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916 Modeling Pharmacological Inhibition of Bruton's Tyrosine Kinase (BTK) as a Therapy for Insulinoma and Pancreatic Ductal Adenocarcinoma
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european journal of cancer 48, suppl. 5 (2012) S25–S288
CII ubiquinone (UbQ)-binding site (Qp ). Mutation of the UbQ-binding S68 within the Qp of the CII’s SDHC subunit suppressed both ROS generation and apoptosis induction in response to MitoVES. In vivo studies indicated that MitoVES also significantly suppresses tumour progress and the associated angiogenesis. Conclusions: We propose that mitochondrial targeting of VES maximises its anti-cancer efficacy, endowing it with a substantial translational relevance. 916 Modeling Pharmacological Inhibition of Bruton’s Tyrosine Kinase (BTK) as a Therapy for Insulinoma and Pancreatic Ductal Adenocarcinoma L. Soucek1 , D. Masso-Vall ´ es ´ 1 , T. Jauset1 , J.R. Whitfield1 , J.J. Buggy2 , N.M. Sodir3 , R. Kortlever3 , L. Brown Swigart3 , G.I. Evan4 . 1 Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain, 2 Pharmacyclics Inc., Sunnyvale, USA, 3 University of California San Francisco, Department of Pathology, San Francisco, USA, 4 University of Cambridge, Department of Biochemistry, Cambridge, United Kingdom Background: Myc, a pleiotropic transcription factor that is deregulated and/or over-expressed in most human cancers, instructs multiple extracellular programs that are required to sustain the complex microenvironment needed for tumor maintenance, including remodeling of tumor stroma, angiogenesis and inflammation. We previously showed in a model of pancreatic Beta cell tumorigenesis that acute Myc activation in vivo triggers rapid recruitment of mast cells to the tumor site and that this is absolutely required for angiogenesis and macroscopic tumor expansion. Moreover, systemic inhibition of mast cell degranulation with sodium cromoglycate induced death of tumor and endothelial cells in established tumors. Hence, mast cells are required both to establish and to maintain the tumors. While this intimates that selective inhibition of mast cell function could be therapeutically efficacious, cromoglycate is not a practical drug for systemic delivery in humans and no other systemic inhibitor of mast cell degranulation has hitherto been available. Materials and Methods: PCI-32765 is a novel inhibitor of Bruton’s tyrosine kinase (Btk) that blocks mast cell degranulation and is currently in clinical trial as a therapy for B cell non-Hodgkin’s lymphoma. We made use of systemic treatment of insulinoma- and pancreatic ductal carcinoma (PDAC)-bearing mice with PCI-32765. Results: Here, we show that systemic treatment of insulinoma-bearing mice with PCI-32765 efficiently inhibits Btk, blocks mast cell degranulation and triggers collapse of tumor vasculature and tumor regression. We also show that PDAC-bearing mice treated with PCI-32765 survive longer and present reduced tumor stroma, suggesting that combinatorial therapy with PCI-32765 and standard of care could be a feasible therapeutic approach. Conclusions: Our data reinforce the notion that mast cell function is required for maintenance of certain tumor types and indicate that the Btk inhibitor PCI32765 may be useful in treating such diseases and likely other more aggressive pancreatic cancers. 918 Multidrug Resistant Tumor Cells-derived Microparticles Modulate P-glycoprotein and Anti-apoptotic Proteins Expression Through Regulation of MicroRNAs and Oncogenic-related Pathways in Sensitive Cancer Cells P. Souza1 , A.L. Souza Cruz2 , L.O. Silva1 , J.P.B. Viola2 , R.C. Maia1 . 1 Instituto ˆ Nacional de Cancer, Lab. Hemato-Oncologia Celular e Molecular, Rio De ˆ Biologia Celular, Rio De Janeiro, Brazil, 2 Instituto Nacional de Cancer, Janeiro, Brazil Background: Multidrug resistance (MDR) prevents the successful clinical cancer treatment due to P-glycoprotein (Pgp) overexpression, a drug efflux pump, and resistance to apoptosis derived by inhibitor of apoptosis proteins (IAPs), such as survivin and XIAP. Oncogenic pathways can regulate Pgp and IAPs genes via transcriptional factors, such as NFkB. Moreover, YB-1 can bind to regulatory regions of Pgp and EGFR genes. Also, miRNAs are involved to Pgp expression in resistant cells. It has been reported that Pgp expression can be acquired through transfer of membrane microparticles (MPs) from donor Pgp+ cells to recipient Pgp− cells. However, changes involved in IAPs and oncogenic pathways induced by Pgp+ MPs on recipient cells are not elucidated. Materials and Methods: K562-Lucena cells (Pgp+ , derived from myeloid leukemia) was co-cultured with MCF7 and A549 cells (Pgp− , derived from breast and lung carcinoma) for 24 or 48 h separated by Transwell inserts. After co-culture or culture with conditioned medium from Pgp+ cells, Pgp, survivin, XIAP, YB-1, IkBa and NFkB expression were assessed by flow cytometry, Western blot, immunofluorescence and qRT-PCR in recipient cells. The miR21, -27a and -451 were assessed by qRT-PCR. Also, apoptosis was assessed by annexin-V/PI staining after drug treatment. All proteins and miRNAs above were analyzed in isolated MPs from Pgp+ cells. Results: It was observed intercellular transfer of Pgp from donor cells to recipient cells which after co-culture became Pgp+ ; also, clusters of Pgp were identified in recipient cells suggesting the presence of MPs. Recipient cells
Sunday 8 − Tuesday 10 July 2012
showed an enhancement of Pgp mRNA expression, and following treatment with drugs cells showed a decreased of apoptosis index. These cells showed peri or nuclear YB-1 localization with no changes in protein expression. Also, it was observed an enhancement of EGFR expression and its peri and nuclear localization, indicating its activation. The negative regulator of NFkB, IkBa, was degraded, and thus NFkB was translocated to the nucleus. Parallel to this, it was observed an enhancement of survivin and XIAP mRNA and protein expression. The miR-21, -27a and -451 were upregulated, supporting the MDR acquisition in recipient cells. No changes were observed in Pgp, XIAP, survivin, YB-1 and IkBa in recipient cells cultured with conditioned medium. In MPs analysis was identified for the first time survivin, XIAP, YB-1 and miR-21 expression, along with Pgp, miR-27a and -451. Conclusions: Sensitive cells acquired non-genetic Pgp and IAPs expression, and consequently, MDR phenotype through resistant cells-derived MPs. Also, YB-1 and NFkB pathways could be involved with upregulation of mRNA Pgp and IAPs, and additionally contribute to activation of EGFR. The presence of miRNAs supports the recipient cells MDR phenotype. These findings contribute to our knowledge for the emergence of MDR in cancer cells and could be helpful for new treatment approaches. 920 The Effects of N-Acetyl-L-Cysteine on Bleomycin Induced Apoptosis Were Determinated in Malignant Testicular Germ Cell Tumours by Flow Cytometry E. Kucuksayan1 , A. Cort1 , S.G. Yucel1 , T. Ozben1 . 1 Akdeniz University, Biochemistry, Antalya, Turkey Background: Bleomycin is used commonly in the treatment of testicular cancer. Bleomycin causes an increase of ROS resulting in oxidative stress and induces apoptosis. Oxidative stress has been shown to induce apoptosis in cancer cells. Therefore, one might suspect that antioxidants may inhibit reactive oxygen species (ROS) and prevent apoptosis of cancer cells. There is an intense argument on the concurrent use of antioxidants with the conventional cancer treatments. This argument was based on the fact that some chemotherapy drugs generate reactive oxygen species (ROS) and antioxidants may prevent cancer cells to be killed by ROS. N-Acetyl-L-Cysteine (NAC) is a compound known to have powerful antioxidant properties. Due to the property, in our study we examined the effects of NAC on oxidative stress created by Bleomycin. The aim of our study was to clarify the molecular mechanism of apoptosis which induced by Bleomycin and the effect of NAC on apoptosis in human testicular cancer cell line. We have choosen the wild-type p53 expressing cell line, NTera-2 (NT 2). Material and Method: Apoptosis was detected using an FITC Annexin-V Apoptosis Detection Kit by using flow cytometry. Control, Bleomycin, NAC and Bleomycin+NAC groups were exemined. We incubated NT 2 cells with different concentrations of Bleomycin (400 mg/ml) and NAC (5 mM) for 6 hours. Results and Discussion: NT 2 cells were incubated with Bleomycin, NAC and Bleomycin+NAC for 6 h (pre-apoptotic time). The percentage of apoptosis was determined by FACS analysis utilizing Annexin V and PI. Incubation of the cells with 400 mg/ml Bleomycin induced apoptosis 48.6% (Q1−1 preapoptotic side and Q2−2 post-apoptotic side) detected compared to the control cells. Incubation with 5 mM NAC did not increase significantly percent of apoptosis compared to the control cells. Pretreatment of the NT 2 cells 400 mg/ml Bleomycin with NAC at 5 mM drastically reduced the proportion of apoptotic cell populations from 48.6% to 29.5% (Q1−1 and Q2−2) compared to Bleomycin group. Our results indicated that Bleomycin mediated apoptosis was suppressed by NAC in NT 2 cells. Conclusion: Finally, we think it is important for prognosis of the disease of the chemotherapeutic treatment of patients with testicular cancer all the experiment results. During treatment Bleomycin kills testicular cancer cells by generated ROS. Because of antioxidants prevent ROS, we believe that the use of antioxidants during treatment with Bleomycin negatively affect the treatment process. 921 Furan-based Saccharide Mimetics Inhibit Tumour Cell Adhesion, Migration and Distinct Stages of Angiogenesis E. Frei1 , G. Marano2 , A. Breuer1 , A. Merling3 , M. Frank4 , R. Schwartz-Albiez3 . 1 Deutsches Krebsforschungszentrum, Preventive Oncology, Heidelberg, Germany, 2 Deutsches Krebsforschungszentrum, Molecular Toxicology, Heidelberg, Germany, 3 Deutsches Krebsforschungszentrum, Translational Immunology, Heidelberg, Germany, 4 Biognos AB, Goetoborg, Sweden Introduction: Tumour cell adhesion to extracellular matrix proteins (ECM) is mediated by protein-protein and protein (lectin)-oligosaccharide interactions. Angiogenesis, which involves stages of endothelial cell adhesion and migration, is vital for tumour growth and metastasis formation. Since similar molecules are involved in both cellular activities, we further developed furanbased saccharide mimetics, which were previously shown to interfere with these cell-ECM interactions (Kim EY. et al. ChemBiochem 2005, 6, 422).
european journal of cancer 48, suppl. 5 (2012) S25–S288
CII ubiquinone (UbQ)-binding site (Qp ). Mutation of the UbQ-binding S68 within the Qp of the CII’s SDHC subunit suppressed both ROS generation and apoptosis induction in response to MitoVES. In vivo studies indicated that MitoVES also significantly suppresses tumour progress and the associated angiogenesis. Conclusions: We propose that mitochondrial targeting of VES maximises its anti-cancer efficacy, endowing it with a substantial translational relevance. 916 Modeling Pharmacological Inhibition of Bruton’s Tyrosine Kinase (BTK) as a Therapy for Insulinoma and Pancreatic Ductal Adenocarcinoma L. Soucek1 , D. Masso-Vall ´ es ´ 1 , T. Jauset1 , J.R. Whitfield1 , J.J. Buggy2 , N.M. Sodir3 , R. Kortlever3 , L. Brown Swigart3 , G.I. Evan4 . 1 Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain, 2 Pharmacyclics Inc., Sunnyvale, USA, 3 University of California San Francisco, Department of Pathology, San Francisco, USA, 4 University of Cambridge, Department of Biochemistry, Cambridge, United Kingdom Background: Myc, a pleiotropic transcription factor that is deregulated and/or over-expressed in most human cancers, instructs multiple extracellular programs that are required to sustain the complex microenvironment needed for tumor maintenance, including remodeling of tumor stroma, angiogenesis and inflammation. We previously showed in a model of pancreatic Beta cell tumorigenesis that acute Myc activation in vivo triggers rapid recruitment of mast cells to the tumor site and that this is absolutely required for angiogenesis and macroscopic tumor expansion. Moreover, systemic inhibition of mast cell degranulation with sodium cromoglycate induced death of tumor and endothelial cells in established tumors. Hence, mast cells are required both to establish and to maintain the tumors. While this intimates that selective inhibition of mast cell function could be therapeutically efficacious, cromoglycate is not a practical drug for systemic delivery in humans and no other systemic inhibitor of mast cell degranulation has hitherto been available. Materials and Methods: PCI-32765 is a novel inhibitor of Bruton’s tyrosine kinase (Btk) that blocks mast cell degranulation and is currently in clinical trial as a therapy for B cell non-Hodgkin’s lymphoma. We made use of systemic treatment of insulinoma- and pancreatic ductal carcinoma (PDAC)-bearing mice with PCI-32765. Results: Here, we show that systemic treatment of insulinoma-bearing mice with PCI-32765 efficiently inhibits Btk, blocks mast cell degranulation and triggers collapse of tumor vasculature and tumor regression. We also show that PDAC-bearing mice treated with PCI-32765 survive longer and present reduced tumor stroma, suggesting that combinatorial therapy with PCI-32765 and standard of care could be a feasible therapeutic approach. Conclusions: Our data reinforce the notion that mast cell function is required for maintenance of certain tumor types and indicate that the Btk inhibitor PCI32765 may be useful in treating such diseases and likely other more aggressive pancreatic cancers. 918 Multidrug Resistant Tumor Cells-derived Microparticles Modulate P-glycoprotein and Anti-apoptotic Proteins Expression Through Regulation of MicroRNAs and Oncogenic-related Pathways in Sensitive Cancer Cells P. Souza1 , A.L. Souza Cruz2 , L.O. Silva1 , J.P.B. Viola2 , R.C. Maia1 . 1 Instituto ˆ Nacional de Cancer, Lab. Hemato-Oncologia Celular e Molecular, Rio De ˆ Biologia Celular, Rio De Janeiro, Brazil, 2 Instituto Nacional de Cancer, Janeiro, Brazil Background: Multidrug resistance (MDR) prevents the successful clinical cancer treatment due to P-glycoprotein (Pgp) overexpression, a drug efflux pump, and resistance to apoptosis derived by inhibitor of apoptosis proteins (IAPs), such as survivin and XIAP. Oncogenic pathways can regulate Pgp and IAPs genes via transcriptional factors, such as NFkB. Moreover, YB-1 can bind to regulatory regions of Pgp and EGFR genes. Also, miRNAs are involved to Pgp expression in resistant cells. It has been reported that Pgp expression can be acquired through transfer of membrane microparticles (MPs) from donor Pgp+ cells to recipient Pgp− cells. However, changes involved in IAPs and oncogenic pathways induced by Pgp+ MPs on recipient cells are not elucidated. Materials and Methods: K562-Lucena cells (Pgp+ , derived from myeloid leukemia) was co-cultured with MCF7 and A549 cells (Pgp− , derived from breast and lung carcinoma) for 24 or 48 h separated by Transwell inserts. After co-culture or culture with conditioned medium from Pgp+ cells, Pgp, survivin, XIAP, YB-1, IkBa and NFkB expression were assessed by flow cytometry, Western blot, immunofluorescence and qRT-PCR in recipient cells. The miR21, -27a and -451 were assessed by qRT-PCR. Also, apoptosis was assessed by annexin-V/PI staining after drug treatment. All proteins and miRNAs above were analyzed in isolated MPs from Pgp+ cells. Results: It was observed intercellular transfer of Pgp from donor cells to recipient cells which after co-culture became Pgp+ ; also, clusters of Pgp were identified in recipient cells suggesting the presence of MPs. Recipient cells
Sunday 8 − Tuesday 10 July 2012
showed an enhancement of Pgp mRNA expression, and following treatment with drugs cells showed a decreased of apoptosis index. These cells showed peri or nuclear YB-1 localization with no changes in protein expression. Also, it was observed an enhancement of EGFR expression and its peri and nuclear localization, indicating its activation. The negative regulator of NFkB, IkBa, was degraded, and thus NFkB was translocated to the nucleus. Parallel to this, it was observed an enhancement of survivin and XIAP mRNA and protein expression. The miR-21, -27a and -451 were upregulated, supporting the MDR acquisition in recipient cells. No changes were observed in Pgp, XIAP, survivin, YB-1 and IkBa in recipient cells cultured with conditioned medium. In MPs analysis was identified for the first time survivin, XIAP, YB-1 and miR-21 expression, along with Pgp, miR-27a and -451. Conclusions: Sensitive cells acquired non-genetic Pgp and IAPs expression, and consequently, MDR phenotype through resistant cells-derived MPs. Also, YB-1 and NFkB pathways could be involved with upregulation of mRNA Pgp and IAPs, and additionally contribute to activation of EGFR. The presence of miRNAs supports the recipient cells MDR phenotype. These findings contribute to our knowledge for the emergence of MDR in cancer cells and could be helpful for new treatment approaches. 920 The Effects of N-Acetyl-L-Cysteine on Bleomycin Induced Apoptosis Were Determinated in Malignant Testicular Germ Cell Tumours by Flow Cytometry E. Kucuksayan1 , A. Cort1 , S.G. Yucel1 , T. Ozben1 . 1 Akdeniz University, Biochemistry, Antalya, Turkey Background: Bleomycin is used commonly in the treatment of testicular cancer. Bleomycin causes an increase of ROS resulting in oxidative stress and induces apoptosis. Oxidative stress has been shown to induce apoptosis in cancer cells. Therefore, one might suspect that antioxidants may inhibit reactive oxygen species (ROS) and prevent apoptosis of cancer cells. There is an intense argument on the concurrent use of antioxidants with the conventional cancer treatments. This argument was based on the fact that some chemotherapy drugs generate reactive oxygen species (ROS) and antioxidants may prevent cancer cells to be killed by ROS. N-Acetyl-L-Cysteine (NAC) is a compound known to have powerful antioxidant properties. Due to the property, in our study we examined the effects of NAC on oxidative stress created by Bleomycin. The aim of our study was to clarify the molecular mechanism of apoptosis which induced by Bleomycin and the effect of NAC on apoptosis in human testicular cancer cell line. We have choosen the wild-type p53 expressing cell line, NTera-2 (NT 2). Material and Method: Apoptosis was detected using an FITC Annexin-V Apoptosis Detection Kit by using flow cytometry. Control, Bleomycin, NAC and Bleomycin+NAC groups were exemined. We incubated NT 2 cells with different concentrations of Bleomycin (400 mg/ml) and NAC (5 mM) for 6 hours. Results and Discussion: NT 2 cells were incubated with Bleomycin, NAC and Bleomycin+NAC for 6 h (pre-apoptotic time). The percentage of apoptosis was determined by FACS analysis utilizing Annexin V and PI. Incubation of the cells with 400 mg/ml Bleomycin induced apoptosis 48.6% (Q1−1 preapoptotic side and Q2−2 post-apoptotic side) detected compared to the control cells. Incubation with 5 mM NAC did not increase significantly percent of apoptosis compared to the control cells. Pretreatment of the NT 2 cells 400 mg/ml Bleomycin with NAC at 5 mM drastically reduced the proportion of apoptotic cell populations from 48.6% to 29.5% (Q1−1 and Q2−2) compared to Bleomycin group. Our results indicated that Bleomycin mediated apoptosis was suppressed by NAC in NT 2 cells. Conclusion: Finally, we think it is important for prognosis of the disease of the chemotherapeutic treatment of patients with testicular cancer all the experiment results. During treatment Bleomycin kills testicular cancer cells by generated ROS. Because of antioxidants prevent ROS, we believe that the use of antioxidants during treatment with Bleomycin negatively affect the treatment process. 921 Furan-based Saccharide Mimetics Inhibit Tumour Cell Adhesion, Migration and Distinct Stages of Angiogenesis E. Frei1 , G. Marano2 , A. Breuer1 , A. Merling3 , M. Frank4 , R. Schwartz-Albiez3 . 1 Deutsches Krebsforschungszentrum, Preventive Oncology, Heidelberg, Germany, 2 Deutsches Krebsforschungszentrum, Molecular Toxicology, Heidelberg, Germany, 3 Deutsches Krebsforschungszentrum, Translational Immunology, Heidelberg, Germany, 4 Biognos AB, Goetoborg, Sweden Introduction: Tumour cell adhesion to extracellular matrix proteins (ECM) is mediated by protein-protein and protein (lectin)-oligosaccharide interactions. Angiogenesis, which involves stages of endothelial cell adhesion and migration, is vital for tumour growth and metastasis formation. Since similar molecules are involved in both cellular activities, we further developed furanbased saccharide mimetics, which were previously shown to interfere with these cell-ECM interactions (Kim EY. et al. ChemBiochem 2005, 6, 422).