Effects Of Sodium Dichloroacetate Combined With Metformin On Neuroblastoma BE-2C Cells

Objective: To investigate the effect of metformin(Met)combined with dichloroacetate(DCA)on MYCN-amplified neuroblastoma cells and its mechanism.Methods: CCK-8 method was used to detect the inhibitory effect of metformin and dichloroacetic acid on the proliferation of MYCN-amplified neuroblastoma cells,and the synergistic effect of the two drugs was judged.The changes of the content of supernatant in culture medium were detected by glucose and lactic acid kit.The expression of Bcl-2,Bax and Cleavead caspase-3 related to apoptosis was changed.Flow cytometry was used to detect the effects of metformin,dichloroacetic acid and combination of the two drugs on neuroblastoma cell apoptosis.Results: Metformin combined with dichloroacetic acid inhibited the proliferation of MYCN-amplified neuroblastoma cells,and its combined use index(CI)was less than 1.Compared with the control group,the glucose consumption and lactic acid production in the metformin group increased significantly(P < 0.05),while the glucose consumption and lactic acid production in the dichloroacetic acid group decreased significantly(P< 0.01),compared with that in the dimethylacetic acid group.Compared with the metformin group,the glucose consumption and lactic acid production in the combination group were significantly reduced(P<0.05),indicating that dichloroacetic acid could reduce the accumulation of lactic acid induced by metformin.Flow cytometry showed that 24 hours after administration,the apoptotic rate of metformin group was significantly higher than that of normal control group(5.89 ± 1.26)(P<0.05).Compared with normal control group(5.89 ± 1.26),the apoptotic rate of dichloroacetic acid group(13.29 ± 4.90)was significantly higher(P<0.05).The apoptotic rate of combined administration(58.26 ± 7.90)was significantly higher than that of single administration(58.26 ± 7.90).There were significant differences between them(P < 0.05).The relative ratio of Bcl-2/Bax in metformin group orsodium dichloroacetate group was significantly lower than that in normal group.The relative ratio of Bcl-2/Bax in combination group was significantly lower than that in metformin group or dichloroacetic acid group(P < 0.05).The expression of Cleavead caspase-3 protein in metformin group or sodium dichloroacetate group was significantly higher than that in normal group(P < 0.05).The expression of caspase-3protein in combined group was higher than that in metformin or sodium dichloroacetate alone group,and the difference was statistically significant(P < 0.01).It is suggested that the increase of BE-2C cell apoptosis induced by metformin combined with sodium dichloroacetate may be related to down-regulation of Bcl-2protein expression,up-regulation of Bax protein expression and enhancement of Cleavead caspase-3 activity.Conclusion: Metformin combined with dichloroacetate has synergistic effect on the proliferation of MYCN-amplified neuroblastoma cells.The mechanism may be related to the reduction of accumulation of lactic acid and the promotion of apoptotic signaling pathway.