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

Abstract

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

Orexin-A stimulates 3β-hydroxysteroid dehydrogenase expression and cortisol production in H295R human adrenocortical cells through the AKT pathway

“…However, data on whether orexins regulate the growth of carcinoma cells are inconlcusive. Certain studies have found that orexins promote the growth of cancer cells (8,10), athough others have shown inhibitory effects (30,31). The different cell types used may be the reason for these conflicting results.…”

Mucosal IL-10 and TGF-β play crucial roles in preventing LPS-driven, IFN-γ–mediated epithelial damage in human colon explants

“…Informed patient consent was obtained, according to the French bioethics law (58), beginning August 6, 2004. Immediately after removal, the tissues were placed in 4°C oxygenated Krebs solution, and the mucosa was carefully stripped from the underlying compartment made of muscularis mucosae and submucosa as previously described (59,60). Fragments of 20-30 mg were cut out and pinned in Sylgard-coated Petri dishes and maintained in culture for 24 or 48 h in 2 ml RPMI 1640 medium (Invitrogen) containing 0.01% BSA and antibiotics (200 μg/ml streptomycin, 200 U/ml penicillin, 1% fungizone; Invitrogen).…”

Aberrant Expression of OX1 Receptors for Orexins in Colon Cancers and Liver Metastases: an Openable Gate to Apoptosis

“…6). We made 2 hits with orexin-A and orexin-B which appear to be robust growth inhibitors (6). Orexins do not alter cell cycle but promote strong apoptosis by inducing cytochrome c release from mitochondria to cytosol and activation of caspase-3 and caspase-7 (6).…”