Mohammadi-Farsani Azadeh, Habibi-Roudkenar Mehryar, Golkar Majid, Shokrgozar Mohammad Ali, Jahanian-Najafabadi Ali, KhanAhmad Hossein, Valiyari Samira, Bouzari Saeid
Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran.
Medical Biotechnology Department, Paramedicine Faculty, Guilan University of Medical Sciences, Rasht, Iran.
EXCLI J. 2018 Jun 25;17:590-597. doi: 10.17179/excli2018-1120. eCollection 2018.
The NGR peptide is one of the well-known peptides for targeting tumor cells. It has the ability to target aminopeptidase N (CD13) on tumor cells or the tumor vascular endothelium. In this study, the NGR peptide was used for targeting A subunit of the Shiga toxin to cancer cells. The cytotoxic effect of the A-NGR fusion protein was assessed on HT1080, U937, HT29 cancer cells and MRC-5 normal cells. For this purpose, cells were treated with different concentrations of A-NGR (0.5-40 µg/ml). The evaluation of cell viability was achieved by MTT assay. Apoptosis was determined by annexin-V/PI double staining flow cytometry. Alterations in the mRNA expression of apoptosis - related genes were assessed by real time RT- PCR. The results showed that A-NGR fusion protein effectively inhibited the growth of HT1080 and U937 cancer cells in comparison to negative control (PBS) but for CD13-negative HT-29 cancer cells, only at high concentrations of fusion protein was inhibited growth recorded. On the other hand, A-NGR had little cytotoxic effect on MRC-5 normal cells. The flow cytometry results showed that A-NGR induces apoptosis. Furthermore, the results of real time RT-PCR revealed that A-NGR significantly increases the mRNA expression of caspase 3 and caspase 9. Conclusively, A-NGR fusion protein has the ability of targeting CD13-positive cancer cells, the cytotoxic effect on CD13-positive cancer cells as well as has low cytotoxic effect on normal cells.
NGR肽是一种著名的靶向肿瘤细胞的肽。它能够靶向肿瘤细胞或肿瘤血管内皮上的氨肽酶N（CD13）。在本研究中，NGR肽用于将志贺毒素A亚基靶向癌细胞。评估了A-NGR融合蛋白对HT1080、U937、HT29癌细胞和MRC-5正常细胞的细胞毒性作用。为此，用不同浓度的A-NGR（0.5-40μg/ml）处理细胞。通过MTT法评估细胞活力。通过膜联蛋白V/PI双染流式细胞术测定细胞凋亡。通过实时RT-PCR评估凋亡相关基因mRNA表达的变化。结果表明，与阴性对照（PBS）相比，A-NGR融合蛋白有效抑制了HT1080和U937癌细胞的生长，但对于CD13阴性的HT-29癌细胞，仅在高浓度融合蛋白时才记录到生长受到抑制。另一方面，A-NGR对MRC-5正常细胞几乎没有细胞毒性作用。流式细胞术结果表明，A-NGR诱导细胞凋亡。此外，实时RT-PCR结果显示，A-NGR显著增加了caspase 3和caspase 9的mRNA表达。总之，A-NGR融合蛋白具有靶向CD13阳性癌细胞的能力，对CD13阳性癌细胞具有细胞毒性作用，并且对正常细胞具有低细胞毒性作用。
