Effects Of EGCG Combined With Glutaminase Inhibitor CB-839 On The Proliferation And Apoptosis Of Imatinib-resistant K562 Cells

Objective:To study the effect of epigallocatechin gallate(EGCG)combined with glutaminase inhibitor CB-839 on the proliferation and apoptosis of Imatinib-resistant K562 cells(K562 / G cells),to provide new ideas of treatment for patients with drug-resistant chronic myelogenous leukemia.Method: This study focused on K562 / G cells.1.CCK8 method was used to detect the effect of different concentrations of CB-839 and EGCG acting alone or in combination on the proliferation activity of K562 / G cells,calculating the cell proliferation inhibition rate of different drug groups.The Chou-Talalay combination index method was used to study the combined application effect of the two drugs.The CalcuSyn software calculated the values of CI(Combination Index)to analyze whether the combination of the two drugs had synergistic effects.The combination with the greatest synergy was selected for subsequent experiments.2.Flow cytometry was used to detect cell cycle distribution and apoptotic rate in the control group,CB-839 group,EGCG group and combination drug group.3.Western blot was used to detect the expressions of Bcl-2,Bax and LC3 B protein in the control group,CB-839 group,EGCG group and combination drug group.Results: 1.CCK8 assay results showed that both different concentrations of CB-839 and EGCG can inhibit the proliferation of K562 / G cells to a certain extent(P<0.05).Combined with EGCG group(40,80,120 ?mol / L)respectively,the CB-839 group(1.25,2.5,5 ?mol / L)acted upon K562 / G cells,and the results revealed that the inhibition rate of cell proliferation in the combined group was significantly higher than that in the control group and the single drug groups(P<0.05).The result of CompuSyn software analysis showed that the CI values of all combination drug groups were <1,indicating that the two drugs had a synergistic effect.When CB-839 was 5 ?mol / L and EGCG was 80 ?mol / L,the CI value was the smallest and the synergy was the largest,so the concentration of the combined drug group was used for subsequent experiments.2.The results of flow cytometry showed that compared with the control group,the application of CB-839 alone had no effect on the K562 / G cell cycle(P>0.05),the proportion of cells in the G2 phase in the EGCG group increased(P<0.05).The proportion of cells in the G2 phase in the combined drug group was significantly higher than that in the control group and the single drug groups(P<0.05).Flow cytometry analysis of apoptosis rate results showed that compared with the control group,there was no significant difference in the apoptosis rate of K562 / G cells in the CB-839 group(P>0.05);the apoptosis rate in the EGCG group was significantly increased(P<0.05);Compared with the control group and the single drug groups,the apoptosis rate was significantly higher in combined drug group(P<0.05).3.Western Blot analysis showed that compared with the control group,the expression of Bax protein and the ratio of Bax / Bcl-2 increased in the combined drug group(P<0.05).Compared with the control group,there was no significant difference in the ratio of LC3B-II/LC3B-I in the CB-839 group and the EGCG group(P>0.05),but decreased in the combined drug group(P<0.05).Conclusion: CB-839 can inhibit the proliferation of K562 / G cells to a certain extent,but cannot induce apoptosis.CB-839 alone had limited antitumor activity,but when it was used in combination with EGCG,its anti-cell proliferation and pro-apoptotic effects were significantly improved,showing a good synergistic effect of two drugs,which may be related to blocking cell cycle,affecting the ratio of Bax/Bcl-2,and regulating autophagy in a combined way.