西达本胺靶向P21调控DHFR表达逆转T淋巴细胞白血病甲氨蝶呤耐药的初步探究

The reversal of methotrexate resistance in T⁃lymphocyte leukemia by targeting P21 with chidamide to regulate DHFR expression

目的探究西达本胺(CDM)靶向P21调控二氢叶酸还原酶(DHFR)表达逆转T淋巴细胞白血病(T⁃ALL)甲氨蝶呤(MTX)的耐药机制,为T⁃ALL靶向治疗提供参考依据。方法将过表达载体(ov⁃DHFR)转染T⁃ALL细胞系Jurkat细胞,Western blot检测细胞中DHFR蛋白表达水平。将转染ov⁃DHFR载体的Jurkat细胞采用不同浓度(5、25和40μmol/L)的MTX处理、0.5μmol/L CDM处理以及不同浓度(5、25和40μmol/L)的MTX联合0.5μmol/L CDM进行处理。采用25μmol/L MTX、0.5μmol/L CDM和25μmol/L MTX+0.5μmol/L CDM联合处理进行增殖、周期和蛋白检测。MTS实验检测转染ov⁃DHFR载体的Jurkat细胞对MTX耐药增殖,并计算相应的半抑制浓度(IC50)值。MTS、流式细胞和Western blot实验分别检测CDM+MTX处理对过表达DHFR的Jurkat细胞的增殖、周期及细胞中耐药蛋白和周期蛋白表达的影响。在MTX+CDM处理的过表达DHFR的Jurkat细胞增加P21抑制剂处理,MTS、流式细胞和Western blot实验分别检测CDM+MTX+P21抑制剂处理对过表达DHFR的Jurkat细胞的增殖、周期及细胞中耐药蛋白和周期蛋白表达的影响。结果与转染阴性对照载体(ov⁃NC)组细胞MTX IC50值相比,转染ov⁃DHFR组Jurkat细胞的MTX IC50值变大。不同浓度的MTX和CDM处理发现25和40μmol/L MTX+CDM组的治疗能够最显著地抑制过表达DHFR的Jurkat细胞的增殖,而且这两组治疗效果相似,因此后续采用25μmol/L MTX+CDM组进行分析。与cell组相比,MTX组、CDM组和MTX+CDM组的过表达DHFR的Jurkat细胞在24、48、72 h这3个时间点,细胞的吸光度值均显著下降,差异均有统计学意义(F=40.180、378.900、510.700,P均<0.001)。G1期的细胞比例均显著上升,而MTX+CDM组中表现最显著(F=7.419,P<0.001)。Cyclin D、p⁃GP、BCRP蛋白表达水平均显著下调,且在MTX+CDM组细胞中表现最显著;此外,P21蛋白表达水平均显著增加,且在MTX+CDM组细胞中表现最显著。与MTX+CDM+PBS组相比,MTX+CDM+P21抑制剂组ov⁃DHFR⁃Jurkat细胞在48、72 h这2个时间点,细胞的吸光度值均显著上升,差异均有统计学意义(F=66.690、68.400,P均<0.001)。与MTX+CDM+PBS组相比,MTX+CDM+P21抑制剂组ov⁃DHFR⁃Jurkat细胞中G1期的细胞比例显著降低,差异有统计学意义(P<0.019);细胞中cyclin D、p⁃GP、BCRP蛋白表达水平均显著上调。结论CDM靶向P21调控DHFR表达逆转T⁃ALL的MTX耐药。

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 40μmol/L)of MTX,0.5μmol/L CDM,as well as combinations of different concentrations(5,25,and 40μmol/L)of MTX with 0.5μmol/L CDM.25μmol/L MTX,0.5μmol/L CDM,and the combination of 25μmol/L MTX with 0.5μmol/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(IC50)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 40μmol/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 25μmol/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.