Liu Shuai, Wang Xiaoqing, Lu Jiaju, Han Liping, Zhang Yongfei, Liu Zheng, Ding Sentai, Liu Zhao, Bi Dongbin, Niu Zhihong
Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
Department of Neurology, Shandong Police Hospital, Jinan, Shandong 250021, P.R. China.
Oncol Lett. 2016 Nov;12(5):3403-3410. doi: 10.3892/ol.2016.5036. Epub 2016 Aug 22.
Renal cell carcinoma (RCC) is resistant to standard radiotherapy. Ubenimex, an aminopeptidase N inhibitor, is widely used as an adjunct therapy after surgery to enhance the function of immunocompetent cells and confer antitumor effects. Our previous study demonstrated that ubenimex induces autophagic cell death in RCC cells. Recently, the molecular mechanism of autophagy induction has been associated with radiosensitivity in RCC cells. In the present study, the ability of ubenimex to enhance RCC cell sensitivity to radiation via the induction of autophagic cell death was determined, and the mechanism of action of this effect was investigated. The 786-O and OS-RC-2 human RCC cell lines were treated with 0.5 mg/ml ubenimex and different doses of irradiation (IR). The cell viability was measured using a colony-formation assay and flow cytometry. Acridine orange (AO)-ethidium bromide (EB) staining was assessed by fluorescence microscopy as an indicator of autophagic cell death. Protein expression was assessed by western blotting. Autophagosomes were evaluated using transmission electron microscopy. RCC cells were used to evaluate the sensitivity to radiation using clonogenic survival and lactate dehydrogenase assays. Furthermore, these parameters were also tested at physiological oxygen levels. The AO-EB staining and flow cytometry of the OS-RC-2 cells indicated that the combined treatment significantly enhanced autophagic cell death compared with ubenimex or IR alone. Therefore, treatment with ubenimex did not significantly alter cell cycle progression but increased cell death when combined with radiation. An Akt agonist could significantly weaken this effect, indicating that ubenimex may act as an Akt inhibitor. Furthermore, the western blot analysis indicated that the combined treatment inhibited the Akt signaling pathway compared with ubenimex treatment or IR alone. Ubenimex may enhance RCC cell sensitivity to radiation by inducing cell autophagy. This induction changes the role of autophagy from protective to lethal , and this switch is associated with the inhibition of the Akt signaling pathway.
肾细胞癌（RCC）对标准放疗具有抗性。乌苯美司是一种氨肽酶N抑制剂，广泛用作术后辅助治疗，以增强免疫活性细胞的功能并赋予抗肿瘤作用。我们之前的研究表明，乌苯美司可诱导肾癌细胞发生自噬性细胞死亡。最近，自噬诱导的分子机制已与肾癌细胞的放射敏感性相关。在本研究中，确定了乌苯美司通过诱导自噬性细胞死亡来增强肾癌细胞对辐射敏感性的能力，并研究了这种作用的机制。用0.5mg/ml乌苯美司和不同剂量的辐射（IR）处理786-O和OS-RC-2人肾癌细胞系。使用集落形成试验和流式细胞术测量细胞活力。通过荧光显微镜评估吖啶橙（AO）-溴化乙锭（EB）染色作为自噬性细胞死亡的指标。通过蛋白质印迹评估蛋白质表达。使用透射电子显微镜评估自噬体。使用克隆形成存活和乳酸脱氢酶试验评估肾癌细胞对辐射的敏感性。此外，还在生理氧水平下测试了这些参数。OS-RC-2细胞的AO-EB染色和流式细胞术表明，与单独使用乌苯美司或IR相比，联合治疗显著增强了自噬性细胞死亡。因此，乌苯美司治疗并未显著改变细胞周期进程，但与辐射联合使用时会增加细胞死亡。Akt激动剂可显著减弱这种作用，表明乌苯美司可能作为Akt抑制剂发挥作用。此外，蛋白质印迹分析表明，与单独的乌苯美司治疗或IR相比，联合治疗抑制了Akt信号通路。乌苯美司可能通过诱导细胞自噬来增强肾癌细胞对辐射的敏感性。这种诱导将自噬的作用从保护性转变为致死性，并且这种转变与Akt信号通路的抑制有关。
