- Email: [email protected]
B1. Nitric oxide induces RKIP expression and sensitizes tumor cells to TRAIL-mediated apoptosis via inhibition of both NF-κB and YY1 and upregulation of DR5
Available online at www.sciencedirect.com
Nitric Oxide 17 (2007) S16–S19 www.elsevier.com/locate/yniox
Oral communication abstracts B1. Nitric oxide induces RKIP expression and sensitizes tumor cells to TRAIL-mediated apoptosis via inhibition of both NF-jB and YY1 and upregulation of DR5 S. Baritaki, K.C. Yeung, D. Chatterjee, S. Huerta-Yepez, D. Spandidos, B. Bonavida Department of Microbiology, Immunology & Molecular Genetics, 10833 Le Conte Avenue, Los Angeles, USA TRAIL, a member of the TNF family, is a new candidate for immunotherapy in the treatment of drug-refractory tumor cells. However, many drug-resistant tumor cells are also resistant to TRAIL. NO donors can reverse resistance to TRAIL via inhibition of NF-jB and downstream inhibition of YY1 repressor activity concomitant with upregulation of DR5 expression (Huerta-Yepez et al., Oncogene, 23: 4993, 2004; Baritaki et al., Mol Cancer Therap, 6(4): 1387, 2007). The mechanism of NOinduced inhibition of NF-jB was examined. We hypothesized that NO may induce the metastasis tumor suppressor gene product Raf kinase inhibitory protein (RKIP) leading to inhibition of NF-jB. Treatment of tumor cells with the NO donor, DETANONOate, resulted in significant induction of RKIP mRNA and protein levels. Also, DETANONOate induced elevated RKIP promoter activity and reduced YY1 promoter activity in tumor cells. The direct role of RKIP in tumor cell sensitization to TRAIL was confirmed with RKIP siRNA. The role of YY1 in the regulation of DR5 and sensitization to TRAIL was corroborated with the used of YY1 siRNA. These findings provide evidence that NO-induced sensitization to TRAIL is mediated, in large part, via induction of RKIP expression and subsequently, inhibition of NF-jB and YY1 activities and upregulation of DR5 expression. The in vitro studies were corroborated in in vivo mice bearing tumor xenografts that were treated with DETANONOate. Hence, NO donors can be used therapeutically to reverse TRAIL resistance when used in combination with TRAIL or anti-DR4/5 monoclonal antibodies. doi:10.1016/j.niox.2007.09.034
B2. Activated CD8+ T cells radiosensitize EMT-6 mammary carcinoma cells through the interferon-gamma/iNOS pathway M. De Ridder, V. Verovski, G. Van Esch, D. Van den Berge, C. Monsaert, K. Law, G. Storme Oncologisch Centrum UZ Brussel, Laarbeeklaan 101, 1090 Brussel, Belgium Activated CD8+ T cells were shown to be major mediators of antitumor immunity, while their effect on radiosensitivity has not yet been explored. The aim of this study was to examine the cytolytic and radiosensitizing activities of CD8+ T cells in 1% oxygen, modeling the immunosuppressive and radioprotective microenvironment of solid tumors. Expanded CD8+ T cells secreted high levels of IFN-gamma and increased the radiosensitivity of syngeneic EMT-6 mammary carcinoma cells up to
doi:10.1016/j.niox.2007.09.033
1.8-fold. This radiosensitization was abrogated by IFN-gamma immunoneutralization and by the metabolic iNOS inhibition in tumor cells. While considerable radiosensitizing effects were observed at a CD8+ T / EMT-6 cell ratio below 1/1, the cytotoxicity of CD8+ T cells was impaired by hypoxia even at a 10/1 ratio. RT-PCR, FACS and ELISA data in agreement revealed down-regulation of IFN-gamma in hypoxic CD8+ T cells. In contrast, hypoxia transcriptionally up-regulated iNOS in EMT-6 tumor cells that were exposed to IFN-g+/CD8+ T cells. The latter was essential for preserving the radiosensitizing effects under hypoxic conditions. These data for the first time demonstrate the radiosensitizing potential of IFNg+/CD8+ T cells and indicate a rationale for combining immunostimulatory and radiosensitizing strategies. doi:10.1016/j.niox.2007.09.035
B3. A study to determine the role of iNOS as a radiosensitizer and potentiator of bioreductive drugs in human tumours B. Fitzpatrick, M. Babur, B. Telfer, E. Chinje, R. Cowen, I. Stratford University of Manchester, Oxford Road, Manchester, United Kingdom NO generated by iNOS is a potent radiosensitizer and is directly cytotoxic. As an adjuvant to radiotherapy, bioreductive drugs preferentially toxic towards hypoxic cells have been developed. We have shown that iNOS can also potentiate these agents in vitro. The aim of this study was to develop a human tumour xenograft model to determine the role of iNOS as a radiosensitizer and potentiator of bioreductive drugs in vivo. We used HT1080 stable cell-line constitutively over expressing iNOS. Their iNOS expression and activity were characterised using iNOS ELISA, citrulline assay and Griess assay. Tumour xenografts were characterised for their growth kinetics, stability of iNOS activity and IHC was used to determine the distribution of iNOS, hypoxia, perfusion and vascularity. HT1080 clonal cell line significantly over expressed iNOS (232.07 U/mg protein compared to 2.56 U/mg protein in WT), which correlated with increased iNOS oxygenase activity (42.8 compared to 3.93 citrulline/min/mg protein). Established iNOS overexpressing cell line and HT1080 WT were studied in vivo showing similar tumour take and growth rates (5.6 and 5.25 days RTV4 respectively), and increased iNOS expression levels were maintained (7.22 U/mg protein compared to 0.24 U/mg protein in WT). HT1080 WT and HT1080 NOS tumour xenografts showed that elevated iNOS did not affect the hypoxic fraction of the tumour, or the level of vascularisation. We are now using this model to investigate the potential of iNOS to potentiate two clinical bioreductive drugs, AQ4N and NLCQ, and radiotherapy in vivo. doi:10.1016/j.niox.2007.09.036
Nitric Oxide 17 (2007) S16–S19 www.elsevier.com/locate/yniox
Oral communication abstracts B1. Nitric oxide induces RKIP expression and sensitizes tumor cells to TRAIL-mediated apoptosis via inhibition of both NF-jB and YY1 and upregulation of DR5 S. Baritaki, K.C. Yeung, D. Chatterjee, S. Huerta-Yepez, D. Spandidos, B. Bonavida Department of Microbiology, Immunology & Molecular Genetics, 10833 Le Conte Avenue, Los Angeles, USA TRAIL, a member of the TNF family, is a new candidate for immunotherapy in the treatment of drug-refractory tumor cells. However, many drug-resistant tumor cells are also resistant to TRAIL. NO donors can reverse resistance to TRAIL via inhibition of NF-jB and downstream inhibition of YY1 repressor activity concomitant with upregulation of DR5 expression (Huerta-Yepez et al., Oncogene, 23: 4993, 2004; Baritaki et al., Mol Cancer Therap, 6(4): 1387, 2007). The mechanism of NOinduced inhibition of NF-jB was examined. We hypothesized that NO may induce the metastasis tumor suppressor gene product Raf kinase inhibitory protein (RKIP) leading to inhibition of NF-jB. Treatment of tumor cells with the NO donor, DETANONOate, resulted in significant induction of RKIP mRNA and protein levels. Also, DETANONOate induced elevated RKIP promoter activity and reduced YY1 promoter activity in tumor cells. The direct role of RKIP in tumor cell sensitization to TRAIL was confirmed with RKIP siRNA. The role of YY1 in the regulation of DR5 and sensitization to TRAIL was corroborated with the used of YY1 siRNA. These findings provide evidence that NO-induced sensitization to TRAIL is mediated, in large part, via induction of RKIP expression and subsequently, inhibition of NF-jB and YY1 activities and upregulation of DR5 expression. The in vitro studies were corroborated in in vivo mice bearing tumor xenografts that were treated with DETANONOate. Hence, NO donors can be used therapeutically to reverse TRAIL resistance when used in combination with TRAIL or anti-DR4/5 monoclonal antibodies. doi:10.1016/j.niox.2007.09.034
B2. Activated CD8+ T cells radiosensitize EMT-6 mammary carcinoma cells through the interferon-gamma/iNOS pathway M. De Ridder, V. Verovski, G. Van Esch, D. Van den Berge, C. Monsaert, K. Law, G. Storme Oncologisch Centrum UZ Brussel, Laarbeeklaan 101, 1090 Brussel, Belgium Activated CD8+ T cells were shown to be major mediators of antitumor immunity, while their effect on radiosensitivity has not yet been explored. The aim of this study was to examine the cytolytic and radiosensitizing activities of CD8+ T cells in 1% oxygen, modeling the immunosuppressive and radioprotective microenvironment of solid tumors. Expanded CD8+ T cells secreted high levels of IFN-gamma and increased the radiosensitivity of syngeneic EMT-6 mammary carcinoma cells up to
doi:10.1016/j.niox.2007.09.033
1.8-fold. This radiosensitization was abrogated by IFN-gamma immunoneutralization and by the metabolic iNOS inhibition in tumor cells. While considerable radiosensitizing effects were observed at a CD8+ T / EMT-6 cell ratio below 1/1, the cytotoxicity of CD8+ T cells was impaired by hypoxia even at a 10/1 ratio. RT-PCR, FACS and ELISA data in agreement revealed down-regulation of IFN-gamma in hypoxic CD8+ T cells. In contrast, hypoxia transcriptionally up-regulated iNOS in EMT-6 tumor cells that were exposed to IFN-g+/CD8+ T cells. The latter was essential for preserving the radiosensitizing effects under hypoxic conditions. These data for the first time demonstrate the radiosensitizing potential of IFNg+/CD8+ T cells and indicate a rationale for combining immunostimulatory and radiosensitizing strategies. doi:10.1016/j.niox.2007.09.035
B3. A study to determine the role of iNOS as a radiosensitizer and potentiator of bioreductive drugs in human tumours B. Fitzpatrick, M. Babur, B. Telfer, E. Chinje, R. Cowen, I. Stratford University of Manchester, Oxford Road, Manchester, United Kingdom NO generated by iNOS is a potent radiosensitizer and is directly cytotoxic. As an adjuvant to radiotherapy, bioreductive drugs preferentially toxic towards hypoxic cells have been developed. We have shown that iNOS can also potentiate these agents in vitro. The aim of this study was to develop a human tumour xenograft model to determine the role of iNOS as a radiosensitizer and potentiator of bioreductive drugs in vivo. We used HT1080 stable cell-line constitutively over expressing iNOS. Their iNOS expression and activity were characterised using iNOS ELISA, citrulline assay and Griess assay. Tumour xenografts were characterised for their growth kinetics, stability of iNOS activity and IHC was used to determine the distribution of iNOS, hypoxia, perfusion and vascularity. HT1080 clonal cell line significantly over expressed iNOS (232.07 U/mg protein compared to 2.56 U/mg protein in WT), which correlated with increased iNOS oxygenase activity (42.8 compared to 3.93 citrulline/min/mg protein). Established iNOS overexpressing cell line and HT1080 WT were studied in vivo showing similar tumour take and growth rates (5.6 and 5.25 days RTV4 respectively), and increased iNOS expression levels were maintained (7.22 U/mg protein compared to 0.24 U/mg protein in WT). HT1080 WT and HT1080 NOS tumour xenografts showed that elevated iNOS did not affect the hypoxic fraction of the tumour, or the level of vascularisation. We are now using this model to investigate the potential of iNOS to potentiate two clinical bioreductive drugs, AQ4N and NLCQ, and radiotherapy in vivo. doi:10.1016/j.niox.2007.09.036