Leptin Counteracts Sodium Butyrate-induced Apoptosis in Human Colon Cancer HT-29 Cells via NF-κB Signaling

Abstract

This study shows that leptin induced a rapid phosphorylation of p42/44 mitogen-activated protein kinase, an enhancement of both NF-κB DNA binding and transcriptional activities, and a concentration-dependent increase of HT-29 cell proliferation. These effects are consistent with the presence of leptin receptors on cell membranes. The leptin induction of cell growth was associated with an increase of cell population in S and G2/M phase compared with control cells found in G0/G1 phase of the cell cycle. Moreover, cyclin D1 immunoreactivity was enhanced in leptin-treated HT-29 cells and this increase was essentially associated with cell population in G0/G1 phase. On the other hand, we observed that sodium butyrate inhibited cell proliferation by blocking HT-29 cells in G0/G1 phase of the cell cycle. Interestingly, at physiological concentration, leptin prevented sodium butyrate-induced morphological nucleus changes, DNA laddering and suppressed butyrate-induced cell cycle arrest. This anti-apoptotic effect of leptin was associated with HT-29 cell proliferation and activation NF-κB pathways. However, the phosphorylation of p42/44 MAP kinase in response to leptin was reduced in butyrate-treated cells. These data demonstrated that leptin is a potent mitogenic factor for intestinal epithelial cells through the MAP kinase and NF-κB pathways. They also showed, for the first time, that leptin promotes colon cancer HT-29 cell survival upon butyrate challenge by counteracting the apoptotic programs initiated by this short chain fatty acid probably through the NF-κB pathways. Although further studies are required to unravel the precise mechanism, these data may have significance in the pathogenesis of colorectal cancer and ulcerative colitis diseases. This study shows that leptin induced a rapid phosphorylation of p42/44 mitogen-activated protein kinase, an enhancement of both NF-κB DNA binding and transcriptional activities, and a concentration-dependent increase of HT-29 cell proliferation. These effects are consistent with the presence of leptin receptors on cell membranes. The leptin induction of cell growth was associated with an increase of cell population in S and G2/M phase compared with control cells found in G0/G1 phase of the cell cycle. Moreover, cyclin D1 immunoreactivity was enhanced in leptin-treated HT-29 cells and this increase was essentially associated with cell population in G0/G1 phase. On the other hand, we observed that sodium butyrate inhibited cell proliferation by blocking HT-29 cells in G0/G1 phase of the cell cycle. Interestingly, at physiological concentration, leptin prevented sodium butyrate-induced morphological nucleus changes, DNA laddering and suppressed butyrate-induced cell cycle arrest. This anti-apoptotic effect of leptin was associated with HT-29 cell proliferation and activation NF-κB pathways. However, the phosphorylation of p42/44 MAP kinase in response to leptin was reduced in butyrate-treated cells. These data demonstrated that leptin is a potent mitogenic factor for intestinal epithelial cells through the MAP kinase and NF-κB pathways. They also showed, for the first time, that leptin promotes colon cancer HT-29 cell survival upon butyrate challenge by counteracting the apoptotic programs initiated by this short chain fatty acid probably through the NF-κB pathways. Although further studies are required to unravel the precise mechanism, these data may have significance in the pathogenesis of colorectal cancer and ulcerative colitis diseases. Leptin, the protein product encoded by the ob gene, is a 16-kDa circulating hormone produced primarily by adipose tissue and is a multifunctional hormone that regulates body weight homeostasis, neuroendocrine function, fertility, immune function, and angiogenesis (1Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11807) Google Scholar, 2Schwartz M.W. Baskin D.G. Kaiyala K.J. Woods S.C. Am. J. Clin. Nutr. 1999; 69: 584-596Crossref PubMed Scopus (203) Google Scholar, 3Masuzaki H. Ogawa Y. Sagawa N. Hosoda K. Matsumoto T. Mise H. Nishimura H. Yoshimasa Y. Tanaka I. Mori T. Nakao K. Nat. Med. 1997; 3: 1029-1033Crossref PubMed Scopus (1107) Google Scholar, 4Ahima R.S. Flier J.S. Annu. Rev. Physiol. 2000; 62: 413-437Crossref PubMed Scopus (1495) Google Scholar). The biological actions of leptin on target tissues are carried out through interaction with its specific receptors, Ob-R. The leptin receptor (Ob-R) 1The abbreviations used are: Ob-R, leptin receptor; MAP, mitogen-activated protein; HT-29, human colon cancer cells, NF-κB, nuclear factor κB transcription factor; PI, propidium iodide; ERK, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; NaB, sodium butyrate; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay. is a member of the gp130 family of cytokine receptors (5Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. May G.G. Wolf E.A. Monroe C. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3238) Google Scholar), which occurs in several receptor variants (Ob-Ra through Ob-Rf) that are generated by alternative splicing of the db leptin receptor gene. These isoforms share the same extracellular domain but differ in the length of the transmembrane/cytoplasmic (6Chen H. Charlat O. Tartaglia L.A. Woolf E.A. Weng X. Ellis S.J. Lakey N.D. Culpepper J. Moore K.J. Breitbart R.E. Duyk G.M. Tepper R.I. Morgenstern J.P. Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1944) Google Scholar, 7Lollmann B. Gruninger S. Stricker-Krongrad A. Chiesi M. Biochem. Biophys. Res. Commun. 1997; 238: 648-652Crossref PubMed Scopus (167) Google Scholar). The long Ob-Rb subtype (Ob-RL) appears as the functional, signal-transducing isoform, responsible for the action of leptin (8Lee G.H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2112) Google Scholar). The long isoform, Ob-Rb, can activate the signal transducers and activators of transcription (STAT) pathways, whereas both Ob-Rb and the short isoform (Ob-Ra) can transduce signals through insulin receptor substrates and through mitogen-activated protein kinase-(MAP kinase) dependent pathways (for review, see Ref. 9Tartaglia L.A. J. Biol. Chem. 1997; 272: 6093-6096Abstract Full Text Full Text PDF PubMed Scopus (1191) Google Scholar). Although, it is currently believed that leptin action is largely mediated by the central nervous system, the expression of Ob-RL in peripheral tissues indicates that it is a direct target of leptin. There is now an emerging role of leptin as a growth factor for several cell types (10Santos-Alvarez J. Goberna R. Sanchez-Margalet V. Cell. Immunol. 1999; 194: 6-11Crossref PubMed Scopus (490) Google Scholar, 11Martin-Romero C. Sanchez-Margalet V. Cell. Immunol. 2001; 212: 83-91Crossref PubMed Scopus (119) Google Scholar, 12Najib S. Sanchez-Margalet V. J. Mol. Endocrinol. 2001; 27: 85-91Crossref PubMed Scopus (92) Google Scholar). In the gut, leptin has been reported to stimulate the proliferation of gastric and intestinal epithelial cells through activation of the MAP kinase-dependent pathways (13Hardwick J.C. Van Den Brink G.R. Offerhaus G.J. Van Deventer S.J. Peppelenbosch M.P. Gastroenterology. 2001; 121: 79-90Abstract Full Text PDF PubMed Scopus (343) Google Scholar, 14Liu Z. Uesaka T. Watanabe H. Kato N. Int. J. Oncol. 2001; 19: 1009-1014PubMed Google Scholar). Besides this leptin growth effect, we have previously demonstrated that leptin can enhance the activity of the brush border Pept-1 transporter (15Buyse M. Berlioz F. Guilmeau S. Tsocas A. Voisin T. Peranzi G. Merlin D. Laburthe M. Lewin M.J. Roze C. Bado A. J. Clin. Investig. 2001; 108: 1483-1494Crossref PubMed Scopus (199) Google Scholar) and the uptake of butyrate via the monocarboxylate carrier MCT-1 in the human intestinal Caco-2 cells (16Buyse M. Sitaraman S.V. Liu X. Bado A. Merlin D. J. Biol. Chem. 2002; 277: 28182-28190Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Butyrate is a short chain fatty acid that is produced in the colonic lumen by bacterial fermentation of carbohydrate and dietary fibers (17Topping D.L. Clifton P.M. Physiol. Rev. 2001; 81: 1031-1064Crossref PubMed Scopus (2319) Google Scholar). In the proximal large bowel, butyrate represents the preferred respiratory fuels in the intestine through β-oxidation. Apart from the function of butyrate as a dominant energy source for colonocytes, its also inhibits cellular proliferation and induces apoptosis by regulating the key proteins controlling the cell cycle (18Coradini D. Pellizzaro C. Marimpietri D. Abolafio G. Daidone M.G. Cell Prolif. 2000; 33: 139-146Crossref PubMed Scopus (71) Google Scholar). In particular, NaB was shown to down-regulate 25 genes including cyclin D1, a key regulator of the G1/S phase and the proliferating cell nuclear antigen PCNA in colonic epithelial cells, arguing for its pro-apoptotic property (19Tabuchi Y. Arai Y. Kondo T. Takeguchi N. Asano S. Biochem. Biophys. Res. Commun. 2002; 293: 1287-1294Crossref PubMed Scopus (53) Google Scholar). In this study we investigated whether leptin could affect sodium butyrate-induced apoptosis in the human colon cancer cell line HT-29 and examine the intracellular mechanisms involved. We demonstrated, for the first time, that leptin promotes intestinal epithelial HT-29 cell survival upon butyrate challenge by counteracting the apoptotic programs initiated by this short chain fatty acid, probably through activation of NF-κB pathways. Reagents—Human leptin was purchased from R&D Systems (R&D Systems Inc., Minneapolis, MN) and stored as stock solution in phosphate-buffered saline solution (PBS) at -20 °C. Antibody sources. Rabbit polyclonal NF-κB p65 and IκB-α antibodies, goat polyclonal anti-Ob-R(C-20) were purchased from Santa Cruz Biotechnology, and mouse monoclonal cyclin D1 was from Cell Signaling Technology, Inc. (Beverly, MA). Fluorescein- and horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma Chemicals. Cell Culture—The human colon cancer cell line, HT-29 were cultured in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum in a humidified atmosphere in 5% CO2 at 37 °C. Subculture of HT-29 cell line was performed by enzymatic digestion (trypsin/EDTA solution: 0.05/0.02%). Cell Growth and Viability—Cells (3 × 103/well) were seeded into 48-well dishes under the growth conditions described above. Three days after seeding, cells were serum-deprived for 24 h and then treated with recombinant human leptin (0–10 nm) for 24 h. The time course effects (24, 48, and 72 h) were in serum-free culture cells using 1 nm of rhLeptin; the medium was renewed every 48, and 72 the cells were by and cell was by with a of in HT-29 was from HT-29 cells, using to the was into then with Ob-Rb The of and for Ob-Rb were on the of the previously for human Ob-Rb leptin receptor which an of was performed under the conditions as 1 at 1 at and 1 at 72 for The was by a at 72 °C. were by in in the presence of cells were on in Dulbecco's medium supplemented with 10% fetal bovine serum for h. were at for with then with were with 5% serum in for were with a goat polyclonal under a for were with a to with for 1 h in by and were with and observed under an was observed in HT-29 cells under the goat serum used in of immune serum of with Ob-Rb and the control Cell and were seeded into (3 × and to that cells through the cell cycle in cells were in serum-free medium from h. were treated with leptin nm) for 24 h. cells were with PBS, and in for at h at -20 °C. cells were with and with mouse monoclonal cyclin D1 with a solution and propidium for at 37 °C. The cell cycle was using a were on a using cells were for of the HT-29 cell population as with and propidium of was used to phosphorylation to the protein found in cells the HT-29 cells were seeded on and for 24 h. the cells were treated with leptin NaB in for time The cells were of the was used for of using a specific the other were used for of with by with horseradish peroxidase-conjugated antibodies and was by at nm with a of The of cells in was then through the The of the and signals were for cell The data are as a of to and cell Res. 19: PubMed Scopus Google Scholar) were to and to a were first with a specific that NF-κB p65 and were further with horseradish peroxidase-conjugated for 1 h. The immune were by of NF-κB to whether the activation of transcription factor NF-κB after of nuclear of NF-κB was by were cultured in and treated with 1 nm leptin for and and 24 The cells were as described and were with a polyclonal p65 by a at were then with propidium were under a of the to NF-κB p65 and the propidium was using the were as described by and Res. 19: PubMed Scopus Google Scholar) from serum-free HT-29 cells treated with 1 nm leptin. of protein were with of NF-κB NF-κB in 1 and for at The binding was on in electrophoretic and then to the was to for and then for at in 5% bovine serum in The immune were using horseradish peroxidase-conjugated at and The of the was in shift on the same nuclear that NF-κB factor to NF-κB and were seeded 1 × in was performed by with of T. S. D. PubMed Scopus Google Scholar). The a for with κB The κB to NF-κB activity the of its 24 h cells were for h and then with leptin nm) with 10% fetal bovine serum for Cell medium was the cells were with PBS, and then with of of the was with and was by a The were performed in and data are of The are as increase of which was to the of activity to 1 and for for was used to the DNA binding activity in the in the presence of binding to the I. A. M. H. M. J. Res. 2001; PubMed Scopus (343) Google Scholar). a the NF-κB was on that nuclear the of to the binding in the The transcription factor is with a specific to by a secondary horseradish peroxidase-conjugated antibodies and is by at nm with a of of DNA binding activity was performed in the presence of an of the binding to that the transcription isoform is binding to the of NF-κB was as of DNA was by the of DNA after h of with 1 nm leptin NaB both HT-29 cells were and in a of and were to the cells and at 37 was to a of 1 by and at for at °C. were and was by and the were at for 1 and at for h at °C. DNA was and in 25 of was to the at 37 for 1 h and in are as They were compared by of by a were were with HT-29 and leptin receptor immunoreactivity was in HT-29 cells and was at the of the cell as shown by with with The was specific leptin receptor immunoreactivity was observed in the presence of the specific Ob-Rb an of was by HT-29 cells leptin receptors on cell membranes. leptin induced a HT-29 cell proliferation with an of nm In HT-29 cell was after h of to leptin nm) sodium butyrate both by HT-29 cell proliferation whereas of the cells with sodium butyrate reduced In the presence of sodium HT-29 cell growth was and by upon leptin challenge These data that leptin as potent on HT-29 cells growth and inhibits sodium data of cell cycle of serum-free control cells that of HT-29 cells are in G0/G1 in S and in G2/M phase. serum-free HT-29 cells were treated with 1 nm leptin for 24 of HT-29 cells were found in G0/G1 in S and in G2/M phase These data that leptin is a potent mitogenic factor for HT-29 cells. These are consistent with of cyclin of shown in cyclin D1 cells were both in serum and leptin-treated cells. cyclin D1 cells were in the of the compared with serum-free control cells, the cyclin D1 cells were in HT-29 cells an expression of cyclin D1 upon of HT-29 cells by leptin. to cyclin cells with the cell cycle phase. In serum-free control HT-29 cells, the of the cyclin D1 was found in cells in G0/G1 but cyclin D1 cells in S and G2/M phase were 24 h after leptin immunoreactivity of cyclin D1 was by and this immunoreactivity was associated with the G0/G1 phase of cell cycle. MAP kinase and NF-κB in HT-29 nm) induced a increase in the of to that leptin pathways in HT-29 cells. NaB effect on phosphorylation but reduced phosphorylation leptin leptin promotes HT-29 cell survival upon butyrate we investigated whether NF-κB could in this nm) induced a increase in NF-κB DNA binding activity in with data in On the other hand, NaB effect on NF-κB DNA binding activity and affect NF-κB DNA binding activity and of NF-κB in HT-29 of and nuclear an increase of transcription factor NF-κB p65 protein in the nuclear from leptin-treated cells compared with control cells In in from leptin-treated cells, IκB-α protein was compared with control that IκB-α is of the This nuclear of NF-κB was by studies using the The transcription factor NF-κB was in the nucleus after of the cells to leptin leptin induces of NF-κB p65 into the nucleus of HT-29 cells. DNA binding activity of the transcription factor NF-κB was also by shown in nuclear from leptin-treated cells to the NF-κB whereas nuclear from control cells DNA binding In the presence of the NF-κB both nuclear from control leptin-treated cells DNA binding these data that leptin promotes the nuclear of NF-κB and its DNA binding activity in HT-29 cells. The in transcriptional activity of NF-κB was by the activity in HT-29 cells with the activity was in cells as compared with control transcriptional NF-κB p65 is to that with serum leptin the transcription factor NF-κB via the leptin receptor in HT-29 cells. in HT-29 of propidium HT-29 cells were in HT-29 cells as a whereas leptin-treated HT-29 cells cell probably to proliferation of the cells with sodium butyrate reduced HT-29 cell proliferation and induced morphological of the by that sodium butyrate cell cycle by blocking the cells in G0/G1 phase of the cell cycle Interestingly, of HT-29 cells with 1 nm of leptin prevented sodium butyrate-induced morphological nucleus and suppressed butyrate-induced cell cycle in the HT-29 cells In leptin prevented the butyrate induction of DNA laddering whereas leptin effect on DNA This anti-apoptotic effect of leptin was also associated with HT-29 cell proliferation observed 24 h and h after butyrate butyrate-treated cells an of DNA in DNA laddering in with the pro-apoptotic property of sodium butyrate Res. 1994; Google Scholar, A. D. C. Int. J. PubMed Scopus Google Scholar). In this we have demonstrated that human colonic HT-29 cells the leptin receptor can and to physiological of leptin by proliferation. In we that leptin promotes HT-29 cell survival from butyrate The leptin of HT-29 cell proliferation that leptin is a growth factor for colonic epithelial cells (13Hardwick J.C. Van Den Brink G.R. Offerhaus G.J. Van Deventer S.J. Peppelenbosch M.P. Gastroenterology. 2001; 121: 79-90Abstract Full Text PDF PubMed Scopus (343) Google Scholar). These data are also to the in that leptin epithelial cell proliferation in the colon (13Hardwick J.C. Van Den Brink G.R. Offerhaus G.J. Van Deventer S.J. Peppelenbosch M.P. Gastroenterology. 2001; 121: 79-90Abstract Full Text PDF PubMed Scopus (343) Google Scholar, K. R. D. V. J. 2002; Full Text PDF PubMed Scopus Google Scholar) and an enhanced DNA in the intestine after with leptin K. R. D. V. J. 2002; Full Text PDF PubMed Scopus Google Scholar). In this we that the leptin induction of cell growth is associated with an increase of HT-29 cell population in S and G2/M phase of the cell cycle. into the cell cycle of from the to the cellular the cell to the and through of DNA and phase and and after S phase and are the of the through these the cyclin D1 is for cell cycle C. Lee A. Rev. 1999; PubMed Scopus Google Scholar). We that cyclin D1 immunoreactivity was enhanced in leptin-treated HT-29 cells, and this increase was essentially associated with cells in G0/G1 phase of the cell cycle. This effect with that leptin-treated cells through with an cell population in S and G2/M phase of the cell for a mitogenic role of leptin. The leptin of HT-29 cell proliferation was by a rapid phosphorylation of proteins that the MAP kinase is to this This is consistent with several activation of MAP kinase growth in response to leptin in cell types including epithelial cells (13Hardwick J.C. Van Den Brink G.R. Offerhaus G.J. Van Deventer S.J. Peppelenbosch M.P. Gastroenterology. 2001; 121: 79-90Abstract Full Text PDF PubMed Scopus (343) Google Scholar), as cells V. J. Mol. Endocrinol. 1999; PubMed Scopus Google cells L. S. Mol. Cell. Endocrinol. 1999; PubMed Scopus Google Scholar), immune cells (10Santos-Alvarez J. Goberna R. Sanchez-Margalet V. Cell. Immunol. 1999; 194: 6-11Crossref PubMed Scopus (490) Google Scholar, 11Martin-Romero C. Sanchez-Margalet V. Cell. Immunol. 2001; 212: 83-91Crossref PubMed Scopus (119) Google Scholar), and T. D. C. A. S. A. 1996; PubMed Scopus Google Scholar). Moreover, we have for the first time, the transcription factor NF-κB as a target of in HT-29 cells. The family of transcription which and have been in the of cell and from apoptosis through of transcription in D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google and S. May M.J. Annu. Rev. Immunol. PubMed Scopus Google Scholar). In cell NF-κB is in the in an with IκB-α signals the phosphorylation and of in the nuclear of NF-κB and DNA transcription S. T. M. J. Biol. 1994; PubMed Scopus Google Scholar, L. G. PubMed Scopus Google Scholar). We demonstrated that leptin induces an increase in protein in the nucleus and the of an IκB-α protein in This nuclear was by an increase of NF-κB DNA binding and transcriptional in an in HT-29 cells. These data that leptin activation of NF-κB pathways may enhance HT-29 cell proliferation through an expression of cyclin This with the of NF-κB binding on the cyclin D1 D. B. M. C. M. J. Lee J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). They are also in line with of cells T. A. 3: PubMed Scopus Google Scholar, M. D. A. A. C. M. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar, C. Mol. Cell. Biol. 1999; 19: PubMed Google Scholar) that NF-κB a role in cell cycle through of cyclin D1 we that leptin induces HT-29 cell growth through activation of NF-κB, which in expression of cyclin leptin the expression of cyclin D1 under conditions to further In this we also demonstrated that leptin HT-29 cells from sodium butyrate-induced This can that leptin butyrate uptake in human intestinal Caco-2 cells through the monocarboxylate transporter MCT-1 (16Buyse M. Sitaraman S.V. Liu X. Bado A. Merlin D. J. Biol. Chem. 2002; 277: 28182-28190Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The sodium butyrate is a product of fermentation of and dietary fibers in the large Butyrate has to the and function of the colonic and may an factor in the pathogenesis of of the colonic F. J. 1995; Full Text PDF PubMed Scopus Google Scholar, A. T. Y. PubMed Scopus Google Scholar). Butyrate is to growth and stimulate the of and colonic cells both in and in to apoptosis A. PubMed Scopus Google Scholar, A. C. Nutr. 1996; PubMed Scopus Google Scholar). with we that sodium butyrate inhibits cell proliferation and induces apoptosis in colon cancer HT-29 cells. Interestingly, we have an anti-apoptotic effect of leptin that was associated with its to stimulate HT-29 cell proliferation. The precise mechanisms by which leptin promotes cell survival upon butyrate challenge However, the reduced phosphorylation of the activation NF-κB, and the of butyrate-treated cells to in response to leptin that NF-κB may the of this We that leptin promotes intestinal epithelial cell survival upon butyrate challenge by counteracting the apoptotic programs initiated by this short chain fatty acid probably through NF-κB pathways. However, we the of pathways in this anti-apoptotic On the other hand, leptin has been shown to apoptosis in human cells through activation of pathways J.S. J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and in adipose tissue C. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). We have for these cell but it dependent on the of cell In we have shown that leptin as a potent and anti-apoptotic cytokine on colon cancer cells. We have NF-κB as an of leptin receptor in intestinal epithelial cells. The mitogenic and anti-apoptotic effects both the NF-κB and pathways. studies are required to unravel the precise mechanisms of the leptin anti-apoptotic We Voisin for the of this