Isolation and purification of novel peptides derived from Sepia ink: Effects on apoptosis of prostate cancer cell PC-3

Novel prostate cancer therapeutics are in high demand. In order to identify potential therapeutic targets, protein from sepia ink was hydrolyzed by utilizing pepsin in an orthogonal array design. Pepsin hydrolysate (SH) obtained at optimal conditions exhibited the highest antitumor activity. Subsequently, a novel antitumor peptide, which was termed SHP, was isolated through ultrafiltration, gel filtration chromatography and reversed phase high-performance liquid chromatography. The amino acid sequence of SHP was identified as Leu-Lys-Glu-Glu-Asn-Arg-Arg-Arg-Arg-Asp with a molecular mass of 1371.53 Da. The results of the proliferation assay revealed that SHP significantly inhibited the proliferation of PC-3 cells in a time-and dose-dependent manner. Acridine orange/ethidium bromide staining indicated significant SHP-induced apoptosis. Furthermore, Annexin V/PI double-staining assays revealed that the percentage of early-stage apoptotic cells increased from 8.85 to 29% following PC-3 exposure to 5, 10 and 15 mg/ml SHP for 24 h. SHP-induced apoptosis was accompanied by the activation of cellular tumor antigen p53 and caspase-3, the upregulation of apoptosis regulator BAX, and the downregulation of apoptosis regulator Bcl-2. These findings suggest that SHP is a novel inducer of apoptosis in vitro and merits further investigation as a possible therapeutic agent for the treatment of cancer.