Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Nitric Oxide Mediates Natural Polyphenol-induced Bcl-2 Down-regulation and Activation of Cell Death in Metastatic B16 Melanoma

2006; Elsevier BV; Volume: 282; Issue: 5 Linguagem: Inglês

10.1074/jbc.m605934200

ISSN

1083-351X

Autores

Paula Ferrer, Miguel Asensi, Sonia Priego, María Benlloch, Salvador Mena, Ángel Ortega, Elena Obrador, Juan M. Esteve, José M. Estrela,

Tópico(s)

Bioactive Natural Diterpenoids Research

Resumo

Intravenous administration to mice of trans-pterostilbene (t-PTER; 3,5-dimethoxy-4′-hydroxystilbene) and quercetin (QUER; 3,3′,4′,5,6-pentahydroxyflavone), two structurally related and naturally occurring small polyphenols, inhibits metastatic growth of highly malignant B16 melanoma F10 (B16M-F10) cells. t-PTER and QUER inhibit bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity. However, the molecular mechanism(s) linking polyphenol signaling and bcl-2 expression are unknown. NO is a potential bioregulator of apoptosis with controversial effects on Bcl-2 regulation. Polyphenols may affect NO generation. Short-term exposure (60 min/day) to t-PTER (40 μm) and QUER (20 μm) (approximate mean values of the plasma concentrations measured within the first hour after intravenous administration of 20 mg of each polyphenol/kg) down-regulated inducible NO synthetase in B16M-F10 cells and up-regulated endothelial NO synthetase in the vascular endothelium and thereby facilitated endothelium-induced tumor cytotoxicity. Very low and high NO levels down-regulated bcl-2 expression in B16M-F10 cells. t-PTER and QUER induced a NO shortage-dependent decrease in cAMP-response element-binding protein phosphorylation, a positive regulator of bcl-2 expression, in B16M-F10 cells. On the other hand, during cancer and endothelial cell interaction, t-PTER- and QUER-induced NO release from the vascular endothelium up-regulated neutral sphingomyelinase activity and ceramide generation in B16M-F10 cells. Direct NO-induced cytotoxicity and ceramide-induced mitochondrial permeability transition and apoptosis activation can explain the increased endothelium-induced death of Bcl-2-depleted B16M-F10 cells. Intravenous administration to mice of trans-pterostilbene (t-PTER; 3,5-dimethoxy-4′-hydroxystilbene) and quercetin (QUER; 3,3′,4′,5,6-pentahydroxyflavone), two structurally related and naturally occurring small polyphenols, inhibits metastatic growth of highly malignant B16 melanoma F10 (B16M-F10) cells. t-PTER and QUER inhibit bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity. However, the molecular mechanism(s) linking polyphenol signaling and bcl-2 expression are unknown. NO is a potential bioregulator of apoptosis with controversial effects on Bcl-2 regulation. Polyphenols may affect NO generation. Short-term exposure (60 min/day) to t-PTER (40 μm) and QUER (20 μm) (approximate mean values of the plasma concentrations measured within the first hour after intravenous administration of 20 mg of each polyphenol/kg) down-regulated inducible NO synthetase in B16M-F10 cells and up-regulated endothelial NO synthetase in the vascular endothelium and thereby facilitated endothelium-induced tumor cytotoxicity. Very low and high NO levels down-regulated bcl-2 expression in B16M-F10 cells. t-PTER and QUER induced a NO shortage-dependent decrease in cAMP-response element-binding protein phosphorylation, a positive regulator of bcl-2 expression, in B16M-F10 cells. On the other hand, during cancer and endothelial cell interaction, t-PTER- and QUER-induced NO release from the vascular endothelium up-regulated neutral sphingomyelinase activity and ceramide generation in B16M-F10 cells. Direct NO-induced cytotoxicity and ceramide-induced mitochondrial permeability transition and apoptosis activation can explain the increased endothelium-induced death of Bcl-2-depleted B16M-F10 cells. Different natural polyphenols show potent antioxidant effects and may have therapeutic applications in oxidative stress-related diseases such as cancer (1Yang C.S. Landau J.M. Huang M.T. Newmark H.L. Annu. Rev. Nutr. 2001; 21: 381-406Crossref PubMed Scopus (1098) Google Scholar, 2Ross J.A. Kasum C.M. Annu. Rev. Nutr. 2002; 22: 19-34Crossref PubMed Scopus (1706) Google Scholar, 3Pervaiz S. FASEB J. 2003; 17: 1975-1985Crossref PubMed Scopus (477) Google Scholar). The cancer-chemopreventive activity of trans-resveratrol (t-RESV 2The abbreviations used are: t-RESV, trans-resveratrol; t-PTER, trans-pterostilbene; QUER, quercetin; B16M-F10, B16 melanoma F10; VCAM-1, vascular adhesion molecule 1; HSE, hepatic sinusoidal endothelium; iNOS, inducible nitric-oxide synthetase; eNOS, endothelial nitric-oxide synthetase; DMEM, Dulbecco's modified Eagle's medium; BCECF-AM, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; CREB, cAMP-response element-binding protein; siRNA, small interfering RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; SMase, sphingomyelinase; AMG, aminoguanidine; HPLC, high pressure liquid chromatography; SNAP, S-nitroso-N-acetyl-dl-penicillamine. 2The abbreviations used are: t-RESV, trans-resveratrol; t-PTER, trans-pterostilbene; QUER, quercetin; B16M-F10, B16 melanoma F10; VCAM-1, vascular adhesion molecule 1; HSE, hepatic sinusoidal endothelium; iNOS, inducible nitric-oxide synthetase; eNOS, endothelial nitric-oxide synthetase; DMEM, Dulbecco's modified Eagle's medium; BCECF-AM, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; CREB, cAMP-response element-binding protein; siRNA, small interfering RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; SMase, sphingomyelinase; AMG, aminoguanidine; HPLC, high pressure liquid chromatography; SNAP, S-nitroso-N-acetyl-dl-penicillamine.; trans-3,5,4′-trihydroxystilbene) was first reported by Jang et al. (4Jang M. Cai L. Udeani G.O. Slowing K.V. Thomas C.F. Beecher C.W. Fong H.H. Farnsworth N.R. Kinghorn A.D. Mehta R.G. Moon R.C. Pezzuto J.M. Science. 1997; 275: 218-220Crossref PubMed Scopus (4437) Google Scholar). However, the potential anticancer properties of t-RESV are strongly limited because of its low bioavailability (5Asensi M. Medina I. Ortega A. Carretero J. Baño M.C. Obrador E. Estrela J.M. Free Radic. Biol. Med. 2002; 33: 387-398Crossref PubMed Scopus (336) Google Scholar). Thus, structural modifications of the t-RESV molecule appeared to be necessary to increase its bioavailability while preserving its biological activity. Recently, we found that trans-pterostilbene (t-PTER; trans-3,5-dimethoxy-4′-hydroxystilbene) and quercetin (QUER; 3,3′,4′,5,6-pentahydroxyflavone) have longer in vivo half-lives compared with t-RESV (6Ferrer P. Asensi M. Segarra R. Ortega A. Benlloch M. Obrador E. Varea M.T. Asensio G. Jorda L. Estrela J.M. Neoplasia. 2005; 7: 37-47Crossref PubMed Scopus (152) Google Scholar). In vitro growth of highly malignant B16 melanoma F10 (B16M-F10) cells is inhibited (56%) by short-term exposure (60 min/day) to t-PTER (40 μm) and QUER (20 μm) (6Ferrer P. Asensi M. Segarra R. Ortega A. Benlloch M. Obrador E. Varea M.T. Asensio G. Jorda L. Estrela J.M. Neoplasia. 2005; 7: 37-47Crossref PubMed Scopus (152) Google Scholar). Intravenous administration of t-PTER and QUER (20 mg/kg/day) to mice inhibits (73%) metastatic growth of B16M-F10 cells in the liver, a common site for metastasis development (6Ferrer P. Asensi M. Segarra R. Ortega A. Benlloch M. Obrador E. Varea M.T. Asensio G. Jorda L. Estrela J.M. Neoplasia. 2005; 7: 37-47Crossref PubMed Scopus (152) Google Scholar). The antimetastatic mechanism involves (a) t-PTER-induced inhibition of vascular adhesion molecule 1 (VCAM-1) expression in the hepatic sinusoidal endothelium (HSE), which decreases B16M-F10 cell adhesion to the endothelium via very late activation antigen 4, and (b) QUER- and t-PTER-induced inhibition of bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity (6Ferrer P. Asensi M. Segarra R. Ortega A. Benlloch M. Obrador E. Varea M.T. Asensio G. Jorda L. Estrela J.M. Neoplasia. 2005; 7: 37-47Crossref PubMed Scopus (152) Google Scholar). Analysis of the bcl-2 family of genes revealed that B16M-F10 cells (high metastatic potential), compared with B16M-F1 cells (low metastatic potential), overexpress bcl-2 preferentially (7Ortega A. Ferrer P. Carretero J. Obrador E. Asensi M. Pellicer J.A. Estrela J.M. J. Biol. Chem. 2003; 278: 39591-39599Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). t-PTER increases expression of pro-death bax (∼2.2-fold) and decreases expression of anti-death bcl-2 (∼2.0-fold) (6Ferrer P. Asensi M. Segarra R. Ortega A. Benlloch M. Obrador E. Varea M.T. Asensio G. Jorda L. Estrela J.M. Neoplasia. 2005; 7: 37-47Crossref PubMed Scopus (152) Google Scholar), whereas QUER increases expression of different pro-death genes (bax, bak, bad, and bid; 1.5–2.5-fold) and decreases expression of all anti-death genes analyzed (bcl-2 (∼7.3-fold), bcl-w (∼1.5-fold), and bcl-xL (∼2-fold)) (6Ferrer P. Asensi M. Segarra R. Ortega A. Benlloch M. Obrador E. Varea M.T. Asensio G. Jorda L. Estrela J.M. Neoplasia. 2005; 7: 37-47Crossref PubMed Scopus (152) Google Scholar). bcl-2 overexpression prevents the QUER- and t-PTER-dependent increase in metastatic B16M-F10 cell death caused by the HSE in vivo (6Ferrer P. Asensi M. Segarra R. Ortega A. Benlloch M. Obrador E. Varea M.T. Asensio G. Jorda L. Estrela J.M. Neoplasia. 2005; 7: 37-47Crossref PubMed Scopus (152) Google Scholar), thus suggesting that Bcl-2 by itself plays a critical role in regulating B16M-F10 resistance against vascular endothelium-induced damage. In agreement with this idea, we also observed that antisense bcl-2 therapy potentiates tumor necrosis factor-α-induced oxidative stress and death in B16M-F10 cells (7Ortega A. Ferrer P. Carretero J. Obrador E. Asensi M. Pellicer J.A. Estrela J.M. J. Biol. Chem. 2003; 278: 39591-39599Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). However, the molecular mechanisms that link polyphenol signaling with bcl-2 expression are unclear. In vitro, t-RESV-driven apoptosis of chronic leukemic B cells has been shown to correlate with activation of caspase-3, a drop in the mitochondrial membrane potential, and reduction in the expression of inducible nitric-oxide synthetase (iNOS) (8Billard C. Izard J.C. Roman V. Kern C. Mathiot C. Mentz F. Kolb J.P. Leuk. Lymphoma. 2002; 43: 1991-2002Crossref PubMed Scopus (133) Google Scholar, 9Quiney C. Dauzonne D. Kern C. Fourneron J.D. Izard J.C. Mohammad R.M. Kolb J.P. Billard C. Leuk. Res. 2004; 28: 851-861Crossref PubMed Scopus (66) Google Scholar). NO is a potential bioregulator of apoptosis because high concentrations of NO or peroxynitrite can induce apoptotic death in different cells types, including tumor cells (10Chung H.T. Pae H.O. Choi B.M. Billiar T.R. Kim Y.M. Biochem. Biophys. Res. Commun. 2001; 282: 1075-1079Crossref PubMed Scopus (458) Google Scholar), although, on the other hand, NO may also act as an anti-apoptotic signal associated with, for example, suppression of mitochondrial cytochrome c release, ceramide generation, and caspase activation (10Chung H.T. Pae H.O. Choi B.M. Billiar T.R. Kim Y.M. Biochem. Biophys. Res. Commun. 2001; 282: 1075-1079Crossref PubMed Scopus (458) Google Scholar). Indeed, NO donors can elevate bcl-2 expression both at the mRNA and protein levels and prevent apoptotic cell death in vitro (11Genaro A.M. Hortelano S. Alvarez A. Martinez C. Bosca L. J. Clin. Investig. 1995; 95: 1884-1890Crossref PubMed Scopus (306) Google Scholar), whereas, paradoxically, also in vitro, NO-induced apoptosis of K-1735 melanoma cells (12Xie K. Wang Y. Huang S. Xu L. Bielenberg D. Salas T. McConkey D.J. Jiang W. Fidler I.J. Oncogene. 1997; 15: 771-779Crossref PubMed Scopus (81) Google Scholar) or human myeloid leukemia U937 cells (13Brockhaus F. Brune B. Exp. Cell Res. 1998; 238: 33-41Crossref PubMed Scopus (86) Google Scholar) is associated with down-regulation of Bcl-2 and caspase activation. Therefore, NO, which may either prevent or induce apoptosis, can also increase or decrease Bcl-2 levels. These apparently controversial facts suggest that different intracellular NO levels may likely determine opposite effects. Whether natural polyphenols such as t-PTER and QUER also cause reduction in iNOS gene expression in metastatic cells (and consequently a decrease in their intracellular NO levels) and whether this is linked to changes in bcl-2 expression is unknown. On the other hand, t-RESV, as well as other polyphenols (e.g. black tea polyphenols), can increase endothelial nitric-oxide synthetase (eNOS) activity and induce accumulation of p53 and p21WAF1/CIP1 in cultured pulmonary artery endothelial cells (14Hsieh T.C. Juan G. Darzynkiewicz Z. Wu J.M. Cancer Res. 1999; 59: 2596-2601PubMed Google Scholar, 15Anter E. Thomas S.R. Schulz E. Shapira O.M. Vita J.A. Keaney Jr., J.F. J. Biol. Chem. 2004; 279: 46637-46643Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Thus, it is possible that natural polyphenols may also alter NO levels in the metastatic microenvironment during interaction of cancer and endothelial cells, which is important because endothelial NO generation was found to be essential in the mechanism of tumor cytotoxicity during B16M-F10 cell adhesion to the vascular endothelium (16Carretero J. Obrador E. Esteve J.M. Ortega A. Pellicer J.A. Sempere F.V. Estrela J.M. J. Biol. Chem. 2001; 276: 25775-25782Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The aim of this study was to investigate the possible relationship between NO and the effect t-PTER and/or QUER (at in vivo bioavailable concentrations) on bcl-2 expression in B16M-F10 cells. Our results show that this polyphenolic association decreases NO production in isolated B16M-F10 cells and increases NO release from the vascular endothelium during B16M-F10/endothelial cell interaction. At both steps, changes in NO levels trigger Bcl-2 down-regulation and activation of death mechanisms in metastatic B16M-F10 cells. Culture of B16M-F10 Cells—Murine B16M-F10 cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen), pH 7.4, supplemented with 10% fetal calf serum (Invitrogen), 10 mm HEPES, 40 mm NaHCO3, 100 units/ml penicillin, and 100 μg/ml streptomycin (17Obrador E. Carretero J. Ortega A. Medina I. Rodilla V. Pellicer J.A. Estrela J.M. Hepatology. 2002; 35: 74-81Crossref PubMed Scopus (78) Google Scholar). Cell integrity was assessed by trypan blue exclusion and leakage of lactate dehydrogenase activity (17Obrador E. Carretero J. Ortega A. Medina I. Rodilla V. Pellicer J.A. Estrela J.M. Hepatology. 2002; 35: 74-81Crossref PubMed Scopus (78) Google Scholar). Measurement of H2O2, Nitrite, and Nitrate—The assay of H2O2 production was based, as reported previously (16Carretero J. Obrador E. Esteve J.M. Ortega A. Pellicer J.A. Sempere F.V. Estrela J.M. J. Biol. Chem. 2001; 276: 25775-25782Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), on the H2O2-dependent oxidation of homovanillic acid (3-methoxy-4-hydroxyphenylacetic acid) to a highly fluorescent dimer (2,2′-dihydroxydiphenyl-5,5′-diacetic acid), which is mediated by horseradish peroxidase (18Ruch W. Cooper P.H. Baggiolini M. J. Immunol. Methods. 1983; 63: 347-357Crossref PubMed Scopus (217) Google Scholar). Nitrite and nitrate determinations were performed as described previously (16Carretero J. Obrador E. Esteve J.M. Ortega A. Pellicer J.A. Sempere F.V. Estrela J.M. J. Biol. Chem. 2001; 276: 25775-25782Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and based on the methodology of Braman and Hendrix (19Braman R.S. Hendrix S.A. Anal. Chem. 1989; 61: 2715-2718Crossref PubMed Scopus (709) Google Scholar). Total NOx (NO−2 plus NO−3) determinations were made by monitoring NO evolution from a measured sample placed into a boiling VCl3/HCl solution (which will reduce both NO−2 and NO−3 to NO). Quantitation was accomplished using a standard curve made up of known amounts of NO−2 and NO−3. Isolation, Identification, and Culture of the HSE—Male C57BL/6J mice (10–12 weeks old) were from Charles River Laboratories, Inc. (Barcelona, Spain). The HSE was separated and identified as described previously (20Vidal-Vanaclocha F. Rocha M. Asumendi A. Barbera-Guillem E. Hepatology. 1993; 18: 328-339Crossref PubMed Scopus (38) Google Scholar). Sinusoidal cells were separated in a 17.5% (w/v) metrizamide gradient. HSE cultures were established and maintained in pyrogen-free DMEM supplemented as described above for the B16M-F10 cells. Differential adhesion of endothelial cells to the collagen matrix and washing allowed complete elimination of other sinusoidal cell types (Kupffer and stellate cells and lymphocytes) from the culture flasks. B16M/Endothelial Cell Adhesion and Cytotoxicity Assays—B16M-F10 cells were loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes, Eugene, OR). (106 cells were incubated in 1 ml of HEPES-buffered DMEM containing 50 μg of BCECF-AM and 5 μl of Me2SO for 20 min at 37 °C.) After washing, BCECF-AM-containing cells were resuspended in HEPES-buffered DMEM without phenol red at a concentration of 2.5 × 106 cells/ml and added (0.2 ml/well) to endothelial cells (plated 24 h before) and also to plastic- or collagen-precoated control wells. The plates were then incubated at 37 °C, and 20 min later, the wells were washed three times with fresh medium and read for fluorescence using a Fluoroskan Ascent FL (LabSystems, Manchester, UK). The number of adherent tumor cells was quantified by arbitrary fluorescence units based on the percentage of the initial number of B16M cells added to the HSE culture (21Anasagasti M.J. Alvarez A. Martin J.J. Mendoza L. Vidal-Vanaclocha F. Hepatology. 1997; 25: 840-846Crossref PubMed Scopus (55) Google Scholar). Damage to B16M cells during their in vitro adhesion to the HSE was measured as described previously (22Anasagasti M.J. Martin J.J. Mendoza L. Obrador E. Estrela J.M. McCuskey R.S. Vidal-Vanaclocha F. Hepatology. 1998; 27: 1249-1256Crossref PubMed Scopus (51) Google Scholar) using tumor cells loaded with calcein acetoxymethyl ester (Molecular Probes). Other reagents used in experiments of tumor cytotoxicity were from Sigma. Reverse Transcription-PCR and Detection of mRNA Expression—Total RNA was isolated using the TRIzol kit (Invitrogen) following the manufacturer's instructions. cDNA was obtained using a random hexamer primer and a MultiScribe reverse transcription kit (TaqMan reverse transcription reagents, Applied Biosystems, Foster City, CA) as described by the manufacturer. A PCR Master Mix and AmpliTaq Gold DNA polymerase (Applied Biosystems) containing specific primers (synthesized by Integrated DNA Technologies according to published sequences available from the GenBank™ Data Bank) were then added: iNOS, 5′-CGGATATCTCTTGCAAGTCCAAA (forward) and 5′-AAGTATGTGTCTGCAGATATG (reverse); eNOS, 5′-CACCAGGAAGAAGACCTTTAAGGA (forward) and 5′-CACACGCTTCGCCATCAC (reverse); bcl-2, 5′-CTCGTCGCTACCGTCGTGACTTCG (forward) and 5′-CAGATGCCGGTTCAGGTACTCAGTC (reverse); and glyceraldehyde-3-phosphate dehydrogenase, 5′-CCTGGAGAAACCTGCCAAGTATG (forward) and 5′-GGTCCTCAGTGTAGCCCAAGATG (reverse). Real-time quantitation of the mRNA relative to glyceraldehyde-3-phosphate dehydrogenase was performed with a SYBR Green I assay and a iCycler detection system (Bio-Rad). Target cDNA was amplified as follows: 10 min at 95 °C and then 40 cycles of amplification (denaturation at 95 °C for 30 s and annealing and extension at 60 °C for 1 min per cycle). The increase in fluorescence was measured in real time during the extension step. The threshold cycle (CT) was determined, and then the relative gene expression was expressed as follows: -fold change = 2–Δ(ΔCT), where ΔCT = CT target – CT glyceraldehyde-3-phosphate dehydrogenase and Δ(ΔCT) =ΔCT treated –ΔCT control. Bcl-2 Analysis—Bcl-2 protein was quantitated in the soluble cytosolic fraction by enzyme immunoassay (23Eissa S. Seada L.S. Clin. Chem. 1998; 44: 1423-1429Crossref PubMed Scopus (65) Google Scholar) using a monoclonal antibody-based assay from Sigma (1 unit of Bcl-2 is defined as the amount of Bcl-2 in 1000 non-transfected B16M-F10 cells). eNOS-deficient Mice—Generation of eNOS-deficient mice was carried out as described previously (24Shesely E.G. Maeda N. Kim H.S. Desai K.M. Krege J.H. Laumback V.E. Sherman P.A. Sessa W.C. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13176-13181Crossref PubMed Scopus (778) Google Scholar). We interbred heterozygous (+/–) eNOS-deficient mice to generate eNOS+/+ and eNOS–/– mice. We used eNOS+/+ and wild-type C57BL/6J mice as controls. Genotyping of the animals was performed by Southern blotting DNA from tail biopsies. The identification of eNOS+/+ and eNOS–/– mice was as described previously (24Shesely E.G. Maeda N. Kim H.S. Desai K.M. Krege J.H. Laumback V.E. Sherman P.A. Sessa W.C. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13176-13181Crossref PubMed Scopus (778) Google Scholar). Briefly, 20-μg samples were digested with BamHI, separated on 1.0% agarose gels, and transferred to nylon-supported nitrocellulose. The blots were then hybridized using a random primer-labeled 1.4-kb eNOS cDNA probe (24Shesely E.G. Maeda N. Kim H.S. Desai K.M. Krege J.H. Laumback V.E. Sherman P.A. Sessa W.C. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13176-13181Crossref PubMed Scopus (778) Google Scholar). A 5.3-kb fragment was diagnostic of the endogenous eNOS locus, and a 6.4-kb fragment was diagnostic of the targeted allele. Measurement of iNOS Activity in B16M-F10 Cells—Conversion of l-arginine to l-citrulline was measured by a modification of a previously described methodology (25Rao C.V. Kawamori T. Hamid R. Reddy B.S. Carcinogenesis. 1999; 20: 641-644Crossref PubMed Scopus (173) Google Scholar). The assay was carried out by adding 100 μg of sample protein to 150 μl of assay buffer (50 mm HEPES, 1 mm dithiothreitol, 1 mm MgCl2, 5 mg/liter pepstatin A, 0.1 mm phenylmethylsulfonyl fluoride, and 3 mg/liter aprotinin, pH 7.4) containing 70 μm arginine, 250,000 dpm l-[3H]arginine, (GE Healthcare, Little Chalfont, UK), 2 mm NADPH, 5 μm tetrahydrobiopterin, 5 μm FAD, and 0.5 mm CaCl2 to measure total NOS activity or in the presence of 1 mm EGTA (without calcium) to determine Ca2+-independent iNOS activity. After 30 min at 37 °C, the reaction was stopped with 100 μl of 1 m trichloroacetic acid. The samples were adjusted to pH 4.6 by adding 500 μl of 20 mm HEPES and applied to Dowex AG 50W-X8 resin columns. l-[3H]Citrulline was eluted and separated by thin-layer chromatography. Radioactivity was counted with an AR-2000 scanner detector (Bioscan, Inc., Washington, D. C.). The results are expressed as pmol of l-[3H]citrulline/mg of protein/min. Western Blot Analysis—Cultured cells were harvested as indicated above and then washed twice with ice-cold Krebs-Henseleit bicarbonate buffer, pH 7.4. Whole cell extracts were made by freeze-thaw cycles in buffer containing 150 mm NaCl, 1 mm EDTA, 10 mm Tris-HCl, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin, pH 7.4. Fifty μg of protein (as determined by the Bradford assay (68Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211864) Google Scholar)) were boiled in Laemmli buffer and resolved by 12.5% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and subjected to Western blotting with anti-cAMP-response element-binding protein (CREB) or anti-phospho-CREB (Ser133) monoclonal antibody (Chemicon International Inc., Temecula, CA). Blots were developed using horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (ECL system, Amersham Biosciences). Gene Silencing—Small interfering RNA (siRNA) transfection experiments were performed with double-stranded RNA designed and synthesized by Eurogentec (Seraing, Belgium). A CREB-specific siRNA sense orientation strand (5′-UACAGCUGGCUAACAAUGGdTdT-3′) was used. Cells were transfected with the siRNA delivery reagent jetSI™ (Eurogentec) at 3μl/μgof siRNA according to the manufacturer's instructions. Transfection efficiency in cells plated on coverslips was determined with fluorescein isothiocyanate-labeled siRNA and determined by cell counting using a TCS-SP2 confocal microscope (Leica Microsystems, Bensheim, Germany) to be 90–95% after 24 and 48 h. Guanylate Cyclase Assay—Enzyme activity (soluble and particulate) was determined as described previously (26Ignarro L.J. Wood K.S. Wolin M.S. Proc. Natl. Acad. Sci. IJ. S. A. 1982; 79: 2870-2873Crossref PubMed Scopus (195) Google Scholar, 27Waldman S.A. Sinacore M.S. Lewicki J.A. Chang L.Y. Murad F. J. Biol. Chem. 1984; 259: 4038-4042Abstract Full Text PDF PubMed Google Scholar). Briefly, the reaction mixture contained 7.5 pmol of Tris-HCl, pH 7.6, 0.75 μmol of creatine phosphate, 0.5 μmol of cGMP, 0.45 μmol of MgCl2, 1.2 μmol of theophylline, 0.6 units of creatine kinase, and 37.5 nmol of [8-3H]GTP (10–12 mCi/mmol; PerkinElmer Life Sciences) in a total reaction volume of 150 μl. The radioactive cGMP produced was isolated as described previously (28Deguchi T. Arnano E. Nakane M. J. Neurochem. 1976; 27: 1027-1034Crossref PubMed Scopus (30) Google Scholar) and measured using a Packard Tri-Carb 2700TR Varisette analyzer (PerkinElmer Life Sciences). Assay of Sphingomyelinases—This procedure was a modification of a previously reported methodology (29Takeda Y. Tashima M. Takahashi A. Uchiyama T. Okazaki T. J. Biol. Chem. 1999; 274: 10654-10660Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). B16M-F10 cells (5 × 106) were separated from the co-cultured HSE as described previously (30Ortega A.L. Carretero J. Obrador E. Gambini J. Asensi M. Rodilla V. Estrela J.M. J. Biol. Chem. 2003; 278: 13888-13897Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), washed twice with ice-cold phosphate-buffered saline, and homogenized in 0.4 ml of lysis buffer (10 mm HEPES/KOH, pH 7.4, 2 mm EDTA, 0.1% CHAPS, 5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 100 μm pepstatin, 0.15 units/ml aprotinin, and 50 mg/ml leupeptin) containing 0.1% Triton X-100. The homogenate was passed through a 25-gauge needle and then centrifuged at 100,000 × g for 1 h at 4°C. The supernatant was used as an enzyme source. The mixture used to assay magnesium-dependent neutral sphingomyelinase (SMase) contained 0.1 m Tris-HCl, pH 7.5, 50 nmol of [N-methyl-14C]sphingomyelin (specific activity, 55 mCi/mmol; GE Healthcare), 10 mm MgCl2, 0.1% Triton X-100, and 200 μg of protein in a final volume of 0.2 ml. For magnesium-independent neutral SMase, MgCl2 was removed from the reaction mixture. For acid SMase, 0.1 m sodium acetate, pH 5.0, was used instead of Tris-HCl. Incubation was carried out at 37 °C for 30 min. The reaction was stopped by adding 1.25 ml of chloroform/methanol (2:1). Then, 0.25 ml of double-distilled water were added to the tubes and vortexed. The tubes were centrifuged at 1000 × g for 6 min to separate the two phases. The clear upper phase was removed, placed in a glass scintillation vial, and counted with a scintillation counter (Packard Tri-Carb 2700TR Varisette). C16-ceramide and C16-dihydroceramide Analysis—The amounts of ceramide and dihydroceramide in B16M-F10 cells were measured as described previously (31Soeda S. Iwata K. Hosoda Y. Shimeno H. Biochim. Biophys. Acta. 2001; 1538: 234-241Crossref PubMed Scopus (12) Google Scholar). Briefly, cancer cell suspensions (5 × 106 cells in 200 μl of DMEM) were vigorously mixed with 4 ml of chloroform/methanol (2:1, v/v) for 20 min. Then, 0.8 ml of distilled water were added to the mixture, and the sample was vortexed and centrifuged. The lower layer was collected, and the chloroform was allowed to evaporate. The residue was dissolved in a solvent and subjected to liquid chromatography-mass spectrometry analysis using a Quattro micro triple-quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with a Shimadzu LC-10ADVP pump and SCL-10AVP controller system with an SIL-10ADVP autoinjector. Samples were analyzed by reverse-phase high pressure liquid chromatography using a Phenomenex ODS column (35 × 2 mm) with 5-μm particle size. In all cases, 40 μl were injected onto the column. The temperature of the column was maintained at 25 °C. Mobile phases were as follows: mobile phase A, 5 mm ammonium formate/methanol/tetrahydrofuran (5:2:3, v/v); and mobile phase B, 5 mm ammonium formate/methanol/tetrahydrofuran (1:2:7, v/v) containing 0.01% formic acid. Elution was carried out at a flow rate of 0.2 ml/min with 70% mobile phase A and 100% mobile phase B for 6.3 min in a linear gradient mode. Cell Death Analysis—Apoptotic and necrotic cell death were distinguished by fluorescence microscopy (32Obrador E. Carretero J. Esteve J.M. Pellicer J.A. Pascual A. Petschen I. Estrela J.M. Free Radic. Biol. Med. 2001; 31: 642-650Crossref PubMed Scopus (37) Google Scholar). For this purpose, isolated cells were incubated with Hoechst 33342 (10 μm; which stains all nuclei) and propidium iodide (10 μm; which stains nuclei of cells with disrupted plasma membranes) for 3 min and analyzed using a Nikon Diaphot 300 fluorescence microscope with excitation at 360 nm. Nuclei of viable, necrotic, and apoptotic cells were observed as blue round nuclei, pink round nuclei, and fragmented blue or pink nuclei, respectively. About 1000 cells were counted each time. DNA strand breaks in apoptotic cells were assayed using a direct terminal transferase dUTP nick end labeling assay (Roche Applied Science) and fluorescence microscopy following the manufacturer's methodology. Assay for in Vitro Invasion of the Hepatic Endothelial Cell Monolayer by B16M-F10 Cells—Invasion of the endothelial cell monolayer by B16M-F10 cells was assayed following the method by Ohigashi et al. (33Ohigashi H. Shinkai K. Mukai M. Ishikawa O. Imaoka S. Iwanaga T. Acedo H. Jpn. J. Cancer Res. 1989; 80: 818-821Crossref PubMed Scopus (30) Google Scholar) with some modificatio

Referência(s)