Orexins acting at native OX₁ receptor in colon cancer and neuroblastoma cells or at recombinant OX₁ receptor suppress cell growth by inducing apoptosis

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 μM), suppression of cell growth was total after 24 h and >70% after 48 or 72 h, with an EC₅₀ 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 OX₁R but not OX₂R was expressed in HT29-D4 cells and mediated orexin-induced Ca²⁺ transients in HT29-D4 cells. The expression of OX₁R 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 OX₁R in mediating apoptosis was further demonstrated by transfecting Chinese hamster ovary cells with OX₁R cDNA, which conferred the ability of orexins to promote apoptosis. A neuroblastoma cell line SK-N-MC, which expresses OX₁R, also underwent growth suppression and apoptosis upon treatment with orexins. Promotion of apoptosis appears to be an intrinsic property of OX₁R regardless of the cell type where it is expressed. In conclusion, orexins, acting at native or recombinant OX₁R, 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.