Nitric Oxide Disrupts H2O2-dependent Activation of Nuclear Factor κB

Tumor necrosis factor α (TNF-α) exerts its effect by two distinct signaling pathways. It can trigger cytotoxicity in sensitive target cells. TNF-α can also promote nuclear factor κB (NF-κB) activity and regulate the expression of genes that interfere with apoptosis and thus conferring resistance to several apoptotic stimuli. We have observed that interferon-γ (IFN-γ) sensitizes human ovarian carcinoma cell lines to TNF-α-mediated apoptosis and further, IFN-γ induces the expression of the inducible nitric-oxide synthase (iNOS) and the generation of nitric oxide (NO). This study examines the role of NO in the sensitization of the ovarian carcinoma cell line AD10 to TNF-α-mediated cytotoxicity. Treatment of AD10 cells with the NOS inhibitor l-NMA blocked the IFN-γ-dependent sensitization whereas NO donors (S-nitroso-N-acetylpenicillamine) sensitized these cells to TNF-α cytotoxicity. Analysis of the activation status of NF-κB upon treatment with NO donors confirmed the inhibitory role of NO on both the NF-κB DNA-binding property and its activation. Moreover, the inhibition of NF-κB nuclear translocation by NO donors directly correlated with the intracellular concentration of H2O2 and was reversed by the addition of exogenous H2O2. These findings show that NO might interfere with TNF-α-dependent NF-κB activation by interacting with O⨪2 and reducing the generation of H2O2, a potent NF-κB activator. Therefore, NO-mediated disruption of NF-κB activation results in the removal of anti-apoptotic/resistance signals and sensitizes tumor cells to cytotoxic cytokines like TNF-α. Tumor necrosis factor α (TNF-α) exerts its effect by two distinct signaling pathways. It can trigger cytotoxicity in sensitive target cells. TNF-α can also promote nuclear factor κB (NF-κB) activity and regulate the expression of genes that interfere with apoptosis and thus conferring resistance to several apoptotic stimuli. We have observed that interferon-γ (IFN-γ) sensitizes human ovarian carcinoma cell lines to TNF-α-mediated apoptosis and further, IFN-γ induces the expression of the inducible nitric-oxide synthase (iNOS) and the generation of nitric oxide (NO). This study examines the role of NO in the sensitization of the ovarian carcinoma cell line AD10 to TNF-α-mediated cytotoxicity. Treatment of AD10 cells with the NOS inhibitor l-NMA blocked the IFN-γ-dependent sensitization whereas NO donors (S-nitroso-N-acetylpenicillamine) sensitized these cells to TNF-α cytotoxicity. Analysis of the activation status of NF-κB upon treatment with NO donors confirmed the inhibitory role of NO on both the NF-κB DNA-binding property and its activation. Moreover, the inhibition of NF-κB nuclear translocation by NO donors directly correlated with the intracellular concentration of H2O2 and was reversed by the addition of exogenous H2O2. These findings show that NO might interfere with TNF-α-dependent NF-κB activation by interacting with O⨪2 and reducing the generation of H2O2, a potent NF-κB activator. Therefore, NO-mediated disruption of NF-κB activation results in the removal of anti-apoptotic/resistance signals and sensitizes tumor cells to cytotoxic cytokines like TNF-α. nuclear factor κB interferon γ inducible nitric-oxide synthase S-nitroso-N-acetylpenicillamine N G-monomethyl-l-arginine tumor necrosis factor lactate dehydrogenase reverse transcription polymerase chain reaction pyrrolidine dithiocarbamate glyceraldehyde-3-phosphate dehydrogenase electrophoretic mobility shift assay tumor necrosis factor receptor fetal bovine serum reactive oxygen species 2′,7′-dichlorofluorescein diacetate. The development of resistance to either the immune system or chemo-immunotherapeutic strategies remains a disadvantage in the therapy of cancer, particularly in cases where recurrences and/or relapses occurred. Apoptosis has been accepted as a distinct pathological mechanism in tumors responding to anticancer therapies. Further, resistance to apoptosis in tumor cells has been recognized as a common pathway to multiple drug resistance (1Dive C. Hickman J.A. Br. J. Cancer. 1991; 64: 192-196Crossref PubMed Scopus (413) Google Scholar, 2Eastman A. Cancer Cells. 1990; 2: 275-280PubMed Google Scholar). Multiple lines of evidence have implicated the activation of the transcription factor NF-κB1 as one of the primary signals in the onset of resistance to many apoptotic stimuli, particularly TNF-α (3Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2509) Google Scholar, 4Wang C.Y. Cusack Jr., J.C. Liu R. Baldwin Jr., A.S. Nat. Med. 1999; 5: 412-417Crossref PubMed Scopus (0) Google Scholar, 5Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2933) Google Scholar). TNF-α is a proinflammatory cytokine that exerts a broad spectrum of biological effects by its interaction with two distinct cell surface receptors, TNFR1 and TNFR2 (6Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (1002) Google Scholar). Most cytotoxic effects of TNF-α are mediated by the TNFR1. It has been demonstrated that, upon interaction with TNF-α, trimerization of TNFR1 takes place and results in cellular signaling leading to the recruitment of the TNFR1-associated death domain protein and the receptor-interacting protein to the receptor complex (7Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1732) Google Scholar). The TNFR1-associated death domain protein interacts with the Fas-associated death domain to initiate the death pathway and engages several proteins such as the TNFR-associated factor-1, the TNFR-associated factor-2, and receptor-interacting protein to initiate the TNF signaling pathways such as the activation of NF-κB (8Wang C.Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2573) Google Scholar). Reactive oxygen species (ROS) have also been implicated in the signaling pathways initiated by TNF-α. Stimulation of mammalian cells with TNF-α triggers the generation of various ROS (9Garcia-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar, 10Hennet T. Richter C. Peterhans E. Biochem. J. 1993; 289: 587-592Crossref PubMed Scopus (261) Google Scholar). Hence, the use of antioxidants results in the inhibition of various TNF-α-related effects such as the activation of transcription factors, gene expression, and cytotoxicity. In addition, the use of exogenous ROS mimics the biological activity of TNF-α (11Suzuki Y.J. Forman H.J. Sevanian A. Free Radical Biol. Med. 1997; 22: 269-285Crossref PubMed Scopus (1259) Google Scholar). These data support the hypothesis that ROS function as second messengers for TNF-α-mediated signaling. In biological systems the most important ROS generated upon TNF-α stimulation are the result of enzymatic partial reduction of oxygen yielding superoxide (O⨪2), which is either immediately reduced by superoxide dismutase to hydrogen peroxide (H2O2) or alternatively reacts rapidly with nitric oxide (NO) to generate ONOO− (12Huie R.E. Padmaja S. Free Radical Res. Commun. 1993; 18: 195-199Crossref PubMed Scopus (2015) Google Scholar, 13Szabo C. Ohshima H. Nitric Oxide. 1997; 1: 373-385Crossref PubMed Scopus (392) Google Scholar, 14Fukuto J.M. Adv. Pharmacol. 1995; 34: 1-15Crossref PubMed Scopus (60) Google Scholar). However, the regulatory role of NO in TNF-α signaling via the disruption of ROS-dependent activation of NF-κB has not been established. Several lines of evidence showed that resistant tumors could be sensitized to TNF-α-mediated cytotoxicity by various cytokines or pharmacological treatments (15Han S.Y. Choung S.Y. Paik I.S. Kang H.J. Choi Y.H. Kim S.J. Lee M.O. Biol. Pharm. Bull. 2000; 23: 420-426Crossref PubMed Scopus (21) Google Scholar, 16Mizutani Y. Bonavida B. Nio Y. Yoshida O. J. Urol. 1994; 151: 1697-1702Crossref PubMed Scopus (27) Google Scholar, 17Safrit J.T. Berek J.S. Bonavida B. Gynecol. Oncol. 1993; 48: 214-220Abstract Full Text PDF PubMed Scopus (13) Google Scholar, 18Safrit J.T. Belldegrun A. Bonavida B. J. Urol. 1993; 149: 1202-1208Crossref PubMed Scopus (14) Google Scholar, 19Morimoto H. Yonehara S. Bonavida B. Cancer Res. 1993; 53: 2591-2596PubMed Google Scholar, 20Frost P.J. Belldegrun A. Bonavida B. Prostate. 1999; 41: 20-30Crossref PubMed Scopus (22) Google Scholar). Recently, we have reported that IFN-γ induced the sensitization of the human ovarian carcinoma AD10 cell line to Fas-mediated apo ptosis and the sensitization was due in part to the generation of nitric oxide by the induction of iNOS in these cells (21Garban H.J. Bonavida B. Gynecol. Oncol. 1999; 73: 257-264Abstract Full Text PDF PubMed Scopus (82) Google Scholar). NO has been identified as a potential second messenger based on its ability to chemically interact with a broad range of regulatory proteins. Furthermore, NO can interact with metal cluster- and thiol-containing proteins (for review, see Ref. 22Stamler J.S. Curr. Top Microbiol. Immunol. 1995; 196: 19-36Crossref PubMed Scopus (196) Google Scholar) resulting in the modification of both the structures and functions of these proteins. Although NO has been shown to react very rapidly with O⨪2, the only biological effect to this chemical reaction has been assigned to the generation of ONOO−, a proposed cytotoxic derivative (23Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6698) Google Scholar, 24Beckman J.S. J. Dev. Physiol. 1991; 15: 53-59PubMed Google Scholar). Herein, we hypothesize that NO is interfering with the TNF-α-mediated signaling by chemically reacting with O⨪2. Since can serve as a precursor to H2O2, which is a proposed activator of the anti-apoptotic transcription factor NF-κB, the reaction of O⨪2 with NO will interfere with the activation of NF-κB and will result in the removal of anti-apoptotic signals and sensitization of the tumor cells to TNF-α cytotoxicity. This study has been designed to test this hypothesis, and the following have been examined: (a) the molecular mechanism by which IFN-γ sensitizes the human ovarian carcinoma cell line to TNF-α-induced cytotoxicity, (b) the specific role of NO in the disruption of TNF-α-mediated generation of H2O2, and, subsequently, (c) the mechanism by which NO can disrupt the TNF-α-dependent NF-κB activation. The AD10 cell line is an adriamycin-resistant, MDR phenotype-expressing subline derived from the human ovarian carcinoma cell line A2780 and was obtained from Dr. Ozols (Fox Chase Cancer Center, Philadelphia, PA). The PC-3 cell line is a metastatic bone-derived human prostatic adenocarcinoma, CRL-1435, obtained from ATCC (American Type Culture Collection, Manassas, VA). Cell cultures were maintained as monolayers on plastic dishes in RPMI 1640 medium (MediaTech, Inc., Herndon, VA), supplemented with 10% heat-inactivated FBS (Gemini Bio-Products, Inc., Calabasas, CA), 1%l-glutamine (Life Technologies, Inc.), 1% pyruvate (Life Technologies, Inc.), 1% nonessential amino acids (Life Technologies, Inc.), and incubated at 37 °C and 5% CO2. For every experimental condition, the cells were cultured in 1% FBS 24 to In cases where and by Dr. was was to the cultured cells to stimulation with cytokines in the For iNOS cultured cells were with of human IFN-γ Inc., For cells were incubated in the of the of or blocked TNF-α-mediated cytotoxicity was TNF-α at the of and in a The lactate dehydrogenase assay was to cytotoxicity T. J. Immunol. PubMed Scopus Google Scholar). in were a and cultured at a serum concentration to for experimental condition, the was with a enzymatic which results in the of a a that is at in an cytotoxicity was the as cytotoxicity of experimental of was and from cells for experimental by a of and (Life Technologies, of was to for at °C with of reverse and (Life Technologies, of of these by was the following TNF-α and TNF-α for in reaction was the following glyceraldehyde-3-phosphate dehydrogenase of specific were the polymerase (Life Technologies, by a for and a at The of were based on of the of for gene the of the The were on and were by of the on a Inc., the domain at the of and on the cells experimental were with (MediaTech, Inc., Herndon, VA). was to the of the cells and incubated on for cells were by to and to a for experimental were generated by two and at were in and nuclear protein were the of Biochem. PubMed Scopus Google Scholar). protein were for interaction by as with Baltimore D. Cell. Full Text PDF PubMed Scopus Google Scholar). The NF-κB was with by with of and removal the DNA-binding the were on and the were and of the DNA-binding reaction was by with of NF-κB or The of specific NF-κB were by of the the cells were cultured in a for in medium supplemented with 1% In the cells were with to stimulation with or TNF-α. H2O2 were the cell 2′,7′-dichlorofluorescein Inc., the medium was with was on an Inc., The intracellular activation of NF-κB was by of AD10 cells with the cultured cells were with of of (Life Technologies, to the cells were a and incubated experimental The was on an The experimental were as the of the for the of in of was to and were to the of on the of the multiple and test were for two were for 5% the role of nitric oxide on the sensitization of the human ovarian carcinoma AD10 cell line to TNF-α-mediated cytotoxicity, we AD10 cells with IFN-γ in the or of potent NOS inhibitor The of AD10 cells to the cytotoxic effect of of TNF-α and was by the of the medium 24 of of AD10 cells to IFN-γ for sensitized the tumor cells to TNF-α-mediated cytotoxicity and the of sensitization with of TNF-α. by IFN-γ was in the of NOS inhibitor l-NMA the specific role of nitric oxide in the sensitization of AD10 we the cytotoxic effect of TNF-α in the of and nitric oxide for with of we observed a in the of AD10 cells to TNF-α-mediated cytotoxicity in a assay that directly correlated with the of we have that IFN-γ sensitized the prostatic cell line to TNF-α-mediated cytotoxicity from to 37 and the sensitization was blocked by the addition of l-NMA to the use of the NO sensitized PC-3 cells to TNF-α cytotoxicity from to The transcription factor has been demonstrated to regulate the gene expression of TNF-α, a in tumor cells that TNF-α that in NF-κB H. B. J. J. Immunol. 2000; PubMed Scopus Google Scholar). Furthermore, has been shown to TNF-α-mediated activation of in several cell and in T. J. B. C. A. R. M. J. Immunol. 1993; 151: Google Scholar). the specific effect of nitric oxide on the expression of TNF-α, we incubated AD10 cells with and for and the cells with TNF-α for The of generated TNF-α were by of the specific TNF-α The expression of TNF-α by AD10 cells was demonstrated and a was observed upon treatment with exogenous TNF-α. Moreover, this of TNF-α was blocked following treatment of the cells with nitric oxide to the of the TNF-α These findings that NO NF-κB and TNF-α results to observed with AD10 cells were obtained with the human prostatic cell line The expression of TNF-α messenger in PC-3 was upon treatment with the role of nitric oxide in the expression of TNF-α. the of NF-κB on TNF-α expression, we the of expression of TNF-α treatment of AD10 cells with and for with TNF-α stimulation for TNF-α gene expression of cells in the of was blocked as was observed following treatment with These results the role of ROS in the activation of the transcription factor NF-κB and the expression of TNF-α. nitric oxide could interfere with the TNF-α-mediated activation of NF-κB, we the NF-κB DNA-binding activity by shown in nuclear from AD10 cells an activity specific for the NF-κB H2O2 also induced specific NF-κB activity in AD10 cells of Further, NF-κB activity was by the of AD10 cells with for to stimulation with TNF-α for The NF-κB activity by was by the addition of H2O2 to as in the AD10 cells. these results that the at which nitric oxide the at which H2O2 is generated stimulation of AD10 cells with TNF-α. AD10 cells a of NF-κB activity that is not by nitric oxide and whereas in cells the NF-κB activity the in the of nitric oxide nitric oxide the generation of H2O2 in AD10 cells with TNF-α, we the intracellular generation of H2O2 the AD10 cells were incubated in the or of and with and TNF-α, for of these experimental a in H2O2 generated by the TNF-α of the cells in the of reduced the of H2O2 generated by these cells These data that nitric oxide is the intracellular of H2O2 by superoxide dismutase via its chemical interaction with TNF-α-induced O⨪2. Nitric oxide has been shown to directly the of NF-κB and its DNA-binding ability due to modification of amino S. Biochem. J. 1997; PubMed Scopus Google Scholar). the effect of nitric oxide on the activation of we an system by κB to the We AD10 cells with the and the cells in the or of of these cells a activation of the gene by TNF-α and H2O2, and the of activation was a function of the The TNF-α-induced activation of the gene was in the of the findings obtained in the NF-κB The inhibitory activity of on the TNF-α-induced activation of the gene was by stimulation with exogenous H2O2 These data the inhibitory effect of nitric oxide on the activation of NF-κB in AD10 cells. We also that AD10 cells were to of NF-κB activation that were not by treatment with nitric the findings observed in the assay in The activation of the transcription factor NF-κB by TNF-α and many has been implicated in the development of resistance of tumor cells to a of cytotoxic TNF-α (3Wang C.Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2509) Google Scholar, 5Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2933) Google Scholar). NF-κB is an transcription factor that has been shown to to of exogenous H2O2 or to reactive oxygen species generated as part of the signaling by many such as TNF-α Y.H. H.B. J. Immunol. 1997; Google Scholar, R. P.A. Free Radical Res. Commun. 1992; PubMed Scopus Google Scholar, R. P.A. J. 1991; PubMed Scopus Google Scholar). We have reported that the sensitization of the human ovarian carcinoma AD10 cell line to Fas-mediated apoptosis is due in part to the generation of nitric or its reaction by iNOS in these cells (21Garban H.J. Bonavida B. Gynecol. Oncol. 1999; 73: 257-264Abstract Full Text PDF PubMed Scopus (82) Google Scholar). In the evidence is for the that that NO also sensitizes tumor cells to TNF-α-mediated cytotoxicity. Further, we a molecular mechanism by which nitric oxide the activation of NF-κB resulting in sensitization of the AD10 cells to TNF-α cytotoxicity. The specific role of nitric oxide in tumor is not established. broad spectrum of has been assigned to either the or the of nitric oxide in tumor cells (for a review, see Ref. S. Pharmacol. 1991; Google Scholar). of nitric oxide has been correlated with and the tumor S. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). In addition, the generation of nitric oxide by tumor cells the activation and or the apoptosis of that can for the immune tumor not Furthermore, of nitric oxide could the activation of and the signals J.S. Cell 1999; PubMed Scopus Google Scholar, J. 2000; PubMed Scopus Google Scholar). However, the effect has also been observed in many systems the generation of of nitric either by iNOS induction or by the use of NO tumor and B. Cancer Res. 2000; Google Scholar). Therefore, the of NO-mediated effects be by many the concentration and of nitric oxide and the of reactive that might the status in the tumor In the human ovarian carcinoma AD10 cell line with the cytokine we observed a of these tumor cells to the cytotoxic effect of TNF-α. IFN-γ also induces iNOS expression in these cells (21Garban H.J. Bonavida B. Gynecol. Oncol. 1999; 73: 257-264Abstract Full Text PDF PubMed Scopus (82) Google Scholar). to TNF-α was by the use of the specific NOS and was by the use of the NO the role of nitric oxide in the sensitization IFN-γ treatment might not be to iNOS expression in cultured cells. The of IFN-γ in the induction of iNOS is to the of the activity of cytokines like TNF-α, or These cytokines and/or the have been shown to the transcription factor NF-κB, the for the induction of the expression of iNOS that might be by the of IFN-γ 1998; Google Scholar). We observed that AD10 cells a of activation of NF-κB and Therefore, the activation of NF-κB in AD10 cells could the treatment with IFN-γ was to iNOS and generate nitric NF-κB has been shown to be a transcription factor TNF-α gene expression in many either as a activator or in with transcription H. B. J. J. Immunol. 2000; PubMed Scopus Google Scholar). the activation of the NF-κB in AD10 cells might the of a expression of TNF-α by these cells and Moreover, TNF-α has been implicated as a cytokine by tumor cells either to anti-apoptotic or cellular A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cell Res. 2000; PubMed Scopus (27) Google Scholar, Cell Immunol. 2000; PubMed Scopus Google Scholar). Therefore, the of a in which the expression of TNF-α is by the TNF-α-mediated activation of NF-κB could a role in the and/or of tumor cells. has been shown to be a potent and specific inhibitor of the expression of TNF-α T. J. B. C. A. R. M. J. Immunol. 1993; 151: Google 1998; PubMed Scopus Google Scholar). AD10 cells a expression of TNF-α, which was by stimulation with exogenous TNF-α and by the nitric oxide we were to the expression of TNF-α In nitric oxide was to the expression of TNF-α in the of exogenous These results the inhibitory role of nitric oxide on TNF-α-induced activation of NF-κB and resulting in the disruption of TNF-α gene TNF-α induces the generation of ROS that serve as second messengers in the activation of pathways to the cell death B. R. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, R. J. 1993; PubMed Scopus Google Scholar, Y. J. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Stimulation of many cell with TNF-α results in the generation of intracellular superoxide T. Richter C. Peterhans E. Biochem. J. 1993; 289: 587-592Crossref PubMed Scopus (261) Google Scholar). In biological O⨪2 is immediately reduced by superoxide dismutase to H2O2 or rapidly reacts with ONOO− C. Ohshima H. Nitric Oxide. 1997; 1: 373-385Crossref PubMed Scopus (392) Google Scholar). Therefore, of O⨪2 will result in a reduced generation of H2O2. This could the activation of NF-κB P.A. T. Immunol. 1994; PubMed Scopus Google Scholar). the generation of H2O2 in AD10 we have a reduction in the of H2O2 generated in the of nitric oxide These results the effect of NO on the O⨪2 generated upon TNF-α NO can O⨪2 by the modification of the activity of the that O⨪2 the cell J. J. 1992; PubMed Scopus Google Scholar, H. H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Further, we have that the addition of NO donors to AD10 cells either the activity of NF-κB or its activation This inhibition was to the by treatment with exogenous H2O2. In nitric oxide not the NF-κB activation in AD10 the with TNF-α gene These results the of at two pathways in the activation of NF-κB in AD10 cells that in to H2O2 and the of nitric oxide to one of these two pathways. The of NF-κB upon NO treatment was not mediated by activation the effect on NF-κB and we could not the inhibitory effect of NO on NF-κB activation by the use of the not have implicated the role of nitric oxide on the activation of NO has been shown to the expression of the NF-κB inhibitory or its cellular by protein H.B. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). to the generation of H2O2 upon TNF-α treatment and the activation NF-κB in AD10 is that regulatory like the induction of with the NF-κB activation. It is that, in the a of both for the inhibitory role of nitric oxide on the TNF-α-induced activation of proposed mechanism implicated in the inhibition of the NF-κB activity by NO is via the of resulting in the disruption of the NF-κB and its DNA-binding ability S. Biochem. J. 1997; PubMed Scopus Google Scholar). However, the in be complex due to the of and proteins the which the modification of In findings that the mechanism by which NO sensitizes the human ovarian carcinoma cell line to TNF-α-mediated apoptosis is due to the specific disruption of the TNF-α-induced generation of H2O2 and the inhibition of the expression of anti-apoptotic These results can be to tumor as observed with the human prostatic cell line shown in the the expression of TNF-α could be by the inhibitory activity that nitric oxide exerts on the TNF-α-induced activation of Furthermore, in an in the of tumor cells to cytokines such as IFN-γ will promote the induction of iNOS by the tumor cells or and which in will result in the generation of nitric Hence, the generated or the NO the O⨪2 and the activation of on these molecular a mechanism of NO-mediated sensitization to apoptosis is We Dr. and Dr. for of this study and with the of the of gene expression and NO-mediated We are to for with the We also for in the of the