| Objective: To investigate the antitumor and inducing apoptosis efficacy of cytotoxin12(CTX-12) from cobra(Naja naja atra) venom in combination of radiotherapy onhypodermal transplantation bladder tumor, and preliminary detecting whether theCTX-12 has the radiosensitizational effect on human bladder cancer.Methods: Establishing Balb/c female nude mice hypodermal transplantation tumormodel with 40 Balb/c female nude mice(6 to 8 weeks of age) bred in grade 3 cleanroom and BIU-87 TCC cells in logarithmic growth phase, BIU-87 belongs to a kindof bladder cancer cell line, being well differentiated from papillary human bladderTumor cell line. 40 Balb/c female nude mice were inoculated with 3×10~6 active cellsof BIU-87. But the tumor should be changed into 0.2 millilitre of the tumor cellssuspension solution before inoculation. After approximately 12 days, whentransplantation bladder tumor volume is to 0.60±0.09 cubic centimeter, 40 nude micewere randomly divided into 4 groups(n=10): CTX-12 plus radiotherapy, CTX-12alone, radiotherapy alone and control group. CTX-12 dose is concordant in everygroup, as radiation dose. control group were treated by the same way with equalvolume solution of 0.9％NaCl(0.15ml). During therapy, measure tumor volume withsliding caliper every three days,and draw tumor growth curve. Six random experimental animals of every goup are sacrificed in the next day, when experimentaltherapy is over. Measures including tumor weight,tumor volume inhibitory ratio,measurement of radiation sensitization effect,HE staining observing pathohistologicalcharacter transplantation bladder tumor cell by microscope,TUNEL systermdetermining apoptotic circumstances,determining the apoptotic morphologicalchanges by transmission electron microscope,using flow cytometry to determine theapoptotic ratio and the survive time(ST). To evaluate the treatment effects of threedifferent means of therapy.Results:Compared with control group, there are inhibitive effect on hypodermaltransplantation bladder tumors growth, but CTX-12 with radiotherapy group wassignificantly suppressed (P＜0.001), and tumor inhibition rate of the three group was77.9％,46.7％,18.2％respectively. Time of tumor growth delay(TGD)(volume from0.6 tol.2cm~3) was beyond 19 days in combination group,10 days and 5 days inradiation group alone and CTX-12 group alone, respectively(P＜0.05). Themorphological changes of apoptosis such as cell shrinkage, chromatin condensation,nuclear fragmentation and apoptotic body were observed by both light and electronmicroscope. The apoptotic morphological and quantitative changes are the mostobvious in combination therapy group. In addition, in CTX-12 plus radiation groupthe apoptotic peak and apoptotic ratio is the highest than other group. Compared withcontrol group, the apoptotic ratio is respectively (50.8±1.45, p＜0.01),(35.9±1.67,P＜0.05),(21.7±0.98, P＜0.05) and (7.64±0.21) in combination therapy group,CTX-12group alone,radiation group alone and control group. Measurement of radiationsensitization effect indicates CTX-12 has radiosensitization effect. Four nude micesurvived in combination group within 90 days, but 2,1 survived only in CTX-12group alone,radiation group alone respectively, however all killed in control group.Conelusion: The cobra venom CTX-12 can enhance significantly radiosensitivity onBalb/c female nude mice hypodermal transplantation tumor model. CTX-12 couldinduce tumor cell apoptosis, which may be one of the possible mechanisms to inhibitbladder tumors. Cytotoxin 12(CTX-12) from cobra(Naja naja atra) venom incombination of radiotherapy can inhibit conspicuously hypodermal transplantation bladder rumor growth, and inducing tumor cell apoptosis may be one of mostimportant pathway of achieving radiosensitization for CTX-12. Transmissionelectronmicroscope is the most accurate method for detecting morphological changesof apoptosis. |
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