Orexin-induced apoptosis: the key role of the seven-transmembrane domain orexin type 2 receptor.

Orexin-A and orexin-B are regulatory peptides involved in the control of feeding, sleep-wakefulness, and exerting various endocrine and metabolic actions. Recently we demonstrated that orexins, acting at OX(1) receptor (OX(1)R), are proapoptotic peptides. The aim of this study was to investigate the role of the receptor subtype OX(2)R in the control of apoptosis. Orexins caused a caspase-dependent cell death by apoptosis and a drastic cell growth inhibition in Chinese hamster ovary cells transfected with OX(2)R cDNA. On addition of either orexin (10(-6) m) for 48 h, apoptosis was demonstrated by DNA fragmentation, chromatin condensation, annexin-V binding, and activation of caspase-3 and caspase-9. Orexins were active on apoptosis and cell growth inhibition in the range of concentrations between 10(-10) and 10(-5) m with an EC(50) of 5 x 10(-8) m peptides. No effect of orexins could be detected in parental Chinese hamster ovary cells. A rat pancreatic acinar cell line, AR42J, which expresses OX(2)R but not OX(1)R, also underwent growth suppression and apoptosis on treatment with orexins. Suppression of AR42J cell growth by 10(-6) m orexin was more than 75% after 24 h. Induction of annexin-V-labeled AR42J cell number was dose dependent, with EC(50) of 5.1 x 10(-8) m orexin-A and 9.8 x 10(-8) m orexin-B. The OX(2)R agonist [Ala (11), d-Leu (15)]orexin-B promoted effects on cell growth and apoptosis, which were similar to those elicited by orexins. The OX(1)R antagonist SB33487 did not alter orexin-induced inhibition of growth or orexin-induced stimulation of apoptosis in AR42J cells. For the first time, we provide functional and pharmacological evidence for a role of the OX(2)R in orexin-induced apoptosis.

Orexins acting at native OX(1) receptor in colon cancer and neuroblastoma cells or at recombinant OX(1) 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 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.