【摘要】：目的:觀察AXL表達(dá)在前列腺癌PC-3和DU145細(xì)胞多西他賽化療耐藥中的作用及可能機(jī)制。方法:應(yīng)用Western印跡測定多西他賽刺激PC-3和DU145后AXL蛋白表達(dá),通過多西他賽濃度遞增間斷刺激PC-3和DU145細(xì)胞,建立耐藥細(xì)胞PC-3-DR和DU145-DR,應(yīng)用Western印跡測定PC-3和DU145細(xì)胞耐藥前后AXL,AXL磷酸化蛋白激酶(p-AXL)及配體蛋白生長阻滯特異性因子-6(Gas6)蛋白表達(dá)。用脂質(zhì)體Lipofectamine 2000將經(jīng)過篩選證實(shí)有效的AXL-shRNA序列和陰性NCshRNA序列轉(zhuǎn)染PC-3和DU145細(xì)胞,應(yīng)用CCK8和流式細(xì)胞儀檢測轉(zhuǎn)染前后加入多西他賽后的細(xì)胞增殖和凋亡情況。應(yīng)用MTT和流式細(xì)胞儀檢測加入AXL抑制劑MP470和多西他賽單獨(dú)及聯(lián)合應(yīng)用的細(xì)胞增殖率、凋亡率和細(xì)胞周期分布。Western印跡檢測AXL抑制劑R428,多西他賽單獨(dú)或聯(lián)合處理耐藥細(xì)胞后ABCB1的表達(dá)情況。結(jié)果:多西他賽刺激PC-3和DU145細(xì)胞后,AXL表達(dá)上升(P0.05);與PC-3和DU145細(xì)胞相比,PC-3-DR和DU145-DR中AXL,p-AXL蛋白表達(dá)明顯上升,Gas6蛋白表達(dá)明顯下降(P均0.05)。AXL轉(zhuǎn)染PC-3和DU145后的細(xì)胞經(jīng)多西他賽處理48 h,細(xì)胞增殖率分別為(51.03±3.16)%和(57.39±2.37)%,明顯高于未轉(zhuǎn)染細(xì)胞(36.41±4.28)%和(45.5±3.93)%(P0.05),轉(zhuǎn)染后細(xì)胞凋亡率分別為(42.37±3.43)%和(39.54±2.39)%,明顯低于未轉(zhuǎn)染細(xì)胞(65.48±3.16)%和(54.98±2.84)%(P0.05)。MP470可明顯抑制細(xì)胞增殖和促進(jìn)耐藥細(xì)胞凋亡,MP470與多西他賽聯(lián)合應(yīng)用后的耐藥細(xì)胞增殖抑制率和凋亡率明顯高于單獨(dú)應(yīng)用,聯(lián)合應(yīng)用后G2/M期細(xì)胞百分?jǐn)?shù)亦明顯高于單獨(dú)應(yīng)用(P均0.05)。R428可明顯降低耐藥細(xì)胞的ABCB1表達(dá),與多西他賽聯(lián)用后,ABCB1的蛋白表達(dá)水平明顯低于單獨(dú)用藥組(P均0.05)。結(jié)論:AXL表達(dá)上調(diào)可促進(jìn)前列腺癌PC-3和DU145細(xì)胞耐藥,AXL抑制可增加細(xì)胞對多西他賽的敏感性,這一作用可能與G2/M期細(xì)胞凋亡率升高和ABCB1表達(dá)降低有關(guān)。
[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 鹵3.16)% and (57.39 鹵2.37)%, respectively, which were significantly higher than those of untransfected cells (36.41 鹵4.28)% and (45.5 鹵3.93)% (P0.05). After transfection, the apoptotic rates were (42.37 鹵3.43)% and (39.54 鹵2.39)%, respectively. Compared with untransfected cells (65.48 鹵3.16)% and (54.98 鹵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.
【作者單位】： 南京醫(yī)科大學(xué)附屬南京明基醫(yī)院泌尿外科;南京醫(yī)科大學(xué)附屬南京醫(yī)院泌尿外科;南京醫(yī)科大學(xué)生物化學(xué)與分子生物學(xué)系;南京醫(yī)科大學(xué)第一附屬醫(yī)院泌尿外科;
【基金】：江蘇省自然科學(xué)基金(BK20161108) 南京市科技發(fā)展項(xiàng)目(201503063) 南京市醫(yī)學(xué)科技發(fā)展項(xiàng)目(YKK15092)~~
【分類號】：R737.25
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本文編號：2404607