Isocorydine Controls Cell Differentiation Through Mesenchymal-to-epithelial Transition In SiHa Cervical Cancer Cell

Background and objective: The cervical cancer is one of the major malignant tumor which result in the death of women. Meanwhile, the morbidity is on the rise and the age tend to be younger. The surgery-based therapies are always assisted by chemotherapy and radiotherapy. However, the surgery will bring about trauma and impaired function, the costly chemotherapy and radiotherapy are always accompanied with severe complication and unsatisfied efficacy either. The recently research show that the main cause of the cervical is the infection of human papillomavirus 16(HPV16), whereas, we haven’t have a efficient way to hold-up the expression and activity of HPV. To restrain the expression and activity of HPV could be a effective way,as well as a quantum jump, in the prevention and therapy of cervical cancer. Isocorydine(ICD) is a kind of isoquinoline we extract from dicranostigma leptopodum. Although our early study demonstrated that the Chinese herb Isocorydine could inhibit the expression of HPV16E6 in Si Ha cell, the mechanism by which lsocorydine suppressing the activity of HPV is still not clear. The present project aims at providing a solid experimental and theoretical foundation for the possible effects of Isocorydine as a new and effective treatment of HPV infection.Methods: The human Si Ha cell line were purchased from the Cell Bank of the China Academy of Sciences.The cell line was cultured with Dulbecco’s Modified Eale’s Medium(DMEM)high glucose containing 10% heat-inactivated fetal bovine serum.The cell line was incubated at 37°C with 5% carbon dioxide. To confirm whether ICD could suppress the development of cervical cancer or not, we exposed the cell to indicated concentrations for indicated hours and MTT assay were performed in the ICD-treated Si Ha cell. In order to find out the mechanism by which ICD suppress the cytoactive of Si Ha cell line,we examined using the Annexin V/PI double staining method and flow cytometry analysis. We also examined the Si Ha cell line with ICD treatment at indicated concentration for 48 h via flow cytometry analysis to comfirm whether ICD could effect cell cycle or not.Meanwhile, we check the result of c DNA Microarray to confirm the specific gene which could influence the Si Ha cervical cacer cell. And the result of Q-PCR verified the authenticity of the c DNA Microarray.The Western blotting was performed in Si Ha cell line to verify the relevant protein expression.Result: The MTT assay result suggested that ICD could significantly inhibit the growth of Si Ha cell line in both dose-dependant and time-dependant manners with the maximum inhibition ratio of 68.69% at 72 h. The percentage of apoptotic cell of each sample was less than 6% which we usually consider as no apoptosis happening.At the same time, we found that ICD cause G1/S cell cycle arrest apparently via flow cytometry analysis. We got the meaningful genes included Caspase 14, CLCA2, NDRG1 and CDH1 through the repeated screening of the c DNA Microarray which show a prominent upregulation. We verified the authenticity of the c DNA Microarray via Q-PCR that confirm the upregulated expression of the three genes. In addition, we check the expression of HPV16E6 through Q-PCR and Western-blot which show a significant downregulation in both expression of gene and protein.The result of Western-blot demonstrate the downregulation of Vimentin and PARP,as well as the upregulation of E-Cadherin、P21 and P53.Conclusion: The ICD restrain the proliferation of Si Ha cervival cell mainly through P53-P21 pathway and force the Si Ha cell transforming into epithelial cell to suppress the invasion and metastasis via regulating the expression of CLCA2, NDRG1 and Caspase14 which aim at undergoing the Mesenchymal-to-Epithelial Transition(MET) to carry out antitumous effect.