Orexins Acting at Native OX1 Receptor in Colon Cancer andNeuroblastoma Cells or at Recombinant OX1 Receptor Suppress CellGrowth by Inducing Apoptosis*

Received for publication, April 14, 2004, and in revised form, August 6, 2004Published, JBC Papers in Press, August 13, 2004, DOI 10.1074/jbc.M404136200

Patricia Rouet-Benzineb‡§, Christiane Rouyer-Fessard‡, Anne Jarry¶, Virgile Avondo‡,Cecile Pouzet�, Masashi Yanagisawa**, Christian Laboisse¶, Marc Laburthe‡,and Thierry Voisin‡§§

From ‡INSERM U410, Neuroendocrinologie et Biologie Cellulaire Digestives and �IFR 02 Claude Bernard,Faculte de Medecine Xavier Bichat, 16 Rue Henri Huchard, BP 416, 75870 Paris Cedex 18, ¶INSERM U539,Faculte de Medecine, 44035 Nantes, France, and **Howard Hughes Medical Institute and Department ofMolecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050

Screening of 26 gut peptides for their ability to inhibitgrowth of human colon cancer HT29-D4 cells grown in10% fetal calf serum identified orexin-A and orexin-B asanti-growth factors. Upon addition of either orexin (1�M), 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 apo-ptosis as demonstrated by morphological changes in cellshape, DNA fragmentation, chromatin condensation, cy-tochrome c release into cytosol, and activation ofcaspase-3 and caspase-7. The serpentine G protein-cou-pled orexin receptor OX1R but not OX2R was expressedin HT29-D4 cells and mediated orexin-induced Ca2�

transients in HT29-D4 cells. The expression of OX1R andthe pro-apoptotic effects of orexins were also indicatedin other colon cancer cell lines including Caco-2, SW480,and LoVo but, most interestingly, not in normal colonicepithelial cells. The role of OX1R in mediating apoptosiswas further demonstrated by transfecting Chinese ham-ster ovary cells with OX1R cDNA, which conferred theability of orexins to promote apoptosis. A neuroblas-toma cell line SK-N-MC, which expresses OX1R, also un-derwent growth suppression and apoptosis upon treat-ment with orexins. Promotion of apoptosis appears to bean intrinsic property of OX1R regardless of the cell typewhere it is expressed. In conclusion, orexins, acting atnative or recombinant OX1R, are pro-apoptotic pep-tides. These findings add a new dimension to the biolog-ical activities of these neuropeptides, which may haveimportant implications in health and disease, in partic-ular colon cancer.

Classical growth factors for colon cancer cells have beenextensively described including agonists of tyrosine kinase re-ceptors 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 hor-mones (3–5), prostaglandins (6), or serine proteases (7, 8) havebeen shown also to promote colon cancer cell proliferation oftenthrough transactivation of the epidermal growth factor recep-tor (6, 8). These GPCRs are expressed in both normal colonicepithelium and colon tumors (9) or even ectopically expressedby cancer cells such as in the case of the neurotensin receptorNT1 (10) or the thrombin receptor protease-activated receptor1 (7). Whatever their expression pattern, they probably allcontribute to the growth of colon tumors because of the pres-ence of abundant ligands in the neuroendocrine environment ofcolonic tumors and/or to the production of receptor ligands bythe tumor itself (11, 12).

Our knowledge of receptor agonist suppressing colon cancercell growth is much more limited apart from a few observationsregarding transforming growth factor-� (13) or Fas ligand (14).We reasoned that among the very rich environment of peptidehormones and neuropeptides in the gut, we should be able tofind natural agonists behaving as suppressors of colon cancergrowth. In order to test this hypothesis, we developed a verysimple assay by using human colon adenocarcinoma cellsHT29-D4 grown in 10% FCS and screened for various peptidesby their ability to inhibit cell growth. We made two dramatichits with orexin-A and orexin-B, which appear to be robustgrowth inhibitors as shown here.

Orexin-A and orexin-B (15), also named hypocretin-1 andhypocretin-2 (16), were discovered in 1998 by orphan receptortechnologies (15) or subtractive cDNA cloning (16). They areencoded by a single gene that drives the synthesis of prepro-orexin that is subsequently matured into the 33-amino acidorexin-A and the 28-amino acid orexin-B, sharing 46% aminoacid identity in humans (reviewed in Ref. 17). Two orexinreceptor subtypes OX1R and OX2R have been cloned (15). Theyare serpentine GPCRs that bind both orexins with poor selec-tivity and are coupled to Ca2� mobilization (15). Orexins wereinitially characterized as neuropeptides restricted to hypotha-lamic neurons that project in the brain to nuclei involved in the

* This work was supported by INSERM. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: INSERM U460, Bat 13, 46 Rue Henri Huchard,BP 416, 75870 Paris Cedex 18, France.

§§ To whom correspondence should be addressed: INSERM U410,Faculte de Medecine Xavier Bichat, 16 Rue Henri Huchard, BP 416,75870 Paris Cedex 18, France. Fax: 33-0-42288765; E-mail: tvoisin@bichat.inserm.fr.

1 The abbreviations used are: GPCR, G protein-coupled receptor; OX1R,human orexin receptor type 1; OX2R, human orexin receptor type 2;TUNEL, TdT-mediated dUTP digoxigenin nick-end labeling; DAPI, 4�,6-diamidine-2�-phenylindole dihydrochloride; RT, reverse transcription;CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; PBS,phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium;FCS, fetal calf serum; LDH, lactate dehydrogenase; Chaps, 3-[(3-cholami-dopropyl)dimethylammonio]-1-propanesulfonic acid; Pipes, 1,4-pipera-zinediethanesulfonic acid; ELISA, enzyme-linked immunosorbent assay;h, human.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 44, Issue of October 29, pp. 45875–45886, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 45875

control of feeding, sleep-awakeness, neuroendocrine homeosta-sis, and autonomic regulation (17). Genetic or experimentalalterations of the orexin system have been shown to be associ-ated with narcolepsy (18, 19). More recent observations indi-cated that orexins and their receptors are not restricted to thehypothalamus but are also expressed in a few peripheral tis-sues (17), including the gastrointestinal tract (20).

Here we show that orexins acting at the OX1R suppress thegrowth of human colon cancer cells HT29-D4 by promotingapoptosis through cytochrome c release from mitochondria andcaspase activation. We further expand upon these data byshowing that activation of native OX1R in other colon cancercell lines and neuroblastoma cells or activation of recombinantOX1R expressed in CHO cells also leads to strong apoptosis andsubsequent growth suppression. The OX1 receptor-mediatedapoptosis therefore appears to be an intrinsic property of thereceptor regardless of the cell type where the receptor isexpressed.

EXPERIMENTAL PROCEDURES

Materials—Orexin-A, orexin-B, and other peptides were from Neo-sytem (Strasbourg, France) with the exception of cholecystokinin-8and gastrin-1 which were from Sigma. Rabbit polyclonal anti-OX1Rantibodies (297980A) were from Alpha Diagnostic International (SanAntonio, TX). Mouse monoclonal anti-cytochrome c antibodies6H2-B4 (immunoprecipitation experiments) and 7H8–2C12 (Westernblot) were from Pharmingen. Mouse monoclonal anti-�-actin antibod-ies (AC-74) were from Sigma. Rabbit polyclonal anti-caspase-3 anti-bodies, anti-cleaved caspase-3 antibodies (Asp-175), anti-caspase-7antibodies, and anti-cleaved caspase-7 antibodies (Asp-198) werefrom Cell Signaling Technology/Ozyme (Saint-Quentin en Yvelines,France). Human cytochrome c ELISA kit was from Bender MedSys-tems (San Bruno, CA). The In Situ Cell Death Detection kit and theM30 antibody were from Roche Diagnostics. The Enzyline LDH kitwas from Biomerieux (Marcy l’Etoile, France). The ATPlite kit wasfrom PerkinElmer Life Sciences. The Guava NexinTM assay was fromGuava Technologies (Hayward, CA).

Cell Culture—Chinese hamster ovary (CHO-K) cells were grown asdescribed (21). Recombinant CHO/hOX1R and CHO/hOX2R cell linesstably expressing human orexin receptor type 1 (OX1R) or humanorexin receptor type 2 (OX2R) were grown in F-12 medium containingL-glutamine, supplemented with 10% FCS, 100 �g/ml streptomycin, 100units/ml penicillin, and 0.7 mg/ml geneticin. The human colon cancercell line HT29-D4 (22) was obtained from Dr. J. Marvaldi (CNRS,Marseilles, France). The human colon cancer cell lines Caco-2, SW480,LoVo, and HCT116 were obtained from American Type Culture Collec-tion (Manassas, VA). HT29-D4, SW480, and HCT116 cell lines weregrown in Dulbecco’s modified Eagle’s medium (DMEM) containing glu-cose (4.5 g/liter) supplemented with 10% FCS, 100 �g/ml streptomycin,and 100 units/ml penicillin. The Caco-2 cell line was grown in DMEMcontaining glucose (4.5 g/liter) supplemented with 20% FCS, 1% non-essential amino acids, 100 �g/ml streptomycin, and 100 units/ml peni-cillin and the LoVo cell line in F-12 medium containing L-glutamine,supplemented with 10% FCS, 100 �g/ml streptomycin, 100 units/mlpenicillin. The human neuroblastoma cell line SK-N-MC was obtainedfrom the American Type Culture Collection. It was grown in minimumEagle’s medium containing Earle’s salts and L-glutamine supplementedwith 10% FCS, 1% nonessential amino acids, 1% sodium pyruvate, 100�g/ml streptomycin, and 100 units/ml penicillin. All cells were main-tained at 37 °C in a humidified 5% CO2/air incubator.

Isolation of Colonic Epithelial Cells from Normal Human Colon—Fresh normal human colons with no digestive disease were collectedwith the assistance of France-Transplant following French bioethicallaw. The colons were removed from small intestine and then immedi-ately carried from the operating room to our laboratory in an isothermicbox on ice. It usually took 30–60 min from the colon collection to thebeginning of the epithelial cell isolation procedure. The colons weregently washed with water, and normal colon epithelial cell isolation wasperformed as reported previously (23).

Explant Culture of Human Colonic Mucosa—Fragments of humannormal sigmoid colon taken at about 10 cm downstream to the tumorwere obtained from a patient undergoing surgery for moderately differ-entiated colon carcinoma. The tissue fragments were processed accord-ing to the Guidelines of the French Ethics Committee for Research onHuman Tissues. A sample, taken adjacent to the explants, was submit-

ted to histological analysis and subsequently reported as normal by thepathologists. Immediately after removal, the tissues were placed in 4 °Coxygenated Krebs solution and processed as described previously (24).The muscularis propria was removed by microdissection under micro-scopic control in a Sylgard-coated Petri dish. Mucosa was then carefullystripped from the underlying compartment made of muscularis muco-sae and submucosa. Fragments of 20–30 mg were cut out and pinned inSylgard-coated Petri dishes and maintained in culture for 24 h in 2 mlof RPMI 1640 medium (Invitrogen) containing 0.01% bovine serumalbumin and antibiotics (200 �g/ml streptomycin, 200 units/ml penicil-lin, 1% fungizone, Invitrogen). The explants were maintained at 37 °Cin a 95% oxygen, 5% carbon dioxide humid atmosphere on a rockingplatform at 30 rpm, in the absence (n � 4) or presence (n � 4) of 1 �M

orexin-B. Before culture (t0), some dissected fragments were frozen forsubsequent analysis. At the end of the 24-h culture, the supernatantswere centrifuged, and aliquots were stored at �80 °C for further anal-ysis. Tissue specimens were cut into several fragments as follows: onewas used for intracellular ATP measurement; one was used for histo-logical examination after formalin fixation/paraffin-embedding, andone was stored at �80 °C. Tissue integrity and cell viability of theexplants were assessed by standard morphological analysis, i.e. byhematoxylin-eosin staining on paraffin sections. Cell viability was alsoassessed by two assays as follows: measurement of the percentage ofLDH released and of the intracellular ATP level. LDH was measuredwith the Enzyline LDH kit both in the supernatant after the 24-hincubation (extracellular LDH or LDHe) and in a small fragment of thetissue (intracellular LDH or LDHi). Percent toxicity, represented bypercentage LDH released, was calculated as (LDHe/(LDHe � LDHi) �100), taking into account the weight of the tissue samples. The LDHrelease was not significantly different upon a 24-h orexin-B treatmentfrom control cultures (8.5 � 1.2 versus 10.5 � 1% respectively, n � 4).Cell viability was also assessed in the mucosal explants by measuringthe intracellular ATP level before (t0) and after the 24-h culture. ATPwas measured in tissue lysates with a luminometric assay ATPlite kitaccording to the manufacturer’s instructions. Orexin-B (1 �M) did notsignificantly modify the ATP level (7 � 0.9 pmol/�g protein, n � 4)compared with the untreated explant cultures (5.5 � 0.6 pmol/�g pro-tein, n � 4). These levels were not significantly different from those ofthe explanted tissue at t0, i.e. before culture (6.2 � 0.7 pmol/�g protein).

Cell Growth Assay—CHO-K cells, CHO/hOX1R cells (both seededat 5 � 104 cells/well), HT29-D4 cells, Caco-2, SW480, LoVo, HCT116,and SK-N-MC cells (seeded at 2 � 105 cells/well) were grown in24-well plates for 24 h in standard culture conditions with 10% FCS(see above). The culture medium was then replaced every 24 h withfresh medium containing or not containing orexins at concentrationsindicated in the legends to the figures. At the end of the treatment,adherent cells were trypsinized, and cells excluding trypan blue werecounted in a hemocytometer.

Cell Cycle Analysis and in Vitro [methyl-3H]Thymidine IncorporationAssay—HT29-D4 cells (3 � 105) were seeded in 25-cm2 dishes andgrown at 70–80% confluency in standard medium. In some experi-ments, cells were maintained in serum-free media for 48 h in order tosynchronize the cell cycle. The culture medium was then replaced withfresh standard medium containing or not containing 1 �M orexins. After24 h, adherent cells were harvested by trypsinization and fixed at�20 °C by 70% ethanol in phosphate-buffered saline (PBS). Cell ali-quots (106 cells) were then treated with 50 �g/ml RNase A for 15 minand stained with 10 �g/ml propidium iodide. Percentages of cells inG0/G1, S, and G2/M phases were determined by flow cytofluorometricanalysis (Beckman Coulter Epics XL-MCL). For [methyl-3H]thymidineincorporation, HT29-D4 cells (105 cells/well) were seeded in 12-wellclusters (Costar) in medium with 10% FCS and cultured for 24 h toallow cell adhesion. The medium was then removed; cells were washedwith PBS and cultured for another 48 h with serum-free medium.Thereafter, orexin-B (1 �M) was added in standard (10% FCS) or serum-free medium, and cells were cultured for 1, 2, 4, 6, or 24 h in thepresence of 0.1 �Ci of [methyl-3H]thymidine per well. The medium wasthen removed, and cells were washed twice with PBS and incubated intrypsin/EDTA for 10 min. They were then harvested in 200 �l ofmedium, incubated for 30 min in 5% trichloroacetate, and centrifugedat 10,000 � g for 4 min. Pellets were washed in 95% ethanol, solubilizedin 10% Triton X-100, and put in 2.5 ml of scintillation fluid for countingincorporated radioactivity. For each point, cells excluding trypan bluewere counted in a hemocytometer. The experiments were performed intriplicate wells and at least repeated twice. Results are expressed indisintegrations/min/106 viable cells.

Characterization of Apoptosis in Cultured Cell Lines—Three meth-ods were used for the characterization of apoptosis in cultured cell lines.

Pro-apoptotic Role of Human OX1R Orexin Receptor45876

For the TUNEL method, cells (7 � 105) were seeded on Lab-Tek cham-ber coverglasses and grown for 24 h in standard culture medium. Theculture medium was then replaced with fresh culture medium contain-ing or not containing orexins. After 24 h, cells were fixed for 30 min in4% paraformaldehyde/PBS at 4 °C followed by permeabilization for 10min with 0.2% Triton X-100 in PBS. Cells were then analyzed by theTUNEL method using the Roche Diagnostics in situ cell death detectionkit according to the manufacturer’s instructions. The samples werevizualized with a confocal laser scanning microscope (LSM 510 META,Zeiss). For in situ chromosome analysis, cells (2 � 105) were seeded onglass coverslips and grown for 24 h in standard medium. The culturemedium was then replaced with fresh culture medium containing or notcontaining orexins. After 24 h, cells were fixed with 80% ethanol for 10min at 4 °C and stained with either propidium iodide (50 �g/ml) orDAPI (10 �g/ml). Apoptotic nuclei were detected by epifluorescencemicroscopy. For DNA fragmentation assay, cells (2 � 106) were seededin culture dishes and grown for 24 h in standard culture medium. Theculture medium was then replaced with fresh culture medium contain-ing or not containing orexins. After 48 h, cells were trypsinized andcollected by centrifugation for 5 min at 1,000 � g. DNA was thenextracted as described (25). Briefly, cells were washed in 1 ml of coldPBS and then lysed in 50 mM Tris-HCl, pH 8.0, containing 2 mM NaCl,10 mM EDTA, 4% SDS, and 0.38 mg/ml of proteinase K. Samples wereincubated for 30 min at 65 °C, followed by an overnight incubation at37 °C. DNA was then precipitated, washed, resuspended in Tris-EDTAbuffer, and incubated for 1 h with 0.1 mg/ml RNase A as described (25).DNA fragmentation was analyzed on 1.5% agarose gels in the presenceof 0.5 mg/ml ethidium bromide.

Cytochrome c Release—HT29-D4 cells were seeded in 75-cm2 culturedishes (106/dish) and grown in standard culture medium for 24 h. Themedium was then replaced by a fresh culture medium containing or notcontaining orexins, and cells were further grown for 24 h. After celllysis, cytochrome c was measured in cytosol (14,000 � g supernatantafter 15 min centrifugation) by using an ELISA kit (see above) accord-ing to the manufacturer’s instructions. Under these experimental con-ditions, the cytosolic fraction did not contain mitochondria as verified byelectronic microscopy.2 Alternatively, cytochrome c was characterizedby immunoprecipitation followed by Western blot. Briefly, cytochrome cin cytosol was immunoprecipitated using the 6H2-B4 monoclonal anti-cytochrome c antibodies (2 �g/ml) and protein G-Sepharose beads. Afteraddition of SDS-PAGE sample buffer to the washed beads, the sampleswere separated by electrophoresis on 10% SDS-polyacrylamide gel andthen transferred to nitrocellulose membrane. Total cytochrome c pres-ent in the cellular extract before cytosolic separation was characterizedas described above. The blot was probed with 1 �g/ml of 7H8–2C12monoclonal anti-cytochrome c antibodies, and immune complexes wererevealed with secondary peroxidase-conjugated antibodies using achemiluminescent kit.

In Situ Caspase Activation—HT29-D4 cells (7 � 105) were seeded onLab-Tek chamber coverglasses and grown for 24 h in standard culturemedium. The culture medium was then replaced with fresh culturemedium containing or not containing 1 �M orexins. After 24 h, caspaseactivation was detected as described (26) using the CaspACETM FITC-VAD-FMK In Situ Marker (Promega Corp., Madison, WI) which bindsto activated caspases. The bound marker was then localized by fluores-cence detection using a confocal microscope.

Immunocytochemical Studies of Cleaved Forms of Caspase-3 andCaspase-7—HT29-D4 cells (7 � 105) were seeded on Lab-Tek chambercover glasses and grown for 24 h in standard culture medium. Theculture medium was then replaced with fresh culture medium contain-ing or not containing orexins. After 24 h, the coverslips were probedwith rabbit polyclonal anti-cleaved caspase-3 antibodies (Asp-175, di-lution 1:100) or anti-cleaved caspase-7 antibodies (Asp-198, dilution1:100) that specifically recognize cleaved enzyme isoforms. These anti-bodies do not recognize full-length caspase-3 or full-length caspase-7 orother cleaved caspases. Fluorescein isothiocyanate (FITC)-conjugatedgoat anti-rabbit immunoglobulin IgG was used as the secondary anti-body. Vectashield mounting medium containing propidium iodide wasadded, and coverslips were observed by confocal microscopy.

Western Immunoblotting Studies of Caspase-3 and Caspase-7—HT29-D4 cells were seeded in 75-cm2 culture dishes (106/dish) andgrown in standard culture medium for 24 h. The culture medium wasthen replaced with fresh culture medium containing or not containingorexins. After 24 h, cells were lysed by adding Chaps cell extract buffer

containing 50 mM Pipes/NaOH (pH 6.5), 2 mM EDTA, 0.1% Chaps, 5 mM

DTT, 2 �g/ml leupeptin, 1 �g/ml pepstatin, 1 �g/ml aprotinin, and 1 mM

phenylmethylsulfonyl fluoride. Cells were resuspended in the bufferand frozen and thawed three times, and the lysate was centrifuged for30 min at 14,000 rpm. After addition of SDS-PAGE sample buffer to thesupernatant (30 �g of protein), the samples were separated by electro-phoresis on 16% SDS-polyacrylamide gel and then transferred to nitro-cellulose membrane. Blots were probed with rabbit polyclonal anti-cleaved caspase-3 antibodies (Asp-175, dilution 1:1000) or anti-cleavedcaspase-7 antibodies (Asp-198, dilution 1:1000) that specifically recog-nize cleaved enzyme isoforms. Subsequently, blots were probed withrabbit polyclonal anti-caspase-3 antibodies (1:1000), which principallydetect the full-length procaspase-3 (32 kDa) and the fragment of cleavedcaspase-3 following cleavage at Asp-175 (17 kDa), or with rabbit poly-clonal anti-caspase-7 antibodies (1:1000), which detect the full-lengthprocaspase-7 (35 kDa). Blots were standardized by using the mousemonoclonal anti-�-actin antibodies (AC-74). Immune complexes wererevealed with secondary peroxidase-conjugated antibodies by using achemiluminescent kit.

Morphological Analysis of Apoptotic Cell Death in Human ColonicMucosa Explants—Morphological analysis of apoptotic cell death wasassessed on paraffin sections by using two assays. The DNA-specific dyeHoechst 33258 (Calbiochem), which visualizes all the steps of the apo-ptotic process, was applied on deparaffinized sections (1 �g/ml inHanks’ balanced salt solution without phenol red, Invitrogen). Sectionswere mounted with the Prolong Antifade medium (Molecular Probes,Eugene, OR). The fluorescence was observed on an Axiovert 200-M-CarlZeiss microscope equipped with an ApoTome slider. Cells were visual-ized with a �63/1.4 oil immersion lens. Image processing was per-formed using an AxioCam MRCCD camera and the AxioVision 4.0software (Carl Zeiss). In addition, immunohistochemical staining ofapoptotic epithelial cells was performed by using the M30 antibody(1:50) as described previously (27). This antibody recognizes a cyto-keratin 18 neoepitope cleaved by caspases and is considered as an earlymarker of apoptosis in epithelial cells (28, 29).

Apoptotic Cell Measurement by Annexin V Labeling—Apoptosis wasanalyzed in SK-N-MC, CHO/hOX1R, and parent CHO-K cell lines usingthe Guava NexinTM kit, which discriminates between apoptotic andnonapoptotic cells. The Guava NexinTM assay utilizes annexin V-phy-coerythrin to detect phosphatidylserines on the external membrane ofapoptotic cells. Cells (2 � 106) were seeded in culture dishes and grownfor 24 h in standard culture medium. The culture medium was thenreplaced with fresh culture medium containing or not containingorexin-B. After 24 h, apoptotic cell staining was performed according tothe manufacturer’s instructions and analyzed with a Guava PCA sys-tem. Results are expressed as the percentage of apoptotic annexinV-phycoerythrin-positive cells and are the means of four analyses.

RT-PCR—For cultured cell lines (HT29-D4, Caco-2, SW480, LoVo,and HCT116) or for epithelial cells isolated from normal human colons,total RNA (RNAT) was extracted from cells by using Trizol® reagent(Invitrogen). Five �g of RNAT were reverse-transcribed by using oli-go(dT) primers. Twenty five percent of the cDNA mixture was amplifiedby using human OX1R sense primer (5�-CCTGTGCCTCCAGACTATG-A-3�) and OX1R antisense primer (5�-ACACTGCTGACATTCCATGA-3�), OX2R sense primer (5�-TAGTTCCTCAGCTGCCTATC-3�) andOX2R antisense primer (5�-CGTCCTCATGTGGTGGTTCT-3�), or �-a-ctin sense primer (5�-ATCTGGCACCACACCTTCTACAATGAGCTGC-G-3�) and �-actin antisense primer (5�-CGTCATACTCCTGCTTGCTG-ATCCACATCTGC-3�). Each of the 30 cycles of amplification consistedof 94 °C for 1 min, 63 °C for 1 min, and 72 °C for 1 min. Amplicons wereseparated by electrophoresis in 1.5% agarose gel, stained with ethidiumbromide, and viewed under ultraviolet illumination. The OX1R ampli-con (500 bp) obtained from HT29-D4 RNAT was subcloned by thepGEM®-T-Easy vector system (Promega Corp.) and sequenced.

Immunocytochemical Detection of OX1R—Cells (2 � 105) were cul-tured on coverslips, washed with cold PBS, and immediately fixed inice-cold 4% paraformaldehyde in PBS for 1 h followed by three washingsin PBS. Samples were incubated for 1 h at 4 °C with 10% newborn calfserum in TBST buffer (10 mM Tris, pH 8, 150 mM NaCl and 0.1% Tween20) and then incubated overnight at 4 °C with rabbit polyclonal anti-bodies against OX1R (dilution 1:100). Staining was revealed usingFITC-conjugated goat serum anti-rabbit IgG by confocal microscopy.

[Ca2�]i Measurements by Confocal Fluorescence Imaging—HT29-D4cells (2.5 � 105cells/cm2) were seeded onto LabTek. When cells achievednear-confluency (24 h before the experiment), the medium was replacedwith free red phenol DMEM without serum. Cells were incubated for 30min at 37 °C (5% CO2 and darkness) in serum-free medium containing6.7 �M Fluo-4 acetoxymethyl ester (Fluo-4AM) and rinsed twice with2 G. Peranzi and M. Ostuni, unpublished results.

Pro-apoptotic Role of Human OX1R Orexin Receptor 45877

assay buffer containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM

MgSO4, 1.2 mM KH2PO4, 6 mM glucose, 1 mM probenicid, and 25 mM

Hepes, pH 7.4. De-esterification of the intracellular fluorophore wasperformed at 37 °C for 30 min. The change of intracellular Ca2� con-centration was examined by using a confocal laser-scanning microscope(LSM 510 META, Zeiss) with a �40 objective. Fluo4-AM was excited bythe 488-nm argon laser line, and emission was collected through a505–530-nm bandpass filter.

Fura-2/AM Loading and Intracellular Calcium Measurement by Flu-orimeter—Intracellular calcium concentrations were measured usingFura-2/AM. HT29-D4 cells or HCT116 cells (5 � 103 cells/ml) wereseeded onto the center of glass coverslips and cultured in DMEM for 4days to 70–80% confluence. These coverslips were then loaded with 5�M of Fura-2/AM in Na-Hepes-buffered saline, pH 7.4 (135 mM NaCl,4.6 mM KCl, 1.2 mM MgCl2, 11 mM Hepes, 11 mM glucose, and 1.5 mM

CaCl2), containing 0.01% pluronic acid for 45–60 min at 37 °C. Theywere then washed in Na-Hepes buffer and placed at 37 °C in a fluorim-eter. The fluorescence was measured with a dual wavelength excitationfluorimeter at 340 and 380 nm for excitation and 510 nm for emission.The cells were challenged first with 1 �M orexin-B followed by a controlchallenge with 0.1 �M neurotensin.

Miscellaneous Procedures—Routine procedures such as radioimmu-noassay of intracellular cAMP content (30) and measurement of proteincontents (31) were performed as described.

Statistical Analysis—All data were expressed as mean � S.E. valuesand analyzed by analysis of variance and Student’s t test for statisticalsignificance. A p value of �0.05 was considered as statistically significant.

RESULTS

Orexins Inhibit Cell Growth in Human Colon Cancer CellsHT29-D4 in Culture—Human colon cancer HT29-D4 cellsgrown in standard medium in the presence of 10% FCS weretreated for 24 h with a variety of peptide hormones or neu-ropeptides present in the gut. Among the 26 peptides tested,orexin-A and orexin-B were the only peptides inhibiting cellgrowth, other peptides being without any effect or even stim-ulating cell growth such as in the case of ghrelin (Table I). Inthe presence of 10% FCS, which triggers a strong mitogeniceffect on HT29-D4 cells, orexin-A and orexin-B elicited a dra-matic decrease in cell number (Fig. 1, A and B). This inhibitionof serum-induced increase in cell number, referred to as sup-pression of cell growth, was almost total after 24 h (Fig. 1, Aand B) of treatment and still extensive after 48 (Fig. 1, A and B)or 72 h (Fig. 1B) of treatment, i.e. �70% suppression. Orexinswere active in the range of concentrations between 1 nM and 1�M, with half-maximal responses being obtained for 5 nM forboth orexins (Fig. 1C). Similar dose-response curves were ob-served after challenging cells with orexins for 24 (Fig. 1C) or48 h (not shown). After 1–2 days of challenge with orexins somemorphological changes in cell shape were observed, in partic-ular the cells looked rounder and shrunken (Fig. 1A, insets fand g). Although no cell detachment was observed, these mor-phological changes were reminiscent of apoptosis.

Orexins Promote Cell Apoptosis but Do Not Alter Cell Prolif-eration in Human Colon Cancer Cells HT29-D4 in Culture—Next we determined whether suppression of HT29-D4 cellgrowth by orexins was related to inhibition of cell proliferationand/or induction of cell apoptosis. Flow cytometric data of cellcycle analysis of control cells cultured in standard mediumwith 10% FCS indicated that 69.5 � 4.5% of HT29-D4 cells arein G0/G1 phase; 15.6 � 2.9% are in S phase, and 13.2 � 2.2%are in G2/M phase (n � 6). When HT29-D4 cells were treatedwith 1 �M orexin-A or orexin-B for 24 h, 69.0 � 5.3 and 70.2 �5.9% of cells were found in G0/G1 phase, 14.8 � 2.2 and 13.4 �2.1% in S phase, and 12.3 � 3.6 and 14.0 � 2.5% in G2/M phase,respectively (n � 6). These data indicated that orexins have nosignificant effect on the HT29-D4 cell cycle. This was furtherconfirmed when experiments were carried out with HT29-D4cells that were synchronized by serum deprivation for 48 h.Indeed treatment of synchronized cells with 10% serum in theabsence or presence of 1 �M orexin-B (Fig. 2A) or orexin-A (data

not shown) provided identical flow cytometric data. Direct as-sessment of DNA synthesis by [methyl-3H]thymidine incorpo-ration into DNA of HT29-D4 cells further supported the ideathat orexins did not alter cell proliferation. Indeed, treatmentof synchronized HT29-D4 cells with orexin-B (1 �M) did notmodify serum-induced [methyl-3H]thymidine incorporationinto DNA (Fig. 2B).

In sharp contrast with the proliferation data, a body of evi-dence supported that HT29-D4 cells undergo apoptosis uponorexin-A or orexin-B treatment. Fluorescence microscopic anal-ysis of DNA-staining patterns with DAPI (Fig. 3A, a–c) re-vealed an increase in apoptotic cell death in orexin-treated cellsas compared with control cells. Moreover, HT29-D4 cells dis-played typical chromatin condensation and fragmentation ofnuclei into small spherical particles upon treatment with orex-ins. DNA fragmentation was ascertained by the TUNEL assay(Fig. 3A, d–f). TUNEL-positive cells were observed uponorexin-A or orexin-B treatment, whereas no labeling was de-tected in control cells. Apoptosis was further indicated by typ-ical DNA ladder corresponding to cleavage of genomic DNAupon cell treatment with orexins (Fig. 3B).

Orexin-induced Cell Apoptosis in Human Colon CancerHT29-D4 Cells Is Associated with Cytochrome c Release andCaspase Activation—Orexin-induced apoptosis was shown tobe associated with cytochrome c release into cytosol. By using acytochrome c ELISA, we showed that cytochrome c levels incytosol were significantly increased 3, 15, and 24 h after chal-lenging the cells with orexin-A or orexin-B (Fig. 4A). These datawere consistent with immunoprecipitation/Western blottinganalysis that also indicated a sharp increase in cytosolic cyto-

TABLE IEffect of gut peptides on serum-induced increase

of HT29-D4 cell numberHuman colon cancer HT29-D4 cells grown in standard medium con-

taining 10% FCS were treated for 24 h with peptide hormones orneuropeptides present in the gut. Cells were seeded at low density in48-well plates and grown at 70–80% confluency (about 90,000 cells perwell). The culture medium was then replaced with fresh medium con-taining 10% FCS without (none) or with 0.1 �M peptides. After 24 h,cells were harvested by trypsinization and counted. Cells produced over24-h period of treatment (number of cells/well) are expressed asmeans � S.E. (six determinations).

Peptide (0.1 �M) 24-h cell production

None 193,559 � 10,634Bombesin 178,773 � 9857Cholecystokinin-8 189,386 � 12,469Galanin 217,723 � 19,847Gastrin 174,448 � 5898Gastric inhibitory polypeptide 176,539 � 9801Ghrelin 287,817 � 27,718a

Glucagon-like peptide-1 183,774 � 6741Glucagon-like peptide-2 176,812 � 9904Glucagon 190,456 � 17,284Growth hormone-releasing factor 187,214 � 7344Neuromedin N 186,017 � 5066Neurotensin 187,657 � 8510Neuropeptide Y 187,975 � 15,033Orexin-A 76,668 � 3711a

Orexin-B 46,717 � 10,741a

Pituitary adenylate cyclase-activating polypeptide

190,032 � 12,101

Pancreastatin 181,506 � 16,306Peptide histidine-methionine 181,808 � 5729Pancreatic polypeptide 192,061 � 15,920Prolactin-releasing peptide 195,098 � 25,148Parathyroid hormone 186,295 � 6265Peptide YY 184,594 � 7799Secretin 192,655 � 10,372Somatostatin-14 208,654 � 13,310Vasoactive intestinal contractor 191,688 � 25,573Vasoactive intestinal peptide 189,207 � 8802

a p � 0.001 versus none.

Pro-apoptotic Role of Human OX1R Orexin Receptor45878

chrome c after a 24-h challenge with orexins (Fig. 4B). Furtherexperiments showed that orexin-induced apoptosis is associ-ated with caspase activation. In situ caspase activation wasfollowed by cleavage of a fluorogenic substrate (see “Experi-mental Procedures”). The fluorescent product was strongly la-beled orexin-B (1 �M)-treated cells (Fig. 5A, b). Similar resultswere obtained with orexin-A (data not shown). A faint fluores-cent labeling was also observed in control cells indicating a lowbackground of caspase activation in the culture conditions used(Fig. 5A, a). To confirm caspase activation in orexin-inducedapoptosis in HT29-D4 cells, we immunodetected cleavage ofeffector caspases downstream of the cytochrome c release, i.e.caspase-3 and caspase-7. By using cleaved caspase-3 (Asp-175)and cleaved caspase-7 (Asp-198) antibodies, endogenous levelsof cleaved caspase-3 (Fig. 5A, d) and cleaved caspase-7 (Fig. 5A,f) were detected in HT29-D4 apoptotic cells upon 24 h of treat-ment with 1 �M orexin-B. Cleaved caspase-3 and caspase-7detected upon HT29-D4 cell treatment with orexin-B were co-localized with fragmented nuclei (Fig. 5A, d and f). Similarresults were obtained with orexin-A (data not shown). Nocleaved caspase-3 (Fig. 5A, c) or caspase-7 (Fig. 5A, e) could bedetected in control untreated cells. Further characterization ofthe effects of orexins on caspase cleavage was obtained byWestern blot. As shown in Fig. 5B, treatment of HT29-D4 cellsfor 24 h with 1 �M orexin-B resulted in the appearance of 19-and 17-kDa forms of cleaved caspase-3 and the 20-kDa form ofcleaved caspase-7 (Fig. 5B, right), whereas no cleaved caspasescould be detected in control cells. Similar data were obtainedwith orexin-A (not shown). The pro-forms of caspase-3 (Fig. 5B,left) and caspase-7 (Fig. 5B, right) were present in both controland orexin-treated cells.

HT29-D4 Cells Express the OX1 Receptor Subtype—Becausereliable orexin tracers are still unavailable (17), the nature oforexin receptor subtype(s) expressed in HT29-D4 cells wasdetermined by RT-PCR and immunocytochemistry. Total

mRNA from HT29-D4 cells or control CHO cell lines expressingrecombinant hOX1R or hOX2R were reverse-transcribed andamplified with specific couples of primers for the two subtypesof orexin receptors. Amplification products of the expected sizewere obtained in HT29-D4 cells with OX1R primers but notOX2R primers (Fig. 6A). Control recombinant CHO cells clearlyexpress OX1R or OX2R transcripts (Fig. 6A), supporting theidea that the absence of OX2R mRNA in HT29-D4 cells was notrelated to limits of RT-PCR technology. The OX1R-amplifiedproduct obtained from HT29-D4 cells was sequenced and foundto be identical to the hOX1R cDNA sequence (GenBankTM

AF0412343). To provide evidence for OX1R protein expressionin HT29-D4 cells, indirect immunofluorescence was performedby using rabbit polyclonal antibodies against hOX1R (Fig. 6B).Strong immunostaining with membrane localization was ob-served. This staining was completely abolished by co-incubat-ing antibodies with the immunogen peptide.

Because orexins were shown previously to consistently in-duce Ca2� transients in orexin receptor-expressing cells (15),we further investigated this second messenger as evidence forthe presence of functional OX1R in HT29-D4 cells. The intra-cellular Ca2� concentration was first monitored under a confo-cal microscope using Fluo-4AM dye. Fig. 6C shows confocalpictures before and after treatment with 1 �M orexin-B. Theneuropeptide clearly induced Ca2� transients as shown byconfocal images. The effect of orexin-B on cytosolic calcium wasfurther tested in Fura-2/AM dye-loaded HT-29-D4 cells by flu-orescence analysis in a classical fluorimeter. Again orexin-Binduced calcium transients with a time course of responsesimilar to that observed with neurotensin (Fig. 6D), a wellknown inducer of calcium transients in human colon cancercells (32). In sharp contrast, orexin-B failed to induce calciumtransients in the human colon cancer HCT116 cells (Fig. 6D)that are not equipped with OX1 receptor (see below). As acontrol, we showed that neurotensin nicely induced calcium

FIG. 1. Effect of orexins on serum-induced increase of HT29-D4 cellnumber. A, a–g show morphological as-pects of HT29-D4 cells cultured with 10%FCS at day 0 (a), after 24 h of culturewithout (b) or with 1 �M orexin-A (c) ororexin-B (d), and after 48 h of culturewithout (e) or with 1 �M orexin-A (f) ororexin-B (g). Photographs were taken un-der reverse phase microscope (magnifica-tion: �10 or 40 in the insets). B showsgrowth curves of HT29-D4 in the presenceof culture medium containing 10% FCS(● ) supplemented with 1 �M orexin-A (Œ)or orexin-B (E). C shows dose-responsecurves of orexin-A (● ) and orexin-B (E) onHT29-D4 cell growth upon 24 h of treat-ment with peptides. The results aremeans � S.E. of three separateexperiments.

Pro-apoptotic Role of Human OX1R Orexin Receptor 45879

transients in HCT116 cells. All these results clearly showedthat functional OX1 receptors are expressed in HT29-D4 cells.

Expression of OX1R and Anti-growth and Pro-apoptotic Ef-fects of Orexins Are Observed in Other Colon Cancer Cell Linesbut Not in Normal Colonic Epithelium—As shown in Fig. 7, theOX1R-mediated effects of orexins are observed in three of fourother human colon cancer cell lines tested. RT-PCR experi-ments showed that amplification products of the expected sizewere obtained in Caco-2, SW480, and LoVo cells with OX1Rprimers (Fig. 7A). In contrast, no OX1R mRNA could be de-tected in the HCT116 cell line. In good agreement with theRT-PCR data, orexin-B strongly inhibited FCS-stimulated cellgrowth in Caco-2, SW480, and LoVo cells but not in HCT116cells (Fig. 7B). Finally, the effect of orexin-B on apoptosis wastested in the four colon cancer cell lines. Apoptosis was clearlyindicated by a typical DNA ladder corresponding to cleavage ofgenomic DNA upon cell treatment with orexin-B in Caco-2,SW480, and LoVo but not HCT116 cells (Fig. 7C). Altogetherthese data indicated that expression of OX1R and OX1R-medi-ated anti-growth and pro-apoptotic effects of orexins are fre-quent in colon cancer because they are observed in four of fivehuman colon cancer cell lines originating from different patienttumors, i.e. HT29-D4, Caco-2, SW480, and LoVo.

In this context, we explored the status of OX1R in normalhuman colonic mucosa. RT-PCR experiments using total RNAextracted from pure epithelial cell preparations isolated fromthree normal human colons failed to indicate OX1R mRNA (notshown) under conditions in which specific amplification prod-ucts were clearly detected in human colon cancer cell lines (seeFigs. 6A and 7A). Because the long term culture of isolatedhuman colonic epithelial cells still remains an elusive task andthe existence of normal intestinal epithelial cell lines is still adebated question, we used explant cultures of dissected humannormal colonic mucosa to assess the effects of orexin on apo-ptosis in normal colon. These mucosal explants (polarized epi-thelial barrier and underlying lamina propria) can be main-tained in good viability conditions, as assessed by amultiparametric approach (see “Experimental Procedures”).Mucosal explants maintained their morphological integrityover a 24-h culture period as shown by standard histology (Fig.8). Spontaneous apoptosis occurred in a minority of epithelialcells (less than 1%). Indeed, a few apoptotic epithelial cellswere visualized by the M30 antibody immunostaining specificfor caspase-3-cleaved cytokeratin 18 and by the Hoechst dye,which reveals DNA condensation and fragmentation. Theywere preferentially located at the tip of the surface epithelium,

FIG. 2. Absence of effect of orexins on HT29-D4 cell proliferation. A represents cytometric flow analysis. Cells were seeded at low densityin a 25-cm2 flask and grown at 70–80% confluency. Cells were maintained in serum-free media for 48 h to synchronize the cell cycle. The culturemedium was then replaced with fresh medium containing no FCS (left) or 10% FCS without (middle) or with (right) 1 �M orexin-B. After 24 h,adherent cells were harvested by trypsinization and subjected to propidium iodide (PI) staining. After counting 10,000 events, the data areexpressed in % of the cell population in different phases (G0/G1, S, and G2/M) of the cell cycle and represent the means � S.E. of duplicate samplesfrom three different experiments ((without FCS: 92.4 � 3.9% in G0/G1, 3.2 � 0.5% in S, and 4.6 � 0.3% in G2/M) (with FCS: 57.5 � 3.3% in G0/G1,23.4 � 1.3% in S, and 16.3 � 0.9% in G2/M) (with FCS and orexin-B: 56.9 � 2.9% in G0/G1, 25.1 � 2.1% in S ,and 16.2 � 0.6% in G2/M)). B showseffect of orexin-B (1 �M) on [methyl-3H]thymidine incorporation in the DNA of HT29-D4 cells grown in the presence of 10% FCS. Cells were seededat 105 cells per well in 12-well plates and grown for 24 h in standard culture medium with 10% FCS. Cells were then grown in serum-free mediumfor 72 h. Finally, cells were cultured for up to 24 h in fresh medium containing 10% FCS and 0.1 �Ci of [methyl-3H]thymidine in the presence (blackbar) or in the absence (open bar) of 1 �M orexin-B. At time 0, cells were 427,000 � 28,000 per well. After 24 h, HT29-D4 cells were 801,000 � 33,900in standard medium and 408,000 � 10,680 in the presence of orexin-B. Values, expressed as disintegrations per min of [methyl-3H]thymidineincorporated per 106 cells, are means � S.E. of triplicate experiments.

Pro-apoptotic Role of Human OX1R Orexin Receptor45880

undergoing exfoliation, and occasionally in the lower region ofthe crypt (Fig. 8). A few lamina propria macrophages underly-ing the epithelial barrier occasionally contained M30 antibody-positive cells. Most interestingly, a 24-h treatment with 1 �M

orexin-B neither altered the morphological integrity of the co-lonic crypts nor increased the number of apoptotic cells (Fig. 8).

The OX1R-mediated Anti-growth and Pro-apoptotic Effects ofOrexins Are Intrinsic Properties of the OX1R and Not Depend-ent on Cell Context—All the actions of orexins described hereinwere observed in human colon cancer cell lines. We next askedthe question of whether the OX1R-mediated anti-growth andpro-apoptotic effects of orexins are restricted to colon cancercells or are intrinsic properties of the OX1R. Because OX1Rswere initially described in the brain (15), we first tested thehuman neuroblastoma cell line SK-N-MC which expressesOX1R (33). As shown in Fig. 9, orexin-A and orexin-B, in therange of concentrations between 1 nM and 1 �M, strongly re-duced SK-N-MC cell growth (Fig. 9A, top). The maximal effectobserved at 1 �M peptides represented 75% inhibition of growthas compared with control cells. Half-maximal inhibitions wereobtained for 5 nM for both orexins (Fig. 9A, top). The neuropep-tides also induced SK-N-MC cell apoptosis as shown by pro-pidium iodide staining, which revealed condensed nuclei uponorexin challenge but not in control cells (Fig. 9B).

To generalize further the role of OX1R in inhibition of cellgrowth and induction of apoptosis, we considered CHO cellsexpressing the recombinant hOX1R. Quite interestingly,orexin-A and orexin-B strongly inhibited CHO/hOX1R cellgrowth (Fig. 9A, middle), whereas no effect of orexins on cellgrowth could be detected in the parent CHO-K cell line (Fig.9A, bottom). Similarly, propidium iodide staining indicatedthat both orexins triggered apoptosis in the CHO/hOX1R cellline (Fig. 9B), whereas parent CHO-K cells did not undergoapoptosis upon orexin treatment (Fig. 9B).

In order to better quantitate the apoptotic rate of SK-N-MCand CHO/hOX1R cells upon orexin challenge, we analyzed an-nexin V binding (Guava nexin assay), which reveals phosphati-dylserine externalization in apoptotic cells. After a 24-h treat-ment of cells with 1 �M orexin-B, the percentage of apoptoticcells strongly increased up to 11 and 27% in SK-N-MC andCHO/hOX1R cells, respectively (Table II). In sharp contrast,orexin-B had no significant effect on the percentage of apoptoticcells in the parent CHO-K cell line (Table II). Altogether theseresults supported the idea that OX1R mediates inhibition of cellgrowth and induction of apoptosis independently of thecell environment.

DISCUSSION

In this work, we discover and characterize a new function ofthe neuropeptides orexins as drastic pro-apoptotic peptides. Weshow that orexins acting at either native (human colon adeno-carcinoma cells or neuroblastoma cells) or recombinant (CHOcells) seven transmembrane domain receptor OX1R have stronganti-growth properties by inducing cytochrome c- and caspase-dependent apoptosis. These data provide the first evidence thatthe OX1R and its natural agonists orexin-A and orexin-B areimportant players in the control of apoptosis. They may havefuture interesting applications in the treatment of apoptosis-resistant cancers such as colon cancer. They also promote thedesign of new studies to understand the physiological role oforexins in relation to apoptosis in OX1R-expressing tissues.

FIG. 3. Analysis of orexin-induced apoptosis in HT29-D4 cells.A, HT29-D4 cells were grown on sterile coverslips and then left un-treated (a and d) and treated with 1 �M orexin-A (b and e) or orexin-B(c and f) for 48 h. Cells were fixed and stained with 0.1 mg/ml DAPI(blue nuclei; a–c) for 30 min and examined and visualized by confocalmicroscopy (original magnification �100). White arrowheads indicatecells undergoing apoptosis (chromatin condensation and nuclear seg-mentation). In situ cell death detection kit (d–f) shows enzymatic in situlabeling of apoptosis-induced DNA strand breaks (in green) in cells thatwere stained with propidium iodide (red nuclei). Cells are examined andvisualized by confocal microscopy. B, typical DNA ladder obtained afteragarose gel electrophoresis staining with ethidium bromide and photo-graphed under UV light (*, 123-bp DNA marker; lane 1, extracted DNAfrom control cells; lane 2, extracted DNA from orexin-A (1 �M)-treatedcells; lane 3, extracted DNA from orexin-B (1 �M)-treated cells).

FIG. 4. Effect of orexins on cytochrome c release in HT29-D4cells. A shows ELISA detection of cytochrome c in cytosol of untreatedcells (open bar) or cells treated with 1 �M orexin-A (black bar) or 1 �M

orexin-B (hatched bar). Cells were grown in standard medium contain-ing 10% FCS. B shows a Western blot of cytochrome c in cell extract(total cytochrome c) and in the cytosol of cells treated without (lane 1)or with 1 �M orexin-A (lane 2) or 1 �M orexin-B (lane 3) for 24 h.

Pro-apoptotic Role of Human OX1R Orexin Receptor 45881

Considering that many growth factors (1–8) but very fewanti-growth factors for human colon cancer cells have beendescribed during the last 2 decades (13, 14, 34), we tested alarge array of gut peptide hormones and neuropeptides fortheir ability to inhibit cell growth in the human colon cancerHT29-D4 cell line cultured in the strong growth-promotingenvironment of 10% FCS. All peptides tested except orexinswere without any effect in inhibiting cell growth. Among thepeptides tested, some were described previously as growth-promoting such as neurotensin (3), glucagon-like peptide 1 (35),or gastrin/cholecystokinin (5). It is not surprising that they areinactive in our assay conditions because growth is alreadystrongly stimulated by FCS. In this context, the ability ofghrelin, a peptide hormone produced by the stomach (36), topromote cell growth even in the presence of 10% FCS is amaz-ing but is outside the scope of the present study. The screeningassay identified only two anti-growth peptides, the closely re-lated orexin-A and orexin-B encoded by the same gene (15, 17)and acting at common receptors (15). Initial experiments es-tablished that orexin-A and orexin-B are equipotent at sup-pressing HT29-D4 cell growth (see Fig. 1) with an ED50 of 5 nM.A body of evidence supports that orexins suppress HT29-D4cell growth (e.g. inhibit serum-induced increase of cell number)

by promoting apoptosis. (i) Orexins do not alter cell prolifera-tion as indicated by flow cytometric data of cell cycle and[methyl-3H]thymidine incorporation into DNA. (ii) Upon celltreatment with orexins, morphological changes in cell shape,

FIG. 5. Effect of orexins on in situ caspase activation andcaspase-3 or caspase-7 cleavage in HT29-D4 cells. A, activatedcaspases are detected in situ by fluorescence labeling (in green) usingcaspase fluorescein isothiocyanate-VAD-fluoromethyl ketone, a cell-permeable caspase inhibitor that binds to activated caspases. HT29-D4cells were grown on coverslips and then left untreated (a) or treatedwith 1 �M orexin-B (b) for 24 h at 37 °C, and then 10 �M of caspasefluorescein isothiocyanate-VAD-fluoromethyl ketone was added and in-cubated in darkness for 20 min. Cells were rinsed with PBS and thenfixed in 10% buffered formalin for 30 min at room temperature underdarkness and were observed by confocal microscopy. Confocal micro-graphs of immunostaining are shown for cleaved caspase-3 (Asp-175)antibody (green) in HT29-D4 cells treated for 24 h without (c) or with (d)1 �M orexin-B. Confocal micrographs of immunostaining are shown forcleaved caspase-7 (Asp-198) antibody (green) in HT29-D4 cells treatedfor 24 h without (e) or with (f) 1 �M orexin-B. c–f, nuclei were stained inred with propidium iodide. B, immunoblot analysis of cell lysates fromHT29-D4 cells treated or not by 1 �M orexin-B using caspase-3 antibod-ies and cleaved caspase-3 antibodies (left) or caspase-7 antibodies andcleaved caspase-7 antibodies (right). A �-actin antibody was used asa control.

FIG. 6. Expression of functional OX1R in HT29-D4 cells. A showsRT-PCR analysis of specific OX1R (left panel) or OX2R (right panel)mRNA issued from HT29-D4 cells (lane 2), CHO/hOX1R cells (lane 3),and CHO/hOX2R cells (lane 6). Controls are shown in lane 4 (absence ofreverse transcription) and lane 5 (without DNA template). RT-PCRanalysis of �-actin mRNA was used as control. Lane 1 corresponds tothe DNA ladder. B shows immunofluorescence localization of OX1R inHT29-D4 cells in the absence (� peptide) and in the presence (� pep-tide) of immunogen peptide. C shows confocal fluorescence imaging ofcalcium flux in HT29-D4 cells in response to 1 �M orexin-B. Cell surfacereceptor activation was monitored through the use of the calcium-sensitive dye Fluo-4AM. Fluorescence was recorded during orexin ad-dition, and the bottom images were taken every 10 s for 150 s. Threeconfocal pictures show fluorescence before (a) and after 45 (b) or 120 s(c) of treatment of HT29-D4 cells with orexin-B. D shows effects oforexin-B and neurotensin (NT) on calcium signaling on human coloncancer HT29-D4 (left) and HCT116 (right) cells. Cells were loaded withFura-2/AM and assayed in medium containing Ca2� using a fluorime-ter. The cells were challenged first with orexin-B (1 �M) followed by asecond control challenge with neurotensin (0.1 �M). All of the com-pounds were given at the arrows. The results are representative ofthree other experiments.

Pro-apoptotic Role of Human OX1R Orexin Receptor45882

reminiscent of apoptosis, are observed, in particular cells lookrounder and shrunken. (iii) Direct evidence for orexin-inducedapoptosis is provided by several techniques including DNA-staining patterns with DAPI, TUNEL assay, and DNA ladder.(iv) Orexins induce cytochrome c release into cytosol and acti-vation of effector caspase-3 and caspase-7. The orexin-inducedapoptosis in HT29-D4 cells is not associated with the observ-able cell detachment in contrast to Fas ligand-induced apo-ptosis (37), which is responsible for massive HT29-D4 cell de-

tachment.3 Because cells do undergo apoptosis without detach-ment, we have to assume that apoptotic cells in culture areeliminated. Although not documented in this paper, a possibleway is phagocytosis by adjacent nonapoptotic epithelial cells, aprocess demonstrated previously (38, 39).

Two human orexin receptors sharing 64% amino acid iden-tity have been cloned (15). They are serpentine GPCRs coupledto Ca2� mobilization (15, 40). Because their molecular phar-macology is poorly developed (17) and reliable orexin tracersare still unavailable, orexin receptors have been mainly char-acterized in tissues or cell lines because of their coupling tocalcium mobilization (15, 40), by studies of receptor mRNAexpression (20, 41), or by immunocytochemical detection ofreceptor proteins (20). In this context, all the currently avail-able tools indicate that HT29-D4 cells are only equipped withOX1R. (i) RT-PCR experiments identified an OX1R transcriptwhose sequence is identical to that of OX1R cDNA. In sharpcontrast, no OX2R transcript is detected in HT29-D4 cells un-der RT-PCR conditions that nicely provide a specific product incontrol OX2R-expressing CHO cells. (ii) the OX1R protein is

3 P. Rouet-Benzineb, C. Rouyer-Fessard, A. Jarry, V. Avondo,C. Pouzet, M. Yanagisawa, C. Laboisse, M. Laburthe, and T. Voisin,unpublished data.

FIG. 7. Expression of OX1R mRNA and anti-growth and pro-apoptotic effects of orexin-B in different human colon cancercell lines. A, total RNA extracted from Caco-2, SW480, LoVo, andHCT116 cells were reverse-transcribed and PCR-amplified with hOX1R(top) or �-actin (bottom) primers. A single PCR-amplified product of theexact predicted size (500 bp) for hOX1R was visualized in Caco-2,SW480, and LoVo cells. B shows the effect of orexin-B on serum-inducedcell number in Caco-2, SW480, LoVo, and HCT116 cell lines. Humancolon cancer cells grown in standard medium containing FCS weretreated for 24 h with orexin-B. Cells were seeded at low density in48-well plates and grown at 70–80% confluency (about 90,000 cells perwell). The culture medium was then replaced with fresh medium con-taining FCS without (�) or with (�) 1 �M orexin-B. After 24 h, cellswere harvested by trypsinization and counted. Cells produced over a24-h period of treatment (number of cells/well) are expressed asmeans � S.E. (four determinations). **, p � 0.001 versus without orexin(�). C shows typical DNA ladders for Caco-2, SW480, LoVo, andHCT116 cell lines treated or not treated for 48 h with 1 �M orexin-B.Agarose gel stained with ethidium bromide was photographed underUV light. *, 123-bp DNA marker; �, extracted DNA from control cells;�, extracted DNA from orexin-B (1 �M)-treated cells.

FIG. 8. Absence of effect of orexin on apoptosis in explantcultures of human normal colonic mucosa. Explants of humannormal colonic mucosa were microdissected and cultured for 24 h in theabsence (control) or presence of 1 �M orexin-B. Morphological integrityof the explants was assessed by standard hematoxylin-eosin (HE) stain-ing on paraffin sections. The crypt morphology was well preserved bothin the control and orexin-treated explants. Epithelial apoptosis wasevaluated by M30 immunostaining of caspase-cleaved cytokeratin 18and by the Hoechst dye. M30-positive cells (*) (brown cytoplasmicstaining) undergoing exfoliation were present near the surface epithe-lium or occasionally in the lower region of the crypts. Hoechst stainingshows a few nuclei undergoing DNA condensation and fragmentationwithin the surface epithelium (*). Original magnification �400 forhematoxylin-eosin and M30, and �630 for Hoechst staining.

Pro-apoptotic Role of Human OX1R Orexin Receptor 45883

immunodetected at the cell surface of HT29-D4 cells by using aspecific anti-OX1R antibody (20). In contrast, specific anti-OX2R antibodies do not immunostain HT29-D4 cells underconditions in which control OX2R-expressing CHO cells arenicely stained.3 The presence of a functional orexin receptor inHT29-D4 cells is otherwise indicated by robust calcium re-sponse observed upon treatment with orexins. The presence ofonly OX1R in HT29-D4 cells strongly suggests that this recep-tor mediates the pro-apoptotic effects of orexins. This OX1Rdoes not discriminate between orexin-A and orexin-B becausethe two peptides are equipotent at suppressing HT29-D4 cellgrowth (see Fig. 1). The pharmacology of orexin receptors isstill in its early stages, and the literature is somewhat incon-sistent regarding the ability of OX1R to discriminate betweenthe two orexins. In calcium mobilization studies using CHOcells expressing recombinant OX1R, orexin-A was shown to befrom 5- to 100-fold more potent than orexin-B (15, 42–44)contributing to the disparity of their reported selective actions.In the same transfected cellular model, binding studies re-sulted in contradictory results. By using 125I-labeled orexin-Aas a tracer, the affinity of orexin-A was found to be 20 timeshigher than orexin-B (15). In contrast, Wieland et al. (33) found

identical affinity for the two orexins in CHO cells expressingOX1R when using 125I-labeled orexin-A as a tracer. More un-expectedly, orexin-B was shown to have even higher affinitythan orexin-A when using 125I-labeled orexin-B as a tracer inCHO cells expressing recombinant OX1R or SK-N-MC neuro-blastoma cells expressing native OX1R (33). In this context,further studies and the development of instrumental agonistsand/or antagonists of OX1R are clearly needed to ascertain thepharmacological profile of OX1R.

The role of OX1R in controlling apoptosis is directly dem-onstrated by the expression of recombinant OX1R in CHO-Kcells that confers the ability of orexins to promote apoptosisin this cell line. This experiment also suggests that the pro-apoptotic role of OX1R is an intrinsic property of the receptorthat is not restricted to the HT29-D4 cell context. In line withthis idea, our work shows that the OX1R-expressing SK-N-MC neuroblastoma cells (33) do undergo apoptosis upontreatment with orexins.

The pro-apoptotic activity of orexins demonstrated here inhuman colon cancer cells HT29-D4, human neuroblastomacells SK-N-MC and CHO cells expressing recombinant OX1Rreceptor, results in massive suppression of cell growth regard-less of the OX1R-expressing cell. The mechanisms of couplingbetween activation of cell surface seven transmembrane do-main OX1R and release of cytochrome c into cytosol remain tobe elucidated. Activation of OX1R is known to result in mobi-lization of intracellular calcium through a Gq-dependent mech-anism (15, 40, 45). This Ca2� response also observed here inHT29-D4 cells is certainly not sufficient to explain the pro-apoptotic effect of orexins, although increases of cytosolic cal-cium are well known to occur during apoptotic cell death (46).Indeed, a variety of GPCRs in human colon cancer cells includ-ing HT-29 cells is known to promote intracellular Ca2� mobi-lization (3, 7, 8, 10). These receptors such as NT1 receptors forneurotensin (3, 10), protease-activated receptors 1 (PAR1) forthrombin (7), protease-activated receptors 2 (PAR2) for trypsin(8), or muscarinic M3 receptors for acetylcholine (47) not onlydo not trigger apoptosis but rather stimulate cell proliferation.In this context, it is worth pointing out that up to now very fewserpentine GPCRs have been shown to inhibit cell growth bypromoting apoptosis e.g. endothelin receptor ETB (48), chemo-

TABLE IIDetection of apoptotic cell by annexin V labeling in SK-N-MC,

CHO/hOX1R, and parent CHO-K cell linesApoptosis was analyzed in cell lines using the Guava Nexin™ kit,

which discriminates between apoptotic and nonapoptotic cells. TheGuava Nexin™ assay utilizes annexin V-phycoerythrin (annexin V-PE)to detect phosphatydylserine on the external membrane of apoptoticcells. Cells (2 � 106) were seeded in culture dishes and grown for 24 hin standard culture medium. The culture medium was then replacedwith fresh culture medium containing or not orexin-B (1 �M). After 24h, apoptotic cell staining was performed according to the manufactur-er’s instructions by using a Guava PCA system. Results are expressedas the percentage of apoptotic annexin V-PEby positive cells and are themeans of four analyses.

Cell line% apoptotic cells (annexin V-positive)

Control Orexin-B (1 �M)

SK-N-MC 0.2 � 0.1 11.4 � 1.2a

CHO/hOX1R 2.1 � 0.8 27.7 � 0.9a

Parent CHO-K 0.8 � 0.3 1.7 � 0.7a p � 0.001 versus control.

FIG. 9. Effect of orexins on cellgrowth and apoptosis in human neu-roblastoma SK-N-MC cells and re-combinant CHO/hOX1R cells. A, effectof orexin-A (● ) or orexin-B (E) on thegrowth of SK-N-MC cells (top), CHO/hOX1R cells (middle), or parent CHO-Kcells (bottom). For SK-N-MC cells grownin standard medium with 10% FCS, thedose response of orexins is shown after24 h of treatment with peptides. ForCHO/hOX1R cells or CHO-K cells grownin 10% FCS, cell counts are shown aftercell treatment without (Œ) or with orexinsfor 1–3 days. B, SK-N-MC cells (top),CHO/hOX1R cells (middle), or parentCHO-K cells (bottom) were grown instandard culture medium containing 10%FCS and then treated for 24 h without(control) or with 1 �M orexin-A (● ) ororexin-B (E). Condensed nuclei (arrows)were then visualized by propidium iodidestaining.

Pro-apoptotic Role of Human OX1R Orexin Receptor45884

kine CXCR4 receptor (49), �-adrenergic receptor (50), angio-tensin AT2 receptor (51), somatostatin SST2 receptor (52), orparathyroid hormone receptor (53).

The OX1R-mediated pro-apoptotic role of orexins describedherein raises the question of its significance in health anddisease. With respect to colon cancer, this study already showsthat expression of OX1R and OX1R-mediated apoptosis arefrequent in colon cancer cells because they are observed in fourof five human colon cancer cell lines originating from differentpatients (9), i.e. HT29-D4, Caco-2, SW480, and LoVo. Mostinterestingly, our data also show that normal human colonicepithelial cells are not equipped with OX1R, resulting in theabsence of the pro-apoptotic effect of orexins in normal humancolon epithelium. These observations suggest that OX1Rs areaberrantly expressed in human colon cancer cells. Whether thisaberrant expression represents an ectopic expression or there-expression of receptors expressed in colonic epithelium dur-ing embryogenesis remains to be determined. Ectopic expres-sion of G protein-coupled receptors in colon cancers has beenreported previously for neurotensin receptor (10) and PAR1 forthrombin (7). However, these receptors were clearly shown topromote cell proliferation and colon cancer cell growth (7, 10).In that respect, this paper reports the first example of anectopic expression of a receptor promoting apoptosis in coloncancer cells. Given the known resistance of colon cancer toapoptosis and chemotherapy (39, 54), OX1R thereby representsan attractive new target for the development of instrumentalorexin agonists in this cancer. On the other hand, because thepro-apoptotic role of OX1R appears to be an intrinsic propertyof the receptor regardless of the nature of the OX1R-expressingcell, we may speculate about the role of orexins in normaltissues expressing OX1R. In that respect, a site of expression oforexin receptors in normal tissues is small intestinal epithe-lium (17, 20). Because this epithelium is a rapidly renewingtissue in which cell homeostasis is regulated by a balanceamong proliferation, growth arrest, differentiation, and apo-ptosis (39), the possibility that orexins, which are neuropep-tides expressed in the small intestinal wall (20), may controlapoptosis and cell homeostasis in this tissue is an attractivehypothesis. A major site of expression of OX1R is the brain andmore specifically the hypothalamus (15, 16). In this context,our studies raise the question of the possible role of orexins inneuronal apoptosis which is a major event during brain devel-opment and maturation (55) and also in neurodegenerativediseases (55).

In conclusion, this work characterizes for the first time thepro-apoptotic and subsequent anti-cell growth properties oforexins acting at the OX1 receptor. Although the molecularmechanisms of the OX1R-mediated pro-apoptotic effect of orex-ins remain to be elucidated, these findings add a new dimen-sion to the biological activities of these neuropeptides that mayhave important implications in health and disease.

REFERENCES

1. Roberts, R. B., Min, L., Washington, M. K., Olsen, S. J., Settle, S. H., Coffey,R. J., and Threadgill, D. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99,1521–1526

2. Reinmuth, N., Fan, F., Liu, W., Parikh, A. A., Stoeltzing, O., Jung, Y. D.,Bucana, C. D., Radinsky, R., Gallick, G. E., and Ellis, L. M. (2002) Lab.Investig. 82, 1377–1389

3. Maoret, J. J., Anini, Y., Rouyer-Fessard, C., Gully, D., and Laburthe, M. (1999)Int. J. Cancer 80, 448–454

4. Ferris, H. A., Carroll, R. E., Rasenick, M. M., and Benya, R. V. (1997) J. Clin.Investig. 100, 2530–2537

5. Dehez, S., Bierkamp, C., Kowalski-Chauvel, A., Daulhac, L., Escrieut, C.,Susini, C., Pradayrol, L., Fourmy, D., and Seva, C. (2002) Cell Growth &Differ. 13, 375–385

6. Pai, R., Soreghan, B., Szabo, I. L., Pavelka, M., Baatar, D., and Tarnawski,A. S. (2002) Nat. Med. 8, 289–293

7. Darmoul, D., Gratio, V., Devaud, H., Lehy, T., and Laburthe, M. (2003) Am. J.Pathol. 162, 1503–1513

8. Darmoul, D., Gratio, V., Devaud, H., Laburthe, M. (2004) J. Biol. Chem. 279,

20927–209349. Zweibaum, A., Laburthe, M., Grasset, E., and Louvard, D. (1991) in Handbook

of Physiology (Schultz, S. G., ed) Vol. IV, pp. 223–255, American Physiolog-ical Society, Bethesda

10. Maoret, J. J., Pospai, D., Rouyer-Fessard, C., Couvineau, A., Laboisse, C.,Voisin, T., and Laburthe, M. (1994) Biochem. Biophys. Res. Commun. 203,465–471

11. Rovere, C., Barbero, P., Maoret, J. J., Laburthe, M., and Kitabgi, P. (1998)Biochem. Biophys. Res. Commun. 246, 155–159

12. Howell, G. M., Ziober, B. L., Humphrey, L. E., Willson, J. K., Sun, L., Lynch,M., and Brattain, M. G. (1995) J. Cell. Physiol. 162, 256–265

13. Fink, S. P., Swinler, S. E., Lutterbaugh, J. D., Massague, J., Thiagalingam, S.,Kinzler, K. W., Vogelstein, B., Willson, J. K., and Markowitz, S. (2001)Cancer Res. 61, 256–260

14. Houghton, J. A., Harwood, F. G., Gibson, A. A., and Tillman, D. M. (1997) Clin.Cancer Res. 3, 2205–2209

15. Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R. M., Tanaka,H., Williams, S. C., Richarson, J. A., Kozlowski, G. P., Wilson, S., Arch,J. R., Buckingham, R. E., Haynes, A. C., Carr, S. A., Annan, R. S., McNulty,D. E., Liu, W. S., Terrett, J. A., Elshourbagy, N. A., Bergsma, D. J., andYanagisawa, M. (1998) Cell 92, 573–585

16. De Lecea, L., Kilduff, T. S., Peyron, C., Gao, X., Foye, P. E., Danielson, P. E.,Fukuhara, C., Battenberg, E. L., Gautvik, V. T., Bartlett, F. S., Frankel,W. N., van den Pol, A. N., Bloom, F. E., Gautvik, K. M., and Sutcliffe, J. G.(1998) Proc. Natl. Acad. Sci. U. S. A. 95, 322–327

17. Voisin, T., Rouet-Benzineb, P., Reuter, N., and Laburthe, M. (2003) Cell. Mol.Life Sci. 60, 72–87

18. Beuckmann, C. T., and Yanagisawa, M. (2002) J. Mol. Med. 80, 329–34219. Overeem, S., Scammell, T. E., and Lammers, G. J. (2002) Curr. Opin. Neurol.

15, 739–74520. Kirchgessner, A. L., and Liu, M. (1999) Neuron 24, 941–95121. Voisin, T., Goumain, M., Lorinet, A. M., Maoret, J. J., and Laburthe, M. (2000)

J. Pharmacol. Exp. Ther. 292, 638–64622. Fantini, J., Abadie, B., Tirard, A., Remy, L., Ripert, J. P., el Battari, A., and

Marvaldi, J. (1986) J. Cell Sci. 83, 235–24923. Salomon, R., Couvineau, A., Rouyer-Fessard, C., Voisin, T., Lavallee, D., Blais,

A., Darmoul, D., and Laburthe, M. (1993) Am. J. Physiol. 264, E294–E30024. Neunlist, M., Toumi, F., Oreschkova, T., Denis, M., Leborgne, J., Laboisse,

C. L., Galmiche, J. P., and Jarry, A. (2003) Am. J. Physiol. 285,G1028–G1036

25. Murgia, M., Pizzo, P., Sandona, D., Zanovello, P., Rizzuto, R., and Di Virgilio,F. (1992) J. Biol. Chem. 267, 10939–10941

26. Zeuner, A., Eramo, A., Peschle, C., and De Maria, R. (1999) Cell Death Differ.6, 1075–1080

27. Charrier, L., Jarry, A., Toquet, C., Bou-Hanna, C., Chedorge, M., Denis, M.,Vallette, G., and Laboisse, C. L. (2002) Cancer Res. 62, 2169–2174

28. Leers, M. P., Kolgen, W., Bjorklund, V., Bergman, T., Tribbick, G., Persson, B.,Bjorklund, P., Ramaekers, F. C., Bjorklund, B., Nap, M., Jornvall, H., andSchutte, B. (1999) J. Pathol. 187, 567–572

29. Caulin, C., Salvesen, G. S., and Oshima, R. G. (1997) J. Cell Biol. 138,1379–1394

30. Laburthe, M., Rousset, M., Boissard, C., Chevalier, G., Zweibaum, A., andRosselin, G. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2772–2775

31. Bradford, M. M. (1976) Anal. Biochem. 72, 248–25432. Bozou, J. C., Rochet, N., Magnaldo, I., Vincent, J. P., and Kitabgi, P. (1989)

Biochem. J. 264, 871–87833. Wieland, H. A., Soll, R. M., Doods, H. N., Stenkamp, D., Hurnaus, R., Lammle,

B., and Beck-Sickinger, A. G. (2002) Eur. J. Biochem. 269, 1128–113534. Dkhissi, F., Lu, H., Soria, C., Opolon, P., Griscelli, F., Liu, H., Khattar, P.,

Mishal, Z., Perricaudet, M., and Li, H. (2003) Hum. Gene Ther. 14,997–1008

35. Buteau, J., Foisy, S., Joly, E., and Prentki, M. (2003) Diabetes 52, 124–13236. St Pierre, D. H., Wang, L., and Tache, Y. (2003) News Physiol. Sci. 18, 242–24637. Liu, J. J., Nilsson, A., Oredsson, S., Badmaev, V., Zhao, W. Z., and Duan, R. D.

(2002) Carcinogenesis 23, 2087–209338. Bursch, W., Hochegger, K., Torok, L., Marian, B., Ellinger, A., and Hermann,

R. S. (2000) J. Cell Sci. 113, 1189–119839. Jones, B. A., and Gores, G. J. (1997) Am. J. Physiol. 273, G1174–G118840. Lund, P. E., Shariatmadari, R., Uustare, A., Detheux, M., Parmentier, M.,

Kukkonen, J. P., and Akerman, K. E. (2000) J. Biol. Chem. 275,30806–30812

41. Johren, O., Neidert, S. J., Kummer, M., Dendorfer, A., and Dominiak, P. (2001)Endocrinology 142, 3324–3331

42. Langmead, C. J., Jerman, J. C., Brough, S. J., Scott, C., Porter, R. A., andHerdon, H. J. (2004) Br. J. Pharmacol. 141, 340–346

43. Smart, D., Jerman, J. C., Brough, S. J., Rushton, S. L., Murdock, P. R., Jewitt,F., Elshourbagy, N. A., Ellis, C. E., Middlemiss, D. N., and Brown, F. (1999)Br. J. Pharmacol. 128, 1–3

44. Okumura, T., Takeuchi, S., Motomura, W., Yamada, H., Egashira, S. S., Asahi,S., Kanatani, A., Ihara, M., and Kohgo, Y. (2001) Biochem. Biophys. Res.Commun. 280, 976–981

45. Darker, J. G., Porter, R. A., Eggleston, D. S., Smart, D., Brough, S. J., Sabido-David, C., and Jerman, J. C. (2001) Bioorg. Med. Chem. Lett. 11, 737–740

46. Rizzuto, R., Pinton, P., Ferrari, D., Chami, M., Szabadkai, G., Magalhaes, P. J.,Di Virgilio, F., and Pozzan, T. (2003) Oncogene 22, 8619–8627

47. Keely, S. J., Uribe, J. M., and Barrett, K. E. (1998) J. Biol. Chem. 273,27111–27117

48. Okazawa, M., Shiraki, T., Ninomiya, H., Kobayashi, S., and Masaki, T. (1998)J. Biol. Chem. 273, 12584–12592

49. Biard-Piechaczyk, M., Robert-Hebmann, V., Richard, V., Roland, J., Hipskind,

Pro-apoptotic Role of Human OX1R Orexin Receptor 45885

R. A., and Devaux, C. (2000) Virology 268, 329–34450. Remondino, A., Kwon, S. H., Communal, C., Pimentel, D. R., Sawyer, D. B.,

Singh, K., and Colucci, W. S. (2003) Circ. Res. 92, 136–13851. Miura, S., and Karnik, S. S. (2000) EMBO J. 19, 4026–403552. Teijeiro, R., Rios, R., Costoya, J. A., Castro, R., Bello, J. L., Devesa, J., and

Arce, V. M. (2002) Cell. Physiol. Biochem. 12, 31–38

53. Turner, P. R., Mefford, S., Christakos, S., and Nissenson, R. A. (2000) Mol.Endocrinol. 14, 241–254

54. Carethers, J. M., Smith, E. J., Behling, C. A., Nguyen, L., Tajima, A.,Doctolero, R. T., Cabrera, B. L., Goel, A., Arnold, C. A., Miyai, K., andBoland, C. R. (2004) Gastroenterology 126, 394–401

55. Lossi, L., and Merighi, A. (2003) Prog. Neurobiol. 69, 287–312

Pro-apoptotic Role of Human OX1R Orexin Receptor45886