Investigation On Anti-cervical Cancer Activity And The Mechanism Of Cyclin-Dependent Kinase4/6 Inhibitor LEE011

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The study of the cytotoxicity of LEE011 on the cervical cancer cellsObjective To detect the effect of LEE011 on cervical cancer cells,including cell proliferation and apoptosis in cervical cancer cell lines C33A and HeLa.Methods The morphological changes of cervical cancer cell lines C33 A and HeLa were observed after 48h treatment with different concentrations of LEE011.Flow cytometry was used to detect the changes of cell cycle and apoptosis of cervical cancer cell lines C33A and HeLa after 48 hours of treatment with LEE011(10?M).Results After 48 hours of treatment with LEE011,C33A cells show pronounced morphologic signs of toxicity,including abnormal shape and appearance,cellular lysis,and destruction.C33A cells have started to show observable signs of toxicity in the form of cells reduction and abnormal cells morphology when treated with 10?M LEE011,and cellular death becomes more pronounced when the drug concentration increased up to 20 ?M.However,HeLa cells show little changes in morphology and little cellular death.The results of flow cytometry showed that after the treatment with LEE011(10 ?M)for 48 hours,a significant G1 cell-cycle arrest accompanied by a reduction in the fraction of cells in S phase and G2/M phase in C33 A cells(P<0.05),compared with the control;however,HeLa cells treated with LEE011 did not have a significant result on cell cycle arrest.The results of flow cytometry showed that the apoptosis rate of C33A cells which were treated with LEE011(10 ?M)for 48 h increased compared with the control group,the difference was statistically significant(P<0.05),while LEE011 treatment did not significantly increase the apoptotic index compared with control,in HeLa cells.Conclusion LEE011 inhibits cell proliferation and promotes apoptosis of C33A in a dose-dependent manner,LEE011 induces cell cycle G1 arrest and cell apoptosis in C33A cells,but has no anti-tumor activity on HeLa.Part ? The study on the molecular mechanisms ofLEE011 against the cervical cancer cellsObjective To explore the molecular mechanism of LEE011 anti-cervical cancer cell activity and the relationship with Rb-E2F signaling pathway.The background expressions of CDK4,CDK6 and cyclin D1 in cervical cancer cell lines C33 A and HeLa cells were detected to determine the feasibility of LEE011 acting on related signaling pathways.The expression levels of CDK4,CDK6,cyclin D1,CDK2 and cyclin E1 were detected,in order to clarify that LEE011 inhibits the expressions of proteins in cell cycle-regulated signaling pathways.The expression levels of related proteins in the apoptotic pathway were also detected to identify the changes in apoptotic pathway during LEE011-induced apoptosis.Methods Cellular immunofluorescence and western blotting were used to detect the background expression of CDK4,CDK6 and cyclin D1 in cervical cancer cell lines C33A and HeLa cells.Western blotting was used to detect the expression levels of related pathway proteins in cervical cancer cells after treated with different concentrations of LEE011(0,5,10,15,20?M).for 48 hours.Results By western blotting and immunofluorescence assay,we found respectively that CDK4,CDK6 and cyclin D1 are highly expressed and are mostly localized in the nucleus with some localized in the cytoplasm of cervical cancer cell lines.Ribociclib induced cell cycle arrest in G0-G1 phase and cell apoptosis,and inhibited C33A cell proliferation in dose-dependent manner following by decreased expression of certain related genes such as CDK4,CDK6,E2F1,P-Rb,and increased Bax expression.However,there were no significant changes in cell cycle regulation-related pathways and apoptotic pathway signaling molecules in LEE011-treated HeLa cells.Conclusion LEE011 may block cell cycle and induce apoptosis through Rb-E2F signaling pathway,and thus exert anti-cervical cancer C33A cell activity;however,this pathway has no obvious change in cervical cancer HeLa cells,which needs further study.Part ? The study on the anti-cervical cancer activityof LEE011 in nude mice xenograft modelsObjective To verify the anti-cervical cancer activity of LEE011 and its mechanism of action in xenograft models,and to evaluate the drug safety and toxicity of LEE011 in vivo.Methods Nude mice were implanted with cervical cancer cell line C33A and HeLa cells to establish a cervical cancer xenograft model.LEE011 treatment group and control group were set up,5 mice in each group.LEE011 treatment group nude mice were given LEE011(200mg/kg/day)by normal gavage,and control group nude mice were given equal amount of solvent with normal diet(0.5%methylcellulose).The body weight,tumor volume and survival of tumor-bearing nude mice were monitored every 3 days for a total of 21 days.The expression of CDK4,CDK6,cyclin D1,Rb and Ki-67 protein in tumor-bearing nude mice was detected by immunohistochemistry.The levels of tumor cell apoptosis in tumor-bearing nude mice were detected by TUNEL method.The levels of biochemical indicators ALT,AST,ALB,ALP,BUN,CR,LDH-L and CK of nude mice were analyzed by biomedical analyzer.The morphological changes of myocardial tissue,liver tissue and renal tissue of nude mice were observed by HE staining.Results The xenograft models were treated with 200mg/kg of LEE011 in 0.5%methylcellulose or vehicle daily during a total of 21 days.LEE011 effectively hindered tumor growth in C33 A xenograft models.The result shown that there are the reduction of kinetic of the tumor volume growth in C33A xenografts treated with LEE011 compared with the untreated control group.Moreover,LEE011 had no significant effect on body weight in C33 A xenografts.For HeLa xenografts,LEE011 did not induce suppression of tumor growth,but caused the mice lost weight.In another hand,we confirmed the suppression of the CDK4/6-cyclin D-Rb-E2F1 pathway by immunohistochemical staining for CDK4,CDK6,cyclin D1,Rb and assessed the anti-proliferative effect by the proliferation marker Ki-67 immunostaining.Furthermore,we also performed TUNEL assay to detect apoptotic cells on the xenografts.We found that the expression of CDK4,CDK6,cyclin D1 and Rb were reduced,and the proportion of apoptotic cells was increased in LEE011 treatment group compared with the control group in C33 A xenografts.However,we did not found the significant effects of LEE011 on the HeLa xenografts.Conclusion LEE011 has anti-cervical cancer activity in cervical cancer cell line C33A nude mice xenograft models,and its mechanism is related to CDK4/6-cyclin D-Rb-E2F signaling pathway;LEE011 does not have significant anti-cervical cancer activity HeLa nude mice xenograft models.LEE011 has no obvious toxicity in cervical xenograft models of cervical cancer.Part ? Bioinformatics analysis of CDK4/6-cyclin D-Rb-E2F expression in different subtypes of cervical cancerObjective To investigate the difference in the anti-tumor activity of LEE011 between cervical cancer cells C33 A and HeLa in vitro and in vivo,and to analyze the expression of related pathway proteins in different subtypes of cervical cancer and to screen or exclude possible factors or biomarkers.Methods RNA secondary sequencing data and corresponding clinical characteristics of cervical cancer patients were downloaded from the TCGA database.According to HPV infection status,it is divided into HPV positive and HPV negative subtypes.The RNA levels of CDK4,CDK6,cyclin D1,Rbl,E2F1,Bax and Bcl-2 genes were compared.The R-package DEseq was used to screen whether the relevant pathway genes were statistically different between the two subtypes.Results A total of 178 cases of cervical cancer patients with tumor samples were analyzed in the TCGA database,of which 169 were positive for HPV infection and 9 were negative for HPV infection.The RNA levels of CDK4,CDK6,cyclin D1,Rbl,E2F1,Bax and Bcl-2 genes in cervical cancer subtypes were compared by Deseq.The differences were not significant according to the principle of |log2FC|>1 and P<0.05.Conclusion The expression levels of CDK4/6-cyclin D-Rb-E2F signaling pathway in tumor tissues of patients with different subtypes of cervical cancer may not be related to the sensitivity of LEE011.Further research is needed to screen biomarkers and analyze the cause of the difference in drug sensitivity.