Zhu Xin, Zhou Mi, Liu Guanyu, Huang Xiaolong, He Weiyang, Gou Xin, Jiang Tao
Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China.
Department of Respiratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China.
Exp Ther Med. 2017 May;13(5):2348-2354. doi: 10.3892/etm.2017.4287. Epub 2017 Mar 30.
The aim of the present study was to investigate the role of autophagy in celecoxib-induced apoptosis in human hormone-insensitive prostate cancer cell line PC3 cells and to explore the underlying molecular mechanism leading to autophagic activation. A cell viability assay was applied to investigate the effect of various concentrations of celecoxib (0, 40, 60, 80, 100 and 120 µmol/l) on PC3 cells for 24 and 48 h, respectively. The 50% inhibitory concentration of celecoxib for 24 h was chosen for subsequent experiments. Annexin V-fluorescein isothiocyanate/propidium iodide double staining flow cytometry, as well as caspase 3 and poly (ADP-ribose) polymerase proteins detected by western blotting, were applied to analyze cellular apoptosis induced by celecoxib. Ultrastructural cellular changes observed by transmission electron microscopy and the level of LC-3 II and P62 detected by western blotting were used to determine the activation of autophagy. It was demonstrated that celecoxib induced apoptosis and activated autophagy in PC3 cells in a dose- and time-dependent manner. Furthermore, flow cytometry and western blotting were applied to elucidate whether the role of autophagy in celecoxib-induced apoptosis is protective or destructive. Blockade of autophagy markedly increased apoptosis, suggesting that celecoxib-activated autophagy was cytoprotective. Additionally, c-jun-N-terminal kinase (JNK) was demonstrated to have a role in autophagic activation, and suppression of JNK was able to reduce autophagy and increase apoptosis. In conclusion, the results of the present study indicate that celecoxib induces apoptosis in PC3 cells; however, celecoxib also activates JNK-mediated autophagy, which exerts cytoprotective effects in prostate cancer PC3 cells. Blockade of autophagy via the JNK-mediated pathway may provide a promising strategy for prostate cancer therapy.
本研究的目的是探讨自噬在塞来昔布诱导人激素不敏感前列腺癌细胞系PC3细胞凋亡中的作用，并探索导致自噬激活的潜在分子机制。应用细胞活力测定法分别研究不同浓度（0、40、60、80、100和120 μmol/l）的塞来昔布作用于PC3细胞24小时和48小时的效果。选择塞来昔布24小时的50%抑制浓度用于后续实验。采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶双染流式细胞术以及通过蛋白质印迹法检测的半胱天冬酶3和聚（ADP-核糖）聚合酶蛋白，来分析塞来昔布诱导的细胞凋亡。通过透射电子显微镜观察细胞超微结构变化以及用蛋白质印迹法检测LC-3 II和P62的水平，来确定自噬的激活情况。结果表明，塞来昔布以剂量和时间依赖性方式诱导PC3细胞凋亡并激活自噬。此外，应用流式细胞术和蛋白质印迹法来阐明自噬在塞来昔布诱导的凋亡中是起保护作用还是破坏作用。阻断自噬显著增加凋亡，表明塞来昔布激活的自噬具有细胞保护作用。此外，已证明c-Jun氨基末端激酶（JNK）在自噬激活中起作用，抑制JNK能够减少自噬并增加凋亡。总之，本研究结果表明，塞来昔布诱导PC3细胞凋亡；然而，塞来昔布也激活JNK介导的自噬，这在前列腺癌PC3细胞中发挥细胞保护作用。通过JNK介导的途径阻断自噬可能为前列腺癌治疗提供一种有前景的策略。
