Classical growth factors for colon cancer cells have been extensively described including agonists of tyrosine kinase receptors such as epidermal growth factor and related proteins (1) or insulin-like growth factors (2). More recently, some G protein-coupled receptor (GPCR) 1 agonists such as peptide hormones (3-5), prostaglandins (6), or serine proteases (7, 8) have been shown also to promote colon cancer cell proliferation often through transactivation of the epidermal growth factor receptor (6, 8). These GPCRs are expressed in both normal colonic epithelium and colon tumors (9) or even ectopically expressed by cancer cells such as in the case of the neurotensin receptor NT1 (10) or the thrombin receptor protease-activated receptor 1 (7). Whatever their expression pattern, they probably all contribute to the growth of colon tumors because of the presence of abundant ligands in the neuroendocrine environment of colonic tumors and/or to the production of receptor ligands by the tumor itself (11, 12).Our knowledge of receptor agonist suppressing colon cancer cell growth is much more limited apart from a few observations regarding transforming growth factor-␤ (13) or Fas ligand (14). We reasoned that among the very rich environment of peptide hormones and neuropeptides in the gut, we should be able to find natural agonists behaving as suppressors of colon cancer growth. In order to test this hypothesis, we developed a very simple assay by using human colon adenocarcinoma cells HT29-D4 grown in 10% FCS and screened for various peptides by their ability to inhibit cell growth. We made two dramatic hits with orexin-A and orexin-B, which appear to be robust growth inhibitors as shown here.Orexin-A and orexin-B (15), also named hypocretin-1 and hypocretin-2 (16), were discovered in 1998 by orphan receptor technologies (15) or subtractive cDNA cloning (16). They are encoded by a single gene that drives the synthesis of preproorexin that is subsequently matured into the 33-amino acid orexin-A and the 28-amino acid orexin-B, sharing 46% amino acid identity in humans (reviewed in Ref. 17). Two orexin receptor subtypes OX 1 R and OX 2 R have been cloned (15). They are serpentine GPCRs that bind both orexins with poor selectivity and are coupled to Ca 2ϩ mobilization (15). Orexins were initially characterized as neuropeptides restricted to hypothalamic neurons that project in the brain to nuclei involved in the

3). Orexins were initially characterized in hypothalamic neurons, which project to and excite many brain areas (4, 5). Orexin neurons have functional interactions with hypothalamic feeding pathways and monoaminergic-cholinergic centers to provide a link between energy balance and the brain mechanisms that coordinate sleep/wakefulness states and motivated behavior such as food seeking (6 -10). In this context, the characterization of orexin deficiency in narcoleptic patients (11) and mutation of orexin receptors in canine (12) and murine (13) narcolepsy emphasized the key role of orexins in the regulation of sleep/wakefulness. More recent studies showed that orexins are not restricted to the hypothalamus but are also expressed in peripheral tissues including adrenals, gastrointestinal tract, or endocrine pancreas (for review see Ref.3). Orexins alter endocrine function (14) by increasing glucagon secretion and decreasing glucose-stimulated insulin release from pancreatic islets (15), suppressing GH secretion (16), or regulating corticotropin release (14).Two orexin receptor subtypes OX 1 R and OX 2 R have been cloned (1, 2). They are serpentine G protein-coupled receptors that bind both orexins with poor selectivity and appear to be coupled to calcium mobilization (1). In humans, OX 1 R and OX 2 R show 64% amino acid identity more particularly present in transmembrane domains whereas the N-terminal extracellular domain and C-terminal tail exhibit very weak sequence identity (3). Orexin receptors have been described in the central nervous system as well as peripheral organs (3,17).Recently we discovered a new unexpected aspect of orexins as potent proapoptotic peptides (18). We showed that orexin-A and orexin-B induce a drastic apoptosis in human colon cancer cell lines in culture resulting in massive reduction of cell growth. The effect was extended to human neuroblastoma cells (18). The OX 1 R but not the OX 2 R is expressed in colon cancer and neuroblastoma cells and was shown to be responsible for the proapoptotic effect of orexins. The role of OX 1 R in mediating apoptosis was further demonstrated by transfecting CHO cells with OX 1 R cDNA, which conferred the ability of orexins to promote apoptosis (18). The OX 1 R-mediated apoptosis has been shown to be associated with cytochrome c release into cytosol and activation of caspase-3 and caspase-7 (18).As mentioned above a second orexin receptor OX 2 R has been cloned (1). Like the OX 1 R, it is expressed in both the central nervous system and peripheral organs (3,17). However, its role in orexin-induced apoptosis is still unknown. In the present paper, we explored the role of OX 2 R with respect to apoptosis. We show that transfection of OX 2 R cDNA in