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Radiation-Induced Lung Apoptosis is Mediated by TNF-Alpha Action
Proceedings of the 47th Annual ASTRO Meeting
Conclusions: Hb levels were significantly increased from baseline during the XRT period, irrespective of tumour localisation and stage. This increase reached the maximum value during week 10 from the baseline and remained significant 2 months post XRT. Quality of life was significantly improved in this group of patients.
137
Anti-TGF Antibodies May Prevent Lung Injury Following High Dose Fractionated Radiation
M. Anscher,1 B. Thrasher,1 Z. Rabbani,1 B. Teicher,2 Z. Vujaskovic1 1 Radiation Oncology, Duke University Medical Center, Durham, NC, 2Genzyme Corporation, Cambridge, MA Purpose/Objective: The cytokine Transforming Growth Factor (TGF) has been shown to play an important role in fibrogenesis associated with radiation (RT) induced normal tissue injury. Antagonists of TGF may have great potential to reduce formation of fibrous tissue and thereby protect normal tissues from the adverse effects of radiation. The purpose of this study is to determine whether an anti-TGF antibody (1D11, Genzyme Corp, Cambridge, MA) can prevent/ameliorate radiation induced lung injury. Materials/Methods: Fractionated and sham lung irradiation of Fischer-344 rats, both tumor (R3230Ac mammary adenocarcinoma) and non-tumor bearing animals, was performed to assess the radioprotective effect of the antibodies, and to insure that the antibodies do not protect tumor from irradiation. Animals were divided into the following groups(12 animals per group): 1) control (sham RT, isotype control antibody); 2) RT (800 cGy ⫻ 5 over 5 days to the right hemithorax); 3) RT plus 0.1 mg/kg i.p. antibody; and, 4) RT plus 1 mg/kg antibody. Animals were anesthetized for RT. Antibodies were administered as a single dose only, immediately after the last dose of RT. Animals were sacrificed at 6 weeks and 6 months after irradiation and lungs were assessed for histologic changes (inflammation and fibrosis), activation of macrophages, expression and activation of TGF and the TGF signal transduction pathway. Endpoints were the differences between groups with respect to both pulmonary toxicity and tumor growth delay. Results: Tumor growth delay studies did not show any evidence of protection of tumor by the anti-TGF antibody at either dose. There were no changes in body weight and no toxicity apparent from the addition of the anti-TGF antibody. At 6 weeks post-RT, there was a significant reduction in macrophages accumulation (p⫽0.041) and alveolar wall thickness (p⫽0.0003) in the animals receiving 1.0 mg/kg of anti-TGF antibody after RT as compared to the RT control antibody group. This significant decrease in inflammatory and histopathologic changes, seen after treatment with the higher dose of anti-TGF antibody (1mg/kg) following lung irradiation, are also associated with a significant inhibition in the expression of active TGF (p⫽0.031, for high dose group only) (Figure). Six month samples are currently under evaluation and these data will be presented at the meeting. Conclusions: Administration of a single dose of 1.0 mg/kg of the anti-TGF antibody 1D11 resulted in decreased morphological changes, inflammatory response, and reduced expression and activation of TGF 6 weeks after 40 Gy to the right hemithorax. Targeting the TGF pathway may be a useful strategy to prevent radiation induced lung injury. Supported by a grant from Genzyme Corporation.
High dose antibody (left bar) significantly decreases TGF beta expression compared to low dose antibody (center bar) or control (right bar)
138
Radiation-Induced Lung Apoptosis is Mediated by TNF-Alpha Action
M. Zhang, F. Kong, J. Qian, M. Chen, T.S. Lawrence Radiation Oncology, University of Michigan, Ann Arbor, MI Purpose/Objective: Radiation-induced lung damage is a major limitation for effective radiotherapy in lung cancer patients. This damage is correlated to an early lung apoptosis mediated by aberrant cytokine production after radiation treatment. In this study, we investigated the role of TNF-alpha in radiation-induced lung apoptosis and the potential protective effect of inhibiting TNF-alpha by using antisense oligonucleotide gene therapy. Materials/Methods: Balb/c mice (6 – 8 week, male) were irradiated with 15 Gy in lung and were measured for expression of TNF-alpha and TNFR1. Lung apoptosis was assessed by TUNEL staining and caspase-3 activation at 3 h after radiation. Inhibition of TNFR1 was established by pre-treatment of mice with antisense oligonucleotide specific for TNFR1 (i.p. 25
S83
S84
I. J. Radiation Oncology
● Biology ● Physics
Volume 63, Number 2, Supplement, 2005
mg/kg, 36 h interval ⫻ 4). Human lung macrophages and micro vascular endothelial cells were used to study TNF-alpha secretion and potential apoptosis in co-culture in response to radiation treatment. TNF-alpha production in lung tissue and culture media were determined by ELISA assay. Radio protection by antisense oligonucleotide was assessed by TUNEL staining and caspase-3 activation in lung. Results: Radiation in lung induced apoptosis in mice in a time- and dose-dependent manner. TUNEL staining and caspase-3 activation in lung were increased maximally at 3 h after 15 Gy of radiation by approximately 5- and 3-fold, correlating to the induction of TNF-alpha transcription and secretion. Pre-treatment of mice with antisense oligonucleotide effectively inhibited TNFR1 gene expression in lung by 50% and significantly decreased radiation-induced TUNEL staining and caspase-3 activation in lung. Radiation increased TNF-alpha production in human lung macrophages by 2 fold and induced caspase-3 activation in pulmonary micro vascular endothelial cells in co-culture with macrophages. Presence of antibody against TNF-alpha in culture media abolished the caspase-3 activation. Conclusions: Ionizing radiation induces lung apoptosis through upregulation of TNF-alpha. This may provide us the rationale for TNF-alpha targeted strategies for lung protection during radiotherapy.
139
Long-Term Administration of a Small Molecular Weight Catalytic Metalloporphyrin Antioxidant AEOL 10150 Protects Lungs From Radiation-Induced Injury
Z.N. Rabbani, I. Batinic-Haberle, S. Poulton, M.S. Anscher, M.W. Dewhirst, Z. Vujaskovic Radiation Oncology, Duke University Medical Ctr, Durham, NC Purpose/Objective: Radiation therapy is an important therapeutic modality in the treatment of several thoracic tumors. Though tumor control is best obtained with higher doses of radiation, clinical dosing protocols are often limited by the radiation tolerance of normal tissues. We have previously demonstrated that following RT, there is a prolonged generation of reactive oxygen species several months after the initial exposure, which correlates with persistent RT-induced damage. Furthermore, we have shown that chronic overexpression of extracellular superoxide dismutase (EC-SOD) in transgenic mice confers protection against RT-induced lung injury. Therefore, the objective of this study was to determine whether long-term administration of a small molecular weight catalytic metalloporphyrin antioxidant AEOL 10150 with SOD mimetic properties increases the tolerance of the lung to ionizing radiation by reducing the severity of radiation-induced pulmonary injury. Materials/Methods: Female Fisher 344 rats received single dose irradiation of 28 Gy to the right hemithorax. Animals were randomly divided into four groups a) RT⫹PBS, b) RT⫹1mg AEOL 10150, c) RT⫹10mg AEOL 10150, and d) RT⫹30mg AEOL 10150. The drug was administered for 10 weeks post-radiation with an osmotic pump implanted subcutaneously. Animals were followed up for 20 weeks after irradiation. Pulmonary function was assessed by monitoring changes in breathing rates. Histopathology was used to measure lung tissue damage and immunohistochemistry was performed to assess macrophage infiltration and activity. Results: Breathing rates increased beginning at 8 weeks and reached a peak 18 weeks after irradiation in animals treated with PBS and 1mg AEOL 10150. The average breathing rates in irradiated groups treated with AEOL 10150 (10 and 30 mg/kg/day) were significantly lower than the group receiving RT⫹PBS and RT⫹1mg AEOL 10150 (p⬍0.05). Lung histology at 20 weeks revealed a significant decrease in structural damage in animals receiving 10 and 30 mg/kg/day after radiation in comparison to RT⫹PBS group (p⫽0.01). Immunohistochemistry demonstrated a significant reduction in macrophage presence and activity in animals receiving AEOL 10150 (10 and 30 mg/kg/day, p⫽0.0006 and 0.016 respectively) after lung irradiation compared to RT⫹PBS. Conclusions: The chronic administration of novel SOD mimetic, AEOL 10150, demonstrates a significant protective effect from radiation-induced lung injury, as assessed by breathing frequency, histopathology, and immunohistochemistry. Such findings support the concept that SOD mimetics may be useful as radioprotective agents based on their ability to scavenge free radicals. This current study demonstrates that the chronic administration of SOD mimetic after exposure to ionizing irradiation might be an effective strategy to prevent/treat radiation induced lung injury. Supported by NIH/NCI R01-CA98452 and 1R-CA96245.
140
Novel Hormone HE2100 Protects Rhesus Macaques From Lethal Total Body Irradiation (TBI)- Induced Severe Neutropenia and Thrombocytopenia
D.R. Stickney, C. Dowding, S. Authier, A. Garsd, N. Onizuka-Handa, J. Frincke Hollis Eden Pharmaceuticals, San Diego, CA Purpose/Objective: HE2100 is a naturally occurring adrenal steroid hormone. Studies of lethally irradiated rodents demonstrated a significant survival advantage for HE2100 treated groups (Whitnall et. al., Int. J. Immunopharm.2000). Previously, (ASTRO-2004) we reported a reduction in lethality in Rhesus macaques following 6.00 Gy of TBI. The objectives of the current studies were to further investigate the effects of this compound to prevent severe radiation induced neutropenia and thrombocytopenia and to enhance survival following lethal irradiation in a non-human primate (NHP) model. We report a meta-analysis of 6 studies involving 108 Rhesus macaques. Materials/Methods: Studies investigated effects of HE2100 in non-human primates (NHP) with induced myelosuppression/ thrombocytopenia from ionizing radiation. NHP studies were performed in 6 separate studies of 108 NHP (untreated/vehicle and 5-36.5 mg/kg HE2100 IM daily once or x5) administered 2–3 hours after 6.00 Gy to 6.34 Gy, cobalt-60 or 6 MV X-ray TBI. No clinically supportive measures i.e. transfusions or antibiotics were given during the study. NHP were continuously monitored for temperature. Blood counts were measured daily during expected neutropenic days. Strict euthanasia criteria were observed to allay animal suffering. Studies were performed under Institutional Animal Control and Use Committee review and approval. Results: In the HE2100 treated groups 13/54 animals died (LD24) compared to 24/54 (LD44) in the untreated or vehicle control groups (p⫽0.0419). The cumulative incidence of days of severe neutropenia (absolute neutrophil count ⬍ 500 cells/microL)
Conclusions: Hb levels were significantly increased from baseline during the XRT period, irrespective of tumour localisation and stage. This increase reached the maximum value during week 10 from the baseline and remained significant 2 months post XRT. Quality of life was significantly improved in this group of patients.
137
Anti-TGF Antibodies May Prevent Lung Injury Following High Dose Fractionated Radiation
M. Anscher,1 B. Thrasher,1 Z. Rabbani,1 B. Teicher,2 Z. Vujaskovic1 1 Radiation Oncology, Duke University Medical Center, Durham, NC, 2Genzyme Corporation, Cambridge, MA Purpose/Objective: The cytokine Transforming Growth Factor (TGF) has been shown to play an important role in fibrogenesis associated with radiation (RT) induced normal tissue injury. Antagonists of TGF may have great potential to reduce formation of fibrous tissue and thereby protect normal tissues from the adverse effects of radiation. The purpose of this study is to determine whether an anti-TGF antibody (1D11, Genzyme Corp, Cambridge, MA) can prevent/ameliorate radiation induced lung injury. Materials/Methods: Fractionated and sham lung irradiation of Fischer-344 rats, both tumor (R3230Ac mammary adenocarcinoma) and non-tumor bearing animals, was performed to assess the radioprotective effect of the antibodies, and to insure that the antibodies do not protect tumor from irradiation. Animals were divided into the following groups(12 animals per group): 1) control (sham RT, isotype control antibody); 2) RT (800 cGy ⫻ 5 over 5 days to the right hemithorax); 3) RT plus 0.1 mg/kg i.p. antibody; and, 4) RT plus 1 mg/kg antibody. Animals were anesthetized for RT. Antibodies were administered as a single dose only, immediately after the last dose of RT. Animals were sacrificed at 6 weeks and 6 months after irradiation and lungs were assessed for histologic changes (inflammation and fibrosis), activation of macrophages, expression and activation of TGF and the TGF signal transduction pathway. Endpoints were the differences between groups with respect to both pulmonary toxicity and tumor growth delay. Results: Tumor growth delay studies did not show any evidence of protection of tumor by the anti-TGF antibody at either dose. There were no changes in body weight and no toxicity apparent from the addition of the anti-TGF antibody. At 6 weeks post-RT, there was a significant reduction in macrophages accumulation (p⫽0.041) and alveolar wall thickness (p⫽0.0003) in the animals receiving 1.0 mg/kg of anti-TGF antibody after RT as compared to the RT control antibody group. This significant decrease in inflammatory and histopathologic changes, seen after treatment with the higher dose of anti-TGF antibody (1mg/kg) following lung irradiation, are also associated with a significant inhibition in the expression of active TGF (p⫽0.031, for high dose group only) (Figure). Six month samples are currently under evaluation and these data will be presented at the meeting. Conclusions: Administration of a single dose of 1.0 mg/kg of the anti-TGF antibody 1D11 resulted in decreased morphological changes, inflammatory response, and reduced expression and activation of TGF 6 weeks after 40 Gy to the right hemithorax. Targeting the TGF pathway may be a useful strategy to prevent radiation induced lung injury. Supported by a grant from Genzyme Corporation.
High dose antibody (left bar) significantly decreases TGF beta expression compared to low dose antibody (center bar) or control (right bar)
138
Radiation-Induced Lung Apoptosis is Mediated by TNF-Alpha Action
M. Zhang, F. Kong, J. Qian, M. Chen, T.S. Lawrence Radiation Oncology, University of Michigan, Ann Arbor, MI Purpose/Objective: Radiation-induced lung damage is a major limitation for effective radiotherapy in lung cancer patients. This damage is correlated to an early lung apoptosis mediated by aberrant cytokine production after radiation treatment. In this study, we investigated the role of TNF-alpha in radiation-induced lung apoptosis and the potential protective effect of inhibiting TNF-alpha by using antisense oligonucleotide gene therapy. Materials/Methods: Balb/c mice (6 – 8 week, male) were irradiated with 15 Gy in lung and were measured for expression of TNF-alpha and TNFR1. Lung apoptosis was assessed by TUNEL staining and caspase-3 activation at 3 h after radiation. Inhibition of TNFR1 was established by pre-treatment of mice with antisense oligonucleotide specific for TNFR1 (i.p. 25
S83
S84
I. J. Radiation Oncology
● Biology ● Physics
Volume 63, Number 2, Supplement, 2005
mg/kg, 36 h interval ⫻ 4). Human lung macrophages and micro vascular endothelial cells were used to study TNF-alpha secretion and potential apoptosis in co-culture in response to radiation treatment. TNF-alpha production in lung tissue and culture media were determined by ELISA assay. Radio protection by antisense oligonucleotide was assessed by TUNEL staining and caspase-3 activation in lung. Results: Radiation in lung induced apoptosis in mice in a time- and dose-dependent manner. TUNEL staining and caspase-3 activation in lung were increased maximally at 3 h after 15 Gy of radiation by approximately 5- and 3-fold, correlating to the induction of TNF-alpha transcription and secretion. Pre-treatment of mice with antisense oligonucleotide effectively inhibited TNFR1 gene expression in lung by 50% and significantly decreased radiation-induced TUNEL staining and caspase-3 activation in lung. Radiation increased TNF-alpha production in human lung macrophages by 2 fold and induced caspase-3 activation in pulmonary micro vascular endothelial cells in co-culture with macrophages. Presence of antibody against TNF-alpha in culture media abolished the caspase-3 activation. Conclusions: Ionizing radiation induces lung apoptosis through upregulation of TNF-alpha. This may provide us the rationale for TNF-alpha targeted strategies for lung protection during radiotherapy.
139
Long-Term Administration of a Small Molecular Weight Catalytic Metalloporphyrin Antioxidant AEOL 10150 Protects Lungs From Radiation-Induced Injury
Z.N. Rabbani, I. Batinic-Haberle, S. Poulton, M.S. Anscher, M.W. Dewhirst, Z. Vujaskovic Radiation Oncology, Duke University Medical Ctr, Durham, NC Purpose/Objective: Radiation therapy is an important therapeutic modality in the treatment of several thoracic tumors. Though tumor control is best obtained with higher doses of radiation, clinical dosing protocols are often limited by the radiation tolerance of normal tissues. We have previously demonstrated that following RT, there is a prolonged generation of reactive oxygen species several months after the initial exposure, which correlates with persistent RT-induced damage. Furthermore, we have shown that chronic overexpression of extracellular superoxide dismutase (EC-SOD) in transgenic mice confers protection against RT-induced lung injury. Therefore, the objective of this study was to determine whether long-term administration of a small molecular weight catalytic metalloporphyrin antioxidant AEOL 10150 with SOD mimetic properties increases the tolerance of the lung to ionizing radiation by reducing the severity of radiation-induced pulmonary injury. Materials/Methods: Female Fisher 344 rats received single dose irradiation of 28 Gy to the right hemithorax. Animals were randomly divided into four groups a) RT⫹PBS, b) RT⫹1mg AEOL 10150, c) RT⫹10mg AEOL 10150, and d) RT⫹30mg AEOL 10150. The drug was administered for 10 weeks post-radiation with an osmotic pump implanted subcutaneously. Animals were followed up for 20 weeks after irradiation. Pulmonary function was assessed by monitoring changes in breathing rates. Histopathology was used to measure lung tissue damage and immunohistochemistry was performed to assess macrophage infiltration and activity. Results: Breathing rates increased beginning at 8 weeks and reached a peak 18 weeks after irradiation in animals treated with PBS and 1mg AEOL 10150. The average breathing rates in irradiated groups treated with AEOL 10150 (10 and 30 mg/kg/day) were significantly lower than the group receiving RT⫹PBS and RT⫹1mg AEOL 10150 (p⬍0.05). Lung histology at 20 weeks revealed a significant decrease in structural damage in animals receiving 10 and 30 mg/kg/day after radiation in comparison to RT⫹PBS group (p⫽0.01). Immunohistochemistry demonstrated a significant reduction in macrophage presence and activity in animals receiving AEOL 10150 (10 and 30 mg/kg/day, p⫽0.0006 and 0.016 respectively) after lung irradiation compared to RT⫹PBS. Conclusions: The chronic administration of novel SOD mimetic, AEOL 10150, demonstrates a significant protective effect from radiation-induced lung injury, as assessed by breathing frequency, histopathology, and immunohistochemistry. Such findings support the concept that SOD mimetics may be useful as radioprotective agents based on their ability to scavenge free radicals. This current study demonstrates that the chronic administration of SOD mimetic after exposure to ionizing irradiation might be an effective strategy to prevent/treat radiation induced lung injury. Supported by NIH/NCI R01-CA98452 and 1R-CA96245.
140
Novel Hormone HE2100 Protects Rhesus Macaques From Lethal Total Body Irradiation (TBI)- Induced Severe Neutropenia and Thrombocytopenia
D.R. Stickney, C. Dowding, S. Authier, A. Garsd, N. Onizuka-Handa, J. Frincke Hollis Eden Pharmaceuticals, San Diego, CA Purpose/Objective: HE2100 is a naturally occurring adrenal steroid hormone. Studies of lethally irradiated rodents demonstrated a significant survival advantage for HE2100 treated groups (Whitnall et. al., Int. J. Immunopharm.2000). Previously, (ASTRO-2004) we reported a reduction in lethality in Rhesus macaques following 6.00 Gy of TBI. The objectives of the current studies were to further investigate the effects of this compound to prevent severe radiation induced neutropenia and thrombocytopenia and to enhance survival following lethal irradiation in a non-human primate (NHP) model. We report a meta-analysis of 6 studies involving 108 Rhesus macaques. Materials/Methods: Studies investigated effects of HE2100 in non-human primates (NHP) with induced myelosuppression/ thrombocytopenia from ionizing radiation. NHP studies were performed in 6 separate studies of 108 NHP (untreated/vehicle and 5-36.5 mg/kg HE2100 IM daily once or x5) administered 2–3 hours after 6.00 Gy to 6.34 Gy, cobalt-60 or 6 MV X-ray TBI. No clinically supportive measures i.e. transfusions or antibiotics were given during the study. NHP were continuously monitored for temperature. Blood counts were measured daily during expected neutropenic days. Strict euthanasia criteria were observed to allay animal suffering. Studies were performed under Institutional Animal Control and Use Committee review and approval. Results: In the HE2100 treated groups 13/54 animals died (LD24) compared to 24/54 (LD44) in the untreated or vehicle control groups (p⫽0.0419). The cumulative incidence of days of severe neutropenia (absolute neutrophil count ⬍ 500 cells/microL)