Effect Of Sodium Cantharidate On Dendritic Cells In Patients With Bladder Cancer And On BIU-87 Cell Proliferation And Apoptosis

Background:Bladder cancer is a common malignant tumor in our urinary system and one of the top ten tumors in the body.Bladder cancer is the fourth most common solid tumor in men and the seventh in women in the world.Bladder cancer is still one of the most common malignant neoplasms of the urinary system.Its morbidity and mortality rate ranks the first place in the incidence of genitourinary tumors in our country.The most common one is bladder urothelial carcinoma,more than 90%of the total.Most of them originate from the transitional epithelium.In recent years,along with the acceleration of industrialization,urbanization and aging,the incidence of bladder cancer in men,women,cities,rural areas and in China has been increasing year by year.In recent years,with the development of traditional Chinese medicine and pharmaceutical preparations,many anti-tumor effects of Chinese herbal medicines have been excavated,so that more traditional Chinese medicines and their derivatives are applied to the treatment of tumors.Cantharidin is the main active ingredient of Cantharidin.Cantharidin has a good affinity with tumor cells,but also has acute irritant toxicity and severe irritation to the urinary system.Strong toxicity limits its clinical application.Sodium cantharidate is a semisynthetic drug produced by the hydrolysis of cantharidin and sodium hydroxide when heated together.The molecular formula is C₁₀H₁₂Na₂O_5,which is less toxic and irritating than cantharidin sodium cantharidate because of its molecular weight Small,easier to enter the cell,anti-tumor effect is also stronger.Objective:To investigate the effect of sodium cantharidate on the differentiation,maturation and function of dendritic cells derived from peripheral blood in patients with bladder cancer and to study the effects of sodium cantharidate about the proliferation and apoptosis of bladder cancer cells BIU-87.Methods:1.Peripheral blood mononuclear cells from patients with advanced stage bladder cancer were studied.Peripheral blood mononuclear cells were isolated by Ficoll paque plus lymphocyte separation and cultured to obtain dendritic cells.The experiment was divided into low dose cantharidin group?10?g/ml?,sodium cantharidinate medium dose group?20?g/ml?and sodium cantharidate high dose group?40?g/ml?and the blank control group.2.The changes of the expression of CDla and CD83 on dendritic cells were detected by flow cytometry.3.To detect the effect of sodium cantharidate on the proliferation of dendritic cells stimulated by MTT assay.4.Detect the inhibitory effect of sodium cantharidate on bladder cancer BIU-87cells by MTT method..5.The clonogenic ability of sodium cantharidinate on BIU-87 cells was tested by colony formation assay.6.The effect of sodium cantharidate on the apoptosis of bladder cancer BIU-87cells was detected by AnexinV-FITC/PI double staining flow cytometry.7.The effect of sodium cantharidate on the mitochondrial membrane potential was examined by flow cytometry.8.Autophagy effects of sodium cantharidate on BIU-87 cells were detected by flow cytometry.9.Western blot was used to detect the expression of Caspase-3 and PARP.Results:1.Compared with the control group,the cells in the sodium cantharidate group became larger in volume and grew longer.In the high-dose cantharidin sodium group,the cells appeared protruding and shaped like branches,which was similar to that of the healthy human.2.In cantharidin high-dose group,dendritic cell phenotype molecules CD83,CDla expression highest.Compared with the control group,the expression of CD83and CDla on the surface of dendritic cell membrane of sodium cantharidate increased,and there were significant differences.3.Catecholam treatment group stimulated allogeneic lymphocyte proliferation index was significantly higher than the control group,the difference was statistically significant?p<0.05?.4.MTT method confirmed that sodium cantharidate can significantly inhibit the growth of bladder cancer cell line BIU-87.5.Colony formation experiments showed that with the increase of the concentration of sodium cantharidin,the number of clonal colony formation decreased by minus,with the concentration was negatively correlated.6.After 24 h,the apoptosis of BIU-87 cells was detected by flow cytometry and double staining.7.Cytometric detection of sodium cantharidinate on mitochondrial membrane potential of BIU-87 cells by flow cytometry showed that sodium cantharidinate could significantly induce the collapse of mitochondrial membrane potential in BIU-87 cells and significantly decrease the membrane potential,increasing the concentration decreased the more obvious membrane potential.8.Autophagy effect of sodium cantharidate on BIU-87 cells was detected by flow cytometry,indicating that autophagy was induced by sodium cantharidinate at different concentrations and the autophagy was gradually enhanced with the increase of concentration,and was positively correlated with the drug concentration Related.9.Western blot analysis showed that sodium cantharidinate significantly increased the expression of Caspase-3 and PARP protein in BIU-87 cells.Conclusion:Sodium cantharidate can induce dendritic cell maturation in bladder cancer patients and increase expression of CD83 and CDla,the phenotypic molecules of dendritic cells.In addition,sodium cantharidate can inhibit the proliferation of bladder cancer cell line BIU-87 and induce cell apoptosis.