The Effect Of Apigenin On Multi-drug Resistance In Human Gastric Carcinoma Cells And Its Mechanisms

Gastric cancer is one of the most common malignancies in China. Multi-drug resistance (MDR) is a major obstacle to successful cancer chemotherapy. The single mechanism,poor selectivity and serious side-effects of current MDR-reversing agents limit their clinical application. Therefore,the search for new anti-MDR substances with high efficacy,low toxicity and minimum side effects is a key in cancer treatment. Apigenin,a natural flavonoid,has been shown to possess anti-tumor effects with low toxicity and high efficacy through multiple mechanisms. A recent study has shown that apigenin modulated MRP1's ATPase activity and therefore stimulated GSH transport. Moreover, a series of apigenin-based flavonoid dimmers are reported to competitively disrupt P-gp induced drug efflux and increase drug accumulation in drug-resistance cancer cells. Therefore,apigenin may play an anti-MDR or MDR-reversing role in gastric cancer.In this study,we investigated the effect of apigenin on cell growth , proliferation and MDR in gastric cancer. We also detected the expression of MDR1 and P-gp. Our study is helpful to elucidate the molecular mechanisms of anti-MDR prosperity of apigenin,and provides an effective modality to overcome MDR in human gastric cancer.【Objectives】1. To investigate the effect of apiginen on cell growth and cell proliferation in multidrug resistant gastric cancer cells;2. To examine the effect of apigenin on drug sensitivity and drug efflux in multidrug resistant gastric cancer cells;3. To explore the possible mechanisms of anti-tumor and MDR-reversing effects of apigenin in multidrug resistant gastric cancer cells;4. To observe the effect of apigenin on anti-cancer capacity of chemotherapeutic drug in gastric cancer cells.【Methods】1. MTT assay was performed to describe the growth of gastric cancer cells treated with apigenin;2. Cell cycle distribution of gastric cancer cells treated with apigenin was studied by flow cytometry;3. Apoptosis of the gastric cancer cells treated with apigenin was examined by Annexin V/PI staining;4. MTT assay was performed to determine the in vitro drug sensitivity of gastric cancer cells treated with apigenin and the IC50 values were calculated.5. Rhodamine-123 efflux was performed to detect P-gp transporter activity of gastric cancer cells treated with apigenin;6. Western blot was used to detect the expression of P-gp in gastric cancer cells treated with apigenin;7. The expression levels of MDR1 mRNA in gastric cancer cells were measured by real time polymerase chain reaction;8. FACS analyzed the changes in cell cycle distributions after the treatments of apigenin, ADM , and the combinative treatments respectively.9. Apoptosis was determined by Annexin-V/PI doubling staining assay after the treatment of apigenin,ADM,and Apigenin plus ADM in gastric cancer cells;10. Real time polymerase chain reaction was performed to investigate the expressions of anti-oncogene p53 and p21 after the treatment of apigenin,ADM,and Apigenin plus ADM in gastric cancer cells.【Results】1The growth of SGC7901/VCR cells was significantly inhibited by Apigenin,which was confirmed by cell growth assay.2. Treatment with apigenin decreased the number of cells of G1 phase,and increasing the cell numbers of G2 and S phases in SGC7901/VCR cells.3. Apigenin treatment resulted in the increase of apoptosis in SGC7901/VCR cells.4. The accumulation and retention of Rhodamine-123 were increased in SGC7901/VCR cells treated with apigenin.5.SGC7901/VCR cells were sensitive to Apigenin.And apigenintreatment increased drug sensitivity to VCR in SGC7901/VCRcells.6. Apigenin treatment decreased the expression of P-gp inSGC7901/VCR cells.7.MDR1 mRNA was down-regulated in SGC7901/VCR cellswith apigenin treatment.8.Both of apigenin and ADM affected the cell cycledistributions by G2 arrest,and apigenin plus ADM increasedthe cell number of G2 arrest.9.Apigenin and ADM respectively induced apoptosis inSGC7901/VCR cells,and the apoptosis was enhanced by thecombination of apigenin plus ADM;10.The mRNA level of p21 was markly increased afterSGC7901/VCR cells treated with apigenin plus ADM,but thelevel of p53 mRNA changed little.【Conclusions】1. Apigenin inhibits drug resistant gastric cancer cellseffectively in vitro through inducing G2 cell cycle arrest andapoptosis.2. Apigenin enhances cell chemosensitivity and inhibits P-gpdrug efflux activity in drug resistant gastric cancer cells.3. Apigenin down-regulates the expression of MDR1 / P-gp,which may be the molecular mechanism of apigenin anti-MDR effect.4. Apigenin enhances the ADM induced cell cycle arrest and apoptosis by increasing p21 expression in p53 independent pathway.