The Anti-Proliferative Effect Of Equol On Gastric Cancer Cells And Its Underlying Molecular Mechanisms

Background and Aims:Gastric cancer, the fourth common cancer worldwide and a leading cause of cancer death in China, constitutes a serious threat to human health. In recent years, cancer chemoprevention has received increasing attention among scientists in medicine. It has been reported that more than 30% of human cancers could be prevented by an alternative strategy of appropriate dietary modification. Therfore, dietary modification is an alternative strategy for lowering gastric cancer risk. It has been suggested that high consumption of fresh vegetables, fruits, and soy products may be associated with a decreased risk for gastric cancer. Among the bioactive substances in soy, the isoflavones have received wide attention because of its biological activities, including cancer and osteoporosis prevention, menopausal symptoms alleviation of women and cardiovascular diseases prevention and treatment. Genistein and daidzein are two major isoflavones in soy, and equol is a metabolite of daidzein produced by gut bacteria. It has been shown that among humans, only 30–50% have the bacteria capable of producing equol and isoflavones may be more effective in its biological effects in equol-producing individuals. Therefore, equol seems to be biologically more active than its precursor, daidzein. Chemopreventive effects of equol have been demonstrated in a wide variety of human tumors, including breast cancer and prostate cancer, however, few studies have been done in gastric cancer. In the present study, we investigated the anti-proliferative and apoptosis-inducing effects of equol in gastric cancer MGC-803 and SGC-7901 cells and its mechanisms. The data in our study suggest that equol holds promise as a novel candidate for gastric cancer chemoprevention and therapy and provide theoretical basis for further equol exploitation.Methods:To demonstrate the anti-proliferative effect of equol on MGC-803 cells, MTS assay was determined to test the effect of equol on the viability of gastric cancer cells, q RT-PCR analysis and immunofluorescent detection of Ki67 were determined to examine the m RNA and protein expression of Ki67（a general marker of cellular proliferation in tumor）. Cells were stained with propidium iodide（PI）, Annexin V/PI or JC-1, then subjected to flow cytometry to examine cell cyle, apoptosis or mitochondrial membrane potential. Futhermore, to explore the molecular mechanisms underlying equol-induced cell cycle arrest and apoptosis, q PCR and western blot were deternmind to examine the expression of cell cycle regulators, apoptosis-related protein and the protein associated with MAPK and AKT signal pathway. Finally, all kinds of specific inhibitors were used to characterize the effect of MAPK and AKT signal pathway on equol-induced cell proliferation inhibition and apoptosisResults:1. Equol effectively inhibited the viability of MGC-803 and SGC-7901 cells and down-regulated the m RNA and protein levels of Ki67 in MGC-803 cells.2. Equol triggered cell cycle arrest at G0/G1 phase in MGC-803 and SGC-7901 cells, obviously downregulated the protein levels of cyclin E1, cyclin D1, CDK2, CDK4, and upregulated the levels of p21WAF1 in MGC-803 cells.3. Equol obviously induced apoptosis of MGC-803 and SGC-7901 cells, which was accompanied by altered levels of cleaved caspase-3, caspase-9 and PARP, Bcl-2 family proteins（Bid、Bcl-x L）, CIAP1 and mitochondrial membrane potential in MGC-803 cells.4. Equol induced sustained ERK activation, but did not affect phosphorylation of JNK and P-38 in MGC-803 cells. Inhibition of ERK by U0126, a MEK inhibitor, partly abolished equol-induced cell viability inhibition and apoptosis, but increased the number of cells in the G0/G1 phase, as compared to equol alone treatment.5. Equol treatment significantly induced sustained AKT dephosphorylation at Thr450（as early as 12h）in a dose and time-dependent manner, However, equol modulated the phosphorylation of AKT at Ser473 and Thr308 in a different way. Equol led to hypophosphorylation of AKT at these two sites before a dephosphorylation. Furthermore, we found that treatment cells with LY294002, a PI3 K inhibitor, decreased the protein levels P-AKT（ Thr308） and P-AKT（Ser473）,but did not affect the phosphorylation of AKT at Thr450. In addition, pretreatment cells with LY294002, slightly enhanced equol-induced cell viability inhibition and apoptosis, and slightly increased the number of cells in the G0/G1 phase, as compared to equol alone treatment.ConclusionsTaken together, these findings indicate that equol exhibits anti-tumor effects on human gastric carcinoma cells by leading to G0/G1 cell cycle arrest and apoptotic death, which might be mediated by inducing sustained ERK activation and AKT dephosphorylation at Thr450. The present study also demostrated the involvement of cell cycle regulators and apoptosis-related protein, including CDK2/4, Cyclin D1/E1, P21^WAF1, Bcl-x L, Bid, c IAPI, Caspase-3,Caspase-9 and PARP cleavage. These findings suggested that equol may be a novel candidate for gastric cancer chemoprevention and therapy.