The Effect Of Glycolytic Inhibitors On Human Gastric Cancer Cell Line MGC-803 And Its Mechanism

[Objective] To investigate the effect of glycolytic inhibitors3-bromopyruvate(BP)and sodium citrate(SCT)on the growth and proliferation of gastric cancer cell line MGC-803 and its mechanism,and to provide the experimental basis for 3-bromopyruvate and sodium citrate using on clinical trial and further treatments of gastric cancer.[Methods] Culture the poorly differentiated human gastric cancer cell line MGC-803,and divided into 8 groups.The corresponding drug containing medium was added(4?6?8 ug/ml of BP,5?10?20 mmol/L of SCT and0.5mmol/L of 5-FU)after cells were attached to the flasks respectively,and the negative control group was incubated with the complete culture medium.The inhibition of proliferation were assessed using MTT assay.Then a flow cytometer was used to analysze the cell cycle repartition and apoptosis.Besides,the content of lactate and ATP and the activity of HK and PFK-1 were measured with Assay Kits respectively by enzymes combined method.Finally,the expression of Bcl-2,Bax,Cyt-C and Suvivin was measured by Real-time fluorescent quantitative PCR and Western blotting.[Results]1.After incubation with BP and SCT respectively,the inhibitory rate of thecell proliferation presentd increasing trend with the concentration of BP and SCT increased,the trend was increasing with the time increased at the same time.The IC50 of BP and SCT on MGC-803 cell was 5.73±0.75ug/ml and10.08±0.87mmol/L respectively.The total number of cells in different concentration groups of BP and SCT and 5-FU group was lower than that in control group,and the morphological rounded cells appeared to be more concentrated than the control group.2.Flow cytometry showed that the apoptotic rate of MGC-803 cells was increased from 10.67% to 76.19% after treatment with different concentration of BP,while it was increased from 15.61% to 83.10% after incubation with different concentration of SCT.Compared to the control group,the difference was statistically significant(p<0.05).The apoptotic rate in the 5-FU group was50.52% and 86.06% for 24 h and 48 h respectively,which was significantly higher than the control group(p<0.05).After incubated with different concentration of BP and SCT for 24 h,the rate of the cells arrested in the G2/M phase of cell cycle was increased from 14.49% to 39.39% in the groups of BP,while it was increased from 13.51% to 41.24% in the groups of SCT,which was significantly different from that of the control group(p<0.05).The rate was21.93% after incubated with 5-FU,which was significantly higher than the control group(p<0.05).3.Both BP and SCT could effectively reduce the production of ATP and lactate in MGC-803 cells.The intracellular ATP content was decreased from30.79umol/gprot to 7.31umol/gprot in the groups of BP,while it was decreased from 28.24umol/gprot to 6.76umol/gprot in the groups of SCT,there was significant difference compared to the control group(p<0.05).The ATP contentwas 21.21umol/gprot and 10.34umol/gprot after incubated for 4h and 8h with5-FU,compared to the control group,the difference was statistically significant(p<0.05).In addition,the content of intracellular lactate was decreased from84.91umol/gprot to 18.92umol/gprot in the groups of BP,and it was decreased from 78.87umol/gprot to 11.44umol/gprot in the groups of SCT,the difference was statistically significant compared to the control group(p<0.05).The intracellular lactate content in the 5-FU group was 106.45umol/gprot and113.98umol/gprot for 24 h and 48 h respectively,which was comparable for that in the control group(p>0.05).4.BP and SCT were able to selectively inhibit the activity of rate-limiting enzymes of glycolysis in MGC-803 cells.The activity of intracellular HK was decreased from 8.67u/gprot to 1.98u/gprot in the groups of BP.Compared to the control group,the difference was statistically significant(p<0.05).However,the intracellular HK activity was comparable for different SCT-dose groups,the5-FU group and the control group(p>0.05).In addition,the activity of intracellular PFK-1 decreased from 19.01u/gprot to 2.51u/gprot after incubation of SCT,which was significantly different from that of control group(p<0.05),but no significant difference was saw then treated with BP and 5-FU for the intracellular PFK-1 activity(p>0.05).5.Both BP and SCT could concentration-dependently inhibit the expression of Bcl-2 and Survivin mRNA and protein in MGC-803 cells.In addition,both BP and SCT could promote the expression of Bax and Cyt-C mRNA and protein in MGC-803 cells in a concentration-dependent manner.5-FU could also inhibit the expression of Bcl-2 and Survivin mRNA and protein and promote the expression of Bax and Cyt-C mRNA and protein in MGC-803 cells.Compared to the control group,the difference was statistically significant(p<0.05).[Conclusion]Both BP and SCT have a remarkable inhibitory effect on the growth and proliferation of human gastric cancer cell line MGC-803.BP and SCT could selectly inhibit the activity of HK and PFK-1 in MGC-803 cells respectively,then the glycolysis was inhibited at the same time,and the production of ATP content in the cells was decreased.Besides,the expression of Bax was up-regulated,and the expression of anti-apoptotic genes Bcl-2 and Suvivin was down-regulated,then the mitochondrial pathway was activated,resulted in Cyt-C releasing,which induced the cell apoptosis and inhibited the proliferation of gastric cancer cells.