阿帕替尼抑制鼻咽癌CNE-2Z细胞的生长及机制研究

Study on Apatinib Inhibiting the Growth of Nasopharyngeal Carcinoma CNE-2Z Cells and Its Mechanism

关键词:  阿帕替尼  鼻咽癌  CNE-2Z细胞  PI3K/Akt  凋亡  增殖

Key words:  apatinib  nasopharyngeal carcinoma  CNE-2Z cells  PI3K/Akt  apoptosis  proliferation

摘要:

目的 探讨阿帕替尼对鼻咽癌CNE-2Z细胞生长抑制作用及其可能机制。方法 将鼻咽癌CNE-2Z细胞与不同浓度阿帕替尼（0，0.5，1，2 μmol·L^-1）共培养48 h，并分别定义为0，0.5，1，2 μmol·L^-1组。MTT法检测细胞生长抑制率，Annexin V-FITC/PI联合流式细胞仪检测细胞凋亡率，Western blotting检测Bcl-2、Bax、caspase-3、caspase-9、PI3K、p-PI3K、Akt、p-Akt蛋白表达，RT-PCR检测p-PI3K、p-Akt mRNA表达。按药物不同，将鼻咽癌CNE-2Z细胞分为4组：对照组、阿帕替尼组、740YP组、阿帕替尼+740YP组，48 h后Annexin V-FITC/PI联合流式细胞仪检测细胞凋亡率，Western blotting检测p-PI3K和p-Akt蛋白表达。结果 MTT检测结果表明，阿帕替尼呈浓度依赖性抑制CNE-2Z细胞的增殖（P<0.05），阿帕替尼对鼻咽癌细胞的半数抑制浓度（IC₅₀值）为1.518 μmol·L^-1。Annexin-V/PI联合流式细胞仪检测结果表明，阿帕替尼呈浓度依赖性诱导CNE-2Z细胞凋亡（P<0.05）。Western blotting检测结果表明，阿帕替尼呈剂量依赖性地促进促Bax、caspase-3和caspase-9的表达（P<0.05），抑制Bcl-2的表达（P<0.05）。阿帕替尼能抑制p-PI3K、p-Akt的蛋白和mRNA表达（P<0.05），但对PI3K、Akt蛋白表达无影响。细胞培养48 h后，对照组细胞凋亡率为（10.5±0.96）%，阿帕替尼组为（25.3±1.67）%，740YP组为（6.30±0.52）%，阿帕替尼+740YP组为（11.9±0.61）%，与对照组相比，阿帕替尼组凋亡率上调（P<0.05），740YP组凋亡率下调（P<0.05），阿帕替尼和740YP联用时，凋亡率比740YP组上调（P<0.05）。与对照组相比，阿帕替尼组p-PI3K、p-Akt蛋白表达下调（P<0.05），740YP组p-PI3K、p-Akt蛋白表达上调（P<0.05），阿帕替尼和740YP联用时，p-PI3K、p-Akt蛋白表达比740YP组下调（P<0.05）。结论 阿帕替尼可通过抑制PI3K/Akt信号转导通路而诱导鼻咽癌CNE-2Z细胞凋亡，从而达到抑制其增殖的目的。

Abstract:

OBJECTIVE To explore the inhibitory effect of apatinib on nasopharyngeal carcinoma CNE-2Z cells and its possible mechanism. METHODS Nasopharyngeal carcinoma CNE-2Z cells combined with different concentrations of apatinib(0, 0.5, 1, 2 μmol·L^-1) were co-cultured for 48 h, and defined as 0, 0.5, 1 and 2 μmol·L^-1 group, respectively. MTT method was used to detect cell growth inhibition rate. Annexin V-FITC/PI combined with flow cytometry were used to detect cell apoptosis rate. Western blotting was used to detect Bcl-2, Bax, caspase-3, caspase-9, PI3K, p-PI3K, Akt, p-Akt protein expression. RT-PCR was used to detect p-PI3K, p-Akt mRNA expression. According to different drugs, the nasopharyngeal carcinoma CNE-2Z cells were diveded into 4 groups:control group, apatinib group, 740YP group, apatinib+740 YP group, 48 h later, Annexin V-FITC/PI combined with flow cytometry were used to detect cells apoptosis rate, Western blotting was used to detect the expression of p-PI3K and p-Akt protein. RESULTS MTT test results showed that apatinib inhibited the proliferation of CNE-2Z cells in a concentration-dependent manner(P<0.05), and the half-inhibitory concentration(IC₅₀ value) of apatinib on nasopharyngeal carcinoma cells was 1.518 μmol·L^-1. Annexin-V/PI combined with flow cytometry showed that apatinib induced apoptosis of CNE-2Z cells in a concentration-dependent manner(P<0.05). The result of Western blotting showed that apatinib promoted the expression of Bax, caspase-3 and caspase-9 in a dose-dependent manner(P<0.05), and inhibited the expression of Bcl-2(P<0.05). Apatinib could inhibit the expression of p-PI3K and p-Akt proteins and mRNA(P<0.05), but has no effect on the expression of PI3K and Akt proteins. After cell culture for 48 h, the apoptosis rate of the control group was (10.5±0.96)%, the apatinib group was (25.3±1.67)%, the 740YP group was (6.30±0.52)%, the apatinib+740YP group was (11.9±0.61)%. Compared with the control group, the apoptosis rate in the apatinib group was increased(P<0.05), while it was decreased in the 740YP group(P<0.05). When apatinib and 740YP were used in combination, the apoptosis rate was higher than that in the 740YP group(P<0.05). Compared with the control group, the expression of p-PI3K and p-Akt protein in the apatinib group was down-regulated(P<0.05), while they were up-regulated in the 740YP group (P<0.05). When apatinib and 740YP was used in combination, the expression of p-PI3K and p-Akt was down-regulated than that in the 740YP group(P<0.05). CONCLUSION Apatinib can induce apoptosis of nasopharyngeal carcinoma CNE-2Z cells by inhibiting the PI3K/Akt signal transduction pathway, thereby achieving the purpose of inhibiting its proliferation.