Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
ABNOBA GmbH, Pforzheim, Germany.
Sci Rep. 2018 Feb 9;8(1):2768. doi: 10.1038/s41598-018-20915-y.
Ribosome inactivating proteins (RIPs) are highly potent cytotoxins that have potential as anticancer therapeutics. Mistletoe lectin 1 (ML1) is a heterodimeric cytotoxic protein isolated from European Mistletoe and belongs to RIP class II. The aim of this project was to systematically study ML1 cell binding, endocytosis pathway(s), subcellular processing and apoptosis activation. For this purpose, state of the art cell imaging equipment and automated image analysis algorithms were used. ML1 displayed very fast binding to sugar residues on the membrane and energy-dependent uptake in CT26 cells. The co-staining with specific antibodies and uptake blocking experiments revealed involvement of both clathrin-dependent and -independent pathways in ML1 endocytosis. Co-localization studies demonstrated the toxin transport from early endocytic vesicles to Golgi network; a retrograde road to the endoplasmic reticulum. The pro-apoptotic and antiproliferative activity of ML1 were shown in time lapse movies and subsequently quantified. ML1 cytotoxicity was less affected in multidrug resistant tumor cell line 4T1 in contrast to commonly used chemotherapeutic drug (ML1 resistance index 6.9 vs 13.4 for doxorubicin; IC: ML1 1.4 ng/ml vs doxorubicin 24000 ng/ml). This opens new opportunities for the use of ML1 as an alternative treatment in multidrug resistant cancers.
核糖体失活蛋白（RIPs）是一种高效的细胞毒素，具有作为抗癌治疗剂的潜力。槲寄生凝集素 1（ML1）是一种从欧洲槲寄生中分离出来的异二聚体细胞毒性蛋白，属于 RIP 类 II。本项目的目的是系统研究 ML1 的细胞结合、内吞途径、亚细胞加工和凋亡激活。为此，使用了最先进的细胞成像设备和自动化图像分析算法。ML1 与细胞膜上的糖残基非常快速地结合，并在 CT26 细胞中进行能量依赖性摄取。用特异性抗体共染色和摄取阻断实验表明 ML1 内吞作用涉及网格蛋白依赖和非依赖途径。共定位研究表明毒素从早期内吞小泡运输到高尔基网络；这是内质网的逆行途径。在时程电影中显示了 ML1 的促凋亡和抗增殖活性，并随后进行了定量。与常用的化疗药物（多柔比星的 ML1 耐药指数为 6.9，阿霉素的 IC 为 1.4 纳克/毫升）相比，ML1 在多药耐药肿瘤细胞系 4T1 中的细胞毒性受影响较小。这为 ML1 在多药耐药癌症中的替代治疗开辟了新的机会。
