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A17. Role of S-nitrosylation in apoptosis resistance and malignant transformation of human lung epithelial cells
Speaker abstracts / Nitric Oxide 17 (2007) S9–S15
S13
Theme 4: NO and Cancer: Apoptosis and Chemo Immunosensitization A16. NO/cGMP regulation of cell death T.R. Billiar University of Pittsburgh Medical Center, UPMC Presbyterian, F1281, 200 Lothrop Street, Pittsburgh, PA 15213, USA The effects of nitric oxide (NO) on cell viability are dependent on the level of NO exposure, cell type, and redox status of the cell. Furthermore, some cell types have established mechanisms to limit NO production by tight post-translational regulation of NO synthase activity. For example, we have shown that monomeric iNOS is sequestered in peroxisomes in hepatocytes. An efficient mechanism for the inhibition of apoptosis by NO/cGMP involves the suppression of the proximal signaling steps involved in apoptosis. Although NO can lead to the inhibition of caspase activity through nitrosation, more proximal events in apoptosis can be suppressed by NO induced cGMP formation. Ligand-induced apoptosis requires the formation of a death-inducing signaling complex (DISC) comprised of adapter proteins and caspase 8. We have found that the DISC is rapidly internalized as part of the initiating events in apoptotic signaling. This internalization can be inhibited by cyclic nucleotides, such as cGMP. Thus, NO activity through cGMP can suppress apoptosis by blocking the most proximal events in apoptotic signaling.
A18. Constitutive intracellular production of NO in human melanoma regulates resistance to apoptosis E. Grimm, C.H. Tang, S. Ekmekcioglu, J. Ellerhorst, V.G. Prieto University of Texas, M.D. Anderson Cancer Center, Department of Experimental Therapeutics, Unit 362, Houston, TX, USA Expression of inducible nitric-oxide synthase protein in melanoma cells from surgical specimens has been reported to provide survival predictive value. Tumors from patients who were untreated as well as those refractory to chemotherapy contained variable levels of iNOS, for which higher quantities were independent predictors of poorer survival (p < 0.001). Molecular data suggest that low levels of nitric oxide (NO), <50 nM, supports antiapoptotic activity. Therefore, we asked whether constitutive NO in melanoma regulated apoptosis, which was tested by employing NO quenchers and donors. Our results indicate that depletion of NO led to cell cycle changes and enhanced cisplatin-induced apoptosis. Curiously, we observed that the depletion of NO inhibited cisplatininduced wild type p53 accumulation and p21 (Waf1/Cip1/Sdi1) expression in melanoma cells and reduced the presence of p53-DNA complexes. Furthermore, dominant negative inhibition of p53 enhanced cisplatin-induced apoptosis. Our data strongly suggest that endogenously produced NO is required for cisplatin-induced p53 activation and p21 (Waf1/Cip1/Sdi1) expression, which can regulate melanoma sensitivity to chemotherapies.
doi:10.1016/j.niox.2007.09.021 doi:10.1016/j.niox.2007.09.023
A17. Role of S-nitrosylation in apoptosis resistance and malignant transformation of human lung epithelial cells Y. Rojanasakul, N. Azad, L. Wang West Virginia University, P.O. Box 9530, Morgantown, WV 26506, USA
A19. Chemo and immuno-sensitizing activities of nitric oxide (NO) and reversal of tumor resistance to apoptotic stimuli: Therapeutic implications B. Bonavida, H. Garban, S. Huerta-Yepez, M. Vega, S. Huerta, S. Baritaki Department of Microbiology, Immunology & Molecular Genetics, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, USA
Resistance to apoptosis and dysregulated nitric oxide (NO) production are key features of many human tumors; however, their relationship and the mechanisms by which they regulate carcinogenesis are not well understood. To determine their role in carcinogenic development, we chronically exposed non-tumorigenic human lung epithelial cells to sub-cytotoxic doses of Cr(VI), a known carcinogen, and examined their transforming activities, apoptosis and NO responses. As compared to their passage-matched controls, the Cr(VI)-treated cells exhibited loss of contact inhibition, colony formation, and cell invasion, characteristics of malignant transformation. We also found that these cells exhibited a significant increase in basal and inducible NO levels and a parallel increase in apoptosis resistance to Cr(VI). Inhibition of NO production reversed the apoptosis resistant phenotype, whereas addition of NO increased the resistance. NO was further shown to exhibit antiapoptotic effect through two independent but closely related mechanisms. First, NO induced S-nitrosylation of antiapoptotic protein Bcl-2, which controls Cr(VI) resistance, and prevented its ubiquitin-proteasomal degradation. Secondly, NO interacted with superoxide which served a key mediator of Bcl-2 ubiquitination. Such interaction effectively reduced the cellular levels of superoxide as well as other oxidative species that are involved in the process. Thus, our results indicate an important role of NO in the development of apoptosis resistant malignant phenotype through Bcl-2 and redox regulation.
Tumor cells develop resistance to cytotoxic drug-induced apoptosis and various sensitizing agents can reverse resistance to chemo-radio-immuno therapies. We have found that nitric oxide donors, like DETANONOate, can sensitize various human tumor cell lines to apoptosis by both chemotherapeutic drugs and immune death ligands. NO-mediated sensitization resulted from inhibition of constitutively activated anti-apoptotic cell survival pathways, such as NF-jB, and gene products in apoptosis regulated by these pathways. For instance, NO-mediated chemosensitization resulted from perturbation of the balance between pro and anti-apoptotic Bcl-2 family and mitochondrial depolarization and activation of type II apoptotic pathway. Likewise, NO-mediated immuno-sensitization to Fas-L and TRAIL apoptosis, for example, resulted from inhibition of the expression and DNA-binding activity of the transcription repressor, Yin Yang 1 (YY1), by inhibition of NF-jB and S-nitrosylation of YY1, respectively. YY1 was shown to negatively regulate the transcription of Fas and DR5 and plays a pivotal role in resistance. In addition, treatment with NO induces the expression of the metastatic cancer gene and immune surveillance cancer gene Raf kinase inhibitory protein (RKIP). RKIP inhibits cell survival pathways (example, Raf 1/MEK and NF-jB) and participates in NO-mediated sensitization. In vivo findings in mice with tumor xenografts corroborate the in vitro findings. The clinical therapeutic implications of NO-mediated sensitization in cancer will be discussed.
doi:10.1016/j.niox.2007.09.022
doi:10.1016/j.niox.2007.09.024
S13
Theme 4: NO and Cancer: Apoptosis and Chemo Immunosensitization A16. NO/cGMP regulation of cell death T.R. Billiar University of Pittsburgh Medical Center, UPMC Presbyterian, F1281, 200 Lothrop Street, Pittsburgh, PA 15213, USA The effects of nitric oxide (NO) on cell viability are dependent on the level of NO exposure, cell type, and redox status of the cell. Furthermore, some cell types have established mechanisms to limit NO production by tight post-translational regulation of NO synthase activity. For example, we have shown that monomeric iNOS is sequestered in peroxisomes in hepatocytes. An efficient mechanism for the inhibition of apoptosis by NO/cGMP involves the suppression of the proximal signaling steps involved in apoptosis. Although NO can lead to the inhibition of caspase activity through nitrosation, more proximal events in apoptosis can be suppressed by NO induced cGMP formation. Ligand-induced apoptosis requires the formation of a death-inducing signaling complex (DISC) comprised of adapter proteins and caspase 8. We have found that the DISC is rapidly internalized as part of the initiating events in apoptotic signaling. This internalization can be inhibited by cyclic nucleotides, such as cGMP. Thus, NO activity through cGMP can suppress apoptosis by blocking the most proximal events in apoptotic signaling.
A18. Constitutive intracellular production of NO in human melanoma regulates resistance to apoptosis E. Grimm, C.H. Tang, S. Ekmekcioglu, J. Ellerhorst, V.G. Prieto University of Texas, M.D. Anderson Cancer Center, Department of Experimental Therapeutics, Unit 362, Houston, TX, USA Expression of inducible nitric-oxide synthase protein in melanoma cells from surgical specimens has been reported to provide survival predictive value. Tumors from patients who were untreated as well as those refractory to chemotherapy contained variable levels of iNOS, for which higher quantities were independent predictors of poorer survival (p < 0.001). Molecular data suggest that low levels of nitric oxide (NO), <50 nM, supports antiapoptotic activity. Therefore, we asked whether constitutive NO in melanoma regulated apoptosis, which was tested by employing NO quenchers and donors. Our results indicate that depletion of NO led to cell cycle changes and enhanced cisplatin-induced apoptosis. Curiously, we observed that the depletion of NO inhibited cisplatininduced wild type p53 accumulation and p21 (Waf1/Cip1/Sdi1) expression in melanoma cells and reduced the presence of p53-DNA complexes. Furthermore, dominant negative inhibition of p53 enhanced cisplatin-induced apoptosis. Our data strongly suggest that endogenously produced NO is required for cisplatin-induced p53 activation and p21 (Waf1/Cip1/Sdi1) expression, which can regulate melanoma sensitivity to chemotherapies.
doi:10.1016/j.niox.2007.09.021 doi:10.1016/j.niox.2007.09.023
A17. Role of S-nitrosylation in apoptosis resistance and malignant transformation of human lung epithelial cells Y. Rojanasakul, N. Azad, L. Wang West Virginia University, P.O. Box 9530, Morgantown, WV 26506, USA
A19. Chemo and immuno-sensitizing activities of nitric oxide (NO) and reversal of tumor resistance to apoptotic stimuli: Therapeutic implications B. Bonavida, H. Garban, S. Huerta-Yepez, M. Vega, S. Huerta, S. Baritaki Department of Microbiology, Immunology & Molecular Genetics, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, USA
Resistance to apoptosis and dysregulated nitric oxide (NO) production are key features of many human tumors; however, their relationship and the mechanisms by which they regulate carcinogenesis are not well understood. To determine their role in carcinogenic development, we chronically exposed non-tumorigenic human lung epithelial cells to sub-cytotoxic doses of Cr(VI), a known carcinogen, and examined their transforming activities, apoptosis and NO responses. As compared to their passage-matched controls, the Cr(VI)-treated cells exhibited loss of contact inhibition, colony formation, and cell invasion, characteristics of malignant transformation. We also found that these cells exhibited a significant increase in basal and inducible NO levels and a parallel increase in apoptosis resistance to Cr(VI). Inhibition of NO production reversed the apoptosis resistant phenotype, whereas addition of NO increased the resistance. NO was further shown to exhibit antiapoptotic effect through two independent but closely related mechanisms. First, NO induced S-nitrosylation of antiapoptotic protein Bcl-2, which controls Cr(VI) resistance, and prevented its ubiquitin-proteasomal degradation. Secondly, NO interacted with superoxide which served a key mediator of Bcl-2 ubiquitination. Such interaction effectively reduced the cellular levels of superoxide as well as other oxidative species that are involved in the process. Thus, our results indicate an important role of NO in the development of apoptosis resistant malignant phenotype through Bcl-2 and redox regulation.
Tumor cells develop resistance to cytotoxic drug-induced apoptosis and various sensitizing agents can reverse resistance to chemo-radio-immuno therapies. We have found that nitric oxide donors, like DETANONOate, can sensitize various human tumor cell lines to apoptosis by both chemotherapeutic drugs and immune death ligands. NO-mediated sensitization resulted from inhibition of constitutively activated anti-apoptotic cell survival pathways, such as NF-jB, and gene products in apoptosis regulated by these pathways. For instance, NO-mediated chemosensitization resulted from perturbation of the balance between pro and anti-apoptotic Bcl-2 family and mitochondrial depolarization and activation of type II apoptotic pathway. Likewise, NO-mediated immuno-sensitization to Fas-L and TRAIL apoptosis, for example, resulted from inhibition of the expression and DNA-binding activity of the transcription repressor, Yin Yang 1 (YY1), by inhibition of NF-jB and S-nitrosylation of YY1, respectively. YY1 was shown to negatively regulate the transcription of Fas and DR5 and plays a pivotal role in resistance. In addition, treatment with NO induces the expression of the metastatic cancer gene and immune surveillance cancer gene Raf kinase inhibitory protein (RKIP). RKIP inhibits cell survival pathways (example, Raf 1/MEK and NF-jB) and participates in NO-mediated sensitization. In vivo findings in mice with tumor xenografts corroborate the in vitro findings. The clinical therapeutic implications of NO-mediated sensitization in cancer will be discussed.
doi:10.1016/j.niox.2007.09.022
doi:10.1016/j.niox.2007.09.024