AXL表达在前列腺癌细胞多西他赛耐药中的作用研究

Expression of AXL enhances docetaxel-resistance of prostate cancer cells

目的:观察AXL表达在前列腺癌PC-3和DU145细胞多西他赛化疗耐药中的作用及可能机制。方法:应用Western印迹测定多西他赛刺激PC-3和DU145后AXL蛋白表达,通过多西他赛浓度递增间断刺激PC-3和DU145细胞,建立耐药细胞PC-3-DR和DU145-DR,应用Western印迹测定PC-3和DU145细胞耐药前后AXL,AXL磷酸化蛋白激酶(p-AXL)及配体蛋白生长阻滞特异性因子-6(Gas6)蛋白表达。用脂质体Lipofectamine 2000将经过筛选证实有效的AXL-shRNA序列和阴性NCshRNA序列转染PC-3和DU145细胞,应用CCK8和流式细胞仪检测转染前后加入多西他赛后的细胞增殖和凋亡情况。应用MTT和流式细胞仪检测加入AXL抑制剂MP470和多西他赛单独及联合应用的细胞增殖率、凋亡率和细胞周期分布。Western印迹检测AXL抑制剂R428,多西他赛单独或联合处理耐药细胞后ABCB1的表达情况。结果:多西他赛刺激PC-3和DU145细胞后,AXL表达上升(P<0.05);与PC-3和DU145细胞相比,PC-3-DR和DU145-DR中AXL,p-AXL蛋白表达明显上升,Gas6蛋白表达明显下降(P均<0.05)。AXL转染PC-3和DU145后的细胞经多西他赛处理48 h,细胞增殖率分别为(51.03±3.16)%和(57.39±2.37)%,明显高于未转染细胞(36.41±4.28)%和(45.5±3.93)%(P<0.05),转染后细胞凋亡率分别为(42.37±3.43)%和(39.54±2.39)%,明显低于未转染细胞(65.48±3.16)%和(54.98±2.84)%(P<0.05)。MP470可明显抑制细胞增殖和促进耐药细胞凋亡,MP470与多西他赛联合应用后的耐药细胞增殖抑制率和凋亡率明显高于单独应用,联合应用后G2/M期细胞百分数亦明显高于单独应用(P均<0.05)。R428可明显降低耐药细胞的ABCB1表达,与多西他赛联用后,ABCB1的蛋白表达水平明显低于单独用药组(P均<0.05)。结论:AXL表达上调可促进前列腺癌PC-3和DU145细胞耐药,AXL抑制可增加细胞对多西他赛的敏感性,这一作用可能与G2/M期细胞凋亡率升高和ABCB1表达降低有关。

Objective ： To explore the effect of the AXL expression on the chemosensitivity of prostate cancer PC-3 and DU145 cells to docetaxel and possible mechanisms. Methods ： Using Western blot, we examined the expressions of the AXL protein, p-AXL and Gas6 in the docetaxel-resistant PC-3 （PC-3-DR） and DU145 （DU145-DR） cells stimulated with gradually increased concentra- tions of docetaxel. We transfected the PC-3 and DU145 cells with negative NC ShRNA and AXL-ShRNA, respectively, which were confirmed to be effective, detected the proliferation, apoptosis and cycle distribution of the cells by CCK8, MTT and flow cytometry af- ter treated with the AXL-inhibitor MP470 and/or docetaxel, and determined the expression of the ABCB1 protein in the PC-3-DR and DU145-DR cells after intervention with the AXL-inhibitor R428 and/or docetaxel. Results ： The expression of the AXL protein in the PC-3 and DU145 cells was significantly increased after docetaxel treatment （ P 〈 0.05 ）. The expressions AXL and p-AXL were re- markably higher （P 〈 0.05 ） while that of Gas6 markedly lower （P 〈 0.05 ） in the PC-3 and DU145 than in the PC-3-DR and DU145- DR cells. The inhibitory effect of docetaxel on the proliferation and its enhancing effect on the apoptosis of the PC-3 and DU145 cells were significantly decreased at 48 hours after AXL transfection （ P 〈 0.05 ）. MP470 obviously suppressed the growth and promoted the apoptosis of the PC-3-DR and DU145-DR ceils, with a higher percentage of the cells in the G2/M phase when combined with docetaxel than used alone （P 〈 0.05）. R428 markedly reduced the expression of ABCB1 in the PC-3-DR and DU145-DR cells, even more sig- nificantly in combination with docetaxel than used alone （ P 〈 0.05 ）. Conclusion ： The elevated expression of AXL enhances the do- cetaxel-resistance of PC-3 and DU145 prostate cancer ceils and AXL intervention improves their chemosensitivity to docetaxel, which may be associated with the increased cell apoptosis in the G2/M phase and decreased expression of ABCB1.