| Objective:To observe the effect of ?-asarone on chemosensitivity of gastric cancer cell lines with different degrees of differentiation in vitro and to investigate the correlation between ?-asarone and PDKs expression in gastric cancer.Methods:1.MTT method was used to observe the inhibitory effect of ?-asarone,cisplatin and the combination on the proliferation of various gastric cancer cell lines,and the synergistic sensitivity of ?-asarone and cisplatin:was determined by using median.effect principle.Real-time PCR was used to detect the expression of ERCC1 and BRCA1 result from?-asarone,cisplatin and the combination intervention in human gastric cancer cells.2.Using lactate dehydrogenase test kit to detect in different concentrations of 0-asarone(120 ?g · mL-1,60 ?g · mL-1,30 ?g · mL-1,15 ? g·mL-1)or in Coc12(200mM 24h)which induced by hypoxia and in H2O2(100mM 1H)induced by oxygen environment,the influence of?-asarone on lactate dehydrogenase production in gastric cancer cells,in order to identify the effect of ?-asarone on the activity of PDK1.3.The effects of ?-asarone on the apoptosis of gastric cancer cell lines which in COCl2(200mM 24h)which induced by hypoxia and in H2O2(100mM 1H)induced by oxygen environment were determined by Annexin V/PI double staining assay.Meanwhile,the periodic block effect were detected by flow cytometry assay.4.The effects of ?-asarone on the expression of PDK1,PDK4,HIF-1 a and c-myc genes in gastric cancer cells were detected by RT-PCR.5.The effects of ?-asarone on the expression of PDK1,p-PDK1,PDK4,HIF-1 a,STAT5 and p-STAT5 proteins were detected by western blot.Results:1.?-asarone has chemotherapeutic effect on gastric cancer cell lines with different differentiation degree.The gastric cancer cell line MGC-803 in which chemotherapy sensitization effect is best.?-asarone can down-regulate the expression of ERCC1 and BRCA1 gene.2.The cell lactate dehydrogenase content was inhibited and was concentration-related after the treatment with different concentrations of ?-asarone on gastric cancer cells MGC-803,SGC-7901,MKN-28.There is no correlation between the different degree of differentiation of gastric cancer cells.The expression of LDH in gastric cancer cells was significantly decreased after the treatment of ?-asarone when under the condition of hypoxia,and the results were statistically significant.3.0-asarone can induce apoptosis of gastric cancer cell lines MGC-803,SGC-7901 and MKN-28 under the hypoxic conditions and aerobic conditions,which can block gastric cancer cells in G2/M phase.and after the treatment with ?-asarone the results showed obvious sub-diploid peak,Suggesting that it has the role of induction of apoptosis.4.?-asarone can down-regulate the expression of PDK1,PDK4,HIF-1 ? and c-myc genes in gastric cancer cell lines.5.?-asarone can inhibit the expression of PDK1,p-PDKl,PDK4,HIF-1?,STAT5 and p-STAT5 protein in gastric cancer cell line when under the hypoxia condition.Conclusion:1.?-asarone have proliferation inhibition on gastric cancer cell lines with different differentiation degree,and was concentration-related;?-asarone which in a certain concentration range has chemosensitization effect on cisplatin and the chemotherapy sensitivity may be related to the inhibition of ERCC1 and BRCA1 gene expression.2.?-asarone can decrease the production of lactate dehydrogenase in gastric cancer cells and in a concentration-dependent manner.?-asarone can decrease the lactate dehydrogenase content of gastric cancer cell lines under hypoxia,and it is similar to H202 group,suggesting that its antitumor mechanism may be related to the inhibition of aerobic glycolysis of tumor cells.3.Under hypoxic conditions ?-asarone can induce the apoptosis of gastric cancer cell lines,gastric cancer cells were blocked in the cell cycle G2/M phase,while the experiment show that after the treatment with ?-asarone the results showed obvious a significant sub-diploid peak,Suggesting that it has the role of induction of apoptosis.4.?-asarone can induce the apoptosis of gastric cancer cells,and its effect may be related to inhibition of PDK1,PDK4,HIF-1?,c-myc gene expression and down-regulation of PDK1,p-PDK1,PDK4,HIF-?,p-STAT5 protein expression.The above genes and proteins are associated with glycolysis,which further suggests that ?-asarone inhibits the proliferation of gastric cancer cells,and the increase of chemotherapy sensitization may be associated with the inhibition of glycolysis during tumor growth. |
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