Nitric Oxide-Induced Down-Regulation of β-Catenin in Colon Cancer Cells by a Proteasome-Independent Specific Pathway
2006; Elsevier BV; Volume: 131; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2006.07.017
ISSN1528-0012
AutoresLaurent Prévotat, Rodolphe Filomenko, Éric Solary, Jean‐François Jeannin, Ali Bettaı̈eb,
Tópico(s)Ubiquitin and proteasome pathways
ResumoBackground & Aims: We have previously reported that nitric oxide could induce the death of colon cancer cells. Because an inappropriate activation of β-catenin has been associated with intestinal cell malignant transformation, we explored whether nitric oxide could affect β-catenin expression and function. Methods: Human colon cancer cell lines were treated with the nitric oxide donor glyceryl trinitrate (GTN) before analyzing β-catenin expression by immunofluorescence, immunoblotting, and immunoprecipitation methods and its transcriptional activity using a luciferase reporter gene driven by a T-cell factor-responsive promotor. Results: GTN induces β-catenin degradation and down-regulates its transcriptional activity in colon cancer cells. This effect is preceded by GTN-induced tyrosine nitration of β-catenin, together with its dephosphorylation on serine 33, 37, and 45 and threonine 41. GTN-induced β-catenin degradation involves proteases that are sensitive to a broad-spectrum caspase inhibitor, z-VAD-fmk, and to serine protease inhibitors N-tosyl-L-phenylalaline chloromethyl ketone (TPCK) and [4-(2-aminoethyl)-benzenesulfonylfluoride] (AEBSF), whereas the ubiquitin/proteasome pathway is not involved. Interestingly, only TPCK and AEBSF restore β-catenin transcriptional activity and preserve β-catenin nuclear localization in GTN-treated colon cancer cells. Conclusions: Exposure of colon cancer cells to nitric oxide unraveled a so-far-unidentified mechanism of β-catenin regulation. The protein is nitrated and dephosphorylated, and its transcriptional activity is reduced through degradation by a TPCK and AEBSF-sensitive protease. Background & Aims: We have previously reported that nitric oxide could induce the death of colon cancer cells. Because an inappropriate activation of β-catenin has been associated with intestinal cell malignant transformation, we explored whether nitric oxide could affect β-catenin expression and function. Methods: Human colon cancer cell lines were treated with the nitric oxide donor glyceryl trinitrate (GTN) before analyzing β-catenin expression by immunofluorescence, immunoblotting, and immunoprecipitation methods and its transcriptional activity using a luciferase reporter gene driven by a T-cell factor-responsive promotor. Results: GTN induces β-catenin degradation and down-regulates its transcriptional activity in colon cancer cells. This effect is preceded by GTN-induced tyrosine nitration of β-catenin, together with its dephosphorylation on serine 33, 37, and 45 and threonine 41. GTN-induced β-catenin degradation involves proteases that are sensitive to a broad-spectrum caspase inhibitor, z-VAD-fmk, and to serine protease inhibitors N-tosyl-L-phenylalaline chloromethyl ketone (TPCK) and [4-(2-aminoethyl)-benzenesulfonylfluoride] (AEBSF), whereas the ubiquitin/proteasome pathway is not involved. Interestingly, only TPCK and AEBSF restore β-catenin transcriptional activity and preserve β-catenin nuclear localization in GTN-treated colon cancer cells. Conclusions: Exposure of colon cancer cells to nitric oxide unraveled a so-far-unidentified mechanism of β-catenin regulation. The protein is nitrated and dephosphorylated, and its transcriptional activity is reduced through degradation by a TPCK and AEBSF-sensitive protease. β-catenin was originally identified as a key component of the cadherin-catenin complex that mediates cell-cell adhesion. It functions to link directly the cadherins to the actin cytoskeleton via α-catenin.1Ben-Ze’ev A. Geiger B. 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Influence of the nitric oxide donor glyceryl trinitrate on apoptotic pathways in human colon cancer cells.Gastroenterology. 2002; 123: 235-246Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar It has been initially suggested that NO could promote invasive and angiogenic properties of tumor cells (for review, see Onier et al31Rao C.V. Nitric oxide signaling in colon cancer chemoprevention.Mutat Res. 2004; 555: 107-119Crossref PubMed Scopus (186) Google Scholar). Actually, in a randomized phase II clinical trial, the NO donor GTN has been demonstrated to improve response rate and survival when combined with anticancer drugs in patients with non–small-cell lung cancer.32Yasuda H. Yamaya M. Nakayama K. Sasaki T. Ebihara S. Kanda A. Asada M. Inoue D. Suzuki T. Okazaki T. Takahashi H. Yoshida M. Kaneta T. Ishizawa K. Yamanda S. Tomita N. Yamasaki M. Kikuchi A. Kubo H. Sasaki H. Randomized phase II trial comparing nitroglycerin plus vinorelbine and cisplatin with vinorelbine and cisplatin alone in previously untreated stage IIIB/IV non-small-cell lung cancer.J Clin Oncol. 2006; 24: 688-694Crossref PubMed Scopus (125) Google Scholar The present study was designed to determine whether NO-induced apoptosis of colon cancer cells was related to degradation of β-catenin and alteration of its transactivation potential. We show that the NO donor GTN nitrates β-catenin on its tyrosine residues, induces its degradation, and reduces its transcriptional activation potential. This degradation does not involve the known pathways, including the ubiquitin/proteasome machinery, caspases, or calpains but a TPCK or AEBSF-sensitive pathway, suggesting an alternative, so far unidentified mechanism, of regulation of β-catenin expression. GTN was purchased from Merck (Lyon, France), the broad-spectrum caspase inhibitor Z (Benzyloxycarbonyl)-Val-Ala-Asp (OMe)-CH2F (fluoromethylketone) (Z-VAD-fmk) from Bachem (Weil am Rhein, Germany), the proteasome inhibitor MG132 from the Peptide Institute (Minoh-shi Ozaka, Japan), the chymotryptic inhibitor TPCK from Roche Diagnostics (Mannheim, Germany), AEBSF from AG Scientific (San Diego, CA), N-Acetyl-Leu-Leu-Met-CHO (ALLM) from BIOMOL (Plymouth Meeting, PA), and all other chemicals and reagents from Sigma (Saint Quentin Fallavier, France) or other local sources. Anti-human β-catenin monoclonal antibody (mAb), anti-iNOS mAb, and anti-GSK-3β mAb were purchased from BD Transduction Laboratories (Le Pont de Claix, France); anti-phospho Ser 33/37/Thr 41-β-catenin, phospho Ser 45/Thr 41-β-catenin human polyclonal antibody (pAb), and anti-phospho Ser 9-GSK-3β pAb from Cell Signaling (Saint Quentin Yvelines, France); anti-casein kinase 1 α pAb and anti-Hsc 70 mAb from Santa Cruz Biotechnology (Santa Cruz, CA); anti-nitrotyrosine, anti-TCF4, and anti-Flag mAbs from Upstate (Mundolsheim, France); 488-alexa goat anti-mouse and 568-alexa goat anti-rabbit antibodies from Molecular Probes (Cergy Pontoise, France); and horseradish peroxidase-conjugated goat anti-rabbit and mouse antibodies from Jackson Immunoresearch Laboratories (West Grove, PA). The human (SW480, HCT116, RKO, and HT29) cells, the murine (CT26) colorectal cancer cells, and the 293T cells were purchased from American Tissue Culture Collection (Manassas, VA). SW480 were grown in 1:1 (vol/vol) Dulbecco’s modified Eagle medium (DMEM), HAM-F10 (Biowhittaker, Fontenay-sous-Bois, France), supplemented with 5% FCS (Gibco BRL, Eriny, France), and 2 mmol/L L-glutamine at 37°C in a dry atmosphere. RKO, HCT 116, HT29, and CT26 cells were cultured in the same medium but in an atmosphere of 95% air and 5% CO2 at 37°C. Cells were routinely detached with 0.125% trypsin-0.1% EDTA and washed once in the culture medium before treatment. Transfection experiments were performed by adding 5 μg of plasmid DNA to 3 × 106 cells for 4 hours in the presence of Superfect transfection reagent (Qiagen, Courtaboeuf, France). Transfected cells were harvested 24 hours later. Cell viability was not affected by the transfection. The vectors used for transfection included pGL3-OT (improved version of TOP-flash) and pGL3-OF (improved version of FOP-flash) containing multimerized wild-type or mutated TCF4-binding sites upstream of the SV40 promoter driving luciferase gene expression,33Shih I.M. Yu J. He T.C. Vogelstein B. Kinzler K.W. The β-catenin binding domain of adenomatous polyposis coli is sufficient for tumor suppression.Cancer Res. 2000; 60: 1671-1676PubMed Google Scholar a kind gift from Drs B. Vogelstein and K.W. Kinzler (Johns Hopkins Oncology Center, Baltimore, MD), pcDNA1-containing murine inducible NO synthase (piNOSL8, Euromedex, Mundolsheim, France), and a plasmid containing Flag-Ubiquitin. Twenty-four hours after transfection of the constructs encoding the reporter luciferase gene, cells were lysed in a luciferase or passive lysis buffer (Promega, Charbonnières, France) at room temperature for 20 minutes, then centrifuged for 10 minutes at 8000g at 4°C before collecting the supernatant for luciferase activity measurement using the Promega “Luciferase Assay Reagent” or “Dual Luciferase Reporter assay system.” As internal controls for normalization of transfection efficacy, we used either the pRSV-β-galactosidase reporter vector (250 ng) or the phRL-TK (150 ng) (Promega). Luciferase activities were measured by using a luminometer (Lumat LB 9507, EG&G Berthold). NO production was determined by measuring the accumulation of nitrites and nitrates in cell culture media using the Griess microassay as described.34Green L.C. Wagner D.A. Glogowski J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. 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Catalytic properties of 26S and 20S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities.J Biol Chem. 1997; 272: 24899-24905Crossref PubMed Scopus (57) Google Scholar Briefly, cells (2 × 106 in 200 μL phosphate-buffered saline [PBS], pH 7.4) were incubated for 30 minutes at 37°C with 100 μmol/L of the cell-permeant fluorogenic substrate N-succinyl-L-leucyl-L-tyrosine-7-amido-4-methyl coumarin (Suc-LLVY-AMC) (Bachem), and fluorescence generated by the cleavage of this substrate was quantified by using a Kontron SFM spectrofluorometer (Kontron AG). Cells (3 × 105/mL culture medium) were treated with 100 or 500 μmol/L GTN for 48 hours at 37°C. After treatment, the whole population of cells including plastic-attached and floating cells was washed in cold PBS and exposed for 15 minutes to 1 μg/mL Hoechst 33342 at 37°C before evaluating changes in cellular nuclear chromatin by fluorescence microscopy. The percentage of apoptotic cells (chromatin condensation and nuclear fragmentation) was determined by counting 300 cells in each sample. Cells (3 × 106) were washed twice with cold PBS, and whole-cell lysates were prepared in boiling buffer (1% sodium dodecyl sulfate [SDS], 1 mmol/L sodium-orthovanadate, 10 mmol/L Tris, pH 7.4) in the presence of a cocktail of protease inhibitors (Roche) for 10 minutes at 4°C. The viscosity of the samples was reduced by ultrasounds. Lysates were harvested, and protein concentration was measured by using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). Fifty micrograms of proteins were incubated in loading buffer (125 mmol/L Tris-HCl, pH 6.8, 10% β-mercaptoethanol, 4.6% SDS, 20% glycerol, and 0.003% bromophenol blue), separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad). After blocking nonspecific binding sites for 2 hours with 8% nonfat milk in 0.1% Tween 20 PBS (TPBS), the membrane was incubated overnight at 4°C with the primary Ab. After 3 washes in TPBS, the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit Abs for 30 minutes at room temperature then washed 3 times in TPBS. Immunoblot was revealed using enhanced chemiluminescence detection kit (Luminol, Santa Cruz) and autoradiography. Nuclear fractions were obtained by lysing the cells (60 × 106) in lysis buffer (10 mmol/L Hepes [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid], pH 7.8, 10 mmol/L KCl, 0.1 mmol/L ethylenediaminetetraacetic acid [EDTA], 0.1 mmol/L ethylene glycol-bis [beta-aminoethyl ether]-N,N,N′,N′-tetraacetic acid [EGTA], 1 mmol/L dithiothreitiol [DTT]) in the presence of complete protease inhibitor mixture. Next, 0.6% NP-40 was added rapidly in the cell lysates, and, after shaking, lysates were centrifuged at 1200g for 10 minutes, and the pellets were washed once in lysis buffer then resuspended in a buffer containing 20 mmol/L Hepes, pH 7.8, 400 mmol/L NaCl, 1 mmol/L EDTA, and 1 mmol/L EGTA in the presence of complete protease inhibitor mixture for 30 minutes on ice. Nuclear extracts were cleared by centrifugation at 20,000g for 30 minutes. Cell nuclear extracts (5 μg) were incubated with 100,000 cpm of 32P-end labelled TCF4 (5′-GCACCCTTTGATCTTACC-3′) consensus oligonucleotide in 10 μL of reaction buffer (5μL buffer A [0.5 mol/L sucrose, 15 mmol/L Tris, pH 7.5, 60 mmol/L KCl, 0.25 mmol/L EDTA, pH 8, 0.125 mmol/L EGTA, pH 5, 0.15 mmol/L spermine, 0.5 mmol/L spermidine, 1 mmol/L (DTT)], 2 μL MgSp [10 mmol/L MgCl2, 80 mmol/L spermidine], 1.5 μL NaPi [10 mmol/L NaPi, 1 mmol/L EDTA], 10 mmol/L DTT, 0.2 μg poly(dI-dC)). After 30 minutes, DNA-protein complexes were separated from free oligonucleotides by electrophoresis in a 4% polyacrylamide gel and detected by using a PhosphorImager (Amersham Pharmacia Biotech, Orsay, France). SW480 cells (3 × 106) were treated with 500 μmol/L GTN for 24 hours then fixed with 2% paraformaldehyde (PFA) in PBS for 10 minutes at 4°C. Fixed cells were washed 3 times with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes, and incubated with primary Abs diluted 1/100 in PBS-1% bovine serum albumin for 45 minutes at room temperature. After washing, cells were incubated for 30 minutes with appropriate 568- or 488-alexa-conjugated Abs 1/1000. Analysis was made by using a fluorescence microscope (Nikon). Nonrelevant isotype-matching Abs were used as negative controls. Total RNA were isolated from SW480 cells with NucleoSpin RNA II kit (Macherey-Nagel). One microgram samples of deoxyribonuclease I–treated total RNA were reverse transcribed then amplified in one step with the QIAGEN OneStep RT-PCR kit (Qiagen, Courtabeuf, France). Sense and antisense primers used to amplify human complementary DNA (cDNA) were as follows: matrilysin: 5′-CATGAGTGAGCTACAGTGGG-3′ (sense) and 5′-TTCTGCAACATCTGGCACTC-3′ (antisense), GAPDH: 5′-GAGTCAAC GGATTTGGTCGT-3′ (sense) and 5′-TTGATTTTGGAGGGATCTCG-3′ (antisense). Tumors were induced in Balb/c mice by subcutaneous injection of 5 × 105 CT26 colon cancer cells in HAM-F10. When these tumors reached an average volume of 50–100 mm3 (10 days), animals were assigned randomly to 1 of 2 groups. Those from the first group were treated with GTN (0.2 mg/kg per day) diluted in 100 μL physiologic serum containing ethanol (25% vol/vol). Those from the other group received a similar volume of vehicle. An intratumour injection was performed every day, and tumor volume was measured daily with an external caliper. We have shown previously that the human SW480 colon cancer cells underwent apoptosis in response to the NO donor GTN.20Millet A. Bettaieb A. Renaud F. Prevotat L. Hammann A. Solary E. Mignotte B. Jeannin J.F. Influence of the nitric oxide donor glyceryl trinitrate on apoptotic pathways in human colon cancer cells.Gastroenterology. 2002; 123: 235-246Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar GTN induces a dose-dependent production of NO in these cells, as determined by measuring nitrite and nitrate production (Figure 1A). Accumulation of NO metabolites was partly prevented by the NO scavenger 2-Pyenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) (Figure 1A), whose lack of complete inhibitory effect might be related to PTIO-mediated NO oxidation resulting in the formation of nitrites.36Pfeiffer S. Leopold E. Hemmens B. Schmidt K. Werner E.R. Mayer B. Interference of carboxy-PTIO with nitric oxide- and peroxynitrite-mediated reactions.Free Radic Biol Med. 1997; 22: 787-794Crossref PubMed Scopus (79) Google Scholar SW480 cells exhibit a constitutively high level of β-catenin/TCF4-mediated transcriptional activity,12Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2896) Google Scholar which was confirmed by using the reporter gene encoding plasmid pGL3-OT, and treatment with 500 μmol/L GTN for 48 hours strongly diminished (∼7-fold) this constitutive activity (Figure 1B). The GTN-induced fall in β-catenin/TCF4-mediated transcriptional activity was significantly recovered by PTIO (Figure 1B). As negative controls, we expressed pGL3-OF, another reporter gene in which TCF4-binding sites were mutated, in SW480 cells, and we expressed pGL3-OT plasmid in RKO cells that do not express β-catenin (Figure 1B, insert).37Deng J. Miller S.A. Wang H.Y. Xia W. Wen Y. Zhou B.P. Li Y. Lin S.Y. Hung M.C. β-Catenin interacts with and inhibits NF-κB in human colon and breast cancer.Cancer Cell. 2002; 2: 323-334Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar We also transfected 293T cells with a plasmid encoding inducible NOS (iNOS) or the corresponding empty vector (Figure 1C, insert) together with pGL3-OT. The expression of iNOS increased the production of nitrites and nitrates (Figure 1C), which was associated with a decrease in β-catenin/TCF4-mediated transcriptional activity, as measured by using the pGL3-OT reporter gene (Figure 1D). Both effects were reversed by the iNOS inhibitor aminoguanidine (Figure 1C and 1D). Altogether, these observations indicated that GTN could decrease the transcriptional activity of β-catenin. A time course analysis performed in SW480 cells exposed to 500 μmol/L GTN indicated that this effect started 12 hours after the beginning of treatment (Figure 1E) and correlated with a transient nitration of β-catenin on tyrosine residues (Figure 1F). A longer exposure of SW480 cells to GTN, eg, 48 hours, induced a dose-dependent increase in the percentage of cells with nuclear chromatin condensation, as identified by Hoechst 33352 staining. A similar effect of GTN was observed in HCT116 cells. This phenotype, which suggested apoptosis, was associated with the appearance of 3 β-catenin fragments, as identified by Western blotting (Figure 2A and B). Because both SW480 cells in which β-catenin is wild type38Nishisho I. Nakamura Y. Miyoshi Y. Miki Y. Ando H. Horii A. Koyama K. Utsunomiya J. Baba S. Hedge P. Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients.Science. 1991; 253: 665-669Crossref PubMed Scopus (1599) Google Scholar and HCT116 cells in which the protein is mutated on serine 4539Polakis P. Wnt signaling and cancer.Genes Dev. 2000; 14: 1837-1851PubMed Google Scholar demonstrate the same changes in the expression of β-catenin when studied by Western blotting, this effect of GTN may not depend on phosphorylation of β-catenin serine 45. The anti-β-catenin mAb detected 2 bands in both cell lines when untreated. The lower band, which was previously shown to be detecte
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