Study On The Antineoplastic Mechanism Of Cantharidin And Norcantharidin For Bladder Tumor

The dried body of the Chinese blister beetle is sold in China as Mylabris,a concoction called cantharidin has been used in traditional Chinese medicine for the treatment of cancer.However,it has irritant effects on the urinary and digestive organs. Norcantharidin,the demethylated form of cantharidin,is easier to be synthesized and is relatively free from sided effects.This traditional Chinese medicine has not been used in treatment of bladder cancer by intravesical instillation.Objective:To study the antineoplastic effects of CA and NCTD on bladder cancer cell lines,EJ cells,T24 cells,MB49 cells and SqCa cells in vitro.To study the influence on blood,bladder and vitals all over the body by infusing CA and NCTD into mouse bladder.To establish a model of orthotopic murine bladder cancer,study the effects of intravesical instillation of CA and NCTD on murine bladder cancer.Content:Study on antineoplastic effects of CA and NCTD on bladder cancer cell lines in vitro;study on intravesical instillation of CA and NCTD for murine bladder cancer.Methods:The inhibitory rate of the cells was measured by MTT assay.Cell apoptotic rate was detected by FCM using Annexin V-FITC/PI detection kit. Morphology of cell apoptosis was observed by Hoechst 33258 fluorescence staining, electron/light microscopy and LSCM.DNA fragmen-tation was assayed by agarose gel electrophoresis.Caspase-3,-8,-9 were assayed with colorimetric assay kits.The expression of Bcl-2 and bax was detected by Western Blot.Telomerase was detected by PCR-TRAP-Silver staining.The change of intracellular free calcium concentration was detected by LSCM.After intravesical instillation of CA and NCTD,Blood subtypes of lymphocyte were detected by FCM.Bladders,hearts,livers,spleens, lungs and kidneys were anatomized out for pathology to observe morphological changes.Orthotopic C57BL/6 murine bladder cancer model was established by intravesical instillation of MB49 cells after argentine nitrate cautery.The diagnosis of bladder cancer was made by routine pathology and electron microscopy.Bladders were anatomized out for pathology to calculate the cure rate.TUNEL detection was made in tumor-loaded mice. Results:CA and NCTD could inhibit the growth of EJ cells,T24 cells and MB49 cells and cause apoptosis significantly.CA could inhibit the growth of SqCa cells and cause apoptosis significantly.The suppression was both in a time- and dose-dependent manner in specified concentration and time scope.CA and NCTD could down-regulate the Bcl-2 expression and up-regulate the Bax expression of T24 cells and MB49 cells,increase the activity of Caspase-3,-8,-9.Concentration of intracellular free calcium increased.There were no significant differences in lymphocyte subtypes between control group and experiment groups.The cure rate in experiment groups was higher than control group,of which differences were significant.There were no apparent degeneration,necrosis,inflammatory leukocyte infiltration and fibrosis in the abovementioned viscera.TUNEL detection showed that there were apparent apoptosis cells in each group.The apparent apoptosis is near to the tumor surface.Conclusions:CA and NCTD,especially CA,have apparent inhibition and apoptosis-inducing effects on bladder cancer cells.CA have apparent inhibition and apoptosis-inducing effects on SqCa cells.CA may induce apoptosis by activating Caspase-3,-8,-9,enhancing the express of bcl-2,tapering the express of bax,and increasing intracellular free calcium concentration.The orthotopic C57BL/6 murine bladder cancer model established by instillation of MB49 cells after argentine nitrate cautery is practical for animal experiment of instillation therapy for bladder cancer. CA and NCTD have apparent antineoplastic effects,of which mechanism may be induction of apoptosis.After instillation of CA and NCTD,heart,liver,kidneys,lungs and spleen are safe without damages.