Yang Jing, Dai Lv-Xia, Chen Ming, Li Bei, Ding Nana, Li Gang, Liu Yan-Qing, Li Ming-Yuan, Wang Bao-Ning, Shi Xin-Li, Tan Hua-Bing
Department of Infectious Disease, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China; Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, P.R. China; Department of Microbiology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China.
Experiment Teaching Center of Clinical Medicine, Chengdu College of Medicine, Chengdu, Sichuan 610500, P.R. China.
Exp Ther Med. 2016 Nov;12(5):2965-2973. doi: 10.3892/etm.2016.3718. Epub 2016 Sep 20.
In order to evaluate the potential application value of cidofovir (CDV) in the prevention of human papillomavirus (HPV) infection and treatment of cervical cancer, the inhibitory effect of CDV on the proliferation of HPV 18-positive HeLa cells in cervical cancer was preliminarily investigated, using cisplatin (DDP) as a positive control. An MTT assay was used to analyze the effects of CDV and DDP on HeLa cell proliferation. In addition, clone formation assay and Giemsa staining were used to examine the extent of HeLa cell apoptosis caused by CDV and DDP. Flow cytometry was also used to detect the shape and size of apoptotic cells following propidium iodide staining, while western blot analysis identified the expression levels of of E6 and p53 proteins in HeLa cells. A cell climbing immunofluorescence technique was used to locate the subcellular position of p53 in HeLa cells. The results demonstrated that CDV and DDP inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Flow cytometry showed that CDV and DDP treatments resulted in cell arrest in the S-phase, and triggered programmed cell death. Furthermore, western blot analysis revealed that CDV and DDP inhibited E6 protein expression and activated p53 expression in HeLa cells. Finally, the immunofluorescence results indicated that CDV and DDP inhibited the nuclear export of p53 by E6 protein, which is required for degradation of endogenous p53 by MDM2 and human papilloma virus E6. In conclusion, CDV and DDP inhibited HeLa cell proliferation in a concentration- and time-dependent manner, reduced the expression of E6 protein, and reinstated p53 protein activity. Thus, CDV regulates cell cycle arrest and apoptosis, and may be a potential cervical cancer therapeutic strategy.
为了评估西多福韦(CDV)在预防人乳头瘤病毒(HPV)感染及治疗宫颈癌方面的潜在应用价值,以顺铂(DDP)作为阳性对照,初步研究了CDV对宫颈癌中HPV 18阳性HeLa细胞增殖的抑制作用。采用MTT法分析CDV和DDP对HeLa细胞增殖的影响。此外,利用克隆形成试验和吉姆萨染色检测CDV和DDP引起的HeLa细胞凋亡程度。还用流式细胞术检测碘化丙啶染色后凋亡细胞的形态和大小,同时通过蛋白质免疫印迹分析确定HeLa细胞中E6和p53蛋白的表达水平。采用细胞爬片免疫荧光技术定位HeLa细胞中p53的亚细胞位置。结果表明,CDV和DDP以浓度和时间依赖性方式抑制HeLa细胞的增殖。流式细胞术显示,CDV和DDP处理导致细胞停滞于S期,并引发程序性细胞死亡。此外,蛋白质免疫印迹分析显示,CDV和DDP抑制HeLa细胞中E6蛋白表达并激活p53表达。最后,免疫荧光结果表明,CDV和DDP抑制E6蛋白介导的p53核输出,而E6蛋白介导的p53核输出是MDM2和人乳头瘤病毒E6降解内源性p53所必需的。总之,CDV和DDP以浓度和时间依赖性方式抑制HeLa细胞增殖,降低E6蛋白表达,并恢复p53蛋白活性。因此,CDV可调节细胞周期停滞和凋亡,可能是一种潜在的宫颈癌治疗策略。