Inhibition of DBTRG proliferation and migration in vitro by THC and cannabidiol: a cellular basis for the effects of Cannabis sativa L. on glioblastoma.
Fiore Giovina1, Montefrancesco Giuseppe2, Fiaschi Anna Ida1, Giorgi Giorgio1, Cerretani Daniela1 1. Dipartimento di Farmacologia “G. Segre”, Università Degli Studi di Siena 2. Ce. S. Di. P. – Centro Studi nelle Dipendenze Patologiche, Università Degli Studi di Siena
Introduction One of the most active areas of research in the cannabinoid field is the study of the potential application of cannabinoids as therapeutic agents; in this contest cannabinoids are investigated as potential antitumoral drugs (inhibitors of tumor angiogenesis and metastasis) and cannabinoid treatment, via induction of apoptosis or cell cycle arrest, affects directly the viability of a wide spectrum of tumor cells in culture. Both CB1 and CB2 receptors have been shown to mediate the antiproliferative actions of THC and related cannabinoids in tumor cells and in gliomas, while the CB2 receptor plays a major role in pro-apoptotic effect of cannabinoids. The aim of this study was to investigate the effect in vitro of THC and cannabidiol on human glioma cell viability, proliferation and migration and to try to elucidate the correlation of these effects with a mechanism of oxidative stress.
Methods Cell Culture DBTRG-05MG cells were obtained from the American Type Culture Collection and were used within seven passages. Cells were maintained routinely in RPMI 1640 medium supplemented with 10% FBS, 2 mmol/liter l-glutamine, 100 IU/ml penicillin, and 100 g/ml streptomycin at 37 °C under 5% CO2-95% air. MTT assay The proliferation rate were determined by MTT assay Cells were plated at a density of 5000 cells/well in 96-well plastic plates, At the end of incubation with THC and cannabidiol, MTT stock solution (5 mg/ml) was added to each well being assayed to equal one tenth the original culture volume and incubated for 4 h. At the end of the incubation period, converted dye was solubilized with acidic isopropanol (0.04–0.1 n HCl in absolute isopropanol). Absorbance of converted dye was measured at a wavelength of 570 nm with background subtraction at 630 nm. Scratch assay The migration of cell population were measured by scratch assay. Cells were plated at a density of 35000 cells/well in 96-well plastic plates, After 24 h were created a “scratch” in the cell monolayer and then stimulated with THC and cannabidiol. The images were been captured at the beginning and after 24 h. MDA assay MDA levels were measured in cell lysates by HPLC method as described by Shara (Shara et al, 1992). The cells were homogenized in mixture (1:1) of 0.04 mol/liter Tris-HCl buffer (pH 7.4) and a solution of acetonitrile, containing 0.1% of butyl hydroxytoluene (4°C), to prevent the artificial oxidation of polyunsaturated free fatty acid during the assay, and centrifuged at 3000 g for 15 min at 4°C. The supernatants were used for MDA-analysis after pre-column derivatization with 2,4dinitrophenylhydrazine. The MDA-hydrazone was quantified by isocratic reversed-phase HPLC and UV detection at 307 nm.
Results
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DBTRG-05MG cells (human glioblastoma multiforme cell line) are sensitive to THC and cannabidiol treatment. Both cannabinoids reduced significantly the cell proliferation, quantified with MTT test, in a concentration and time dependent manner (Figures 1, 2). Indeed the results obtained indicate that cannabidiol is the most potent inhibitor of cancer cell growth with IC50 of the 20 µM, whereas THC showed a IC50 of the 40 µM.
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Figure 1: Concentration-response curves of DBTRG-05MG cells after 72 h of treatment with Cannabidiol (red) and THC (blue). Each data represents the mean ± SD of six experiments, each performed in triplicate.
Since glioma cells express both cannabinoid CB1 and CB2 receptors in the membrane, we also evaluated their engagement in the antiproliferative effect of THC and cannabidiol (Figure 3). The inhibition of cell proliferation was only partially antagonized by the selective cannabinoid receptor antagonists AM251 (CB1) and AM630 (CB2), in fact the pre-treatment of the DBTRG – 05MG cells with AM251 and AM630 reduced the THC effect on the proliferation rate of 50%. On the contrary, the cannabidiol effect was only antagonized by AM251 with a decrease of the proliferation rate of 66%, whereas the pre-treatment of the DBTRG – 05MG cells with AM630 failed to antagonize the inhibitory effect of cannabidiol.
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Figure 2: Time-response bar charts of DBTRG-05MG cells after 24-48-72 h of treatment with Cannabidiol (red) and THC (blue). Each data represents the mean ± SD of six experiments, each one in triplicate. * P<0.05; * * P<0.01
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Figure 3: Effects of THC and cannabidiol on the cell proliferation in presence of their selective antagonists. Each data represents the mean Âą SD of six experiments, each one in triplicate. * * P<0.01; * * * P<0.01;
The cell migration was also investigate by scratch assay, an in vitro method that mimics to some extent migration of cells in vivo. The images (Figure 4) captured at the beginning and after 24 h incubation period showed a significant inhibition of the migration of the cells treated with THC and cannabidiol toward the scratch, indeed the images captured after 72 h (data not shown) of incubation with THC and cannabidiol showed that the cells treated with THC tented to close the scratch even if they migrated at a slower rate compared with the control cells, on the contrary the cells incubated with cannabidiol showed that the cells were partially moved from the edge toward the center of the scratch.
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Figure 4: Analysis of DBTRG-05MG cell migration by in vitro scratch assay
As an indicator of oxidative stress was chosen lipid peroxidation, a well established mechanism of cellular injury. Lipid peroxides, derived from polyunsaturated fatty acids, are unstable and decompose to form a complex series of compounds, of which the most abundant is malondialdehyde (MDA). The results obtained shows that THC and cannabidiol treatment for 6 h induces a significant increase of MDA levels (Figure 5).
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Figure 5: MDA levels in DBTRG-05 MG cell lysates after 6h of THC and cannabidiol treatment
Conclusions In conclusion the present study demonstrates that Cannabis sativa L. constituents inhibit in vitro the proliferation and migration of glioblastoma cells, and these events could be correlated to an oxidative stress pathway where lipid peroxidation represents the last step of the cell injury. The mechanisms of this action is not clear at the moment and the role of CB1 and CB2 receptors is debated. Our results show that selective cannabinoid receptor antagonists only partially antagonize the inhibition of cell proliferation induced by THC and cannabidiol treatments. This effect could be due to an alternative pathway related to reactive oxygen species generation (Massi et al, 2004) that increase MDA levels and induces oxidative stress and cell death. Since the effects of cannabidiol, observed in this study, are more potent respect to THC and it is the one of the nonpsychoactive cannabinoid compounds of the Cannabis sativa, the cannabidiol could be represent a potential antitumoral agent for treating malignant glioblastoma.
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