Study On The Anti-cervical Cancer Mechanism Of Nitidine Chloride And Its Reversal Of HeLa/DDP Cell Resistanc

Cervical cancer is a very common malignant tumor of female reproductive system.Due to the particularity of its pathogenesis,it not only harms women’s health,but also threatens human reproduction.Persistent infection by High-risk human papillomavirus（HR-HPV）is the main cause of cervical cancer.In HR-HPV-positive cervical cancer,E6 a proto-oncoprotein encoded by HPV binds to ubiquitin ligase（E3）E6-associated protein（E6AP）to form an E6/E6AP dimer.The dimer can mediate ubiquitination and degradation of p53,a tumor suppressor protein,which leads to the impairment of the tumor suppressor function of p53.Therefore,which can reduce the ubiquitination level of p53 by inhibiting the expression level of E6AP and its binding level with p53 to achieve the role of restoring the protein level and cancer inhibition function of p53,which is of great significance for the study of new strategies for the treatment of cervical cancer.Nitidine chloride（NC）is the main active component of Zanthoxylum nitidum（Roxb.）DC.and has a wide range of pharmacological activities.However,there are few reports related studies and unclear mechanism of action in cervical cancer chemotherapy.Objective:The purpose of this study was to explore the role of NC in HR-HPV-positive cervical cancer cell lines He La and Si Ha,and to clarify the effect of NC on the ubiquitination modification level of p53 protein mediated by E6AP in cervical cancer cells,as well as the effect of NC on the drug resistance of cervical cancer cells to cisplatin（DDP）.Methods:（1）Disease ontology（DO）was used to predict the effect in disease of NC.（2）The inhibitory effect of NC on the growth of cervical cancer cells was detected by CCK-8 assay and colony formation assay.（3）TUNEL assay and Western blot assay were used to investigate the effect of NC on apoptosis and apoptosis-related protein expression of cervical cancer cells.（4）The influence of NC on the protein level of E6AP,the interaction between E6AP and p53,the ubiquitination modification level of p53 protein and the stability of p53 protein were detected by Western blot assay,immunoprecipitation assay and ubiquitination assay.（5）He La cisplatin resistant cells were induced by high-dose shock method,and the reverse drug resistance and mechanism of NC were detected by CCK-8 assay and Western blot assay.Results:（1）NC was correlated with a variety of tumors,including malignant tumors of female reproductive system;（2）Compared with the control group（Con group）,the inhibitory effect of NC on He La and Si Ha was decreased with the increase of Inhibitory concentration 50(IC₅₀)of NC,while the inhibitory effect of NC on cells was increased with the increase of drug administration concentration.（3）NC can promote apoptosis of cervical cancer cells,and up-regulate the activation levels of apoptosis-related proteins Caspase-3,Caspase-8,Caspase-9 and PARP in cervical cancer cells.（4）NC can down-regulate the expression level of E6AP protein in cervical cancer cells,inhibit the combination of E6AP and p53,reduce the ubiquitination modification level of p53,slow down its degradation rate,increase its protein stability,and up-regulate the level of p53 protein.（5）We have successful construction of cervical cancer cisplatin（DDP）resistant strain He La/DDP,and found that NC can reverse the drug resistance of He La/DDP cells,and down-regulate the expression level of multi-drug resistance（MDR1）protein in He La/DDP cells.Conclusion:NC can inhibit the expression of E6AP and p53 ubiquitination mediated by E6AP and restore the level of p53 protein and tumor inhibition function to exert anti-cervical cancer activity.NC can reverse He La/DDP cell resistance in cervical cancer.