Investigation On The Effect Of HPV Infection On Anti-cervical Cancer Activity Of LEE011 And Its Mechanism

Part Ⅰ The relationship between the expression of cyclin D1 and the prognosis of cervical cancer was analyzed by tissue microarray Objective To explore the expression difference of Cyclin D1 in cervical cancer and adjacent tissues,nucleus and cytoplasm,and to further analyze the relationship between the expression level of Cyclin D1 and prognosis of cervical cancer.Methods Immunohistochemical staining was used to detect the difference of Cyclin D1 expression in cervical cancer tissue microarray.Chi-square test was used to analyze the difference of Cyclin D1 expression in cervical cancer and adjacent tissues.Chi-square test was used to analyze the correlation between the expression level of Cyclin D1 and clinical indicators(age,pathological grade,TNM stage).Kaplan-Meier survival curve was used to analyze the relationship between Cyclin D1 expression level and survival prognosis of cervical cancer patients,including overall survival and disease-free survival.Log-rank statistical test was used for univariate analysis of survival,and statistically significant variables in univariate analysis were included in COX multivariate regression analysis.Results There was no significant difference in Cyclin D1 antibody expression between cancer and adjacent cancer.The expression of Cyclin D1 antibody in cytoplasm and nucleus was correlated with age and pathological grade(P<0.05);Overall survival analysis: High expression of Cyclin D1 antibody in cytoplasm and nucleus showed poor prognosis,and the difference was significant.Analysis of disease-free survival: The expression of Cyclin D1 antibody in the nucleus showed a low disease-free survival rate with high expression,and the difference was significant.COX multivariate regression analysis showed that cytoplasm,Cyclin D1 expression,age and TNM stage were significantly correlated with prognosis,which could be regarded as independent factors affecting prognosis of cervical cancer patients.Nucleus,age and TNM stage were significantly correlated with prognosis,which could be used as independent factors influencing prognosis of cervical cancer patients.Conclusion Cyclin D1 is expressed in both cytoplasm and nucleus of cervical cancer cells,and the higher the expression level,the worse the prognosis.Therefore,Cyclin D1 may be a reliable predictor of cervical cancer prognosis.Part Ⅱ Effect of LEE011 on HPV positive cervical cancer cellsObjective To investigate the activity of LEE011 against HPV positive cervical cancer cells,including the proliferation ability of HPV positive cervical cancer cell lines Si Ha and Ca Ski,and the ability of LEE011 to induce cell cycle arrest and promote cell apoptosis.Methods The morphological changes of HPV positive cervical cancer cell lines SiHa and Ca Ski were observed under a common light microscope after 48 h treatment with LEE011 at different concentrations.Flow cytometry was used to detect the changes in cell cycle and apoptosis of HPV-positive cervical cancer cells Si Ha and Ca Ski treated with LEE011(10u M)for 48 h,and the effects of LEE011 treatment on proliferation of Si Ha and Ca Ski were detected by clonal formation method.Results After treatment with LEE011 at different concentrations for 48 h,the morphology of HPV positive cervical cancer cell lines Si Ha and Ca Ski did not change significantly with the increase of LEE011 concentration.Flow cytometry showed that the cell cycle and apoptosis of HPV positive cervical cancer cell lines Si Ha and Ca Ski did not change significantly after 48 h treatment with LEE011(10u M).After 14 days of treatment with LEE011(10u M),the number of cell clones formed in HPV-positive cervical cancer cells Si Ha and Ca Ski was counted.There was no significant difference in the number of cell clones formed in HPV-positive cervical cancer cells Si Ha and Ca Ski from the control group without LEE011 treatment.Conclusion HPV positive cervical cancer cell lines Si Ha and Ca Ski cells are not sensitive to LEE011 antitumor activity.Part Ⅲ The mechanism of HPV virus oncoprotein E7 regulating the activity of LEE011 against HPV positive cervical cancer cellsObjective To investigate the mechanism of HPV oncoprotein E7 resistance to LEE011 in HPV-positive cervical cancer cells and its relationship with Cyclin D1-CDK4/6-Rb-E2F1 signaling pathway.Methods E7 knockdown model of HPV positive cervical cancer He La cells and E7 over expression model of HPV negative cervical cancer C33 A cells were constructed.LEE011(10u M)was used to treat the knock down model and over expression model with the control group for 48 h.Flow cytometry was used to detect the changes of cell cycle and apoptosis.Western blotting was used to detect the changes of related protein expression levels in the Cyclin D1-CDK4/6-Rb-E2F1 pathway.The expression levels of cell cycle related proteins(CDK4,CDK6,Cyclin D1,CDK2,Cyclin E1,P16,Rb,P-Rb,E2F1)and apoptosis related proteins(Bax and Bcl-2)were detected.Results Western blotting experiment verified that E7 knockdown model He La-E7-sh and control group He La-E7-sh NC were successfully constructed,E7 over expression model C33A-E7-OE and control group C33A-E7-NC of C33 A cells were successfully constructed.There were no significant differences in cell cycle and apoptosis between E7 knock down model group and over expression group by flow cytometry.After LEE011 treatment,the proportion of cells with cell cycle arrest in G1 phase increased in E7 knock down group,while the proportion of cells in S phase and G2/M phase decreased,with statistical difference(p < 0.05).The proportion of apoptosis increased in E7 knock down group,and the difference was statistically significant(p< 0.05).After LEE011 treatment,there was no significant difference in cell cycle and apoptosis rate between the E7 over expression group and the control group.Western blotting results showed that after 48 h of LEE011 treatment group and E7 knock down group,the expression of Cyclin D1 was increased and the expression of CDK4 was decreased in E7 knock down group compared with the control group.The expression levels of transcription-related proteins Rb,P-Rb and E2F1 were decreased.The expression of anti-apoptosis protein Bcl-2 was decreased,and the expression of pro-apoptosis protein Bax was increased.After LEE011 treatment,there were no significant changes in the expression levels of proteins related to cell cycle and apoptosis pathways in the E7 overexpression group and the control group.Conclusion E7 protein affects the antitumor effect of LEE011,a selective cell cycle dependent kinase 4/6 inhibitor.Knocking down E7 protein level can increase the sensitivity of HPV positive cervical cancer cells to LEE011.Overexpression of E7 protein reduced the sensitivity of HPV negative cervical cancer C33 A to LEE011.The possible mechanism is that E7 reduces the level of phosphorylated Rb by binding Rb and interferes with LEE011’s effect on the Cyclin D1-CDK4/6-Rb-E2 F signaling pathway,thus affecting cell cycle and apoptosis.Part Ⅳ Study on anti-HPV-positive cervical cancer activity of LEE011 in nude mouse xenograft modelObjective To investigate the effect and mechanism of LEE011 on HPV oncoprotein E7 knockdown model He La-E7-sh in nude mouse transplanted tumor model,and to evaluate the drug safety and toxicity of LEE011 in vivo.Methods Nude mice were planted with E7 knockout model He La-E7-sh cells and control group He La-E7-sh NC cells in armpit to establish xenograft model of HPV positive cervical cancer cells.LEE011 treatment group and control group were set up with 6 mice in each group.LEE011 treatment group was given LEE011(200mg/kg/day)by intragastric administration in addition to normal diet,and control group was given the same amount of solvent(0.5% methyl cellulose)in addition to normal diet.The body weight,tumor volume and growth status of tumor-bearing nude mice were monitored twice a week.The expression of CDK4,CDK6,Rb and Ki67 proteins in tumor tissues of nude mice were detected by immunohistochemical staining.TUNEL assay was used to detect the apoptosis of tumor cells in tumor-bearing nude mice.The toxic and side effects of drugs on the hemocytes of tumor bearing nude mice were analyzed by eyeball method.Results In the nude mouse transplanted tumor model of He La-E7-sh cells in the knockdown group,the growth rate of the drug group was slower than that of the solvent group,and the volume and weight of the transplanted tumor were significantly smaller than that of the control group,with statistical significance(P < 0.05).There were no significant differences in growth rate,graft volume,final weight and body weight in nude mice between the knockdown model He La-E7-sh cells and the control group He La-E7-sh NC cells.In the nude mouse model of He La-E7-sh NC cells in the control group,there were no significant differences in the growth rate,volume and final weight of transplanted tumor,and body weight of nude mice between the drug group and the solvent group.Immunohistochemical staining results showed that the expression levels of CDK4,CDK6,Rb and Ki67 proteins in the nude mouse tumor model of He La-E7-SH cells in the knockdown group were lower than those in the solvent group,and the difference was statistically significant(p<0.05).However,there was no significant difference in the expression levels of relevant immunohistochemical proteins in the nude mouse xenograft model of He La-E7-sh cells and the nude mouse xenograft model of He La-E7-sh NC cells in the knockout model and the nude mouse xenograft model of He La-E7-sh NC cells in the drug group and solvent group.TUNEL assay showed that in the nude mouse transplanted tumor model of He La-E7-sh cells in the knockdown group,the apoptosis level of tumor cells in the nude mouse tumor tissue of the drug group increased,and the difference was statistically significant(p<0.05).There was no significant change in apoptosis level in the knockdown model of He La-E7-sh cells,the control group of He La-E7-sh NC cells,and the drug group and solvent group of He LaE7-sh NC cells.Blood analysis of nude mice showed that LEE011 had no significant changes in blood cell indexes of tumor-bearing nude mice.Conclusions LEE011 has anti-cervical cancer activity in E7 knock down model of HPV positive cervical cancer He La cells in nude mouse xenograft model,but not in He La-E7-sh NC xenograft model,the mechanism of action may be related to Cyclin D1-CDK4/6-Rb-E2F1 signaling pathway.LEE011 has high safety due to no significant hematotoxicity in nude mice transplanted with cervical cancer.