关键词: 人乳头瘤病毒16, 细胞增殖, 细胞凋亡, 细胞周期, 自噬, 紫檀芪, 永生化人宫颈上皮细胞

Abstract: 【Abstract】 Objective To investigate the effect of pterostilbene on the growth, apoptosis and autophagy of a human papillomavirus type 16 （HPV-16）-immortalized cervical epithelial cell line H8. Methods H8 cells were treated with pterostilbene at different concentrations of 0 （control group）, 25, 50, 75, 100 μmol/L for 24 and 48 hours. Cell counting kit-8 （CCK8） assay was performed to evaluate the cellular proliferative activity, flow cytometry was conducted to detect apoptosis and cell cycle, monodansylcadaverine （MDC） staining and fluorescence microscopy were performed to detect autophagy, and Western blot analysis was conducted to determine the expression of the cell cycle-related protein cyclinD1, apoptosis-related proteins caspase-3 and caspase-9, autophagy-related proteins Beclin1, microtubule-associated protein 1 light chain 3 （LC3）-Ⅱ/Ⅰ, ATG5 and P62, as well as HPV oncoproteins E6 and E7. Statistical analysis was carried out by using one-way analysis of variance, repeated measures analysis of variance and least significant difference-t test. Results After 48-hour treatment with pterostilbene at different concentrations of 0, 25, 50, 75, 100 μmol/L, the relative cellular proliferation rate significantly differed among the groups （100.00% ± 1.56%, 99.02% ± 4.97%, 93.59% ± 2.01%, 81.28% ± 4.90%, 69.17% ± 7.56%, respectively; F = 77.22, P < 0.05）, and gradually decreased along with the increase in the concentration of pterostilbene; compared with the control group, the pterostilbene groups all showed significantly decreased cellular proliferation rate （all P < 0.05）. After 24-hour treatment with pterostilbene, the proportions of H8 cells at G1, G2 and S phases significantly differed among the above groups （F = 7 845.00, 51.14, 266.50, respectively, all P < 0.05）; compared with the control group, the pterostilbene groups showed significantly increased proportions of H8 cells at G1 and G2 phases （all P < 0.05）, but significantly decreased proportions of H8 cells at S phase （P < 0.05）. After 48-hour treatment with pterostilbene, the apoptosis rate was significantly higher in the 25-, 50-, 75- and 100-μmol/L pterostilbene groups （14.66% ± 0.22%, 13.50% ± 0.49%, 14.56% ± 0.19%, 15.30% ± 0.76%, respectively） than in the control group （11.58% ± 0.50%, all P < 0.05）. After 24-hour treatment with pterostilbene, MDC staining showed only a small number of H8 cells with bright dot-like fluorescence in the control group, but increased number of autophagosome-positive H8 cells with bright dot-like fluorescence in the pterostilbene groups. Western blot analysis revealed that there were significant differences in the protein expression of cyclin D1, caspase-3, caspase-9, Beclin1, LC3-Ⅱ/Ⅰ, ATG5, P62, E6 and E7 among the control and pterostilbene groups after 24- and 48-hour treatment with pterostilbene （all P < 0.05）. The treatment with pterostilbene could down-regulate the expression of cyclin D1, E6 and E7, and up-regulate the expression of caspase-3, caspase-9, Beclin1, LC3-Ⅱ/Ⅰ, ATG5 and P62, with significant differences between the control group and most pterostilbene groups in expression of the above proteins（all P < 0.05）. Conclusion Pterostilbene can inhibit the proliferation of H8 cells, promote their apoptosis and autophagy, and down-regulate the expression of oncogenes E6 and E7.

Key words: Human papillomavirus 16, Cell proliferation, Apoptosis, Cell cycle, Autophagy, Pterostilbene, Immortalized human cervical epithelial cells