AXL表达在前列腺癌细胞多西他赛耐药中的作用研究
发布时间：2019-01-08 12:39
【摘要】：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(P0.05);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)%(P0.05),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)%(P0.05)。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。
[Abstract]:Aim: to investigate the role of AXL expression in docetaxel chemotherapeutic resistance of prostate cancer PC-3 and DU145 cells and its possible mechanism. Methods: Western blot was used to detect the expression of AXL protein in PC-3 and DU145 stimulated by docetaxel. PC-3 and DU145 cells were stimulated by increasing docetaxel concentration. PC-3-DR and DU145-DR, resistant cells were established. The expression of AXL,AXL phosphorylated protein kinase (p-AXL) and ligand protein growth retardation factor 6 (Gas6) in PC-3 and DU145 cells before and after drug resistance were detected by Western blot. Liposome Lipofectamine 2000 was used to transfect AXL-shRNA and negative NCshRNA sequences into PC-3 and DU145 cells. The proliferation and apoptosis of PC-3 and DU145 cells were detected by CCK8 and flow cytometry. Cell proliferation rate, apoptosis rate and cell cycle distribution were detected by MTT and flow cytometry. AXL inhibitor R428 was detected by Western blot. Expression of ABCB1 in resistant cells treated with docetaxel alone or in combination. Results: after docetaxel stimulated PC-3 and DU145 cells, the expression of AXL increased (P0.05). Compared with PC-3 and DU145 cells, the expression of AXL,p-AXL protein increased significantly in PC-3-DR and DU145-DR, and the expression of Gas6 protein decreased significantly (P0. 05). AXL transfected PC-3 and DU145 cells were treated with docetaxel for 48 h. The cell proliferation rates were (51.03 卤3.16)% and (57.39 卤2.37)%, respectively, which were significantly higher than those of untransfected cells (36.41 卤4.28)% and (45.5 卤3.93)% (P0.05). After transfection, the apoptotic rates were (42.37 卤3.43)% and (39.54 卤2.39)%, respectively. Compared with untransfected cells (65.48 卤3.16)% and (54.98 卤2.84)% (P0.05), MP470 significantly inhibited cell proliferation and promoted apoptosis of drug-resistant cells. The inhibition rate of proliferation and apoptosis of drug-resistant cells treated with MP470 combined with docetaxel was significantly higher than that of single administration. The percentage of cells in G _ 2 / M phase was significantly higher than that in control group (P < 0.05). R428 could significantly reduce the expression of ABCB1 in drug-resistant cells, and after combined with docetaxel, R428 could significantly reduce the expression of ABCB1 in drug-resistant cells. The protein expression level of ABCB1 was significantly lower than that of the control group (all P 0. 05). Conclusion: upregulation of AXL expression can promote drug resistance in PC-3 and DU145 cells of prostate cancer, and inhibition of AXL can increase the sensitivity of cells to docetaxel, which may be related to the increase of apoptosis rate in G 2 / M phase and the decrease of ABCB1 expression.
【作者单位】： 南京医科大学附属南京明基医院泌尿外科;南京医科大学附属南京医院泌尿外科;南京医科大学生物化学与分子生物学系;南京医科大学第一附属医院泌尿外科;
【基金】：江苏省自然科学基金(BK20161108) 南京市科技发展项目(201503063) 南京市医学科技发展项目(YKK15092)~~
【分类号】：R737.25
，
本文编号：2404607
[Abstract]:Aim: to investigate the role of AXL expression in docetaxel chemotherapeutic resistance of prostate cancer PC-3 and DU145 cells and its possible mechanism. Methods: Western blot was used to detect the expression of AXL protein in PC-3 and DU145 stimulated by docetaxel. PC-3 and DU145 cells were stimulated by increasing docetaxel concentration. PC-3-DR and DU145-DR, resistant cells were established. The expression of AXL,AXL phosphorylated protein kinase (p-AXL) and ligand protein growth retardation factor 6 (Gas6) in PC-3 and DU145 cells before and after drug resistance were detected by Western blot. Liposome Lipofectamine 2000 was used to transfect AXL-shRNA and negative NCshRNA sequences into PC-3 and DU145 cells. The proliferation and apoptosis of PC-3 and DU145 cells were detected by CCK8 and flow cytometry. Cell proliferation rate, apoptosis rate and cell cycle distribution were detected by MTT and flow cytometry. AXL inhibitor R428 was detected by Western blot. Expression of ABCB1 in resistant cells treated with docetaxel alone or in combination. Results: after docetaxel stimulated PC-3 and DU145 cells, the expression of AXL increased (P0.05). Compared with PC-3 and DU145 cells, the expression of AXL,p-AXL protein increased significantly in PC-3-DR and DU145-DR, and the expression of Gas6 protein decreased significantly (P0. 05). AXL transfected PC-3 and DU145 cells were treated with docetaxel for 48 h. The cell proliferation rates were (51.03 卤3.16)% and (57.39 卤2.37)%, respectively, which were significantly higher than those of untransfected cells (36.41 卤4.28)% and (45.5 卤3.93)% (P0.05). After transfection, the apoptotic rates were (42.37 卤3.43)% and (39.54 卤2.39)%, respectively. Compared with untransfected cells (65.48 卤3.16)% and (54.98 卤2.84)% (P0.05), MP470 significantly inhibited cell proliferation and promoted apoptosis of drug-resistant cells. The inhibition rate of proliferation and apoptosis of drug-resistant cells treated with MP470 combined with docetaxel was significantly higher than that of single administration. The percentage of cells in G _ 2 / M phase was significantly higher than that in control group (P < 0.05). R428 could significantly reduce the expression of ABCB1 in drug-resistant cells, and after combined with docetaxel, R428 could significantly reduce the expression of ABCB1 in drug-resistant cells. The protein expression level of ABCB1 was significantly lower than that of the control group (all P 0. 05). Conclusion: upregulation of AXL expression can promote drug resistance in PC-3 and DU145 cells of prostate cancer, and inhibition of AXL can increase the sensitivity of cells to docetaxel, which may be related to the increase of apoptosis rate in G 2 / M phase and the decrease of ABCB1 expression.
【作者单位】： 南京医科大学附属南京明基医院泌尿外科;南京医科大学附属南京医院泌尿外科;南京医科大学生物化学与分子生物学系;南京医科大学第一附属医院泌尿外科;
【基金】：江苏省自然科学基金(BK20161108) 南京市科技发展项目(201503063) 南京市医学科技发展项目(YKK15092)~~
【分类号】：R737.25
，
本文编号：2404607
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