Study On The Effect And Mechanism Of Arsenite Combined With BCL-2 Inhibitor In Leukemia

Objective:To investigate the use of arsenic trioxide(ATO)and BCL-2 inhibitorvenetoclax(VEN)alone or in combination,to study their effect on the proliferation and apoptosis of HL60 cell lines and their Possible mechanisms.Methods:1.Taking the human leukemia cell lines HL60 as the research objects,the cells were treated with different concentrations of ATO and VEN and the cell proliferation inhibition rate was detected by CCK8 method at 24 h,48h and 72 h,and the half maximal inhibitory concentration(IC50)of the two drugs was determined.HL60 cells were treated with the combination of the two drugs,and the cell proliferation inhibition rate was detected by CCK8 method at 24 h,48h and 72 h.2.After determining the IC50 value of the two drugs,the IC50 value close to 48 h of the two drugs was selected as the concentration of the combined drug.The concentrations in the experimental group were ATO3μmol/L,VEN0.1μmol/L,VEN0.5μmol/L,VEN1μmol/L,ATO3μmol/L+VEN0.1μmol/L,ATO3μmol/L+VEN0.5μmol/L,ATO3μmol/L+VEN1μmol/L.After 48 h of drug treatment,the cells in each group were stained by Swiss Giemsa staining method to observe cell morphology.3.Flow cytometry(FCM)was used to detect the effects of ATO group,VEN group and two drug combination group on the apoptosis rate of HL60 cells after their use at 48 h time node.4.FCM was used to detect the effects of ATO group,VEN group and two drug combination group on the cell cycle after treatment of HL60 cells at 48 h time node.5.Real-Time PCR was used to detect the expression of BCL-2 m RNA,MCL-1m RNA,BAX m RNA and BIM m RNA in the ATO group,VEN group and the two drug combination group after treatment of HL60 cells at the 48 h time node.Results:1.CCK8 test results showed that the proliferation inhibition rates of HL60 cells in ATO single drug,VEN single drug and combined drug groups were all increased compared with the control group(P < 0.01,the differences were statistically significant).The inhibition rate in combination group increased more significantly than that in single drug group(P < 0.01,the difference was statistically significant).ATO single drug inhibited HL60 cell proliferation in a time and concentration dependent manner within 72 h at a concentration range of0μmol/L-8μmol/L.The IC50 values of ATO at 24 h,48h and 72 h were 18.721μmol/L,3.693μmol/L and 1.477μmol/L.The inhibitory effect of single drug VEN single drug inhibited HL60 cell proliferation in a time and concentration dependent manner within 48 h at a concentration range of 0μmol/L-4μmol/L(P < 0.01,the difference was statistically significant).The IC50 values of VEN at 24 h,48h and 72 h were 1.649μmol/L,0.78μmol/L and0.156μmol/L.2.Swiss Giemsa staining method showed that the size of cells in the control group was basically uniform,mostly round,with uniform nuclear chromatin and large nuclear/plasma ratio.Compared with the control group,the number of cells in the ATO group,VEN group and the two-drug combination group was significantly reduced,with different cell sizes and irregular morphology.Some cells were shrinked,the nucleus/plasma ratio of the control group was reduced,which was more significant in the two-drug combination group.3.The apoptosis results showed that the apoptosis rate of ATO group,VEN group and two-drug combination group was increased compared with the control group,and the increase was more significant in the two-drug combination group(P < 0.01,the difference was statistically significant).Compared with ATO group and VEN group of the same concentration,the apoptosis rate of the two drug combination group was higher than that of the single drug group(P < 0.01,the difference was statistically significant).4.Cell cycle results showed that compared with the control group,the proportion of G2/M phase cells increased in the ATO group,VEN group and the two-drug combination group,while the proportion of S phase cells decreased,and the change was more significant in the two-drug combination group(P < 0.01,the difference was statistically significant).Compared with ATO group and VEN group of the same concentration,the proportion of G0/G1 and G2/M phase cells in the two-drug combination group was significantly increased,while the proportion of S phase cells was significantly decreased(P < 0.01,the difference was statistically significant).5.Real-Time PCR results showed that: After 48 h of drug intervention in HL60 cells of each group,the expressions of BCL-2 and MCL-1 genes in ATO group,VEN group and two drug combination group were all decreased compared with the control group,while the expressions of BAX and BIM genes were increased compared with the control group.The decrease of BCL-2 and MCL-1 genes in the combined drug group was more obvious than that in the single drug group.The increase of BAX and BIM gene expression was more obvious than that in combined drug group(P < 0.01,the difference was statistically significant).Conclusion:1.Both ATO and VEN could inhibit the proliferation of HL60 cells in a time-dose dependent manner.2.ATO and VEN can promote the apoptosis of HL60 cells.3.Both ATO and VEN could inhibit the proliferation cycle of HL60 cells.4.The combined action of ATO and VEN on HL60 cells can synergically inhibit proliferation and promote apoptosis.The possible mechanism is to obstruct the normal cell cycle.ATO can down-regulate the expressions of BCL-2 and MCL-1 genes and up-regulate the expressions of BAX and BIM genes,thereby enhancing the effect of VEN.