The effect of Bruceine D on high-risk human papillomavirus 16 infected cell and its possible mechanism
1.Department of Gynecology and Obstetrics, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325027; 2.Department of Gynecology and Obstetrics, the Frist Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015
PAN Liuliu1,ZHENG Feiyun2,ZHU Haiyan2, et al. The effect of Bruceine D on high-risk human papillomavirus 16 infected cell and its possible mechanism[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2015, 45(2): 89-.
Abstract：Objective: To investigate the Bruceine D’s (BD) effect on human papillomavirus (HPV) type 16 infected cell in vitro. Methods: The Ect1/E6E7 was cultivated with BD (1, 5, 10, 15 and 30 μmol/L) for 24, 48 and 72 h, the anti-proliferation effect was measured with MTT colorimetric assay; The Ect1/E6E7 was cultivated with BD (1, 5 and 10 μmol/L) for 24, 48 and 72 h, the cell growth cycle change was measured with cell cycle staining solution, the morphologic changes of cell apoptosis stained with Hoechst 33258 in the Ect1/E6E7 cell line were observed under fluorescence microscope treated with BD at concentration of 5 μmol/L for 48 h. Flow cytometry of Annexin V-FITC/PI assay was used to evaluate the apoptosis rate of Ect1/E6E7 cell line treated with BD at different concentrations (1, 5 and 10 μmol/L) in vitro. Cervical cancer cells Caski which contain complete HPV 16 E6, E7 gene, as a positive control, by real-time quantitative PCR to detect the mRNA expression of HPV E6, E7 of those two cells treated with BD. Results: Immortalized human ectocervical Ect1/E6E7 cell line treated with BD showed obviously morphological changes, and with the prolongation of drug action and the increase of drug concentration, the morphological changes became more apparent. BD can inhibit the proliferation of Ect1/E6E7 cell in dose-dependent and time-dependent manner (P<0.05). Ect1/E6E7 cell growth cycle was stagnated in the G1 phase. Hochest fluorescence staining presented the Ect1/E6E7 cells nucleus appeared apoptotic morphological change, and apoptotic bodies can be observed. Flow cytometry showed the apoptosis rate were (6.25±0.76)%, (20.21±1.32)%, (8.61±1.59)%, respectively, after 48 hours cultured with 1, 5, 10 μmol/L of BD (P<0.05). Real-time PCR results showed that after cultivating with 1, 5, 10 μmol/L BD for 48 h, the expression of HPV16 E6, E7 mRNA of Ect1/E6E7 cell were decreased, but compared with the control group, the difference was not statistically significant (P>0.05). The expression of HPV16 E6, E7 mRNA of Caski cell were decreased, the difference was statistically significant (P<0.05) compared with the control group. Conclusion: BD can inhibit the proliferation of Ect1/E6E7 cells significantly, the possible mechanism may be associated with the expression decline of HPV16 E6, E7 mRNA, which promote cell apoptosis.
