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C10. Heparan sulfate, NO and cancer
S22
Poster abstracts / Nitric Oxide 17 (2007) S20–S29
Among all artemisinin derivatives, artesunate revealed the highest cytotoxicity against human CEM-CCRF leukemia cells. Measurement of ROS showed that artesunate produced the highest amount of ROS. In RAW264.7 mouse macrophages, which are the well-established model cell line to analyze NO generation, artesunate produced the highest NO amount. The IC50 value of RAW264.7 cells for artesunate was 3 lM. In order to associate the cytotoxic effects with specific alteration in gene expression related to NO and ROS metabolism and signalling microarray analyses were conducted. RAW cells were treated with 3 and 30 lM of artesunate using DMSO as vehicle control followed by microarray analysis. We conclude that the cleavage of the endoperoxide bridge of artemisinins play a crucial role for the cytotoxic activity. Relevant signalling pathways related to NO and ROS will be discussed.
reduce tumour load in vivo with no observed side-effects. In growth-inhibited cells, primed HS is degraded by NO to oligosaccharides that are transported to the nucleus. Accordingly, NO depletion reduces nuclear localization of the oligosaccharides and abolishes the growth-inhibitory effect of the xyloside (2-4). Cancer cells accumulate misfolded carbonylated proteins. Recently, we have reported that conjugates between NO-generated HS oligosaccharides and carbonylated proteins colocalize and coprecipitate with proteasomes in both carcinoma and neuroblastoma cells (5). References 1. Fransson et al., Cell. Mol. Life Sci. 61 (2004) 1016, 2. Mani et al., Cancer Res. 58 (1998) 1099, 3. Mani et al., Glycobiology 14 (2004) 387, 4. Jacobsson et al. J. Med. Chem. 49 (2006) 1932, 5. Mani et al. J. Biol. Chem. 282 (2007) 21934. doi:10.1016/j.niox.2007.09.055
doi:10.1016/j.niox.2007.09.053
C9. Influence of nitric oxide and tissue oxygenation on invasive capacity of urinary bladder cancer C. Ewe, N.P. Wiklund, P.J. de Verdier Department of Molecular Medicine and Surgery, Forskningscentrum M3:02, Urologylab, Stockholm, Sweden Abstract Background: High-risk invasive bladder tumours and carcinoma in situ (CIS) may be treated with the immunotherapeutic drug bacillus Calmette-Guerin (BCG), which is shown to increase activity of nitric oxide synthases and highly elevate the concentration of nitric oxide (NO) in the bladder of patients after treatment. The transcription factor Hypoxia-inducible factor 1 (HIF1)’s stabilization is dependent of the oxygen levels in the cell, but may also be affected by the NO levels which the cell is exposed to. Objective: Bladder cancer cell-lines derived from transitional cell carcinomas (J82, DAG-1, and T24) and control cell-lines with high (HT1080) and low (NIH3T3) invasive properties are used in a chemoinvasion assay to study how the invasive capacity of urinary bladder cancer is influenced by exogenously added NO (DETA-NO; 0.01–3 mM) and altered levels of tissue oxygenation (normoxia (21%), hypoxia (3%) and anoxia (0%)). Results and conclusions: Low levels of NO enhance cell proliferation whereas high levels are cytotoxic. Under anoxic (0%) conditions the studied cell-lines seem more sensitive to the cytotoxic effect of NO compared to when exposed to atmospheric (21%) oxygen levels. The response curves are similarly shaped, but during anoxic conditions the curves are shifted to give an earlier response. We have also seen that low levels of exogenously added NO (0.01–0.1 mM) have an effect on the invasion capacity of some cell lines during anoxic conditions. doi:10.1016/j.niox.2007.09.054
C10. Heparan sulfate, NO and cancer L.A. Fransson, F. Cheng, U. Ellervik, K. Mani Lund University, Experimental Medical Science, BMC A13, Lund, Sweden Many growth factors are activated by cell-surface heparan sulfate (HS). Altered expression of HS-containing proteoglycans, especially glypicans (Gpc), is correlated with neoplastic transformation. When expression of a Gpc is silenced in tumour cells, both growth-factor response and tumour formation are depressed. We have shown that Gpc-1 can be S-nitrosylated by NO in the core protein. During endocytosis and recycling of Gpc-1 via endosomes and the Golgi, NO is released and cleaves HS chains at specific sites. This reaction can be enhanced by vitamin C (reviewed in 1). We have also shown that specific naphthyl-xylosides, while serving as primers for HS synthesis, selectively inhibit tumour cell-growth in vitro by inducing apoptosis. Perorally administered xylosides also
C11. Hypoxia increases the shedding of MHC class I chain-related molecule (MIC) from the surface of tumour cells: Involvement of nitric oxide signalling Charles H. Graham a,b,c, Nianping Hu a, Karim Sheikhi a, Eugene Chung a, Hugh Pross d, D. Robert Siemens a,b a Department of Anatomy and Cell Biology, Queen’s University, Kingston, Ontario, Canada b Department of Urology, Queen’s University, Kingston, Ontario, Canada c Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario, Canada d Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario, Canada The major histocompatibility complex class I chain-related (MIC) molecules play important roles in tumour immunosurveillance through their interaction with the NKG2D receptor on NK and cytotoxic T cells. Furthermore, shedding of MIC from the cell membrane represents a potential mechanism of tumour escape from NKG2D-mediated immunosurveillance. Tumour hypoxia is associated with a poor clinical outcome for cancer patients and with aggressive tumour cell phenotypes. We show that hypoxia contributes to tumour cell shedding of MIC through a mechanism involving impaired NO signalling. While hypoxia increased MIC shedding in DU145 prostate cancer cells, activation of NO signalling inhibited the hypoxia-mediated MIC shedding. Similar to incubation in hypoxia, inhibition of endogenous NO signalling increased MIC shedding. Parallel studies showed a hypoxia-mediated increase in tumour cell resistance to lysis by IL-2 activated peripheral blood lymphocytes (PBLs), and an NO-mediated attenuation of this hypoxia-mediated resistance to lysis. Furthermore, inhibition of NO production led to resistance to PBL-mediated lysis. Inclusion of a blocking anti-MIC antibody resulted in lower cytotoxic activity against DU145 cells. In the presence of this antibody, hypoxia did not cause a further increase in resistance to PBL-mediated killing and activation of NO signalling did not restore sensitivity to lysis. These findings suggest that the hypoxic tumour microenvironment contributes to impaired immunosurveillance and that activation of NO signalling is of potential use in cancer immunotherapy. doi:10.1016/j.niox.2007.09.056
C12. Performances and applicability of electrochemical NO-sensor for in vivo detection of nitric oxide in tumor-bearing mice S. Griveau, C. Dume´zy, J. Seguin, G.G. Chabot, D. Scherman, F. Bedioui INSERM, U640, CNRS, UMR8151, Universite´ Rene´ Descartes Paris 5, Ecole Nationale Supe´rieure de Chimie de Paris, Faculty of Pharmacy, Chemical and Genetical Pharmacology Laboratory, 11 Rue Pierre et Marie Curie, Paris, France
Poster abstracts / Nitric Oxide 17 (2007) S20–S29
Among all artemisinin derivatives, artesunate revealed the highest cytotoxicity against human CEM-CCRF leukemia cells. Measurement of ROS showed that artesunate produced the highest amount of ROS. In RAW264.7 mouse macrophages, which are the well-established model cell line to analyze NO generation, artesunate produced the highest NO amount. The IC50 value of RAW264.7 cells for artesunate was 3 lM. In order to associate the cytotoxic effects with specific alteration in gene expression related to NO and ROS metabolism and signalling microarray analyses were conducted. RAW cells were treated with 3 and 30 lM of artesunate using DMSO as vehicle control followed by microarray analysis. We conclude that the cleavage of the endoperoxide bridge of artemisinins play a crucial role for the cytotoxic activity. Relevant signalling pathways related to NO and ROS will be discussed.
reduce tumour load in vivo with no observed side-effects. In growth-inhibited cells, primed HS is degraded by NO to oligosaccharides that are transported to the nucleus. Accordingly, NO depletion reduces nuclear localization of the oligosaccharides and abolishes the growth-inhibitory effect of the xyloside (2-4). Cancer cells accumulate misfolded carbonylated proteins. Recently, we have reported that conjugates between NO-generated HS oligosaccharides and carbonylated proteins colocalize and coprecipitate with proteasomes in both carcinoma and neuroblastoma cells (5). References 1. Fransson et al., Cell. Mol. Life Sci. 61 (2004) 1016, 2. Mani et al., Cancer Res. 58 (1998) 1099, 3. Mani et al., Glycobiology 14 (2004) 387, 4. Jacobsson et al. J. Med. Chem. 49 (2006) 1932, 5. Mani et al. J. Biol. Chem. 282 (2007) 21934. doi:10.1016/j.niox.2007.09.055
doi:10.1016/j.niox.2007.09.053
C9. Influence of nitric oxide and tissue oxygenation on invasive capacity of urinary bladder cancer C. Ewe, N.P. Wiklund, P.J. de Verdier Department of Molecular Medicine and Surgery, Forskningscentrum M3:02, Urologylab, Stockholm, Sweden Abstract Background: High-risk invasive bladder tumours and carcinoma in situ (CIS) may be treated with the immunotherapeutic drug bacillus Calmette-Guerin (BCG), which is shown to increase activity of nitric oxide synthases and highly elevate the concentration of nitric oxide (NO) in the bladder of patients after treatment. The transcription factor Hypoxia-inducible factor 1 (HIF1)’s stabilization is dependent of the oxygen levels in the cell, but may also be affected by the NO levels which the cell is exposed to. Objective: Bladder cancer cell-lines derived from transitional cell carcinomas (J82, DAG-1, and T24) and control cell-lines with high (HT1080) and low (NIH3T3) invasive properties are used in a chemoinvasion assay to study how the invasive capacity of urinary bladder cancer is influenced by exogenously added NO (DETA-NO; 0.01–3 mM) and altered levels of tissue oxygenation (normoxia (21%), hypoxia (3%) and anoxia (0%)). Results and conclusions: Low levels of NO enhance cell proliferation whereas high levels are cytotoxic. Under anoxic (0%) conditions the studied cell-lines seem more sensitive to the cytotoxic effect of NO compared to when exposed to atmospheric (21%) oxygen levels. The response curves are similarly shaped, but during anoxic conditions the curves are shifted to give an earlier response. We have also seen that low levels of exogenously added NO (0.01–0.1 mM) have an effect on the invasion capacity of some cell lines during anoxic conditions. doi:10.1016/j.niox.2007.09.054
C10. Heparan sulfate, NO and cancer L.A. Fransson, F. Cheng, U. Ellervik, K. Mani Lund University, Experimental Medical Science, BMC A13, Lund, Sweden Many growth factors are activated by cell-surface heparan sulfate (HS). Altered expression of HS-containing proteoglycans, especially glypicans (Gpc), is correlated with neoplastic transformation. When expression of a Gpc is silenced in tumour cells, both growth-factor response and tumour formation are depressed. We have shown that Gpc-1 can be S-nitrosylated by NO in the core protein. During endocytosis and recycling of Gpc-1 via endosomes and the Golgi, NO is released and cleaves HS chains at specific sites. This reaction can be enhanced by vitamin C (reviewed in 1). We have also shown that specific naphthyl-xylosides, while serving as primers for HS synthesis, selectively inhibit tumour cell-growth in vitro by inducing apoptosis. Perorally administered xylosides also
C11. Hypoxia increases the shedding of MHC class I chain-related molecule (MIC) from the surface of tumour cells: Involvement of nitric oxide signalling Charles H. Graham a,b,c, Nianping Hu a, Karim Sheikhi a, Eugene Chung a, Hugh Pross d, D. Robert Siemens a,b a Department of Anatomy and Cell Biology, Queen’s University, Kingston, Ontario, Canada b Department of Urology, Queen’s University, Kingston, Ontario, Canada c Department of Pharmacology and Toxicology, Queen’s University, Kingston, Ontario, Canada d Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario, Canada The major histocompatibility complex class I chain-related (MIC) molecules play important roles in tumour immunosurveillance through their interaction with the NKG2D receptor on NK and cytotoxic T cells. Furthermore, shedding of MIC from the cell membrane represents a potential mechanism of tumour escape from NKG2D-mediated immunosurveillance. Tumour hypoxia is associated with a poor clinical outcome for cancer patients and with aggressive tumour cell phenotypes. We show that hypoxia contributes to tumour cell shedding of MIC through a mechanism involving impaired NO signalling. While hypoxia increased MIC shedding in DU145 prostate cancer cells, activation of NO signalling inhibited the hypoxia-mediated MIC shedding. Similar to incubation in hypoxia, inhibition of endogenous NO signalling increased MIC shedding. Parallel studies showed a hypoxia-mediated increase in tumour cell resistance to lysis by IL-2 activated peripheral blood lymphocytes (PBLs), and an NO-mediated attenuation of this hypoxia-mediated resistance to lysis. Furthermore, inhibition of NO production led to resistance to PBL-mediated lysis. Inclusion of a blocking anti-MIC antibody resulted in lower cytotoxic activity against DU145 cells. In the presence of this antibody, hypoxia did not cause a further increase in resistance to PBL-mediated killing and activation of NO signalling did not restore sensitivity to lysis. These findings suggest that the hypoxic tumour microenvironment contributes to impaired immunosurveillance and that activation of NO signalling is of potential use in cancer immunotherapy. doi:10.1016/j.niox.2007.09.056
C12. Performances and applicability of electrochemical NO-sensor for in vivo detection of nitric oxide in tumor-bearing mice S. Griveau, C. Dume´zy, J. Seguin, G.G. Chabot, D. Scherman, F. Bedioui INSERM, U640, CNRS, UMR8151, Universite´ Rene´ Descartes Paris 5, Ecole Nationale Supe´rieure de Chimie de Paris, Faculty of Pharmacy, Chemical and Genetical Pharmacology Laboratory, 11 Rue Pierre et Marie Curie, Paris, France