional gene regulation. A hairpin fold-back structure from the precursor transcript separates the miRNAs from other small RNAs with expression confirmation of about 22 nucleotide-long mature sequence. Presently, based on deep-sequencing data, 1600 human miRNA precursors have been deposited into miRBase v196. The nomenclature of these miRNAs is based on a “mir” or “miR” prefix with identifying numbers assigned sequentially at the time of discovery. “mir” represents a precursor miRNA whereas “miR” denotes a mature miRNA sequence. Similar or identical sequences can be given the same number.

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miRNA genes reside either in intergenic regions, within introns of coding or non-coding genes or within exons of non-coding genes7. Approximately one third of miRNAs are intergenic and most of all miRNA loci contain clustered miRNAs (miRBase v19). The majority of miRNAs are transcribed as long primary transcripts by RNA polymerase II and many are capped and polyadenylated8,9. Analysis of miRNAs residing in intergenic primary transcripts indicates that such transcripts are shorter than protein-coding transcripts with transcriptional start sites about 2 kb upstream of the pre-miRNA and polyadenylation signals 2 kb downstream10. A subset of miRNAs is transcribed by RNA polymerase III. This cluster of miRNAs is located among Alu rich regions on chromosome 1911. Pri-miRNAs fold into hairpin structures containing imperfectly base-paired stems and are processed into 60- to 100-nt hairpins known as pre-miRNAs. The premiRNAs are exported from the nucleus to the cytoplasm by exportin 5, where they, in general, are cleaved by the endonuclease Dicer to yield imperfect miRNAmiRNA* duplexes12. The miRNA strand is selected to become a mature miRNA, whereas, most often, the miRNA* strand is degraded. The mature miRNA that is added to the RNA-induced silencing complex (RISC) identifies the potential precise targets and activates posttranscriptional gene silencing13. On the other hand, an alternative biogenesis pathway was revealed in which miR-451 enters RISC by direct loading of the pre-miR into RISC after Drosha processing, by skipping further processing by Dicer14. Transcription of miRNAs occurs through RNA polymerase II and subsequent processing is mediated by the nuclear ribonuclease III (RNase III) enzyme Drosha to form precursor miRNAs (70–100 nucleotides). Following transportation to the cytoplasm by exportin5, a further cleavage occurs via another RNase III enzyme, Dicer, to form the mature miRNA. miRNAs modulate both physiological and pathological pathways by post-transcriptionally inhibiting the expression of a multitude of target genes. Much work has been done on the role of miRNAs in human disease, especially in cancers and infections.

Use of miRNA as Biomarkers in Clinical Diagnosis miRNAs exhibit strict developmental and tissuespecific expression patterns in organ and immune system development. For example, miR-1 is involved Ann. SBV, Jan-Jun 2016;5(1)

in mammalian heart development15, miR-375 regulates pancreatic insulin secretion16, miR-181 influences the differentiation of hematopoietic cells toward the B-cell lineage17, and miR-430 is required for zebrafish brain development18. These studies highlight the participation of miRNAs in diverse cellular processes. Hence, it is not surprising that dysregulation of miRNA function is associated with many human diseases such as diabetes, neurological disorders, and cancer.

miRNA as Biomarker for Cancer miRNAs play a critical role in the development of cancer and can influence cancer-promoting and cancersuppressing genes. The first documentation of a miRNA abnormality in cancer stemmed from studies of human chromosome 13q1419. In cancer, miRNA expression variations are evident across different stages of cancer progression20. In tumorigenesis, over-expression of certain miRNA down-regulates tumor suppressor genes. These miRNAs can be exploited as potential biomarkers due to their tissue specificity and to tumor type and its origin21. An increasing number of miRNAs are now identified and utilized as prognostic miRNAs to predict drug response. Glioblastoma The primary brain tumor, glioma arises from glial cells of the central nervous system (CNS). Even after aggressive treatment like surgical resection and chemotherapy, glioblastoma multiforme patients show the least promising prognosis, where the reappearance is frequent and mean survival is only 12–15 months22. The intricacy in determining an explicit biomarker for glioma lies in part with the complex heterogeneous nature of the cancer itself. miRNA signatures have been recognized in both glioblastoma tissue and in blood circulation of glioblastoma patients. Recently, deep sequencing method has produced one of the largest sets of miRNA profiles for glioblastoma and control brain tissue. This study identified 33 up-regulated miRNA in the glioblastoma tissue and 40 down-regulated. In addition, 18 novel miRNAs and 16 novel miRNA-3ps were identified (miRNA-3p, miR-3676, miR-204, miR-539, miR-758, miR-382, miR-1271miR-98, miR-1307, miR-181b1-miR-873, miR-212, miR-135a-2, miR511-1, miR-301a, miR-381, miR-487a)23. Breast Cancer Profiling studies of miRNA have led to the categorization of miRNAs that are abnormally expressed in human Page 21

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breast cancer, with down regulation of miR-125b, miR-10b and miR-145 and up-regulation of miR21, miR-9 and miR-155. When comparing breast cancer tissue with the normal tissues, 29 differentiallyexpressed miRNAs were acknowledged, and a subset of 15 miRNAs could be used to discriminate tumor from normal. In recent years, the discovery of upregulation of miR-10b in promoting invasion and metastasis in most cancers, exhibited a downregulation in metastatic breast cancers, which is validated by migration and invasion assay24. miR-9 up-regulation in breast cancer cells, directly targets CDH1, the E-cadherinencoding mRNA, leading to increased cell motility and invasiveness25. Interestingly, miR-378(*) was recognized as a molecular switch in cancer cell bioenergetics pathway, also known as the Warburg effect, by the regulation of ERBB2 expression26. On the other hand, increasing the expression of few tumor suppressor miRNAs can alleviate development of breast tumors27. For example, the expression of tumor suppressor miR-127 down-regulates the expression profile of proto-oncogene BCL6, a potential target of miR-12728. Colorectal Cancer The major reason for the failure of treating advanced colorectal cancer is chemoresistance29. Several miRNAs have been found to be associated (miR-192, miR-215, miR-140, miR-129, let-7, miR-181b, miR-200 s) with chemoresistance by regulating key cell death pathways such as apoptosis and autophagy. Several important miRNA were described that regulate targets such as Bcl2, thymidylate synthase, dihydrofolate reductase, histone deacetylase, and E2F. For example, miR215 was identified to suppress the expression of both thymidylate synthase and dihydrofolate reductase30. In addition, the expression of miR-215 was directly regulated by p53. The expression of miR-215 was significantly associated with colorectal cancer patient survival. Another miRNA, miR-140, was found to modulate chemosensitivity by suppressing HDAC4 expression, and the levels of miR-140 and miR-215 were elevated in colon cancer stem cells31. Furthermore, miR-194 was identified to regulate BMI-1 protein expression (BMI-1 is involved in epithelial to- mesenchymal transition)32. Moreover, miR-502 regulates autophagy in colon cancer by targeting Rab1B33. Taken together, these miRNAs can be utilized in predicting patient’s prognosis and survival. Page 22

miRNA as Biomarker for Diabetes Mellitus (DM) The circulating miRNA (serum and plasma) characterizes a unique form of disease initiation and development. Due to a variety of pathogenesis of diverse types of DM, the differential regulatory roles of miRNA leading to disease outcomes, including β-cell deficiency and insulin resistance, have recently been defined. In type 1 DM, the role of miRs in controlling β-cell genesis, β-cell death (miR-21), insulin production (miR-30d, miR204, miR-124a) and α/ β-cell mass balance (miR-375) or its susceptibility to immunemediated β-cell destruction has been described34-39. In regard to type 2 DM, insulin resistance miRNAs, the key regulators for homeostasis, has been characterized based on the differential expression of the candidate miRNA in insulin targeted DM patients. Some of the insulin sensitivity related miRNA in adipocytes (miR-21, miR-29, miR-93, miR-103, miR-143, miR-320), muscle (miR-1, miR-106b, miR-133a, miR-223), and liver (let-7, miR-130a-3p, miR-143, miR-181a, miR-802) were essential in maintaining physiological homeostasis and energy balance40-42. Studies by Zampetaki et al.43 showed decreased levels of 10 miRNAs in plasma of diabetic patients (miR15a, miR-20b, miR-21, miR-24, miR-126, miR-191, miR-197, miR-223, miR-320 and miR-486).

miRNA as Biomarker for Neurodegenerative Disease Neurodegenerative diseases include several central nervous system disorders characterized by the progressive loss of neural tissues and CNS damage. Hence, early diagnosis is essential to maximize the effectiveness of disease-modifying therapies. In recent years, much effort has been taken to recognize the neuropathological, biochemical, and genetic biomarkers of the diseases so that the diagnosis could be established in the earlier stages. The biomarkers for Alzheimer’s, Parkinson disease and other neurodegenerative diseases must be reliable and specific, and they should be useful in guiding us to make more accurate diagnosis and better treatment of the diseases. Alzheimer’s Disease (AD) miRNAs has been demonstrated as potential noninvasive biomarkers from blood and serum for a wide variety of human pathologies44. A deregulation of miRNA expression might be involved in Ann. SBV, Jan-Jun 2016;5(1)

neurological dysfunction or neurodegenerative processes. Interestingly, Liang et al.45 showed a specific expression signature pattern of brain and blood mononuclear cells as a useful biomarker for AD and other neurological diseases. Though altered miRNA expression patterns have been extensively investigated in AD patients’ tissue samples or cell cultures46, yet less information on circulating miRNAs in AD is known. A recent serum profiling of AD patients provided first evidence that expression changes of circulating miRNAs may be valuable biomarkers for AD 47. Recently, Leidinger et al. 48 has reported 140 unique differentially expressed miRNAs between AD patients and healthy controls. Further, Lugli et al.49 has reported that the expression of microRNAs in plasma fraction enriched in exosomes showed twenty miRNAs with significant differences in the AD group (miR23b-3p, miR-24-3p, miR-29b-3p, miR-125b-5p, miR-138-5p, miR-139-5p, miR-141-3p, miR150-5p, miR-152-3p, miR-185-5p, miR-338-3p, miR-342-3p, miR-342-5p, miR-548at-5p, miR659-5p, miR-3065-5p, miR-3613-3p, miR-3916, miR-4772-3p, miR-5001-3p). Recently, in CIDRF, one of the Scientist, Dr.Doulathunnisa had been working on to determine the effect of artificial sweeteners on miRNA expression that leads to the regulation of AD. Schizophrenia A hemizygous deletion of a 1.5–3-Mb region of chromosome 22 can lead to the 22q11 deletion syndrome (22q11DS), which is characterized by multiple physical and psychiatric abnormalities. A previous study determined that ~30% of 22q11DS patients may develop schizophrenia50. miR-25 and miR-185 are regulators of the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA2), which is responsible for loading Ca2+ into the endoplasmic reticulum. Earls et al., found that miR-25 and miR185 were depleted in mouse models of 22q11DS and restoration of these miRNAs to presynaptic neurons rescued the long-term potentiation of DGCR8 +/− mice 51. The authors concluded that miRNA-dependent SERCA2 dysregulation is a pathogenic event in 22q11DS and schizophrenia. Gardiner et al.52 investigated the expression profile of miRNA in PBMCs of 112 patients and identified 83 miRNAs that were significantly downregulated in the schizoaffective group on chromosome 14q32. Similarly, Lai et al.53 identified a signature of seven miRNAs in an initial cohort of 30 patients with Ann. SBV, Jan-Jun 2016;5(1)

schizophrenia which included the upregulated miR34a, miR-449a, miR-564, miR-548d, miR-572 and miR-652, and downregulated miR-432.

miRNA as Biomarkers for Pulmonary Disease Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis. Some studies have identified a group of miRNAs that are expressed specific and play regulatory roles in the interaction between M.tuberculosis and host cells; these include miR-223, miR-144, and miR-421. In a collaborative project between CIDRF and Pulmonary medicine, sputum and serum miR-144 levels among newly diagnosed pulmonary tuberculosis patients before and after treatment will be correlated.

Use of miRNA as Prognostic Markers microRNAs (miRNAs), endogenous small noncoding RNAs, are found to be detected in plasma, serum, saliva and urine. The diagnosis and prognosis of different pathological conditions are linked with individual miRNAs and their signature patterns54. Tumor-specific miRNAs have been identified in cancer patients55. Plasma miRNAs derived from tissues have been used as biomarkers for injury56. Also, alterations in circulating miRNAs have been found in cardiovascular diseases, diabetes melletius, neurodegenerative disease, as well as autoimmune diseases57-59. A list of miR as prognostic markers has been listed in Table 1. Cardiovascular Disease Circulating miRNAs have been investigated as possible novel prognostic markers for prediction of CHD progression and adverse outcomes. Several studies have explored associations between cardiac function and miRNAs as a predictor of future adverse outcomes in CHD. miR-134, -198, and -370, a miR signature pattern was detected by Hoekstra et al.60 in view to differentiate unstable from stable angina pectoris suggesting a potential prognostic tool in cardiovascular disease. miR-370 has been identified as a possible prognostic biomarker in an earlier study in mice. Also, increased miR-370 expression levels had been found in response to induced ischemia61. Zampetaki et al.62 has reported three signature miRNAs ((miR-126, miR197 and miR-223) for the prediction of myocardial infarction (MI), where miR-126 levels are found to be positive, while the other miRs are inversely associated with future MI. Page 23

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Figure 1. miRNA profiling techniques ( Pritchard et al, 201254, reproduced with permission)

Table 1. List of miRNA as prognostic marker in cancerous tissues.

miRNA miR-205; Let-7F miR-145 miR-124 miR-23b

Cancer type

Ovarian cancer

miR-200 and miR-203, miR-30a and miRMetastatic breast cancer 155

References Zheng et al., 201367 Zhang et al., 201368 Wang et al., 201369 Madavan et al., 201670

let-7c, let-7e, miR-30c, miR-622, and miRcastration-resistant prostate 1285, miR-141 and miR-375; exosomal miRHuang et al., 201571 cancer 1290 and miR-375 miR-148b, miR-376c, miR-409-3p, and Primary breast cancer miR-801

Cuk et al., 201372

miR-25 and miR-223, miR-574-5p and miR1254, miR-21, miR-126, miR-486-5p, and non-small-cell lung cancer miR-210

Shen et al., 201173

miR-17-3p and miR-92, miR-601 and miRColorectal cancer 760, miR-221, miR-29b

Wang et al., 201274, Inoue et al., 201575

miR-328

Yuan et al., 201676

Glioblastoma

Diabetes Mellitus miRNAs are being available as biomarkers for monitoring of disease onset and progression. They have a great tendency to serve as accurate diagnostic and prognostic markers, as well as being viable therapeutic targets for treating diabetes complications. A large number of specific miRNAs have Cancer As an important factor in tumorigenesis, microRNAs (miRNAs) are anticipated as potential biomarkers for early cancer detection and accurate prognosis as well as targets for more efficient treatment. Alterations in miRNAs lead to resistance for anticancer drugs and are well-known to be dysregulated in cancer; current Page 24

literature revealed that miRNA levels in biological samples may be interrelated with chemotherapy response. miRNA expression profiles vary between normal tissues and cancerous cells derived from the same organ, and also between cancer types miRNAs can either function as oncogenes nor tumor suppressors, thereby leading to various pathways in tumorigenesis63. They may be used for prognostic purposes and they also constitute novel targets for cancer treatment. Recently, the evidence for the roles of miRNAs in determining drug sensitivity/resistance has been emerging. miRNA that can be used as prognosis in different types of cancer have been listed in table. Also, human mesenchymal stromal/stem cells generally have the potency to differentiate into Ann. SBV, Jan-Jun 2016;5(1)

various mesenchymal cell lineages that makes them a challenging cell source for the use in tissue repair strategies. Georgi et al.64 has recently investigated that profiling of hMSC donors for specific panel of miRNAs could serve as a prognostic marker for selecting donors with high differentiation potential to improve hMSCbased tissue repair approach. Peripheral blood miRNA has shown expression patterns that serve as a challenging prognostic tool in primary CNS lymphoma patients65.

miRNA Profiling miRNA expression profiling is mainly been done to identify miRNAs that play a critical role in organismal development, establishment and maintenance of tissue differentiation as biomarkers and serve as reagents for the reprogramming of cell fate in stem cell applications. miRNA profiling has attracted many researchers to Ann. SBV, Jan-Jun 2016;5(1)

work in various research areas of biology and medicine. miRNA profiling is defined as the measurement of the relative abundance of a cohort of miRNAs, ranging from a group of several miRNAs of specific biological interest to comprehensive profiling of all miRNAs in a given species (typically numbering in the several hundred)66. Assaying such small RNA molecules poses some inherent challenges, but technological advances in recent years have overcome many of these barriers, and a wide range of approaches and platforms is now available for miRNA profiling (Figure 1). Quantitative reverse Transcription PCR-based Methods. One major approach relies on reverse transcription of miRNA to cDNA, followed by qPCR with realtime monitoring of reaction product accumulation Page 25

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(known as ‘realtime PCR’). An appealing aspect of this approach is the ease of incorporation into the workflow for laboratories that are familiar with realtime PCR. In order to scale this approach for miRNA profiling, reactions are carried out in a highly parallel, high-throughput form (that is, hundreds of qRT-PCR reactions measuring different miRNAs using the same reaction conditions). Two common strategies used for priming the reverse transcription reaction to generate cDNA are enzymatic addition of a poly (A) tail and generation of a reverse transcription primer binding site using a stem–loop primer. A hurdle in performing highly parallel qRT-PCR is that optimal reaction conditions may vary substantially between miRNAs owing to sequence-specific differences in primer annealing. Although different vendors have sought to solve this problem using various approaches, one effective strategy has been the incorporation of locked nucleic acids (LNAs) into primers to standardize optimal miRNA primer hybridization conditions for the hundreds of PCR assays that are to be run simultaneously. Hybridization-based Methods The first method to perform parallel analysis of large number of miRNAs is done by microarrays, where different approaches for fluorescent labelling of the miRNA in a biological sample for subsequent hybridization to DNA- based probes on the array will be performed. One commonly used labelling approach is the enzymatically catalysed ligation of a fluorophoreconjugated nucleotide or short oligonucleotide to the terminal 3′-OH of the miRNA using T4 RNA ligase. Another enzymatic-labelling approach involves 3′ tailing of the miRNA (for example, with poly (A), following which a fluorophore-conjugated oligo-nucleotide may be ligated using a splinted ligation. Alternative chemical approaches to miRNA labelling exist, that includes chemical alkylation-based labelling along the miRNA and approaches based on platinum coordination chemistry with nucleic acids. It should also be kept in mind that other cellular RNAs, in addition to miRNAs, may be labelled by both enzymatic and chemical approaches, which can contribute to background signal as well as to cross-hybridization with specific miRNA probes.

RNA-sequencing The advent of next-generation sequencing platforms has enabled a third major approach for miRNA expression profiling. The general approach begins with the preparation of a small RNA cDNA library from the RNA sample of interest, followed by the ‘massively parallel’ sequencing of millions of individual cDNA molecules from the library. Bioinformatic analysis of the sequence reads identifies both known and novel miRNAs in the data sets and provides relative quantification using a digital approach (that is, the number of sequence reads for a given miRNA relative to the total reads in the sample is an estimate of relative abundance of the miRNA). The major advantages of next-generation sequencing for miRNA profiling are detection of both novel and known miRNAs and precise identification of miRNA sequences (for example, RNA-seq can readily distinguish between miRNAs that differ by a single nucleotide, as well as isomiRs of varying length). However, it should be noted that RNA-seq-based miRNA-profiling studies typically identify a plethora of small RNAs of novel sequence (that is, putative miRNAs), but not all of these may be bona fide miRNAs. Potential limitations of next-generation sequencing include the high cost, although this is dropping with the introduction of newer versions of the instruments, and the use of DNA ‘barcoding’, which permits multiplexing of many samples in a single run.

Conclusion Taking into account of the use of miRNA as biomarkers for clinical diagnostics and prognostics values outlined in this review will allow for more precise understanding for their potential use in clinical applications. Further elucidation of miRNA biogenesis and functionality will enable the development of more specific and sensitive assays. Enhancing the art of performing research and implying its application in clinical set-up will lead to exciting novel gene regulators. Also, their specific functions will augment the opportunities to safely pursue them as therapeutic modalities.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.

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miRBase Sequence Database. Available online: http://microrna.sanger.ac.uk/sequences (accessed on 23 May 2014). Kozomara A, Griffiths-Jones S. miRBase: Integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res. 2011; 39:D152–57. Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC et al. A uniform system for microRNA annotation. RNA. 2003; 9:277–9. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell. 1993; 75:855–62. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993; 75:843–54. Griffiths-Jones S, Saini HK, van Dongen S, Enright AH. miRBase: tools for microRNA genomics. Nucleic Acids Res. 2008; 36:D154–8. Kim VN, Han J, Siomi MC. Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol. 2009; 10:126–39. Baskerville S, Bartel DP. Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA. 2005; 11:241–7. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 2007; 129:1401–14. Saini HK, Griffiths-Jones S, Enright AJ. Genomic analysis of human microRNA transcripts. Proc Natl Acad Sci USA. 2007; 104:17719–24. Borchert GM, Lanier W, Davidson BL. RNA polymerase III transcribes human microRNAs. Nat Struct Mol Biol. 2006; 13:1097–101. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009; 136:215–33. Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell. 2003; 115:209–16. Cheloufi S, Dos Santos CO, Chong MM, Hannon GJ. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature. 2010; 465:584–9. Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature. 2005; 436:214-20. Li X. MiR-375, a microRNA related to diabetes. Gene. 2014; 533:1-4. Chen CZ, Li L, Lodish HF, Bartel DP. MicroRNAs modulate hematopoietic lineage differentiation. Science. 2004; 303:83–6. Giraldez AJ, Cinalli RM, Glasner ME, Enright AJ, Thomson JM, et al. MicroRNAs regulate brain morphogenesis in zebrafish. Science. 2005; 308:833-8. Döhner H, Stilgenbauer S, Benner A, Leupolt E, Kröber A, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000; 343:1910-6. Mishra PJ. MicroRNAs as promising biomarkers in cancer diagnostics. Biomark Res. 2014; 2:19. Mishra PJ, Merlino G. MicroRNA reexpression as differentiation therapy in cancer. J Clin Invest. 2009; 119:2119–23. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 2009; 10:459–66. Hua D, Mo F, Ding D, Li L, Han X, et al. A catalogue of glioblastoma and brain microRNAs identified by deep sequencing. OMICS. 2012; 16:690-9. Ma L, Teruya-Feldstein J, Weinberg RA. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature. 2007; 449:682–8 Ma L, Young J, Prabhala H, Pan E, Mestdagh P, et al. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol. 2010; 12:247-56. Eichner LJ, Perry MC, Dufour CR, Bertos N, Park M, et al. miR-378 (*) mediates metabolic shift in breast cancer cells via the PGC-1β/ERRγ transcriptional pathway. Cell Metab. 2010; 12:352-61. Lowery AJ, Miller N, McNeill RE, Kerin MJ. MicroRNAs as prognostic indicators and therapeutic targets: potential effect on breast cancer management. Clin Cancer Res. 2008; 14:360-5. Saito Y, Liang G, Egger G, Friedman JM, Chuang JC, et al. Specific activation of microRNA-127 with downregulation of the proto-oncogene BCL6 by chromatin-modifying drugs in human cancer cells. Cancer Cell. 2006; 9:435-43. Ju J, Jiang J, Fesler A. miRNA: the new frontier in cancer medicine. Future Med Chem. 2013; 5:983–5. Song B, Wang Y, Titmus MA, Botchkina G, Formentini A, et al. Molecular mechanism of chemoresistance by miR-215 in osteosarcoma and colon cancer cells. Mol Cancer. 2010; 9:96. Karaayvaz M, Pal T, Song B, Zhang C, Georgakopoulos P, et al. Prognostic significance of miR-215 in colon cancer. Clin Colorectal Cancer. 2011; 10:340–7. Zhai H, Karaayvaz M, Dong P, Sakuragi N, Ju J. Prognostic significance of miR-194 in endometrial cancer. Biomark Res. 2013; 1:12. Zhai H, Song B, Xu X, Zhu W, Ju J. Inhibition of autophagy and tumor growth in colon cancer by miR-502. Oncogene. 2013; 32: 1570–9. Lynn FC, Skewes-Cox P, Kosaka Y, McManus MT, Harfe BD, et al. MicroRNA expression is required for pancreatic islet cell genesis in the mouse. Diabetes. 2007; 56: 2938–45. Poy MN, Hausser J, Trajkovski M, Braun M, Collins S, et al. miR-375 maintains normal pancreatic alpha- and beta-cell mass Proc Natl Acad Sci USA. 2009; 106: 5813–8. Tang X, Muniappan L, Tang G, Ozcan S. Identification of glucose-regulated miRNAs from pancreatic beta cells reveals a role for miR-30d in insulin transcription. RNA. 2009; 15: 287–93. Mi QS, He HZ, Dong Z, Isales C, Zhou L. MicroRNA deficiency in pancreatic islet cells exacerbates streptozotocin-induced murine autoimmune diabetes. Cell Cycle. 2010; 9: 3127–9. Ruan Q, Wang T, Kameswaran V, Wei Q, Johnson DS, et al. The microRNA-21-PDCD4 axis prevents type 1 diabetes by blocking pancreatic beta cell death. Proc Natl Acad Sci USA. 2011; 108: 12030–5. Xu G, Chen J, Jing G, Shalev A. Thioredoxin-interacting protein regulates insulin transcription through microRNA-204. Nat Med. 2013; 19: 1141–6. Ling HY, Hu B, Hu XB, Zhong J, Feng SD, et al. miRNA-21 reverses high glucose and high insulin induced insulin resistance in 3T3-L1 adipocytes through targeting phosphatase and tensin homologue. Exp Clin Endocrinol Diabetes. 2012; 120:553–9. Chen YH, Heneidi S, Lee JM, Layman LC, Stepp DW, et al. miRNA-93 inhibits GLUT4 and is overexpressed in adipose tissue of polycystic ovary syndrome patients and women with insulin resistance. Diabetes. 2013; 62: 2278–86. Frost RJ, Olson EN. Control of glucose homeostasis and insulin sensitivity by the Let-7 family of microRNAs. Proc Natl Acad Sci USA. 2011; 108:21075–80. Zampetaki A, Kiechl S, Drozdov I, Willeit P, Mayr U, et al. Plasma microRNA profiling reveals loss of endothelial miR-126 and other microRNAs in type 2 diabetes. Circ Res. 2010; 107: 810–17. Wang K, Yuan Y, Cho JH, McClarty S, Baxter D, et al. Comparing the microRNA spectrum between serum and plasma. PLoS One. 2012; 7:e41561. Liang Y, Ridzon D, Wong L, Chen C. Characterization of microRNA expression profiles in normal human tissues. BMC Genomics. 2007; 8:166. Long JM, Lahiri DK. microRNA-101 downregulates Alzheimer’s amyloid-beta precursor protein levels in human cell cultures and is differentially expressed. Biochem Biophys Res Commun. 2010; 404: 889-95. Geekiyanage H, Jicha GA, Nelson PT, Chan C. Blood serum miRNA: non-invasive biomarkers for Alzheimer’s disease. Exp Neurol. 2011; 235: 491-6. Leidinger P, Backes C, Deutscher S, Schmitt K, Mueller SC, et al. A blood based 12-miRNA signature of Alzheimer disease patients. Genome Biol. 2013; 14:R78. Lugli G, Cohen AM, Bennett DA, Shah RC, Fields CJ, et al. Plasma exosomal miRNAs in persons with and without Alzheimer disease: Altered expression and prospects for Biomarkers. PLoS One. 2015; 10:e0139233. Williams NM. Molecular mechanisms in 22q11 deletion syndrome. Schizophr Bull. 2011; 37:882-9. Earls LR, Fricke RG, Yu J, Berry RB, Baldwin LT, et al. Age-dependent microRNA control of synaptic plasticity in 22q11 deletion syndrome and schizophrenia. J Neurosci. 2012; 32: 14132-44. Gardiner E, Beveridge NJ, Wu JQ, Carr V, Scott RJ, et al. Imprinted DLK1-DIO3 region of 14q32 defines a schizophrenia-associated miRNA signature in peripheral blood mononuclear cells. Mol Psychiatry. 2012; 17:827-40. Lai CY, Yu SL, Hsieh MH, Chen CH, Chen HY, et al. MicroRNA expression aberration as potential peripheral blood biomarkers for schizophrenia. PLoS One. 2011; 6:e21635.

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Annals of SBV 54. Pritchard CC, Cheng HH, Tewari M. MicroRNA profiling: approaches and considerations. Nat Rev Genet. 2012; 13:358-69. 55. Zubakov D, Boersma AW, Choi Y, van Kuijk PF, Wiemer EA, et al. MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation. Int J Leg Med. 2010; 124:217–26. 56. Jeyaseelan K, Lim KY. MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke. 2008; 39:959–66. 57. Tanaka M, Oikawa K. Down-regulation of miR-92 in human plasma is a novel marker for acute leukemia patients. PLoS One. 2009; 4:e5532. 58. Wang K, Zhang S. Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci USA. 2009; 106: 4402–07. 59. Wang GK, Zhu JQ. Circulating microRNA: A novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J. 2010; 31:659–66. 60. Tijsen AJ, Creemers EE. miR423-5p as a circulating biomarker for heart failure. Circ Res. 2010; 106: 1035–9. 61. Igaz I, Szőnyi M, Varga P, Topa L. Potential relevance of microRNAs in the diagnostics of inflammatory bowel diseases. Orv Hetil. 2014; 155: 487–91. 62. Hoekstra M, van der Lans CA, Halvorsen B, Gullestad L, Kuiper J, et al. The peripheral blood mononuclear cell microRNA signature of coronary artery disease. Biochem Biophys Res Commun 2010; 394:792-7. 63. Xu CF, Yu CH, Li YM. Regulation of hepatic microRNA expression in response to ischemic preconditioning following ischemia/reperfusion injury in mice. Omics 2009; 13:513-20. 64. Zampetaki A, Willeit P, Tilling L, Drozdov I, Prokopi M, et al. Prospective study on circulating MicroRNAs and risk of myocardial infarction. J Am Coll Cardiol 2012; 60:290-9 65. Croce CM, Calin GA. miRNAs, cancer, and stem cell division. Cell. 2005, 122:6-7. 66. Georgi N, Taipaleenmaki H, Raiss CC, Groen N, Portalska KJ, et al. MicroRNA Levels as prognostic markers for the differentiation potential of human mesenchymal stromal cell donors. Stem Cells Dev. 2015; 24:1946-55. 67. Zheng H, Zhang L, Zhao Y, Yang D, Song F, et al. Plasma miRNAs as diagnostic and prognostic biomarkers for ovarian cancer. PLoS One. 2013; 8:e77853. 68. Zhang H, Wang Q, Zhao Q, Di W. MiR-124 inhibits the migration and invasion of ovarian cancer cells by targeting SphK1J. Ovarian Res. 2013; 6:84. 69. Wang YQ, Guo RD, Guo RM, Sheng W, Yin LR. MicroRNA-182 promotes cell growth, invasion, and chemoresistance by targeting programmed cell death 4 (PDCD4) in human ovarian carcinomas J Cell Biochem. 2013; 114: 1464–73. 70. Madhavan D, Peng C, Wallwiener M, Zucknick M, Nees J, et al. Circulating miRNAs with prognostic value in metastatic breast cancer and for early detection of metastasis. Carcinogenesis. 2016 Jan 19. 71. Huang X, Yuan T, Liang M, Du M, Xia S, et al. Exosomal miR-1290 and miR-375 as prognostic markers in castration-resistant prostate cancer. Eur Urol. 2015; 67:33-41. 72. Cuk K, Zucknick M, Heil J, Madhavan D, Schott S, et al. Circulating microRNAs in plasma as early detection markers for breast cancer. Int J Cancer. 2013; 132:1602-12. 73. Shen J, Todd NW, Zhang H, Yu L, Lingxiao X, et al. Plasma microRNAs as potential biomarkers for non-small-cell lung cancer. Lab Invest. 2011; 91:579-87. 74. Wang Q, Huang Z, Ni S, Xiao X, Xu Q, et al. Plasma miR-601 and miR-760 are novel biomarkers for the early detection of colorectal cancer. PLoS One. 2012; 7:e44398. 75. Inoue A, Yamamoto H, Uemura M, Nishimura J, Hata T, et al. MicroRNA-29b is a novel prognostic marker in colorectal cancer. Ann Surg Oncol. 2015;22 Suppl 3:S1410-8 76. Yuan J, Zheng Z, Zheng Y, Lu X, Xu L, et al. microRNA-328 is a favorable prognostic marker in human glioma via suppressing invasive and proliferative phenotypes of malignant cells. Int J Neurosci. 2016; 126:145-53.

Therapeutic Epigenetics – A Boon to the Future? Benet Bosco Dhas D, Scientist Central Inter-Disciplinary Research Facility Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India Email: benetbiotech@gmail.com

Abstract 

uccessful completion of the Human Genome Project gave the hope for development of S novel therapeutics, diagnostics for the welfare of humankind. Individual genetic studies and genome wide association studies revealed the genetic risk factors for various diseases which can be used in predetermination. This eventually led to the growth of pharmacogenomics that confers individual drug dosage adjustment preventing from adverse effects. However, it addresses only the hitches raised by the underlying genetic sequence but not external factors that influences the genotypic and phenotypic expression. Epigenetic research deals with these factors and studies the modifications caused along with their phenotype. These modifications are reversible which can be used as target for therapeutics, thus improving the treatment strategies of various diseases. In this review, we attempt to discuss the use of epigenetic modifications as drug targets and their mechanism of action.

Key Words: DNA methylation, DNMT inhibitors, HDACi, HATi, miRNA

Introduction For the past two decades, genomics was ruling the medical research, deciphering disease pathophysiology, risk factors, prognostic strategies and much more. But still, it could not answer several questions raised by the research community like the influence of environmental stress in differential gene expression. This eventually led to the development of “Epigenetics”, which explains the genetic behavior apart from underlying nucleotide sequence. By definition, epigenetics is the study of factors (chemicals, proteins, environmental stress, etc.) that influence differential gene expression in cells without changing the nucleotide sequence.

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The journey of epigenetics started less than a century before, when C. H. Waddington coined the term in 19421. Epigenetics always answers a scientific question in three different contexts, DNA methylation, histone modifications and influence of micro RNA (miRNA). A simple example for epigenetic changes is the process of cellular differentiation in a eukaryotic system2. Epigenetics was found to play important roles in disease pathogenesis3, drug resistance4 and prognosis/ diagnosis5. Apart from the somatic heritable nature of epigenetic changes, the property that motivate researchers is that these changes are reversible, which led to the development of novel therapeutics 6. The use of any drug or factors that influence epigenetic changes and benefits the medical treatment is broadly Page 29

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known as “Epigenetic therapy”. In a similar definition, the drugs that alters or reverses the underlying epigenetic changes in a diseased conditions are also included in epigenetic therapy. In this review, we attempt to discuss the research conducted so far, on therapeutic epigenetics, which may have an impact on future medical treatment strategies.

Epigenetics in Diseases One of the epigenetic mechanism, DNA methylation, which was extensively studied, was found to be associated with several diseases such as Rett syndrome7, diabetes8, cancer9 and systemic lupus erythematosus10. In most of the diseases, hypomethylation of CpG islands in promoter region of specific genes and decreased DNMT1 (DNA methyl transferase 1), DNMT3B (DNA methyl transferase) expression were observed11. Methylation and acetylation are the two important modifications that histones undergo, that led differential gene expression. Histone Acetyl Transferase (HATs) and Histone Deacetylases (HDACs) are involved in histone acetylation, whereas Histone Methyl Transferases (HMTs) and Histone Demethylases (HDMs) influences histone methylation12. Acetylation of histones was found to be associated with diseases such as Rubinstein-Taybi syndrome13, asthma14, cancer15 and diabetes8. On the other hand, histone methylation was also significantly associated with Sotos syndrome16, Huntington’s disease13 and cancer15. The third epigenetic mechanism, miRNA, influences differential gene expression through complementary binding with coding messenger RNA (mRNA) and subsequent deactivation with the help of Dicer protein and other associated proteins17. miRNAs like miR-10118, miR-14319, miR-2920 had decreased expression levels in cancer, whereas expression levels of miR-2121 and miR-15522 are found to be increased. The association of miRNA levels was also studied in relation with diabetic conditions (both type I and type II), wherein miR-14423, miR-146a24, miR-2925 and miR27a26 are widely demonstrated with promising results.

Pharmacoepigenomics The successful discoveries made through pharmacogenomics pooled polymorphic allelic data associated with drug response and efficacy under various diseased conditions. For example, cardiovascular patients with CYP2C19 mutant

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variants should undergo clopidogrel dose adjustment to get therapeutic effect or to avoid adverse effects27. Pharmacoepigenomics emerged as an idea to advance the further understanding of drug response and efficacy through in depth molecular analysis, in the early 1990s. Pharmacoepigenomics is the study of epigenetic alterations and the factors involved, in relation with drug response in any diseased condition. The first identified pharmacoepigenomic phenomenon was methylation changes in the drug metabolizing enzyme, CYP2E1, in relation to birth28. Only in the last decade, it was found that tobacco consumption regulates the methylation levels of CYP1A1 gene promoter29. Eventually, several researchers studied the influence of epigenetic changes in drug response in various disease, especially cancer30. Recently, promoter hypomethylation in IGFBP3 was found to be associated with cisplatin response in nonsmall-cell-lung cancer31. Pharmacoepigenomics is also used to predict the outcomes after a chemotherapy. The outcomes of patients with early stage breast cancer after adjuvant tamoxifen therapy can be assessed through PITX2 promoter methylation32. Low recurrence rates of bladder cancer was associated with CDKN2A hypermethylation after interleukin-2 therapy33. In whole, pharmacoepigenomics can also be applied in the development of novel diagnostic/prognostic markers, predictive markers and therapeutic targets, eventually improving the treatment strategies34. DNA methylation as Therapeutic Target Global methylation studies showed that DNA methylation (both hyper- and hypo-) have significant roles in disease pathogenesis, progression and outcomes. The most widely studied disease in relation with DNA methylation is cancer. Earlier it was suspected that DNA hypomethylation is the only phenomenon occurring in carcinogenesis35, but later it was understood that both hypermethylation and hypomethylation of specific genes influences the disease pathophysiology36. DNA methylation can be targeted using enzyme inhibitors like 5-azacytdine that binds to DNMTs and prevent further methylation during replication37. 5-Azacytidine (Vidaza) and its deoxy analogue, 5-aza2’-deoxycytidine (Dacogen) were approved by the US Food and Drug Administration for the treatment of MDS38,39. Treatment with 5-azacytidine improved the survival rate of MDS patients up to 20%40. It was also studied in patients with acute myeloid leukemia (AML), Ann. SBV, Jan-Jun 2016;5(1)

whereas the deoxy analogue was studied in chronic myelomonocytic leukemia (CMML) patients41,42. A recent epigenome wide association study by Ronn et al. revealed altered DNA methylation levels in type 2 diabetes. Some of the genes that are differentially methylated include TCF7L2, IRS1, PPARG and THADA, involved in pathways of cancer, MAPK signaling and axon guidance43. DNA methylation can also be used as potential therapeutic targets in infectious diseases. In our recent study, significant difference in global methylation was found in newborns with sepsis when compared to nonseptic babies44. Epigenome wide association studies revealed protocadherin beta gene hypermethylation which was correlated with decreased leukocyte adhesion, a physiological process of neonatal sepsis45,46. These epigenetic changes can be targeted with novel drugs, reversing to the original state. For example, curcumin, the natural and edible pigment present in Curcuma longa (turmeric) and genistein, another phytochemical compound, showed reversal of hypermethylation of RARβ2 promoter in cervical cancer cell lines47. In a mouse model of Alzheimer’s disease (AD), the methylating agent, Betaine, was found to improve memory48.

Histone Modifications as Epigenetic Target The histone modifications like acetylation and methylation can be reversed by using appropriate enzyme inhibitors. HDAC inhibitors (HDACi) such as Phenylbutyrate and Suberoylanilide hydroxamic acid (SAHA) were used in MDS and AML, improving the hematological parameters49,50.

Valproic acid (HDACi) was found to be useful in the treatment epilepsy, bipolar disorder51, cancer52 and AD53. Ricobaraza et al., showed that sodium phenylbutyrate improved memory in AD mouse model54. The wellknown class III HDACs, also known as Sirtuins (SIRTs), play a role as epigenetic targets in AD and cancer55. Inhibition of HAT p300 using C646was found to reduce the acetylated and phosphorylated tau protein levels, in vitro56. Curcumin also showed HAT inhibiting activity in AD57.

microRNA as Epigenetic T arget RNA therapeutics targeting the non-coding region of amyloid β precursor protein using erythromycin antibiotic, paroxetine antidepressant and N-acetyl cysteine was found to reduce extracellular amyloid β in AD mouse model58.

Limitation of Therapeutic Epigenetics The target of epigenetic therapy are the genes and pathways affected by the epigenetics mechanisms which triggers a caution of non-specificity. If one attempts to reverse the methylation pattern of a silenced gene (hypermethylated) through some drugs, it may nonspecifically effect on other silenced genes like oncogenes. Hence there is an urge to develop technology for gene specific targets for therapeutic epigenetics.

Conclusion With the extensive bench side knowledge developed through genomic and epigenetic research on disease pathogenesis and progression, its time to implement them bed-side. Development of novel genetic and epigenetic therapeutics will pave betterment of medical treatment strategies.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Waddington CH. The epigenotype. Endeavour. 1942;1:18–20. Reik W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature. 2007; 447: 425–32. Glant TT, Mikecz K, Rauch TA. Epigenetics in the pathogenesis of rheumatoid arthritis. BMC Med. 2014;12:35-9. Brown R, Curry E, Magnani L, Wilhelm-Benartzi CS, Borley J. Poised epigenetic states and acquired drug resistance in cancer. Nat Rev Cancer. 2014;14:747–53. Ho S, Johnson A, Tarapore P, Janakiram V, Zhang X, Leung Y. Environmental epigenetics and its implication on disease risk and health outcomes. ILAR J. 2012;53:289–305. Tompkins JD, Hall C, Chen VC, Li AX, Wu X, Hsu D, et al. Epigenetic stability, adaptability, and reversibility in human embryonic stem cells. Proc Natl Acad Sci U S A. 2012 ;109:12544-9. Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004;429:457–63. Villeneuve LM, Natarajan R. The role of epigenetics in the pathology of diabetic complications. Am J Physiol Renal Physiol. 2010;299:F14–F25. Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis. 2010;31:27–36. Javierre BM. Changes in the pattern of DNA methylation associate with twin discordance in systemic lupus erythematosus. Genome Res. 2010;20:170–9. Feng J, Fan G. The role of DNA methylation in the central nervous system and neuropsychiatric disorders. Int Rev Neurobiol. 2009;89:67–84. Keppler BR, Archer TK. Chromatin-modifying enzymes as therapeutic targets – Part 1. Expert Opin Ther Targets. 2008; 12: 1301–12. Urdinguio RG, Sanchez-Mut JV, Esteller M. Epigenetic mechanisms in neurological diseases: genes, syndromes, and therapies. Lancet Neurol. 2009;8:1056–72. Adcock IM, Ito K, Barnes PJ. Histone deacetylation: an important mechanism in inflammatory lung diseases. COPD. 2005;2:445–55.

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Annals of SBV 15. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128:683–92. 16. Berdasco M, Ropero S, Setien F, Fraga MF, Lapunzina P, et al. Epigenetic inactivation of the Sotos overgrowth syndrome gene histone methyltransferase NSD1 in human neuroblastoma and glioma. Proc Natl Acad Sci USA. 2009;106:21830–5. 17. Rana TM. Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol. 2007;8: 23–36. 18. Varambally S, Cao Q, Mani RS, Shankar S, Wang X, et al. Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer. Science. 2008;322:1695–9. 19. Ng EK, Tsang WP, Ng SS, Jin HC, Yu J, et al. MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer. Br J Cancer. 2009;101:699–706. 20. Fabbri M, Garzon R, Cimmino A, Liu Z, Zanesi N, et al. MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B. Proc Natl Acad Sci USA. 2007;104:15805–10. 21. Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer. 2006;6: 857–66. 22. Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell, 2006;9: 189–98. 23. Karolina DS, Armugam A, Tavintharan S, Wong MTK, Lim SC, et al. MicroRNA 144 impairs insulin signaling by inhibiting the expression of insulin receptor substrate 1 in type 2 diabetes mellitus. PLoS One. 2011; 6: e22839. 24. Rong Y, Bao W, Shan Z, Liu J, Yu X, et al. Increased microRNA-146a levels in plasma of patients with newly diagnosed type 2 diabetes mellitus. PLoS One. 2013; 8: e73272. 25. Slusarz A, Pulakat L. The two faces of miR-29. J Cardiovasc Med (Hagerstown). 2015; 16: 480–90. 26. Wang TT, Chen YJ, Sun LL, Zhang SJ, Zhou ZY, et al. Affection of single-nucleotide polymorphisms in miR-27a, miR-124a, and miR-146a on susceptibility to type 2 diabetes mellitus in Chinese Han people. Chin Med J (Engl) 2015; 128: 533–9. 27. Saab YB, Zeenny R, Ramadan WH. Optimizing clopidogrel dose response: a new clinical algorithm comprising CYP2C19 pharmacogenetics and drug interactions. Ther Clin Risk Manag. 2015;11:1421-7. 28. Ingelman-Sundberg M, Gomez A. The past, present and future of pharmacoepigenomics. Pharmacogenomics. 2010;11:625-7. 29. Anttila S, Hakkola J, Tuominen P. Methylation of cytochrome P4501A1 promoter in the lung is associated with tobacco smoking. Cancer Res. 2003;63:8623–8. 30. Nakajima M, Iwanari M, Yokoi T. Effects of histone deacetylation and DNA methylation on the constitutive and TCDD-inducible expressions of the human CYP1 family in MCF-7 and HeLa cells. Toxicol Lett. 2003;144:247-56. 31. Ibanez de Caceres I, Cortes-Sempere M, Moratilla C, Machado-Pinilla R, et al. IGFBP-3 hypermethylation-derived deficiency mediates cisplatin resistance in non-small-cell lung cancer. Oncogene. 2010;29:1681–90. 32. Martens JW, Margossian AL, Schmitt M, Foekens J, Harbeck N. DNA methylation as a biomarker in breast cancer. Future Oncol. 2009;5:1245–56. 33. Jarmalaite S, Andrekute R, Scesnaite A, Suziedelis K, Husgafvel-Pursiainen K, et al. Promoter hypermethylation in tumour suppressor genes and response to interleukin-2 treatment in bladder cancer: a pilot study. J Cancer Res Clin Oncol. 2010;136:847-54. 34. Claes B, Buysschaert I, Lambrechts D. Pharmaco-epigenomics: discovering therapeutic approaches and biomarkers for cancer therapy. Heredity. 2010;105:152–60. 35. Lapeyre JN, Becker FF. 5-Methylcytosine content of nuclear DNA during chemical hepatocarcinogenesis and in carcinomas which result. Biochem Biophys Res Commun. 1979;87:698–705. 36. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med. 2003;349:2042–54. 37. Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004;429:457–63. 38. Issa JP, Kantarjian HM, Kirkpatrick P. Azacitidine. Nat Rev Drug Discov. 2005;4:275 – 6. 39. Kantarjian H, IssaJP, Rosenfeld CS. Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study. Cancer. 2006;106:1794- 803. 40. Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Finelli C, et al. Efficacy of azacitidine compared with that of conventional care regimens in the treatment of higher-risk myelodysplastic syndromes: a randomised, open-label, phase III study. Lancet Oncol. 2009;10:223–32. 41. Silverman LR, McKenzie DR, Peterson BL, Holland JF, Backstrom JT, et al. Further analysis of trials with azacitidine in patients with myelodysplastic syndrome: studies 8421, 8921, and 9221 by the Cancer and Leukemia Group B. J Clin Oncol. 2006;24:3895–903. 42. Kantarjian HM, O’Brien S, Shan J, Aribi A, Garcia-Manero G, et al. Update of the decitabine experience in higher risk myelodysplastic syndrome and analysis of prognostic factors associated with outcome. Cancer. 2007;109:265–73. 43. Dayeh T, Volkov P, Salö S, Hall E, Nilsson E, et al. Genome-wide DNA methylation analysis of human pancreatic islets from Type 2 diabetic and non-diabetic donors identifies candidate genes that influence insulin secretion. PLoS Genet. 2014;10:e1004160. 44. Dhas BB, Antony HA, Bhat V, Newton B, Parija SC. Global DNA methylation in neonatal sepsis. Indian J Pediatr. 2015;82:340-4. 45. Dhas BB, Antony HA, Bhat V, Parija SC. Functional annotation of protocadherin beta genes hypermethylation and their significance in neonatal sepsis. Int J Cur Res Rev. 2015;7:23-7. 46. Dhas BB, Antony HA, Bhat V, Kalaivani S, Parija SC. Comparison of genomic DNA methylation pattern among septic and non-septic newborns - An epigenome wide association study. Genomics Data. 2015;3: 36–40. 47. Jha AK, Nikbakht M, Parashar G, Shrivastava A, Capalash N, et al. Reversal of hypermethylation and reactivation of the RARβ2 gene by natural compounds in cervical cancer cell lines. Folia Biol (Praha). 2010;56:195-200. 48. Miwa M, Tsuboi M, Noguchi Y, Enokishima A, Nabeshima T, et al. Effects of betaine on lipopolysaccharide-induced memory impairment in mice and the involvement of GABA transporter 2. J Neuroinflammation. 2011; 8:153-65. 49. Gore SD, Weng LJ, Zhai S, Figg WD, Donehower RC, et al. Impact of the putative differentiating agent sodium phenylbutyrate on myelodysplastic syndromes and acute myeloid leukemia. Clin Cancer Res. 2001;7:2330–9. 50. Garcia-Manero G, Yang H, Bueso-Ramos C, Ferrajoli A, Cortes J, et al. Phase 1 study of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in patients with advanced leukemias and myelodysplastic syndromes. Blood. 2008;111: 1060–6. 51. Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem. 2001; 276:36734-41. 52. Göttlicher M, Minucci S, Zhu P, Krämer OH, Schimpf A, et al. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J. 2001; 20:6969-78. 53. Krämer OH, Zhu P, Ostendorff HP, Golebiewski M, Tiefenbach J, et al. The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2. EMBO J. 2003; 22:3411-20. 54. Ricobaraza A, Cuadrado-Tejedor M, Pérez-Mediavilla A, Frechilla D, Del Río J, et al. Phenylbutyrate ameliorates cognitive deficit and reduces tau pathology in an Alzheimer’s disease mouse model. Neuropsychopharmacology. 2009;34:1721-32. 55. Albani D, Polito L, Forloni G. Sirtuins as novel targets for Alzheimer’s disease and other neurodegenerative disorders: experimental and genetic evidence. J Alzheimers Dis. 2010; 19:11-26. 56. Min SW, Cho SH, Zhou Y, Schroeder S, Haroutunian V, et al. Acetylation of tau inhibits its degradation and contributes to tauopathy. Neuron. 2010; 67:953-66. 57. Balasubramanyam K, Varier RA, Altaf M, Swaminathan V, Siddappa NB, et al. Curcumin, a novel p300/CREB-binding protein-specific inhibitor of acetyltransferase, represses the acetylation of histone/nonhistone proteins and histone acetyltransferase-dependent chromatin transcription. J Biol Chem. 2004; 279:51163-71. 58. Tucker S, Ahl M, Cho HH, Bandyopadhyay S, Cuny GD, Bush AI, et al. RNA therapeutics directed to the non-coding regions of APP mRNA, in vivo anti-amyloid efficacy of paroxetine, erythromycin, and N-acetyl cysteine. Curr Alzheimer Res. 2006; 3:221-7.

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“Memory” in the Mammalian Brain Jaichandar Subramanian, Scientist Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Email: jai_sub@mit.edu

Abstract 

Understanding the nature of memory storage is one of the holy grails of modern neuroscience. It has long been recognized that memory storage would involve structural changes in the brain. The development of fluorescence labeling and in vivo imaging techniques have shed unprecedented light on how sub-cellular structures in the brain are modified in an experience dependent manner. Here, I review some of the recent findings on the nature of memory traces in the mammalian brain.

Key Words: Memory, Neuroscience, Synaptic imaging, Synapse.

Introduction The ability to remember and adapt to the environment is critical for survival. A part of this ability comes from the genetic and epigenetic information that we inherit. They reflect “memories” of our ancestors’ past that provided them with a survival advantage. The brain provides an additional substrate to store relatively more “real time” information that is relevant to an individual’s experience. Information in the brain is stored in interconnected population of neurons. Within this network of neurons, the ability or the strength of individual connections (synapses) to influence the activity of the connected neurons varies widely. During a novel experience, the new information can be stored by changing either the pattern of synaptic connectivity between neurons or the strength of existing synapses (Figure 1)1. Donald Hebb’s cell assembly theory provides a framework for understanding how neuronal activity can shape synaptic connectivity patterns or strengths. He posited that synapses are selectively strengthened between neurons that are coactive in response to the encoded information2. The “Hebbian” Ann. SBV, Jan-Jun 2016;5(1)

school of thought has arguably been the most important guiding framework for much of the neuroscience research on information storage in the brain. In this review, I focus on the recent progress, enabled by in vivo imaging, in our understanding of experience dependent changes on synaptic plasticity and its relevance to information storage in the mammalian brain. Experience plays a profound role in sculpting neuronal connectivity during development. In the developing mammalian brain, Hubel and coworkers elegantly demonstrated how visual experience modifies the structural and functional properties of the visual cortex. They found that, in the feline and primate binocular visual cortices, neurons are selectively activated by inputs to one eye or the other. Neurons that are responsive to each eye are organized as alternating columns, also referred to as ocular dominance columns3,4. When one of the eyes was deprived of visual experience by suturing the eye-lid (monocular deprivation or MD) for several weeks, the neurons that previously responded to inputs to this eye switched their response to the open eye inputs3. To test if such experience dependent functional alteration is accompanied by structural changes, Hubel and co-workers injected a radiolabeled amino acid tracer Page 33

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Figure 1. Experience dependent circuit remodeling. A simplified schematic of a neural circuit consisting of four neurons (A-D) in which A, B and C are part of a network (left). The arrow indicates synaptic connections and the width of the arrow represents synaptic strength. Learning or exposure to new experience rearranges the connectivity pattern and strength resulting in a network comprising of A, B and D.

in synapse formation and elimination, respectively. Visual deprivation enhances branch retractions, resulting in increased synapse loss10.Also, in a subset of inhibitory neurons that possess dendritic spines, removal of visual input caused rapid reduction in their spine density11. Deprivation induced synapse loss onto inhibitory neurons could reduce the inhibitory tone which then can create a permissive environment for plasticity in excitatory neurons. Thus, both excitatory and inhibitory neurons in the cortex exhibit plasticity to adapt to changes in sensory experience.

Dendritic spines possess the postsynaptic components of a synapse. The presynaptic components are contained within the axonal boutons. The axonal boutons are also dynamic in an experience and cell type dependent manner. In the layer 1 of mouse somatosensory cortex, the axonal afferents from the thalamus are more stable than the ones from layer 612. However, the relevance of such differential remodeling of different synapse types to animal’s experience is still unknown. Interestingly, changing the visual experience by introducing lesions focally in the retina led to massive restructuring of axons in the visual cortex that received information from the

Figure 2. Invivo imaging of synapses. A. (Left) Cranial window (5mm). (Middle)Visual cortex identified by visual evoked hemodynamic response. (Right) Z projection of the entire volume of layer 2/3 dendrites. Scale bar – 50 mm. Dendrite in white box is zoomed in B (Scale bar – 10 mm). C. Same dendritic segments imaged repeatedly over a period of many weeks (Scale bar – 8 mm). The solid white triangle represents a stable spine. The solid and open yellow triangles indicate gain and loss of dendritic spines, respectively.

in one of the eyes of the animals and subsequently, performed autoradiograms of the tangentially sectioned visual cortex. Consistent with the functional data, they found alternating patches of labeled and unlabeled axons corresponding to the inputs from labeled and unlabeled eyes, respectively. MD, followed by dye injection in the open eye, showed an expansion of the area occupied by labeled inputs carrying information from the open eye with a concomitant reduction in the area occupied by deprived eye inputs. They noted that such alterations are restricted to a period in development, termed as critical period, beyond which changes to experience would not result in structural changes3.

Sensory Experience Dependent Structural Plasticity in Adults Animals continue to learn and remember in adulthood and whether it involved structural changes, as envisioned by Hebb, remained unknown. Visualization of synaptic structure in the mammalian brain slices through light and electron microscopy was consistent with the idea of new synapse formation with learning. With the advent of fluorescence labeling technology and twophoton imaging, it became possible to study synaptic changes associated with experience in the brain of living mammals. Two photon imaging, through a glass cranial window or thinned skull, of excitatory neurons sparsely labeled with green or yellow fluorescence protein (GFP Page 34

or YFP) in the adult mouse brain revealed that dendritic spines, tiny protrusions on the membrane that harbor synapses, are largely stable but a fraction of them turn over(appear and disappear) over a period of days (Figure 2)5,6. Altering animals’ experience by trimming their whiskers in a checkerboard pattern resulted in an enhancement of spine turnover in the barrel cortex, presumably enabling adaptive remodeling of neural circuits (Figure 3)6. Such experience dependent changes in spine turnover are not limited to whisker sensation but has since been found to be ubiquitous for all senses. In the visual cortex, MD causes an increase in spine formation in deep cortical neurons but not in the superficial neurons7. The spines formed during MD persisted even after restoring binocular vision. Moreover, repeating MD did not further increase new spine formation. These results revealed that structural changes of dendritic spines in excitatory neurons could serve as memory traces in adulthood7. In contrast to excitatory neurons, most inhibitory neurons lack dendritic spines and the synapses are located in the dendritic shaft. Earlier observations on excitatory neurons revealed that, though dendritic spines are dynamic in adulthood, the dendrites that harbor them are quite stable6. In contrast, the dendrites of inhibitory neurons are dynamic even in adults8. Interestingly, of all the layers in the visual cortex, dendritic plasticity of interneurons is limited to the superficial layer 2/39. These interneurons exhibit branch extensions and retractions resulting Ann. SBV, Jan-Jun 2016;5(1)

lesioned area13. Thus, both pre and postsynaptic sites remodel in response to changes in animals’ experience.

Learning and Memory Associated Structural Plasticity Experience dependent appearance and disappearance of synapses are not limited to sensory modalities. Mice that learned to perform anew motor task showed an increase in spine formation in the neurons of motor cortex. This was followed by elimination of some of Ann. SBV, Jan-Jun 2016;5(1)

the spines that existed before the training. Practicing the same task did not further increase spine formation whereas learning another new motor task promoted addition of new spines. Further, the number of new spines formed correlated with the extent of task acquisition, thus revealing a direct link between spine formation and learning14. Spine formation and elimination have also been shown to correlate with acquisition of fear. In a paradigm, referred to as fear conditioning, mice are Page 35

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Figure 3. Spine dynamics and circuit remodeling. An illustration of a simple circuit consisting of three neurons. The neuron, indicated in red, loses and gains spines at different locations. Spine loss and gain in the ‘red’ neuron alters its connectivity with cells A and B.

the excitatory synapses tend to appear and disappear at different locations of a neuron, reflecting circuit rearrangement, whereas inhibitory synapses are formed and removed at the same sites, suggesting a role in functional gating of activity at these sites18.

revealed a common core phenotype, an increase in the dynamics of spines containing PSD95 but lacking gephyrin, presumably carrying intracortical synapses20.

Direct visualization of spine and synaptic dynamics can provide a better understanding of etiology of many diseases associated with the nervous system. In a mouse model of Huntington’s disease, visualization of dendritic spine dynamics over a period of 6 weeks revealed an increase in spine formation, but the newly formed spines could not persist as stable spines. Such abnormal remodeling of spines preceded the onset of motor symptoms and therefore, could be causal to symptoms in Huntington’s disease19. More recently, labeling of synapses in various mouse models of autism

Recent developments in synaptic imaging described above allow examination of both excitatory and inhibitory synapse remodeling with unprecedented resolution. It has also revealed heterogeneity in dendritic spines based on their synaptic content. However, we are still oblivious to how synaptic distribution and dynamics of a neuron relates to the source of their afferent inputs. The stage is now set to integrate information from multiple levels, such as molecules, circuits and experience, and study their influence on neurons at the resolution of single synapses.

Conclusion

References

exposed to a tone followed by a brief electric foot shock. Subsequently, mice respond by freezing upon exposure to the same tone. In the auditory cortex, pairing of a tone with a shock significantly increased formation of new spines and these spines persisted for long periods of time, presumably serving as a memory trail15. Interestingly, in the frontal association cortices, fear conditioning led to elimination of spines and the level of freezing correlated with the percentage of spines eliminated. In contrast, extinction of fear, achieved by repeated safe exposure to the same tone used for fear conditioning, resulted in new spine formation and these spines are specifically removed upon reconditioning of fear with the same stimulus16. The above studies make a strong case for spine remodelling as structural correlates of memory. However, a causal link between spine changes and memory storage was not made in these studies. If new spine formation stores new memories then specifically removing those spines after memory formation should erase the associated memory. Recently, a novel optical probe, named AS-PaRac1, was developed to address this question. This probe localizes to activated synapses and contains photoactivatable Rac1, a small GTPase whose activation causes spine shrinkage. Consistent with a causal role for new spines in storing memories, optical activation of AS-PaRac1selectively eliminated spines that were formed following motor learning and consequently, resulted in loss of the relevant motor memory17. Page 36

Multi-color Synaptic Imaging in vivo Though dendritic spines are good surrogates for excitatory synapses, they do not represent the synapse themselves. Some spines may not have synapses or may have synapse with different levels of maturity. Further, they only represent excitatory synapses. A significant fraction of synapses made on excitatory neurons are inhibitory and they lack a structural surrogate. Recently, direct visualization of synapses was achieved by fluorescence labeling of proteins residing in excitatory (PSD95) and inhibitory synapses (gephyrin)18. Simultaneous expression of YFP, PSD95mCherry and Teal-Gephyrin enabled visualization of dendritic spines, mature excitatory synapses and inhibitory synapses, respectively. A vast majority of the dendritic spines contained PSD95 but a significant fraction (~20%) were devoid of it. Post-hoc electron microscopy revealed that these spines carry synapses that might be immature. Surprisingly, an equally large fraction of dendritic spines (~20%) had both PSD95 and gephyrin. These spines are dually innervated by both excitatory and inhibitory synapses (DIS).Such a heterogeneous spine population also exhibited differential remodeling properties. For instance, PSD95 lacking spines are highly dynamic whereas the DIS are extremely stable structures. Interestingly, within the DIS, the excitatory synapses are more stable but the inhibitory synapses appear and disappear on a day-to-day basis. Overall, in over a period of a week, Ann. SBV, Jan-Jun 2016;5(1)

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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Music Therapy in Neonatology: What is Known and What is Unknown?

Music and the Neonate Music Therapy in Neonatology: What is Known and What is Unknown? Parin N Parmar, Visiting Faculty Sumathy Sundar, Director Centre for Music Therapy Education and Research (CMTER), Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India. Email: parinmnparmar@gmail.com

Abstract 

usic therapy is rapidly gaining acceptance in clinical setting due to its emerging supportive M evidence and harmless nature of the therapy. Understanding the effects of sounds (both musical sounds and “noise”) on neonatal development, physiology, and behaviour has made it possible to apply music for therapeutic purposes in neonates.

Music therapy has showed positive effects on heart rates, blood pressure, respiratory rates, oxygen saturation, stress-reduction, pain-control, hospital stay, etc. in neonates admitted in NICUs in different studies. In NICUs, masking of ambient noise is also an important effect. Music therapy positively influences neonatal feeding behaviour and weight gain. Recorded instrumental music and vocal music (including mother’s voice) is used more commonly, however, live music therapy by qualified music therapists seems to be more promising due to obvious reasons. Mothers’ singing seems to be a convenient, acceptable and cost-effective option. Music therapy to mothers also benefits neonates. This article aims to discuss applications of music therapy in neonatology. Limitations, unanswered questions, and need for further research in Indian settings are also discussed.

Key Words: music therapy, neonates, mother’s voice.

Introduction For centuries, musical activities have been part of most, if not all, traditions, cultures, and societies. Effects of music on human mind and body are known since ancient times. However, it is only for last few decades that there has been steady growth of scientific literature exploring influence of music on health and diseases. Scientifically, music can be considered as organized sound (vocal, instrumental, or both) and music therapy is defined, by the American Music Therapy Association (AMTA), as clinical and evidence-based use of music interventions to accomplish individualized goals within a therapeutic relationship by a credentialed Page 38

professional who has completed an approved music therapy program.1 There are many applications of music therapy in neonatology, both for office practice and in NICUs. Some of them may require sophisticated equipment and qualified music therapists, fortunately, some of them do not and hence integration of music therapy into neonatology seems to be at least partially possible for pediatricians and neonatologists. This articles aims to review practical aspects of music therapy in neonates which can be helpful to practicing paediatricians and neonatologists. Ann. SBV, Jan-Jun 2016;5(1)

For those who are unfamiliar to the field of music therapy, positive health effects of music are often perceived as “only psychological” or physiological only. However, many researchers have assessed effects of music on brains of neonates, who are naturally not conditioned to music and emotions, unlike adults. Neonates not only have innate beat perception;2 they also have ability to separate pitch of sound from timbre.3 Neural architecture of even 1-day-old neonates is sensitive to changes in musical tone.4 In a study by Perani et al,4 instrumental piano music has shown to activate bilateral superior temporal gyrus (including primary auditory cortex), bilateral secondary auditory cortex, right insula, right amygdala-hippocampal complex, and some other areas. Interestingly, activation of auditory areas in left hemisphere was weaker than those in right hemisphere with the music, while altered music (the original piano music altered by modification of tones) causes less activation of auditory areas in right hemisphere than in left hemisphere. Structural manipulations of music also activated left inferior frontolateral cortex, and possibly Broca’s area. Effects of music on Broca’s area and limbic structures suggest role of music for linguistic and emotional development in infants.

Music Therapy in NICU A preterm neonate admitted in NICU is a special case, who is under significant stress from inconvenient sounds (rather noise), unpleasant touches, painful procedures, and much more.5 Such stressful stimuli induce adverse behavioral and autonomic responses in neonates and may increase heart rate, blood pressure, and respiratory rate; reduce oxygen saturation and increase likelihood of apnea episodes.5-7 Music therapy has shown to reduce heart rate, blood pressure, and respiratory rate in preterm neonates in different studies.8-10 The type of music used by different researchers is recorded vocal music and recorded instrumental music (including music by Mozart).10-12 Live music therapy in NICU by trained music therapist has an added advantage of application of entrainment (synchronization of a physiologic rhythm with an external stimulus), which can adapt music therapy to an infant’s vitals.9 These findings suggest potential application of music therapy in neonates with cardiorespiratory disorders. However, a single study Ann. SBV, Jan-Jun 2016;5(1)

specifically assessing effects of recorded sedative music on premature infants with respiratory disorders did not show significant difference in heart rate, respiratory rate, and oxygen saturation as compared to control group.13 Hence, at present, it is known that music therapy affects vitals in stressed premature neonates admitted in NICUs, but its usefulness in those with cardiorespiratory disorders needs further research. In addition to effects of autonomic nervous system, music therapy in NICU has shown reduced stress, reduced number and length of crying episodes, and reduced length of hospital stay.14-17 Music has been a great pain reliever for all ages and a study by Butt and Kisilevsky, use of recorded music positively modulated pain-related autonomic and behavioural responses in preterm neonates following heel lance.18 This is even more important for neonates in NICUs since they undergo many painful procedures including, venepuncture, lumbar puncture, etc. Music therapy is good for sleep in premature infants and live music therapy in NICUs has shown even better effects on neonatal sleep than use of recorded music.19 Sound level in NICU environment deserves a special consideration here. Although they vary depending upon design of the NICU, time of day, activities and staff in the NICU, etc. reported sound levels in NICU range from 49.5 to 89.5 dB20, which exceeds the recommended level by the American Academy of Pediatrics, i.e., 45 dB. Sounds of different equipments, various alarms, etc. can further increase the “noise” in the NICU. Music does provide an important masking effect, but it should be made sure that the music used for therapy itself does not cross the safe limits. This is one of the factors that favors live music therapy by a music therapist over recorded music used in NICUs.

Music Therapy, Feeding, and Weight Gain in Neonates Importance of establishment of breastfeeding, and of sucking-swallowing coordination in neonates, especially preterm neonates is well known. Sucking can be considered as the first rhythmic behavior, which also contributes to neurological development by facilitating internally regulated rhythms.21 In a study by Standley, contingent recorded music consisting of recorded lullabies has shown to increase non-nutritive sucking in premature neonates.22 Another study by the same author showed multiple benefits of musical stimulation by reciprocal lullaby singing, including weight gain.23 A study by Caine showed that musical Page 39

Music Therapy in Neonatology: What is Known and What is Unknown?

Annals of SBV

stimulation in stable premature and low-birth weight neonates increased formula and caloric intake, reduced initial weight loss, and increased daily average weight as compared to controls.17

to 60 days after discharge.27 A recent study from India has shown positive effect of music therapy on amount of expressed breast milk in mothers of premature newborns.28

newborns should be easily welcomed and accepted by most of families.

Compared to recorded music, live music therapy should be more effective in improving feeding behavior in neonates. This is because music elements, both instrumental and vocal, can be catered to neonate’s vital parameters and behavior. Sucking behavior has been shown to vary with rhythm sound variations in live music therapy and several studies have shown improvement in sucking behavior, caloric intake, and weight gain using live music therapy.9, 19

Stress-reducing effects of music therapy in adults are well known, and so as adverse effects of psychological stress on breast milk secretion. Hence it is rational to believe that music therapy may be effective in increasing breast milk secretions in mothers, especially stressed mothers like mothers of premature neonates and primiparous mothers.

The concept of fetal memory is not new.29Several studies have assessed effects of maternal music exposure during pregnancy and labor on neonatal behavior after birth. In a study By Tabarro, mothers positively reported alerting response and calming response by neonates to the music that the mothers were exposed to from fifth month of gestation onwards.30 An open-label randomized controlled trial showed significant improvement in neonatal behavior, especially with respect to orientation and habituation, as a result of maternal music exposure

It is interesting to note that positive effect of music therapy on neonatal weight gain may not be only secondary to improved feeding, but also due to reduced energy expenditure.24

Mother’s Voice and the Neonate It is well known that a neonate recognizes his/her mother’s voice early and shows preference towards mother’s voice over other voices. In environment of NICU, exposure to mother’s voice has potential implications both for preterm and term neonates. A recent review concluded potentially positive developmental effects of exposure to mother’s voice and has suggested future directions for research.25 Segall reported significant effect of mother’s voice on heart rate pattern in premature neonates in NICU environment.26 Considering importance of infant-mother bonding, effects of live mother’s voice or singing on neonates need to be explored, although many of the studies mentioned in this article have used recorded mother’s voice as music. In addition, mother’s singing could be a costeffective music therapy intervention in neonates which is an important factor in resource-limited settings.

Music Therapy for Mothers of Neonates Although this article does not aim to discuss effects of music therapy in mothers, effects of music therapy on increasing breast-feeding rates needs to be mentioned. A recently conducted randomized controlled trial in Rio de Janeiro showed a significant increase in breastfeeding rates among mothers of premature neonates; the effect was statistically significant up to the first follow-up visit between 7-15 days, and also showed statistically insignificant positive influence up Page 40

Music Therapy and Cautions in Indian Context There is sufficient evidence to believe that music therapy has positive effects on neonates. However, quantification of the benefits and practical applications are yet unclear. Due to different types of music therapy interventions used by different researchers, it is difficult to recommend a uniform music therapy practice for a specific clinical condition at present. Some of important unanswered questions include minimum age at which music therapy should be started, type of music therapy (recorded/live, instrumental/vocal/mother’s voice), method of delivering music therapy (speakers/ headphones), type of music (classical/lullabies/specific sounds), etc. Music therapy is gaining rapid acceptance in medical community as it is understood as having no side effects. This is true in most circumstances; however, this cannot be taken for granted for neonates, especially premature neonates. It is known that hair cells in internal ears of neonates are highly sensitive to loud sounds, and safe sound levels of therapeutic music for neonates must be established. Another important consideration is use of headphones to deliver music therapy in neonates, which could be a source of infections for premature neonates. Indian scenario needs a special consideration. Music that has been used by Western researchers may not be acceptable to Indian parents due to sociocultural factors. Music therapy to be considered as an evidencebased science in India, scientific studies using Indian music in Indian hospital settings are necessary to make recommendations for music therapy practice. However, a brighter aspect is that music is a part of many Indian cultures and customs; and that mothers singing lullabies is not a new concept in many Indian societies. Hence scientific application of music for Ann. SBV, Jan-Jun 2016;5(1)

Future: “Fetal Music Therapy”?

during pregnancy.31 Of course, effect of music on fetal development and on fetal behavior is an interesting field of research.

Conclusion Music therapy has many applications in neonatology, both in NICUs and for health neonates. Potential effects of music on neonates include those on autonomic nervous system, those on brain development, those on linguistic, behavioral, and psychosocial development, and others. In many countries, music therapy is rapidly gaining acceptance in NICUs, but more research is needed to recommend evidence-based use of music for neonates in Indian settings.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

American Music Therapy Association. AMTA Member Sourcebook. Silver Spring, MD: American Music Therapy Association, Inc.; 2005. Winkler I, Haden GP, Ladinig O, Sziller I, Honing H. Newborn infants detect the beat in music. Proc Natl Acad Sci U S A. 2010; 106: 2468-71. Haden GP, Stefanics G, Vestergaard MD, Denham SL, Sziller I, et al. Timbre-independent extraction of pitch in newborn infants. Psychophysiol. 2009; 46: 69-71. Perani D, Saccuman MC, Scifo P, Spada D, Andreolli G, et al. Functional specializations for music processing in the human newborn brain. Proc Natl Acad Sci U S A. 2010; 107: 4758-63. Maroney DI. Recognizing the potential effect of stress and trauma on premature infants in the NICU: How are outcomes affected? J Perinatol. 2003; 23: 679-83. Wachman EM, Lahav A. The effects of noise on preterm infants in the NICU. Arch Dis Child Fetal Neonatal Ed. 2011; 96: F305-9. Gitto E, Pellegrino S, Manfrida M, Aversa S, Trimarchi G, et al. Stress response and procedural pain in the preterm newborn: the role of pharmacological and nonpharmacological treatments. Eur J Pediatr. 2012; 171: 927-33. Da Silva CM, Cacao JMR, Silva KCS, Marques CF, Merey LSF. Physiological responses of preterm newborn infants submitted to classical music therapy. Rev Paul Pediatr. 2013; 31: 30-6. Loewy J, Stewart K, Dassler AM, Telsey A, Homel P. The effects of music therapy on vital signs, feeding, and sleep in premature infants. Pediatrics. 2013; 131: 902-18. Tramo MJ, Lense M, Ness CV, Kagan J, Settle MD, et al. Effects of music on physiological and behavioral indices of acute pain and stress in premature infants: Clinical trial and literature review. Music Med. 2011; 3: 72-83. Coleman JM, Pratt RR, Stoddar RA, Gerstmann DR, Abel H. The effects of male and female singing and speaking voices on selected physiological and behavioral measures of premature infants in the intensive care unit. Int J Arts Med. 1998; 5: 4-11. Cassidy JW. The effect of decibel level of music stimuli and gender on head circumference and physiological responses of premature infants in the NICU. J Music Ther. 2009; 46: 180-90. Calabro J, Wolfe R, Shoemark H. The effects of recorded sedative music on the physiology and behaviour or premature infants with a respiratory disorder. Aus J Music Ther. 2003; 14: 3-19. Allen KA. Music Therapy in the NICU: Is there evidence to support Integration for procedural support? Adv Neonatal Care. 2013;13:349-52. Keith DR, Russell K, Weaver BS. The effects of music listening on inconsolable crying in premature infants. J Music Ther. 2009; 46: 191-203. Standley J. Music therapy research in the NICU: an updated meta-analysis. Neonatal Netw. 2012; 31: 311-6. Caine J. The effect of music on the selected stress behaviors, weight, caloric and formula intake, and length of hospital stay of premature and low birth weight neonates in a newborn intensive care unit. J Music Ther. 1991; 28: 180-92. Butt ML, Kisilevsky BS. Music modulates behaviour of premature infants following heel lance. Can J Nurs Res. 2000;31:17-39. Arnon S, Shapsa A, Forman L, Regev R, Bauer S, et al. Live music is beneficial to preterm infants in the neonatal intensive care unit environment. Birth. 2006; 33: 131-36. Matook SA, Sullivan MC, Salisbury A, Miller RJ, Lester BM. Variations of NICU sound by location and time of day. Neonatal Netw. 2010; 29: 87-95. Goff DM. The effects of nonnutritive sucking on state regulation in preterm infants. Dissertation Abstracts International. 1985; 46: 2835. Standley JM. The effect of contingent music to increase non-nutritive sucking of premature neonates. Pediatr Nurs. 2000;26:493-5, 498-9. Standley JM. The effect of music and multimodal stimulation on responses of premature infants in neonatal intensive care. Pediatr Nurs. 1998; 24: 532-8. Lubetzky R, Mimouni FB, Dollberg S, Reifen R, Ashbel G, et al. Effect of music by mozart on energy expenditure in growing preterm infants. Pediatrics. 2010; 125: e24-8. Krueger C. Exposure to maternal voice in preterm infants: A review. Adv Neonatal Care. 2010; 10: 13-20. Segall M. Cardiac responsivity to auditory stimulation in premature infants. Nurs Res. 1972; 21: 15-9. Vianna MNS, Barbosa AP, Carvalhaes AS, Cunha AJLA. Music therapy may increase breastfeeding rates among mothers of premature newborns: a randomized controlled trial. J Pediatr (Rio J). 2011;87:206-12. Jayamala AK, Preethi BL, Pradeep GCM, Jaisri G. Impact of music therapy on breast milk secretion in mothers of premature newborns. J Clin Diag Res. 2015; 9: CC04-6. Hepper PG. Fetal memory: Does it exist? What does it do? Acta Paediatr Suppl. 1996;416:16-20.. Tabarro CS, de Campos LB, Galli NO, Novo NF, Pereira VM. Effect of the music in labor and newborn. Rev Esc Enferm USP. 2010;44:445-52. Arya R, Chansoria M, Konanki R, Tiwari DK. Maternal music exposure during pregnancy influences neonatal behaviour: An open-label randomized controlled trial. Int J Pediatr. 2012; 2012: 901812.

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Osteoprotective Effect of Few Indian Herbs: An Update

fruits, nuts, seeds, mushroom, etc.) have identified certain food types that inhibited bone resorption2.

Herbs and Bone

Osteoprotective Effect of Few Indian Herbs: An Update Veni Subramanyam, Scientist 1 Srinivasan Narasimhan, Professor 2 1

Central Inter-Disciplinary Research Facility,

Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India 2

Faculty of Allied Health Sciences

Chettinad Academy of Research & Education (CARE),Kelambakkam, Chennai 603 103, India Email: vsubramcidrf@gmail.com

Abstract 

atural herbs have been widely used in orthopedic clinical practice in India, China and N other countries since ancient times. The increase of aging population and the prevalence of osteoporosis, demands new therapeutic agents and nutritional supplements for the promotion of bone health. The Indian diet includes rich medicinal herbs. This review intends to highlight scientiﬁc information on naturally-occurring herbs like onion, garlic, clover, walnut, beans etc., consumed regularly in Indian diet which has been documented to possess osteoprotective properties. Focus has been put on literature available in the last ten years on Indian medicinal plants used for bone metabolic disorders like osteoporosis.

Key Words: Indian medicinal plants, herbs and bone, osteoprotective.

Introduction Traditional herbal medicines have been used for the treatment of various diseases, since they are considered less toxic and free from side effects when compared with synthetic drugs1. Natural herbs have been widely used in orthopedic clinical practice in India, China and other countries since ancient times. India being one of the richest countries in herbal resources, the food items enacts to be medicine and it is vice versa. This review intends to highlight scientiﬁc information on naturally-occurring herbs consumed regularly in Indian diet which have been documented to possess osteoprotective properties.

Page 42

The common vegetables, salads and herbs commonly consumed in the diet significantly inhibited bone resorption at a dose of 1 g/day. These included: arugula, broccoli, cucumber, cabbage, red cabbage, dill, garlic, wild garlic, leeks, lettuce, onions, Italian parsley, common parsley and tomatoes. Women (45-55 years) who had consumed high amounts of fruits and vegetables in childhood showed higher bone mineral density (BMD) of the femoral neck than those that had consumed medium/low amounts. Pubertal children who have consumed fruit and vegetables >3 times per day showed better bone health, and the radius in particular. Not only in human beings, experimental studies in rats maintained of human diet (vegetables,

Ann. SBV, Jan-Jun 2016;5(1)

Cissus quadrangular Linn. (C. q) belongs to family Vitaceae, is an indigenous medicinal plant of India. It is known as ‘asthisanghara’ in Sanskrit, meaning “which will strengthen the bones” and perandai in Tamil. The use of this plant by the common folk for promoting fracture healing process is an old practice. Commonly known as “bone setter” or “bone knitter”, the plant is referred to as “Asthesamdhani” in Sanskrit and “Hadjod” in Hindi because of its ability to join bones. It has been prescribed in ancient Ayurvedic texts by Bhava Prakash and Chakra Dutta as a general tonic especially for the fractured patient. Since then it has been in extensive use by bone setters both for external application and as an internal medicine to be taken with milk. The stem part of C. q has been reported to contain triterpenes including α and β amyrins, β-sitosterol, ketosteroid, β-carotene and vitamin C. The plant has also been shown to have antioxidant flavanoid, quercetin. These phytoconstituents are known to induce osteoblastic differentiation of mesenchymal cells. Maternal treatment with C. q is effective against diabetes-induced delayed fetal skeletal ossification3, 4 . The effect of C. q in inducing alkaline phosphatase (ALP) activity was found to be mediated through Mitogen Activated Phosphate (MAPK) activity in murine osteoblastic cells5.

C. quadrangularis influenced fracture healing by increasing metabolism and uptake of minerals calcium, sulphur and strontium. C. quadrangularis improved in early regeneration of all connective tissues involved in the healing and quicker mineralization of callus during fracture healing. Bone remodeling includes fibroblastic phase (first week), collagen phase (second week) and osteochondroital phase (third and fourth weeks). Bone healing duration examined in fractured rats envisaged the quickest bone remodeling process with eventual time reduction. This hastening in the fracture healing was attributed to the stimulation of all the cells of mesenchyma origin, namely the fibroblasts, chondroblasts and osteoblasts (OB). C. quandrangularis builds up the chemical composition of the fractured bone namely mucopolysachrides, collagen, calcium (Ca) and phosphorus (P)6. Petroleum ether extracts of C. q stimulated osteoblastogenesis and mineralization in bone marrow mesenchymal cells (BMSC) and murine osteoblastic cell lines4, 5. In vitro studies have shown that ethanolic extracts of C. q increased mRNA and proteins Ann. SBV, Jan-Jun 2016;5(1)

related to the bone formation pathway and (Insulin like growth factor) IGF-I, IGF-II, and IGF binding protein7, 8. C. quandrangularis can reduce ovariectomy (OVX) induced bone loss and it does this in the long bones in a site-specific manner with more effects on the cancellous bone of femur followed by tibia. It probably reduces bone resorption primarily by down regulating pro inflammatory cytokines TNF-α, IL-1β, and IL6 which are increased after OVX in mice9. It is also effective in improving histological, biomechanical and biochemical changes of trabecular and cortical tibial bone in diabetic rats with increase in ALP, Type I collagen (COL-I) and decrease in tartrate resistant acid phosphatase (TRAP)3. In single blind clinical controlled human trial, serum PTH level was in peak at 21st day of fracture healing, osteoblastic activity was also maximum at the end of third week as evidenced in animals. It is also having influence on accelerating the fracture healing process and further it helps in reducing period of immobilization and early rehabilitation10. Pomegranate is the fruit of Punica granatum L. (Punicaceae) that has been used extensively in the folk medicine of many cultures. It is one of the oldest edible fruits. India is a native land of the pomegranate which is grown in coastal and mountainous areas. The presence of estrogenic compounds in pomegranate seeds makes it a potential alternative or supplement to hormone replacement therapy (HRT) in postmenopausal women. In addition to estrogenic activity, it also has antioxidant activity. Pomegranate contains other steroids such as testosterone and β-sitosterol in seeds. Isoflavones (genistein and daidzein), anthocyanins, ascorbic acid, ellagic acid, gallic acid, caffeic acid, catechin, Epigallocatechin gallate (EGCG), quercetin, rutin, numerous minerals, particularly iron and amino acids are in pomegranate juice11. The ethanolic extract of pomegranate significantly enhanced OB growth and differentiation markers (ALP activity and collagen content) and inhibits TNF-α induced IL-6 and NO production in MC3T3-E1cells12. Pomegranate consumption was able to significantly prevent the decrease in BMD and bone microarchitecture impairment and thus preventing the bone loss associated with OVX in mice. Moreover, the exposure of RAW264.7 cells to serum harvested from mice that had been given a pomegranate-enriched diet, elicited reduced osteoclast (OC) differentiation and bone resorption, as shown by the inhibition of the major OC markers. In addition, pomegranate substantially stimulated ALP activity at day 7, mineralization at day 21 and the transcription level of osteogenic markers in MC3T3-E1cell line13. Page 43

Osteoprotective Effect of Few Indian Herbs: An Update

Annals of SBV

Onion (Allium cepa) belongs to the family Alliaceae. Onion is an important source of valuable phytonutrients as flavonoids, fructooligosaccharides (FOS) and thiosulfinates and other sulphur compounds14. Onion extract inhibited loss of bone in an osteoporotic rat model15. γ-L-glutamyl-trans-S-1-propenyl-L-cysteinesulfoxide (a γ-glutamyl peptide) isolated from onion inhibited bone resorption in rats in vivo and OC in vitro16. Water solution of onion powder inhibited the receptor activated nuclear factor κB ligand (RANKL) plus macrophage-colony stimulating factor (M-CSF)induced differentiation of BMSC and RAW 264.7 macrophage cells to OCs17. Onion administration provided significant changes in the levels of ALP, free radicals, total antioxidant capacity, antioxidants, BMD in humans and also decreased in vitro osteoclastogenesis, thus showed a positive modulatory effect on the bone loss by improving antioxidant activities18. Garlic (Allium sativum Linn.) which belongs to the family Alliaceae is a common spicy flavoring agent used since ancient times. Garlic is gaining attention as being beneficial to bone. The estrogenic activity of garlic acid helps in the maintenance of skeletal health in the same manner as estradiol (E2)19. Garlic acid significantly reduced the increase in urinary levels of hydroxyl proline, Ca, PO4, creatinine and serum levels of TRAP and ALP which occurred after OVX and reversed the reduction in BMD20. The immunomodulatory effect of garlic, as well as the modulation of (Interleukin) IL-1, IL-6, (Tumor necrosis factor) TNF-α production was studied in a double-blind randomized controlled clinical trial in postmenopausal women21. Cinnamon belongs to the family Lauraceae. Cinnamon is an evergreen tree, which has been traditionally harvested in Asian countries. Various species of cinnamon are grown in various parts of southern India and a remarkable quantity is produced from Kerala. The Cinnamomum cassia blume (C. cassia) bark has been found to contain cinnamaldehyde (CA) and 2-Methoxy cinnamaldehyde (2-MCA) as its active components22. CA and MCA of C. zeylanicum reduce OC-like cell formation by inhibiting nuclear factor T cell activator 1 (NFATc1) expression. MCA exhibited remarkable inhibition rates of 95% at 2 µM on bone in pit resorption assay. CA and MCA inhibited RANKLinduced osteoclastogenesis23. The ethanolic extract of C. cassia (CCE) has estrogenic activity and the estrogenic compound competes with estrogen (E) ligands for binding to estrogen receptors, ERα and ERβ. CCE bind with ERβ with greater affinity than ERα. Further, CCE increased the survival of MC3T3-E1 cells and increased Page 44

the ALP activity, COL-I synthesis, osteocalcin (OCN) secretion and bone nodule formation. Thus, CCE has anabolic effects on preosteoblasts. However, the extract inhibited TNF-α-induced secretion of IL-6 and nitrite production by MC3T3-E1 cells24. The bark extract inhibited OC activity through suppression of NAFTc mediated signal transduction23. Thus, CCE extract can inhibit bone resorption.

Table 1. List of medicinal plants used for bone fracture healing is given below 43-45.

S. No.

Medicinal plants

Common name

Vernacular Tamil name

Uses Leaves and bark are used for bone fracture

Alangium salviifolium Alangiaceae

Sage Leaved Alandi Alangium

Wedelia calendulacea (Less.) known also as pila bhangra is a perennial herb with erect stems, 20-40 cm. high. The presence of isoflavones and wedelolactone, which are known to act as phytoestrogens are suggested to be responsible for the antiosteoporotic activity 25, 26.

Allophylus serratus (Sapindaceae )

Indian Allophylus

Siruvalli

Stem and leaves paste applied for bone fracture

Amorphophallus cam. Panulates (Araceae)

Yam

Senaikizhangu

Leaf extract is used in preparation of medicated oil for bone fracture

Withaferin A (WFA) from leaves of Withania somnifera commonly known as aswagandha prevented bone loss by reducing expression of OC genes TRAP and RANK and increase in bone turnover marker, OCN. In OVX rats the increase in inflammatory cytokine, TNF-α was reduced with WFA treatment comparable to E2 administration. At cellular level, WFA promoted differentiation of BMSC and increased mineralization by inducing expression of osteoblastogenic genes. WFA treatment prevents bone loss that is comparable to alendronate and E227.

Bambu satulda Roxb. (Bambusaceae)

Bamboo

Moongil

Stem and leaves paste applied for bone fracture

Butea monosperma (Fabaceae)

Flame of the forest

Palasu

bark is used for bone fracture

Calotropis gigantea (Linn.) R.Br.exAit. (Apocynaceae)

Crown flower

Erukku

Roasted leaves are bandaged locally

Cassia auriculata L., (Caesalpiniaceae)

Matura tea tree

Avarai

Leaf paste mixed with egg albumin is applied on the fractured or dislocated area daily once for a week.

Cassia occidentalis L. (Fabaceae)

Coffee weed

NattamTakarai Paeyaavarai

The plant parts are used for healing bone fracture

Commiphoramukuli (Bursaraceae)

Guggul

Guggal

Used in treatment of Arthritis and fracture healing

Dodonaea viscose L. (Sapindaceae)

Hop Bush

Virali

Leaf paste with egg albumin and lime are applied to aid in bone setting

Indian goose berry

Nelli

Induce osteoclast apoptosis through downregulating the expression of IL-6 and NF-κB

Erythrina variegate (E.v) (Kalyanamurungai in Tamil), a member of Leguminosae Family is a showy, spreading tree, a legume with brilliant red blossoms. Commonly known as ‘Indian coral tree’ in Asia. Highly valued ornamental tree described as one of the gems of the floral world. Its bark and leaves are used in India, China, and Southeast Asia, to treat rheumatic joint pain, spasm of the limbs as well as lower back and knee pain, and to stimulate lactation and menstruation in women. Rats treated with the alcoholic extract prevented the OVXinduced increase in the serum OCN, ALP, and urinary deoxypyridoline (DPD) levels. Histomorphometric analysis of the proximal end of the tibia showed that the extract prevented the E deficiency-induced decrease in trabecular thickness and trabecular area, as well as restored the increase in trabecular separation in a dose-dependent manner. An ethanolic extract of this plant has been shown to prevent the bone loss in OVX rats and these effects were attributed to the genistein derivatives present in the extract including 6-prenylgenistein, 8-prenylgenistein. 6,8-diprenylgenistein isolated from E.v demonstrated stimulatory effects on osteogenesis in UMR 106 cells27. E.v suppressed the up-regulation of cathepsin K mRNA and the down-regulation of OPG mRNA Ann. SBV, Jan-Jun 2016;5(1)

Emblica officinalis L. (phyllanthaceae)

Jatropha gossypifolia L. (Euphorbiaceae)

Bellyache Bush

Amanaku

Root extract is given orally

Moringa oleifera Lam. (Moringaceae)

Drumstick

Murungai

Bark paste is applied locally

Murrayapaniculata Linn. ( Rutaceae)

Orange Jasmine

Vengarai

Leaves pounded with egg albumin are applied as a plaster

Ann. SBV, Jan-Jun 2016;5(1)

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Osteoprotective Effect of Few Indian Herbs: An Update

Annals of SBV

Table 1 contd.

Figure1. The schematic diagram represents the mechanism of action of herbs discussed in this review.

Medicinal plants

Common name

Ormocarpum cochinchinense (Lour.) (Fabaceae)

South Indian Caterpillar Bush

Elumbotti

Leaves used for curing bone fracture

Panax Notoginseng (Araliaceae)

Ginseng

Aswagandha

Root is used for bone fracture

Phaseolusmungo (Fabaceae)

Black gram

Ulundhu

Applied on animal bone fractures for bone setting

18.

Prunus cerasoides (Rosaceae)

Himalayan cherry

Patumugam

Fresh bark paste is applied as plaster

Senecio aureus (Asteraceae)

Sarsaparilla

Nannari

Plant decoction is used to treat fractures

Sida acuta Burm. (Malvaceae)

Morning mallow

Vattathirippi

Leaf paste along with white egg yolk is applied

Taxus wallichiana Planch. (Taxaceae)

Beetal

Vetrilai

Bark paste is applied locally

Terminalia arjuna (Roxb) W&A (Combretaceae)

Arjun

Marudam

Bark is used for bone fracture healing.thedecotion or ksheerpak is taken orally to hasten healing

HeartTinospora cordifolia (willd.) leaved hook (Meninspermaceae) moonseed

Shindhilkodi amirtavalli

Stem paste is used as bandage for treatment of bone fracture and dislocation of bones

Zingiber officinale (Zingiberaceae)

Ginger

Ingi

To treat osteoporosis

Ziziphus oenopliaÂ (L.) Mill., (Rhamnaceae)

Jackal jujube

Elandhai

Leaves used to plaster over fractured bone

S. No.

Vernacular Tamil name

in the tibia of OVX rats. TRAP-positive cell numbers were significantly decreased in RANKL-induced RAW 264Âˇ7 cells when cultured with E.v extract28. Wal-nut (Jugulans regia Linn.) methanolic fruit extract stimulates the mineralization of matrix secreated by OBs (KS483 preosteoblast-like cell line, derived from Page 46

Uses

fetal mouse calvaria, which is a non-transformed stable subclone of a parental cell line KS4) when compared with treatment groups of ellagic acid (EA) and ellagic acid derivatives from walnut29. Phaseolus vulgaris (PV) commonly known as French beans is a member of Fabaceae family. Beans contribute Ann. SBV, Jan-Jun 2016;5(1)

to improving bone health. Compared with the OVX rats, methanolic extract of PV significantly decreased serum ALP and reduced serum TRAP and urinary Ca levels. It caused an increase in BMD, bone mechanical strength, increased bone Ca and improved bone microarchitecture30. The bean hull extract (BHE) given in aqueous solution stimulated bone formation and inhibited bone resorption or activity in mice. BHE supplementation (800 mg/kg) significantly increased BMD and trabecular thickness in the third lumbar of vertebrae, decreased serum TRAP and PTH concentrations in mice31. Dried plum, or prunes (Prunus domestica L.) a rich source of polyphenolic compounds is most effective fruit in both preventing and reversing bone loss in two models, androgen deficient male rats and OVX female rat models. It improved the biomechanical parameters like BMD, cortical load, trabecular architectural properties such as trabecular number connectivity density and trabecular separation studied by micro computed tomography. It also enhanced bone recovery during reambulation following skeletal unloading and had comparable effects to PTH in ovx Ann. SBV, Jan-Jun 2016;5(1)

rats. A 3-month clinical trial also indicated that the consumption of dried plum daily by postmenopausal women significantly increased serum markers of bone formation, total ALP, bone-specific ALP (BALP) and IGF-I32. Dried plum entitled its action by upregulating bone morphogenetic protein 4 (BMP4) and IGF-I while down-regulating Nfatc1 in OVX rats33. Camellia sinensis (Tea) is native to mainland South and Southeast Asia, but is today cultivated across the world, in tropical and subtropical regions. It is an evergreen shrub or small tree that is usually trimmed to below two meters (six feet) when cultivated for its leaves. Tea is manufactured in four basic forms viz green tea, white tea, black tea and oolong tea. The major constituents in tea are polyphenols and flavonoids. The four major flavonoids in green tea are the catechins i.e. epicatechin (EC), epigallocatechin (EGC), epicatechingallate (ECG) and EGGC. EGCG is richest in the leaf bud and occurs first in the leaves. The usual concentration of total polyphenols in dried green tea leaves is about 8% to 12%. Other compound of interest in dried green tea leaves include gallic acid, quercetin, kaempferol, myricetin, caffeic acid and chlorogenic acid. Black tea Page 47

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extract (BTE) was effective in preserving and restoring skeletal health by reducing the number of active OCs. BTE supplementation reduced oxidative stress of mononuclear cells, serum levels of IL-6, TNF-α and RANKL. The bone breaking force, histological and histomorphometric analyses also restored the delitorious effect with BTE supplementation in OVX rats. This study suggests that BTE has both protective and restorative actions against OVX-induced mononuclear cell oxidative stress and associated bone loss34. There are 5 main possible mechanisms of action through which green tea protects bone health. Green tea acts on antioxidant stress, anti-inflammation, osteoblastogenesis osteoclastogenesis and osteoimmunology. Green tea elucidates its effect [1] by mitigating bone loss through antioxidative stress action: green tea polyphenols (GTP) supplementation improved cellular antioxidant enzymes and diminished oxidative stress damage, and also had a beneficial effect on bone mass and microarchitecture in rats. EGCG in GTP decreased the formation of oxidative stress-induced Ca stone deposit formation in rats because of EGCG’s antioxidative effects. [2] by mitigating bone loss through anti-inflammatory action: GTP has also been proven to be beneficial in the prevention and treatment of a number of inflammatory diseases. A low-grade systemic chronic inflammation occurring in atherosclerosis leading to inflammation can also result in systemic bone loss by elevation of proinflammatory mediators, such as TNF-α, IL-β, γ-interferon and prostaglandin E2 (PGE2) which act directly on bone or indirectly to increase osteoclastogenesis, prevent OC apoptosis, and/or inhibit OB activity. The protective effect of EGCG is due to its ability to decrease lipid peroxidation, oxidative stress and the production of NO radicals by inhibiting the expression of inducible NO synthase. [3] by enhancing osteoblastogenesis: the components in green tea support osteoblastogenesis by increasing OB survival, proliferation, differentiation and bone formation, [4] by suppressing osteoclastogenesis: The bioactive components in green tea decreases the action of OCs in vivo and reduce osteoclastogenesis in vitro. The effects of green tea include suppressing bone resorption, increasing apoptosis of OCs, and inhibiting the formation of OCs, and [5] probably through osteoimmunological action: GTPs may modulate osteoimmunological activity first, by inhibiting differentiation of OCs through RANKL signaling, and second, by modulating the production of cytokines by immune cells34. Matrix metallo protein (MMP)-2 and MMP-9 activities were lower in theaflavin-3,3’digallate (TFDG). TFDG and EGCG inhibited the formation and differentiation of OC via inhibition of Page 48

MMPs. TFDG may suppress actin ring formation more effectively than EGCG. Five flavanes, isolated from Huangshan Maofeng tea, showed effects on proliferation of osteoblastic cells and ameliorated H2O2-induced C2C12 mouse myoblast cell apoptosis. (-)-Epicatechin increased ALP activity and hydroxyproline content, (-)-Epiafzelechin significantly increased the area of mineralized bone nodules. The flavanes are effective in promoting osteblastic proliferation, differentiation and protecting against apoptosis in C2C12 cells. Thus tea has anti-osteoporotic potential linked to antioxidative activity35. Carthamus tinctorious L. commonly known as safflower or false saffron belongs to the family Asteraceae. Oral administration of safflower seed oil at a dose of 1 ml/kg to OVX rats for 30 days significantly increased IGF-I, IGF-II, IGFBP-3 and BALP levels. The histopathological study suggested that safflower seeds have a possible role in the improvement of OVXinduced osteoporosis in rats36. The aqueous safflower seed extract supplementation in rats for 3 weeks after fracture increased BMP-2 gene expression in vivo and also increased BMP-2 expression in MG-63 cells in vitro. Aqueous extract of safflower seed has promoted bone nodule formation, Ca uptake, ALP activity and intracellular concentration of Ca2+and proliferation37, 38 of MC3T3-E1 cells. Safflower seeds are rich in Calcium, potassium and Phosphorus. A significant increase in the levels of serum IGF-I in rats was observed after treatment with methanolic extract of safflower seeds for 1 week and a strong correlation between femur length and IGF-I was observed39. Clover is native to Europe, western Asia and northwest Africa, but planted and naturalized in many temperate areas including America and Australia. Red clover, Trifolium pratense belongs to Fabaceae family. In the homeopathic Materia Medica, T. pratense is used in the treatment of menopause symptoms, maintenance/ improvement of cardiovascular health and for the reported benign effects on the breast, endometrium and neural structure besides for its safety40. The isoflavones biochanin A and genistein present in the leaves have estrogenic activity. Red clover extract (RCE) contain 40% isoflavones by weight (genistein, daidzein, biochanin A and formononetin present as hydrolyzed aglycones). As for the bone-preserving property, the effects of red clover have not been examined as extensively as for soy. Methanol extract of T. pratense shows significant competitive binding to ERα and β. Isoflavones significantly increased bone mineral content, mechanical strength of the tibia, femoral weight, Ann. SBV, Jan-Jun 2016;5(1)

femoral density and prevented the rise of serum ALP levels in OVX rats. In addition, the treatment with isoflavones, It also significantly reduced the number of OC compared to OVX rats. The findings suggested that T. pratenseis flavones are effective in reducing bone loss induced by OVX, probably by reduction of the bone turnover via. inhibition of bone resorption41. Luteolin-7-O-glycoside (LG), major constituents of the another clover species T. alexandrinum L. LG derived from aqueous methanol extract of T. alexandrinum L. has estrogenic effect, significantly inhibited the bone turnover markers, increased BALP, OCN, COL-I, N-terminal and C-telopeptide of type II collagen levels and promoted bone formation42. The Plant-based drugs are used globally for healing different illnesses in traditional systems of medicines. The medicinal plants used for bone healing are listed in Table 1. Eco-friendly and bio-friendly plant based approach for the healthy living is appreciated and followed throughout the world. The employment of plants in ethnomedicine is on rise worldwide. The scarce scientific evidences for the medicinal properties on bone healing is being attempted since two decades. The gist of herbs used for bone healing and its mechanism of action is given in Figure 1. ALP-Alkaline phosphatase , BMP-BoneMorphogenetic Protein, Ca-Calcium, COL-I-Type I collagen, IFNInterferon, IL-Interleukin, M-CSF-Macrophage-Colony Stimulating Factor, MMP-Matrix Metallo Protein, OBOsteoblast, OC-Osteoclast, OCN-Osteocalcin, OPG-

Osteoprotgerin, P-Phosphorus, PGE2-Prostaglandin E2 , S-Sulphur, Sr-Strontium, RANK-Receptor Activator of Nuclear Factor κB, RANKL-Receptor Activator of Nuclear Factor κb ligand, TRAP-Tartrate Resistant Acid Phosphatase.

Conclusion Breakthrough research, evidences and knowledge in medicinal herbs helps us to maintain bone health. Bone-building herbs, reduces the fracture risk naturally. Natural products for the management of osteoporosis are largely phytoestrogens, which include isoflavones, lignins, flavonoids, and coumestans that share structural and functional similarities with naturally occurring or synthetic estrogens. Phytoestrogens exhibit estrogenlike effects at various reproductive and non-reproductive tissues. Traditional medicines have been re-evaluated by clinicians, because these medicines have fewer side effects and because they are more suitable for longterm use when compared to chemically synthesized medicines. Most of plant-derived medicines have been developed on the basis of traditional knowledge in health care and in many cases. There is a correlation between the indications of pure substances and those of respective crude extracts used in traditional medicine. Many herbs were scientifically validated on its efficacy in last few decades in their osteoprotective effects. Thus the natural herbs promoting bone health may be targets for therapeutic approach towards enhancing bone formation or deducing bone loss in maintaining bone health.

References 1.

Nadkarni KM, editor. [Indian Materia Medica]; Dr. KM Nadkarni’s Indian Materia Medica: with ayurvedic, unani-Tibbi, siddha, allopathic, homeopathic, naturopathic & home remedies, appendices & indexes. 1. Popular Prakashan India; 1996. 2. Mühlbauer RC, Lozano A, Reinli A, Wetli H. Various selected vegetables, fruits, mushrooms and red wine residue inhibit bone resorption in rats. J Nutr. 2003; 133: 3592-7. 3. Rao SS, Ranganath Pai Karkala S, Potu BK, Bhat KM. Beneficial Effect of Cissus quadrangularis Linn. on osteopenia associated with streptozotocin-induced type 1 Diabetes Mellitus in male wistar rats . Adv Pharmacol Sci. 2014; 2014:483051. 4. Potu BK, Bhat KMR, Rao MS, Nampurath GK, Chamallamudi MR, et al. Petroleum ether extract of Cissus quadrangularis (linn.) enhances bone marrow mesenchymal stem cell proliferation and facilitates osteoblastogenesis. Clinics. 2009. 64:993-8. 5. Parisuthiman D, Singhatanadgit W, Dechatiwongse T, Koontongkaew S. Cissus quadrangularis extract enhances biomineralization through up-regulation of MAPKdependent alkaline phosphatase activity in osteoblasts. In Vitro Cell Dev Biol Anim. 2009; 45:194-200. 6. Srivastava MG, Sourabh, Nagori BP. Pharmacological and therapeutic activity of Cissus quadrangularis: An overview. Int J Pharm Tech Res CODE (USA) 2010; 1298–310. 7. Muthusami S, Ramachandran I, Krishnamoorthy S, Govindan R, Narasimhan S. Cissus quadrangularis augments IGF system components in human osteoblast like SaOS-2 cells. Growth Horm IGF Research. 2011; 21: 343-48. 8. Muthusami S, Senthilkumar K, Vignesh C, Ilangovan R, Stanley J, et al. Effects of Cissus quadrangularis on the proliferation, differentiation and matrix mineralization of human osteoblast like SaOS-2 cells. J Cell Biochem. 2011; 112: 1035-45. 9. Banu J, Varela E, Bahadur AN, Soomro R, Kazi N, Fernandes G. Inhibition of bone loss by Cissus quadrangularis in mice: A preliminary report. J Osteoporos. 2012; 21:101206. 10. Rasale PL, Raut SY, Narkhede HP, Muddeshwar MG. Biochemical and hormonal evaluation of Cissus quadrangularis in accelerating healing process of bone fracture: a clinical study. Int J Res Ayur Pharm. 2014; 5: 461-69. 11. Jurenka JS. Therapeutic applications of pomegranate (Punica granatum L.): a review. Altern Med Rev. 2008; 13: 128-44. 12. Kim ND, Mehta R, Yu W, Neeman I, Livney T, et al. Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer. Breast Cancer Res Treat. 2002; 71: 203-17.

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Annals of SBV 13. Spilmont M, Léotoing L, Davicco MJ, Lebecque P, Miot-Noirault E, et al. Pomegranate peel extract prevents bone loss in a preclinical model of osteoporosis and stimulates osteoblastic differentiation in vitro. Nutrients. 2015; 7:9265-84. 14. Okamoto T. Safety of quercetin for clinical application. Int J Mol Med. 2005; 16: 2755-58. 15. Huang TH, Mühlbauer RC, Tang CH, Chen HI, Chang GL, et al. Onion decreases the ovariectomy-induced osteopenia in young adult rats. Bone 2008; 42: 1154-63. 16. Wetli HA, Brenneisen R, Tschudi I, Langos M, Bigler P, Sprang T. A gamma-glutamyl peptide isolated from onion (Allium cepa L.) by bioassay guided fractionation inhibits resorption activity of osteoclasts. J Agric Food Chem. 2005; 53: 3408-14. 17. Tang CH, Huang TH, Chang CS, Fu WM, Yang RS. Water solution of onion crude powder inhibits RANKL-induced osteoclastogenesis through ERK, p38 and NF-kappaB pathways. Osteoporos Int. 2009; 20: 93-103. 18. Law YY, Chiu HF, Lee HH, Shen YC, Venkatakrishnan K, Wang CK. Consumption of onion juice modulates oxidative stress and attenuates the risk of bone disorders in middle-aged and post-menopausal healthy subjects. Food Funct. 2016; 17:902-12. 19. Mukherjee M, Das AS, Das D, Mukherjee S, Mitra S, Mitra C. Effects of garlic oil on postmenopausal osteoporosis using ovariectomized rats: comparison with the effects of lovastatin and 17-beta-estradiol. Phytother Res. 2006; 20: 21-7. 20. Mukherjee M, Das AS, Das D, Mukherjee S, Mitra S, Mitra C. Role of peritoneal macrophages and lymphocytes in the development of hypogonadal osteoporosis in an ovariectomized rat model: possible phytoestrogenic efficacy of oil extract of garlic to preserve skeletal health. Phytother Res. 2007; 21: 1045-54. 21. Mozaffari-Khosravi H, Hesabgar HA, Owlia MB, Hadinedoushan H, Barzegar K, Fllahzadeh. The effect of garlic tablet on pro-inflammatory cytokines in postmenopausal osteoporotic women: a randomized controlled clinical trial. J Diet Suppl. 2012; 9: 262-71 22. Lee KH, Choi EM. Stimulatory effects of extract prepared from the bark of Cinnamomum cassia blume on the function of osteoblastic MC3T3-E1 cells. Phytother Res. 2006; 20: 952-60 23. Tsuji-Naito K. Aldehydic components of cinnamon bark extract suppresses RANKL-induced osteoclastogenesis through NFATc1 down regulation. Bio org Med Chem. 2008; 16: 9176-83. 24. Lee YS, Choi CW, Kim JJ, Ganapathi A, Udayakumar R, Kim SC. Determination of mineral content in methanolic safflower (Carthamus tinctorius L.) seed extract and its effect on osteoblast markers. Int J Mol Sci. 2009; 10: 292-305. 25. Annie S, Prabhu RG, Malini S. Activity of Wedelia calendulacea Less. in post-menopausal osteoporosis. Phytomed. 2006; 13: 43-8 26. Khedgikar V, Ahmad N, Kushwaha P, Gautam J, Nagar GK, et al. Preventive effects of withaferin A isolated from the leaves of an Indian medicinal plant Withania somnifera (L.): comparisons with 17-β-estradiol and alendronate. Nutrition. 2015; 31: 205-13. 27. Zhang Y, Li XL, Lai WP, Chen B, Chow HK, et al. Anti-osteoporotic effect of Erythrina variegate L. in ovariectomized rats. J Ethnopharmacol. 2007; 109:165-9. 28. Zhang Y, Li Q, Li X, Wan HY, Wong MS. Erythrina variegata extract exerts osteoprotective effects by suppression of the process of bone resorption. Brit J Nutr. 2010; 104: 965-71. 29. Papoutsi Z, Kassi E, Chinou I, Halabalaki M, Skaltsounis LA, et al. Wal nut extract (Juglans regia L.) and its component ellagic acid exhibit anti-inflammatory activity in human aorta endothelial cells and osteoblastic activity in the cell line KS483. Brit J Nutr. 2008; 99: 715-22. 30. Shirke SS, Jadhav SR, Jagtap AG. Osteoprotective effect of Phaseolus vulgaris L. in ovariectomy-induced osteopenia in rats. Menopause. 2009; 16: 589-96. 31. Cao JJ, Gregoire BR, Sheng X, Liuzzi JP. Pinto bean hull extract supplementation favorably affects markers of bone metabolism and bone structure in mice. Food Res Int. 2010; 43: 560-66. 32. Hooshmand S, Arjmandi BH. Viewpoint: dried plum, an emerging functional food that may effectively improve bone health. Ageing Res Rev. 2009; 8:122-7. 33. Smith BJ, Bu SY, Wang Y, Rendina E, Lim YF, et al. A comparative study of the bone metabolic response to dried plum supplementation and PTH treatment in adult, osteopenic ovariectomized rat. Bone. 2014; 58:151-9. 34. Shen C, Chyu M, Wang J. Tea and bone health: steps forward in translational nutrition. Am J Clin Nutr. 2013; 98: 1694–99S. 35. Zeng X, Tian J, Cai K, Wu X, Wang Y, et al. Promoting osteoblast differentiation by the flavanes from Huangshan Maofeng tea is linked to a reduction of oxidative stress. Phytomed. 2014; 21:217-24 36. Alam MR, Kim SM, Lee JI, Chon SK, Choi SJ, et al. Effects of safflower seed oil in osteoporosis induced-ovariectomized rats. Am J Chin Med. 2006; 34: 601-12. 37. Jang HO, Park YS, Lee JH, Seo JB, Koo KI, et al. Effect of extracts from safflower seeds on osteoblast differentiation and intracellular calcium ion concentration in MC3T3-E1 cells. Nat Prod Res. 2007; 21: 787-97. 38. Kim KW, Suh SJ, Lee TK, Ha KT, Kim JK, et al. Effect of safflower seeds supplementation on stimulation of the proliferation, differentiation and mineralization of osteoblastic MC3T3-E1 cells. J Ethnopharmacol. 2008; 115: 42-9. 39. Lee YS, Choi CW, Kim JJ, Ganapathi A, Udayakumar R, Kim SC. Determination of mineral content in methanolic safflower (carthamus tinctorius l.) seed extract and its effect on osteoblast markers. Int J Mol Sci. 2009; 10: 292–305 40. Occhiuto F, Zangla G, Samperi S, Palumbo DR, Pino A, et al. The phytoestrogenic isoflavones from Trifolium pratense L. (Red clover) protects human cortical neurons from glutamate toxicity. Phytomed. 2008; 15: 676-82. 41. Occhiuto F, Pasquale RD, Guglielmo G, Palumbo DR, Zangla G, et al. Effects of phytoestrogenic isoflavones from red clover (Trifolium pratense L.) on experimental osteoporosis. Phytother Res. 2007; 21: 130-4. 42. Ammar NM, El-Hawary SS, Mohamed DA, El-Halawany AM, El-Anssary AA, et al. Estrogenic activity including bone enhancement and effect on lipid profile of Luteolin7-O-glucoside Isolated from Trifolium alexandrinum L. in ovariectomized rats. Phytother Res. 2016; 30:768-73. 43. Chhavi S, Sushma D, Ravinder V, Anju D, Asha S. Recent update on proficient bone fracture reviving herbs. Int Res J Pharm.2011; 2: 3-5. 44. Pradesh A. Indigenous phytotherapy for bone fractures from Eastern Ghats. Ind J Trad Knowledge. 2011; 10: 550-3. 45. Kumar MD, John KM, Karthik S. The bone fracture–healing potential of Ormocarpum cochinchinense, methanolic extract on albino wistar rats. J herb, spices & med plants. 2013; 19:1-10.

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Glioblastoma: Evolving Niches and Challenges Pooja Pratheesh, Scientist Central Inter-Disciplinary Research Facility, Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India Email: poojapratheesh@gmail.com

Abstract 

lioblastoma multiforme (GBM) are the most refractory brain tumors characterized by G aggressive growth and metastatic potential along with the resistance to any kind of therapy. These tumors show high heterogeneity both at the molecular and histological levels. Glioblastoma differ from low grade gliomas as they exhibit a necrotic core and microvascular proliferation making them one of the most hypoxic as well as angiogenic tumors. The differential niches within the GBM microenvironment not only regulate the metabolic needs, tumor survival and invasion, they are also responsible for cancer stem cell (CSC) maintenance. This review discusses the cellular and functional diversity of the GBM niches and the cross-talk between them.

Key Words: Glioblastoma multiforme, neovascularization, hypoxia, necrosis, niche.

Introduction Glioblastoma multiforme (GBM) develop with a well formed yet inefficient neo-vascular system compared to the normal brain tissue1. Intra-tumoral necrosis due to insufficient vascular supply is a hallmark feature of GBM and sometimes it may involve molecular or genetic changes intrinsic to the tumor2. The overall prognosis for GB has changed little since the 1980s, despite major improvements in neuroimaging, neurosurgery, radiotherapy, and chemotherapy techniques (Figure 1). Evidence suggests that hypoxia is a well-recognized component of the tumor microenvironment and plays a role in the malignant transformation of cells and subsequent tumor growth. It has been linked to poor patient outcome and resistance to therapies in different cancer types. In a number of human cancers, hypoxia predicts the chances of metastases3, tumor relapse, resistance to chemo- as well as radiation therapy5, and poor prognosis. In fact, the degree of necrosis within a GBM correlates inversely with the survival of the Ann. SBV, Jan-Jun 2016;5(1)

patient. Hypoxic stress has been linked to phenotypic changes in tumors wielded through genomic instability, anoikis, altered gene expression, and neo-angiogenesis. Figure 1. Diagrammatic representation of the survival estimation of GBM

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Hypoxia in Glioblastoma Recent advancements in genomic sequencing and analysis have stratified GBM into different molecular subtypes, of which the mesenchymal (MES) and proneural (PN) subtypes appear to be the most pronounced6. Interestingly, high levels of necrosis were observed in tumors of patients having a mesenchymal subtype. The MES phenotype in GBMs has been associated with refractory tumors and elevated metastatic potential7. GBM cells surrounding necrotic zones with hypoxic core express high levels of the mesenchymal transcription factors like C/EBP-β and C/EBP-δ. Incidentally, the expression of these transcription factors is associated with a poor disease prognosis8. GBMs invariably display swift cell proliferation coupled with insufficient vascularization leading to generation of niches with scarce oxygen supply. This chronic exposure to extremely low levels of oxygen produces necrotic zones surrounded by densely packed hypoxic tumor cells called “pseudopalisading” GBM cells. These “pseudopalisading” GBM cells are shown to express hypoxia-regulated genes that control angiogenesis, extracellular matrix degradation, and invasive behavior9. The cellular responses to hypoxia are generally mediated by the hypoxia-inducible factor (HIF) family of

transcription factors. HIFs function as heterodimers composed of an oxygen-sensitive HIFα subunit and a constitutively expressed HIFβ subunit. Under normoxia HIFα undergoes proteasomal degradation by ubiquitination mediated by the von Hippel–Lindau (vHL) tumor suppressor gene product. However, under hypoxic conditions, the interaction between HIFα and vHL is abolished leading to the stabilization of the HIFα subunit. This allows its dimerization with HIFβ and subsequent binding to hypoxia responsive elements (HREs) in the promoters of hypoxia-regulated genes10. Hence, the transcription of hundreds of downstream genes that can modulate cell survival, mobility, metabolism and angiogenesis in order to restore oxygen homeostasis are well regulated (Figure 2) Two HIFα subunits, HIF1α and HIF2α, though structurally similar in their DNA binding and dimerization domains, are known to have nonoverlapping biological roles and require different levels of oxygen for activation. HIF-1α and HIF-2α regulate different subsets of genes, although they do share common targets such as VEGF and GLUT1. In arginine homeostasis, HIF-1α induces iNOS expression and increases nitric oxide production from arginine, whereas HIF-2α stimulates arginase expression, and suppresses NO production. Therefore, identification of differential roles of HIF-1α and HIF-2α is essential to

Figure 2. Regulation of HIF-1α under normoxic and hypoxic conditions

understand the molecular mechanisms of the metabolic consequences of hypoxia in cancers Another feature of GBM is extensive and abnormal angiogenesis that leads to disorganized and leaky blood vessels. This is predominantly induced by the remarkable elevation of vascular endothelial growth factor (VEGF) activity. It has been demonstrated that hypoxia induces both increased transcription and decreased degradation of VEGF mRNA in solid tumors11. The vascular abnormalities in GBM can cause the disruption of the blood–brain barrier (BBB) as a result of which the circulating immune cells and derived chemokines and cytokines enter the brain. Monocytes, neutrophils, and myeloid-derived suppressor cells (MDSC) 12, 13 are all commonly found in the perivascular tumor niche. These cells discharge immune-suppressive functions, and interact with tumor cells and cancer stem cells (CSCs), thereby promoting tumor propagation and progression. Several studies from GBM and other tumor types have provided evidence that hypoxic tumor niches including peri-necrotic regions of human glioblastoma biopsies are rich in CSCs14. Hypoxia promotes stem-ness through the activation of genes implicated in self-renewal and dedifferentiation, and shields tumor cells and CSCs from chemo- and radiotherapy. The increased stem-ness of the tumor cells can be measured by an increase in neurosphere forming ability of CSCs, with increased expression of several cancer stem cell markers, including CD133, SOX2, OCT4, and nestin15, 16. The necrotic cell death at the center of the hypoxic niche releases pro-inflammatory cytokines/chemikines into the surrounding tissue microenvironment that tunes inflammatory cells such as tumor associated macrophages (TAMs) and neutrophils to lose their original function of removing necrotic debris and transform them into immune-suppressive and angiogenesis-promoting cells. In addition, hypoxia appears to enhance trans-differentiation of CSCs into endothelial-like cells, and promotes their incorporation into tumor vessels17. Micro vascular hyperplasia often observed in close proximity to pseudopalisading necrosis enables tumor cells and CSC to grow towards newly formed vasculature and thus contributes to a vicious cycle.

The Invasive GBM Niche GBMs not only migrate away from hypoxic regions within the tumor mass, but have the propensity to invade normal tissue as well. GBM infiltrate as single cells or move along white-matter tracts and basement membranes, including those of blood vessels to invade normal brain parenchyma18, 19. It is notable that Page 52

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several membrane metalloproteinases (MMPs) have been associated with GBM invasion mode of GBM2022 . A search for MMP-inducing factors in tumour cells led to the identification of CD147, also known as EMMPRIN (extracellular matrix metalloproteinase inducer), a highly glycosylated cell-surface transmembrane protein which stimulates MMP synthesis in neighboring fibroblasts and tumor cells23 (Figure 3). CD147 is highly expressed in various human carcinoma tissues and cell lines, correlating with tumour progression under experimental and clinical conditions. Glioma cells have been shown to over- express CD147 and its expression is associated with more aggressive tumor type and poor prognosis. The best characterized function of CD147 is its ability to induce the expression of MMPs, including MMP-1, MMP-2, MMP-3, MMP- 9 and MMP-11 in stromal cells. In view of the high expression of CD147 in malignant tissues and its potential as a target for cancer therapy, many studies have investigated how CD147 modulates MMP in cancer. As a trans membrane glycoprotein, CD147 forms homo-oligomers in both heterotypic and homotypic cell–cell interactions to induce Figure 3. Transcriptional control of membrane metalloproteinases (MMPs) by CD147.

production of MMPs 24. Moreover, full-length EMMPRIN is released by tumor cells via vesicle shedding25. Secreted soluble CD147 in conditioned medium is equally capable of inducing MMP production, either from surrounding fibroblasts or tumor cells themselves. Our laboratory at Central Inter-Disciplinary Research Facility is actively engaged in elucidating the role played by CD147 in glioblastomas pathology. Upregulation of CD147 in solid tumors imparts them Page 53

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higher viability by facilitating the metastasis, and by enabling the glycolytic switch that allows cancer cells to survive under low oxygen tension. We are working with human GBM cell lines under normoxic and hypoxic conditions to elucidate the role played by CD147 in their metabolic reprogramming as well as enhanced metastatic capacity.

Conclusion There is a growing support for the concept of presence of structurally and functionally distinct tumor niches in GBMs and other invasive tumor types with hypoxic features. The high proliferative rate of GBMs and subsequent neovascularization converts these niches into hypoxic and subsequent perivascular tumor niches. These

distinct tumor niches undergo dynamic flux in a temporal and spatial manner creating tumor microenvironments to accommodate the aggressive growth of GBM into normal tissue. The prevailing view so far has focused on the signaling events in a tumor as a whole. However given the cellular and functional diversity of the vasculature, the cross-talk between the tumor and non-tumor niche may vary widely. This opens up new avenues for exploration into the crucial dialogues between these “in-flux” tumor and non-tumor compartments, the type of tumor niche that will be generated out of this cross-talk, and how these distinct niches might react to cancer therapies. The forthcoming knowledge will provide key information that may help in identifying new opportunities for designing therapeutic strategies aimed at all tumor niches in GBMs to enhance survival of cancer patients.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Blasberg RG, Kobayashi T, Horowitz M, Rice JM, Groothuis D, et al. Regional blood flow in ethylnitrosourea-induced brain tumors.Ann Neurol. 1983; 14:189-201. Raza SM, Lang FF, Aggarwal BB, Fuller GN, Wildrick DM, et al. Necrosis and glioblastoma: a friend or a foe? A review and a hypothesis. Neurosurgery. 2002; 51:2-12. Sundfør K, Lyng H, Rofstad EK. Tumour hypoxia and vascular density as predictors of metastasis in squamous cell carcinoma of the uterine cervix. Br J Cancer. 1998 ;78:822-7. Höckel M, Schlenger K, Höckel S, Aral B, Schäffer U, et al. Tumor hypoxia in pelvic recurrences of cervical cancer. Int J Cancer. 1998; 79:365-9. Sanna K, Rofstad EK. Hypoxia-induced resistance to doxorubicin and methotrexate in human melanoma cell lines in vitro. Int J Cancer. 1994; 58:258-62. Huse JT, Phillips HS, Brennan CW. Molecular sub-classification of diffuse gliomas: seeing order in the chaos. Glia. 2011; 59:1190-9. Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, et al. Cancer Genome Atlas Research Network.Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 2010; 17:98-110 Cooper LA, Gutman DA, Chisolm C, Appin C, Kong J, et al. The tumor microenvironment strongly impacts master transcriptional regulators and gene expression class of glioblastoma. Am J Pathol. 2012; 180:2108-19. Brat DJ, Castellano-Sanchez AA, Hunter SB, Pecot M, Cohen C, et al. Pseudopalisades in glioblastoma are hypoxic, express extracellular matrix proteases, and are formed by an actively migrating cell population. Cancer Res. 2004; 64:920-7. Keith B, Simon MC. Hypoxia-inducible factors, stem cells, and cancer. Cell. 2007; 129:465-72. Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol. 1996; 16:4604-13. Liang J, Piao Y, Holmes L, Fuller GN, Henry V, et al. Neutrophils promote the malignant glioma phenotype through S100A4. Clin Cancer Res. 2014; 20:187-98. Feng X, Szulzewsky F, Yerevanian A, Chen Z, Heinzmann D, et al. Loss of CX3CR1 increases accumulation of inflammatory monocytes and promotes gliomagenesis. Oncotarget. 2015; 6:15077-94. Soeda A, Park M, Lee D, Mintz A, Androutsellis-Theotokis A, et al. Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1alpha. Oncogene. 2009; 28:3949-59. Bar EE, Lin A, Mahairaki V, Matsui W, Eberhart CG. Hypoxia increases the expression of stem-cell markers and promotes clonogenicity in glioblastoma neurospheres. Am J Pathol. 2010; 177:1491-502. Soda Y, Marumoto T, Friedmann-Morvinski D, Soda M, Liu F, et al. Transdifferentiation of glioblastoma cells into vascular endothelial cells. Proc Natl Acad Sci U S A. 2011; 108:4274-80. Cuddapah VA, Robel S, Watkins S, Sontheimer H. A neurocentric perspective on glioma invasion. Nat Rev Neurosci. 2014;15:455-65. Westphal M, Lamszus K. The neurobiology of gliomas: from cell biology to the development of therapeutic approaches. Nat Rev Neurosci. 2011; 12:495-508. Yoshida S, Shibata M, Yamamoto S, Hagihara M, Asai N, et al. Homo-oligomer formation by basigin, an immunoglobulin superfamily member, via its N-terminal immunoglobulin domain. Eur J Biochem. 2000; 267:4372-80. Deryugina EI, Soroceanu L, Strongin AY. Up-regulation of vascular endothelial growth factor by membrane-type 1 matrix metalloproteinase stimulates human glioma xenograft growth and angiogenesis. Cancer Res. 2002; 62:580-8. Choe G, Park JK, Jouben-Steele L, Kremen TJ, Liau LM, et al. Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype. Clin Cancer Res. 2002; 8:2894-901. Du R, Petritsch C, Lu K, Liu P, Haller A, et al. Matrix metalloproteinase-2 regulates vascular patterning and growth affecting tumor cell survival and invasion in GBM. Neuro Oncol. 2008; 10254-64. Kaushik DK, Hahn JN, Yong VW. EMMPRIN, an upstream regulator of MMPs, in CNS biology. Matrix Biol. 2015; 44:138-46. Taylor PM, Woodfield RJ, Hodgkin MN, Pettitt TR, Martin A, et al. Breast cancer cell-derived EMMPRIN stimulates fibroblast MMP2 release through a phospholipase A(2) and 5-lipoxygenase catalyzed pathway. Oncogene. 2002; 21:5765-72. Sidhu SS, Mengistab AT, Tauscher AN, LaVail J, Basbaum C. The microvesicle as a vehicle for EMMPRIN in tumor-stromal interactions. Oncogene. 2004; 23:956-63.

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Glioblastoma Multiforme Metabolism: Fuel to the Flame Preethi Sridharan, PhD Scholar Central Inter-Disciplinary Research Facility, Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India E-mail: preethiresearcher@gmail.com

Abstract 

lioblastoma Multiforme (GBM) is an aggressive, lethal brain tumor. Cellular metabolism G is the major process affected during tumorigenesis. It is recently revealed that oncogenic signaling pathways are unswervingly involved in metabolic reprogramming of tumors. In GBM, metabolic pathways are reprogrammed and the underlying mechanisms causing these changes are yet to be unraveled. Interestingly, the pentose phosphate pathway in GBM shows differential association with glycolysis. This review discusses about the key metabolic enzymes and their association with several pathways in GBM highlighting the potential therapeutic targets.

Key Words: Glioblastoma multiforme, Glycolysis, Glutamine metabolism, Lipid metabolism, Pentose phosphate pathway.

Introduction GBM, the most aggressive of the gliomas, is a collection of tumors arising from glial cells or their precursors within the brain. The most common grading system uses the scale of I to IV, of which, grade I is benign, grade II is low grade glioma whereas, grade III and IV are considered to be high grade and malignant. GBMs draw significance because of their poor prognosis (less than a year). The histological features that distinguish GBM from other grades are the presence of necrosis and increased vasculature around the tumor. GBM is characterized by a heterogeneous cell population which makes it genetically unstable, highly infiltrative, angiogenic and resistant to chemotherapy 1. Moreover, GBMs show altered cell metabolism. In recent years, the understanding of the regulation of tumor metabolism has much improved. Evidence show that tumor cells reprogram their metabolism to meet high energy demands which markedly elevate biosynthetic processes Ann. SBV, Jan-Jun 2016;5(1)

and energy production, which in turn promote rapid tumor growth and division (Figure 1). Thus, targeting metabolism has become a promising strategy for cancer treatment 2. Figure 1. GBM metabolism in a nutshell

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Glycolysis The most popular metabolic remodeling described in tumor cells is an increase in glucose uptake, the enhancement of glycolytic capacity and the high lactate production, along with the almost inefficient oxidative phosphorylation even in the presence of high oxygen tension (Warburg effect)3. GBMs upregulate glycolysis more than three times that of normal brain tissue as measured by increased lactate: pyruvate ratio. Several in vitro studies have demonstrated large variability in mitochondrial respiration and glucose dependency in cell lines derived from GBM tissues and xenografts. The ability to modulate mitochondrial respiration is an important component to tumor cell survival under hypoxic conditions4-6. In addition to enhanced glucose uptake and aerobic glycolysis, GBM cells also exhibit altered glutamine catabolism, particularly within myc expressing cells7, 8. Hexokinase Hexokinase (HK) catalyzes the first rate limiting step in glycolysis. HK has four isoforms I-IV showing differential tissue expression. In normal human brain and in low-grade gliomas HK-I is predominantly expressed, whereas in high grade gliomas, HK-II is highly expressed9, 10. Regional variation in HK-II expression is observed in GBMs, showing higher expression in the highly proliferating and apoptosis-resistant perinecrotic central regions, suggesting HK-II may provide a survival and proliferative advantage in vivo, especially within tumor microenvironments. The depletion of HK-II in vitro showed potential inhibition of aerobic glycolysis and promoted normal oxidative glucose metabolism followed by decreased lactate production and increased expression of Oxidative Phosphorylation (OXPHOS) proteins with enhanced O2 consumption. The high affinity kinases HK1 and HK-II are inhibited by excess Glyceraldehyde-6- Phosphate (G6P). They are associated with the mitochondria, are partly responsible for the increased glycolytic tumor activity and are implicated in cell survival. Many inhibitors of HK affecting the glycolytic flux like 2-deoxy-D-glucose (2DG), 3-bromopyruvate and Ionidamine have shown promising effects in preclinical studies. Pyruvate Kinase Pyruvate Kinase (PK) catalyzes the final step of glycolysis converting the phosphoenolpyruvate (PEP) to pyruvate and generating ATP. Four PK forms are identified PKL Page 56

& PKR, PKM1 & PKM2. M1 and M2 isoforms are produced by alternative splicing containing exon 9 and exon 10 and exhibit different functional activities. PKM1 is constitutively expressed whereas, PKM2 can be regulated by fructose-1,6-bisphosphate. PKM1 expression is down-regulated by the splicing repressors while PKM2 is mainly expressed in cancer cells11, and is involved in EGFR signaling pathway in GBM12. Upon its activation by EGFR via phosphorylation at Ser37 by ERK1/2, PKM2 was shown to translocate from the cytoplasm to the nucleus where it binds to importin α5. After entering into the nucleus, PKM2 promotes the transcription of C-MYC which further upregulates glucose transporters (GLUT) and HK-II expression to promote glycolysis13. In GBM cells, activation of EGFR by EGF induces PKCε monoubiquitylation that recruits and phosphorylates IKKβ, to promote the interaction of activated Rel A with HIF-1α. This complex binds to the PKM2 promoter and transcribes it14. It was observed that the PKM1 expressed in normal brain samples exhibited high levels of PK activity whereas a significantly lower activity level was observed in all gliomas including GBMs15. Recently; it was shown that in adult glioma spheroids, PKM2-Oct4 interaction inhibited the stemness property, promoted differentiation and enhanced the sensitivity to cell death16. Isocitrate Dehydrogenase Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to produce α-Keto Glutarate ( α-KG),generating NADH in mitochondria or NADPH in cytoplasm. Five IDH genes have been identified. IDH1 and IDH2 mutations were first reported in low grade gliomas and secondary GBMs. IDH1 mutation occur 80% and 85% of astrocytomas and secondary GBMs respectively. IDH1/2 mutants gain new enzyme activity, which catalyzes α-KG to 2-hydroxyglutarate (2-HG), a metabolite mostly produced by error during normal metabolism and leads to its accumulation in GBM patients with IDH1/2 mutations. These mutations led to a hypermethylation phenotype and alteration in cellular metabolism in response to hypoxic and oxidative stress17. However, as of now, the ability to target GBMs with IDH mutations remains limited, and more investigations are necessary to optimize the therapeutic strategies targeting IDH mutant subsets of GBMs.

Glutamine Metabolism In addition to glucose, amino acids can also act as a fuel for cancer cells and funnel into the tricarboxylic acid Ann. SBV, Jan-Jun 2016;5(1)

cycle (TCA)18. Glutamine is hydrolyzed to glutamate by glutaminase (GLS) and is further converted to α-KG that finally enters the TCA cycle. In the presence of mutant IDH1, α-KG is converted to 2-HG thus affecting the TCA cycle19. The NMR spectra showed that the glutamine concentration was significantly higher in GBM patients than in control subjects20. The conversion of glutamate to glutamine by glutamine synthetase (GS) is compromised in GBM patients that improved their survival rate 21. In GBM cell cultures, it has been reported that around 60% of the glutamine is converted to alanine22. Glutaminolysis also generates NADPH which is used for fatty acid biosynthesis via the activity of malate dehydrogenase. Glutaminase Phosphate-activated glutaminase, the enzyme converting glutamine to glutamate plays a remarkable role in tumor biology. There are two genes encoding for GLS: the GLS encoding kidney-type (K-type) isozymes and GLS2 encoding liver-type (L-type) isozymes. The L-type is expressed in brain. Brain tumors show differential expression of GLS transcripts depending on their cellular origin. GLS isoforms play opposite roles in tumorigenesis, as the expression of K-type is correlated with greater cell proliferation while that of L-type with low proliferation and quiescent/resting cells. Knocking down GLS in GBM cells led to the reversion of the transformed phenotype. The similar effects were obtained by the overexpression of GLS2 gene in GBM 23.

Lipid Metabolism Lipids, such as fatty acids, cholesterol, triglycerides, cholesterol esters, phospholipids and spingolipids are the important component of biological membranes. Other than their role as structural component, they also function as energy resource and as signaling molecules to maintain cell growth. Lipid metabolism is largely impaired in cancers. In GBM, higher levels of unsaturated fatty acids are observed compared with normal brain. Key proteins such as SREBP-1, Acetyl-CoA carboxylase (ACC), Fatty acid synthase (FAS) and low-density lipoprotein receptor (LDLR) are up-regulated in GBMs. The oncogenic signaling pathway EGFR/PI3K/Akt regulates the lipid metabolic reprogramming. Recent studies have shown that fatty acyl-CoA synthetase VL3 (ACSVL3) is involved in maintenance of GBM stem cell and in their tumorinitiating capacity in neurospheres24. Ann. SBV, Jan-Jun 2016;5(1)

Sterol Regulatory Element-Binding Protein 1 (SREBP-1) SREBP-1 is the transcription factor that regulates de novo fatty acid synthesis. It has three isoforms SREBP1a, -1c and -2 that play differential role in regulation of lipid metabolism. SREBP-1 plays a major role in energy metabolism including fatty acid and glucose metabolism while, SREBP-2 activates cholesterol synthesis. SREBPs are synthesized in inactive form bound to ER. These are activated by sequential proteolytic cleavage and translocate to Golgi complex. Eventually, their N-terminal domain is released into the nucleus where it activates the target genes. It was observed that SREBP-1 is highly up-regulated in GBM cell lines and its N-terminal domain is present in the nucleus of tumor cells in patients’ tissues25. Inhibition or reduction of SREBP-1 by pharmacologic agents significantly induced GBM cell death 26. Thus, SREBP-1 is considered a promising molecular target for GBM therapy. Low-density lipoprotein receptor (LDLR): Cholesterol is the major component in cell membranes. Extra cholesterol is esterified to form cholesterol esters. Maintenance of cholesterol levels is important for cell morphology and its function. In human blood, cholesterol is transported from liver to the rest of the body by lipoproteins such as low density lipoproteins (LDL). The LDLR is a cell surface protein that binds LDL and transports it inside the cell. LDLR is found to be highly expressed in GBM ensures that tumor cells obtain sufficient cholesterol for their rapid growth and division. Its upregulation could explain the accumulation of cholesterol esters in GBM. LDLR is shown to be upregulated by EGFR/PI3K/Akt pathway which was found to be mediated by SREBP-1 and not -2 in GBM cells26. Pentose Phosphate Pathway (PPP) Glucose is a common fuel entering cells through a glucose transporter and being phosphorylated to form glucose-6-phosphate (G-6-P), which can be further metabolized by both glycolysis and the PPP. PPP has oxidative and the nonoxidative pathway with G6PD, transketolase and transaldolase catalyzing their rate limiting steps. Glycolysis and Gluconeogenesis coordinate with the PPP to control the production of NADPH and Ribose 5-Phosphate (R-5-P), which determines whether the oxidative or non-oxidative pathway Page 57

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of PPP would be activated. GBM shows a pentose phosphate flux rate of approximately 4% of the total glucose f lux. Both the activity and the regulation of glucose-6-phosphate dehydrogenase (G6PDH) are altered in GBMs 27. It was recently reported that the PPP enzyme expression increased in rapidly dividing GBM cells, whereas glycolytic enzymes were elevated in migrating cells 28. It was also reported that the knockdown of G6PD reduced GBM cell proliferation while that of ALDOC (encoding Aldolase c) knockdown decreased migration 28.

Conclusion Metabolic reprogramming is a key feature of oncogenesis and the recent studies have revealed that the glucose, glutamine and lipid metabolism are largely impaired in GBM facilitating malignancy while the role of PPP still remains obscured. Targeting molecules and enzymes that metabolically reprogram GBM can be a novel and potential therapeutic approach. Further understanding of the metabolic alterations in GBM will helps in developing more promising approaches to abrogate GBM malignancy and to overcome GBM resistance to current therapeutic approaches.

Cancer Stem Cells – A Brief Overview Sam Vijay Kumar J, Scientist Central Inter-Disciplinary Research Facility, Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India Email: samvijaykumar@cidrf.res.in

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

Ramirez YP, Weatherbee JL, Wheelhouse RT, Ross AH. Glioblastoma multiforme therapy and mechanisms of resistance. Pharmaceuticals (Basel). 2013; 6:1475-506. Ru P, Williams TM, Chakravarti A, Guo D. Tumor metabolism of malignant gliomas. Cancers (Basel). 2013; 5:1469-84. Warburg O, Wind F, Negelein E. The metabolism of tumors in the body. J Gen Physiol.1927; 8:519-30. Parliament MB, Franko AJ, Allalunis-Turner MJ, Mielke BW, Santos CL, et al. Anomalous patterns of nitroimidazole binding adjacent to necrosis in human glioma xenografts: possible role of decreased oxygen consumption. Br J Cancer. 1997; 75:311-8. Franko AJ, Parliament MB, Allalunis-Turner MJ, Wolokoff BG. Variable presence of hypoxia in M006 human glioma spheroids and in spheroids and xenografts of clonally derived sublines. Br J Cancer. 1998; 78:1261-8. Allalunis-Turner MJ, Franko AJ, Parliament MB. Modulation of oxygen consumption rate and vascular endothelial growth factor mRNA expression in human malignant glioma cells by hypoxia. Br J Cancer. 1999; 80:104-9. DeBerardinis RJ, Mancuso A, Daikhin E, Nissim I, Yudkoff M, et al. Beyond aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proc Natl Acad Sci U S A. 2007; 104:19345-50. Wise DR, DeBerardinis RJ, Mancuso A, Sayed N, Zhang XY, et al. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc Natl Acad Sci U S A. 2008; 105:18782-7. Wolf A, Agnihotri S, Micallef J, Mukherjee J, Sabha N, et al. Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme. J Exp Med. 2011; 208:313-26. Agnihotri S, Wolf A, Munoz DM, Smith CJ, Gajadhar A, et al. A GATA4-regulated tumor suppressor network represses formation of malignant human astrocytomas. J Exp Med. 2011; 208:689-702. Mazurek S, Boschek CB, Hugo F, Eigenbrodt E. Pyruvate kinase type M2 and its role in tumor growth and spreading. Semin Cancer Biol. 2005;15:300-8. Yang W, Zheng Y, Xia Y, Ji H, Chen X, et al. ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat Cell Biol. 2012; 14:1295-304. Yang W, Xia Y, Ji H, Zheng Y, Liang J, et al. Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation. Nature. 2011; 480:118-22. Yang W, Xia Y, Cao Y, Zheng Y, Bu W, et al. EGFR-induced and PKCε monoubiquitylation-dependent NF-κB activation upregulates PKM2 expression and promotes tumorigenesis. Mol Cell. 2012; 48:771-84. Mukherjee J, Phillips JJ, Zheng S, Wiencke J, Ronen SM, et al. Pyruvate kinase M2 expression, but not pyruvate kinase activity, is up-regulated in a grade-specific manner in human glioma. PLoS One. 2013;8:e57610. Morfouace M, Lalier L, Oliver L, Cheray M, Pecqueur C et al. Control of glioma cell death and differentiation by PKM2-Oct4 interaction. Cell Death Dis. 2014; 5:e1036. Koh J, Cho H, Kim H, Kim SI, Yun S, et al. IDH2 mutation in gliomas including novel mutation. Neuropathology. 2015; 35:236-44. Tanaka K, Sasayama T, Irino Y, Takata K, Nagashima H, et al. Compensatory glutamine metabolism promotes glioblastoma resistance to mTOR inhibitor treatment. J Clin Invest. 2015 ;125:1591-602. Ohka F, Ito M, Ranjit M, Senga T, Motomura A, et al. Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation. Tumour Biol. 2014; 35:5911-20. Kallenberg K, Bock HC, Helms G, Jung K, Wrede A, et al. Untreated glioblastoma multiforme: increased myo-inositol and glutamine levels in the contralateral cerebral hemisphere at proton MR spectroscopy. Radiology. 2009; 253:805-12. Rosati A, Marconi S, Pollo B, Tomassini A, Lovato L, et al. Epilepsy in glioblastoma multiforme: correlation with glutamine synthetase levels. J Neurooncol. 2009; 93:319-24. DeBerardinis RJ, Mancuso A, Daikhin E, Nissim I, Yudkoff M, et al. Beyond aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proc Natl Acad Sci U S A. 2007; 104:19345-50. Cheng T, Sudderth J, Yang C, Mullen AR, Jin ES, et al. Pyruvate carboxylase is required for glutamine-independent growth of tumor cells. Proc Natl Acad Sci U S A. 2011; 108:8674-9. Sun P, Xia S, Lal B, Shi X, Yang KS, et al. Lipid metabolism enzyme ACSVL3 supports glioblastoma stem cell maintenance and tumorigenicity. BMC Cancer. 2014; 14:401. Guo D, Prins RM, Dang J, Kuga D, Iwanami A, et al. EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy. Sci Signal. 2009; 2:ra82. Guo D, Reinitz F, Youssef M, Hong C, Nathanson D, et al. An LXR agonist promotes glioblastoma cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway. Cancer Discov. 2011; 1:442-56. Loreck DJ, Galarraga J, Van der Feen J, Phang JM, Smith BH, et al. Regulation of the pentose phosphate pathway in human astrocytes and gliomas. Metab Brain Dis. 1987;2:31-46. Kathagen-Buhmann A, Schulte A, Weller J, Holz M, Herold-Mende C, et al. Glycolysis and the pentose phosphate pathway are differentially associated with the dichotomous regulation of glioblastoma cell migration versus proliferation. Neuro Oncol. 2016 Feb 24. pii: now024. [Epub ahead of print]

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Abstract 

ancer is a disease where there is aberrant cellular behaviour characterized by uncontrolled C growth and cellular signalling. Cancer though is viewed as a homogeneous pathology, does not show uniformity at the cellular level - there is difference in the characteristics within cells of a tumour. A major caveat in understanding the biology of cancer is the paucity of information on the origin and perpetuation of cancers. Towards salvaging these two models of cancer genesis and progression have been proposed: ‘Stochastic’ and ‘Cancer stem cell’ theories. The stochastic model holds that all cells in a tumour are identical while the cancer stem cell theory supports the existence of a subset of cells called cancer stem cells in a tumour that are responsible for the origin and perpetuation of the disease. Cancer stem cells are implicated in various aspects of cancer including metastasis, recurrence and therapeutic resistance. Though cancer stem cells have been reported from many cancers methods to identify and characterize them still rely on animal transplantation models along with surface protein studies. However better techniques of characterization of these cells would play a positive role in elucidating these cells better. The characterization of cancer stem cells would play an important role in the research and clinical management of the disease.

Key Words: Cancer, Stem cells, Perpetuation, Stem cell theory, Markers.

Stem Cells Human body is made up of 1012 to 1016 cells. Recently with a bibliographical and mathematical approach the total number of cells in the human body was averaged to be 3.72 x 1013 1. A preprint data from Weizmann Institute, Israel, hints at a revised estimate of the number of human cell in a ‘reference man’ of 70kg to be 3 x 1013 2. An estimated 108 cells die in an adult per day3 and have to be replaced daily. With such huge number of cells turned over it is of paramount importance to maintain the number and quality of cells. Homeostasis is important as there is constant loss of differentiated cells because of their limited lifespan. This is verified Ann. SBV, Jan-Jun 2016;5(1)

under in vitro conditions where cultured normal somatic cells undergo only certain number of cell divisions beyond which they undergo senescence and apoptosis; a phenomenon called ‘Hayflick’s limit’4. Loss of cells and the subsequent need for replenishment in the body might also be due to normal wear and tear, injury or degeneration. The production and replacement of cells undergoing senescence and apoptosis with healthy and viable cells is a process that is well regulated, where a small population of cells called ‘stem cells’ serve as the reservoir, which can give rise to proliferating progenitors and terminally differentiated cells. Page 59

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Stem cells are a small subset of cells within any kind of tissue in the body, that have the capacity for long term self-renewal, asymmetric cell division, and differentiation into one or more lineages5, 6. These cells subscribe to a hierarchical model7,8 wherein a small fraction of stem cells having the above mentioned capacities maintain their cell numbers by self-renewing and when required, as in tissue injury or homeostasis, give rise to daughter cells that can proliferate extensively to accommodate the need for high cell numbers (Figure 1). Examples of well-studied systems in the body for the hierarchical stem cell model are the haematopoietic system9 and gut10. Figure 1. Stem Cell Hierarchy

A stem cell has the capacity for long-term self-renewal and thus maintaining the stem cell pool. This stem cell can divide and give rise to transit amplifying cells which are committed progenitor cells which differentiate into terminally differentiated cells of the tissue of origin. After their limited life span these cells undergo senescence, apoptosis and death. Thus stem cells are characterized by three properties: [1] long term self-renewal, [2] extensive proliferation and [3] differentiation into multiple lineages.

Generally stem cells are categorized as ‘Embryonic stem cells’ and ‘Adult stem cells’6. Embryonic stem cells were first described more than three decades ago11,12 and are seen in the inner cell mass of the blastocyst. These cells are pluripotent and can form any organ of the body but not the whole organism. While embryonic cells are predominantly responsible for the complete development of the foetus, ‘Adult stem cells’ or ‘Somatic stem cells’ take active role in meeting the body’s cell requirements after birth. These adult stem cells have been demonstrated in almost all Page 60

of the tissues of the human body including rapidly dividing intestinal tract13-15, bone marrow16 as well as slowly recycling tissues like muscle17. One of the ways of identifying normal adult stem cells from various human tissues is by their surface markers for e.g. normal colonic stem cells are reported to be positive for Lgr5 expression on the surface which is a Leucine rich protein receptor13,18, Haematopoietic stem cells are characterized by CD34+CD38-19,20. Underlying these differences are multiple molecular signalling pathways involved in a well regulated physiology from stem cell to differentiated cell distribution in any tissue.

Cancer The World Health Organization (WHO) defines cancer as a generic term for a large group of diseases that can affect any part of the body, the defining features of which are the rapid creation of abnormal cells that grow beyond their usual boundaries, and invade adjacent parts of the body and spread to other organs21. Cancer is the second largest non-communicable disease; a leading cause of death around the world with 70% of related mortality seen in low and middle income countries. While 30% of cancers could be prevented, deaths due to cancer worldwide are projected to increase to an estimated 12 million deaths in 2030 (The global burden of disease: 2004 update, WHO). There were 7.6 million deaths related to cancer in 2008 alone. One in four deaths in the United States is due to cancer22. In India cancer incidence is reported to be increasing23. Six hallmark features of cancer were described by Hanahan and Weinberg more than a decade ago: Selfsufficiency in growth signals, insensitivity to anti-growth signals, resistance to apoptosis, limitless reproductive potential, sustained angiogenesis, and tissue invasion and metastasis24. Over the years the understanding of the hallmarks have been furthered with the addition of two more features: re-programming of energy metabolism and evasion of immune destruction25. There is also the emerging concept of metabolic hallmarks of cancer that serves to elucidate the disease from the perspective of energy and nutrition. A recent perspective describes six metabolic hallmarks of cancer26 that serves to elucidate more on the energy dynamics in cancer. These hallmarks are also believed to be influenced by the extracellular matrix associated with tumour cells27. There is also the contrarian view that that proposes that carcinogenesis is due to interaction of cells with extracellular matrix28. Taken together, all these point to the growing complexity of the disease and its origin which ultimately has therapeutic ramifications. Ann. SBV, Jan-Jun 2016;5(1)

Cancer Perpetuation Though there are many reports describing the aberrant cellular and molecular signalling in cancers and in the process of carcinogenesis, the putative cell that succumbs to the initial transformation event is yet to be elucidated clearly. There is paucity of information on the exact origin of the disease. In spite of the various hallmark features of cancer and the factors associated with carcinogenesis being described in many reports, the initial transformation event which results in tumorigenesis is still not clear. Towards reckoning this lack of clarity about the biology and genesis of cancer two broad models have been posited. These two models that attempt to explain carcinogenesis or the initiation of cancer are the ‘stochastic’ and the ‘cancer stem cell’ models29,30 (Figure 2). Figure 2. Theories of cancer genesis

The stochastic model states that every cell in a tumour has the capability to re-initiate and maintain a tumour whereas the cancer stem cell model attributes this capacity only to a limited subset of cells within the tumour bulk.

Stochastic Theory The ‘stochastic model’ does not subscribe to the speciality or uniqueness of any particular cell subtype and states that every cell in a tumour bulk is equally endowed with the potential to propagate the tumour and form new tumours. This model associates randomly accumulating genetic changes in the DNA along with micro environmental selections with carcinogenesis and cancer progression. Thus this model aims to address tumours as a heterogeneous mixture of cells Ann. SBV, Jan-Jun 2016;5(1)

but without any biological difference which could have any implication on tumour progression or recurrence. Thus this model attributes no inherent differences between the constituent cells in a tumour to maintain the disease.

Cancer Stem Cell Theory On the other hand, the Cancer Stem Cell (CSC) model is a hierarchical model31,32 where a particular cell called the ‘cancer stem cell’ has the capacity to self-renew and proliferate thus giving rise to a host of transit amplifying cells which form the bulk of the heterogeneous tumour mass; this feature parallels the normal stem cell biology in a tissue, where they maintain their cell number in low frequency but also proliferate and give rise to terminally differentiated cells as in case of an injury or normal homeostasis. The CSC model posits the existence of a small subpopulation of CSC within a tumour that exclusively harbours the capacity to initiate and propagate the disease. This model attributes the qualities of tumour initiation, maintenance and propagation to only a biologically distinct subpopulation of cells occurring in low frequency within a tumour called CSC33. A corollary of this has been that these CSC are also responsible for metastasis and for resistance to conventional chemotherapy and radiotherapy. Like normal stem cells which self-renew and proliferate to produce terminally differentiated cells, CSC also have the capability to maintain their numbers by self-renewal and, by proliferation, give rise to a host of transit amplifying cells which form the heterogeneous tumour bulk34 (Figure 3). To surmise, this model subscribes to the theory that a tumour arising from a cancer stem cell is composed of cancer stem cells in low frequency and the non-stem cells as the tumour bulk.

Cancer Stem Cells Markers One of the earliest hints for the cancer stem cell theory came from the publication of the ‘Trophoblast theory of cancer’ in the year 190235. This theory states that cancer is a germ cell disorder wherein remnant foetal trophoblasts in the adult get activated to form cancer upon sufficient activation by environmental and chemical cues thus hinting at the stemness nature in cancer. Though this theory was not promptly accepted at that time, in retrospect its proponent John Beard is considered one of the pioneers of the present day theory of cancer stem cells36. Page 61

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Figure 3. Stem cells in tumours and normal tissue

Figure 4 . Plausible mechanisms of cancer stem cell genesis

The stem cell hierarchy in the tumour tissue parallels that of normal tissue. Like normal tissue the cancer stem cells model also states the existence of a small subset of stem cells which gives rise to the heterogeneous tumour populations and the atypical cells of the tumour.

Many plausible mechanisms for the origin of CSC have been put forth29,37-39. One of the ways suggested for the origin of CSC is said to be the activation of a normal resident stem cell. In a report by Dean37 activation of a stem cell is proposed to be a good target for subsequent genetic hits leading to a complete transformation resulting in an autonomous growth and acquisition of cancer cell phenotype. This cell is transformed while retaining the stemness property. Activation of a stem cell according to this theory can occur in ways: (a) the stem cell might be naturally dividing as in an embryo or haematopoietic system thus rendering it vulnerable to accrue genetic errors during replication, (b) hormonal activation of stem cells can be another way as in the case of oestrogen and ovarian cells and lastly (c) tissue damage caused by injury, inflammation, infection or chemical exposure like asbestos etc. In all these conditions a resident stem cell is activated and stimulated to divide thus increasing the probability of acquiring genetic mutations due to errors of DNA repair. Even the dedifferentiation of a mature cell with concomitant acquisition of self-renewal ability can result in the formation of a cancer stem cell. Another way of a cell acquiring CSC phenotype described by Dean37 as well as in Costea et al38 is the dedifferentiation of mature differentiated cell which acquires the ability of selfrenewal. Apart from direct stem cell transformation and dedifferentiation Costea et al38 have also reported two additional ways in which the origin of CSC can be envisaged to happen especially in the context of oral cancers (a) fusion of a mutated haematopoietic stem Page 62

similar results indicating the involvement of a subset of cells in the process of carcinogenesis41,42. The actual term ‘cancer stem cell’ was popularized after Carney et al43 demonstrated the tumorigenicity of patient derived lung cancer cells grown as tumour cell colonies on soft agarose, when injected into athymic nude mice. It was just around the same time that the involvement of stem cell compartment in leukaemogenesis was reported44,45. The first definitive description of such a cell was in acute myeloid leukaemia, where it was shown that cells which were characterized to be CD34+/CD38-, when transplanted into SCID mice, could stably re-initiate and sustain the disease46. There have subsequently been many reports trying to prove and identify these cells in tumours of diverse origin including breast (CD44+/ CD24-/Lineage-)47, pancreas (CD44+/CD24+/ESA+)48, colon (CD133+, CD44 and Lgr5+)49-53, and prostate (CD44+/α2β1 Integrin-hi/CD133+)54. CSC reported in many cancers corresponds to the stem cell theory in being of low percentages in the tissue. Ricci-Vittiani et al55 and Dalerba et al56 showed that the percentages of CSC in colon identified by CD133 and CD44 respectively were around 0.7 and 2.5%. In the samples analyzed by O’Brien et al49, CSC identified by Lgr5 were also found to be as high percentage as high as 24.5% in one of the samples. Similarly CSC in Figure 5. Quiescence in stem cells

Plausible mechanisms of the origin of cancer stem cells include (a) Genetic mutation in a resident tissue stem cell, (b) De-differentiation of a mature differentiated cell in a tissue (c) Fusion of a mutated haematopoietic stem cell with a mature differentiated cell and (4) Senescence by-pass wherein a senescent giant cell undergoes multiple genetic changes and results in a transformed cancer stem cell.

cell with a keratinocyte (differentiated cell) can result in a heterokaryon and might give rise to a CSC and (b) Exposure of senescent cells to chemicals can also result CSC like cells (Figure 4). In 1937 one of the early proofs for the existence of cancer initiating property within subpopulation of cells came of the work of Furth and co-workers40, where inoculation of single cells from inbred leukaemic mice into mice of the same type resulted only a small fraction (5%) of animals developing leukaemia. In the early nineteen fifties some studies with solid tumours also yielded Ann. SBV, Jan-Jun 2016;5(1)

breast, identified by CD44+C,CD24-,ESA+ was found to be 2-4%47. CSC in AML identified by CD34+CD38was reported to be 0.2%57. CSC percentages have been reported in many other cancers including liver58, pancreas48,59, prostate60, kidney61. It is generally accepted that the percentages of CSC in tumours are low because these cells are quiescent, slow cycling and express high levels of anti-apoptotic proteins29,39, all of which are also implicated in resistance to conventional chemotherapy. Quiescence is one of the properties that are seen in stem cells and essential for maintaining their numbers. Quiescence enables the stem cells to go into a reversible state of minimal metabolic behaviour without cell division, the deregulation or loss of which could affect the number of resident stem cell numbers in a tissue and can lead to depletion of the same. G0 phase in the cell cycle is an irreversible state where cells like those undergoing senescence or differentiation, while stem cells go into quiescence which is a reversible G0 phase62 (Figure 5). As indicated previously in Costea et al38 it is at this point that malignant transformation can occur where a senescent cell or differentiated cell can through multiple genetic hits acquire the property to enter the cell cycle again thus activating their ability for quiescence . There are many factors that regulate quiescence. This confers the cells with the ability to escape any cytotoxic agent acting via DNA replication mechanism or cell division protein inhibition. Quiescence has been well described in hematopoietic stem cells63,64 and the various factors having an influence on quiescence. Consequently characterization of these cells is of paramount importance in the context of therapeutic oncology; and these cells unlike other tumour cells need unique techniques of isolation and characterization which include in situ, in vitro and in vivo approaches.

Normally a cell undergoing cell cycle goes into G0 phase upon reaching a point of senescence (a dysfunctional state reached because of limited life span or accumulated errors) or differentiation into mature cells. This is an irreversible step except for the cancer stem cell genesis. Normal stem cells have the property of entering and leaving the G0 phase as dictated by the homeostatic cues. Both normal and cancer stem cells by entering into quiescence are shielded from cytotoxic agents targeting cell cycle components.

Ann. SBV, Jan-Jun 2016;5(1)

Cancer Stem Cells Characterization In Situ Identification using Surface Markers Identification of CSC using surface markers is one of the widely employed techniques because of the availability of a repertoire of antibodies. One of the techniques exploiting the surface antigen chemistry of cells is flow cytometry which depends on treating live or fixed cells with monoclonal antibodies tagged with fluorescent tags65,66 such as fluorescein isothiocyanate

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(FITC), phycoerythrin (PE), allophycocyanin (APC) or peridinin-chlorophyll protein Cy5.5 (PerCP Cy5.5). Identification using surface markers gives the advantage of specificity and sensitivity. The use of flow cytometry assisted cell surface profiling gives the advantage of qualitative as well as quantitative analysis of cells based on surface proteins. This approach would not only help elucidate the presence or absence of markers; this can also successfully identify fluctuations in the marker expression. Flow cytometry based surface profiling has been used in studying CSC in cancers affecting various tissues including oral mucosa67, colon68 and breast47. Fluorescence microscopy69 is another important tool that is successfully used for the study of CSC. This technique gives us the flexibility of analyzing a fixed and stored tissue and gives us the advantage of analyzing single cells visually. Immunofluorescence based study of CSC has been employed for studying the topographical distribution of cells of our interest in a tissue. Thus Immunofluorescence microscopy helps us to correlate the location, frequency and distribution of stained cells with the tissue histology. This makes it a vital tool for studying CSC.Surface markers and their utility in studying CSC have been used in another technique: magnetic activated cell sorting (MACS). It uses surface protein differences to differentially enrich or deplete cells of concern. The best functional study for CSC is animal transplantation and MACS serves to purify cells of our interest based on surface markers. Thus surface marker based identification is one fruitful avenue for the study of different types of CSC. In Vitro Assays for Cancer Stem Cells Many techniques and ways have been reported to isolate and study cancer stem cells. Chiou et al suggest three ways in which CSC can be isolated and studied: Immunophenotyping by flow cytometry, Hoechst 33343 exclusion based side population (SP) assay and Sphere formation70. Immunophenotyping by flow cytometry is certainly a strong tool for studying CSC because of the ease of performing and also because of the availability of a wide range of antibodies against a wide range of surface proteins. This technique easily gives signature surface profiles across multiple samples and hence is a valuable tool in the study of CSC as well. Hoechst 33342 is a DNA binding dye which can be used to stain live cells. Hoechst 33342 dye based SP Page 64

assay exploits the fact that ABC transporters especially ABCG2 efflux this dye from cells. Thus there would be differential staining between stem cells expressing high levels of ABCG2 and non-stem cells showing low levels of this transporter when treated with this chemical71. This protocol was first established for bone marrow derived haematopoietic stem cells which is successfully adapted to other types of stem cells and also to cancer stem cells. Sphere formation assay is another important way to study CSC from any tissue. This technique relies on the fact that non stem cells fail to survive and grow under an anchorage independent or serum starved condition with growth factors, while normal stem cells and CSC not only remain viable in these conditions but also form spheres of cells indicating their ability to proliferate and clonally expand. The ability of these cells to selfrenew can also be inferred by generating secondary spheres from dissociated primary spheres72. CSC have been successfully identified by label retaining assays like the DNA intercalating bromodeoxyuridine (BrdU) assay, as these cells have a low turnover thus labelling the long term non-dividing cells64. Tritiated thymidine, which exhibits a similar DNA binding ability, has also been used to label slow cycling cells or quiescent cells73. There has been lot of technological advancement and histone protein based labelling systems have been developed like H2B-green fluorescent protein (H2B-GFP)74 where the histone protein are genetically modified by the addition of a green fluorescent protein for easy visualization. Some other approaches that identify CSC include RNA content, lack of proliferation markers, elevated antiapoptotic proteins etc. These techniques though pick up slow cycling cells and help elucidate the quiescent behaviour it has become apparent that other techniques to identify CSC are used in tandem to get significant results. In Vivo Assays for Cancer Stem Cells Normal stem cells are endowed with properties of selfrenewal and lineage capability. CSC, paralleling on normal stem cells, have properties of self-renewal and tumour propagation. A technique that can successfully demonstrate these features would certainly be of a huge impact75. Thus amidst all the other techniques serial orthotopic xenotransplantation is still hailed as the gold standard in experimental CSC biology75. Though no xenotransplantation model exactly replicates the Ann. SBV, Jan-Jun 2016;5(1)

host tissue environment, they give the advantage of studying the putative CSC under question in an environment that gives a milieu and microenvironment at least distantly similar to the native tissue from which the CSC is derived. One of the concerns to bear with while using this assay is the inherent difference in the transplantation site especially the lack of stromal cell signals, which are also described to impact the development and propagation of CSC73. This caveat can be addressed and improved by co-engrafting the putative CSC with stromal cells. Another factor to be aware of is the cell preparation and the process of transplantation; these can to some degree introduce some mechanical stress but in any case this would be true of other assays as well. Xenotransplantation is nevertheless considered the best functional test for CSC. Another important development is the newer and better models of in vivo experimentation that are being designed from time to time for tumour transplantation studies76. Development of mice models that are more immunocompromised than SCID and NOD/ SCID show greater transplantability with cancer cells. This suggests altogether a different perspective on the methodologies to estimate the frequencies of

CSC. Quintana et al77 have shown this in melanoma by injecting single melanoma cells into two types of immunocompromised animals (NOD/SCID and NOD/ SCID with Interleukin 2 deficiency) and showing that the former model can underestimate the frequency of CSC. Thus a combined and informed way of isolating and studying CSC is imperative.

Conclusion Development and perpetuation of cancers is highly debated with both stochastic and stem cell model of cancer being identified as plausible models to explain the genesis. Cancer stem cell model has been repeatedly supported by multiple studies attempting the isolation of cancer stem cells from tumours. However a unique, all-encompassing marker for isolation, characterization of CSC has not yet been available which engenders the necessity for the use of multiple techniques – both in vitro and in vivo – in the analysis of CSC. Thus elucidation of CSC would require a host of techniques and further validation of presently available techniques towards harnessing the knowledge of stem cells in cancer research and therapy.

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A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007;445:106-10. Saigusa S, Inoue Y, Tanaka K, Toiyama Y, Matsushita K, et al. Clinical significance of LGR5 and CD44 expression in locally advanced rectal cancer after preoperative chemoradiotherapy. Int J Oncol. 2012;41:1643-52. Vermeulen L, Todaro M, de Sousa Mello F, Sprick MR, Kemper K, et al. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad Sci U S A. 2008;105:13427-32. Kemper K, Prasetyanti PR, De Lau W, Rodermond H, Clevers H, et al. Monoclonal antibodies against Lgr5 identify human colorectal cancer stem cells. Stem Cells. 2012;30:2378-86. Todaro M, Gaggianesi M, Catalano V, Benfante A, Iovino F, et al. CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis. Cell Stem Cell. 2014;14:342-56. Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res. 2005;65:10946-51. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, et al. Identification and expansion of human colon-cancer-initiating cells. Nature. 2007;445:111-5. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A. 2007;104:10158-63. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature Med. 1997;3:730-7. Suetsugu A, Nagaki M, Aoki H, Motohashi T, Kunisada T, et al. Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/progenitor cells. Biochem Biophys Res Commun. 2006;351:820-4. Li C, Lee CJ, Simeone DM. Identification of human pancreatic cancer stem cells. Methods Mol Biol. 2009;568:161-73. Lawson DA, Xin L, Lukacs R, Xu Q, Cheng D, et al. Prostate stem cells and prostate cancer. Cold Spring Harb Symp Quant Biol. 2005;70:187-96. Bruno S, Bussolati B, Grange C, Collino F, Graziano ME, et al. CD133+ renal progenitor cells contribute to tumor angiogenesis. Am J Pathol. 2006;169:2223-35. Cheung TH, Rando TA. Molecular regulation of stem cell quiescence. Nat Rev Mol Cell Biol. 2013;14:329-40. Passegue E, Wagers AJ. Regulating quiescence: new insights into hematopoietic stem cell biology. Dev Cell. 2006;10:415-7. Glauche I, Moore K, Thielecke L, Horn K, Loeffler M, et al. Stem cell proliferation and quiescence--two sides of the same coin. PLoS Comput Biol. 2009;5:e1000447. Mahnke YD, Roederer M. Optimizing a Multicolor Immunophenotyping Assay. Clin Lab Med. 2007;27:469-85. Brown M, Wittwer C. Flow Cytometry: Principles and Clinical Applications in Hematology. Clin Chem. 2000;46:1221-9. Dalley AJ, AbdulMajeed AA, Upton Z, Farah CS. Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75 NTR in malignancy. J Oral Pathol Med.42:37-46. Ernst A, Aigner M, Nakata S, Engel F, Schlotter M, et al. A gene signature distinguishing CD133hi from CD133- colorectal cancer cells: essential role for EGR1 and downstream factors. Pathology.43:220-7. Lichtman JW, Conchello J-A. Fluorescence microscopy. Nat Meth. 2005;2:910-9. Chiou SH, Yu CC, Huang CY, Lin SC, Liu CJ, et al. Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma. Clin Cancer Res. 2008;14:4085-95. Goodell MA, McKinney-Freeman S, Camargo FD. Isolation and characterization of side population cells. Methods Mol Biol. 2005;290:343-52. Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer. 2008;8:755-68. Pedersen EA, Shiozawa Y, Mishra A, Taichman RS. Structure and function of the solid tumor niche. Front Biosci (Schol Ed). 2011;4:1-15. Fuchs E, Horsley V. Ferreting out stem cells from their niches. Nat Cell Biol. 2011;13:513-8. Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CHM, Jones DL, et al. Cancer Stem Cells - Perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res. 2006;66:9339-44. Beckhove P, Schutz F, Diel IJ, Solomayer EF, Bastert G, et al. Efficient engraftment of human primary breast cancer transplants in nonconditioned NOD/Scid mice. Int J Cancer. 2003;105:444-53. Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, et al. Efficient tumour formation by single human melanoma cells. Nature. 2008;456:593-8.

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Mannose Binding Lectin, Genetic Variations, Deficiency and Disease Associations Farzana Begum Liakath, Senior Research Fellow Central Inter-Disciplinary Research Facility, Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India Email: farzana.liakath@gmail.com

Abstract 

annose-binding lectin (MBL) is an important arm of innate immunity and plays a vital M role in the first line of host defense. Genetic variation in MBL2 have been shown to associate with many infectious diseases, autoimmune and inflammatory disorders such as malaria, leishmaniasis, leprosy, tuberculosis, filariasis, HIV, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). MBL has been shown to bind with glycoconjugates on the surface of mannose rich microbes and deficiency of MBL has been associated with susceptibility and modulating the severity in bacterial, fungal, protozoan and viral infections. Many different approaches are being used to define ‘MBL deficiency’. It is more relevant in young children in whom immune system fails to mount an effective response to carbohydrate antigens. MBL replacement therapy has been tried in the past for patients with MBL deficiency. Currently, production of recombinant MBL is underway and provides a hope for children with innate immune disorders.

Key Words: Mannose binding lectin, Genetic variation, MBL deficiency, MBL therapy.

Introduction One of the fundamental components of innate immune system is the complement cascade which functions through both antibody-dependent and – independent manner providing protection against invading pathogens1. An effective immune response is mediated through the complement cascade involving interactions between cellular and humoral immunity which includes phagocytosis, chemotaxis, cell adhesion, B-cell differentiation and regulation of both B and T cell responses2. The initiation of the complement cascade is well studied and three activation mechanisms are known to be involved which include the classical, alternative and lectin pathways. The lectin pathway is the most recently discovered and is considered to be the most ancient of the three activation pathways3. The initiating complexes of lectin pathway comprise of Ann. SBV, Jan-Jun 2016;5(1)

separate recognition and enzyme components similar to the C1 complex of the classical pathway. Recognition components such as MBL and serum ficolins of the lectin pathway bind directly to carbohydrate moieties like N-acetyl glucosamine or mannose on pathogens and activate three enzymes, MBL-associated serine proteases (MASPs-1 to -3) to activate complement4.

MBL Structure and Function MBL belongs to the C-type lectin family synthesized by liver and circulates in serum. Being an acute phase protein, its level rises during inflammatory conditions. As part of the collectin family characterized by collagen and lectin domains, the carbohydrate recognition domain (CRD) is required for binding ligand surfaces in a calcium dependent manner 5, 6. In humans, it is encoded by MBL2 located on the long arm of Page 67

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chromosome 10 at 10q11.2-q21containing four exons coding four identical peptide chains of 32- kD subunit which associate to form higher oligomers (trimershexamers) of a 96-kD triple helix circulating in serum. Among four exons, exon 1 encodes for a cysteine rich N-terminal region (confers stabilization) and 7 repeats of a glycine-rich motif, which contains repeated sequences of two glycines and this region is critical for the triple helix formation of the collagen structure (Figure 1). Figure 1. Schematic representation of MBL2 and MBL subunit.

MBLMediated Complement Activation Upon binding of the CRD complex to carbohydrate moieties present on the surface of invading pathogens, the proenzyme form of MASPs 1 and 2 is activated by cleaving the domains of complement control protein-2 (CCP-2) and serine protease producing heavy and light chains11. This activates complement (C) by MASP-2 to cleave C2 and C4 which causes the transformation of C3 into C3a and C3b12. This cleaving process then stimulates the downstream complement cascade. Thus the MBL pathway initiates complement activation in the same way as the classical pathway, forming a C3 convertase from C2b bound to C4b (Figure 2).

Figure 3. Pictorial representation of normal MBL (MBL-S) from an A/A (wild type) genotype which circulates in the bloodstream complexed with sMAP and MASPs leading to opsonization and complement activation and deficient MBL (MBL-D) derived from a 0/0 (mutant) genotype which has a reduced capacity to build high order oligomers, fails to complex with MASPs and to activate complement.

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MBL Structure for Effective Immune Function of MBL Though both MASP-1 and MASP-2 were considered to be responsible for activation of C2, one report suggests that MASP-2 may be the major initiator of the complement pathway as evidenced by its interaction with C4. Findings also suggest that MASP-1 cleaves C2 but not C4 and MASP-1 probably helps in complement activation mediated by MBP13.

Ann. SBV, Jan-Jun 2016;5(1)

The search for genetic variations as a cause for MBL dysfunction began when defective opsonisation was associated with low serum MBL level in an in vitro assay14. MBL2 genetic polymorphisms can affect the serum levels as well as configuration and function (Figure 3). The first study of genetic defects in the MBL gene was reported in three children in the UK with recurrent infections who had an opsonic defect and low serum MBL concentrations. Sequence analysis showed a mutation at base 230 of exon 1 causing a change of codon 54 from GGC to GAC resulting in replacement of glycine with an aspartic acid residue disrupting the formation of the normal triple helix, and rendering the molecule vulnerable to degradation15. Subsequently, a SNP in codon 57 identified in a Gambian population termed variant C which substitutes a glutamic acid instead of glycine (GGA to GAA) and another SNP at codon 52 termed variant D representing a substitution of arginine by cystein (CGT to TGT) were also observed8,16. Two promoter variants H and L at position -550 are in linkage disequilibrium with the X and Y variants at position -221 to produce three haplotypes HY, LY and LX. Haplotype HY is reported to be associated with the highest plasma levels of MBL, LY haplotype with intermediate and LX haplotype with the lowest plasma MBL level8.

Figure 2 . Overview of the main components and effector actions of complement pathway.

Exon 2 encodes part of the collagenous domain which contains enzyme MASP binding site and a short α-helical coiled-coil domain is encoded by exon 37. Exon 4 transcribes the most important CRD domain containing Calcium binding site and C-terminal end by which MBL binds to a wide range of pathogens such as Gram-positive and Gram-negative bacteria, parasites and viruses by recognizing D-mannose, N-acetylD-glucosamine and L-fructose sugar motifs on the surface of microorganisms. After post-translational modification, the final functional MBL contains subunits ranging from dimers to hexamers but the majority exists as trimers and tetramers8. Studies using high resolution force microscopy on recombinant MBL identified for the first time that the stable MBL structure was broken into an elongated state with separation of the ligand-binding domains confirming the large conformational changes happening in MBL while interacting with surface-immobilized ligands. This report highlights the importance of surface topography in immune recognition9, 10.

MBL2 – promoter and exon1 polymorphisms

During early childhood, children deficient in MBL experience a substantial increase in infections indicating the importance of the MBL pathway in host defense. Low serum MBL levels are associated with opsonic defects and recurrent infections in infants14. Especially in the pediatric population, MBL exerts greatest influence during “window of vulnerability” between the decline in passive immunity by the mother and before the development of a fully functional adaptive immune system. Ann. SBV, Jan-Jun 2016;5(1)

The promoter polymorphisms and three point mutations in codon 52, 54 and 57 exhibit a pattern of linkage disequilibrium and thus combination of SNPs at both promoter and exon1 occur in a nonrandom fashion. Though 28 possible combinations can occur due to two SNPs linked as haplotypes, only seven haplotypes have been reported till now. These include HYPA, LYQA, LYPA, LXPA, HYPD, LYQC and LYPB17-32. Studies in various populations and age groups indicate that MBL serum levels largely depend on the MBL2 genotype18. Factors other than genetic variation, which influence serum MBL, include age, hormonal status and immune activation. MBL levels vary with age, increasing in the first months of life and falling at about the age of 12 due to hormonal changes33. MBL levels also vary hugely based on the ethnicity both in children and adults, as described in Table 1.a and 1.b. Lower MBL levels have been found in preterm neonates with comparable levels in cord blood34. A cohort study of 95 patients with autoimmune thyroid disorders suggested that Page 69

Mannose Binding Lectin, Genetic Variations, Def iciency and Disease Associations

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Table 1. MBL levels in healthy controls from different ethnic background.

(A) In adults: Country/Ethnicity

Age group (years)

Serum MBL level (ng/ml)

Reference

Hungary

50-60

Median (IQR) - 1027 (253-2120)

(17)

Australian

not given

Mean (range) - 1940 (0-8810)

(18)

Finnish

44-60

Mean – 3970

(19)

Swedish

50-60

Mean – 1680

(20)

Caucasians, African-American

60-70

Mean (Caucasians) - 1672; Mean (African Americans) – 1158

(21)

Egyptians Chinese

30-38 16-20 31-40 41-57

Mean=619 Mean = 2050 Mean = 2160 Mean = 1466

(22) (23)

Age group (years)

Serum MBL level (ng/ml)

Reference

Chinese

0 to 6

Median (p2.5-p97.5)=2536 (161-5070)

(24)

Han Chinese

0 to 2

Cord blood (median)=1462

(25)

Newborns (median)=1597

3 to 11 1 to 15 13 to 16 2 to 3 9 to 10 0 to 18

The first MBL product for therapeutic use was isolated from plasma of Danish blood donors by the Statens serum institute (SSI) Copenhagen, Denmark 38. The first patient to receive MBL replacement therapy was a two year old girl who had suffered debilitating and recurrent infection from the age of 4 month. She had opsonic defect and very low MBL level. The girl was given daily infusion (2 mg) of MBL for 3 consecutive

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Children (median)=2536 Turkish Hungary Dutch Caucasian Chinese Polish Netherlands

Future Directions - recombinant MBL Therapy

Median (range) = 2950 (1.5-4048.5) Mean±SD = 1411 ± 576 Median = 1650 Median (IQR)=1065 (862-1452) Median (range) = 1762 (41-8544) Median (IQR)= 1090 (420-2700)

(26) (27) (28) (29) (30) (31)

13. 14. 15. 16. 17. 18. 19. 20.

MBL levels were found to negatively correlate with thyroid stimulating hormone (TSH) in patients with autoimmune hypo- or hyperthyroidism, irrespective of the genotype35.

MBL Deficiency Though there have been a number of studies describing the genotype/phenotype or serum levels / disease Page 70

days and this treatment was repeated after 10 days. The MBL concentration in her blood reached normal values after each infusion and the opsonic activity of her plasma was temporally restored to normal. She remained free from recurrent or abnormal infection during the 8 yr since she received this treatment 39. Jensenius and his group first produced recombinant MBL at the University of Aarhus Denmark. A human endothelial kidney cell line was transiently transfected and cultured in proteinfree medium40. MBL replacement therapy to help patients with MBL deficiency has undergone phase I clinical trials41. Phase II and III trials and production of recombinant MBL for replacement therapy are currently underway. Thus MBL replacement therapy should be carefully chosen and will be restricted to a few carefully selected patients until proof of efficacy is established by more controlled clinical trials.

References

(B) In children: Country/Ethnicity

factor for infectious diseases. A recent study indicates MBL-2 polymorphisms were associated with increased risk for bacterial infections in children with B acute lymphoblastic leukemia36. In severe meningococcal or pneumococcal infections, ‘MBL deficient’ children with serum MBL < 500 ng/mL were found to a have a higher risk of death 37.

association, a clear understanding of MBL deficiency is still lacking. MBL2 genotype and corresponding serum MBL levels show a wide range making it difficult to correlate genotype/phenotype associations. Cut-off levels to define an MBL ‘deficient’ state and ‘MBL deficient genotype’ hugely vary ranging from <50 ng/ml to <1000 ng/ml depending on the age and ethnicity. In pediatric populations, low MBL or ‘MBL deficiency’ seem to be an important predisposing Ann. SBV, Jan-Jun 2016;5(1)

21. 22. 23. 24. 25.

Porter RR, Reid KB. The biochemistry of complement. Nature. 1978; 275: 699-704. Carroll MC. The complement system in regulation of adaptive immunity. Nat Immunol. 2004; 5: 981-6. Dodds AW. Which came first, the lectin/classical pathway or the alternative pathway of complement? Immunobiology. 2002; 205: 340-54. Turner MW. Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol Today. 1996; 17:532-40. Jack DL, Klein NJ, Turner MW. Mannose-binding lectin: targeting the microbial world for complement attack and opsonophagocytosis. Immunol Rev. 2001; 180:86-99. Sastry K, Herman GA, Day L, Deignan E, Bruns G, et al. The human mannose-binding protein gene. Exon structure reveals its evolutionary relationship to a human pulmonary surfactant gene and localization to chromosome 10. J Exp Med. 1989; 170:1175-89. Wallis R, Shaw JM, Uitdehaag J, Chen CB, Torgersen D, et al. Localization of the serine protease-binding sites in the collagen-like domain of mannose-binding protein: indirect effects of naturally occurring mutations on protease binding and activation J Biol Chem. 2004; 279:14065-73. Madsen HO, Garred P, Kurtzhals JA, Lamm LU, Ryder LP, et al. A new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein. Immunogenetics. 1994; 40: 37-44. Dong M, Xu S, Oliveira CL, Pedersen JS, Thiel S, et al. Conformational changes in mannan-binding lectin bound to ligand surfaces. J Immunol. 2007; 178:3016-22. Heitzeneder S, Seidel M, Förster-Waldl E, Heitger A. Mannan-binding lectin deficiency - Good news, bad news, doesn’t matter? Clin Immunol. 2012; 143: 22-38. Fujita T. Evolution of the lectin-complement pathway and its role in innate immunity. Nat Rev Immunol. 2002; 2:346-53. Møller-Kristensen M, Thiel S, Sjöholm A, Matsushita M, Jensenius JC. Cooperation between MASP-1 and MASP-2 in the generation of C3 convertase through the MBL pathway Int Immunol. 2007; 19:141-9. Chen CB, Wallis R. Two mechanisms for mannose-binding protein modulation of the activity of its associated serine proteases. J Biol Chem. 2004; 279:26058-65. Super M, Thiel S, Lu J, Levinsky RJ, Turner MW Association of low levels of mannan-binding protein with a common defect of opsonisation. Lancet 1989; 2(8674):1236-9. Sumiya M, Super M, Tabona P, Levinsky RJ, Arai T, et al. Molecular basis of opsonic defect in immunodeficient children. Lancet. 1991; 337:1569-70. Lipscombe RJ, Beatty DW, Ganczakowski M, Goddard EA, Jenkins T, et al. Mutations in the human mannose-binding protein gene: frequencies in several population groups. Eur J Hum Genet. 1996; 4:13-9. Altorjay I, Vitalis Z, Tornai I, Palatka K, Kacska S, et al. Mannose-binding lectin deficiency confers risk for bacterial infections in a large Hungarian cohort of patients with liver cirrhosis. J Hepatol. 2010; 53:484-91. Minchinton RM, Dean MM, Clark TR, Heatley S, Mullighan CG. Analysis of the relationship between mannose-binding lectin (MBL) genotype, MBL levels and function in an Australian blood donor population. Scand J Immunol. 2002; 56:630-41. Aittoniemi J, Koskinen S, Laippala P, Laine S, Miettinen A. The significance of IgG subclasses and mannan-binding lectin (MBL) for susceptibility to infection in apparently healthy adults with IgA deficiency. Clin Exp Immunol. 1999; 116:505-8. Saevarsdottir S , Ding B , Steinsson K , Grondal G , Valdimarsson H , et al. Mannan Binding Lectin (MBL) genotypes coding for high MBL serum levels are associated with rheumatoidfactor negative rheumatoid arthritis in never smokers. Arthritis Res Ther. 2011; 13: R65. Zanetti KA, Haznadar M, Welsh JA, Robles AI, Ryan BM, et al. 3’-UTR and functional secretor haplotypes in mannose-binding lectin 2 are associated with increased colon cancer risk in African Americans. Cancer Res. 2012; 72:1467-77. Esmat S, Omran D, Sleem GA, Rashed L. Serum mannan-binding lectin in egyptian patients with chronic hepatitis C: its relation to disease progression and response to treatment. Hepat Mon. 2012; 12: 259-64. Ip WK, To YF, Cheng SK, Lau YL. Serum mannose-binding lectin levels and mbl2 gene polymorphisms in different age and gender groups of southern Chinese adults. Scand J Immunol. 2004; 59: 310-4. Chen J, Xu Z, Ou X, Wang M, Yang X, et al. Mannose-binding lectin polymorphisms and recurrent respiratory tract infection in Chinese children. Eur J Pediatr. 2009; 168: 1305-13. Ou X, Li Q, Wang M, Chen J, Wang LJ, et al. Determination of the serum mannose binding lectin levels in 738 Han ethnic group children. Zhonghua Er Ke Za Zhi. 2008; 46: 610-2.

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Annals of SBV 26. Cosar H, Ozkinay F, Onay H, Bayram N, Bakiler AR, et al. Low levels of mannose-binding lectin confers protection against tuberculosis in Turkish children. Eur J Clin Microbiol Infect Dis. 2008 ; 27: 1165-9. 27. Gergely P Jr, Pazár B, Nagy ZB, Gombos T, Rajczy K, et al. Structural polymorphisms in the mannose-binding lectin gene are associated with juvenile idiopathic arthritis. J Rheumatol. 2009 ; 36: 843-7. 28. Dolman KM, Brouwer N, Frakking FN, Flatø B, Tak PP, et al. Mannose-bnding lectin deficiency is associated with early onset of polyarticular juvenile rheumatoid arthritis: a cohort study. Arthritis Res Ther. 2008; 10: R32. 29. Tao R, Hua CZ, Hu YZ, Shang SQ. Genetic polymorphisms and serum levels of mannose-binding lectin in Chinese pediatric patients with common infectious diseases. Int J Infect Dis. 2012; 16: e403-7. 30. Bak-Romaniszyn L, Szala A, Sokolowska A, Mierzwa G, Czerwionka-Szaflarska M, et al. Mannan-binding lectin deficiency in pediatric patients with inflammatory bowel disease. Scand J Gastroenterol. 2011; 46:1275-8. 31. Israëls J, Frakking FN, Kremer LC, Offringa M, Kuijpers TW, et al. Mannose-binding lectin and infection risk in newborns: a systematic review. Arch Dis Child Fetal Neonatal Ed. 2010;95: F452-61. 32. Garred P, Larsen F, Seyfarth J, Fujita R, Madsen HO. Mannose-binding lectin and its genetic variants. Genes Immun. 2006; 7:85-94. 33. Aittoniemi J, Miettinen A, Laippala P, Isolauri E, Viikari J, et al. Age-dependent variation in the serum concentration of mannan-binding protein. Acta Paediatr. 1996; 85: 906-9. 34. Frakking FN, Brouwer N, Zweers D, Merkus MP, Kuijpers TW, Offringa M, et al. High prevalence of mannose-binding lectin (MBL) deficiency in premature neonates. Clin Exp Immunol. 2006; 145: 5-12. 35. Potlukova E, Jiskra J, Freiberger T, Limanova Z, Zivorova D, et al. The production of mannan-binding lectin is dependent upon thyroid hormones regardless of the genotype: a cohort study of 95 patients with autoimmune thyroid disorders. Clin Immunol. 2010; 136: 123-9. 36. Pana ZD, Samarah F, Papi R, Antachopoulos C, Papageorgiou T, et al. Mannose binding lectin and ficolin-2 polymorphisms are associated with increased risk for bacterial infections in children with B acute lymphoblastic leukemia. Pediatr Blood Cancer. 2014; 61: 1017-22. 37. Eisen DP, Dean MM, Boermeester MA, Fidler KJ, Gordon AC, et al. Low serum mannose-binding lectin level increases the risk of death due to pneumococcal infection. Clin Infect Dis. 2008; 47: 510-6. 38. Valdimarsson H, Stefansson M, Vikingsdottir T, Arason GJ, Koch C, et al. Reconstitution of opsonizing activity by infusion of mannan-binding lectin (MBL) to MBLdeficient humans. Scand J Immunol. 1998; 48:116-23. 39. Valdimarsson H. Infusion of plasma-derived mannan-binding lectin (MBL) into MBL-deficient humans. Biochem Soc Trans. 2003; 31:768-9. 40. Jensenius JC, Jensen PH, McGuire K, Larsen JL, Thiel S. Recombinant mannan-binding lectin (MBL) for therapy. Biochem Soc Trans. 2003; 31:763-7. 41. Petersen KA, Matthiesen F, Agger T, Kongerslev L, Thiel S, et al. Phase I safety, tolerability, and pharmacokinetic study of recombinant human mannan-binding lectin. J Clin Immunol. 2006; 26: 465-75.

Adipose Tissue Hypoxia in Obesity Akshayavardhani A, PhD Research Scholar Central Inter-Disciplinary Research Facility, Sri Balaji Vidyapeeth - Mahatma Gandhi Medical College and Research Institute Campus Pillaiyarkuppam, Puducherry - 607403, India Email: akshi.anbu@gmail.com

Abstract  Obesity is linked to a variety of metabolic disorders, such as insulin resistance and atherosclerosis.

Adipose tissue plays a vital in life cycle of animals as well as humans. Free fatty acid is the key for heat production and energy source in the fasting stages. Adipose tissue are of two types white adipose tissue (WAT) and brown adipose tissue (BAT). Adipose tissue composed of altered cellular composition and functions. The cells present within the WAT respond to hypoxia, by inhibiting the differentiation of pre-adipocytes to adipocytes and instead being transformed into leptinsecreting cells. Due to nutrient deprivation there is a dynamic change in adipose tissue through adipocyte hypertrophy and hyperplasia accomplish the energy homeostasis. AT remodeling is a constant process that speed up over production of extracellulllar membrane components (ECM) overproduction and condensed angiogenic remodeling, increased state of immune cell infiltration and consequent proinflammatory responses. Molecular mechanism underlying adipose tissue remodeling has an impact on identification of novel s trategies treat hypoxia induced adipose tissue remodeling.

Key Words: Adipose tissue, Obesity, Hypoxia, Macrophage infiltration.

Introduction Obesity Obesity is considered as abnormal accumulation body fat it has negative effect on health. Obesity is measured by body mass index (BMI), measured by a person's weight (in kilograms) divided by the square of height (in meters). Chronic inflammatory disease, with dysregulated innate immune system leading to a cluster of metabolic disorders, including increased risk of insulin resistance, Type 2 Diabetes (T2DM), Hypertension, Dyslipidemia, Cardiovascular diseases leads to tumors1 (Colon cancer, Breast cancer, Gastrointestinal cancer). Factors which are leading to Page 72

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obesity neither genetic nor environmental. Variation in certain genes predisposes to obesity. Current scenario obesity due to high fat/sugar diet. Mouse models have been created to study obesity and its comorbidities. Obesity is attributed to hypertrophy and hyperplasia of adipocytes. These adipocytes become hypertrophic obesity conditions, during hypertrophy their size increases up to 140–180µm in diameter2 which is beyond the oxygen limit. For that reason, hypertrophic adipocytes undergo less oxygen supply and turn hypoxic.3

Hypoxia : Impact on Adipose Tissue Hypoxia is defined as a moderate availability of oxygen to body tissues adapt to low O2 tension have been Page 73

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extensively investigated.4,5 Molecular oxygen (O2) level is crucial to maintain normal tissue functions, cellular energy production to regulate intracellular signal transduction pathways. Hypoxia is one of the innermost mechanisms which hypothesize the development of inflammation and following metabolic dysfunction of White adipose tissue (WAT) in obesity.6 Increase of WAT occurs due to obesity that results in chronic adipose tissue hypoxia. Adipose tissue expands; it is unable to balance the tissue oxygenation and becomes chronically hypoxic. In the case of WAT, it was anticipated that as fat mass expands in obese conditions adipose tissue became larger which forms the vasculature and therefore, O2 deprivation occur.7 White adipocytes reveals wide-ranging of adaptations to low PO2 metabolically. The key regulator of hypoxic transcription factor is HIF-1 gene. Other transcriptions which are involved in large number of genes which sensitive to adipocytes. Hypoxic conditions widen a rapid increase in adipose tissue mass which show low PO2 increasing tissue would be unique to obesity 8

differentiation to adipocytes. Expression of the PPARγ nuclear transcription factor is down regulated in preadipocytes by hypoxia, through Hypoxia Induced Factor (HIF)-1 alpha.12 PPARγ expression is also inhibited by hypoxia in mature adipocytes and this may explain, at least in part, the hypoxia-induced changes in the expression of genes such as adiponectin. These effects may in turn upregulate C/EBP homologous protein (CHOP) in adipose tissue of obese animals and in 3T3-L1 adipocytes exposed to hypoxia. The down-regulation of PPARγ and inhibition of adipocyte differentiation in a low O2 environment may inhibit fat cell recruitment in obesity. This would be consistent with the concept that fat cell number stays constant in adult obese (and normal weight) subjects as the number of adipocytes is set in childhood and adolescence.13

Other than being the precursors of mature adipocytes, pre-adipocytes also act as inflammatory cells expressing and releasing a range of inflammation related factors, Hypoxia: Effect on the Stromal Vascular particularly in response to stimulation by macrophagederived mediators. Hypoxia leads to the stabilization Cells, Macrophages and Pre-Adipocytes of HIF-1α, leading to its accumulation. This in turn Hypoxia has been shown to increase the secretion of modulates the expression of several genes that are key cytokines as well as of VEGF from the cells of hypoxia-sensitive in adipocytes, including VEGF, the stromal-vascular fraction (SV) of human adipose FABP4 (aP2), and GLUT1, PAI-1, IL-4, and IL-6.14 tissue. The cytokines include TNF-α, IL-6, IL-10, and CCL-2 (MCP-1). The SV fraction is composed of One of the most intriguing aspects of the response of predifferent types of cell and effectively all the cells within adipocytes to hypoxia relates to leptin expression. Preadipose tissue rae involved in the release of cytokines adipocytes are considered not to express the LEP gene. and adipokines. It is therefore difficult to attribute the Its expression generally occurs around 3–4 days after hypoxic response in the SV fraction as a whole to a the induction of differentiation. Hence, the expression specific cell type. However, the effects were reported to of leptin is a marker of adipocyte differentiation. In be enhanced in SV fractions enriched in the macrophage several cell types, including trophoblast-derived BeWo marker. This implies that macrophages are the major cells and breast cancer cell lines, which normally show source of inflammatory cytokines within the SV under very little or no leptin expression, exposure to hypoxia hypoxic conditions.9 leads to the induction of leptin synthesis. Similarly, incubation of human pre-adipocytes in low PO2 results Direct studies on macrophages, or macrophage cell in the marked induction of leptin gene expression.15 lines, indicate that they respond strongly to hypoxia Thus hypoxia turns pre-adipocytes into leptin-secreting with a stimulation of the production of a range of endocrine cells. Pre-adipocyte-derived leptin may play cytokines and other inflammation-related factors.10 In a specific local autocrine/paracrine role within adipose a recent study, hypoxia has been reported to up-regulate tissue. TLR-4 (Toll-like receptor 4) expression via HIF-1 in RAW264.7 cells. Interrogation of microarray datasets Obesity Induced Macrophage Infiltration does not suggest, however, any such hypoxia-induced in Adipose Tissue up-regulation of TLR-4 expression in adipocytes.11 Pre-adipocytes, as the precursors of adipocytes are AMacrophage infiltration is increased in white adipose key cells within adipose tissue, and their response to tissue some of the authors reported that the adipose hypoxic conditions has been explored. A major effect tissue macrophages are responsible for the majority of of low O2 tension on pre-adipocytes is to inhibit their inflammatory cytokine production and that these cells Page 74

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are derived from the bone marrow. During weight gain adipose tissue expands macrophages are recruited adipose tissue macrophages secrete cytokines and they have higher inflammatory response. These cytokines contribute propagation of macrophages secreting chemokines.16 Adipose tissue macrophages results in the formation of crown like structures in dead adipocytes. Macrophages majorly involved in adipose tissue remodeling occurs were fewer crown like structures originate in adipose tissue expansion Macrophage infiltration is increased in white adipose tissue some of the authors reported that the adipose tissue macrophages are responsible for the majority of inflammatory cytokine production and that these cells are derived from the bone marrow. These adipose tissue macrophages results in the formation of crown like structures in dead adipocytes. Macrophage majorly involved in adipose tissue remodeling occurs were fewer crown like structures originate in adipose tissue expansion. To understand the paracrine loop between adipocytes and macrophages here is a physiological relevance and it was stated that the pathophysiological

macrophages significances of obesity is further studied in animal models.17 Accepting the molecular mechanisms underlying increased macrophage infiltration into obese adipose tissue may direct novel identification of adipocyte-derived chemokine(s) and even therapeutic strategies to prevent or treat obesity-induced adipose tissue inflammation.

Conclusion Adipose tissue hypoxia focuses on many therapeutic strategies that provide a pivotal role played by it in adipose tissue remodeling in obesity. To satisfy the O2-signaling pathways or by reversing hypoxia, via HIFs or other regulatory factors, which presents novel opportunity and targets. Adipose tissue is skilled by affecting multiple tissues and organs by virtue of a large number of adipocytokines and as a result, influences a diversity of physiologic and pathophysiologic processes. Hence, it is important to identify effective therapeutic strategies for obesity and associated disorders

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Hosogai N, Fukuhara A, Oshima K, Miyata Y, Tanaka S, et al . Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation. Diabetes. 2007; 56:901-11. Brook CG, Lloyd JK, Wolf OH. Relation between age of onset of obesity and size and number of adipose cells. Br Med J. 1972; 2:25–7. Helmlinger G, Yuan F, Dellian M, Jain RK. Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat Med. 1997; 3:177–82 TrayhurnP. Hypoxia and adipocyte physiology: implications for adipose tissue dysfunction in obesity. Annu Rev Nutr. 2014; 34:207–36. Stern JS, Batchelor BR, Hollander N, Cohn CK, Hirsch J .Adipose cell size and immunoreactive insulin levels in obese and normal weight adults. Lancet. 1972; 2:948–51. Ye J. Emerging role of adipose tissue hypoxia in obesity and insulin resistance. Int J Obes (Lond). 2008; 33:54–66. Brahimi Horn MC, Pouysségur J. Oxygen, a source of life and stress. FEBS Lett. 2007; 581:3582–91. Rausch ME, Weisberg SP, Vardhana P, Tortorielllo DV. Obesity in C57BL/6J mice is characterised by adipose tissue hypoxia and cytotoxic T cell infiltration. Int J Obes (Lond). 2008; 32:451–63. O’Rourke R, White A, Metcalf M, Olivas A, Mitra P, et al. Hypoxia-induced inflammatory cytokine secretion in human adipose tissue stromovascular cells. Diabetologia. 2011; 54: 1480–90. Lewis JS, Lee JA, Underwood JC, Harris AL, Lewis CE. Macrophage responses to hypoxia: relevance to disease mechanisms. J Leukoc Biol. 1999; 66: 889–900. Kim SY, Choi YJ, Joung SM, Lee BH, Jung YS, et al. Hypoxic stress up-regulates the expression of Toll-like receptor 4 in macrophages via hypoxia-inducible factor. Immunology. 2010; 129:516–24. Yun Z, Maecker HL, Johnson RS, Giaccia AJ. Inhibition of PPAR-2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia. Dev Cell 2002; 2: 331–41. Hosogai N, Fukuhara A, Oshima K, Miyata Y, Tanaka S, et al. Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation. Diabetes. 2007; 56: 901–11. Wang B, Wood IS, Trayhurn P. Hypoxia induces leptin gene expression and secretion in human preadipocytes: differential effects of hypoxia on adipokine expression by preadipocytes. J Endocrinol. 2008; 198: 127–34. Wu MH, Chen KF, Lin SC, Lgu CW, Tsai SJ. Aberrant expression of leptin in human endometriotic stromal cells is induced by elevated levels of hypoxia inducible factor1. Am J Pathol. 2007; 170: 590–8. Weisberg SP, Hunter D, Huber R, Lemieux J, Slaymaker S, et al. CCR2 modulates inflammatory and metabolic effects of high-fat feeding. J Clin Invest. 2006; 116:115–24. Ito A, Suganami T, Yamauchi A, Degawa-Yamauchi M, Tanaka, et al. Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue. J Biol Chem. 2008; 283, 35715–23.

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Post-Mortem Examination: Combining Conventional Autopsy Technique with Virtual Autopsy

Switzerland. Michel Thali, forensic pathologist and project manager for Virtopsy, says that, “when an autopsy is done it destroys the 3-D geometry of the body”1.

Post-Mortem Examination: Combining Conventional Autopsy Technique with Virtual Autopsy - Concerted Effort by the Departments of Forensic Medicine & Toxicology and Radiology Dipayan Deb Barman, Assistant Professor Vijaya Kumar Nair G, Professor and HOD Department of Forensic Medicine and Toxicology, Sri Sathya Sai Medical College and Research Institute, Sri Balaji Vidyapeeth, Kancheepuram - 603108, India Email: drvkng@gmail.com

Abstract  Post-mortem examination or autopsy is a pivotal technique for helping the law enforcement

agencies and as well as medical science in ascertaining the cause of death. The purpose for carrying out an autopsy is for either medico-legal or pathological reasons, with the intention to determine cause of death, time of death, manner of death and identification e.g. in mass disaster, as well as documentation and expert testimony. In addition to the conventional method of a meticulous dissection of the deceased body nowadays with the help of advanced radiological imaging the body can be scanned and this can give a lot of information which will help immensely in the administration of justice. This technique is called virtual autopsy or virtopsy. This paper is an attempt to explore the scope of collaboration of departments of forensic medicine and radiology in the field of virtual autopsy.

Key Words: Forensic medicine, Forensic radiology, Multi slice computed tomography, Virtual autopsy, Post-mortem examination

Introduction Autopsy procedures have become increasingly challenging in modern days due to the evolvement of newer methods of crime and unknown pathological conditions which could be the putative cause of death. This paper is an attempt to explore the scope of collaboration between departments of forensic medicine and radiology in the field of virtual autopsy. Exploration of body cavities which is done during conventional autopsy sometimes faces resistance due to the social, customary and religious issues. The term, “Virtopsy” came from the term virtual autopsy which means autopsy carried out using modern medical, imaging and measuring technology. Virtual autopsy or digital autopsy is a new radiologic technique that uses a combination of postmortem multi-slice computed tomography (MSCT) and magnetic resonance imaging (MRI). Page 76

The advancement of MSCT and MRI technology with improved contrast and resolution technique as well the options for 2D and 3D reconstruction, provides an observer-independent, objective and reproducible forensic assessment. Minimally invasive forensic autopsy with the aid of imaging technique can look deeper and precise inside the body cavity with three dimensional views, which provides all the information like position and dimensions of the wound, including other pathological conditions in the body.

Historical Aspects of Virtual Autopsy Richard Dirnhofer, former Director of Forensic Medicine University, Berne, Switzerland, was credited for the development of this excellent technology. His work was carried on further by Michel Thali and his colleagues at the University of Berne’s Institute of Forensic Medicine, Ann. SBV, Jan-Jun 2016;5(1)

Imaging Modalities used in a Virtual Autopsy Before discussing the imaging modalities used in virtual autopsy we have to understand the limitations of doing a conventional autopsy. In a conventional autopsy the external injuries are noted and photographs are taken to compare them later with the findings observed during the dissection of the body. Photographs even though can give an excellent idea about the type of injury, their location, approximate size, and nature (simple or grievous) of injury; it has the limitation of being only 2D. In the context of size of a wound, especially in stab wounds, which are caused by sharp pointed instruments or weapon like knife/dagger and firearm wounds it sometimes becomes impossible to establish the actual depth and track of a wound. In case of gunshot wounds, which forms a track inside the body, the chance of creating a false track sometimes becomes inevitable if the person doing the procedure is not aptly skilled. In India where there is a lack of forensic medicine experts, most autopsies are performed by MBBS doctors, which can be a factor in inaccurate findings that hampers with justice.Virtual autopsy employs a combination of the medical imaging technologies and as well as technology which is used in other fields of science such as: −− 3-D surface scan which is used in the automobile designing can be used to map the exterior of the body. It provides and documents the three dimensional image of the body surface area in details −− Multi-slice computed tomography(MSCT) −− Magnetic Resonance imaging Both MSCT and MRI technique can give excellent and accurate view of the interior of the body. Histopathological samples can be accurately obtained from the body using CT guided biopsy technique. The condition and pathologic findings of different organs can be seen and understood in depth using these techniques. The body can be examined slice by slice in the desired plane according to the need. The time since death can also be estimated using MRI with spectroscopy by measuring the metabolites in the brain during the process of postmortem decomposition of the body. Examination of the heart is vital in every autopsy to rule out any cardiac cause of death, especially in cases like sudden cardiac death in a young individual without any documented history of cardiac disease. It may not be possible to study the cardiac muscle pathology during a conventional autopsy Ann. SBV, Jan-Jun 2016;5(1)

and hence 3-D angiography technique is used to confirm or refute any underlying coronary artery disease2-4. In case of any injury to the blood vessel, there will be spillage of dye to the surrounding tissues, making it visible in the CT images.

Comparison of the advantages of Virtual Autopsy over Conventional Autopsy In more than 100 virtual autopsies performed by Michel Thali and colleagues4, 5 the results were comparable with those of conventional autopsy. Various forensically pertinent parameters were considered during the comparison such as presence of fractures and foreign bodies, as well as tissue and organ trauma. While performing post-mortem angiography and biopsy procedures, Michel Thali and colleagues observed that in cadavers where there is no respiratory movements and cardiovascular activities, the results were much better. MSCT images provide information about the general pathology of the body and can give detailed information about trauma or injuries. MRI on the other hand is used to focus on specific areas of the body, providing details about soft tissues, muscles and organs. Digital autopsy provides a 3D geometric documentation of injuries on the body surface and internal injuries in the living as well as in deceased cases. Surface scanner is the means for measuring and depicting the images in 3-dimensional views with precision. The body is scanned in all angles using a sensor which takes pictures using two cameras. The computer then gives image of the body in three dimensional views which can be rotated as per requirement without any distortion for collection of the findings. In case of gunshot injuries where sometimes traditional autopsy may not yield very subtle findings, the use spiral CT and MRI examinations with the subsequent 2D multi-planar reformation and 3D shaded surface display reconstruction, the entire gunshot created complex skull fractures and brain injuries (such as wound channels and deeply-driven bone splinters) could be documented in complete and graphic detail. Thali et al., in their study by fusing CT and MRI found vital reaction to gunshot seen as air emboli in the heart and blood vessels and the classic pattern of blood aspiration to the lung. Gunshot residues deposited within and under the skin were visible using imaging modalities6. According to one study by Plattner it was reported that in a case of virtual autopsy in a death due to drowning, massive vital decompression with pulmonary barotraumas and lethal gas embolism were identified in the radiological images. In this study, MSCT and MRI were found superior to conventional autopsy in their ability to demonstrate the extent and distribution of Page 77

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gas accumulation in intra-parenchymal blood vessels of internal organs as well as in areas of the body7. A rapidly evolving facility in the western countries is digital mortuary. The concept of digital mortuary is a novel one where the digital morgue stores the body structure of each case as a 3D data set consisting of sectioning planes of the whole body obtained from MSCT or MRI figures. According to Takatsu et al., a retrospective observation with a detailed quantitative analysis of the structural damage of the body has become possible using very high dimension medical imaging and medical virtual reality8.Foetal autopsy is another important area, where in addition to conventional autopsy, the application of virtual autopsy can yield valuable and accurate results. However there is a decrease in number of peri-natal autopsy and importantly, a large majority of parents often refuse to give their consent to perform autopsy because of concerns about disfigurement of their deceased child during the procedure 9, 10. The reasons justifying the autopsy, with the need to know the cause of death or the identity of an unknown deceased individual overrules this emotional involvement. Therefore, the families and relatives of the victim often remain in a conflicting situation with the forensic examiners on whether to give permission or not11.In virtual autopsy, an interdisciplinary approach by a team comprising of a radiologist, forensic pathologist and pathologist is required so that the findings of the conventional autopsy can be coupled with virtual autopsy and this can be analyzed, stored in the form digital data and will later be reported in the same database and specified according to the ICD10. The risk of infection to autopsy surgeon and staff always exists in a conventional autopsy. Nowadays, with

emergence of newer and deadly infections coming, it very hazardous doing a conventional autopsy as the infectivity from the body is always unknown and infection can easily spread from a fresh dead body as well as a highly putrefied body. Virtopsy is a much safer procedure that does not involve any blood-shed or exposure to contaminated body fluid. The main advantage of virtopsy is visualization of 3D anatomical structures thoroughly, in real time, without damaging the body12. However in comparison to the conventional autopsy virtual autopsy has some disadvantages. Rüegger et al., in their study found that in imaging based autopsy, the tissue for microbiological and histopathological examination cannot be collected, which may result in poor accuracy of MRI, since infection may be a primary cause of death13.

Conclusions In the era of scientific advancements where progress is happening by leaps and bounds in order to solve the questions pertinent to forensic medicine and science investigations, the use of both conventional autopsy and radiological imaging techniques can be the best mechanism, whereby many difficult domains may be reached. There are differences in the ante-mortem radiological findings as well as the post-mortem finding which need more intensive study. In India, it has not yet been possible to use imaging technology to aid in postmortem examination due to restraints of resources and priority for the dead. However, future implementation of these modalities will greatly augment Forensic Medicine study and investigation.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

The future is Virtopsy: Sequence format (Neue Zuricher Zeitung) Telecast,2006, www.Virtopsy.com (accessed on 20 May 2016) Thali MJ, Yen K, Vock P, Ozdoba C, Kneubuehl BP, et al. Image-guided virtual autopsy findings of gunshot victims performed with multi-slice computed tomography and magnetic resonance imaging and subsequent correlation between radiology and autopsy findings. Forensic Sci Int. 2003;138:8-16 Thali MJ, Braun M, Buck U, Aghayev E, Jackowski C, et al. VIRTOPSY—scientific documentation, reconstruction and animation in forensic: individual and real 3D data based geometric approach including optical body/object surface and radiological CT/MRI scanning. J Forensic Sci. 2005; 50:428-42 Dirnhofer R, Jackowski C, Vock P, Potter K, Thali MJ. VIRTOPSY: minimally invasive, imaging-guided virtual autopsy. Radiographics. 2006; 26:1305-33. Buck U, Naether S, Braun M, Bolliger S, Friederich H, et al. Application of 3D documentation and geometric reconstruction methods in traffic accident analysis: with high resolution surface scanning, radiological MSCT/MRI scanning and real data based animation. Forensic Sci Int. 2007;170:20-8. Thali MJ, Barun M, Dirnhofer R. Optical 3D surface digitizing in forensic medicine: 3D documentation of skin and bone injuries. Forensic Sci Int. 2003:137:203-8. Plattner T, Thali MJ, Yen K, Sonnenschein M, Stoupis C, et al. Virtopsy-postmortem multislice computed tomography (MSCT) and magnetic resonance imaging (MRI) in a fatal scuba diving incident. J Forensic Sci. 2003; 48:1347- 55. Takatsu A, Suzuki N, Hattori A, Shigeta A. The concept of the digital morgue as a 3D database. Leg Med (Tokyo). 1999;1:29-33. McHaffie HE, Fowlie PW, Hume R, Laing IA, Lloyd DJ, et al. Consent to autopsy for neonates. Arch Dis Child Fetal Neonatal Ed. 2001;85:F4–F7. Breeze ACG, Statham H, Hackett GA, Jessop FA, Lees CC. Perinatal postmortems: what is important to parents and how do they decide? Birth. 2012;39:57–64. Maldonado MT. Psicologia da gravidez. 17ª ed. São Paulo: Saraiva; 2009 Junior R, Souza P, Coudyzer W, Thevissen P, et al. Virtual autopsy in forensic sciences and its applications in the forensic odontology. Rev Odonto Cienc. 2012; 27:5-9. Rüegger CM, Bartsch C, Martinez RM, Ross S, Bolliger SA et al. Minimally invasive, imaging guided virtual autopsy compared to conventional autopsy in foetal, newborn and infant cases: study protocol for the paediatric virtual autopsy trial. Paediatrics 2014, 14:15

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Annals of SBV Sri Balaji Vidyapeeth

(Deemed University, Accredited by NAAC with 'A' Grade)

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