Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Suppression of NF-κB Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL

2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês

10.1074/jbc.m306111200

ISSN

1083-351X

Autores

Mamta Chawla‐Sarkar, Joseph A. Bauer, Joseph A. Lupica, Bei Morrison, Zhuo Tang, Rhonda K. Oates, Alexandru Almasan, Joseph A. DiDonato, Ernest C. Borden, Daniel J. Lindner,

Tópico(s)

Folate and B Vitamins Research

Resumo

We have previously demonstrated the anti-tumor activity of nitrosylcobalamin (NO-Cbl), an analog of vitamin B12 that delivers nitric oxide (NO) and increases the expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. The specific aim of this study was to examine whether NO-Cbl could sensitize drug-resistant melanomas to Apo2L/TRAIL. Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by TUNEL and confirmed by examining levels and activity of key mediators of apoptosis. The activation status of NF-κB was established by assaying DNA binding, luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity. NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. Treatment with NO-Cbl followed by Apo2L/TRAIL induced apoptosis in Apo2L/TRAIL-resistant tumor cells, characterized by cleavage of caspase-3, caspase-8, and PARP. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα and inhibition of NF-κB DNA binding activity. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. XIAP, an inhibitor of apoptosis, was inactivated by NO-Cbl. NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of NO-Cbl and Apo2L/TRAIL capitalizes on the tumor-specific properties of both agents and represents a promising anti-cancer combination. We have previously demonstrated the anti-tumor activity of nitrosylcobalamin (NO-Cbl), an analog of vitamin B12 that delivers nitric oxide (NO) and increases the expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. The specific aim of this study was to examine whether NO-Cbl could sensitize drug-resistant melanomas to Apo2L/TRAIL. Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by TUNEL and confirmed by examining levels and activity of key mediators of apoptosis. The activation status of NF-κB was established by assaying DNA binding, luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity. NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. Treatment with NO-Cbl followed by Apo2L/TRAIL induced apoptosis in Apo2L/TRAIL-resistant tumor cells, characterized by cleavage of caspase-3, caspase-8, and PARP. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα and inhibition of NF-κB DNA binding activity. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. XIAP, an inhibitor of apoptosis, was inactivated by NO-Cbl. NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of NO-Cbl and Apo2L/TRAIL capitalizes on the tumor-specific properties of both agents and represents a promising anti-cancer combination. Withdrawal: Suppression of NF-κB survival signaling by nitrosylcobalamin sensitizes neoplasms to the anti-tumor effects of Apo2L/TRAIL.Journal of Biological ChemistryVol. 294Issue 48PreviewVOLUME 278 (2003) PAGES 39461–39469 Full-Text PDF Open Access Apoptosis is the rigorously controlled process of programmed cell death. Current trends in cancer drug design focus on selective targeting to activate the apoptotic signaling pathways within tumors while sparing normal cells (1Reed J.C. Cancer Cell. 2003; 3: 17-22Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). The tumor specific properties of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) 1The abbreviations used are: Apo2L/TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; GTN, glycerol trinitrate; IKK, inhibitor κB kinase, NF-κB nuclear factor κB; NO-Cbl, nitrosylcobalamin; NOC-18 (DETA NONOate, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; PARP, poly (ADP-ribose) polymerase; SNAP, S-nitroso-N-acetyl-md,l-penicillamine; SNP, sodium nitroprusside; SRB, sulforhodamine B; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling; XIAP, X-linked inhibitor of apoptosis. have been widely reported (2Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2664) Google Scholar, 3Pitti R.M. Marsters S.A. Ruppert S. Donahue C.J. Moore A. Ashkenazi A. J. Biol. Chem. 1996; 271: 12687-12690Abstract Full Text Full Text PDF PubMed Scopus (1655) Google Scholar, 4Walczak H. Miller R.E. Ariail K. Gliniak B. Griffith T.S. Kubin M. Chin W. Jones J. Woodward A. Le T. Smith C. Smolak P. Goodwin R.G. Rauch C.T. Schuh J.C. Lynch D.H. Nat. Med. 1999; 5: 157-163Crossref PubMed Scopus (2236) Google Scholar, 5Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1111) Google Scholar). Apo2L/TRAIL has been used as an anti-cancer agent alone and in combination with other agents (6Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. McMurtrey A.E. Hebert A. DeForge L. Koumenis I.L. Lewis D. Harris L. Bussiere J. Koeppen H. Shahrokh Z. Schwall R.H. J. Clin. Investig. 1999; 104: 155-162Crossref PubMed Scopus (2008) Google Scholar, 7Frese S. Brunner T. Gugger M. Uduehi A. Schmid R.A. J. Thoracic & Cardiovasc. Surg. 2002; 123: 168-174Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 8Gliniak B. Le T. Cancer Res. 1999; 59: 6153-6158PubMed Google Scholar, 9Mizutani Y. Nakanishi H. Yoshida O. Fukushima M. Bonavida B. Miki T. Eur. J. Cancer. 2002; 38: 167-176Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 10Yamanaka T. Shiraki K. Sugimoto K. Ito T. Fujikawa K. Ito M. Takase K. Moriyama M. Nakano T. Suzuki A. Hepatology. 2000; 32: 482-490Crossref PubMed Scopus (193) Google Scholar) including ionizing radiation (11Chinnaiyan A.M. Prasad U. Shankar S. Hamstra D.A. Shanaiah M. Chenevert T.L. Ross B.D. Rehemtulla A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1754-1759Crossref PubMed Scopus (488) Google Scholar, 12Kim M.R. Lee J.Y. Park M.T. Chun Y.J. Jang Y.J. Kang C.M. Kim H.S. Cho C.K. Lee Y.S. Jeong H.Y. Lee S.J. FEBS Lett. 2001; 505: 179-184Crossref PubMed Scopus (47) Google Scholar, 13Di Pietro R. Secchiero P. Rana R. Gibellini D. Visani G. Bemis K. Zamai L. Miscia S. Zauli G. Blood. 2001; 97: 2596-2603Crossref PubMed Scopus (95) Google Scholar). Apo2L/TRAIL can initiate apoptosis in cells that overexpress the survival factors Bcl-2 and Bcl-XL, and may represent a treatment strategy for tumors that have acquired resistance to chemotherapeutic drugs (14Walczak H. Bouchon A. Stahl H. Krammer P.H. Cancer Res. 2000; 60: 3051-3057PubMed Google Scholar). Apo2L/TRAIL binds its cognate receptors and activates the caspase cascade utilizing adapter molecules such as FADD (5Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1111) Google Scholar, 15Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1562) Google Scholar). TRAIL receptors, type II membrane-bound proteins, are members of the tumor necrosis factor (TNF) superfamily of receptors (2Wiley S.R. Schooley K. Smolak P.J. Din W.S. Huang C.P. Nicholl J.K. Sutherland G.R. Smith T.D. Rauch C. Smith C.A. Immunity. 1995; 3: 673-682Abstract Full Text PDF PubMed Scopus (2664) Google Scholar). To date, five Apo2L/TRAIL receptors have been identified (5Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1111) Google Scholar). Two receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) mediate apoptotic signaling, and three non-functional receptors, DcR1, DcR2, and osteoprotegerin (OPG) may act as decoy receptors (5Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1111) Google Scholar). Agents that increase expression of DR4 and DR5 may exhibit synergistic anti-tumor activity when combined with Apo2L/TRAIL (16Gibson S.B. Oyer R. Spalding A.C. Anderson S.M. Johnson G.L. Mol. Cell. Biol. 2000; 20: 205-212Crossref PubMed Scopus (230) Google Scholar). Recently we demonstrated the anti-tumor effects of nitrosylcobalamin (NO-Cbl), an analogue of vitamin B12 (cobalamin, Cbl) coordinated with nitric oxide (NO) as a ligand (17Bauer J.A. Morrison B.H. Grane R.W. Jacobs B.S. Dabney S. Gamero A.M. Carnevale K.A. Smith D.J. Drazba J. Seetharam B. Lindner D.J. J. Natl. Cancer Inst. 2002; 94: 1010-1019Crossref PubMed Google Scholar). Anti-tumor activity correlated with the expression of the transcobalamin II receptor (TCII-R) on the plasma membrane of tumor cells. NO-Cbl is an ideal candidate to be used in combination with Apo2L/TRAIL because NO-Cbl induced the mRNAs of DR4, DR5, and Apo2L/TRAIL in ovarian carcinoma cells (17Bauer J.A. Morrison B.H. Grane R.W. Jacobs B.S. Dabney S. Gamero A.M. Carnevale K.A. Smith D.J. Drazba J. Seetharam B. Lindner D.J. J. Natl. Cancer Inst. 2002; 94: 1010-1019Crossref PubMed Google Scholar). Treatment of leukemia cells with Apo2L/TRAIL resulted in increased Apo2L/TRAIL mRNA and protein, suggesting autocrine regulation that can function in a positive feedback loop (18Herr I. Posovszky C. Di Marzio L.D. Cifone M.G. Boehler T. Debatin K.M. Oncogene. 2000; 19: 4255-4262Crossref PubMed Scopus (39) Google Scholar). Transfecting ovarian carcinoma cells with a non-functional, dominant negative DR5 receptor (DR5Δ) (19Gong B. Almasan A. Cancer Res. 2000; 60: 5754-5760PubMed Google Scholar) abrogated increases in DR4, DR5, and Apo2L/TRAIL when treated with NO-Cbl. 2J. Bauer, R. Grane, and D. Lindner, unpublished results. This suggested that a functional Apo2L/TRAIL receptor was necessary for the autoinduction of Apo2L/TRAIL, and that DR5Δ interfered with positive feedback signaling. Cytokines of the TNF superfamily, upon receptor ligation, simultaneously induce an apoptotic signal (mediated via caspase-8) in addition to a survival signal (mediated by activation of NF-κB) (20Bharti A.C. Aggarwal B.B. Biochem. Pharmacol. 2002; 64: 883-888Crossref PubMed Scopus (473) Google Scholar). NF-κB is a transcription factor that generally functions to suppress apoptosis (20Bharti A.C. Aggarwal B.B. Biochem. Pharmacol. 2002; 64: 883-888Crossref PubMed Scopus (473) Google Scholar, 21Bours V. Bentires-Alj M. Hellin A.C. Viatour P. Robe P. Delhalle S. Benoit V. Merville M.P. Biochem. Pharmacol. 2000; 60: 1085-1089Crossref PubMed Scopus (204) Google Scholar). Binding of TNF-α (22DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1917) Google Scholar) or Apo2L/TRAIL (23Baetu T.M. Kwon H. Sharma S. Grandvaux N. Hiscott J. J. Immunol. 2001; 167: 3164-3173Crossref PubMed Scopus (98) Google Scholar) to their cognate receptors results in the phosphorylation and activation of the inhibitor of κB-kinase (IKK). In its quiescent state, NF-κB is complexed to the inhibitor of κB (IκB) in the cytoplasm (24DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar). Activated IKK phosphorylates serine 32 and serine 36 of IκB (25DiDonato J. Mercurio F. Rosette C. Wu-Li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). Once phosphorylated, IκB is ubiquitinated and targeted for proteolysis as it remains complexed to NF-κB (24DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar). Within the proteosome IκB is degraded, while NF-κB is not, allowing NF-κB to translocate to the nucleus where it binds to NF-κB response elements, which activate transcription of target genes (22DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1917) Google Scholar, 24DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar). NF-κB stimulates transcription of genes such as Bcl-XL and cIAP that function as survival factors (26Chen C. Edelstein L.C. Gelinas C. Mol. Cell. Biol. 2000; 20: 2687-2695Crossref PubMed Scopus (698) Google Scholar, 27LaCasse E.C. Baird S. Korneluk R.G. MacKenzie A.E. Oncogene. 1998; 17: 3247-3259Crossref PubMed Scopus (947) Google Scholar). Therefore, agents that inhibit NF-κB may have anti-tumor activity. NO is a ubiquitous, multifaceted signaling molecule (28Gross S.S. Wolin M.S. Annu. Rev. Physiol. 1995; 57: 737-769Crossref PubMed Scopus (821) Google Scholar, 29Anggard E. Lancet. 1994; 343: 1199-1206Abstract PubMed Scopus (769) Google Scholar) that inhibits NF-κB DNA binding activity (30DelaTorre A. Schroeder R.A. Kuo P.C. Biochem. Biophys. Res. Commun. 1997; 238: 703-706Crossref PubMed Scopus (110) Google Scholar, 31Matthews J.R. Botting C.H. Panico M. Morris H.R. Hay R.T. Nucleic Acids Res. 1996; 24: 2236-2242Crossref PubMed Scopus (463) Google Scholar) and suppresses the cell survival function of NF-κB (32Kang J.L. Park W. Pack I.S. Lee H.S. Kim M.J. Lim C.M. Koh Y. J. Appl. Physiol. 2002; 92: 795-801Crossref PubMed Scopus (70) Google Scholar, 33D'Acquisto F. Maiuri M.C. de Cristofaro F. Carnuccio R. Naunyn-Schmiedebergs Arch. Pharmacol. 2001; 364: 157-165Crossref PubMed Scopus (25) Google Scholar). Sulfasalazine, an anti-inflammatory agent, inhibits NF-κB activity, enhancing Apo2L/TRAIL-induced apoptosis in human leukemia cells (34Goke R. Goke A. Goke B. Chen Y. Cell. Immunol. 2000; 201: 77-82Crossref PubMed Scopus (59) Google Scholar). Furthermore, Apo2L/TRAIL-induced apoptosis was increased in prostate carcinoma cells that were infected with a mutant IκB, supporting the role of NF-κB as a TRAIL-induced survival factor (35Eid M.A. Lewis R.W. Abdel-Mageed A.B. Kumar M.V. Int. J. Oncol. 2002; 21: 111-117PubMed Google Scholar). The use of NO-Cbl to deliver nitric oxide and suppress the survival arm of NF-κB is a promising strategy to enhance the anti-tumor effects of Apo2L/TRAIL in resistant tumors. In this study we pretreated cells with NO-Cbl to inhibit NF-κB activity and enhance the apoptotic signal of Apo2L/TRAIL. Our specific aims were to: 1) measure the anti-tumor effects of NO-Cbl and Apo2L/TRAIL in Apo2L/TRAIL-resistant cell lines, and to 2) determine the mechanism by which NO-Cbl inhibits NF-κB activation. Synthesis of Nitrosylcobalamin—Nitrosylcobalamin was synthesized as previously described (17Bauer J.A. Morrison B.H. Grane R.W. Jacobs B.S. Dabney S. Gamero A.M. Carnevale K.A. Smith D.J. Drazba J. Seetharam B. Lindner D.J. J. Natl. Cancer Inst. 2002; 94: 1010-1019Crossref PubMed Google Scholar, 36Bauer J.A. Anticancer Drugs. 1998; 9: 239-244Crossref PubMed Scopus (31) Google Scholar). Hydroxocobalamin (vitamin B12a) acetate (George Uhe Company, Paramus, NJ) was dissolved in dichloromethane (Burdick and Jackson, Muskegon, MI) and exposed to CP grade NO gas (Praxair, Wickliff, OH) at 150 psi. The reaction proceeds in a closed system within a high-pressure gas cylinder (Praxair, Cleveland, OH). The system was nitrogen-purged daily and evacuated prior to NO exposure. The NO gas was scrubbed prior to entering the system using a stainless steel cylinder (Swagelok, Abbott valve and fitting, Solon, OH) containing NaOH pellets. The solid NO-Cbl product was collected following rotary evaporation of the solvent and stored at –80 °C prior to use. Cell Culture and Cytokine Treatments—Human melanoma tumor cell lines, WM9 and WM3211 (Wistar Institute, Philadelphia, PA), and A375(ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with heat-inactivated 10% fetal bovine serum (FBS; HyClone, Logan, UT) and 1% Antibiotic-Antimycotic (Invitrogen). Cells were maintained in 5% CO2 at 37 °C in a humidified tissue culture incubator. Primary non-tumorigenic melanoma cell lines (DMN-1 and CMN-1, A. Gudkov, CCF, Cleveland, OH), and human foreskin fibroblasts (HFF; CCF, Cleveland, OH) were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum. Cells were confirmed as mycoplasma free. All experiments were performed using trimeric recombinant human Apo2L/TRAIL (37Lawrence D. Shahrokh Z. Marsters S. Achilles K. Shih D. Mounho B. Hillan K. Totpal K. DeForge L. Schow P. Hooley J. Sherwood S. Pai R. Leung S. Khan L. Gliniak B. Bussiere J. Smith C.A. Strom S.S. Kelley S. Fox J.A. Thomas D. Ashkenazi A. Nat. Med. 2001; 7: 383-385Crossref PubMed Scopus (636) Google Scholar) (Genentech Inc., San Francisco, CA) and were independently confirmed using recombinant Apo2L/TRAIL from another source (Peprotech Inc, NJ). Apo2L/TRAIL (Genentech Inc.), consisted of >99% trimeric protein with Zn+2, which is necessary for optimal biologic activity of Apo2L/TRAIL (37Lawrence D. Shahrokh Z. Marsters S. Achilles K. Shih D. Mounho B. Hillan K. Totpal K. DeForge L. Schow P. Hooley J. Sherwood S. Pai R. Leung S. Khan L. Gliniak B. Bussiere J. Smith C.A. Strom S.S. Kelley S. Fox J.A. Thomas D. Ashkenazi A. Nat. Med. 2001; 7: 383-385Crossref PubMed Scopus (636) Google Scholar). Sulforhodamine B Cell Growth Assay—Cells were harvested with 0.5% trypsin/0.53 mm EDTA, washed with phosphate-buffered saline and resuspended in media containing 10% fetal bovine serum. Cells were plated in 96-well plates in 0.2-ml aliquots containing 10,000 cells. Cells were allowed to adhere to the plate for 4 h and then NO-Cbl was added in different dilutions (25, 50 and 100 μm) to the assay plate. Replicates of four were performed for each treatment. After 16 h, recombinant human Apo2L/TRAIL was added at different concentrations (25–100 ng/ml). Growth was monitored by the sulforhodamine B (SRB; Sigma Chemical) colorimetric assay (38Skehan P. Storeng R. Scudiero D. Monks A. McMahon J. Vistica D. Warren J.T. Bokesch H. Kenney S. Boyd M.R. J. Natl. Cancer Inst. 1990; 82: 1107-1112Crossref PubMed Scopus (8938) Google Scholar). After 40 h growth, the medium was removed, and the cells were fixed with 10% trichloroacetic acid and stained with SRB. Bound dye was eluted from the cells with 10 mm Tris-HCl (pH 10.5) and absorbance was measured at 570 nm using a Lab systems Multiskan RC 96-well plate reader (Lab Systems Multiscan RC, Thermo Lab Systems, Franklin, MA). To quantify the growth of the cells, the experimental absorbance values (A exp) were compared with initial absorbance readings representing the starting cell numbers (A ini). To determine the starting cell number, an additional 96-well plate was seeded with cells and fixed at the beginning of the experiment. The absorbances derived from the initial plate and from the untreated cells at the end of the growth period (A fin) were defined as 0 and 100% growth, respectively. The percentage control growth (100% × [A exp – A ini]/[A fin – A ini]) is expressed as a percentage of untreated controls. In Vivo Experiments—The Institutional Animal Care and Use Committee at the Cleveland Clinic Foundation approved all procedures for animal experimentation. 5-week-old NCR male athymic nude homozygous (nu/nu) mice (Taconic, Germantown, NY) were inoculated with A375 tumors. There were four experimental groups (untreated, single agents, and the combination) n = 8. Cultured tumor cells (4 × 106) were inoculated into flanks in the mid-axillary line. NO-Cbl was given twice daily (50 mg/kg s.c.) and recombinant trimeric Apo2L/TRAIL (50 mg/kg s.c.) (37Lawrence D. Shahrokh Z. Marsters S. Achilles K. Shih D. Mounho B. Hillan K. Totpal K. DeForge L. Schow P. Hooley J. Sherwood S. Pai R. Leung S. Khan L. Gliniak B. Bussiere J. Smith C.A. Strom S.S. Kelley S. Fox J.A. Thomas D. Ashkenazi A. Nat. Med. 2001; 7: 383-385Crossref PubMed Scopus (636) Google Scholar) was administered every other day, starting on day 2. Tumor volume was measured three times a week using the formula for a prolate spheroid: (4/3) πab2 where 2a = major axis, 2b = minor axis. Formalin-fixed sections were processed by the Cleveland Clinic Histology Core. Sections were stained with hematoxylin and eosin and evaluated for pathologic changes in a blinded fashion. TUNEL Assay—A375 cells were cultured for 36 h and exposed to various treatments (control, NO-Cbl, Apo2L/TRAIL, and NO-Cbl + Apo2L/TRAIL). Apoptotic cells were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling) staining using a commercially available kit (APO-BRDU kit, BD PharMingen, San Diego, CA). Cells were processed according to the manufacturer's recommended protocol. The percentage of fluorescein isothiocyanate-positive cells was analyzed by fluorescent-activated cell scanning (FACS, Becton Dickinson, Facsvantage, San Diego, CA). Gel Electrophoresis and Immunoblot Analyses—Whole cell lysates were prepared in 1× lysis buffer (50 mm Tris-Cl, pH 8.0, 1% Triton X-100, 10% glycerol, 1 mm EDTA, 250 mm NaCl, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin) for subsequent immunoblotting studies. SDS-PAGE was conducted by using the Laemmli buffer system and 12% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes by the semidry method (Trans Blot S.D., BioRad, Hercules, CA). Binding of the primary and secondary antibodies was performed according to standard protocols (39Chawla-Sarkar M. Leaman D.W. Jacobs B.S. Borden E.C. J. Immunol. 2002; 169: 847-855Crossref PubMed Scopus (80) Google Scholar). Membranes were immunoblotted with pAb to caspase-3, caspase-8, XIAP (BD PharMingen), PARP (BioMOL), FLIP (Calbiochem), pIκBα, IκBα (Cell Signaling), cIAP-1, anti-IKKα/β (Santa Cruz Biotechnology) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Pierce). Immunoreactive bands were visualized by using enhanced chemiluminescence (PerkinElmer). Equal protein loading was confirmed by reprobing with monoclonal anti-actin antibody (Sigma Chemical Co.). All immunoblots in this study were repeated 3 times with reproducible results. Electrophoretic Mobility Shift Assay (EMSA)—A375 cells were treated with NO donors (NO-Cbl, NOC-18, SNAP, 100 μm, 16 h), or with Apo2L/TRAIL (100 ng/ml) or TNF-α (20 ng/ml) for 15 min and 1 h, or with NO donors (16 h) followed by Apo2L/TRAIL or TNF-α (15 min and 1 h). Plates were washed twice with ice-cold phosphate-buffered saline. Cells were resuspended in cold 1× lysis buffer (20 mm HEPES, 20 mm NaF, 1 mm Na3VO4, 1 mm EDTA, 1 mm dithiothreitol, 100 mm NaCl, 10% glycerol, and protease inhibitors) as previously described (40Li X. Commane M. Nie H. Hua X. Chatterjee-Kishore M. Wald D. Haag M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10489-10493Crossref PubMed Scopus (142) Google Scholar) and incubated on ice for 30 min followed by centrifugation at 4 °C at 10,000 rpm for 10 min. Supernatants were transferred to fresh tubes and protein concentrations were assessed using the Bradford method (BioRAD protein assay, BioRad). The NF-κB consensus binding sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′) from the IFN-β gene promoter was end-labeled with [γ-32P]dATP (3000 Ci/mol) using T4 polynucleotide kinase. DNA binding reactions were performed in 20 μl reaction volumes for 20 min at 25 °C containing 10 μg of protein, 20 mm HEPES, 10 mm KCl, 0.1% Nonidet P-40, 0.5 mm dithiothreitol, and 10% glycerol. Complexes were separated from the free probe on 6% nondenaturing polyacrylamide gels in 0.5× TBE buffer at 200 V for 2 h. Gels were dried and exposed to film. To verify the identity of the band observed lysates from A375 cells stimulated for 15 min with TNF-α (20 ng/ml) were incubated with anti-NF-κB p50 or p65 antibodies (Santa Cruz Biotechnology). Dual Luciferase NF-κB Reporter Assay—The NF-κB-luciferase (NF-κB-luc) reporter plasmid, containing a 2xNF-κB response element fused to luciferase, has been previously characterized (41Elewaut D. DiDonato J.A. Kim J.M. Truong F. Eckmann L. Kagnoff M.F. J. Immunol. 1999; 163: 1457-1466PubMed Google Scholar). Renilla luciferase (pRL-TK, Promega, Madison, WI) was co-transfected to normalize for transfection efficiency. A375 cells were co-transfected with 20 μg of NF-κB-luc and 10 μg of pRL-TK using Lipofectamine plus (Invitrogen). After transfection cells were allowed to recover overnight and were plated in 6-well plates. Cells were pretreated with NO-Cbl (100 μm) for 16 h followed by TNF-α (10 ng/ml) or Apo2L/TRAIL (100 ng/ml) for 4 h. Cells were then harvested in 1× passive lysis buffer and luciferase activity was measured according to the manufacturer's protocol (Promega, Madison, WI) using a Wallac 1420 multilabel counter (PerkinElmer). Fold induction of NF-κB-luciferase for each treatment was based on untreated values normalized to the fold induction of pRL-TK reporter values. The assays were performed in triplicate. IκB kinase (IKK) assay—Whole cell extracts (300 μg) were supplemented with 150 μl of Buffer A (20 mm HEPES, pH 7.9, 20 mm β-glycerophosphate, 10 mm NaF, 0.1 mm orthovanadate, 5 mm p-nitrophenyl phosphate (pNPP), 10 mm 2-mercaptoethanol, 0.5 mm phenylmethylsulfonyl fluoride, and protease mixture), 2 μl of normal rabbit serum, and mixed by rotation at 4 °Cfor1has previously described (42DiDonato J.A. Methods Enzymol. 2000; 322: 393-400Crossref PubMed Google Scholar). A 50% slurry of protein G-Sepharose (80 μl) (Amersham Biosciences) prepared in Buffer A (without mercaptoethanol or phenylmethylsulfonyl fluoride) was added and mixed by rotation at 4 °C for 1 h. Protein G-Sepharose was removed by centrifugation at 800 × g for 1 min and discarded. Anti-IKKα monoclonal antibody (0.5 μg, BD PharMingen), or anti-β-actin epitope antibody was added to the supernatant and mixed by rotation at 4 °C for 2 h. A 50% slurry of protein G-Sepharose (60 μl) prepared in Buffer C (Buffer A plus 50 mm NaCl and 10 mm MgCl2, without mercaptoethanol and phenylmethylsulfonyl fluoride) was added and mixed by rotation in the cold for 30 min. Protein G immunopellets were collected by centrifugation at 800 × g for 30 s, washed three times with Buffer B (Buffer A plus 250 mm NaCl), and once with Buffer C (Buffer A plus 50 mm NaCl and 10 mm MgCl2). Immunopellets were resuspended in 30 μl of kinase buffer with 0.1 mm orthovanadate, 50 μm unlabeled ATP, 5 μCi of [γ-32P]ATP, 2 mm dithiothreitol, and 2 μg of recombinant GST-IκBα1–54 (22DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1917) Google Scholar) and incubated at 30 °C for 30 min. Reactions were stopped by the addition of 15 μl of 4× SDS-PAGE loading buffer (200 mm Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 0.2% 2-mercaptoethanol), heated at 95 °C for 10 min, and resolved by SDS-PAGE on a 12% acrylamide gel by standard procedures. Gels were rinsed, stained with Bio-Safe Coomassie (BioRad) to visualize protein bands, rinsed, photographed, then dried and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, New York) to detect substrate phosphorylation. IKK activation was quantified by PhosphorImage analysis on a Storm-840 imager using Image Quant v 4.2 software (Molecular Dynamics, Amersham Biosciences). Statistical Analysis—Median effect analysis was used to characterize the interaction between NO-Cbl and Apo2L/TRAIL (43Chou T.C. Talalay P. Adv. Enzyme Regulat. 1984; 22: 27-55Crossref PubMed Scopus (6007) Google Scholar). A combination index (CI) >1 indicates antagonism, CI = 1 indicates additivity, and CI <1 indicates synergy. Differences in mean tumor volume between groups were compared using the unpaired two-tailed Student's t test, using a pooled estimator of variance to determine statistical significance. Anti-tumor Effects of NO-Cbl, Apo2L/TRAIL, and the Combination in Vitro and in Vivo—To test our hypothesis that NO-Cbl would enhance the anti-cellular effects of Apo2L/TRAIL against malignant Apo2L/TRAIL-resistant cell lines, we measured the antiproliferative effects of three melanoma lines A375, WM9, and WM3211 (previously shown to be resistant to Apo2L/TRAIL) (39Chawla-Sarkar M. Leaman D.W. Jacobs B.S. Borden E.C. J. Immunol. 2002; 169: 847-855Crossref PubMed Scopus (80) Google Scholar). Three non-malignant human cell lines CMN1 and DMN1 (normal melanocytes) and primary human foreskin fibroblasts (HFF) were examined to demonstrate the tumor-specific effects of NO-Cbl and Apo2L/TRAIL. We used the SRB antiproliferative assay, used by the National Cancer Institute (NCI) to evaluate new chemotherapeutic agents (38Skehan P. Storeng R. Scudiero D. Monks A. McMahon J. Vistica D. Warren J.T. Bokesch H. Kenney S. Boyd M.R. J. Natl. Cancer Inst. 1990; 82: 1107-1112Crossref PubMed Scopus (8938) Google Scholar). Median effect analysis was used to characterize the interaction between NO-Cbl and Apo2L/TRAIL (43Chou T.C. Talalay P. Adv. Enzyme Regulat. 1984; 22: 27-55Crossref PubMed Scopus (6007) Google Scholar). Cells were pretreated with NO-Cbl for 16 h followed by Apo2L/TRAIL for 24 h. Sequential drug treatment resulted in synergistic antiproliferative activity in all three malignant cell lines (Fig.

Referência(s)