Experimental Investigation On Antioxidant And Anticarcinogenesis Activities Of Resveratrol In Vitro

ObjectiveResveratrol (3,4,5-trihydroxy-trans-stilbene), a phytoalexin found in grape skins, peanuts, and red wine, has been reported to exhibit a wide range of biological and pharmacological properties. It has been speculated that dietary resveratrol could be an explanation for the so-called 'French paradox' as it may act as an antioxidant, promote nitric oxide production, inhibit platelet aggregation, and increase high-density lipoprotein cholesterol, and thereby serve as a cardioprotective agent. Recently, it has been demonstrated that resveratrol can function as a cancer chemopreventive agent, and there has been a great deal of experimental effort directed toward defining this effect. It has been shown that resveratrol interferes with signal transduction pathways, modulates cell cycle-regulating proteins, and is a potent inducer of apoptosis in multiple carcinoma cell lines. The present study was designed to evaluate the effect of resveratrol on the growth of gliomas C6 in vitro and possible mechanisms of its anti-tumor activities.Methods1 Cell growth curve: Gliomas C6 cells were incubated at 37 °C in an atmosphere containing 50 ml/L CO₂ for l⁸days, and the viable cells were determined by MTT assay each day for an average value. According to the results, drawing cell growth curve of C6.2 Cytotoxicity assay: Gliomas C6 cells in 96-well culture plate were divided intocontrol group, 0.1% dimethyl sulfoxide (DMSO) vehicle group, and experimental groups treated with Res at various concentrations (0.01 > 0.1 > 1.0^ 2.0? 5.0> 10.0> 20.0> 50.0> 100^ 200.Qwmol/L). All of the cells were incubated at 37 °C in an atmosphere containing 50 ml/L CO2 for 96 h followed by MTT assay. Evaluating the effect of resveratrol on the growth of gliomas C6 in vitro, IC30 and IC50 of Res on C6 cells were obtained from the concentration-effect curve.3 Cell morphology: Gliomas C6 cells in 24-well culture plate were divided into control group, 0.1% dimethyl sulfoxide (DMSO) vehicle group, and experimental groups treated with Res at IC30 and ICso, incubated for 96h, and observed using phase-contrast microscope.4 Cell mitotic index: Gliomas C6 cells in 24-well culture plate were divided into control group, 0.1% dimethyl sulfoxide (DMSO) vehicle group, and experimental groups treated with Res at IC30 and IC50, incubating for l⁸d. Slides were examined under a microscope and at least 1000 cells were counted on each slide for determination of mitotic index every day.5 Test of the antioxidative effect: Gliomas C6 cells were divided into control group, 0.1% dimethyl sulfoxide (DMSO) vehicle group, and experimental groups treated with Res at IC30 and IC50, incubated for 96h, and determined changes in the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and the level of maleic dialdehyde (MDA).6 RT-PCR: Gliomas C6 cells were divided into control group, 0.1% dimethyl sulfoxide (DMSO) vehicle group, and experimental groups treated with Res at IC30 and IC50, incubated for 96h, and detected the relative mRNA levels of the box, bcl-2 andp53 with the semi-quantitative RT-PCR technique.7 Immunohistochemistry: Gliomas C6 cells were divided into control group, 0.1%dimethyl sulfoxide (DMSO) vehicle group, and experimental groups treated with Res at IC30 and IC50, incubated for 96h, detected the expressions of Bcl-2 immunohistochemially.Results:1 Cell growth curve: From 1² days, cells were in lag phase, from 3-7 days, cells were in log phase, and after 7 days, cells were in stationary phase.2 Cytotoxicity assay: Res at concentrations of under 5.0jMmol/L, no obvious effects on the growth of gliomas C6 cells were observed (P>0.05);But Res at concentrations of 5.0²00.0/amol/L can dose-dependently inhibit the growth of gliomas C6 cells^O.05) (rs = -0.980, P=0.000);IC30 and IC50 of Res are 11.0|#mol/L and 31.2/^mol/L respectively.3 Cell morphology: Compared with control group, the cells of Res-treated groups were both reduced quantitatively, the morphology was abnormal or wizened, and the number of apoptosis was up-regulated.4 Cell mitotic index: The mitotic index of each group reached the peak at about 4 day;In both of Res-treated groups, the mitotic activites of gliomas C6 cells were reduced (F<0.05);And at 3,4,5 day, the mitotic activites of IC50 group were lower than IC30group(P<0.05).5 Test of the antioxidative effect: In IC30 group, the levels of MDA were reduced(P<0.05), there were no obvious effects on the activites of GSH-Px and SOD(P>0.05);In IC50 group, the levels of MDA were reduced(P<0.05), the activites of SOD were up-regulated, there were no obvious effects on the activites ofGSH-Px(P>0.05);6 RT-PCR: In both of Res-treated groups, no obvious effects on the relative mRNA levels of bcl-2 were observed (P>0.05), but the relative mRNA levels of boxwere up-regulated and the values of bcl-2/bax were lowered (P<0.05);In IC50 group, the relative mRNA levels of ^53 were up-regulated (P<0.05), but there were no obvious effects in IC30 group (P>0.05).7 Immunohistochemistry: In both of Res-treated groups, there were no obvious effects on the expressions of Bcl-2(P>0.05).Conclusions:1 In this study, Res at concentrations of 5.0²00.0/Mmol/L can dose-dependently inhibit the growth of gliomas C6 cells.2 Res can induce apoptosis, inhibit the activities of mitosis;That demonstrate Res can block cell cycle, and reduce proliferation of C6 cells.3 After gliomas C6 cells treated with Res, the levels of MDA were reduced, and the activites of SOD were enhanced.4 Res can promote the expressions of anti-oncogene bax and p53, and reduce the value of bcl-2/bax.5 Res can function as a cancer chemopreventive agent through inducing apoptosis, blocking cell cycle, reducing proliferation, increasing the antioxidative abilities, and promoting the mRNA levels of anti-oncogene.