Effect And Mechanism Of Harmine Sensitization Of Bcl-2 Inhibitor Against Non-small Cell Lung Cancer

Objective: Chemotherapy is currently the main treatment for non-small cell lung cancer(NSCLC),and tumor resistance is the main cause of poor chemotherapy efficacy.A large number of studies have found that the increase of anti-apoptotic protein Bcl-2 expression is usually associated with NSCLC chemotherapy tolerance and poor prognosis.Bcl-2 protein small molecule inhibitor is a new research direction of current anti-tumor drugs.However,high expression of Mcl-1 often causes solid tumor resistance of Bcl-2 inhibitors(ABT-199/ABT-737).Harmine is an inhibitor of DYRK1 A kinase,which has anti-tumor proliferation and induces tumor cell apoptosis.The aim of this study was to investigate the anti-NSCLC activity of Harmine-sensitized Bcl-2 inhibitors and the specific molecular mechanism of down-regulating Mcl-1 protein expression,and to provide theoretical basis and experimental data for expanding the clinical application of the two drugs.Methods: SRB method was used to investigate the proliferation inhibition effect of Harmine,ABT-199/ABT-737 alone or in combination on NSCLC cells(A549,NCI-H460,NCI-H1299 and Primary 1#,Primary 2#,Primary 3# and Primary 4#).The effects of colony formation assay on the proliferation of NSCLC cells were observed.The changes of cell morphology after treatment were observed by microscopy.The effects of Harmine and Bcl-2 inhibitors on tumor cell apoptosis were detected by flow cytometry.Western Blotting was used to detect the expression of related proteins such as Cleaved-PARP,Caspase,Cleaved-Caspase-3,DYRK1 A,Mcl-1,Bcl-2 and Bcl-xl.The expression of target protein(Mcl-1)was amplified by plasmid transfection.RT-PCR was used to detect the effects of si DYRK1 A or Harmine on DYRK1 A and Mcl-1 m RNA levels;si RNA was used to silence DYRK1 A gene expression.Results: SRB assay showed that Harmine could significantly enhance the sensitivity of Bcl-2 inhibitor to the proliferation of NSCLC cells in vitro.The average CI values of Harmine combined with ABT-199 in A549,NCI-H460 and NCI-H1299 cells were 0.80,0.85 and 0.68,respectively.The average CI values of Harmine and ABT-737 in the three cell lines were 0.87,0.77 and 0.61,respectively;It also had a good combination effect in primary NSCLC cells.The average CI values of Harmine combined with ABT-199 in Primary 1 #,Primary 2 #,Primary 3 # and Primary 4 # cells were 0.81,0.86,0.85 and 0.84,respectively.The average CI values of Harmine combined with ABT-737 in four primary NSCLC cells were 0.73,0.87,0.67 and 0.81,respectively.Therefore,the combination of Harmine and Bcl-2 inhibitor has a good synergistic inhibitory effect on the proliferation of NSCLC cells.By using the cell cloning experiment,we found that the clonal inhibition ability of NSCLC cells in the combined group was significantly higher than that in the single group.At the same time,the combination of Harmine and Bcl-2 inhibitor could induce apoptosis of A549,NCI-H460 and Primary 3 # cells,accompanied by the activation of Caspase-3 and PARP.On A549 cell line,after 48 h of drug treatment,the apoptosis rate of Harmine single use group was 21.40±10.04%,the apoptosis rate of ABT-199 single use group was 34.05±1.91%,and the apoptosis rate of ABT-737 alone group was 34.31.00±0.28 %,and the apoptotic rate of the combined group reached 86.05±3.18% and 76.55±1.20%,respectively.On the NCI-H460 cell line,the apoptosis rate of the Harmine single use group was 38.55±7.00%,the apoptosis rate of the ABT-199 single use group was 19.25±0.35%,and the apoptosis rate of the ABT-737 single use group was 10.40±0.42%.The apoptotic rate of the combined group reached 77.10±8.63% and 72.55±13.51%,respectively.The apoptosis rate of Primary 3# cells treated with Harmine(5 mM)and ABT-199(5 mM)was 14.80%,35.8%,while the apoptotic rate of the combined group reached 52.7%.The combination of Harmine and Bcl-2 inhibitor could promote the down-regulation of Mcl-1 protein.Overexpression of Mcl-1 could weaken the inhibitory effect of ABT-199 on proliferation.The survival rate of control group was 18.83 ±0.92% when treated with 2.5 mM ABT-737,while the survival rate of Primary 3 # transfected with p TOPO-Mcl-1 plasmid was 49.46 ±2.21%.Compared with the control group,The survival rate of Primary 3 # cells with high expression of Mcl-1 was significantly higher.Harmine could promote the degradation of Mcl-1 protein in NSCLC cells,thus significantly down-regulated the expression of Mcl-1 protein.The level of Mcl-1 protein in NSCLC cells was down-regulated by si RNA knockdown of DYRK1 A,and the anti-NSCLC activity of Bcl-2 inhibitor was weakened by overexpression of DYRK1 A.Conclusion: 1.Harmine,a DYRK1 A kinase inhibitor,potentiates the proliferation of Bcl-2 inhibitors against NSCLC cells.2.DYRK1 A may be involved in Harmine to promote the degradation of Mcl-1 protein,thereby enhancing the role of Bcl-2 inhibitors.