In Vitro Cytotoxicity Following Specific Activation Of A Bladder Cancer-associated Momoclonal Antibody Conjugated To A Cytotoxin From Naja Naja Atra

OBJECTIVE The inhibiting tumor cell proliferate actives of four cytotoxins (CTX-10,CTX-11,CTX-12 and CTX13) were tested using two human bladder tumor cell lines(BIU-87 and EJ). The CTX-12 which owned the strongest activity was chose and the immunotoxin(IT) was prepared by the CTX-12 conjugated to a bladder cancer-associated monoclonal antibody (BDI-1). The antitumor actives of IT on BIU-87 and EJ cell lines were tested. METHODS The inhibiting tumor cell proliferate actives of four cytotoxins were tested by crystal violet colorimetric analysis and [3H]-TdR incubated analysis. CTX-12 and BDI-1 were covalent coupled by chemical methods with heterobifunctional cross-linking reagents (SPDP). The disulfide-linked BDI-1-CTX-12 complexes were detected by SDS-gradient polyacrylamide gel electrophoresis. Using DAB dyeing to analyze the IT binding to BIU-87 and EJ cell lines and using [3H]-TdR incubated analysis to analyze the IT's inhibiting tumor cell proliferate active. RESULTS All of the four CTXs showed favorable inhibiting bladder tumor cell proliferate actives and the inhibitory effects showed a definite dose-effect relationship. The ICso(6h incubation) of CTX-10,CTX-ll,CTX-12 and CTX13 on BIU-87 was 6.03, 6.27, 2.25 and 2.89mg/l, the IC50(6h incubation) on EJ was 8.02, 7.89, 3.05 and 2.76 mg/1 respectively. SDS-PAGE demonstrated that CTX-12 and BDI-1 can be conjugated. The IT owned a specific binding to BIU-87 and EJ cell lines,whereas it can't bind to colonic carcinoma cell lines (Lovo).The IT can inhibited proliferation of malignant tumor cell lines. The IC50(6h incubation) of it on BIU-87, EJ and Lovo cell lines was 97.12, 85.28 and 193.4 mg/ 1 respectively, whereas, if the IT replaced by RPMI1640 after incubated 30 min and continued incubating to 6h, the IC50 on BIU-87,EJ and Lovo cell lines was 94.64, 81.28 and 865 mg/1 respectively. CONCLUSION Four CTXs owned favorable inhibiting bladder tumor cell proliferate actives. The IT was prepared by conjugating CTX-12 with BDI-1 showed selective cytotoxicity against human cancer cell lines. The IT and four CTXs are largely potential anticancer drug for bladder carcinoma, the result is a good foundation for the further in vivo and clinical study.