西多福韦对人宫颈癌细胞CaSki内人乳头瘤病毒16抑制作用研究

Anti-HPV16 activity of cidofovir on cervical carcinoma cells(CaSki)

目的从抗病毒角度探讨西多福韦对宫颈癌细胞CaSki内人乳头瘤病毒16(HPV16)的抑制作用及对细胞周期的影响。方法用MTT法检测西多福韦对细胞的毒性;用实时定量PCR法检测其对病毒E6、E7 mRNA水平的影响;用Western blot方法检测其对病毒蛋白E6、E7和细胞抑癌蛋白p53、pRb表达水平的影响;用流式细胞法检测其对宫颈癌细胞周期的影响。结果西多福韦对宫颈癌细胞毒性较正常细胞大。可使HPV16阳性宫颈癌细胞CaSki内E6、E7 mRNA和蛋白水平降低,最大抑制率分别为(33.38±8.00)%、(28.32±2.73)%和98.92%、97.46%;可以使p53、pRb蛋白水平升高,最大浓度时可以上调12.06和3.53倍;对HPV16阴性宫颈癌细胞C-33A p53蛋白表达无影响,但可提高pRb蛋白水平;可导致CaSki和C-33A细胞发生S期阻滞,最高浓度组细胞相对对照组S期分别增加22.83%和67.64%。结论西多福韦可以在对细胞无毒的浓度下,抑制宫颈癌细胞内的HPV16,诱导宫颈癌细胞发生S期阻滞。

Objective To investigate the antiviral activity of cidofovir （CDV） against HPV type 16 in the CaSki cervical cancer cell line and its effects on the cell cycle. Methods Cytotoxicities of CDV in CaSki, C-33A and HEL were assessed by MTT assay. The mRNA and protein levels of E6 and E7 oncogene were analyzed by quantitative real-time PCR （qRT-PCR） and Western blot. p53 and pRb protein levels were also detected by Western blot. The effect of CDV on cell cycle was analyzed by flow cytometry. Results MTT assay showed that cytotoxicity of CDV was much greater in cervical carcinoma cells than in normal cells. In HPV16-positive CaSki cervical carcinoma cell line, qRT-PCR results showed that E6 and E7 mRNA levels were decreased. Meanwhile, Western blot analysis revealed that E6 and E7 protein levels had the same trend with mRNA. However, p53 and pRb protein expressions were found to be increased simultaneously. Moreover, pRb protein expression was found to be increased in the HPV16-negative C-33A cervical carcinoma cell line, while the level of p53 protein didn’t change. In addition, flow cytometry assay showed CDV could induce S phase arrest in both cervical carcinoma cell lines. Conclusion This study showed that cidofovir has antiviral activity through suppressing E6 and E7 oncogene expressions and could induce S phase arrest at nontoxic concentration.