重组融合蛋白C-CPE-ETA′对卵巢癌细胞的细胞毒作用

Target-specific cytotoxic activity of recombinant fusion toxin C-CPE-ETA′ against CLDN-3, 4-overexpressing ovarian cancer cells

目的 探讨重组融合蛋白C-CPE-ETA′与CLDN-3和CLDN-4高表达卵巢癌细胞的特异性结合能力和细胞毒作用.方法 应用real-time RT-PCR方法检测3种卵巢癌细胞系CAOV3、OVCAR3、SKOV3和20例上皮性卵巢癌组织中CLDN-3和CLDN-4的mRNA表达水平.构建质粒pC-CPE-ETA′,转入E.coli BL21（DE3）plysS细菌中,获得重组融合蛋白C-CPE-ETA′的表达,经His-Bind树脂色谱柱纯化.应用考马斯蓝电泳和Western blot方法,验证重组融合蛋白C-CPE-ETA′成功表达、纯化.应用流式细胞仪、JC-1染色和四甲基偶氮唑蓝（MTT）法,在CLDN-3和CLDN-4高表达卵巢癌细胞中分别枪测C-CPE-ETA′的特异性膜结合能力、促凋亡及细胞毒作用.结果 real-time RT-PCR结果显示,卵巢癌细胞CAOV3、OVCAR3和SKOV3高表达CLDN-3和CLDN-4.20例上皮性卵巢癌组织中分别有18例高表达CLDN-3,12例高表达CLDN-4.考马斯蓝电泳和Western blot检测到相对分子质量为58 000的重组融合蛋白C-CPE-ETA′被表达并纯化.C-CPE-ETA′蛋白能够特异性结合到CLDN-3和CLDN-4高表达的卵巢癌细胞CAOV3、OVCAR3和SKOV3,进而引起促凋亡和细胞毒作用,C-CPE-ETA′蛋白对卵巢癌细胞CAOV3、OVCAR3和SKOV3的IC50值分别为7.364、8.110和22.340ng/ml,而对CLDN-3和CLDN-4缺乏表达的HUVEC细胞,即使C-CPE-ETA′浓度达10μg/ml也无明显细胞毒作用.结论 重组融合蛋白C-CPE-ETA′对CLDN-3和CLDN-4高表达的卵巢癌细胞具有显著的特异性细胞毒作用,为卵巢癌靶向治疗新策略提供了实验基础.

Objective The aim of this study was to explore the possibility of creating a toxin,C-CPE-ETA′, by fusing C-terminal high affinity binding domain of CPE（C-CPE）with a truncated form of Pseudomonas aeruginosa exotoxin A（ETA＇）and to examine whether C-CPE-ETA＇ could specifically target CLDN-3,4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer.Methods CLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell liues and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA′ into E. coli BL21（DE3）plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells. Results Quantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3,OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0%（18/20）and 60.0%（12/20）of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA′ was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA＇ was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA′ was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC50 was 7. 364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22. 340 ng/ml for SKOV3cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA＇ at a concentration up to 10 μg/ml. Conclusions The C-CPE-ETA＇ protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.