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1053 New Strategies to Inhibit Tumor Cell Proliferation and Tumor Metastasis by S100P Monoclonal Antibodies
S254
european journal of cancer 48, suppl. 5 (2012) S25–S288
(LINE-1) methylation level in tumor, a surrogate of global methylaiton, predicts prognosis and response to oral fluoropyrimidines of CRC patients. In this study, the mechanism underling the association between LINE-1 methylation and sensitivity to 5-FU was investigated. Material and Method: CRC cells were used to analyze LINE-1 methylation level, LINE-1 mRNA expression and sensitivity to 5-FU by quantitative methylation specific PCR, Northern blotting and colony forming assay, respectively. Expression of phospho-histone H2AX was analyzed by Western immuno-blotting. RNA interference was employed to knockdown LINE-1 expression. Results and Discussion: LINE-1 methylation level in CRC cells was correlated with LINE-1 mRNA expression, in which hypomethylated CRC cell expressed high level of LINE-1 mRNA. The LINE-1-hypomethylated CRC cell also showed higher sensitivity to 5-FU. LINE-1 mRNA expression was increased after 5FU exposure in the cells showing LINE-1 hypomethylation, which is associated with upregulation of phospho-histone H2AX, a marker of DNA double strand break (DSB). These results suggest that 5FU action to induce DSB and exert cytotoxic effect is mediated, in part, by augmentation of LINE-1 expression in the CRC cell with LINE-1 hypomethylation because LINE-1 gene encodes endonuclease, a possible player to induce DSB. To investigate whether the increased expression of LINE-1by 5-FU is directly involved in the 5-FU action, LINE-1-expressing CRC cell was treated with 5-FU following the knockdown of LINE-1 by RNA interference. From the suggested mechanism that the LINE-1 expression has a role in 5-FU action, we expected that the knockdown of LINE-1 suppressed the cytotoxic effect of 5-FU. Contrary to our expectations, the knockdown of LINE-1 did not suppress but rather enhanced the activity of 5-FU. The study provided the novel strategy to enhance the effect of 5-FU although it did not reveal the mechanism underling the association between LINE-1 methylation and sensitivity to 5-FU. Conclusion: The LINE-1 expression itself has no significant role in the association between LINE-1 methylation level and the sensitivity to 5-FU. Suppression of LINE-1 is a possible strategy to enhance the activity of 5-FU in CRC with LINE-1 hypomethylation. 1052 Polymersomes − a Novel Drug Delivery System for the Delivery of Chemotherapeutic Agents to Oral Cancer Cells H.E. Colley1 , V. Hearnden1 , D. Cecchin2 , S. Armes3 , G. Battaglia2 , C. Murdoch1 , M.H. Thornhill1 . 1 The University of Sheffield, School of Clinical Dentistry, Sheffield, United Kingdom, 2 The University of Sheffield, Department of Biomedical Science, Sheffield, United Kingdom, 3 The University of Sheffield, Department of Chemistry, Sheffield, United Kingdom Introduction: Polymersomes are synthetic block co-polymers that selfassemble in water to form membrane-enclosed nanovesicles. Polymersomes have the potential to encapsulate and carry chemotherapeutic drugs into cells thereby reducing the off-target toxicity that often compromises anticancer treatment. Here, we assess the in vitro efficiency of polymersomes to encapsulate chemotherapeutic agents for the effective delivery to head and neck squamous cancer cells (HNSCC) cultured as monolayers and as three dimensional tumour spheroids. Materials and Methods: HNSCC were grown as monolayers or as multi-cellular tumour spheroids (MCTS), to model an expanding tumour island. Polymersomes loaded with rhodamine as a tracking molecule or chemotherapeutic agents (doxorubicin and/or paclitaxel) were applied to the cells for increasing lengths of time, and the cell viability, uptake and retention of polymersomes analysed. The effectiveness of chemotherapeutic-loaded polymersomes to kill HNSCC in monolayer and their efficacy in causing growth inhibition and damage of MCTS was also analysed. Furthermore, cellular uptake was investigated to determine the mechanism by which polymersomes are internalised by cells. Results and Discussion: Polymersomes loaded with rhodamine were internalised by HNSCC within 10 minutes of administration and maximal delivery was achieved within 30 minutes. When delivered to MCTS, polymersomes were internalised by over 80% of cells within 120 hours and importantly are shown to penetrate into hypoxic regions. Both doxorubicin and paclitaxel were loaded into polymersomes with high encapsulation efficiencies. Furthermore, both drugs can be dually loaded to enable combinational delivery. Loaded polymersomes demonstrated similar killing of HNSCC grown as monolayers compared to the same concentration of free drug. When delivered to MCTS, loaded polymersomes caused growth inhibition and extensive damage of the tumour spheroids. Current work involves the study of the cellular uptake mechanisms involved in the internalisation of the polymersomes to further the knowledge of this novel drug delivery system. Conclusion: Polymersomes offer a novel drug delivery system for the effective delivery of chemotherapeutics for the treatment of head and neck cancer.
Sunday 8 − Tuesday 10 July 2012
1053 New Strategies to Inhibit Tumor Cell Proliferation and Tumor Metastasis by S100P Monoclonal Antibodies J. Hernandez1 , M. Masa1 , R. Messeguer1 , F. Mitjans1 , J. Adan1 , J. Martinez1 , T. Coll1 , L. Padilla1 , S. Dakhel1 , R. Hervas1 . 1 Leitat Technological Center, Biomed Division, Barcelona, Spain Background: Metastasis represents the most devastating stage of malignancy and the leading death cause of cancer. An accurate description of the cellular and molecular mechanisms that underlie this multistep process would greatly facilitate the rational development of therapies that effectively allow metastatic disease to be controlled and treated. In the last years, intensive research in this field has shed light on some molecular targets as the novel metastatic factor S100P protein and its promising role as a key player in metastasis formation and poor clinical outcome. The identification and functional characterization of specific regulators of S100P might be exploited for therapeutic applications. Materials and Methods: Monoclonal antibodies able to neutralize the extracellular activity of S100P were obtained in our lab as therapeutic agents. In order to better elucidate the pivotal role of S100P in tumor cell proliferation and survival, we used either the proliferation measured by BrdU incorporation or MTT assay in human pancreatic BxPC3 and fibrosarcoma HT1080 cells. In addition, we developed a pancreatic orthotopic and a subcutaneous in vivo tumor model using BxPC3-luciferase cell line to understand the dynamic process of human metastasis progression and tumor growth and to validate our therapeutic mAbs as anti-metastatic and anti-tumoral drugs. Results: Here we address the mechanism of S100P in tumor proliferation showing an increased level of DNA synthesis induced by the extracellular activity of the protein and mAbs anti-S100P neutralized this effect. In addition, S100P induced an increased survival of fibrosarcoma HT1080 cells treated with Doxorubicin in a dose-dependent manner and neutralizing mAbs suppressed this activity. Moreover, a dramatic suppression of liver metastasis was observed after intraperitoneal administration of blocking antibodies to S100P in the orthotopic BxPC3 pancreatic tumor model. Relating to these results we also observed a reduction in the total-score staging from animals treated with our mAbs compared with Cromolyn (the unique described inhibitor of extracellular S100P) (p < 0.05). In a subcutaneous tumor model we also observed a tumor grow delay when animals were treated with blocking mAbs. Conclusion: As pointed out by our latest results, clinical therapeutic strategies directed to block the extracellular role of S100P protein by monoclonal antibodies could be a promising therapeutic strategy to decrease tumor growth and metastasis formation in cancer patients. Together with the utilization of chemotherapeutic agents, our studies provide new insights which could have significant implications for the development of novel therapeutic regimens targeting the metastasis. 1054 Evaluation of Hypoxia Inducible Factor 1-Alpha (HIF1A) After Treatment With Melatonin in Breast Cancer Cell Line D. Pires de Campos Zuccari1 , B.V. Jardim2 , J.R. Lopes2 , T.F. Borin1 , G.B. Gelaleti2 , M.G. Moschetta1 , C. Leonel2 , L.C. Ferreira2 , L.B. Maschio1 , ˜ Jose´ do Rio Preto, N.N. Gon¸calves1 . 1 Faculdade de Medicina de Sao ˜ Jose´ do Rio Preto SP, Brazil, Department of Molecular Biology, Sao 2 ˜ Jose´ do Rio Universidade Estadual Paulista, Department of Biology, Sao Preto SP, Brazil Introduction: Breast cancer can cause death in patients due to tumor growth and metastasis development. The hypoxia could limit tumor angiogenesis preventing its growth and progression however, tumor cells induce the transcription of HIF1A that promotes angiogenesis. Several researches focus on developing new therapeutic agents to inhibit angiogenesis factors, such as melatonin, a hormone secreted by the pineal gland. The aim of this study was to evaluate the change in HIF1A protein expression in breast cancer cell line, after the induction of hypoxia and treatment with melatonin. Material and Method: MDA-MB-231 cell line was cultured in DMEM (high glucose) and incubated at 37ºC in 5% CO2 . At first, the cells were divided into 7 groups to measure the cell viability by MTT assay: Group I: no treatment (Control), Group II received 100 mM of CoCl2 to hypoxia, and Groups III to VII also received CoCl2 and different concentrations of melatonin (0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM) for 24 hours. Once established the optimal concentration of melatonin, the cells were divided in 3 groups to assess the expression of HIF1A by immunocytochemistry and Real-Time PCR: Group A (control), Group B with only CoCl2 and Group III with CoCl2 and 1 mM of melatonin. The quantification of immunoexpression was performed by optical densitometry (ImageJ software), demonstrating mean optical density (MOD). The results were compared by ANOVA, followed by the Bonferroni test. Results and Discussion: The results showed that all groups treated with melatonin under hypoxia conditions had reduced cell viability. The concentration of 1 mM showed decrease of 48.13%, being considered the optimal concentration of melatonin for treatment in vitro. By immunocytochemistry,
european journal of cancer 48, suppl. 5 (2012) S25–S288
(LINE-1) methylation level in tumor, a surrogate of global methylaiton, predicts prognosis and response to oral fluoropyrimidines of CRC patients. In this study, the mechanism underling the association between LINE-1 methylation and sensitivity to 5-FU was investigated. Material and Method: CRC cells were used to analyze LINE-1 methylation level, LINE-1 mRNA expression and sensitivity to 5-FU by quantitative methylation specific PCR, Northern blotting and colony forming assay, respectively. Expression of phospho-histone H2AX was analyzed by Western immuno-blotting. RNA interference was employed to knockdown LINE-1 expression. Results and Discussion: LINE-1 methylation level in CRC cells was correlated with LINE-1 mRNA expression, in which hypomethylated CRC cell expressed high level of LINE-1 mRNA. The LINE-1-hypomethylated CRC cell also showed higher sensitivity to 5-FU. LINE-1 mRNA expression was increased after 5FU exposure in the cells showing LINE-1 hypomethylation, which is associated with upregulation of phospho-histone H2AX, a marker of DNA double strand break (DSB). These results suggest that 5FU action to induce DSB and exert cytotoxic effect is mediated, in part, by augmentation of LINE-1 expression in the CRC cell with LINE-1 hypomethylation because LINE-1 gene encodes endonuclease, a possible player to induce DSB. To investigate whether the increased expression of LINE-1by 5-FU is directly involved in the 5-FU action, LINE-1-expressing CRC cell was treated with 5-FU following the knockdown of LINE-1 by RNA interference. From the suggested mechanism that the LINE-1 expression has a role in 5-FU action, we expected that the knockdown of LINE-1 suppressed the cytotoxic effect of 5-FU. Contrary to our expectations, the knockdown of LINE-1 did not suppress but rather enhanced the activity of 5-FU. The study provided the novel strategy to enhance the effect of 5-FU although it did not reveal the mechanism underling the association between LINE-1 methylation and sensitivity to 5-FU. Conclusion: The LINE-1 expression itself has no significant role in the association between LINE-1 methylation level and the sensitivity to 5-FU. Suppression of LINE-1 is a possible strategy to enhance the activity of 5-FU in CRC with LINE-1 hypomethylation. 1052 Polymersomes − a Novel Drug Delivery System for the Delivery of Chemotherapeutic Agents to Oral Cancer Cells H.E. Colley1 , V. Hearnden1 , D. Cecchin2 , S. Armes3 , G. Battaglia2 , C. Murdoch1 , M.H. Thornhill1 . 1 The University of Sheffield, School of Clinical Dentistry, Sheffield, United Kingdom, 2 The University of Sheffield, Department of Biomedical Science, Sheffield, United Kingdom, 3 The University of Sheffield, Department of Chemistry, Sheffield, United Kingdom Introduction: Polymersomes are synthetic block co-polymers that selfassemble in water to form membrane-enclosed nanovesicles. Polymersomes have the potential to encapsulate and carry chemotherapeutic drugs into cells thereby reducing the off-target toxicity that often compromises anticancer treatment. Here, we assess the in vitro efficiency of polymersomes to encapsulate chemotherapeutic agents for the effective delivery to head and neck squamous cancer cells (HNSCC) cultured as monolayers and as three dimensional tumour spheroids. Materials and Methods: HNSCC were grown as monolayers or as multi-cellular tumour spheroids (MCTS), to model an expanding tumour island. Polymersomes loaded with rhodamine as a tracking molecule or chemotherapeutic agents (doxorubicin and/or paclitaxel) were applied to the cells for increasing lengths of time, and the cell viability, uptake and retention of polymersomes analysed. The effectiveness of chemotherapeutic-loaded polymersomes to kill HNSCC in monolayer and their efficacy in causing growth inhibition and damage of MCTS was also analysed. Furthermore, cellular uptake was investigated to determine the mechanism by which polymersomes are internalised by cells. Results and Discussion: Polymersomes loaded with rhodamine were internalised by HNSCC within 10 minutes of administration and maximal delivery was achieved within 30 minutes. When delivered to MCTS, polymersomes were internalised by over 80% of cells within 120 hours and importantly are shown to penetrate into hypoxic regions. Both doxorubicin and paclitaxel were loaded into polymersomes with high encapsulation efficiencies. Furthermore, both drugs can be dually loaded to enable combinational delivery. Loaded polymersomes demonstrated similar killing of HNSCC grown as monolayers compared to the same concentration of free drug. When delivered to MCTS, loaded polymersomes caused growth inhibition and extensive damage of the tumour spheroids. Current work involves the study of the cellular uptake mechanisms involved in the internalisation of the polymersomes to further the knowledge of this novel drug delivery system. Conclusion: Polymersomes offer a novel drug delivery system for the effective delivery of chemotherapeutics for the treatment of head and neck cancer.
Sunday 8 − Tuesday 10 July 2012
1053 New Strategies to Inhibit Tumor Cell Proliferation and Tumor Metastasis by S100P Monoclonal Antibodies J. Hernandez1 , M. Masa1 , R. Messeguer1 , F. Mitjans1 , J. Adan1 , J. Martinez1 , T. Coll1 , L. Padilla1 , S. Dakhel1 , R. Hervas1 . 1 Leitat Technological Center, Biomed Division, Barcelona, Spain Background: Metastasis represents the most devastating stage of malignancy and the leading death cause of cancer. An accurate description of the cellular and molecular mechanisms that underlie this multistep process would greatly facilitate the rational development of therapies that effectively allow metastatic disease to be controlled and treated. In the last years, intensive research in this field has shed light on some molecular targets as the novel metastatic factor S100P protein and its promising role as a key player in metastasis formation and poor clinical outcome. The identification and functional characterization of specific regulators of S100P might be exploited for therapeutic applications. Materials and Methods: Monoclonal antibodies able to neutralize the extracellular activity of S100P were obtained in our lab as therapeutic agents. In order to better elucidate the pivotal role of S100P in tumor cell proliferation and survival, we used either the proliferation measured by BrdU incorporation or MTT assay in human pancreatic BxPC3 and fibrosarcoma HT1080 cells. In addition, we developed a pancreatic orthotopic and a subcutaneous in vivo tumor model using BxPC3-luciferase cell line to understand the dynamic process of human metastasis progression and tumor growth and to validate our therapeutic mAbs as anti-metastatic and anti-tumoral drugs. Results: Here we address the mechanism of S100P in tumor proliferation showing an increased level of DNA synthesis induced by the extracellular activity of the protein and mAbs anti-S100P neutralized this effect. In addition, S100P induced an increased survival of fibrosarcoma HT1080 cells treated with Doxorubicin in a dose-dependent manner and neutralizing mAbs suppressed this activity. Moreover, a dramatic suppression of liver metastasis was observed after intraperitoneal administration of blocking antibodies to S100P in the orthotopic BxPC3 pancreatic tumor model. Relating to these results we also observed a reduction in the total-score staging from animals treated with our mAbs compared with Cromolyn (the unique described inhibitor of extracellular S100P) (p < 0.05). In a subcutaneous tumor model we also observed a tumor grow delay when animals were treated with blocking mAbs. Conclusion: As pointed out by our latest results, clinical therapeutic strategies directed to block the extracellular role of S100P protein by monoclonal antibodies could be a promising therapeutic strategy to decrease tumor growth and metastasis formation in cancer patients. Together with the utilization of chemotherapeutic agents, our studies provide new insights which could have significant implications for the development of novel therapeutic regimens targeting the metastasis. 1054 Evaluation of Hypoxia Inducible Factor 1-Alpha (HIF1A) After Treatment With Melatonin in Breast Cancer Cell Line D. Pires de Campos Zuccari1 , B.V. Jardim2 , J.R. Lopes2 , T.F. Borin1 , G.B. Gelaleti2 , M.G. Moschetta1 , C. Leonel2 , L.C. Ferreira2 , L.B. Maschio1 , ˜ Jose´ do Rio Preto, N.N. Gon¸calves1 . 1 Faculdade de Medicina de Sao ˜ Jose´ do Rio Preto SP, Brazil, Department of Molecular Biology, Sao 2 ˜ Jose´ do Rio Universidade Estadual Paulista, Department of Biology, Sao Preto SP, Brazil Introduction: Breast cancer can cause death in patients due to tumor growth and metastasis development. The hypoxia could limit tumor angiogenesis preventing its growth and progression however, tumor cells induce the transcription of HIF1A that promotes angiogenesis. Several researches focus on developing new therapeutic agents to inhibit angiogenesis factors, such as melatonin, a hormone secreted by the pineal gland. The aim of this study was to evaluate the change in HIF1A protein expression in breast cancer cell line, after the induction of hypoxia and treatment with melatonin. Material and Method: MDA-MB-231 cell line was cultured in DMEM (high glucose) and incubated at 37ºC in 5% CO2 . At first, the cells were divided into 7 groups to measure the cell viability by MTT assay: Group I: no treatment (Control), Group II received 100 mM of CoCl2 to hypoxia, and Groups III to VII also received CoCl2 and different concentrations of melatonin (0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM) for 24 hours. Once established the optimal concentration of melatonin, the cells were divided in 3 groups to assess the expression of HIF1A by immunocytochemistry and Real-Time PCR: Group A (control), Group B with only CoCl2 and Group III with CoCl2 and 1 mM of melatonin. The quantification of immunoexpression was performed by optical densitometry (ImageJ software), demonstrating mean optical density (MOD). The results were compared by ANOVA, followed by the Bonferroni test. Results and Discussion: The results showed that all groups treated with melatonin under hypoxia conditions had reduced cell viability. The concentration of 1 mM showed decrease of 48.13%, being considered the optimal concentration of melatonin for treatment in vitro. By immunocytochemistry,