Anticancer Effects Of Resveratrol By Regulating Activities Of Antioxidant Enzymes

Background and Objectives:Resveratrol (RSV) is a natural polyphenol that is known as a potent chemopreventive and chemotherapeutic molecule. RSV affects cancer cells through different mechanisms of action, most importantly, by inducing apoptosis. However the mechanism of RSV action is away from completely understood. Studies revealed that RSV can serve as either an antioxidant or pro-oxidant reagent depending on the specific microenvironment. This study focused on the effects of RSV on the activities and expression levels of antioxidant enzymes in the cancer cells, as well as apoptosis inducted in cancer cells to clarify RSV’s actions on antioxidative axis.Materials and Methods:Prostate cancer PC-3cells, hepatic cancer HepG2cells, breast cancer MCF-7cells and non-cancerous HEK293T cells were treated with a wide range of RSV concentrations (10-100μM) for24-72hours. In another set of experiments, three Chronic myelogenous (CML) cell lines K-562, KT-1/A3, KT-1/A3R and one acute myelogenous (AML) cell line HL-60were treated with a wide range of RSV concentrations (10-100μM) for24-72hours. Cell growth was estimated by trypan blue staining assay; activities of the antioxidant enzymes were measured spectrophotometrically; expression levels of the antioxidant enzymes were quantified by digitalizing the protein band intensities on Western blots; and the percentage of apoptotic cells was determined by flow cytometry. The content of hydrogen peroxide (H2O2) inside the cells was measured by spectrophotometric method.Results:RSV showed dose-and time-dependent growth inhibition in tumor cells. It was significantly effective against PC-3and HepG2, but not against MCF-7, even at10μM and25μM concentration. However,50μM of RSV showed significant growth inhibitory effect in MCF-7, as well as HEK293T cells.25μM of RSV was identified as the most effective dosage against PC-3, HepG2and MCF-7cells, without affecting HEK293T cells.Treatment of RSV at low concentration significantly increased the activity of antioxidant enzyme superoxide dismutase (SOD) in PC-3, HepG2and MCF-7cells, but not in HEK293T cells. Another antioxidant enzyme catalase (CAT) activity was increased in HepG2, but no effect was found on the activity of antioxidant enzyme glutathione peroxidase (GPX) by RSV treatment. RSV-induced SOD2expression was observed in cancer cells, whereas the expressions of SOD1, CAT and GPX1were not affected. The production of H2O2was significantly increased in PC-3and HepG2at lower concentrations of RSV, and induced production of H2O2was seen in MCF-7and HEK293T when treated with higher concentrations of RSV. The apoptosis was increased by RSV treatment in cancer cells, especially PC-3and HepG2.In all tested leukemic cells, RSV showed dose dependent growth inhibition, and HL-60was found more sensitive to the RSV than K-562, KT-1/A3and KT-1/A3R. After24hours of treatment of RSV at a concentration of less than50μM, the cell growth was not significantly changed. However,48hours treatment of25μM RSV showed significant growth inhibitory effect in HL-60cells, but not in the other cells. The growth of HL-60cells was inhibited by10μM RSV when treated for72hours. RSV showed growth inhibitory effect in K-562cells only when treated with50μM for72hours. In KT-1/A3or KT-1/A3R cells, RSV showed no significant effect when treated with≤50μM. However,100μM of RSV was found lethal to all the tested leukemic cell lines.The SOD activity in HL-60cells was significantly increased (by24%) after RSV treatment for72hours at25μM concentration. When the K-562treated with25μM of RSV, the SOD activity was not significantly increased (only by11%). The CAT activity was slightly increased in K-562cells and slightly decreased in HL-60cells, but none of the effects was significant. In addition, the GPX activity was not significantly changed by RSV in either HL-60or K-562cells. Western blot analysis showed the expression of SOD2in HL-60increased (by36%) significantly when treated with25μM RSV for 72hours, but the effect on K-562was non-significant when the cells were treated with the same conditions. However, in HL-60or K-562cells, the effect of25μM RSV on SOD1, CAT and GPX1was not significant. The flow cytometric analysis revealed that the percentage of apoptotic cells was increased more potently in HL-60cells (6-fold) than in K-562cells (1.5fold) when treated with RSV. RSV also showed significant effect on the production of H2O2in HL-60cells. Treatment of10μM of RSV for72hours decreased the H2O2content slightly in cancer cells, but25μM or higher concentrations of RSV significantly increased H2O2production in HL-60cells.Conclusions:This study demonstrates:(1) RSV inhibits PC-3, HpG2, MCF-7cancer cell growth with minimal effect on non-cancerous cells.(2) RSV is more growth inhibitory against HL-60AML cells than CML cells.(3) Mechanistically, the disproportional up-regulation of SOD, CAT and GPX expression and their enzymatic activity in PC-3, HepG2, MCF-7tumor cells, and HL-60leukemic cells result in mitochondrial accumulation of H2O2, which in turn induces cancer cell apoptosis. On the other hand, slight but proportional up-regulation of anti-oxidative enzymes and reducing oxidative stress could explain RSV’s protective effect on non-cancerous cells.