Survivin belongs to the inhibitor of apoptosis protein family, which is consistently overexpressed in most cancer cells but rarely expressed in normal adult tissues. Therefore, the detection and inhibition of survivin are regarded as attractive strategies for cancer-specific treatment. In this study, we designed and synthesized 7-19 residues of inner centromere protein (INCENP)-derived small peptides (INC peptides) as novel survivin-targeting agents. The INC peptides showed binding affinity for the human survivin protein (K d = 91.4-255 nmol L −1 ); INC 16-22 , which contains residues 16-22 of INCENP, showed the highest affinity (91.4 nmol L −1 ). Confocal fluorescence imaging showed consistent colocalization of FITC-INC 16-22 and survivin in cell lines. Nona-arginine-linked INC 16-22 (r9-INC 16-22 ) rendered INC 16-22 cells penetrable and strongly inhibited cell growth of MIA PaCa-2 cells (52% inhibition at 1.0 µmol L −1 ) and MDA-MB-231 cells (60% inhibition at 10 µmol L −1 ) as determined by MTT assays. The exposure of MIA PaCa-2 cells to 40 µmol L −1 r9-INC 16-22 apparently reduced the intracellular protein expression levels of survivin. However, cleaved caspase-3 was significantly increased in cells treated with r9-INC 16-22 , even at 10 µmol L −1 , compared to untreated cells. Flow cytometry revealed that r9-INC 16-22 strongly induced apoptosis in MIA PaCa-2 cells. These results indicate that the cytotoxic effects of r9-INC 16-22 could be mediated mainly through the disruption of survivin-dependent antiapoptotic functions and partly because of the direct degradation of the survivin protein. Our findings suggest that INC peptides can act as useful scaffolds for novel cancer imaging and anticancer agents. K E Y W O R D S anticancer, apoptosis, cancer imaging, peptide, survivin 1 | INTRODUC TI ON Survivin, the smallest protein (16.5 kDa) among the inhibitor of apoptosis protein (IAP) family, is one of the most cancer-specific proteins 1,2 ; it is highly overexpressed in most cancers, but barely detected in the majority of normal cells. 3-6 Survivin is involved in at least 2 essential cellular processes, inhibition of apoptosis and regulation of the cell cycle. 5 Furthermore, it participates in another antiapoptotic effect by S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Fuchigami T, Ishikawa N, Nozaki I, et al. Discovery of inner centromere protein-derived small peptides for cancer imaging and treatment targeting survivin.

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Survivin is overexpressed in most cancer cells but is rarely expressed in normal adult tissues. It is associated with poor prognosis and resistance to radiation therapy and chemotherapy. In this study, we designed and synthesized borealin-derived small peptides (Bor peptides) to function as survivin-targeting agents for the diagnosis and treatment of cancers. These peptides exhibited binding affinities for recombinant human survivin (K d = 49.6–193 nM), with Bor65–75 showing the highest affinity (K d = 49.6 nM). Fluorescence images of fluorescein isothiocyanate-labeled Bor65–75 showed its co-localization with survivin expression in the human pancreatic cancer cell line, MIA PaCa-2. In the WST-1 assay, cell penetrable nona-d-arginine-conjugated Bor65–75 (r9-Bor65–75) inhibited the growth of MIA PaCa-2 cells and MDA-MB-231 cells (89 and 88% inhibition at 10 μM, respectively), whereas it had almost no effect on the human mammary epithelial cell line, MCF-10A, that inherently does not have high survivin expression. Flow cytometry with annexin V and propidium iodide staining revealed that r9-Bor65–75 induced apoptosis in MIA PaCa-2 cells in a dose-dependent manner. An increase in cleaved poly ADP-ribose polymerase protein expression was observed in MIA PaCa-2 cells exposed to r9-Bor65–75 by western blotting, suggesting that r9-Bor65–75 inhibits cell proliferation by inducing apoptosis. In vivo, r9-Bor65–75 significantly suppressed tumor growth in MIA PaCa-2 xenograft mice, without any marked weight loss. Hence, Bor peptides are promising candidates for the development of cancer imaging and anticancer agents targeting survivin.

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