Orexins Acting at Native OX1 Receptor in Colon Cancer and Neuroblastoma Cells or at Recombinant OX1 Receptor Suppress Cell Growth by Inducing Apoptosis

Abstract

Screening of 26 gut peptides for their ability to inhibit growth of human colon cancer HT29-D4 cells grown in 10% fetal calf serum identified orexin-A and orexin-B as anti-growth factors. Upon addition of either orexin (1 microM), suppression of cell growth was total after 24 h and >70% after 48 or 72 h, with an EC(50) of 5 nm peptide. Orexins did not alter proliferation but promoted apoptosis as demonstrated by morphological changes in cell shape, DNA fragmentation, chromatin condensation, cytochrome c release into cytosol, and activation of caspase-3 and caspase-7. The serpentine G protein-coupled orexin receptor OX(1)R but not OX(2)R was expressed in HT29-D4 cells and mediated orexin-induced Ca(2+) transients in HT29-D4 cells. The expression of OX(1)R and the pro-apoptotic effects of orexins were also indicated in other colon cancer cell lines including Caco-2, SW480, and LoVo but, most interestingly, not in normal colonic epithelial cells. The role of OX(1)R in mediating apoptosis was further demonstrated by transfecting Chinese hamster ovary cells with OX(1)R cDNA, which conferred the ability of orexins to promote apoptosis. A neuroblastoma cell line SK-N-MC, which expresses OX(1)R, also underwent growth suppression and apoptosis upon treatment with orexins. Promotion of apoptosis appears to be an intrinsic property of OX(1)R regardless of the cell type where it is expressed. In conclusion, orexins, acting at native or recombinant OX(1)R, are pro-apoptotic peptides. These findings add a new dimension to the biological activities of these neuropeptides, which may have important implications in health and disease, in particular colon cancer.