The reversal of methotrexate resistance in T-lymphocyte leukemia by targeting P21 with chidamide to regulate DHFR expression
Objective To investigate the mechanism of chidamide(CDM)targets P21 to regulate dihydro-folate reductase(DHFR)expression and reverse methotrexate(MTX)resistance in acute T lymphoblastic leukemia(T-ALL).Methods The overexpression vector(ov-DHFR)was transfected into the T-ALL cell lines Jurkat,and the protein expression level of DHFR was detected by western blot.Jurkat cells transfected with the ov-DHFR vector were treated with different concentrations(5,25,and 40 μmol/L)of MTX,0.5 μmol/L CDM,as well as combinations of different concentrations(5,25,and 40 µmol/L)of MTX with 0.5 μmol/L CDM.25 μmol/L MTX,0.5 μmol/L CDM,and the combination of 25 μmol/L MTX with 0.5 μmol/L CDM were used for the studies of proliferation,cell cycle,and protein expression.The proliferation of Jurkat cells transfected with ov-DHFR vector was detected by MTS assay and 50％inhibitory concentration(1C50)to MTX was calculated.The effects of CDM+MTX treatment on the proliferation,and cycle of DHFR-overexpressed Jurkat cells and the expression of drug-resistant protein and cyclin were detected by MTS,flow cytometry and western blot assay,respectively.MTX+CDM treated Jurkat cells overexpressing DHFR were treated with P21 inhibitor.The effects of CDM+MTX+P21 inhibitor treatment on the proliferation and cycle of DHFR-overexpressed Jurkat cells and the expression of drug-resistant protein and cyclin were detected by MTS,flow cytometry and western blot assay,respectively.Results The MTX IC50 value of Jurkat cells transfected with ov-DHFR was significantly higher than that of ov-NC group.Treatment with different concentrations of MTX and CDM revealed that the 25 and 40 µmol/L MTX+CDM combination most significantly inhibited the proliferation of Jurkat cells overexpressing DHFR.Moreover,the therapeutic effects of these two groups were similar.Therefore,subsequent analysis was performed using the 25 μmol/L MTX+CDM combination.Compared with cell group,the absorbance value of Jurkat cells overexpressing DHFR in MTX group,CDM group and MTX+CDM group decreased significantly at 24,48 and 72 h,but the expression was most significant in MTX+CDM group,the differences were statistically significant(F=40.180,378.900,510.700;all P＜0.001).The proportion of cells in G1 phase increased significantly,and the MTX+CDM group was the most significant(F=7.419,P＜0.001).The expression levels of cyclin D,p-GP and BCRP were significantly down-regulated,and the expression levels were most significant in MTX+CDM group.In addition,the expression level of P21 protein was significantly increased,and the expression was most significant in the MTX+CDM group.Compared with the MTX+CDM+PBS group,the absorbance of Jurkat cells overexpressing DHFR in the MTX+CDM+P21 inhibitor group increased significantly at 48 and 72 h,the differences were statistically significant(F=66.690,68.400;all P＜0.001),the proportion of cells in G1 phase decreased significantly(P＜0.019)and the expression levels of cyclin D,p-GP and BCRP in the cells were significantly up-regulated.Conclusion CDM targets P21 to regulate DHFR expression and reverse MTX resistance in T lymphocyte leukemia.
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