A-NGR fusion protein induces apoptosis in human cancer cells

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.