Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran; Molecular Biology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Science, Tehran, Iran.
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Eur J Pharmacol. 2020 Mar 5;870:172912. doi: 10.1016/j.ejphar.2020.172912. Epub 2020 Jan 10.
Immunotoxin therapy is one of the immunotherapy strategies providing a new, effective and high potency treatment against various cancers. Breast cancer is the most common cancer among women in many countries. The EPH receptors are a large part of tyrosine kinase receptors family and play an effective role in tumor development and angiogenesis. Among EPH receptors, EPHA2 is more commonly well-known and widely expressed in many cancers like breast cancer. In this study, we evaluated the specification of a designed immunotoxin formed by EPHA2-specific scfv linked with PE38KDEL on EPHA2-overexpressing breast cancer cell line. This new scfv-based recombinant immunotoxin was studied in terms of features such as binding potency, cytotoxicity effects, apoptosis induction ability, and internalization. The flow cytometry results showed that the immunotoxin can significantly (approximately 99%) bind to EPHA2-overexpressing breast cancer cell line (MDA-MB-231) in a low concentration (2.5 ng/ul) while cannot significantly bind to the normal cell line (HEK-293) or even EPHA2-very low expressing cell line (MCF-7). Using the MTT assay and Annexin V/Propidium iodide (PI) double staining method by flow cytometry, we observed significant killing and apoptosis induction of the MDA-MB-231 cells at different concentrations. Immunotoxin tracking by confocal microscopy at 2 h and 6 h revealed a massive presence of immunotoxin in the cytoplasm. Finally, given the in vitro results, it seems that this immunotoxin is competent enough to serve as a good candidate for in vivo studies to further explore the possibility of breast cancer treatment.
免疫毒素治疗是免疫治疗策略之一，为治疗各种癌症提供了一种新的、有效且高效的方法。乳腺癌是许多国家女性中最常见的癌症。EPH 受体是酪氨酸激酶受体家族的重要组成部分，在肿瘤的发展和血管生成中发挥着重要作用。在 EPH 受体中，EPHA2 更为常见，在许多癌症中广泛表达，如乳腺癌。在这项研究中，我们评估了由与 PE38KDEL 连接的 EPHA2 特异性 scfv 组成的设计免疫毒素在 EPHA2 过表达乳腺癌细胞系中的特异性。这种新型基于 scfv 的重组免疫毒素在结合效力、细胞毒性作用、凋亡诱导能力和内化等方面进行了研究。流式细胞术结果表明，免疫毒素在低浓度（2.5ng/ul）下能显著（约 99%）结合 EPHA2 过表达乳腺癌细胞系（MDA-MB-231），而不能显著结合正常细胞系（HEK-293）甚至 EPHA2 低表达细胞系（MCF-7）。通过 MTT 测定和流式细胞术 Annexin V/PI 双染色法，我们观察到不同浓度的 MDA-MB-231 细胞的显著杀伤和凋亡诱导作用。用共聚焦显微镜在 2 小时和 6 小时对免疫毒素进行跟踪，发现大量免疫毒素存在于细胞质中。最后，鉴于体外结果，这种免疫毒素似乎有足够的能力作为体内研究的良好候选物，以进一步探索治疗乳腺癌的可能性。
