Effect Of 3-BrPA On Apoptosis Of Colon Cancer HCT-8 Cells,Drug Resistant Cell Line HCT-8/V And Its Mechanism

Objectives To explore the apoptosis-inducing effect and the possible mechanism of 3-bromopyruvate(3-Br PA)on colon cancer HCT-8 cells and drug resistant cell line HCT-8/V.Methods MTT assay was used to investigate the effect of 3-Br PA on the survival rate of colon cancer HCT-8 cells and drug resistant cell line HCT-8/V;Hoechst 33342 staining was used to study the changes of cell apoptosis;JC-1 staining was used to study the change of cell mitochondrial membrane potential;Annexin V-FITC/PI double staining was used to study the change of apoptosis rate;Real-time PCR was used to analyze the m RNA level of multidrug resistance,glucose metabolism and apoptosis-related genes(MDR1,HK2,Bax,Bcl-2,and procaspase-3)in colon cancer cells;Western blot was used to analyze the protein level of multidrug resistance,glucose metabolism and apoptosis-related protein(MDR1,HK2,Bax,Bcl-2,and procaspase-3)in colon cancer cells.Results 1 The survival rate of colon cancer HCT-8 and drug resistant cell line HCT-8/V was significantly decreased after treatment with 3-Br PA for 24 h,48h and 72h(P<0.05);2 The morphology of the cell became round,the membrane was intact,the volume was reduced,and the nucleus was shrinking after colon cancer HCT-8 and HCT-8/V cells treated with 3-Br PA for 24 h;3 The results of JC-1 staining showed that the mitochondrial membrane potential of colon cancer cells was decreased after treated with 3-Br PA for 12 hours;4 Annexin V-FITC/PI results showed that the apoptosis rate of colon cancer cells treated with 3-Pr PA for 48 h increased significantly in a dose-dependent manner(P<0.05);5 Real-time PCR results showed that MDR1 m RNA expression was decreased in colon cancer drug resistant cell line HCT-8/V treated with 3-Br PA for 24 hours(P<0.05);the m RNA expression of HK2,Bcl-2,and procaspase-3 decreased in colon cancer HCT-8 and HCT-8/V cells,and the expression of Bax m RNA increased(P<0.05);6 Western blot results showed that the expression of P-gp in colon cancer drug resistant cell line HCT-8/V was decreased after treatment with 3-Br PA for 24 h(P<0.01);The protein expression of HK2,Bcl-2 and procaspase-3 decreased in colon cancer HCT-8 and HCT-8/V cells,and the expression of Bax protein increased(P<0.01).Conclusions 1 3-Br PA can decrease the survival rate and induce apoptosis of colon cancer HCT-8 and HCT-8/V cells;2 3-Br PA can decrease MDR1 m RNA and P-gp protein expression in colon cancer drug resistant cell line HCT-8/V;3 The inhibition of the key enzyme of glucose metabolism and the promotion of apoptosis of the mitochondrial pathway may be part of the mechanism of apoptosis induced by 3-Br PA in colon cancer HCT-8 cells and drug resistant cell line HCT-8/V.