Cinobufotalin Inhibits Proliferation Of Endometrial Cancer And Its Mechanisms

Background and ObjectiveEndometrial cancer,a group of epithelial malignant tumors occurring in endometrium,has been increasing globally in recent years with a younger trend.There’s a great effect and prognosis of surgical treatment in early endometrial cancer.For non-early endometrial cancer,it needs adjuvant therapy to improve prognosis.At present,problems such as chemotherapeutic drug resistance do exist in clinic,so it is of great significance to find some new adjuvant treatment drugs.A large number of studies have found that natural active ingredients of animals and plants can inhibit tumorigenesis and development.Toad toxin,an effective component of traditional Chinese medicine Bufo crisp,has been reported to have anti-tumor effects.And Cinobufotalin is one of the well-studied monomer component of Toad toxin,while its effect on endometrial cancer remains to be explored.This study aims to clarify whether cinobufotalin can inhibit the proliferation of endometrial cancer and its actional mechanism.Contents and Methods1.Different concentrations of cinobufotalin and cisplatin were used to stimulate endometrial cancer cells RL95-2 and HEC-1-B,and the half inhibition rates of bufalin and cisplatin were examined and compared.The proliferation ability of endometrial cancer cells RL95-2 and HEC-1-B was detected by MTT and EDU methods after cells were stimulated with gradient concentration of cinobufotalin.2.Subcutaneous tumorigenesis of nude mice model was used to detect the effect of cinobufotalin on proliferation and to explore the appropriate concentration of drug administration.Intraperitoneal tumorigenesis model was used to compare the survival time of cinobufotalin with cisplatin and its combination.3.Use of RT-PCR,Western Blot and co-IP experiments to explore the downstream pathway molecule of cinobufotalin that gives play to the biological effect,thus identify the effective functional pathway of cinobufotalin.Result1.The results of MTT and EDU experiments showed that cinobufotalin can inhibit the proliferation of endometrial cancer cells RL95-2 and HEC-1-B in a concentration-and time-dependent manner.And compared with cisplatin,the half inhibitory concentration of cinobufotalin was lower than that of cisplatin.2.Subcutaneous tumorigenesis in nude mice showed that cinobufotalin inhibited endometrial cancer growth in vivo.When the dosage was more than 8 mg/kg,the liver function of nude mice was damaged,and 4 mg/kg was the appropriate safe concentration for nude mice.Survival analysis of intraperitoneal tumorigenesis experiment showed that the survival time of cinobufotalin combined with cisplatin was longer than that of cinobufotalin alone and cisplatin alone.3.Results of RT-PCR,Western Blot and immunohistochemistry showed that cinobufotalin inhibited the proliferation of endometrial cancer by inducing ENKUR gene expression.Co-IP and Western Blot experiments showed that ENKUR interacted with GSK3beta to inhibit WNT/beta-catenin pathway activity,thereby inhibiting endometrial cancer proliferation.Cinobufotalin inhibits endometrial cancer proliferation by up-regulating ENKUR expression and then inhibiting WNT/beta-catenin pathway activity.Conclusion1.Cinobufotalin monomer can inhibit the proliferation of endometrial cancer in vivo and in vitro,which provides preclinical research evidence and lays a good theoretical foundation for the clinical application of cinobufotalin in endometrial cancer treatment.2.ENKUR is a potential target of cinobufotalin in endometrial cancer,which can inhibit WNT/beta-catenin pathway activity and provide a new potential therapeutic target for disease diagnosis and treatment.