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Receptor-interacting Protein 1(rip1) Enhances Radiosensitization via Radiation-induced Necrotic Cell Death
I. J. Radiation Oncology d Biology d Physics
S568
2861
Volume 75, Number 3, Supplement, 2009
Sulfoquinovocylacylglycerol (SQAG) is a Novel Potent Antiangiogenic Radiosensitizer
1,2
K. Ohta , Y. Mori1, M. Ishima1, H. Murata3, F. Sugawara3, K. Sakaguchi3, M. Miura1 Tokyo Medical and Dental University, Tokyo, Japan, 2Cango corp., Yotsuya, Shinjyuku-ku, Tokyo, Japan, 3Tokyo University of Science, Tokyo, Japan 1
Purpose/Objective(s): Despite that much effort has been made for the development of radiosensitizers, useful drugs are still unavailable in usual clinics of radiotherapy. Antiangiogenic agents are thought to be promising in cancer therapy and many drugs possessing such properties have been developed. Recently, a consensus is being made that the combination of such drugs with radiotherapy is quite effective. Sulfoquinovosylacylglycerol (SQAG) is sulfoglycolipids, which we originally extracted from sea urchins or sea algae. We also succeeded in chemical synthesis of the agent. In this study, we show that SQAG could be a unique antiangiogenic radiosensitizer by pre-clinical study. Materials/Methods: Several human tumor cell lines (SAS, TE-8, A549, SW480, and DU145), bovine aortic endothelial cells (BAEC), and human umbilical vein endothelial cells (HUVEC) were used in this study. Angiogenic activities of vascular endothelial cells were evaluated by capillary formation in Matrigel, invasion through Matrigel, and angiogenesis in a HUVEC-fibroblasts co-culture system. In vivo studies were carried out in solid tumors subcutaneously transplanted into KSN nude mice. Irradiation was performed using an X-ray therapeutic machine (225 kV, 15 mA, 1 mm Cu filtration). Results: The agent by itself had an antiangiogenic activity at high doses, however, it synergistically inhibited angiogenesis at low doses when combined with ionizing radiation using different in vitro methods. The agent remarkably enhanced the radio response of several human tumors transplanted into nude mice, accompanied by a significant reduction in the vascularity of the tumors as evaluated by immunohistochemical staining of CD31. Collagen IV, which constitutes the basal membrane of the vessels, was significantly reduced by the combined treatment. The effect of the combined treatment on vascular normalization as evaluated by the time course of a-sma expression and hypoxic fractions as determined by pimonidazole staining will also be described. Conclusions: We conclude that SQAG could be a potent antiangiogenic radiosensitizer. Pre-clinical studies including the optimization of drug structure and pharmacokinetics are currently underway. Author Disclosure: K. Ohta, National Institute of Biomedical Innovation, Japan, B. Research Grant; Cango corp. Japan, C. Other Research Support; Y. Mori, National Institute of Biomedical Innovation, B. Research Grant; M. Ishima, None; H. Murata, None; F. Sugawara, National Institute of Biomedical Innovation, B. Research Grant; Cango Corp., C. Other Research Support; K. Sakaguchi, National Institute of Biomedical Innovation, B. Research Grant; Cango Corp., C. Other Research Support; M. Miura, National Institute of Biomedical Innovation, B. Research Grant; Cango Corp., C. Other Research Support.
2862
Receptor-interacting Protein 1(rip1) Enhances Radiosensitization via Radiation-induced Necrotic Cell Death
D. Jung, K. Kim, B. Lu Vanderbilt University Ingram Cancer Center, Nashville, TN Purpose/Objective(s): The receptor-interacting protein 1(RIP1) have revealed as essential sensors of cellular stresses pathway. In this study, we tested whether RIP1 protein affects necrotic cell death via radiation as using mouse embryonic fibroblasts (MEFs) WT and RIP1 KO cells. Materials/Methods: Clonogenic assay was used to determine radiosensitivity of WT and RIP1 -/- cells. Necrosis was determined with HMGB1 release and Propidium iodide (PI) staining. SiRNA RIP1 (human) and siRNA control were transiently transfected with 25nM of siRNAs using Lipofectamine 2000. Protein levels of HMGB1 and Actin were determined by western blotting. Results: RIP1 deficiency, in both RIP1 KO cells and WT cell treated with Necrostatin-1(Nec-1), reduced radiation sensitivity compared to WT cells (DER= 0.75, p=0.03, student’s t test, n=3) and WT cells treated with DMSO (DER= 0.80, p=0.001, student’s t test, n=3) respectively. Importantly, we found that the transfection of siRNA RIP1 with radiation in non-small cell lung cancer, H460 cells (DER= 0.75, p=0.021, student’s t test, n=3) mediates less radiation sensitivity when compared with siRNA control. Therefore, this less radiation sensitivity may involve in defects of necrosis in RIP deficiency cells. HMGB1 proteins were significantly released to media compared to those in RIP1 KO cells. WT cells were significantly stained with PI compared to those in RIP1 KO cells. Furthermore, WT cells treated with Necrostatin-1(Nec-1) inhibited PI staining when compared with WT cell untreated with Nec-1(12± 1%, p=0.006, t test, n=3). RIP1 KO cells treated or untreated with Necrostatin-1(Nec-1) has no significant PI staining. Conclusions: We demonstrate here that RIP1 is essential for radiation-induced necrotic cell death. Author Disclosure: D. Jung, None; K. Kim, None; B. Lu, None.
2863
Radioimmunoconjugates: Pre-clinical Evaluation of Monoclonal Antibodies Targeting the Epidermal Growth Factor (EGFR) for Radioimmunodiagnostic and Radioimmunotherapeutic Applications
G. L. Ray1, K. E. Baidoo1, K. J. Wong2, M. Williams3, K. G. Garmestani1, M. W. Brechbiel1, D. E. Milenic1 1
Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 2Molecular Imaging Program, CCR, NCI, NIH, Bethesda, MD, 3 Molecular Imaging Program, CCR, NCI, NIH, Bethesda, MD, United States, Bethesda, MD
Purpose/Objective(s): The studies described herein are the first to evaluate the in vitro and in vivo properties of 111In-CHX-A’’panitumumab for radioimmunotherapy (a- and b--emitters) and radioimmunoimaging (SPECT and PET). Materials/Methods: Twenty-seven human carcinoma cell lines were analyzed for EGFR-expression by flow cytometry. Panitumumab was conjugated with CHX-A’’-DTPA and radiolabeled with 111In. Radioimmunoassays were performed to evaluate the
S568
2861
Volume 75, Number 3, Supplement, 2009
Sulfoquinovocylacylglycerol (SQAG) is a Novel Potent Antiangiogenic Radiosensitizer
1,2
K. Ohta , Y. Mori1, M. Ishima1, H. Murata3, F. Sugawara3, K. Sakaguchi3, M. Miura1 Tokyo Medical and Dental University, Tokyo, Japan, 2Cango corp., Yotsuya, Shinjyuku-ku, Tokyo, Japan, 3Tokyo University of Science, Tokyo, Japan 1
Purpose/Objective(s): Despite that much effort has been made for the development of radiosensitizers, useful drugs are still unavailable in usual clinics of radiotherapy. Antiangiogenic agents are thought to be promising in cancer therapy and many drugs possessing such properties have been developed. Recently, a consensus is being made that the combination of such drugs with radiotherapy is quite effective. Sulfoquinovosylacylglycerol (SQAG) is sulfoglycolipids, which we originally extracted from sea urchins or sea algae. We also succeeded in chemical synthesis of the agent. In this study, we show that SQAG could be a unique antiangiogenic radiosensitizer by pre-clinical study. Materials/Methods: Several human tumor cell lines (SAS, TE-8, A549, SW480, and DU145), bovine aortic endothelial cells (BAEC), and human umbilical vein endothelial cells (HUVEC) were used in this study. Angiogenic activities of vascular endothelial cells were evaluated by capillary formation in Matrigel, invasion through Matrigel, and angiogenesis in a HUVEC-fibroblasts co-culture system. In vivo studies were carried out in solid tumors subcutaneously transplanted into KSN nude mice. Irradiation was performed using an X-ray therapeutic machine (225 kV, 15 mA, 1 mm Cu filtration). Results: The agent by itself had an antiangiogenic activity at high doses, however, it synergistically inhibited angiogenesis at low doses when combined with ionizing radiation using different in vitro methods. The agent remarkably enhanced the radio response of several human tumors transplanted into nude mice, accompanied by a significant reduction in the vascularity of the tumors as evaluated by immunohistochemical staining of CD31. Collagen IV, which constitutes the basal membrane of the vessels, was significantly reduced by the combined treatment. The effect of the combined treatment on vascular normalization as evaluated by the time course of a-sma expression and hypoxic fractions as determined by pimonidazole staining will also be described. Conclusions: We conclude that SQAG could be a potent antiangiogenic radiosensitizer. Pre-clinical studies including the optimization of drug structure and pharmacokinetics are currently underway. Author Disclosure: K. Ohta, National Institute of Biomedical Innovation, Japan, B. Research Grant; Cango corp. Japan, C. Other Research Support; Y. Mori, National Institute of Biomedical Innovation, B. Research Grant; M. Ishima, None; H. Murata, None; F. Sugawara, National Institute of Biomedical Innovation, B. Research Grant; Cango Corp., C. Other Research Support; K. Sakaguchi, National Institute of Biomedical Innovation, B. Research Grant; Cango Corp., C. Other Research Support; M. Miura, National Institute of Biomedical Innovation, B. Research Grant; Cango Corp., C. Other Research Support.
2862
Receptor-interacting Protein 1(rip1) Enhances Radiosensitization via Radiation-induced Necrotic Cell Death
D. Jung, K. Kim, B. Lu Vanderbilt University Ingram Cancer Center, Nashville, TN Purpose/Objective(s): The receptor-interacting protein 1(RIP1) have revealed as essential sensors of cellular stresses pathway. In this study, we tested whether RIP1 protein affects necrotic cell death via radiation as using mouse embryonic fibroblasts (MEFs) WT and RIP1 KO cells. Materials/Methods: Clonogenic assay was used to determine radiosensitivity of WT and RIP1 -/- cells. Necrosis was determined with HMGB1 release and Propidium iodide (PI) staining. SiRNA RIP1 (human) and siRNA control were transiently transfected with 25nM of siRNAs using Lipofectamine 2000. Protein levels of HMGB1 and Actin were determined by western blotting. Results: RIP1 deficiency, in both RIP1 KO cells and WT cell treated with Necrostatin-1(Nec-1), reduced radiation sensitivity compared to WT cells (DER= 0.75, p=0.03, student’s t test, n=3) and WT cells treated with DMSO (DER= 0.80, p=0.001, student’s t test, n=3) respectively. Importantly, we found that the transfection of siRNA RIP1 with radiation in non-small cell lung cancer, H460 cells (DER= 0.75, p=0.021, student’s t test, n=3) mediates less radiation sensitivity when compared with siRNA control. Therefore, this less radiation sensitivity may involve in defects of necrosis in RIP deficiency cells. HMGB1 proteins were significantly released to media compared to those in RIP1 KO cells. WT cells were significantly stained with PI compared to those in RIP1 KO cells. Furthermore, WT cells treated with Necrostatin-1(Nec-1) inhibited PI staining when compared with WT cell untreated with Nec-1(12± 1%, p=0.006, t test, n=3). RIP1 KO cells treated or untreated with Necrostatin-1(Nec-1) has no significant PI staining. Conclusions: We demonstrate here that RIP1 is essential for radiation-induced necrotic cell death. Author Disclosure: D. Jung, None; K. Kim, None; B. Lu, None.
2863
Radioimmunoconjugates: Pre-clinical Evaluation of Monoclonal Antibodies Targeting the Epidermal Growth Factor (EGFR) for Radioimmunodiagnostic and Radioimmunotherapeutic Applications
G. L. Ray1, K. E. Baidoo1, K. J. Wong2, M. Williams3, K. G. Garmestani1, M. W. Brechbiel1, D. E. Milenic1 1
Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 2Molecular Imaging Program, CCR, NCI, NIH, Bethesda, MD, 3 Molecular Imaging Program, CCR, NCI, NIH, Bethesda, MD, United States, Bethesda, MD
Purpose/Objective(s): The studies described herein are the first to evaluate the in vitro and in vivo properties of 111In-CHX-A’’panitumumab for radioimmunotherapy (a- and b--emitters) and radioimmunoimaging (SPECT and PET). Materials/Methods: Twenty-seven human carcinoma cell lines were analyzed for EGFR-expression by flow cytometry. Panitumumab was conjugated with CHX-A’’-DTPA and radiolabeled with 111In. Radioimmunoassays were performed to evaluate the