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931: Photodynamic therapy as an option for osteosarcoma
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
Moreover, MET inhibitors impede efficient activation of the DNA repair machinery and cell cycle arrest, eventually leading to cell death. Association of MET inhibitors with ionizing radiation significantly enhanced the efficacy of radiation in blocking the growth of xenografts. Conclusions: We showed that, in a subset of GB, ionizing radiation positively selects the METpos cancer stem cell subpopulation. We found that the MET oncogene is crucial to mediate the intrinsic radioresistance of GB stem cells and that MET inhibition improves cell radiosensitivity by interfering with the DNA repair machinery both in vitro and in vivo. Thus, in a subset of GB, MET can be proposed as a therapeutic target in association with radiotherapy to achieve eradication of the cancer stem cell compartment. No conflict of interest. 929 Early NFKB signalling pathway activation in Ramos B cells in response to ionizing radiation C. Di Nisio1 , S. Sancilio1 , V. Di Giacomo1 , M. Rapino2 , L. Sancillo3 , R.A. Rana3 , R. Di Pietro3 . 1 Universita` G. D’Annunzio, Department of Pharmacy, Chieti, Italy, 2 CNR, Institute of molecular Genetics, Chieti, Italy, 3 Universita` G. D’Annunzio, Department of Medicine and Ageing Sciences, Chieti, Italy Background: The aim of the present study was to investigate the effect of ionizing radiation (IR) on intracellular signalling pathways regulating apoptosis, necrosis, mitotic cell death and proliferation in Ramos B cells, an Epstein–Barr virus (EBV)-negative cell line derived from an American Burkitt’s lymphoma. Materials and Methods: Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), cell cycle analysis, Annexin-V/Propidium Iodide (PI) detection of apoptotic and necrotic cells in flow cytometry and clonogenic assays were performed on Ramos B cells grown in suspension and irradiated with single doses of 3 and 5 Gy (X-rays). We carried out three independent experiments. Results and Discussion: Compared to unirradiated cells, the significant radiation dose-dependent S phase decrease and G2 /M phase accumulation observed in Ramos B cells 24 h after IR exposure could be interpreted as an attempt to repair radiation injury. Annexin-V/PI staining showed a moderate increase in early apoptotic cells 24 h after both doses. 16 days after IR exposure, clonogenic assays displayed a reduction in clonogenic survival after 3 Gy dose radiation and a complete loss of clonogenicity after 5 Gy dose administration. These effects followed an early activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFKB) complex signalling pathway: 30 min after IR exposure, real-time RT-PCR analysis highlighted a significant radiation dose-dependent up-regulation of NFKB1 and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) gene expression compared to control. 3 or 5 Gy doses did not modify v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) and caspase-3 mRNA levels compared to control. A significant increase in X-linked inhibitor of apoptosis (XIAP) gene expression compared to control was observed only after 5 Gy dose treatment. NFKB regulates the expression of anti-apoptotic gene products. The radiation dose-dependency of IKBKB and XIAP gene expression suggests that the attempt to counteract IR-induced damage is higher after 5 Gy than after 3 Gy dose exposure. The apparent inconsistency of our molecular findings with clonogenic assay data could be due to the occurrence of mitotic cell death, a caspaseindependent process. Conclusion: Hence, IR could initially induce the activation of NFKB/XIAP signalling pathway, which leads to cell protection; over time, IR might determine mitotic cell death. No conflict of interest. 930 Effect of radiotherapy on macrophages A.T. Pinto1 , H. Osorio ´ 3 , M.L. Pinto1,4 , A.P. Cardoso1,2 , C. Monteiro1 , M. Marques5 , R. Seruca3,6 , M.B. Barbosa1,4 , S. Rocha7 , M.J. Oliveira1,6 . 1 INEB, Institute of Biomedical Engineering, Oporto, Portugal, 2 FEUP − Faculty of Engineering, Porto, Portugal, 3 IPATIMUP − Institute of Molecular Pathology and Immunology, University of Porto, Portugal, 4 ICBAS − Institute of Biomedical Sciences Abel Salazar, Porto, Portugal, 5 Department of ˜ Joao, ˜ Porto, Portugal, 6 Department of Radiotherapy, Hospital de Sao Pathology and Oncology, Faculty of Medicine, Porto, Portugal, 7 Centre for Gene Regulation and Expression, University of Dundee, Scotland Introduction: In colorectal cancer, neoadjuvant radiotherapy improves locoregional control but has little or no effect on the development of distant metastases, which constitute the major cause of death. Ionizing radiation is known to enhance the activities of tumour-associated host cells that support invasion and metastasis. Therefore, research has been developed to clarify the underlying mechanisms and to improve the efficacy of radiotherapy. Attention has also been paid to tumour-associated macrophages, which may modulate cancer cell response to radiotherapy.
Material and Methods: The effect of ionizing radiation on macrophage differentiation/behaviour and the molecular mechanisms underlying macrophagemediated cancer progression are still unknown. In order to address this question, we subjected human macrophages to conventional doses of ionizing radiation, as used for colorectal cancer patient’s treatment. Results and Discussion: We observed that after 1 or 5 days of irradiation, macrophage viability and metabolic activity were not affected, but the levels and size of gH2AX foci increased, reflecting DNA double-strand breaks. DNA damage induces a DNA repair response, which is highly dependent on NF-kB activation. Therefore, the expression and phosphorylation status of NF-kB and target activation are now being evaluated. Results also evidenced that a single 2 Gy irradiation dose was sufficient to increase tyrosine phosphorylation of EGFR, which became concentrated at areas of basal podosomes. Curiously, we observed that the phosphorylation of other receptor tyrosine kinases (RTKs), like PDGFR, Tie and Tyk2, was decreased. We are also currently focused on the effect of ionizing radiation on macrophage proteome. Therefore, 2D gels performed with total protein extracts from non-irradiated and 10 Gy-irradiated macrophages were analyzed and distinct cytoskeleton and metabolism-related proteins were identified. Conclusions: In summary, our results demonstrate that the ionizing radiation doses used are not affecting macrophage metabolic activity, but are inducing DNA damage and altering the phosphorylation of some RTKs. Current research on NF-kB signaling pathway and identification of proteins differentially expressed in macrophages upon ionizing radiation, will complement these findings. Furthermore, this work will allow us to understand how ionizing radiation modulates macrophage behavior and how this affects macrophage/cancer cell interactions, opening new perspectives for therapeutic intervention. No conflict of interest. 931 Photodynamic therapy as an option for osteosarcoma G. Chohfi de Miguel1 , M. Laranjo2 , A.M. Abrantes2 , R. Teixo2 , T. Rocha1 , 3 A.C. Serra3 , M. Pineiro ˜ , A.M. Rocha-Gonsalves3 , M.F. Botelho2 , D.G. Priolli1 . 1 ˜ Francisco University, Postgraduate Program in Health Science, Sao Bragan¸ca Paulista, Brazil, 2 Faculty of Medicine of University of Coimbra, Biophysics Unit − IBILI − CIMAGO, Coimbra, Portugal, 3 University of Coimbra, Chymiotechan Department of Chemestry, Coimbra, Portugal Background: Osteosarcoma (OS), a malignant tumor characterized by a direct formation of bones or osteoid, is the second most common primary bone neoplasm. This type of cancer affects both children and adults and it grows predominantly on metaphyseal region in young adults or flat bones on patients over 50 years. It has an unknown etiology, and the clinical symptoms comprises local pain and in the surrounding area, such as adjacent joint, unrelieved by rest or analgesic. Furthermore, it is a tumor with low responses to chemotherapy, leading to a variety of tumor cell necrosis, with a better prognosis of surgery for complete removal of the tumor. Frequently, tumors are located on so-called hard to access locations or in areas impossible to access surgically. Therefore, the development of new therapeutic strategies is mandatory to pursue the success on OS treatment. Photodynamic therapy has emerged as a successful treatment for several types of cancer, particularly melanoma, lung or esophageal carcinoma, and might be promising to treat OS. Materials and Methods: The human osteosarcoma cells MNNG/HOS were cultured and incubated with several concentrations of the sensitizer BBr2HPP, a bromated hydroxyphenyl porphyrin, previously synthetized by us, ranging from 50nM to 10mM. After 24 h incubation the medium was replaced by sensitizer-free medium and the cultures were irradiated until reached a total of 10J. The analysis of cell proliferation was made 24 h after irradiation using the MTT assay. The results allowed to establish dose response curves, obtained using Origin 8.0. In order to evaluate the variation in total protein content the SRB assay was performed. To determine the capacity of cells submitted to PDT to form colonies the clonogenic assay was performed. Results: The results from the MTT assay showed that the metabolic activity was inversely proportional to concentration of the photosensitizer and allowed to establish a dose–response curve. It was possible to calculate the concentration of sensitizers that inhibits the proliferation of cultures in 50% (IC50) that corresponds to 84nM. The preliminary results from the SRB assay indicate that when MNNG/HOS cell cultures are submitted to PDT with a PS concentration of 500nM, the protein content diminishes to 5.59±3.71%. Conclusions: The PS evaluated showed good photodynamic properties and cytotoxic effect in human osteosarcoma cells, corroborating previous work from our group in other types of cancer, being the induced cell death with concentrations in the order at nanomolar level. The potential of PDT with this sensitizer will be explored in vitro, and in vivo preclinical studies will be developed. No conflict of interest.
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
Moreover, MET inhibitors impede efficient activation of the DNA repair machinery and cell cycle arrest, eventually leading to cell death. Association of MET inhibitors with ionizing radiation significantly enhanced the efficacy of radiation in blocking the growth of xenografts. Conclusions: We showed that, in a subset of GB, ionizing radiation positively selects the METpos cancer stem cell subpopulation. We found that the MET oncogene is crucial to mediate the intrinsic radioresistance of GB stem cells and that MET inhibition improves cell radiosensitivity by interfering with the DNA repair machinery both in vitro and in vivo. Thus, in a subset of GB, MET can be proposed as a therapeutic target in association with radiotherapy to achieve eradication of the cancer stem cell compartment. No conflict of interest. 929 Early NFKB signalling pathway activation in Ramos B cells in response to ionizing radiation C. Di Nisio1 , S. Sancilio1 , V. Di Giacomo1 , M. Rapino2 , L. Sancillo3 , R.A. Rana3 , R. Di Pietro3 . 1 Universita` G. D’Annunzio, Department of Pharmacy, Chieti, Italy, 2 CNR, Institute of molecular Genetics, Chieti, Italy, 3 Universita` G. D’Annunzio, Department of Medicine and Ageing Sciences, Chieti, Italy Background: The aim of the present study was to investigate the effect of ionizing radiation (IR) on intracellular signalling pathways regulating apoptosis, necrosis, mitotic cell death and proliferation in Ramos B cells, an Epstein–Barr virus (EBV)-negative cell line derived from an American Burkitt’s lymphoma. Materials and Methods: Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), cell cycle analysis, Annexin-V/Propidium Iodide (PI) detection of apoptotic and necrotic cells in flow cytometry and clonogenic assays were performed on Ramos B cells grown in suspension and irradiated with single doses of 3 and 5 Gy (X-rays). We carried out three independent experiments. Results and Discussion: Compared to unirradiated cells, the significant radiation dose-dependent S phase decrease and G2 /M phase accumulation observed in Ramos B cells 24 h after IR exposure could be interpreted as an attempt to repair radiation injury. Annexin-V/PI staining showed a moderate increase in early apoptotic cells 24 h after both doses. 16 days after IR exposure, clonogenic assays displayed a reduction in clonogenic survival after 3 Gy dose radiation and a complete loss of clonogenicity after 5 Gy dose administration. These effects followed an early activation of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFKB) complex signalling pathway: 30 min after IR exposure, real-time RT-PCR analysis highlighted a significant radiation dose-dependent up-regulation of NFKB1 and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) gene expression compared to control. 3 or 5 Gy doses did not modify v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) and caspase-3 mRNA levels compared to control. A significant increase in X-linked inhibitor of apoptosis (XIAP) gene expression compared to control was observed only after 5 Gy dose treatment. NFKB regulates the expression of anti-apoptotic gene products. The radiation dose-dependency of IKBKB and XIAP gene expression suggests that the attempt to counteract IR-induced damage is higher after 5 Gy than after 3 Gy dose exposure. The apparent inconsistency of our molecular findings with clonogenic assay data could be due to the occurrence of mitotic cell death, a caspaseindependent process. Conclusion: Hence, IR could initially induce the activation of NFKB/XIAP signalling pathway, which leads to cell protection; over time, IR might determine mitotic cell death. No conflict of interest. 930 Effect of radiotherapy on macrophages A.T. Pinto1 , H. Osorio ´ 3 , M.L. Pinto1,4 , A.P. Cardoso1,2 , C. Monteiro1 , M. Marques5 , R. Seruca3,6 , M.B. Barbosa1,4 , S. Rocha7 , M.J. Oliveira1,6 . 1 INEB, Institute of Biomedical Engineering, Oporto, Portugal, 2 FEUP − Faculty of Engineering, Porto, Portugal, 3 IPATIMUP − Institute of Molecular Pathology and Immunology, University of Porto, Portugal, 4 ICBAS − Institute of Biomedical Sciences Abel Salazar, Porto, Portugal, 5 Department of ˜ Joao, ˜ Porto, Portugal, 6 Department of Radiotherapy, Hospital de Sao Pathology and Oncology, Faculty of Medicine, Porto, Portugal, 7 Centre for Gene Regulation and Expression, University of Dundee, Scotland Introduction: In colorectal cancer, neoadjuvant radiotherapy improves locoregional control but has little or no effect on the development of distant metastases, which constitute the major cause of death. Ionizing radiation is known to enhance the activities of tumour-associated host cells that support invasion and metastasis. Therefore, research has been developed to clarify the underlying mechanisms and to improve the efficacy of radiotherapy. Attention has also been paid to tumour-associated macrophages, which may modulate cancer cell response to radiotherapy.
Material and Methods: The effect of ionizing radiation on macrophage differentiation/behaviour and the molecular mechanisms underlying macrophagemediated cancer progression are still unknown. In order to address this question, we subjected human macrophages to conventional doses of ionizing radiation, as used for colorectal cancer patient’s treatment. Results and Discussion: We observed that after 1 or 5 days of irradiation, macrophage viability and metabolic activity were not affected, but the levels and size of gH2AX foci increased, reflecting DNA double-strand breaks. DNA damage induces a DNA repair response, which is highly dependent on NF-kB activation. Therefore, the expression and phosphorylation status of NF-kB and target activation are now being evaluated. Results also evidenced that a single 2 Gy irradiation dose was sufficient to increase tyrosine phosphorylation of EGFR, which became concentrated at areas of basal podosomes. Curiously, we observed that the phosphorylation of other receptor tyrosine kinases (RTKs), like PDGFR, Tie and Tyk2, was decreased. We are also currently focused on the effect of ionizing radiation on macrophage proteome. Therefore, 2D gels performed with total protein extracts from non-irradiated and 10 Gy-irradiated macrophages were analyzed and distinct cytoskeleton and metabolism-related proteins were identified. Conclusions: In summary, our results demonstrate that the ionizing radiation doses used are not affecting macrophage metabolic activity, but are inducing DNA damage and altering the phosphorylation of some RTKs. Current research on NF-kB signaling pathway and identification of proteins differentially expressed in macrophages upon ionizing radiation, will complement these findings. Furthermore, this work will allow us to understand how ionizing radiation modulates macrophage behavior and how this affects macrophage/cancer cell interactions, opening new perspectives for therapeutic intervention. No conflict of interest. 931 Photodynamic therapy as an option for osteosarcoma G. Chohfi de Miguel1 , M. Laranjo2 , A.M. Abrantes2 , R. Teixo2 , T. Rocha1 , 3 A.C. Serra3 , M. Pineiro ˜ , A.M. Rocha-Gonsalves3 , M.F. Botelho2 , D.G. Priolli1 . 1 ˜ Francisco University, Postgraduate Program in Health Science, Sao Bragan¸ca Paulista, Brazil, 2 Faculty of Medicine of University of Coimbra, Biophysics Unit − IBILI − CIMAGO, Coimbra, Portugal, 3 University of Coimbra, Chymiotechan Department of Chemestry, Coimbra, Portugal Background: Osteosarcoma (OS), a malignant tumor characterized by a direct formation of bones or osteoid, is the second most common primary bone neoplasm. This type of cancer affects both children and adults and it grows predominantly on metaphyseal region in young adults or flat bones on patients over 50 years. It has an unknown etiology, and the clinical symptoms comprises local pain and in the surrounding area, such as adjacent joint, unrelieved by rest or analgesic. Furthermore, it is a tumor with low responses to chemotherapy, leading to a variety of tumor cell necrosis, with a better prognosis of surgery for complete removal of the tumor. Frequently, tumors are located on so-called hard to access locations or in areas impossible to access surgically. Therefore, the development of new therapeutic strategies is mandatory to pursue the success on OS treatment. Photodynamic therapy has emerged as a successful treatment for several types of cancer, particularly melanoma, lung or esophageal carcinoma, and might be promising to treat OS. Materials and Methods: The human osteosarcoma cells MNNG/HOS were cultured and incubated with several concentrations of the sensitizer BBr2HPP, a bromated hydroxyphenyl porphyrin, previously synthetized by us, ranging from 50nM to 10mM. After 24 h incubation the medium was replaced by sensitizer-free medium and the cultures were irradiated until reached a total of 10J. The analysis of cell proliferation was made 24 h after irradiation using the MTT assay. The results allowed to establish dose response curves, obtained using Origin 8.0. In order to evaluate the variation in total protein content the SRB assay was performed. To determine the capacity of cells submitted to PDT to form colonies the clonogenic assay was performed. Results: The results from the MTT assay showed that the metabolic activity was inversely proportional to concentration of the photosensitizer and allowed to establish a dose–response curve. It was possible to calculate the concentration of sensitizers that inhibits the proliferation of cultures in 50% (IC50) that corresponds to 84nM. The preliminary results from the SRB assay indicate that when MNNG/HOS cell cultures are submitted to PDT with a PS concentration of 500nM, the protein content diminishes to 5.59±3.71%. Conclusions: The PS evaluated showed good photodynamic properties and cytotoxic effect in human osteosarcoma cells, corroborating previous work from our group in other types of cancer, being the induced cell death with concentrations in the order at nanomolar level. The potential of PDT with this sensitizer will be explored in vitro, and in vivo preclinical studies will be developed. No conflict of interest.