[Show abstract][Hide abstract] ABSTRACT: The transcription factor NF-E2-related nuclear factor 2 (Nrf2) regulates expression of genes that protect cells from oxidative damage. Here, we characterized nitric oxide (*NO)-induced Nrf2-Kelch-like ECH-associated protein 1 (Keap1) signaling and its role in counteracting *NO-induced apoptosis of human colon cancer HCT116 cells. Nrf2 was localized in the cytoplasm in control cells; *NO triggered its rapid nuclear accumulation, transcriptional activation, and up-regulation of HO-1, NQO1, and GCL, but not GST A4 and P1 subunits. Nrf2 accumulation in the nucleus was also associated with enhanced transcription and posttranscriptional modifications. (S)-nitrosation of Keap1 may contribute to nuclear accumulation of Nrf2 by facilitating its dissociation from Keap1, thus initiating *NO-mediated Nrf2-Keap1 signaling. *NO-mediated induction of ARE-dependent genes occurred well before apoptosis, as judged by caspase 3 activation. Collectively, these results show that the Nrf2-Keap1 signaling pathway mediates protective cellular responses to mitigate *NO-induced damage and may contribute to the relative resistance of HCT116 to *NO-induced cytotoxicity.
Proceedings of the National Academy of Sciences 09/2009; 106(34):14547-51. DOI:10.1073/pnas.0907539106 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here we investigated the cytotoxicity of JS-K, a prodrug designed to release nitric oxide (NO(*)) following reaction with glutathione S-transferases, in multiple myeloma (MM). JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient-derived MM cells. JS-K induced apoptosis in MM cells, which was associated with PARP, caspase-8, and caspase-9 cleavage; increased Fas/CD95 expression; Mcl-1 cleavage; and Bcl-2 phosphorylation, as well as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) release. Moreover, JS-K overcame the survival advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells. Mechanistic studies revealed that JS-K-induced cytotoxicity was mediated via NO(*) in MM cells. Furthermore, JS-K induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by neutral comet assay, as well as H2AX, Chk2 and p53 phosphorylation. JS-K also activated c-Jun NH(2)-terminal kinase (JNK) in MM cells; conversely, inhibition of JNK markedly decreased JS-K-induced cytotoxicity. Importantly, bortezomib significantly enhanced JS-K-induced cytotoxicity. Finally, JS-K is well tolerated, inhibits tumor growth, and prolongs survival in a human MM xenograft mouse model. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.
[Show abstract][Hide abstract] ABSTRACT: Toxicity induced by nitric oxide (NO(*)) has been extensively investigated in many in vitro and in vivo experimental models. Recently, our laboratories found that both concentration and cumulative total dose are critical determinants of cell death caused by NO(*). Here, we report results of studies designed to define total dose thresholds and threshold effects for several NO(*)-induced toxicity and cellular responses and to determine impacts of p53 on them. We exposed human lymphoblastoid TK6 cells harboring wild-type p53 and isogenic p53-null NH32 cells to NO(*) delivered by a membrane delivery system. Cells were exposed at a steady state concentration of 0.6 microM for varying lengths of time to deliver increasing cumulative doses (expressed in units of microM min), and several end points of cytotoxicity and mutagenesis were quantified. Threshold doses for NO(*)-induced cytotoxicity were 150 microM min in TK6 cells and 300 microM min in NH32 cells, respectively. Threshold doses for NO(*)-induced apoptosis were identical to those for cytotoxicity, but mitochondrial depolarization thresholds were lower than those for cytotoxicity and apoptosis in both cell types. To gain insight into underlying mechanisms, cells of both types were exposed to sublethal (33% of cytotoxicity threshold), cytotoxicity threshold, or toxic (twice the cytotoxicity threshold) doses of NO(*). In TK6 cells (p53), the sublethal threshold dose induced DNA double-strand breaks, but nucleobase deamination products (xanthine, hypoxanthine, and uracil) in DNA were increased only modestly (<50%) by toxic doses. Increased mutant fraction at the thymidine kinase gene (TK1) locus was observed only at the toxic dose of NO(*). Treatment of NH32 cells with NO(*) at the threshold or toxic dose elevated mutagenesis of the TK1 gene, but did not cause detectable levels of DNA double-strand breaks. At similar levels of cell viability, the frequency of DNA recombinational repair was higher in p53-null NH32 cells than in wild-type TK6 cells. NO(*) treatment induced p53-independent cell cycle arrest predominately at the S phase. Akt signaling pathway and antioxidant proteins were involved in the modulation of toxic responses of NO(*). These findings indicate that exposure to doses of NO(*) at or above the cytotoxicity threshold dose induces DNA double-strand breaks, mutagenesis, and protective cellular responses to NO(*) damage. Furthermore, recombinational repair of DNA may contribute to resistance to NO(*) toxicity and potentially increase the risk of mutagenesis. The p53 plays a central role in these responses in human lymphoblastoid cells.
Chemical Research in Toxicology 03/2006; 19(3):399-406. DOI:10.1021/tx050283e · 4.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glutathione (GSH) plays an important role in cellular defense response in many in vitro and in vivo models. Here we investigated its role in NO()-induced toxicity in cell culture and mouse models. Wild-type (TK6) and p53-null (NH32) human lymphoblastoid cells were treated with NO(.) at a steady-state concentration of 0.6 muM, similar to the level estimated to occur in inflamed tissues. In both cell types, GSH was depleted by this exposure in a dose- and time-dependent manner. Contrary to expectations, prior depletion of GSH by treatment with l-buthionine-SR-sulfoximine did not potentiate NO(.)-induced cell killing or DNA deamination in TK6 cells. In activated RAW264.7 murine macrophages producing NO(.), intracellular GSH content did not change, although gamma-glutamate-cysteine ligase was upregulated. NO(.) overproduction in RcsX lymphoma-bearing SJL mice resulted in significantly elevated GSH levels in various organs. Administration of the NO(.) synthase inhibitor N-methylarginine abolished the increase in GSH in these animals. Collectively, these data indicate a multifaceted and complex involvement of GSH in responses of cells and tissues to toxic levels of NO(.). NO(.) treatment effectively depleted GSH levels in human lymphoblastoid cells, but this alteration was not a critical initiating factor for NO(.)-mediated toxicity. Murine macrophages maintained GSH homeostasis when exposed to endogenously produced NO(.). In RcsX lymphoma-bearing mice, upregulation of de novo synthesis of GSH appeared to be a response to the toxic effects of NO(.).
Free Radical Biology and Medicine 01/2006; 39(11):1489-98. DOI:10.1016/j.freeradbiomed.2005.07.011 · 5.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Unphysiologically high levels of nitric oxide (NO*) are mutagenic and may contribute to carcinogenesis. Proapoptotic and anitiapoptotic functions of NO* have been reported in various in vivo and in vitro experimental models. The complexity of biological responses induced is a consequence of the multiple chemical pathways through which NO* causes damage to critical cellular macromolecules. The extent and kinetics of apoptotic and other responses are highly dependent on steady-state NO* levels, cumulative total dose and cell type. Steady-state and total dose thresholds have been defined, both of which must be exceeded for the induction of apoptosis and other responses in human lymphoblastoid cells. DNA damage, protein modifications, p53 activation and mitochondrial respiratory inhibition contribute to NO*-mediated apoptosis via mitochondrial and Fas receptor pathways. Multifaceted cellular defense systems including glutathione, antioxidant enzymes and Nrf2-Keap1 signaling participate in protective responses to mitigate damage by toxic levels of NO*.
Cancer Letters 09/2005; 226(1):1-15. DOI:10.1016/j.canlet.2004.10.021 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Loss of p53 function by inactivating mutations results in abrogation of NO*induced apoptosis in human lymphoblastoid cells. Here we report characterization of apoptotic signaling pathways activated by NO* in these cells by cDNA microarray expression and immunoblotting. A p53-mediated transcriptional response to NO* was observed in p53-wild-type TK6, but not in closely related p53-mutant WTK1, cells. Several previously characterized p53 target genes were up-regulated transcriptionally in TK6 cells, including phosphatase PPM1D (WIP1), oxidoreductase homolog PIG3, death receptor TNFRSF6 (Fas/CD95), and BH3-only proteins BBC3 (PUMA) and PMAIP1 (NOXA). NO* also modulated levels of several gene products in the mitochondria-dependent and death-receptor-mediated apoptotic pathways. Inhibitors of apoptosis proteins X-chromosome-linked inhibitor of apoptosis, cellular inhibitor of apoptosis protein-1, and survivin were significantly down-regulated in TK6 cells, but not in WTK1 cells. Smac release from mitochondria was induced in both cell types, but release of apoptosis-inducing factor and endonuclease G was detected only in TK6 cells. Fas/CD95 was increased, and levels of the antiapoptotic proteins Bcl-2 and Bcl-x/L were reduced in TK6 cells. Activation of procaspases 3, 8, 9, and 10, as well as Bid and poly(ADP-ribose) polymerase cleavage, were observed only in TK6 cells. NO* treatment did not alter levels of death receptors 4 and 5, Fas-associated death domain or proapoptotic Bax and Bak proteins in either cell line. Collectively, these data show that NO* exposure activated a complex network of responses leading to p53-dependent apoptosis via both mitochondrial and Fas receptor pathways, which were abrogated in the presence of mutant p53.
Cancer Research 06/2004; 64(9):3022-9. DOI:10.1158/0008-5472.CAN-03-1880 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO(*)) is mutagenic and, under appropriate conditions of exposure, also induces apoptosis in many in vitro and in vivo experimental models. Biochemical and cellular mechanisms through which NO(*) induces apoptosis are incompletely understood, but involve p53/mitochondria-dependent signaling pathways. In this study, we exposed human lymphoblastoid cells harboring either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) to NO(*), delivered by diffusion through Silastic tubing. Cells were exposed for 2 h at constant rates of 100-533 nM/s, similar to levels estimated to occur in vivo in inflamed tissues. DNA double-strand breaks and fragmentation detected 8-48 h after NO(*) treatment were more extensive in TK6 cells than in WTK-1 cells, whereas NO(*)-induced mutant fractions in both HPRT and TK1 genes were significantly lower in TK6 cells than in WTK-1 cells (P < 0.01-0.05). Treatment of TK6 cells with NO(*) caused extensive apoptosis, but this response was delayed and greatly reduced in magnitude in WTK-1 cells. Mitochondrial membrane depolarization and cytochrome c release were induced in both cell types. However, elevation of apoptotic protease-activating factor-1 (Apaf-1) protein and reduction of X-chromosome-linked inhibitor of apoptosis (XIAP) protein were observed only in TK6 cells. These results indicate that p53 status is an important modulator of NO(*)-induced mutagenesis and apoptosis, and suggest that levels of the Apaf-1 and XIAP proteins, but not mitochondrial depolarization and cytochrome c release, are regulated by p53 in these human lymphoblastoid cells. Thus, Apaf-1 and XIAP may play important roles in the regulation of p53-mediated apoptotic responses.
Proceedings of the National Academy of Sciences 08/2002; 99(16):10364-9. DOI:10.1073/pnas.162356399 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SIN-1 (3-morpholinosydnonimine), the active metabolite of the vasodilator drug molsidomine, decomposes spontaneously in solution. In the presence of oxygen, NO* and O(2)(*-) are released, generating peroxynitrite, a potent oxidizing agent, at a constant rate over a 2 h period. We utilized this system to investigate mechanisms of peroxynitrite-induced cytotoxicity, genotoxicity, apoptosis, and mitochondrial damage in two human lymphoblastoid cell lines carrying either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) genes. Treatment of TK6 cells with 5 mM SIN-1 for 1.5 h resulted in 28 +/- 6% survival 24 h later. Exposure in the presence of different radical scavengers significantly increased survival, as follows: cytochrome c, 96 +/- 3%; Tiron, 69 +/- 0%; SOD plus catalase, 83 +/- 5%; carboxy-PTIO, 87 +/- 3%; and uric acid, 87 +/- 2%. D-mannitol was ineffective in reducing lethality, as were SOD and catalase when added individually or in heat-inactivated form. Spontaneous as well as SIN-1-induced mutant fractions (MF) in both HPRT and TK genes were significantly higher in WTK-1 cells than in TK6 cells (p < 0.05-0.01). Exposure to 2.5 mM SIN-1 induced time-dependent apoptosis in TK6 cells, but not in WTK-1 cells. Mitochondrial membrane depolarization was also observed in both cell lines after SIN-1 treatment. Neutral comet assay demonstrated that SIN-1 treatment resulted in higher levels of DNA double-strand breaks in TK6 cells than in WTK-1 cells. Collectively, these data show that SIN-1 can be used as an effective peroxynitrite generator in cell culture experiments under these experimental conditions, in which it induced a greater apoptotic response but was less potent as a mutagen in TK6 cells compared with WTK-1 cells. Thus, p53 status was an important determinant of SIN-1 induced mutagenesis and apoptosis in these two human lymphoblastoid cell lines.
Chemical Research in Toxicology 04/2002; 15(4):527-35. DOI:10.1021/tx010171x · 4.19 Impact Factor