Role of fruit & vegetable in cancer therapy

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Roles of fruits and vegetables in cancer therapy Introduction Fruit and vegetables are high in nutrients that are potentially protective against cancer. They also play an important role in weight management. As obesity is a known risk factor for cancer of the colon, breast (in post-menopausal women),endometrium, kidney and oesophagus, fruit and vegetables may also protect against cancer indirectly by helping to maintain a healthy body weight. Although there has been a slight weakening of the evidence supporting the role of fruit and vegetables in reducing the risk of some cancers, overall the evidence is suggestive of a protective effect. Recent studies show that fruit and vegetables are protective against oral, laryngeal, oesophageal, colorectal and lung cancers.The association of fruit and vegetable consumption on stomach cancer risk remainsin conclusive, however fruit may possibly be protective. Fruit consumption also appears to provide protection against bladder cancer.Fruit and vegetable consumption does not appear to be associated with a lower risk of prostate, breast or ovarian cancer. However, one meta-analysis suggests that tomato consumption may reduce the risk of prostate cancer.The Cancer Council supports that recommend eating plenty of fruit and vegetables, and the population recommendation of at least two serves of fruit and five serves of vegetables daily.Because the knowledge is still incomplete about the ways in which nutrients may reduce cancer risk, The Cancer Council also recommends that people eat a variety of different fruit and vegetables to obtain maximum benefits. Fruit and vegetables are best consumed fresh and whole (i.e. not in a supplement form) and consumption of both cooked and raw vegetables are recommended. For people already diagnosed with cancer, there is some evidence that a diet high in fruit and vegetables is not of significant benefit, but is unlikely to be harmful. The Cancer Council recommends the general community guidelines of two serves of fruit and five serves of vegetables daily for cancer survivors. Because the knowledge is still incomplete about the ways in which nutrients may reduce cancer risk, The Cancer Council also recommends that people eat a variety of different fruit and vegetables to obtain maximum benefits. Fruit and vegetables are best consumed fresh and whole (i.e. not in a supplement form) and consumption of both cooked and raw vegetables are recommended.

1.1.Background The International Agency for Research on Cancer (IARC) concluded that 5-12% of cancers could be attributed to low fruit and vegetable consumption. 1 Australian data suggests that 2% of cancers were attributable to low consumption of fruit and vegetables. 2 In terms of health care costs, it has been estimated that low vegetable intake (<4 serves per day) accounts for 17% of the cost of bowel cancer, 2% of the cost for breast cancer, and 9% of the cost of lung and of prostate cancer.3 Twenty one percent of the cost of lung cancer and 4% of the cost of breast cancer has been attributed to lower fruit intake (<3 serves per day). 3The protective effects of fruit and vegetables against cancers, as well as other diseases such as coronary heart disease and type 2 diabetes, has led to the promotion of fruit and vegetables consumption as a national public health priority.4Fruit and vegetables also play an important role in weight management due to their low energy density, high fibre content and capacity to displace higher energy foods from the diet.

Obesity is a known risk factor for cancer of the colon, breast (in postmenopausal women), endometrium, kidney and oesophagus.5 Emerging evidence suggests that obesity is also linked to

cancer of the pancreas, gallbladder, thyroid, ovary and cervix as well as non-Hodgkinâ&#x20AC;&#x2122;s lymphoma and multiple myeloma. 6 Therefore fruit and vegetables may reduce the risk of cancer directly through the provision of specific anti-carcinogenic agents and indirectly through their role in weight management.

1.2.The Impact of Diet on the Development of Cancer Dietary factors may account for approximatively 35% of cancer death, similar to the impact of smoking7. Such a close relationship between diet and cancer is well illustrated by the large variations in rates of specific cancers among countries as well as by the spectacular changes observed in the cancer incidence rates in migrating populations 8. Among specific aspects of the diet that are the most closely associated with cancer, a large number of ecological, case-control and cohort epidemiological studies have consistently associated increased consumption of fruits and vegetables with a decreased risk for a wide variety of cancer, particularly those of epithelial origin9. In these studies, people consuming the least fruit and vegetables are about two-fold more susceptible to develop some cancers than those having the highest consumption of these foods.

These studies thus suggest that fruits and vegetables represent an essential source of molecules with chemopreventive properties, an hypothesis strengthened by many experimental data obtained using cellular and animal models in which molecules isolated from several food sources were found to induce tumor cell death as well as to reverse the development of several cancers10 .

2. Fruits and Vegetables are an Important Source of Anticancer Agents Fruit and vegetables are high in nutrients that are potentially protective against cancer. Anticarcinogenic activity may be provided by nutrients such as fibre, vitamins, minerals, antioxidants and phytochemicals, which are chemicals found in plants such as flavonoids, carotenoids and lignans. It is probably a combination of these nutrients and phytochemicals found together in whole foods that helps to reduce the risk of chronic diseases rather than one anti-cancer component, although many different mechanisms have been proposed11.Single nutrients identified from the analysis of epidemiological studies have usually been unsuccessful when investigated further in trials, making the whole food approach more appropriate to prevention advice.Fruit and vegetables in particular have attracted much research attention for their cancer protective effects, and accordingly, many cancer associations worldwide, together with a number of National Dietary Guideline committees have recommended a daily intake of 5-7 serves of fruits and vegetables to reduce cancer risk. In spite of considerable evidence linking the consumption of fruits and vegetables to a reduction of cancer risk, the identification of the biologically active molecules that are responsible for the chemopreventive properties of these foods is still a matter of intensive investigation.Research carried out during the last years has shown that fruits and vegetables arerich sources of phytochemicals, non-nutritive molecules that play essential roles in various aspects of plant physiology, especially in their defense mechanisms against insects and various microorganisms. A wide variety of phytochemicals have been described to date and are classified by protective function, physical characteristics and chemical characteristics. The three major classes of phytochemicals are the polyphenols (flavonoids, isoflavones, anthocyanins, catechins, etc.), terpenes (including the subclasses carotenoids and limonoids) and the thiols (including the subclasses indoles, dithiolthiones and isothiocyanates).These molecules are responsible for most of the color, odor and astringency of fruits and vegetables, and are present in significant amounts in plant-based foods : a daily intake of a mixture of fruits, vegetables and drinks such as green tea and red wine contains about 1 to 2

g of these phytochemicals, corresponding to the ingestion of about 5,000 to 10,000 different compounds12.One of the best characterized biological activity of phytochemicals is their antioxidant properties13. For example, a medium-sized apple, which contains 10 mg of vitamin C has an antioxidant potential similar to that of 2350 mg of this vitamin 14, a property mostly due to its high content in polyphenols such as flavonoids and procyanidins15. However, although the antioxidant properties of phytochemicals have recently received enormous attention, there is concluding evidence that these compounds possess several additional anticancer properties that play crucial roles in the chemopreventive properties of fruits and vegetables (16;17).

2.1. How do Fruits and Vegetables Prevent Cancer?

The recognition of fruits and vegetables as a source of anticancer molecules has led to considerable interest in the identification and characterization of the mechanisms by which naturally occurring phytochemicals found in the diet are capable of inhibiting,retarding or reversing carcinogenesis16. To date, at least four major mechanisms have been proposed to account for the chemopreventive properties of phytochemicals: 2.1.1. Stimulation of the Hostâ&#x20AC;&#x2122;s Defense Mechanisms against DNA Damaging Events. A number of factors can induce damage to DNA and initiate cancer. Free radicals, environmental or diet-associated chemicals, UV irradiation or some viruses all have the capacity to cause significant damage to the cells that may ultimately lead to cancer 18. Most, if not all environmental carcinogens are metabolized once they enter the body by the so-called Phase I metabolism, a physiological reaction primarily catalyzed by the cytochrome P450 enzymes 19.

However, this reaction often converts procarcinogens into highly reactive chemical intermediates that can bind and alter the function of key cellular macromolecules such as DNA. A second group of enzymes, known as Phase II enzymes, conjugate these recative intermediates with a number of endogenous factors, resulting in the production of water-soluble products that can be excreted by the body 20.There is considerable evidence that several chemopreventive phytochemicals elicit their anticancer effects by modulating these enzymatic systems, either by reducing their carcinogenic potential (through the inhibition of the Phase I enzymes) or by increasing the excretion of the carcinogens, through increase of Phase II enzymes activity 20. The most well-documented example of such a mechanism of action is theremarkable anticarcinogenic activity of isothiocyanates, compounds that are found in high amounts in cruciferous vegetables.

Isothiocyanates inhibit tumorigenesis induced by a wide variety of chemical carcinogens, this effect being related to their reduction of genetic damage as a result of the inhibition of Phase I enzymes and activation of Phase II enzymes 21. A number of phytochemicals that modulate the hostâ&#x20AC;&#x2122;s defense mechanism against DNA-damaging molecules are also found in several other fruits and vegetables, including garlic and its related members of the Allium family, as well as citrus fruits. The presence of these biologically molecules causing the reduction of the oncogenic potential represents an efficient first-line defense against cancer that certainly contribute to the chemopreventive properties of fruits and vegetables.

2.1.2. Cytoxicity against Tumor Cells. In vitro experimental systems using cells isolated from various human tumors are being widely used to study the anticancer properties of dietary-derived phytochemicals. A large number of molecules have been shown to cause significant damage to cancer cells, leading to growth arrest and, in several cases, to the induction of apoptosis. Of particular interest is the strong proapoptotic activity of several isothiocyanates from cruciferous

vegetables, such as phenethyl isothiocyanate (PEITC) 22, as well as the potent cytotoxic effect of curcumin against several tumor cell lines23. How these cytotoxic properties contribute to the chemopreventive effects of these phytochemicals remains unclear but could play an essential role by preventing the growth of cells that have already acquire an initiated phenotype (precancerous cells).

2.1.3. Antiangiogenic Properties. Tumor angiogenesis, the process by which tumor cells stimulate the formation of a new blood vessel network that is necessary for their growth and progression, plays an essential role in tumor growth24. As a consequence, inhibition of angiogenesis has been proposed to represent an effective mean to interfere with tumor progression, leading to extensive efforts aimed at the identification of antiangiogenic molecules25. Interestingly, many phytochemicals have recently been shown to possess strong antiangiogenic activities26.For example, an abundant polyphenol found in green tea, epigallocatechin gallate (EGCG), was found to potently inhibit a crucial receptor involved in angiogenesis (VEGFR-2), this inhibitory effect occurring at concentrations readily achievable by the consumption of moderate amounts of green tea 27. This inhibitory effect is not restricted to green tea catachins since a potent VEGFR inhibitory activity was also recently identified for delphidin, an abundant blueberry anthocyanidine36. In a similar manner, ellagic acid, a phenolic acid found in high quantities in some fruits, such as raspberries and strawberries, also interfere with VEGFR-2 and also strongly inhibits the activity of another receptor found in perivascular cells, PDGFR28. This combined inhibitory effect of receptor tyrosine kinases that are both essential for angiogenesis leads to the inhibition of angiogenesis in both in vitro and in vivo assays (27,28). As will be discussed in more details below, there is increasing evidence that the antiangiogenic effects of these phytochemicals may play a crucial role in the chemopreventive effect of these molecules.

2.1.4. Increase in Intestinal Absorption or Inhibition of Hepatic Metabolism. Another manner by which dietary-derived phytochemicals may influence tumor progression involve an indirect mechanism, in which a given molecule, with little intrinsic anticancer property, may greatly influence the biodisponibility of another phytochemical with potent anticancer activity.

Perhaps the best example of this indirect synergistic mechanism is the effect of piperine, a black pepper component, on the serum concentration of curcumin. In the absence of piperine, curcumin is poorly absorbed and rapidly excreted. However, when co-administered with piperine, the absorption of curcumin is increased about a thousand-fold 29, an effect possibly related to the modulation of intestinal proteins, such as P-gp and CYP3A4, both involved in the metabolism ofxenobiotics30.

2.2. Views on Fruit and Vegetables in Cancer Prevention Reports Several major reports that have investigated the relationship between fruit and vegetable intake and cancer risk show that there has been a shift in the strength of the evidence over the last 10 years. These reports indicate that the evidence appears to be somewhat weaker than previously thought (Table 1). On the whole,evidence that vegetables are protective is stronger than for fruits, but this may simply reflect the generally greater consumption of vegetables worldwide or the different mix of nutrients obtained from them.

Table 1: Conclusions from the major cancer prevention reports regarding the cancer protective effect of fruit (f) and vegetables (v)

Organisation Review WCRF/AICR (1997)31

Highest Evidence Convincing Mouth (f&v) Pharynx (f&v) Oesophagus (f&v) Stomach (f&v) Colon & rectum (v) Lung (f&v)

Moderate Evidence Probable Larynx (f&v) Pancreas (f&v) Breast (f&v) Bladder (f&v)

COMA (1998)32

Oesophagus (f&v)

Stomach (f&v) Colon & rectum (v)

WHO/FAO (2003)33

Oral Cavity (f&v) Oesophagus (f&v) Stomach (f&v) Colon & rectum (f&v)

IARC (2003)1

Oesophagus (f&v) Stomach (f) Colon & rectum (v)

Lower Evidence Possible Ovaries (f&v) Cervix (f&v) Endometrium (f&v) Thyroid (f&v) Liver (v) Prostate (v) Kidney (v) Breast (f&v)

Mouth (f&v) Pharynx (f&v) Larynx (f&v) Kidney (f&v) Colon and rectum (f) Bladder (f) Stomach (v) Lung (v) Ovary (v)

In 1997, the World Cancer Research Fund and the American Institute of Cancer Research (WCRF/AICR) jointly published an extensive, global review of the role of food and nutrition in the prevention of cancer.8 The conclusions by WCRF/AICR relating to the protective effect of fruits and vegetables can be seen in Table 2.Many of the views expressed in the 1997 WCRF/AICR report were also reflected inan extensive review published the previous year,which found that fruit and vegetables appeared to protect against cancer of the mouth, pharynx, oesophagus,stomach, colon, pancreas, endometrium and lung34.This review also noted that vegetables in general, and raw vegetables in particular, appear to be the outstanding protective categories34.They specifically identified allium vegetables (including onions, garlic and leeks), carrots, green vegetables in general, cruciferous vegetables (such as cabbage, cauliflower, broccoli, brussel sprouts, Chinese cabbage and bok choy) and tomatoes as strongly protective.

Table 2: WCRF/AICR conclusions relating the cancer protective effect of particular types of fruits and vegetables31.

Fruit or Vegetable Convincing Probable Possible Vegetables

Colon

Prostate Liver

Kidney Cruciferous vegetables

Colon & rectum Thyroid

Allium vegetables

Stomach

Colon

Green vegetables

Lung

Oesophageal Colon Breast

Green leafy vegetables

Stomach

Raw Vegetables

Stomach

Tomatoes

Stomach

Carrots

Citrus fruits

Mouth/oral Pharyngeal

Lung Stomach Lung Bladder

Stomach

Mouth/oral Rectum

Mouth/oral Oesophageal

The United Kingdom Department of Health Committee of the Medical Aspects of the Food Supply (COMA) reviewed the evidence concerning the potential protection afforded by fruit and vegetables against the development of cancer in 1998 32.No strong association was found between fruit and vegetable consumption and cancer at any site, while a moderate association was noted for cancers of the stomach, colon and rectum (Table 1). This is in contrast to WCRF/AICR, who rated the evidence of an association for cancer of the mouth, pharynx, stomach, colon, rectum and lung as convincing (Table 1). Although they did not agree on the strength of evidence for each, these two major international organisations did agree that increased consumption of fruits and vegetables is associated with a reduced risk for cancers of the oesophagus, stomach, colon, rectum and breast (Table 1). The different conclusions reached in the WCRF/AICR and COMA reports relate to their relative weighting of different types of evidence. In the 1998 COMA report, prospective cohort studies carried more weight than case control or ecological studies, and mechanistic studies of induced tumours in standard animal models were not included. In 1997, the WCRF/AICR expert review panel examined in vitro studies and animal trials as well as ecological, case-control and cohort studies in human populations. At the time of the WCRF report, there were only a limited number of cohort studies that had reported on fruit and vegetable intake and cancer risk. Results published in 2001 from the EPIC study showed that significant health gains are made from even a small increase in fruit and vegetable intake35. Increasing intakes of fruit and vegetables by just 50g a day (equivalent to 2/3 cup cooked vegetables or 1/3 of a piece of fruit) was associated with a reduction in cancer risk of around 20% 35. In 2003, The World Health Organisation (WHO) suggested that a high intake of fruit and vegetables probably reduces the risk of cancers of the oral cavity, oesophagus,

stomach and colorectum (Table 1)33. WHO recommended an intake of at least 400g of fruit and vegetables daily (in addition to potatoes). However, they found that support for a broad and strong protective effect of higher vegetable and fruit intake had weakened with results from recent prospective studies. Similarly, IARC found that a high intake of vegetables probably reduces the risk of oesophageal and colorectal cancer, while a high intake of fruit probably reduces the risk of oesophageal and stomach cancer (Table 1) 1. However the IARC rated the evidence for an association between vegetable intake and cancer of the mouth, pharynx, larynx, kidney, stomach, lung and ovary and the evidence for an association between fruit intake and cancer of the mouth, pharynx, larynx, kidney, colorectum and bladder as possible 1.The conclusions from WHO and IARC differ from the previous reviews by WCRF/AICR and COMA because of the inclusion and consideration of more prospective studies published since these original reviews were undertaken. In recent years it has been increasingly apparent that the protective association between fruit and vegetable intake and cancer is much stronger in casecontrol studies than in cohort studies. Furthermore, randomised controlled trials involving fruit and vegetable intake, notably for colorectal cancer, have not demonstrated any benefit. Casecontrol studies are known to be prone to recall and selection bias, however the length of followup in cohort studies can also potentially limit the interpretation of their results. In 2004, the IARC reviewed the evidence relating to cruciferous vegetables, isothiocyanates and indoles and found that for human studies36: • There is limited evidence that eating cruciferous vegetables reduces the risk for cancers of the stomach and lung.

• There is inadequate evidence that eating cruciferous vegetables reduces the risk for cancers at all other sites.

• There is inadequate evidence to assess the independent effects on human. cancer risk of isothiocyanates and indoles, as opposed to their combined effects with other compounds in cruciferous vegetables. The recent evidence supporting a direct role for fruit and vegetables in cancer prevention appears to be mixed. In 2007, WCRF will publish an update of their conclusions regarding fruit and vegetables in their Report on Food, Nutrition Physical Activity and the Prevention of Cancer. The report will review all the available science relating to cancer prevention. It is hoped that this report will further clarify the role of fruit and vegetables in cancer prevention. In the meantime, The Cancer Council NSW has sought to review the recent literature regarding the role of fruit and vegetables and cancer protection to help clarify the discrepancies that appear in the literature.

3. CANCER AT DIFFERENT SITES IN THE BODY 3.1. BREAST CANCER WHO IS AT RISK? Breast cancer is the second most common site of cancer in women (22%) with lung cancer incidence being 25%. It affects 1 out of 36 women in South Africa during their lifetime. Men can also get breast cancer, but this condition is very rare. As this disease is less well studied in men, this discussion is limited to breast cancer as seen in women. Breast cancer is the most common cause of death in women between the ages of 35 and 55 years. Breast cancer is thus a very important disease for women and the incidence of breast cancer in South Africa is increasing annually37. The most recent statistics show that the incidence rate is 25 per 100 000. One million women are diagnosed with breast cancer per year worldwide; while an additional 1/2 million die

of breast cancer every year.Within certain ethnic groups, breast cancer rates may double or triple over time and as they migrate and adopt new lifestyles. In formerly low-risk countries, such as Japan for instance, as the diet become more westernized the incidence of breast cancer increases.

The same patterns of increases have been observed with migrants from low-risk to higher-risk areas, for example Japanese migrants to Hawaii. These patterns of change underscore the importance of the environment and nutrition in breast cancer risk.

WHAT CAUSES BREAST CANCER? The exact cause of breast cancer is not clear yet, but it is known that certain risk factors are linked to the disease. Some risk factors, such as smoking, can be controlled, but others like a person's family history, can't be changed. While all women are at risk for breast cancer, the factors listed below can increase the chances of a woman developing the disease38. ♦ Gender ♦ Age ♦ Race

Women have a much greater risk of developing the disease than men.

Risk increases with age.

Caucasian women are slightly more at risk than black women, but black women are more likely to die of this cancer. Asian and Hispanic women have a lower risk. The incidence of breast cancer in Europe and the USA is 5 times more than in Japan and China.

♦ Family history Mother or sister affected.

Two first-degree family members ♦ Personal history A woman with cancer in one breast has a greater chance of developing cancer in the other breast or in another part of the same breast. Certain types of previous abnormal biopsy results can be linked to a slightly higher risk as well. ♦ Exposure Radiation. ♦ Reproductive factors Early menarche (before age of 12 years).Women who had no children or had their first pregnancy after the age of30.

Late menopause. ♦ Socio demographic factors High income group.

High social class status.

Jewish religion. ♦ Birth control pills It is still not clear what role birth control pills might play in breast cancer risk. A recent study found that women using birth control pills have a slightly higher risk and women who stopped using the pill for 10 years or more do not seem to have any increased risk.

Women should discuss the risk and benefits of birth control pills with their doctor. ♦ Hormone replacement therapy Long-term use (5 years or more) may slightly increase the risk. A woman's breast cancer risk returns to that of the general population within 5 years of stopping the therapy. ♦ Physical activity Low activity levels.

â&#x2122;Ś DIETARY FACTORS: Table 3: Dietary factors associated with a higher incidence of breast cancer Weight gain (obesity)

Energy intake above the level that is needed daily is positively associated with breast cancer. There is an increased risk of breast cancer for women that are overweight, especially for women after menopause and those who do not use hormone replacement therapy. The connection between weight and breast cancer is complex. The risk seems to be higher for women who gained weight as adults, but not for those who have been overweight since childhood. Extra fat around the waist affects the risk more than the same amount of fat around the hips and thighs.

The role of fat intake in weight gain is an important aspect to consider, as well as changes in metabolic rate, physical activity and overall dietary intake.

Dietary fat Intake

As a major contributor to total energy, dietary fat can also increase breast cancer risk. The importance of dietary fat as an influence independent of energy intake is however unclear.Some studies suggest that olive oil intake contributes to a lower breast cancer risk.

Alcohol Intake

Women who have 1 alcoholic drink per day have a small (11%) increased risk compared to those who have 2 drinks per day whom have 25% more risk compared to the risk of nondrinkers.

Fruit and Vegetables Other

High levels of consumption (more than 5 portions per day) have been associated with lower rates of breast cancer.

Other dietary factors that may have an association with breast cancer risk are currently and continuously under study. Examples of other factors that might have an association with breast cancer rates include: Omega-3 fatty acids (fish oils), the active substances in fruit and vegetables (phyto-chemicals), vitamin A, vitamin D and xenoestrogens (soy products) may play a role in prevention of breast cancer.

3.2. PROSTATE CANCER WHO IS AT RISK? Cancer of the prostate gland is one of the most common fatal cancers and among the leading causes of cancer deaths in men over the age of 55.

RISK FACTORS OF PROSTATE CANCER Following a typical western diet high in animal (saturated) fat is associated with a higher risk of prostate cancer. Excessive consumption of red meat has also been found to be positively associated with advanced prostate cancer38.

Lycopene (the substance that provides the red colour in fruits and vegetables), found in foods such as tomato, tomato products, watermelon and red grapefruit, is associated with a reduced risk of prostate cancer as is Vitamin E supplementation37.

3.3. GASTROINTESTINAL CANCERS 3.3.1. COLORECTAL CANCER Colorectal cancer affects the colon and the rectum.

WHO IS AT RISK? Excluding skin cancer, colorectal cancer ranks second after lung cancer in men, and breast cancer in women. It affects up to 6% of men and women by the age of 75 years in western societies.

RISK FACTORS FOR COLORECTAL CANCER Cancer promoting dietary factors Although many dietary factors have been associated with colon cancer, strong associations for an increased risk have been reported with excessive consumption of red and processed meat (containing nitates, nitrites and nitrosamines, and made rich with heterocyclic amines during the cooking process) and high total fat intake. Overweight in men, and low levels of physical activity in men and women are linked with a higher risk, which has been found to increase with age.

Excessive alcohol consumption has also been linked to an increased risk. The malnutrition associated with alcoholism is also likely to be important in the increased risk reported 38. More specifically, excessive beer consumption has been linked to an increased risk for colorectal cancer. It is thus wise to follow the recommendation of no more than 2 drinks per day for men and 1 for women. Patients with chronic ulcerative colitis have a high risk of developing colon cancer and frequently have low levels of folate. Folate supplements result in a 60% lower incidence of colon cancer in these patients.

Protective dietary factors In terms of protective dietary factors, the frequent consumption of fruits and vegetables has been the most consistent in terms of preventive measures. In this regard, vegetarians are generally known to be at a lower risk. Although the predominant data available suggests that the intake of high-fiber foods protect the body from the development of colorectal cancer, many questions remain to be answered before this relationship can be clarified. Fruit and vegetables not only provide a high intake of dietary fiber, but also potentially protective phytonutrients 37.

Furthermore, consuming diets rich in these sources also means displacing potentially cancerpromoting foods that are rich in energy and high in fat. The evidence for dietary fiber as being protective would appear to be less conclusive than that for a high consumption of fruit and vegetables. Other protective dietary factors implicated, but still under study, include folate, calcium, vitamin D, antioxidants and non-nutritive compounds in vegetables such as phyto-

oestrogens and glucosinolates. The benefit of regular exercise in reducing the risk of colon cancer has been demonstrated in a number of studies.

3.3.2. OESOPHAGUS CANCER RISK FACTORS Studies have indicated that alcohol has a causal role in the development of cancer, especially for cancers of the mouth, pharynx and esophagus. It appears to have an increased effect on the tissues directly exposed to it during its consumption. The malnutrition associated with alcoholism is also likely to be important in the increased risk for certain cancers due to the lack of protective factors found in a varied diet. In this regard, such micronutrient deficiencies as niacin, riboflavin, zinc and selenium has been associated with this malignancy. Additionally, an association has been found between the drinking of very hot drinks, such as coffee and tea and the risk of esophageal cancer. Contamination of food by toxic substances takes place during charcoal broiling, frying and smoking of meats. An association has been found between an increased risk of esophageal cancer and the frequent intake of smoked and fried foods.

Moreover, certain fungi (moulds), when present in foods, such as maize and wheat products are well known to produce toxins, known as mycotoxins. Mycotoxins have been found to cause a variety of ill effects in southern Africa, of which oesophageal cancer is one. A high consumption of fruit and vegetables are associated with a decreased risk37.

3.3.3. STOMACH CANCER WHO IS AT RISK? After being the leading cause of cancer death worldwide until the 1980's, stomach cancer has been declining since the 1960's due to improvement in food storage and preservation and the recognition of other factors that play a major role in its development. Recent studies have shown that Helicobacter pylori infection results in lesions and ulcers of the stomach, thus producing conditions that promote cancer development in the stomach.

DIETARY FACTORS ASSOCIATED WITH STOMACH CANCER An association has been found between an increased risk for stomach cancer and frequent intake of smoked, pickled and fried foods. Some studies have shown that a high salt intake also increases the risk. In terms of micronutrients, low blood levels of vitamin C are known to be associated with the progression of stomach cancer. A high consumption of fruit and vegetables appears to provide a protective effect. Emerging evidence also indicates that regular consumption of green tea may possibly reduce the risk of stomach cancer38.

3.4. LUNG CANCER WHO IS AT RISK? Over the span of a century, lung cancer has changed from a rare disease to a leading cause of death.With the role of smoking in lung cancer well established, research has focussed primarily on environmental and genetic factors that can determine its risk.

RISK FACTORS FOR LUNG CANCER Environmental and lifestyle factors: â&#x20AC;˘ When smoking is stopped, lung cancer risk is still present even after 15-20 years of abstention, but not at the predicted high level of risk among those that continue to smoke.

• Exposure to environmental pollutants as well as occupational exposure to certain substances such as radon (found in underground mines), arsenic, asbestos, chromium, chloromethyl ethers, nickel and polycyclic aromatic hydrocarbons.

• Indoor air polluted with smoke from wood or coal burning and cooking fumes.

Dietary factors: • Fruit and vegetables have repeatedly been shown to have a protective effect against lung cancer. The evidence for the protective effects of vegetables appears to be stronger than that for fruit.

• Fat intake is associated with lung cancer risk in men, but not women.

• Clinical trials done to test the hypothesis of the benefit of beta-carotene supplementation as a possible protective factor, particularly focussing on lung cancer, have shown that high dose betacarotene supplements are not beneficial in reducing cancer risk, and probably increase lung cancer risk among high-risk individuals such as those who smoke or those who have a history of occupational exposure to known lung carcinogens.

3.5. ORAL CANCER A meta-analysis of 15 case-control studies and one cohort study found a statistically significant reduction in oral cancer risk for each portion of fruit (combined adjusted odds ratio (OR)= 0.51, 95% CI= 0.40-0.65) and vegetable consumed (combined adjusted OR= 0.50, 95% CI= 0.380.65).15 Similar results were found when results were pooled from 12 studies that adjusted for age, sex, smoking and alcohol intake (OR for fruit= 0.49, 95% CI= 0.39-0.63; OR for vegetables= 0.43; 95% CI= 0.31-0.59)39. Interestingly citrus fruit appeared to provide greater protection than overall fruit consumption (OR= 0.38, 95% CI 0.26-0.56) 39. A meta-analysis completed in 2003 found that in case-control studies (nine studies on fruit and seven studies on vegetable consumption), both fruit (RR= 0.53, 95% CI= 0.37-0.76) and vegetables (RR= 0.84, 95% CI= 0.67-1.07) provided protection against oral cancer, with fruit providing a statistically significant result40.

3.6. LARYNGEAL CANCER In the only meta-analysis identified for laryngeal cancer, fruit consumption provided a significant protective effect (RR= 0.73, 95% CI= 0.64-0.84), while vegetables appeared to provide a very small non-significant reduction in risk (RR= 0.92, 95% CI= 0.83-1.02) in casecontrol studies (five studies on fruit and seven studies on vegetable consumption) 40. This metaanalysis was very limited, as out of the eight case-controls studies identified, one was excluded from the analysis on both fruit and vegetables, while two were excluded from the analysis on fruit. The authors point out that the first study was excluded because study subjects were classified into only two categories of consumption, however it is not known why the other two studies were excluded.

3.7. OVARIAN CANCER In the only meta-analysis identified, total fruit and vegetable intake was not significantly associated with ovarian cancer risk41. Twelve cohort studies were included in this analysis. The pooled multivariate RR comparing the highest versus the lowest quartiles of intake showed little association for total fruit (RR= 1.11, 95% CI= 0.89-1.37) and total vegetable (RR= 0.88, 95% CI= 0.71-1.09) consumption with ovarian cancer cases that occurred within the first five years.25

When fruit and vegetable intake were represented as per 100g/day (which is approximately one serve per day), the RR values for total fruits, total vegetables and total fruits and vegetables had no association with ovarian cancer41. When grouped according to classes of fruit and vegetables, no statistically significant association was found. A marginally significant association with the consumption of green leafy vegetables for a 100g increment was found (pooled multivariate RR= 0.88, 95% CI=0.76-1.00)41.

3.8. BLADDER CANCER Fruit consumption provided significant protection against bladder cancer (RR= 0.81, 95% CI= 0.73-0.91), which was consistent across five case-control (RR= 0.82, 95% CI= 0.70-0.94) and three cohort studies (RR= 0.80, 95% CI= 0.65-0.99)40. Vegetable consumption showed a nonsignificant inverse association across all studies (RR= 0.91, 95% CI= 0.82-1.00), case-control (RR= 0.90, 95% CI= 0.78-1.03) and cohort studies (RR= 0.92, 95% CI= 0.75-1.14)1.

4. Factors Reduce Risk of Cancer Some of the major groupings of dietary factors associated with reduced risks of cancer are discussed below:4.1. Plant phenolics The term plant phenols encompasses a wide variety of naturally occurring compounds which are structurally related to the extent that they all contain one or more benzene rings, each with one or more hydroxyl group substitutions. Under this general rubric are included (1)the simple phenols eg gallic acid (tea), p-cresol (raspberry and blackberry, vanillin (vanilla); (2) the hydroxycinnamic acid derivatives, eg chlorogenic acid, a major component of coffee; and (3) the flavonoids including catechins from tea, anthocyanins which determine the colours of many flowers and fruits, and the ubiquitous flavones and flavonols.

In broad terms these substances are important for:• Their antioxidant properties, ie their ability to scavenge naturally occurring free radicals before they can damage macromolecules directly or indirectly involved in either cell proliferation (relevant to carcinogenesis) or lipid metabolism (relevant to cardiovascular disease).

• Blocking the formation of carcinogenic nitrosamines arising from the reaction of dietary nitrates/nitrites with secondary amines and amides in the stomach.

• Their capacity to act as electrophile traps. In much the same manner in which they can scavenge nucleophilic free radicals, many plant phenols can also absorb highly reactive electrophiles thereby preventing damage to cellular components • Inhibiting the generation of prostaglandins from arachidonic acid, and thereby retarding a ‘promotional’ phase of carcinogenesis.

4.2. Isothiocyanates Cabbages, watercress and other cruciferous vegetables owe their sharp taste in part to isothiocyanates arising from the action of the enzyme myrosinase on glucosinolate conjugates.

These isothiocyanates have been observed to inhibit chemically induced cancers in a variety of animal models. The mechanism involves the inhibition of the so-called Phase I enzymes whose normal function is to prepare foreign molecules for detoxification and excretion but which

sometimes generate highly reactive intermediates capable of far more damage than the original substrates.

4.3. Phytoestrogens There is still no agreement on how to define a phytoestrogen. In the context of this report any phytochemical which can substitute for, or block the action of, natural, endogenously produced steroid sex hormones may be regarded as a phytoestrogen. A recent definition of phytoestrogens as “plant derived compounds that can regulate gene expression that is mediated by an Estrogen Response Element, in a manner either comparable or apparently antagonistic to 17b-oestradiol, as a result of binding to the oestrogen receptor” 42, is too restrictive, and precludes a number of effects which are being increasingly linked in the literature with phytoestrogens. A confusing aspect of the usage of the term ‘phytoestrogenic’ is the fact that compounds to which this adjective is commonly applied may either mimic the effects of natural endogenous oestrogen, or they may block them and the same compound may be an agonist in one situation and an antagonist in others. A substance which is sufficiently similar to endogenous oestrogens to occupy an oestrogen binding site, but not sufficiently similar to reliably induce the event which normally ensues when natural oestrogen binds, would clearly be acting as an antagonist.

However, when natural oestrogen is either absent, or present only in very low concentrations (postmenopausally, for example), the limited ability of a phytoestrogen to induce the secondary event may still be significant, and warrant its reclassification as an agonist. Given the epidemiologic evidence of an increasing risk of breast cancer with increasing cumulative oestrogen exposure it is perhaps not surprising that historically, our interest in the relevance of phytoestrogens to breast cancer stemmed from their ability to act as antagonists of oestradiol.

Oestrogens should not just be linked with unfavourable long-term health outcomes, however.

Premenopausally women have much more favourable risk factor profiles for cardiovascular disease than men – and this may be due to direct and indirect effects of oestradiol. There is also considerable contemporary interest in the potential for phytoestrogens, acting as oestrogen agonists, to alleviate the morbidity (eg hot flushes) associated with the cessation of oestradiol production at the menopause, and to slow or halt the bone-loss which can eventually lead to osteoporosis. Major classes of phytoestrogenic substances in the diet include flavonoid compounds (flavones, isoflavones, flavanones), lignans, and coumestrol from legume sprouts.

While flavonoid compounds are widespread in foods of plant origin, the most significant compounds with oestrogenic activity in this class are genistein and daidzein, found in largest amounts in the soybean43, and formononetin from clovers. Lignans, which are characterised chemically by a 2,3-dibenzylbutane structure are also widespread in plant foods, although the flaxseed contains concentrations which are two orders of magnitude higher than any other known source44. Oestrogenic activity is critically dependent on the metabolism of these compounds by the microflora in the large bowel where daidzein may be either generated from formononetin or metabolised to equol (45,46), and the “mammalian lignans” enterolactone and enterodiol are generated from less oestrogenic precursors such as matairesinol and secoisolariciresinol 47.

Descriptive studies examining urinary excretion or plasma levels of phytoestrogens in groups with different experiences of hormone dependent cancers have been summarised by Adlercreutz and Mazur48. While phytoestrogen intakes are highest in the populations with the lowest cancer risk, this evidence remains circumstantial and experience with the correlational studies of per capita fat consumption and breast cancer should have taught us to regard this kind of evidence as encouraging, but open to many alternative interpretations. An especially exciting finding, from a

case-control study conducted in Western Australia by Ingram et al, of substantially reduced risks of breast cancer associated with high urinary excretion of phytoestrogens is currently in press 49.

Angiogenesis, the process by which new capillaries develop from pre-existing vessels, and on which ‘solid’ cancers are critically dependent for growth, has been shown to be sensitive to phytoestrogens especially genistein50. Sex hormone binding globulins (SHBG) are circulating proteins which are synthesised in the liver, and which exhibit a high affinity for both oestradiol and testosterone. Since the biological activity of steroid hormones bound to SHBG is very low, their bioavailability is determined to a significant extent by the circulating levels of SHBG 51.

Indeed SHBG concentrations increase in response to rising levels of either sex hormone and hence appear to be acting as regulators. The notion that phytoestrogens might stimulate synthesis of SHBG and thereby significantly reduce the bioactivity of endogenous oestrogens has been championed by Adlercreutz and coworkers52 but their human work was based on very heterogeneous groups of participants, and other studies, including unpublished work of our own, have failed to observe any dietary dependence of SHBG53. The observation that some phytoestrogens can inhibit growth in tumours with and without oestrogenm receptors underscores the potential importance of mechanisms unrelated to phytoestrogenic activity in the prevention of malignancy. Genistein, for example, appears to be able to inhibit the tyrosineprotein kinase intimately involved in determining the activity of proteins which regulate cell proliferation54; to inhibit topoisomerase II55; and to arrest the cell division cycle around the G2 to M phases56. Within the context of breast cancer, an important property of phytoestrogens may be their ability to inhibit the cytochrome P450 aromatase, which catalyses the final step in the synthesis of oestrogen and oestrone from testosterone and androstenedione respectively 57. For prostate cancer, the ability of phytoestrogens to inhibit the reductase which converts testosterone to its bioactive form in the prostate, dihydrotestosterone may be an important chemopreventive mechanism58. Certainly this reductase has been the target of chemotherapeutic drugs such as finasteride that are currently undergoing Phase III clinical trials in the US. In many ways the phytoestrogen and cancer story is a case-study in the contemporary status of the nutritional epidemiology of cancer. Weak ecologic data shows that countries whose populations consume the largest amounts of phytoestrogen-rich foods also have the lowest incidence of hormone dependent cancers. Laboratory studies in animal models of breast and prostate cancer together with observations of the effects of phytoestrogens on cell lines either in vitro or implanted, have been encouraging, and have assisted in the identification of a considerable number of mechanisms, although the relative importance of these mechanisms individually is largely undetermined, and the ability of many phytoestrogens to act as weak agonists of endogenous oestrogens is confusing. Some of these mechanisms directly involve oestrogen signal transduction pathways, but others clearly do not. The evidence from analytical human epidemiology is both sparse and of variable quality but generally supportive of the hypothesis that phytoestrogens may be chemopreventive agents. The information is still not sufficiently convincing, either with respect to their anti-carcinogenic properties or the ‘doses’ needed to achieve them, in order to make dietary recommendations of a public health nature.

4.4. Monoterpenes Monoterpenes such as limonene and perillylalcohol (found in the essential oils of citrus fruits, cherry, spearmint, dill and caraway and also used as flavouring agents) can inhibit the biochemical modifications required to incorporate proteins into cell membranes. Many proteins whose functionality depends on their location within membranes play important regulatory roles

and it has been demonstrated that monoterpenes can prevent the incorporation into membranes of the growth signaling ras proteins which become damaged and lose control early in the carcinogenesis process.

4.5. Organosulphur compounds Garlic and other Allium species (onions, leeks) contain organosulphides such as diallyl sulphide which can inhibit chemically induced cancers in laboratory animals. A definitive mechanism has not been established yet – but there is growing evidence that these compounds have differential effects on the Phase I enzymes (cytochrome P450 isozymes) involved in the activation/detoxification/excretion of ‘foreign’ dietary substances (59,60).

4.6. Dietary fibre The term dietary fibre is a rubric for dietary components entering the large bowel having survived the digestive processes in the stomach and small intestine. Non-starch polysaccharides make up the major component of dietary fibre. People who consume diets rich in fibre typically exhibit high stool weights and low (rapid) transit times through the gut, which is the basis for hypotheses that fibre simply reduces the extent to which epithelia in the gut are exposed to carcinogens such as the secondary bile acids produced by the bacterial action on the primary bile acids required for the dispersal of dietary fats. Possibly more important, however, is the capacity of the bacterial flora in the large bowel to ferment non-starch polysaccharides. The short-chain fatty acids (SCFA) generated by fermentation include butyric acid, which, in addition to being a preferred energy substrate for colonocytes, is also capable of inducing aberrant cells to ‘differentiate’ and resume a quiescent state most closely related, in functional terms, to the mature colonocyte. Fermentation may also release sequestered minerals (like calcium) and reduce bowel pH; with both effects acting in concert to precipitate harmful bile acids. In recent years it has been increasingly appreciated that significant amounts of dietary starch may also resist digestion in the upper alimentary tract, and contribute to the fermentable substrates in the large bowel. Unripe bananas and cold cooked potatoes are rich sources of ‘resistant’ starch.

Importantly, the SCFA mixture arising from the fermentation of resistant starch appears to be particularly rich in butyric acid.

4.7. Phytates (inositol phosphates) The outer layer (bran) component of most mature nuts and seeds is rich in phytates, which are well known for their ability to sequester metals and/or minerals. It is not known whether this property is relevant to the mechanism by which phytates appear to be able to inhibit carcinogenesis initiated by the polyaromatic hydrocarbon dimethyl benzanthracene in laboratory animals61.

4.8. Indoles Indole-3-carbinol (derived from the glucosinolate glucobrassicin in Brassica vegetables) has been intensely studied by Bradlow and associates for its apparent ability to divert oestrogen metabolism along a pathway which results in greater production of a less bioactive 2-hydroxy metabolite instead of the more problematic 16-alpha-hydroxy metabolite. This property might be protective against breast cancer.

4.9. Carotenoids

Responsible for the colouring, carotenoids are found in a variety of orange/yellow fruits and vegetables as well as some dark green leafy vegetables (spinach, cabbage and brussels sprouts).

The most well known example is beta-carotene. Like many carotenoids, beta-carotene is a powerful antioxidant (a striking example being the protection it offers the algae from which it is commercially harvested against harmful ultraviolet radiation from the sun). It is also a precursor of vitamin A (retinol) and retinoic acid which have been demonstrated to have the ability to induce differentiation of neoplastic and preneoplastic cells. Intervention trials in populations at risk of skin, cervix, colon and lung cancer have failed to demonstrate any health benefits, however. The alpha-carotenoid lycopene (to which tomatoes owe their red colour) is a very powerful antioxidant which has been associated with reduced risk of prostate cancer.

4.10. Folic acid The vitamin folic acid, from green leafy vegetables, oranges and orange juice, and the outer layers of many seeds and grains, plays an important metabolic role in the synthesis of DNA, and in situations requiring the transfer of a methyl group to a biological acceptor molecule.

Methylation of DNA itself appears to be an important mechanism for controlling the expression of many genes, including those involved in cell proliferation â&#x20AC;&#x201C; and abnormal methylation states of DNA (usually low methylation) have been associated with a number of neoplastic and preneoplastic conditions.

4.11. Vitamins C and E and selenium The water-soluble antioxidant vitamin C is present in many fruits and vegetables, especially citrus and peppers.. It can prevent the formation of carcinogeniic nitrosamines from nitrite and secondary amines in the stomach. It reduces the mutagenicity of gastric juices, and plays a role in immune function. It also regenerates the intracellular fat soluble antioxidant vitamin E (a collective term for a number of tocopherols and tocotrienols). Vitamin e in turn may also keep selenium, another antioxidant. in a reduced state.

5. Role of chemopreventive agents in cancer therapy Tumorigenesis or carcinogenesis is a multi-step process that is induced primarily by carcinogens leading to the development of cancer. Extensive research in the last few years has revealed that regular consumption of certain fruits and vegetables can reduce the risk of acquiring specific cancers. Phytochemicals derived from such fruits and vegetables, referred to as chemopreventive agents include genistein, resveratrol, diallyl sulfide, S-allyl cysteine, allicin, lycopene, capsaicin, curcumin, 6-gingerol, ellagic acid, ursolic acid, silymarin, anethol, catechins and eugenol.

Because these agents have been shown to suppress cancer cell proliferation, inhibit growth factor signaling pathways, induce apoptosis, inhibit NF-kB, AP-1 and JAKSTAT activation pathways, inhibit angiogenesis, suppress the expression of anti-apoptotic proteins, inhibit cyclooxygenase2,they may have untapped therapeutic value Tumorigenesis is a multistep process that begins with cellular transformation, progresses to hyperproliferation and culminates in the acquisition of invasive potential, angiogenic properties and establishment of metastatic lesions 62. This process can be activated by any one of the various environmental carcinogens (such as cigarette smoke, industrial emissions, gasoline vapors), inflammatory agents (such as tumor necrosis factor [TNF] and H2O2), tumor promoters (such as phorbol esters and okadaic acid). This multistep process of carcinogenesis consists of three phases: tumor initiation, promotion and progression phases.

Several population based studies indicate that people in South East Asian countries have a muchlower risk of acquiring colon, gastrointestinal, prostate, breast and other cancers when compared to their Western counterparts (see Table 4). It is very likely that constituents of their diet such as garlic, ginger, soy, curcumin, onion, tomatoes, cruciferous vegetables, chillies and

green tea play an important role in their ability to avoid these cancers. These dietary agents are believed to suppress the transformative, hyperproliferative and inflammatory processes that initiate carcinogenesis. These inhibitory influences may ultimately suppress the final steps of carcinogenesis, namely angiogenesis and metastasis.These dietary agents have been classified as chemopreventive agents since their ability to delay the onset of the carcinogenic process has been studied extensively. Because these chemopreventive agents are derived from natural sources, they are considered pharmacologically safe. The focus of the current review, although brief, is to evaluate the untapped therapeutic potential of these chemopreventive agents in the setting of several molecular targets that are currently under investigation (Fig. 1).

Table 4: Comparison of cancer incidence in USA and India USA Cancer Breast Prostate Colon/rectum Lung Head and neck Liver Pancreas Stomach Melanoma Testis Bladder Kidney Brain,nervous system Thyroid Endometrial cancers Ovary Multiple myeloma Leukemia Non-Hodgkin lymphoma

India

Cases

Death

Cases

Death

660 690 530 660 140 41 108 81 145 21 202 115 65

160 130 220 580 44 44 103 50 27 1 43 44 47

79 20 30 38 153 12 8 33 1.8 3 15 6 19

41 9 18 37 103 13 8 30 1 1 11 4 14

55 163

5 41

12 132

3 72

76 50 100 180

50 40 70 90

20 6 19 17

12 5 17 15

Showing cases per 1 million persons calculated on the basis of current consensus: endometrial cancers include Cervix uteri and Corpus uteri.GLOBOCAN 2000: Cancer Incidence, Mortality and Prevalence Worldwide, Version 1.0. IARC Cancer Base No. 5. Lyon, IARC Press, 2001 5.1. Chemopreventive agents as inhibitors of the NF-kB activation pathway NF-kB is a family of closely related protein dimmers that bind to a common sequence motif in the DNA called the kB site63. The molecular identification of its p50 subunit (v-REL) as a

member of the reticuloendotheliosis (REL) family of viruses provided the first evidence that NFkB is linked to cancer. Research over the past decade has revealed that NF-kB is an inducible transcription factor for genes involved in cell survival, cell adhesion, inflammation, differentiation and growth. In most resting cells, NF-kBis sequestered in the cytoplasm by binding to the inhibitory IkB proteins which blocks the nuclear localization sequences of NF-kB. NF-kB is activated by a varietyof stimuli such as carcinogens, inflammatory agents, tumor promoters including cigarette smoke, phorbol esters, okadaic acid, H2O2 and TNF. These stimuli promote dissociation of IkBa through phosphorylation, ubiquitination and its ultimate degradation in the proteasomes. This process unmasks the nuclear localization sequence of NFkB, facilitating its nuclear entry, binding to kBregulatory elements and activating transcription of target genes. Many of the target genes that are activated are critical to the establishment of early and late stages of aggressive cancers such as expression of cyclin D1, apoptosis suppressor proteins such as Bcl-2 and Bcl-XL and those required for metastasis and angiogenesis such as matrix metalloproteases (MMP) and vascular endothelial growth factor (VEGF).

Fig. 1.Molecular targets of chemopreventive agents in cancer The chemopreventive phytochemicals that are the focus of this review are shown schematically in Fig. 2. Most of these chemopreventives, such as curcumin, catechins, silymarin, caffeic acid phenethyl ester (CAPE), sanguinarine, anethole, emodin, piceatannol, resveratrol, capsaicin, ursolic acid, betulinic acid, flavopiridol and oleandrin are known to block the NF-kB activation process(64-77). Thus, although the maintenance of appropriate levels of NF-kB activity is crucial for normal cellular proliferation, constitutive NF-kB activation is involved in the enhanced growth properties as seen in several cancers 78. Dietary intake of these safe and nontoxic chemopreventives may thus be beneficial for patients whose tumors express persistently high

levels of activated NF-kB such as nonsmall cell lung carcinoma, thyroid, colon, breast, stomach, squamous head and neck carcinomas.

Fig. 2. Chemopreventive agents known to suppress tumorigenesis and their dietary sources.

5.2.Chemopreventive agents as inhibitors of the AP-1 activation pathway Activated protein-1 (AP-1) is another transcription factor that regulates the expression of several genes that are involved in cell differentiation and proliferation. Functional activation of the AP-1 transcription complex is implicated in tumor promotion as well as malignant transformation.

This complex consists of either homo or heterodimers of the members of the JUN and FOS family of proteins79. This AP-1 mediated transcription of several target genes can also be activated by a complex network of signaling pathways that involves external signals such as growth factors, mitogen-activated protein kinases (MAPK), extracellular-signal regulated protein kinases (ERK) and c-Jun N-terminal kinases (JNK). Some of the target genes that are activated

by AP-1 transcription complex mirror those activated by NF-kB and include cyclin D1, Bcl-2, Bcl-XL, VEGF, MMP and urokinase-type plasminogen activator (uPA). Expression of genes such as MMP and uPA especially promotes angiogenesis and invasive growth of cancer cells.

Most importantly, AP-1 can also promote the transition of tumor cells from an epithelial to

mesenchymal morphology which is one of the early steps in tumor metastasis. These oncogenic properties of AP-1 are primarily dictated by the dimer composition of the AP-1 family proteins and their post-transcriptional and translational modifications. Several phytochemicals such as curcumin, capsaicin, resveratrol and green tea catechins have been shown to suppress the AP-1 activation process(71,77). An AP-1 blockade has been shown to interfere with the transmission of proliferative signals induced by peptide growth factors as well as steroid growth factors such as estrogens79. These results suggest that chemopreventive agents specifically targeting AP-1 or its activating kinases could be promising agents for the treatment of several cancers 5.3. Chemopreventive agents as inhibitors of cell proliferation and initiators of apoptosis During the past 8 years, several reports were published which showed that activation of NF-kB promotes cell survival and proliferation and down regulation of NF-kB sensitizes the cells to apoptosis. How NF-kB promotes these proliferation and cell survival mechanisms has become increasingly clear. Expression of several genes including Bcl-2, Bcl-XL, cIAP, survivin, cyclin D1, TRAF1, TRAF2 have been reported to be up-regulated by NF-kB 63. The proteins coded by these genes function primarily by blocking the apoptosis pathway. Several studies have demonstrated that NF-kB activation promotes cell survival and proliferation mechanisms and that suppression of NF-kB leads to an abrogation of these mechanisms. Similarly, c-Jun is primarily a positive regulator of cell proliferation since c-Jun deficient fibroblasts have a marked proliferation defect in vitro and in vivo. c-Jun protein, once fully activated by JNK kinases induces the transcription of positive regulators of cell cycle progression such as cyclin D1 and represses the negative regulators such as the tumor suppressor p53 and the cyclin dependent kinase inhibitor p16 (INK4A). Moreover, activated and oncogenic AP-1 can antagonize apoptosis in several tumors. Several phytochemicals that are known to inhibit the NF-kB or the AP-1 activation process, most notably curcumin, green tea, 6-gingerol and resveratrol can cause a significant suppression of cell proliferation and sensitizes cells for apoptosis (77,80). Most notably, phytochemicals such as curcumin is also known to down regulate the expression of apoptosis suppressor proteins, such as Bcl-2 and Bcl-XL in several cancer cell lines.

5.4. Chemopreventive agents as inhibitors of COX-2 Numerous preclinical studies point to the importance of regulation of cyclooxygenase-2 (COX2) expression in the prevention and, most importantly, in the treatment of several malignancies.

This enzyme is overexpressed in practically every premalignant and malignant conditions involving the colon, liver, pancreas, breast, lung, bladder, skin, stomach, head and neck and esophagus81. Overexpression of this enzyme is a consequence of While several inhibitors of angiogenesis are in clinical trials, it is very important to note here that several safe chemopreventive phytochemicals are already known to target these pathways. These include curcumin, resveratrol and catechins(83-86). First, curcumin can interfere with the activity of MMP2 and 9, reducing the degradation of ECM which forms the basis of angiogenic switch 86. Thus, it can also interfere with the release of angiogenic and other growth factors that are stored in the ECM. By inhibiting several growth factor receptors such as EGFR and VEGFR, it can also significantly impact upon the mechanisms of angiogenic switch and vessel cooption that are necessary for the sprouting growth of new blood vessels in the tumor 87. By interfering with the non-receptor tyrosine kinases such as Src and FAK, agents such as curcumin, genistein and green tea components can interfere with the downstream PI-3 Kinase signaling responsible for the induction of the angiogenic target genes such as COX-2, VEGF, IL-8 and the MMPs (88,89). By

inhibiting another member of the MMP family, namely MMP-2, curcumin may have a negatively impact upon the MMP-2 mediated degradation of the lamin-5 isoform which is implicated in the formation of loose and primitive looking meshwork formed by aggressive cancers such as melanoma and prostate cancers. This plasticity of the cancer cells mimicking the endothelial cells is mainly brought out by the capacity of the cancer cells to express endothelium associated genes such as VE-Cadherin, Src, FAK and PI-3 Kinases, all of which are good targets for these chemopreventive agents. Recent findings showed that curcumin can also inhibit another member of the MMP family, aminopeptidase N (APN) which is implicated in the angiogenic switch process90. Most notably, curcumin and to a lesser extent genistein can also interfere with the expression of VEGF by processes other than hypoxia, such as transforming growth factor (TGF)-b release, COX-2 overexpression, hydrogen peroxide release from bone cells, constitutive and aberrant EGFR and Src signaling and most importantly, by aberrant NF-kB signaling in established cancers. Chemopreventive phytochemicals such as curcumin, genistein . and catechins may work through down regulation of EGFR and HER-2/neu activity, resulting in a reduced expression of COX-2. Resveratrol has also been shown to down-regulate COX-2 expression82. Our laboratory recently showed the suppression of 7,12 dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in rats by resveratrol and this correlated with inhibition of NF-kB, COX-2 andMMP-983.

5.5. Chemopreventive agents as chemosensitizers and radiosensitizers Recent reports point out that these safe and nontoxic cancer chempreventive phytochemicals can function as sensitizers, augmenting the effectiveness of cancer chemotherapy and radiotherapy.

This sensitization is thought to occur at various levels. First, by directly competing with the ATP binding site of the MDR or MRP drug efflux pumps, curcumin can inhibit the pump and increase the intracellular concentrations of the chemotherapeutic drugs such as vinblastine or vincristine.

Second, by functioning as efflux substrates for pumps such as MDR or MRP, chemopreventives such as genistein and green tea components (EGCG) can saturate and hence titrate out the pumps, increasing the amount of the chemotherapeutic drug within the cell. This type of competition with the MDR or MRP substrates in effect sensitizes the cancer cell for a better cell kill by chemotherapeutic agents. Third, curcumin can interfere with the functioning of pumps such as the MRP which require a steady supply of reduced glutathione (GSH), since it is known to be an inhibitor of GSH synthetase. This type of inhibition might enhance the sensitivity of these cancer cells overexpressing MRP to chemotherapeutic agents such as vincristine, arsenicals and platinum derivatives by impairing their efflux91. One other clinical strategy that is currently being pursued is to target c-Jun expression to reduce intracellular GSH levels. Stable increases in the c-Jun expression are associated with the AP-1 mediated increase in the GSH synthetase levels92. Since curcumin targets the same elements, it would be a strong inhibitor, reducing the intracellular GSH at the transcriptional level 93. Expression of glutathione S-transferase Pi (GSTPi) is also correlated with the resistance of cancer cells to chemotherapeutic agents. In a recent study, curcumin efficiently inhibited the TNF- and phorbol ester induced AP-1 and NF-kB transcription factor binding to the sites located on the GST-Pi gene promoter in K562 leukemia cells93. This process efficiently reduced the GST-Pi levels causing an interference with drug resistance and ultimate apoptosis. Chemopreventive agents such as curcumin also can sensitize cancer cells to other traditional chemotherapeutic agents such as etoposide and camptothecin in another capacity. Topo II poisons stabilize the cleavable complexes, an intermediate product of the TopoII catalyzed reaction. Accumulation of these cleavable complexes is thought to lead to

cell death. Conversely, a decrease in the number of cleavable complexes could confer drug resistance. Proteasome inhibition has recently been found to decrease this inducible resistance by inhibiting the TopoII depletion by hypoxia or glucose starvation. Moreover, the observation that proteasomal inhibitors such as lactacystin significantly enhanced the anti-tumor activity of etoposide in xenografts in vivo strongly suggested that the TopoII depletion occurred through a proteasomal mechanism94. With this rationale, several proteasomal inhibitors such as PS-341 are currently showing promise in phase II clinical trials. It is worth noting that curcumin has recently been shown to inhibit cellular proteasome activity in a concentration dependent manner with a parallel increase in the accumulation of ubiquitinated proteins. This agent may be able to inhibit the proteasomes by inhibiting the ubiquitin isopeptidase activity, as shown in recent studies95. This mode of proteasome mediated sensitization of cancer cells to drugs such as etoposide and camptothecin by curcumin will be therapeutically beneficial for patients with several different types of cancer. This expectation is based on inhibition of degradation of TopoII by the proteasomes, resulting in more DNA cleavable complexes. Most of the chemotherapeutic agents and gamma irradiation commonly administered to cancer patients have been found to activate NF-kB. The activation of NF-kB can lead to resistance to apoptosis. Activation of these survival processes occurs in parallel to the induction of the apoptosis by the activation of several caspases by the same agents. In this respect, co-administration of chemopreventive agents such as curcumin will activate the apoptotic pathways in these cancer cells while at the same time down regulating the cell survival pathways, mediated by PI-3 Kinase and AKT proteins. This is generally accomplished without activating the anti-apoptotic pathways which in effect alters the Bcl-2:Bax ratio, contributing to the sensitization effect. This explains, in a nutshell, the sensitizing or potentiating effect of these chemopreventives to achieve a better target cell kill than what can be achieved by chemotherapy or radiotherapy alone. Other mechanisms by which curcumin and other chemopreventive agents may enhance the cytotoxicity of chemo and radiotherapies include the induction of p21Cip1/waf1. Recently, resveratrol was found to mediate chemosensitization through downregulation of survivin, a cell survival gene96. Similarly, curcumin was found to induce radiosensitization of prostate cancer cells through the suppression of NF-kB activation 97. In general, these chemopreventive agents bring out significant sensitization effects by overcoming therapyinduced prosurvival gene expression in several cancers.

6. Role of phytochemicals in the prevention of cancer Evidence suggests that dietary antioxidants can reduce cancer risk. Block et al 98 established this in an epidemiologic review of 200 studies that examined the relationship between fruit and vegetable intake and cancers of the lung, colon, breast, cervix, esophagus, oral cavity, stomach, bladder, pancreas, and ovary. In 128 of 156 dietary studies, the consumption of fruit and vegetables was found to have a significant protective effect. The risk of cancer for most cancer sites was twice as high in persons whose intake of fruit and vegetables was low compared with those with high intake. Significant protection was found in 24 of 25 studies for lung cancer. Fruit was significantly protective in cancers of the esophagus, oral cavity, and larynx. In 26 of 30 studies, there was a protective effect of fruit and vegetable intake with respect to cancers of the pancreas and stomach and in 23 of 38 studies for colorectal and bladder cancers. A prospective study involving 9959 men and women (age 15â&#x20AC;&#x201C;99 y) in Finland showed an inverse association between the intake of flavonoids and the incidence of all sites of cancer combined 99. After a 24-y follow-up, the risk of lung cancer was reduced to 50% in the highest quartile of flavonol intake.

Consumption of quercetin in onions and apples was found to be inversely associated with lung cancer risk in Hawaii100. The effect of onions was particularly strong against squamous cell carcinoma. Boyle et al 101 showed that increased plasma levels of quercetin following a meal of onions were accompanied by increased resistance to strand breakage by lymphocyte DNA and decreased levels of some oxidative metabolites in the urine. Carcinogenesis is a multistep process, and oxidative damage is linked to formation of tumors through several mechanisms (102,103) . Oxidative stresses induced by free radicals cause DNA damage, which, when left unrepaired, can lead to base mutation, single and double strand breaks, DNA cross-linking, and chromosomal breakage and rearrangement. The anticancer properties of a wide variety of fruits and vegetables that are available to consumers in order to elaborate what could be called the optimum diet to prevent cancer. This strategy is based on the screening of fruits and vegetable extracts for their ability to act on a variety of cellular and molecular events that are crucial for tumor progression (Fig. 3). At the molecular level, these include determination of the antioxidant activity of the extracts using the ORACFL assay and monitoring of the effect of the extracts on xenobioticmetabolizing systems, with particular emphasis on cytochrome P-450 and induction of the Phase II detoxification enzymes. At the cellular level, the aim of the project is to determine the cytotoxic and antiangiogenic properties of the extracts in order to identify foods with the highest anticancer potential against several tumor cell lines. As shown in Fig. 2, there is considerable variation in the cytotoxic properties of a given food extract depending on the origin of the cancer cell line This potentially cancer-inducing oxidative damage might be prevented or limited by dietary antioxidants found in fruit and vegetables. Studies to date have demonstrated that phytochemicals in common fruit and vegetables can have complementary and overlapping mechanisms of action, including modulation of detoxification enzymes, scavenging of oxidative agents, stimulation of the immune system, regulation of gene expression in cell proliferation and apoptosis, hormone metabolism, and antibacterial and antiviral effects (104, 105).

Fresh fruits and vegetables,from the market

Juice extractor

Clarification by centrifugation

Sterilization(filtration)

Antioxydant Index

Cancer cell Proliferation

Cancer cell migration

Cancer cell Apoptosis

Growth factor Inhibiton

Fig. 3. Experimental approach to monitor the anticancer properties of fruits and vegetables.

7. Role of gene regulation in the anticancer activity of carotenoids Fruits and vegetables play an important role in the prevention of degenerative diseases including cancer106. Among plant constituents, carotenoids have been implicated as cancer-preventive agents107.Most earlier studies were related to β-carotene, but in recent years, additional carotenoids found in the human diet have also begun to receive attention. Our research focuses primarily on tomato carotenoids, particularly lycopene, thus, in this paper, we will relate mainly to those studies that deal with this carotenoid.

7.1. Human intervention studies with carotenoids The epidemiological and laboratory studies reviewed above suggest a cancer-preventive activity for lycopene. Similar earlier studies on β-carotene led to collaborative large intervention studies with synthetic β-carotene. However, the use of a single plant-derived compound in human prevention studies has not been successful, revealing either no beneficial effect 108or even a negative effect (109,110). These results led to the hypothesis that a single micronutrient cannot replace the power of the concerted action of multiple compounds derived from a diet rich in fruits and vegetables. So, it is not surprising that more recent studies used a natural mixture of phytonutrients. Two small-scale, preliminary intervention studies in prostate cancer patients were carried out with natural tomato preparations. In one, Bowen et al. 111showed that after dietary intervention, serum and prostate lycopene concentrations were increased, whereas oxidative DNA damage both in leukocytes and in prostate tissue was significantly lower. Furthermore, serum levels of prostate-specific antigen (PSA) decreased after the intervention (p < .001). In the other study, Kucuk et al.112 reported that supplementation with tomato extract in men with prostate cancer positively modulates the grade and volume of prostate intraepithelial neoplasia and tumors and the levels of both serum PSA and biomarkers of cell growth and differentiation.

7.2. MECHANISM OF CANCER CELL GROWTH INHIBITION AT THE PROTEIN EXPRESSION LEVEL The mechanisms underlying the anticancer activity of lycopene and other carotenoids may involve changes in pathways leading to cell growth or cell death. These include hormone and growth factor signaling, regulatory mechanisms of cell cycle progression, cell differentiation, and apoptosis. Examples of carotenoid effects on some of these pathways will be described below, with emphasis being placed on the changes in protein expression associated with these effects.

7.2.1. Gap junctional communication One of the earliest discoveries related to carotenoids and modulation of protein level was made

by Bertram and colleagues who found that carotenoids increase gap junctional communication between cells and induce the synthesis of connexin43, a component of the gap junction structure(113,114). This effect was achieved independently of provitamin-A or the antioxidant

properties of the carotenoids. Loss of gap junctional communication may be important for malignant transformation, and its restoration may reverse the malignant process.

7.2.2. Growth factor signaling Growth factors, either in the blood or as part of autocrine or paracrine loops, are important for cancer cell growth. Recently, IGF-I has been implicated as a major cancer risk factor. It was reported that high blood levels of IGF-I existing years before malignancy detection, predict an increase in risk for breast, prostate, colorectal, and lung cancer (115–118). Accordingly, two possible strategies might be used to reduce IGF-related cancer risk: reduction in IGF-I blood levels, and interference with IGF-I activity in the cancer cell. Preliminary results of our studies on the former strategy suggest that tomato phytonutrients lower IGF-I blood levels. In addition, we have recently shown that lycopene inhibits the mitogenic action of IGF-I in human cancer cells.

In mammary cancer cells, lycopene treatment markedly reduced IGF-I stimulation of both tyrosine phosphorylation of insulin receptor substrate-1 and DNA binding capacity of the AP-1 transcription factor119. These effects were not associated with changes in the number or affinity of IGF-I receptors, but rather with an increase in membrane-associated IGFbinding proteins (IGFBPs). This finding can explain the suppression of IGF-I-signaling by lycopene, as based on our previous studies120showing that membrane-associated IGFBP-3 inhibits IGF-I receptor signaling in an IGF-dependent manner.

7.2.3. Cell cycle progression Growth factors have a major effect in promoting cell cycle progression, primarily during G1 phase. We have shown that lycopene treatment of MCF-7 mammary cancer cells slowed down IGF-I-stimulated cell cycle progression119, which was not accompanied by either apoptotic or necrotic cell death. Lycopene-induced delay in progression through G1 and S phases was also observed in other cancer cell lines (leukemic, endometrial, lung, and prostate) tested in laboratory (and an unpublished work). A similar effect of another carotenoid, α-carotene, was demonstrated in GOTO human neuroblastoma cells. Likewise, β-carotene induced a cell-cycle delay in G1 phase in normal human fibroblasts. Cell cycle transition through a late G1 checkpoint is governed by a mechanism known as the “pRb pathway” 121. The central element in this pathway, retinoblastoma protein (pRb), is a tumor suppressor that prevents premature G1/S transition via physical interaction with transcription factors of the E2F family. The activity of pRb is regulated by an assembly of cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors.

Phosphorylation of pRb by Cdks results in the release of E2F, which leads to the synthesis of various cell growth-related proteins. Cdk activity is modulated in both a positive and a negative manner by cyclins and Cdk inhibitors, respectively. It is well documented that growth factors affect the cell cycle apparatus primarily during G1 phase, and that the D-type cyclins are the main elements acting as growth factor sensors122. Moreover, cyclin D1 is known as an oncogene and is found to be overexpressed in many breast cancer cell lines as well as in primary tumors 123.

In a recent study124, we have demonstrated that cancer cells arrested by serum deprivation in the presence of lycopene are incapable of returning to the cell cycle after serum re-addition. This inhibition correlated with reduction in cyclin D1 protein levels that resulted in inhibition of both cdk4 and cdk2 kinase activity and in hypophosphorylation of pRb. Abundance of the Cdk inhibitor p21Cip1/Waf1 was reduced while p27Kip1 levels were unchanged. Inhibition of cdk4 was directly related to a lower amount of cyclin D1-cdk4 complexes while inhibition of cdk2 action was related to a shift of the inhibitor p27Kip1 molecules from cdk4 complexes to cyclin E-cdk2 complexes.

7.2.4. Differentiation-related proteins

Induction of differentiation to mature cells with distinct functions similar to nonmalignant cells has been proposed as an alternative to cytotoxic chemotherapy and may be useful for chronic chemoprevention. Differentiation therapy has been quite effective in treating acute promyelocytic leukemia and is currently being investigated for treatment of solid tumors.

Differentiation inducers that are presently under laboratory and clinical investigation include vitamin D and its analogs, retinoids, polyamine inhibitors, and others. We have shown that lycopene alone induces differentiation of HL-60 promyelocytic leukemia cells 125. A similar effect was described also for other carotenoids such as β-carotene, lutein, and the saffron carotenoids (125,126,127) . The differentiation effect of lycopene was associated with elevated expression of several differentiation-related proteins, such as cell surface antigen (CD14), oxygen burst oxidase (as measured by phorbol ester-stimulated reduction of nitroblue tetrazolium) 125, and chemotactic peptide receptors (unpublished work). The mechanism of the differentiating activity of lycopene and its ability to synergize with 1,25(OH)2D3 in this effect 125 is largely unclear.

However, in a similar study, we have recently shown that the differentiation-enhancing effect of another phytonutrient, carnosic acid from rosemary, is associated with induction of multiple differentiation-related proteins, such as Cdk inhibitor, p21Cip1, early growth response gene-1 (EGR-1), and Cdk5 and its activator protein, p35Nck5a (128,129). Most importantly, carnosic acid and its combinations with 1,25(OH)2D3 and retinoic acid transcriptionally activated the expression of nuclear hormone receptors, such as vitamin D3 receptor (VDR), retinoic acid receptor (RARα), and retinoid X receptor (RXRα) 128. This may represent a molecular basis for synergy between phytonutrients and differentiation inducers. The possibility that lycopene as well as other carotenoids and/or their derivatives may affect nuclear signaling pathways is an attractive suggestion, but requires experimental proof.

7.3. MODULATION OF TRANSCRIPTION BY CAROTENOIDS Our main question is, by what mechanism do carotenoids affect so many and diverse cellular pathways as described above? The changes in the levels of many proteins suggest that the initial effect involves modulation of transcription. As illustrated below, such modulation can occur at the level of ligand-activated nuclear receptors or other transcription factors.

7.3.1. Retinoic acid receptor (RAR) Initially, the structure similarity between lycopene and β-carotene suggested that lycopene or some of its oxidized derivatives may activate retinoid-like receptors. To test this assumption, Stahl and colleagues130 synthesized a hypothetical oxidation product of lycopene, acyclo-retinoic acid, the open chain analog of retinoic acid. However, we found that although acyclo-retinoic acid was able to trans-activate RARα, the growth-inhibitory effect of lycopene was not mediated directly via this classical retinoid receptor131. In addition, Stahl 130 concluded that acyclo-retinoic acid does not have a role in gap junctional communication. Muto and colleagues 132synthesized acyclo-retinoic acid and tested its biological activity as part of a series of acyclic retinoids, but did not observe transactivation by this compound in the RAR or RXR reporter gene systems 133.

However, they did find that other acyclic retinoids, lacking one or two double bonds (geranyl geranoic acid and 4,5-didehydro geranyl geranoic acid), caused transactivation of the reporter gene comparable to that achieved by retinoic acid. It is interesting to note that these acyclic retinoids may be potential derivatives of phytoene and phytofluene (two carotenoids that are present in tomatoes). These studies suggest that carotenoids, their oxidized derivatives, and other phytonutrients interact with a network of transcription factors that are activated by different ligands at low affinity and specificity (Fig. 4). The activation of several transcription factor systems by different compounds may lead to the synergistic inhibition of cell growth. In addition

to the retinoid receptors, other candidate transcription systems that may participate in this network are the peroxisome proliferator-activated receptors (PPARs) (134–136), the antioxidant response element (ARE) (137,138), AP-1139, the xenobiotic receptors 140 and yet unidentified orphan receptors.

Fig.4. Hypothetical network of transcription factors that may be activated by various carotenoids or their derivatives. RA—retinoic acid.

7.3.2. Peroxisome proliferator-activated receptor (PPAR) These nuclear receptors have a key role in the differentiation of adipocytes, but recently their role in cancer cell growth inhibition and differentiation has also been demonstrated. PPARγ is expressed at significant levels in human primary and metastatic breast adenocarcinomas, colon cancer cells, and liposarcomas (141–143). Colon cancer in humans was found to be associated with loss-of-function mutations in PPARγ143. Ligand activation of PPARγ in cultured breast cancer cells 141and in liposarcomas in vivo 142causes changes in gene expression associated with a more differentiated, less malignant state. Human prostate cancer cells expressed PPARγ at prominent levels while normal prostate tissues had a very low expression (135,136). Activation of this receptor with specific ligands, such as troglitazone, exerts an inhibitory effect on the growth of prostate cancer cells and favorable changes in PSA dynamics in prostate cancer patients136. The presence of PPARγ receptors in various cancer cells, their activation by fatty acids, prostaglandins, and related hydrophobic agents in the μM range makesthis liganded transcription factor an interesting target for carotenoid derivatives. To support this hypothesis, we recently compared the relative efficacy of several carotenoids found in tomatoes in transactivation of the PPAR response element. Preliminary results suggest that lycopene, phytoene, phytofluene, and β-carotene transactivate PPARγ in mammary cancer cells.

7.3.3. Xenobiotic and other orphan nuclear receptors Orphan receptors include gene products that are structurally related to nuclear hormone receptors, but lack known physiological ligands. Thus, like all the recognized nuclear receptors,

they should have multiple regulatory roles, some of which may be related to diet-derived compounds. Mammals encounter numerous foreign chemicals (xenobiotics), such as ingested food, environmental pollutants, carcinogens, and drugs, which are metabolized and eliminated mainly by cytochrome P450 (CYP) enzymes 140. CYP enzymes are induced by various xenobiotic substrates, including phytonutrients, through response element of several orphan nuclear receptors, such as steroid and xenobiotic receptor/pregnane X receptor (SXR/PXR) and constitutive androstane receptor (CAR) 140. St. John’s wort, the herbal remedy used widely for the treatment of depression, illustrates the possible role of phytonutrients in this system. It has recently been found that its active compound, hyperforin, is a potent ligand for PXR, which promotes expression of CYP 3A4144.

7.3.4. Antioxidant response element (ARE) Induction of phase 2 enzymes that neutralize reactive electrophiles and act as indirect antioxidants appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through ARE found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs), NAD(P)H:quinone oxidoreductase (NQO1)145, and thioredoxin146. Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80 % in Nrf2-deficient mice compared with wild-type mice 145.

Under basal conditions, Nrf1 and Nrf2 are located in the cytoplasm and are bound to the inhibitory protein, Keap1. Upon challenge with inducing agents, they are released from Keap1 and translocate to the nucleus (147,148). Within the nucleus, these basic region leucine zipper transcription factors are recruited to ARE as heterodimers with either small Maf proteins, FosB, c-Jun, or JunD. Several studies have shown that dietary antioxidants, such as terpenoids 149; phenolic flavonoids (e.g., green tea polyphenols and epigallocatechin-3-gallate 150; and isothiocyanates, may work as anticancer agents by activating this transcription system. To illustrate, an isothiocyanate compound from Japanese horseradish extract induced both nuclear localization of Nrf2 (which binds to ARE) and expression of phase 2 enzyme genes. These effects were completely abrogated inNrf2-deficient mice151.

7.3.5 Activator protein-1 (AP-1) As mentioned above, the activation of the AP-1 transcriptional complex is a middle-term event (1–2 h) in the mitogenic signaling pathway of IGF-I and other growth factors 152. The AP-1 complex consists of protein from the Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) families which associate as homo- (Jun/Jun) or heterodimers (Jun/Fos). These proteins are often induced by mitogenic stimuli and tumor-promoting agents and bind to an activator protein-1 (AP-1) site, known also as the TPA response element (TRE), on the promoter of many genes that are related to cell proliferation, such as cyclin-D. Interestingly, as just discussed, some of these proteins participate in the ARE transcription complex as well. It has recently been shown that this transcriptional system is modulated by carotenoids. The AP-1 family members can form different complexes that bind to AP-1 sites.These proteins differ considerably in their ability to activate transcription of target genes. A complex that is composed of weak transactivators will induce less-transcriptional activity than one containing more potent transactivators. Preliminary results from our studies suggest that lycopene and retinoic acid reduce growth factor-induced stimulation of AP-1 transcriptional activity by altering the composition of AP-1 complexes that bind to DNA. Wang et al.139, reported that the expression of c-Jun and c-Fos genes in lungs of ferrets supplemented with high-dose β-carotene and exposed to

tobacco smoke was elevated by three- to four-fold. In addition, they observed a strong proliferative response in lung tissue and squamous metaplasia as well as an increase in the level of a cell proliferation marker (proliferatingcell nuclear antigen, PCNA). In β-carotenesupplemented animals, this increase was enhanced further by tobacco smoke. Their report offers a possible explanation for the enhancing effect of β-carotene supplementation on lung carcinogenesis in smokers as has been reported in large intervention studies (109,110).The beneficial effect of a diet rich in fruits and vegetables appears to depend on the concerted action of multiple micronutrients, which act either additively or synergistically. In this model, carotenoids, their derivatives, and other phytonutrients may activate the same transcription factor, producing an additive effect.

8. Role of whole food The hypothesis that dietary antioxidants lower the risk of chronic disease has been developed from epidemiologic studies that consistently show that consumption of whole foods, such as fruit and vegetables, is strongly associated with reduced risk ofchronic diseases. Therefore, it is reasonable for scientists to identify the bioactive compounds responsible and hope to find the “magic bullet” to prevent those chronic diseases. The key question here is whether a purified phytochemical has the same health benefit as the phytochemical present in whole food or a mixture of foods. It is now believed that dietary supplements do not have the same health benefits as a diet rich in fruit and vegetables because, taken alone, the individual antioxidants studied in clinical trials do not appear to have consistent preventive effects. The isolated pure compound either loses its bioactivity or may not behave the same way as the compound in whole foods. For example, numerous investigations have shown that the risk of cancer is inversely related to the consumption of green and yellow vegetables and fruit. Because carotene is present in abundance in these vegetables and fruit, it has been extensively investigated as a possible cancer-preventive agent. However, the role of carotenoids as anticancer supplements has recently been questioned as a result of several clinical studies (153–156). In one study, the incidence of nonmelanoma skin cancer was unchanged in patients receiving a carotene supplement 153. In other studies, smokers gained no benefit from supplemental carotene 154with respect to lung cancer incidence and possibly even suffered a deleterious effect, with a significant increase in lung cancer and total mortality (155,156). Vitamin C supplementation also has been shown not to lower the incidence of cancer and heart disease (157,158).Recently reported that phytochemical extracts from fruit have strong antioxidant and antiproliferative effects and proposed that the combination of phytochemicals in fruit and vegetables is critical to powerful antioxidant and anticancer activity (159–161).For example, the total antioxidant activity of phytochemicals in 1 g of apples with skin is equivalent to 83.3 mol vitamin C equivalents that is, the antioxidant value of 100 g apples is equivalent to 1500 mg of vitamin C. This is much higher than the total antioxidant activity of 0.057 mg of vitamin C (the amount of vitamin C in 1 g of apples with skin). In other words, vitamin C in apples contributed only < 0.4% of total antioxidant activity 159. Thus, most of the antioxidant activity comes from phytochemicals, not vitamin C. The natural combination of phytochemicals in fruit and vegetables is responsible for their potent antioxidant activity. Apple extracts also contain bioactive compounds that inhibit tumor cell growth in vitro. Phytochemicals in 50 mg apple with skin per milliliter (on a wet basis) inhibit tumor cell proliferation by 42%.

Phytochemicals in 50 mg apple without skin per milliliter inhibit tumor cell proliferation by 23%. The apple extracts with skin significantly reduced the tumor cell proliferation when compared with the apple extracts without skin. Also studied the total antioxidant activity and

synergy relationships between different fruit combinations, with results showing that plums had the highest antioxidant activity and that combinations of fruit resulted in greater antioxidant activity that was additive and synergistic. We proposed that the additive and synergistic effects of phytochemicals in fruit and vegetables are responsible for their potent antioxidant and anticancer activities, and that the benefit of a diet rich in fruit and vegetables is attributed to the complex mixture of phytochemicals present in whole foods (159â&#x20AC;&#x201C;161). This partially explains why no single antioxidant can replace the combination of natural phytochemicals in fruit and vegetables in achieving the health benefits. There are 8000 phytochemicals present in whole foods. These compounds differ in molecular size, polarity, and solubility, and these differences may affect the bioavailability and distribution of each phytochemical in different macromolecules, subcellular organelles, cells, organs, and tissues. Pills or tablets simply cannot mimic this balanced natural combination of phytochemicals present in fruit and vegetables. Our work suggests that to improve their nutrition and health, consumers should be getting antioxidants from a diverse diet and not from expensive nutritional supplements, which do not contain the balanced combination of phytochemicals found in fruit and vegetables and other whole foods. More important, obtaining antioxidants from dietary intake by consuming a wide variety of foods is unlikely to result in consumption of toxic quantities because foods originating from plants contain many diverse types of phytochemicals in varying quantities. Furthermore, the health benefits of the consumption of fruit and vegetables extend beyond lowering the risk of developing cancers and cardiovascular diseases; this consumption also has preventive effects on other chronic diseases such as cataracts, age-related macular degeneration, central neurodegenerative diseases, and diabetes.

CONCLUSIONS Fruit and vegetables are high in nutrients that are potentially protective against cancer. Increasing the consumption of fruit and vegetables, and whole grains is a practical strategy for consumers to optimize their health and to reduce the risk of chronic diseases. Presents evidence that chemopreventive agents can be used not just to prevent cancer but also to treat cancer. Because of their pharmacological safety, most chemopreventive agents can be used in combination with chemotherapeutic agents to enhance the effect at lower doses and thus minimize chemotherapyinduced toxicity. The beneficial effect of a diet rich in fruits and vegetables appears to depend on the concerted action of multiple micronutrients, which act either additively or synergistically.

The evidence supporting the role of fruit and vegetables in reducing the risk of some cancers, overall the evidence is suggestive of a protective effect.

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