A Laboratory Study On The Antitumor Effect In Vivo And Inducing Apoptosis In Vitro On Human Bladder Cancer Cell Lines Of The Membrane Toxin From Cobra Venom

Objective: To investigate the antitumor effect of the membrane toxin 12(MT-12) from cobra(Naja naja atra) venom on subcutaneous bladder tumor, and 5 methods were used to detect whether the MT-12 can induce apoptosis in E-J and BIU-87 bladder cancer cell lines. Methods: E-J cell suspension were implanted into the subcutaneous tissue of back, and establish Balb/c nude mice ethotopic transplantation tumor model with human bladder cancer. When the diameter of tumor reached 6 mm, 24 nude mice were randomly divided into 4 groups(n=6): high dose group(0.8mg/kg), middle dose group(0.4mg/kg), low dose group(0.2mg/kg) and control group. MT-12 solution of different dose was injected subcutaneously to the perimeter of tumor once every other days, and control group were treated by the same way with equal volum solution of 0.9% NaCl. All animals were sacrificed at next day after the 10th treatment. The antitumor efficacy was evaluated using tumor growth curve and tumor weight. E-J and BIU-87 cells were cultured in vitro, and exposed to 3 μ g/ml concentration of MT-12. HE staining, Hoechst 33342 staining, transmission electronmicroscope were used to determine the apoptotic morphological changes. Apoptotic rates were quantified by flow cytometry(FCM) using Annexin-V FITC/PI dual staining method. Agarose gel electrophoresis was carried out to detect DNA fragmentation. Results: The inhibitive effect of MT-12 on the subcutaneous tumors was obvious, and tumor inhibition rate of the three doses' group (0.8mg/kg, 0.4mg/kg and 0.2mg/kg) were 93.2%, 36.9% and 13.6% respectively. Compared with control group respectively, the tumor growth in high and middle doses' group was significantly suppressed (P<0.001), while tumor weight of low dose's mice was not obviously decreased (P>0.05). Compared with each other, the tumor weight of three doses' group was different (P<005). The morphological changes of apoptosis such as cell shrinkage, chromatin condensation, nuclear fragmentation and apoptotic bodywere observed by both light and electron microscope. The dual staining flow cytometry analysis showed that the early apoptotic rates for 12, 24 and 36 hours were 0.51%, 1.49%, 2.29% for E-J cells respectively, and 59%, 32.78%, 5.19% for BIU-87 cells respectively. The early apoptotic rates of control group for two cell lines were 0.03% and 0.77% respectively. Typical DNA ladder pattern was not detected using agarose gel electrophoresis. Conclusion: The cobra venom MT-12 is very potent in inhibiting the growth of bladder tumor in vivo, and there were obvious dose-effect correlations. MT-12 could induce apoptosis on E-J and BIU-87 cell lines, which is one of the possible mechanisms to inhibit bladder tumors. Transmission electronmicroscope is the most accurate method for detecting morphological changes of apoptosis. FCM using Annexin-V FITC/PI dual staining is a specific, sensitive, accurate and quantitative method for analyzing apoptotic cells, and can differentiate early and late apoptotic cells.