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215 Targeting thioredoxin reductase ‐ a possible new treatment for malignant mesothelioma
S52
Sunday, October 22, 2006 / Poster Session: Novel agents/novel targets I
dian FFS were 2.3 and 4.1 months and the median OS were 4.2 and 9.0 months in chemo-naïve and previously-treated patients, respectively. Conclusions: Sorafenib demonstrated modest activity in this phase II trial but did not meet its primary statistical endpoint. Ongoing correlative science studies including expression of p-ERK 1/2, baseline VEGF and PDGF levels, and B-raf mutations, are being performed to help identify patient subsets who may benefit (PR or SD) from sorafenib.
212
TGF-beta inhibition augments adenoviral-IFNbeta (Ad.IFNβ gene therapy in a murine mesothelioma model
S.S. Kim 1 , J. Sun 1 , A. Workman 1 , M. Corbley 2 , L. Sung 2 , W. Lee 2 , L. Ling 2 , S.M. Albelda 1 . 1 University of Pennsylvania, Philadelphia, USA; 2 Biogen-Idec, San Diego, CA Background: Immunogene therapy with Ad.IFN β (a CD8+ T-cell dependent approach), is less effective against larger MM murine tumors, likely due to the fact that as the tumor grows, it secretes or induces secretion of cytokines, like TGF-β, that create an immunosuppressive tumor micro-environment (TME). We hypothesized that inhibition of TGF-β would augment the efficacy of intratumoral Ad.IFN β in AB12 (murine MM) tumors. Methods: AB12 tumors were grown on the flanks of Balb/c mice. TGF-β effects were inhibited using SM16, a novel orally available, small molecule TGF-β type I receptor kinase inhibitor (from Biogen-Idec). The effect of combining oral SM16 with intratumoral (it)Ad-INF β therapy (10e9 pfu of virus) on large tumors was measured. Changes in the TME were demonstrated by measuring cytokine mRNA expression levels and cytokine/chemokine bead assays. Changes in cell trafficking were studied using flow cytometry on tumors. Results: AB12 tumors showed constitutive phospho-Smad2 levels that was abolished for 3 hrs by a single i.p. bolus of 20mg/kg SM16. AB12 tumorbearing mice treated with SM16 and Ad-INF β alone showed some decrease in tumor volume, but mice treated with SM16 plus Ad-INF β were significantly smaller than mice treated with single therapies. Flow cytometry demonstrated increased CD8 cell infiltration in the combination group compared to control tumors and tumors treated with SM16 or Ad.IFN β alone. The mechanism likely involves changes in tumor microenvironment. Tumors from the SM16 treated mice showed increased expression levels of mRNA for immunostimulatory cyto/chemokines (IP-10, RANTES, TNF-α, INF-γ, IL-12) along with increased protein levels of RANTES, IL-6, MIP-1a. Conclusions: Blockade of the TGF-β signal pathway alters the tumor microenvironment and augments the tumor inhibitory effects of Ad-INF β therapy in AB12 tumor. This approach may have utility in our ongoing clinical trials using intrapleural Ad.IFN β or with other immuntherapies.
213
Asbestos in the past, nanomaterials in the future?
V.C. Sanchez, P. Weston, X. Liu, R.H. Hurt, A.B. Kane. Brown University, Providence, USA Background: Macrophages are the first line of defense in the immune system. Their primary functions are engulfment and elimination of virus, bacteria and foreign bodies as well as phagocytosis of cell debris. Macrophages express several types of receptors on their membrane surfaces, conferring on them the capacity to recognize a wide number of molecules, particles and cell types. Upon phagocytosis of foreign bodies by macrophages, reactive oxygen species and free radicals can be generated by Fenton chemistry, Haber-Weiss cycling or homolytic rupture of carbon-hydrogen bonds. As a consequence, lipid peroxidation, protein oxidation, DNA damage and cell death can occur. In parallel, released chemokines and cytokines can initiate the inflammatory process. Nanomaterials have been proposed as an alternative for drug delivery and gene therapy because they can be designed for attachment of specific molecules and functional groups. Nanomaterials interfere with conventional methods for the study cellular toxicity because of their unique chemical and physical properties. They aggregate in physiological solutions making it difficult to determine doses that reflect real exposure. It is unclear if macrophages can recognize and engulf nanomaterials by specific receptors or by other types of interactions. In addition, aggregated nanomaterials interfere with some widely-used endpoints for acute toxicity. Methods: In order to evaluate nanomaterial cytotoxicity, we used murine bone marrow derived macrophages as a model system. Since crocidolite asbestos fibers share some chemical and physical properties with nanomaterials and its toxic effects are well characterized in this system, we compared the following endpoints: rate of uptake, cellular localization, citotoxicity, mitochondrial membrane permeability, frustrated phagocytosis and lysosomal integrity. Results: In this study, we present several alternatives for evaluation of cytotoxicity of carbon nanofibers synthesized as model materials in our laboratory as well as samples of commercial multiwalled carbon nanotubes.
214
COX-2 specific inhibitors demonstrate a cytotoxic effect independent of COX-2 protein status in vitro
S.L. O’Kane, L. Cawkwell, J. Greenman, M.J. Lind. University of Hull, Hull, UK Background: Cells expressing COX-2 have been shown to demonstrate increased proliferation and reduced apoptosis. COX-2 has been reported to be overexpressed in a variety of tumours including malignant pleural mesothelioma (MPM). COX-2 can be inhibited by both non-specific and specific inhibitors, which bind with differing specificity. We aimed to assess the cytotoxic effect of COX-2 specific inhibitors on three commercially available MPM cell lines. Methods: The cells were treated with a selection of COX-2 inhibitors at a concentration range of 1-250μM. MTT assays were used to analyze the cytotoxic effect of the inhibitors at selected time intervals after addition of drug (2hr, 24hr, 48hr and 72 hr). COX-2 protein expression status was determined by western blotting, and mRNA status by RT-PCR. Results: The IC50 values obtained were as follows: MSTO-211H cells with DuP-697 (231μM) and with PTPBS (180μM); NCI-H2052 cells with DuP-697 (86μM) and with PTPBS (236μM). IC50 values were not achieved using the any inhibitors on the cell line NCI-H2452 or using the inhibitor NS-398 in any cell line. All three MPM cell lines tested negative for COX-2 using western blotting analysis and expressed varying amounts of COX-2 mRNA. Conclusions: COX-2 specific inhibitors have a cytotoxic effect on some MPM cells. This appears to be independent of COX-2 protein status as COX-2 expression was not detected in the cell lines. Further analysis is required to determine the pathway(s) affected by these inhibitors.
215
Targeting thioredoxin reductase – a possible new treatment for malignant mesothelioma
G. Nilsonne 1 , A. Stein 1,2 , A.-K. Rundlöf 1 , A.P. Fernandes 1 , M. Björnstedt 1 , A. Hjerpe 1 , K. Dobra 1 . 1 Karolinska University Hospital in Huddinge, Sweden; 2 University of Dresden, Dresden, Germany Malignant mesothelioma retains the ability of normal mesothelial cells to transdifferentiate between an epithelioid and a fibroblast-like phenotype. Thus epithelioid and sarcomatoid tumor components may arise in different areas of the same tumor. Presence of sarcomatoid cells is correlated to increased resistance to therapy and a worse prognosis. Recent studies using microarray technology indicate that these two components are fundamentally different in terms of their biology and present new potential target molecules. Thioredoxin reductase (TrxR1) is a redox enzyme with protective and regulatory functions. It is highly overexpressed in malignant mesothelioma. We have shown that sodium selenite inhibits the activity of TrxR1 and induces apoptosis through the formation of ROS. This effect is most pronounced in the sarcomatoid mesothelioma cells. Furthermore, selenite appears to potentiate the effects of doxorubicin in the epithelioid cells. The present study aims to investigate the mechanisms whereby selenite exerts its actions. Immunocytochemical analysis of selenite treated cells showed activation of p53. FACS analysis showed activation of Bax, whereas caspase-3 was only partially activated. M-30 Apoptosense ELISA analysis for specifically cleaved cytokeratin 18 by caspases also showed a partial caspase activity induced by selenite. Inhibition of caspase activity by the pan-caspase inhibitor z-VAD-fmk did not attenuate the proapoptotic effects of selenite. These findings suggest a non-classical apoptosis mechanism where caspases are not necessarily the ultimate effectors. Sodium selenite is an interesting new possibility for the treatment of malignant mesothelioma. Further investigations of its efficacy, mechanism of action, and synergistic effects with other drugs, will be undertaken at our laboratory. For this purpose, we are currently establishing a SCID mouse model of malignant mesothelioma.
216
Preclinical evaluation of histone deacetylase inhibitors in malignant mesothelioma
S. Sharma, Y. Giraud, P. Dino, N. Vogelzang. Nevada Cancer Institute, Las Vegas, USA Background: Histones are part of the core proteins of nucleosomes, and acetylation and deacetylation of these proteins play a role in the regulation of gene expression. Acetylation neutralizes the charge of histones and generates a more open DNA conformation promoting expression of the corresponding genes. HDAC inhibitors (HDAIs) have been shown to induce differentiation, cell cycle arrest or apoptosis in cultured tumor cells, and to inhibit the growth of tumors in animal models. Various HDAIs are in clinical trials and there are preliminary data for efficacy in mesothelioma. Methods: An in vitro investigation was carried out to study the effect of various HDAIs in mesothelioma. Eight mesothelioma cell lines were selected. Single agent efficacy of the Hydroxamate class of HDAIs and combination therapy with chemotherapies like cisplatin and pemetrexed and other targeted agents e.g.
Sunday, October 22, 2006 / Poster Session: Novel agents/novel targets I
dian FFS were 2.3 and 4.1 months and the median OS were 4.2 and 9.0 months in chemo-naïve and previously-treated patients, respectively. Conclusions: Sorafenib demonstrated modest activity in this phase II trial but did not meet its primary statistical endpoint. Ongoing correlative science studies including expression of p-ERK 1/2, baseline VEGF and PDGF levels, and B-raf mutations, are being performed to help identify patient subsets who may benefit (PR or SD) from sorafenib.
212
TGF-beta inhibition augments adenoviral-IFNbeta (Ad.IFNβ gene therapy in a murine mesothelioma model
S.S. Kim 1 , J. Sun 1 , A. Workman 1 , M. Corbley 2 , L. Sung 2 , W. Lee 2 , L. Ling 2 , S.M. Albelda 1 . 1 University of Pennsylvania, Philadelphia, USA; 2 Biogen-Idec, San Diego, CA Background: Immunogene therapy with Ad.IFN β (a CD8+ T-cell dependent approach), is less effective against larger MM murine tumors, likely due to the fact that as the tumor grows, it secretes or induces secretion of cytokines, like TGF-β, that create an immunosuppressive tumor micro-environment (TME). We hypothesized that inhibition of TGF-β would augment the efficacy of intratumoral Ad.IFN β in AB12 (murine MM) tumors. Methods: AB12 tumors were grown on the flanks of Balb/c mice. TGF-β effects were inhibited using SM16, a novel orally available, small molecule TGF-β type I receptor kinase inhibitor (from Biogen-Idec). The effect of combining oral SM16 with intratumoral (it)Ad-INF β therapy (10e9 pfu of virus) on large tumors was measured. Changes in the TME were demonstrated by measuring cytokine mRNA expression levels and cytokine/chemokine bead assays. Changes in cell trafficking were studied using flow cytometry on tumors. Results: AB12 tumors showed constitutive phospho-Smad2 levels that was abolished for 3 hrs by a single i.p. bolus of 20mg/kg SM16. AB12 tumorbearing mice treated with SM16 and Ad-INF β alone showed some decrease in tumor volume, but mice treated with SM16 plus Ad-INF β were significantly smaller than mice treated with single therapies. Flow cytometry demonstrated increased CD8 cell infiltration in the combination group compared to control tumors and tumors treated with SM16 or Ad.IFN β alone. The mechanism likely involves changes in tumor microenvironment. Tumors from the SM16 treated mice showed increased expression levels of mRNA for immunostimulatory cyto/chemokines (IP-10, RANTES, TNF-α, INF-γ, IL-12) along with increased protein levels of RANTES, IL-6, MIP-1a. Conclusions: Blockade of the TGF-β signal pathway alters the tumor microenvironment and augments the tumor inhibitory effects of Ad-INF β therapy in AB12 tumor. This approach may have utility in our ongoing clinical trials using intrapleural Ad.IFN β or with other immuntherapies.
213
Asbestos in the past, nanomaterials in the future?
V.C. Sanchez, P. Weston, X. Liu, R.H. Hurt, A.B. Kane. Brown University, Providence, USA Background: Macrophages are the first line of defense in the immune system. Their primary functions are engulfment and elimination of virus, bacteria and foreign bodies as well as phagocytosis of cell debris. Macrophages express several types of receptors on their membrane surfaces, conferring on them the capacity to recognize a wide number of molecules, particles and cell types. Upon phagocytosis of foreign bodies by macrophages, reactive oxygen species and free radicals can be generated by Fenton chemistry, Haber-Weiss cycling or homolytic rupture of carbon-hydrogen bonds. As a consequence, lipid peroxidation, protein oxidation, DNA damage and cell death can occur. In parallel, released chemokines and cytokines can initiate the inflammatory process. Nanomaterials have been proposed as an alternative for drug delivery and gene therapy because they can be designed for attachment of specific molecules and functional groups. Nanomaterials interfere with conventional methods for the study cellular toxicity because of their unique chemical and physical properties. They aggregate in physiological solutions making it difficult to determine doses that reflect real exposure. It is unclear if macrophages can recognize and engulf nanomaterials by specific receptors or by other types of interactions. In addition, aggregated nanomaterials interfere with some widely-used endpoints for acute toxicity. Methods: In order to evaluate nanomaterial cytotoxicity, we used murine bone marrow derived macrophages as a model system. Since crocidolite asbestos fibers share some chemical and physical properties with nanomaterials and its toxic effects are well characterized in this system, we compared the following endpoints: rate of uptake, cellular localization, citotoxicity, mitochondrial membrane permeability, frustrated phagocytosis and lysosomal integrity. Results: In this study, we present several alternatives for evaluation of cytotoxicity of carbon nanofibers synthesized as model materials in our laboratory as well as samples of commercial multiwalled carbon nanotubes.
214
COX-2 specific inhibitors demonstrate a cytotoxic effect independent of COX-2 protein status in vitro
S.L. O’Kane, L. Cawkwell, J. Greenman, M.J. Lind. University of Hull, Hull, UK Background: Cells expressing COX-2 have been shown to demonstrate increased proliferation and reduced apoptosis. COX-2 has been reported to be overexpressed in a variety of tumours including malignant pleural mesothelioma (MPM). COX-2 can be inhibited by both non-specific and specific inhibitors, which bind with differing specificity. We aimed to assess the cytotoxic effect of COX-2 specific inhibitors on three commercially available MPM cell lines. Methods: The cells were treated with a selection of COX-2 inhibitors at a concentration range of 1-250μM. MTT assays were used to analyze the cytotoxic effect of the inhibitors at selected time intervals after addition of drug (2hr, 24hr, 48hr and 72 hr). COX-2 protein expression status was determined by western blotting, and mRNA status by RT-PCR. Results: The IC50 values obtained were as follows: MSTO-211H cells with DuP-697 (231μM) and with PTPBS (180μM); NCI-H2052 cells with DuP-697 (86μM) and with PTPBS (236μM). IC50 values were not achieved using the any inhibitors on the cell line NCI-H2452 or using the inhibitor NS-398 in any cell line. All three MPM cell lines tested negative for COX-2 using western blotting analysis and expressed varying amounts of COX-2 mRNA. Conclusions: COX-2 specific inhibitors have a cytotoxic effect on some MPM cells. This appears to be independent of COX-2 protein status as COX-2 expression was not detected in the cell lines. Further analysis is required to determine the pathway(s) affected by these inhibitors.
215
Targeting thioredoxin reductase – a possible new treatment for malignant mesothelioma
G. Nilsonne 1 , A. Stein 1,2 , A.-K. Rundlöf 1 , A.P. Fernandes 1 , M. Björnstedt 1 , A. Hjerpe 1 , K. Dobra 1 . 1 Karolinska University Hospital in Huddinge, Sweden; 2 University of Dresden, Dresden, Germany Malignant mesothelioma retains the ability of normal mesothelial cells to transdifferentiate between an epithelioid and a fibroblast-like phenotype. Thus epithelioid and sarcomatoid tumor components may arise in different areas of the same tumor. Presence of sarcomatoid cells is correlated to increased resistance to therapy and a worse prognosis. Recent studies using microarray technology indicate that these two components are fundamentally different in terms of their biology and present new potential target molecules. Thioredoxin reductase (TrxR1) is a redox enzyme with protective and regulatory functions. It is highly overexpressed in malignant mesothelioma. We have shown that sodium selenite inhibits the activity of TrxR1 and induces apoptosis through the formation of ROS. This effect is most pronounced in the sarcomatoid mesothelioma cells. Furthermore, selenite appears to potentiate the effects of doxorubicin in the epithelioid cells. The present study aims to investigate the mechanisms whereby selenite exerts its actions. Immunocytochemical analysis of selenite treated cells showed activation of p53. FACS analysis showed activation of Bax, whereas caspase-3 was only partially activated. M-30 Apoptosense ELISA analysis for specifically cleaved cytokeratin 18 by caspases also showed a partial caspase activity induced by selenite. Inhibition of caspase activity by the pan-caspase inhibitor z-VAD-fmk did not attenuate the proapoptotic effects of selenite. These findings suggest a non-classical apoptosis mechanism where caspases are not necessarily the ultimate effectors. Sodium selenite is an interesting new possibility for the treatment of malignant mesothelioma. Further investigations of its efficacy, mechanism of action, and synergistic effects with other drugs, will be undertaken at our laboratory. For this purpose, we are currently establishing a SCID mouse model of malignant mesothelioma.
216
Preclinical evaluation of histone deacetylase inhibitors in malignant mesothelioma
S. Sharma, Y. Giraud, P. Dino, N. Vogelzang. Nevada Cancer Institute, Las Vegas, USA Background: Histones are part of the core proteins of nucleosomes, and acetylation and deacetylation of these proteins play a role in the regulation of gene expression. Acetylation neutralizes the charge of histones and generates a more open DNA conformation promoting expression of the corresponding genes. HDAC inhibitors (HDAIs) have been shown to induce differentiation, cell cycle arrest or apoptosis in cultured tumor cells, and to inhibit the growth of tumors in animal models. Various HDAIs are in clinical trials and there are preliminary data for efficacy in mesothelioma. Methods: An in vitro investigation was carried out to study the effect of various HDAIs in mesothelioma. Eight mesothelioma cell lines were selected. Single agent efficacy of the Hydroxamate class of HDAIs and combination therapy with chemotherapies like cisplatin and pemetrexed and other targeted agents e.g.