Preparation of a claudin-targeting molecule using a C-terminal fragment of Clostridium perfringens enterotoxin
Preparation of a claudin-targeting molecule using a C-terminal fragment of Clostridium perfringens enterotoxin
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DOI：
10.1124/jpet.105.093351
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发表时间：
2006-01-01
影响因子：
3.5
通讯作者：
Watanabe, Y
中科院分区：
文献类型：
--
作者：
Ebihara, C;Kondoh, M;Watanabe, Y
虽然大多数恶性肿瘤是上皮来源的癌，但尚未开发出将抗肿瘤剂特异性且有效地递送至癌的方法。最近的报道表明，上皮细胞过度表达claudin-3和claudin- 4，这是上皮细胞紧密连接的膜蛋白。这表明claudin可以作为肿瘤治疗的靶点，但目前还没有一种方法将药物输送到表达claudin的细胞。在本研究中，我们评估了产气荚膜梭菌肠毒素（C-CPE）的有效的claudin-4结合C-末端片段是否允许靶向表达claudin-4的细胞。我们将C-CPE与蛋白合成抑制因子（PSIF）融合，PSIF缺乏假单胞菌外毒素的细胞结合结构域。这种融合蛋白C-CPE-PSIF对表达内源性密蛋白-4的MCF-7人乳腺癌细胞具有细胞毒性，但对缺乏内源性密蛋白-4的小鼠成纤维细胞L细胞没有毒性。C-CPE-PSIF对MCF-7细胞的细胞毒作用可被C-CPE而非牛血清白蛋白所减弱。此外，C-CPE的紧密连接蛋白-4结合区域的缺失降低了C-CPE-PSIF的细胞毒性。最后，我们发现C-CPE-PSIF对表达claudin-4的L细胞有毒性，但对正常L细胞或表达claudin-1、-2或-5的细胞没有毒性。这些结果表明，使用C-CPE肽可以提供一种将药物靶向表达紧密连接蛋白的细胞的新方法。
Although most malignant tumors are epithelia-derived carcinomas, methods for specific and effective delivery of antitumor agents to carcinomas have not been developed. Recent reports indicate that epithelia overexpress claudin-3 and - 4, which are integral membrane proteins of epithelial tight junctions. This suggests that claudins can be targeted for tumor therapy, but there is not currently a method for delivering drugs to claudin-expressing cells. In the present study, we evaluated whether a potent claudin-4-binding C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) would allow targeting to claudin-4-expressing cells. We fused C-CPE to the protein synthesis inhibitory factor (PSIF), which lacks the cell binding domain of Pseudomonas exotoxin. This fusion protein, C-CPE-PSIF, was cytotoxic to MCF-7 human breast cancer cells, which express endogenous claudin-4, but it was not toxic to mouse fibroblast L cells, which lack endogenous claudin-4. The cytotoxicity of C-CPE-PSIF was attenuated by pretreating the MCF-7 cells with C-CPE but not bovine serum albumin. Also, deletion of the claudin-4-binding region of C-CPE reduced the cytotoxicity of C-CPE-PSIF. Finally, we found that C-CPE-PSIF is toxic to L cells expressing claudin-4 but not to normal L cells or cells expressing claudin-1, -2, or -5. These results indicate that use of the C-CPE peptide may provide a novel way to target drugs to claudin- expressing cells.