北京大学学报(医学版) ›› 2018, Vol. 50 ›› Issue (5): 778-784. doi: 10.19723/j.issn.1671-167X.2018.05.003

• 论著 • 上一篇    下一篇

氯喹抑制肾癌细胞活性促进舒尼替尼诱导的细胞凋亡

孙静1,2 ,宋卫东3,闫思源2,席志军3△   

  1. (1. 首都医科大学基础医学院病理学系, 北京100069; 2. 中国科学院微生物研究所, 北京100101; 3. 北京大学第一医院泌尿外科,北京大学泌尿外科研究所, 北京100034)
  • 出版日期:2018-10-18 发布日期:2018-10-18
  • 通讯作者: 席志军 E-mail: xizhijun@hsc.pku.edu.cn
  • 基金资助:
     国家自然科学基金(81272829)和真菌学国家重点实验室开放课题 (O5KF061013)

Chloroquine inhibits viability of renal carcinoma cells and enhances sunitinib-induced caspase-dependent apoptosis

SUN Jing1,2, SONG Wei-dong3, YAN Si-yuan2, XI Zhi-jun3△   

  1. (1. Department of Pathology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China; 2. Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; 3. Department of Urology, Peking University First Hospital,Institute of Urology,Peking University,Beijing 100034, China)
  • Online:2018-10-18 Published:2018-10-18
  • Contact: XI Zhi-jun E-mail: xizhijun@hsc.pku.edu.cn
  • Supported by:
    Supported by the National Natural Science Foundation of China (81272829) and Open Project Program of State Key Laboratory of Mycology (O5KF061013)

摘要: 目的:研究氯喹(经常用于抑制自噬体与溶酶体的融合,是一种晚期自噬的抑制剂)是否能抑制肾癌细胞的增殖以及对舒尼替尼(sunitinib, ST)诱导的细胞凋亡的影响。方法:以肾癌细胞系786-O和ACHN为模型,利用3-(4,5-二甲基)-5-(3羧甲基苯环)-2-(4-硫基苯)-2H-四唑盐复合物检测法[3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, MTS]观察氯喹对细胞活性的影响;透射电子显微镜、免疫杂交等手段检测氯喹影响ST诱导的凋亡、自噬的情况。应用早期自噬抑制剂3-甲基腺嘌呤(3-methyladenine, 3-MA)、RNA干扰技术敲降自噬相关蛋白Ulk1 (unc-51-like kinase 1)和微管相关蛋白1轻链3融合蛋白(microtubule associated protein 1 light chain 3 fusion protein, LC3)确定早期自噬对ST引起的细胞凋亡的影响。结果:氯喹和ST均可以抑制786-O和ACHN细胞的增殖,氯喹可促进ST诱导的细胞凋亡。与氯喹不同,抑制早期自噬减少了ST诱导的细胞凋亡和活性丢失。结论:舒尼替尼既可以引起细胞自噬,也可以促进细胞的凋亡;早期、晚期自噬对ST诱导的细胞凋亡作用不同,氯喹可以作为一种潜在的抗肾癌药物进行相关的临床研究。

关键词: 氯喹, 舒尼替尼, 肾细胞癌, 自噬, 凋亡

Abstract: Objective: To determine whether chloroquine (CQ), an often used inhibitor of late autophagy and autophagosome/lyosome fusion, can inhibit proliferation of renal carcinoma cells and investigate its effect on sunitinib (ST)-induced apoptosis. Methods: Renal carcinoma cell line 786-O and ACHN had been used as cellular model and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay was carried out to detect the cell viability in response to CQ or ST treatment. Both transmission electron microscope and immunoblotting had been employed to observe apoptotic and autophagic process. To examine the involvement of autophagy in ST-dependent apoptosis, autophagy had been inhibited either chemically or genetically via utilizing autophagy inhibitor or specific small interference RNA (siRNA) targeted to either Ulk1(unc-51-like kinase 1) or LC3 (microtubule associated protein 1 light chain 3 fusion protein), two essential autophagic proteins. Results: Both ST and CQ induced cell viability loss, indicating that either of them could inhibit renal cancer cell proliferation. Clone formation experiments confirmed the aforementioned results. Furthermore, the combined ST with CQ synergistically promoted the loss of cell viability.By transmission electron microscopy and immunoblotting, we found that the ST induced both autophagy and caspasedependent apoptosis. While 3-MA, an early autophagy inhibitor, reduced the ST-induced cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), a substrate of caspase 3/7 and often used marker of caspase-dependent apoptosis, CQ promoted the ST-dependent PARP-1 cleavage, indicating that the early and late autophagy functioned differentially on the ST-activated apoptotic process. Moreover, the knock down of either Ulk1 or LC3 decreased the ST-caused apoptosis.Interestingly, we observed that rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin) and an inducer of autophagy, also showed to inhibit cell viability and increased the cleavage of PARP-1 in the ST-treated cells, suggesting that autophagy was likely to play a dual role in the regulation of the ST-induced apoptosis. Conclusion: ST activates both apoptotic and autophagic process in renal carcinoma cells. Although autophagy precedes the ST-induced apoptosis, however, early and late autophagy functions differentially on the apoptotic process induced by this compound. Additionally, ST can coordinate with the inducer of autophagy to inhibit the cell proliferation. Further research in this direction will let us illuminate to utilize CQ as a potential drug in the treatment of renal carcinoma.

Key words: Chloroquine, Sunitinib, Renal carcinoma, Autophagy, Apoptosis

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