Song Zhen-Tao, Zhang Leshuai W, Fan Li-Qiang, Kong Jing-Wen, Mao Jun-Hua, Zhao Jian, Wang Fu-Jun
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology.
School for Radiological and Interdisciplinary Sciences (RAD-X) and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou.
Anticancer Drugs. 2018 Sep;29(8):736-747. doi: 10.1097/CAD.0000000000000649.
Cyclosporin A (CsA) is a calcium antagonist and can enhance the efficacy of some protein drugs, but its mechanism remains unknown. In this study, MAP30, a ribosome-inactivating protein reported to have apoptotic effects on cancer cells, was fused with S3, an epidermal growth factor receptor (EGFR)-targeting peptide. In addition, CsA was used to investigate whether it can further promote the apoptotic effects of S3 fused MAP30 (MAP30-S3). Our result showed that the internalization of FITC-labeled MAP30-S3 was increased significantly by S3 in HeLa cells. Unexpectedly, MAP30-S3 only showed a minor decrease in the viability of EGFR-overexpressing cancer cells, including HeLa, SMMC-7721, and MGC803 (IC50>5 μmol/l). However, 2 μmol/l CsA significantly increased the cytotoxicity of MAP30-S3, especially for HeLa cells (IC50=40.3 nmol/l). In comparison, CsA did not further decrease the cytotoxicity of MAP30-S3 on MRC-5, an EGFR low-expressing cell line from normal lung tissue, indicating that CsA did not affect the cancer-targeting specificity of MAP30-S3. Our results also showed that CsA further increased the apoptotic activity of MAP30-S3 in HeLa cells. CsA could promote the endosomal escape of FITC-MAP30-S3 with a diffused pattern in the cytoplasm. Five endocytic inhibitors were used to investigate the cellular uptake mechanism of MAP30-S3, and the results showed that the endosomal escape-enhancing effect of CsA on MAP30-S3 may be associated with the clathrin-dependent endocytic pathways. Our study suggested that CsA could be a novel endosomal escape enhancer to potentiate the intracellular release of anticancer protein drugs, resulting in their improved therapeutic efficacy.
环孢素A（CsA）是一种钙拮抗剂，能够增强某些蛋白质药物的疗效，但其作用机制尚不清楚。在本研究中，将据报道对癌细胞具有凋亡作用的核糖体失活蛋白MAP30与表皮生长因子受体（EGFR）靶向肽S3融合。此外，使用CsA来研究其是否能进一步促进S3融合MAP30（MAP30-S3）的凋亡作用。我们的结果表明，在HeLa细胞中，S3显著增加了异硫氰酸荧光素（FITC）标记的MAP30-S3的内化。出乎意料的是，MAP30-S3在包括HeLa、SMMC-7721和MGC803在内的EGFR过表达癌细胞中的活力仅略有下降（半数抑制浓度[IC50]>5 μmol/L）。然而，2 μmol/L的CsA显著增加了MAP30-S3的细胞毒性，尤其是对HeLa细胞（IC50=40.3 nmol/L）。相比之下，CsA并未进一步降低MAP30-S3对来自正常肺组织的EGFR低表达细胞系MRC-5的细胞毒性，这表明CsA不影响MAP30-S3的癌症靶向特异性。我们的结果还表明，CsA进一步增加了HeLa细胞中MAP30-S3的凋亡活性。CsA能够促进FITC-MAP30-S3的内体逃逸，使其在细胞质中呈弥散分布。使用五种内吞抑制剂来研究MAP30-S3的细胞摄取机制，结果表明CsA对MAP30-S3的内体逃逸增强作用可能与网格蛋白依赖性内吞途径有关。我们的研究表明，CsA可能是一种新型的内体逃逸增强剂，可增强抗癌蛋白药物的细胞内释放，从而提高其治疗效果。
