Synthetic protection short interfering RNA screen reveals glyburide as a novel radioprotector.

To assist in screening existing drugs for use as potential radioprotectors, we used a human unbiased 16,560 short interfering RNA (siRNA) library targeting the druggable genome. We performed a synthetic protection screen that was designed to identify genes that, when silenced, protected human glioblastoma T98G cells from gamma-radiation-induced cell death. We identified 116 candidate protective genes, then identified 10 small molecule inhibitors of 13 of these candidate gene products and tested their radioprotective effects. Glyburide, a clinically used second-generation hypoglycemic drug, effectively decreased radiation-induced cell death in several cell lines including T98G, glioblastoma U-87 MG, and normal lung epithelial BEAS-2B and in primary cultures of astrocytes. Glyburide significantly increased the survival of 32D cl3 murine hematopoietic progenitor cells when administrated before irradiation. Glyburide was radioprotective in vivo (90% of C57BL/6NHsd female mice pretreated with 10 mg/kg glyburide survived 9.5 Gy total-body irradiation compared to 42% of irradiated controls, P = 0.0249). These results demonstrate the power of unbiased siRNA synthetic protection screening with a druggable genome library to identify new radioprotectors.

Mitochondrial targeting of a catalase transgene product by plasmid liposomes increases radioresistance in vitro and in vivo.

To determine whether increased mitochondrially localized catalase was radioprotective, a human catalase transgene was cloned into a small pSVZeo plasmid and localized to the mitochondria of 32D cl 3 cells by adding the mitochondrial localization sequence of MnSOD (mt-catalase). The cell lines 32D-Cat and 32D-mt-Cat had increased catalase biochemical activity as confirmed by Western blot analysis compared to the 32D cl 3 parent cells. The MnSOD-overexpressing 32D cl 3 cell line, 2C6, had decreased baseline catalase activity that was increased in 2C6-Cat and 2C6-mt-Cat subclonal cell lines. 32D-mt-Cat cells were more radioresistant than 32D-Cat cells, but both were radioresistant relative to 32D cl 3 cells. 2C6-mt-Cat cells but not 2C6-Cat cells were radioresistant compared to 2C6 cells. Intratracheal injection of the mt-catalase-plasmid liposome complex (mt-Cat-PL) but not the catalase-plasmid liposome complex (Cat-PL) increased the resistance of C57BL/6NHsd female mice to 20 Gy thoracic irradiation compared to MnSOD-plasmid liposomes. Thus mitochondrially targeted overexpression of the catalase transgene is radioprotective in vitro and in vivo.

Ethyl pyruvate, a potentially effective mitigator of damage after total-body irradiation.

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to improve survival and ameliorate organ damage in animal models of sepsis, ischemia/reperfusion injury and hemorrhagic shock. Incubating IL3-dependent mouse hematopoietic progenitor cell 32Dcl3 cells before or after irradiation with 10 mM EP increased resistance to radiation as assessed by clonogenic radiation survival curves, decreased release of mitochondrial cytochrome C into the cytoplasm, and decreased apoptosis. EP inhibited radiation-induced caspase 3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in 32Dcl3 cells in a concentration-dependent fashion. EP was given i.p. to C57BL/6NHsd mice irradiated with 9.75 Gy total-body irradiation (TBI). This treatment significantly improved survival. The survival benefit was apparent irrespective of whether treatment with EP was started 1 h before TBI and continued for 5 consecutive days after TBI or the compound was injected only 1 h before or only for 5 days after TBI. In all of the in vitro and in vivo experiments, ethyl lactate, an inactive analogue of EP, had no detectable radioprotective or mitigating effects. EP may be an effective radioprotector and mitigator of the hematopoietic syndrome induced by TBI.

Effects of MnSOD-plasmid liposome gene therapy on antioxidant levels in irradiated murine oral cavity orthotopic tumors.

Intraoral manganese superoxide dismutase (SOD2)-plasmid liposome (PL) radioprotective gene therapy prolongs the survival of mice with orthotopic oral cavity tumors within the irradiated field. To determine whether the mechanism involved effects in antioxidant pool, C57BL/6J mice bearing orthotopic oral cavity squamous cell carcinoma SCC-VII tumors received intraoral or intravenous MnSOD-PL gene therapy 24 h prior to 18 Gy irradiation to the head and neck region. Glutathione (GSH) levels and levels of radiation-generated nitric oxide and peroxynitrite were measured in orthotopic tumors and in adjacent oral mucosa. MnSOD-PL transfection of the SCC-VII tumor cells, but not normal embryo fibroblasts, produced acute radiosensitization. Furthermore, SCC-VII tumor cells demonstrated increased relative hydrogen peroxide (the product of MnSOD superoxide dismutation)-induced apoptosis in vitro. Radiation decreased levels of GSH and increased GPX in both tumor and normal cells in vitro, effects that were blunted by MnSOD-PL treatment. In vivo irradiation decreased GSH and GPX more effectively in tumors, and the decrease was not reversed by MnSOD-PL therapy. Intravenous but not intraoral administration of epitope-tagged hemagglutinin MnSOD-PL resulted in significant uptake in orthotopic tumors and decreased the levels of radiation-induced nitric oxide and peroxynitrite. Thus normal tissue radioprotective MnSOD-PL gene therapy radiosensitizes tumor cell lines in vitro and has a therapeutic effect on orthotopic tumors in part through its effects on tumor antioxidants.

Reduced irradiation pulmonary fibrosis and stromal cell migration in Smad3-/- marrow chimeric mice.

Pulmonary irradiation fibrosis involves migration to the lungs of bone marrow origin myofibroblast progenitor cells (marrow stromal cells (MSCs)). Smad3-/- mice display decreased ionizing irradiation-induced skin fibrosis, defective osteochondrogenesis and other abnormalities thought to be associated with a defective stromal cell response(s) to transforming growth factor-beta (TGFFbeta). Clonal bone marrow stromal cell lines were derived from the adherent layer of continuous bone marrow cultures of homozygous deletion recombinant negative Smad3-/- mice and Smad3+/+ littermates. Quantitation in an Automated Cell Tracking System of the in vitro single cell migratory capacity over five days demonstrated a significant decrease in locomotion in microns per 24 h of Smad3-/- compared to Smad3+/+ clonal MSC lines. Reexpression by retroviral vector transfection of the Smad3 but not control ds-red transgene restored in vitro migratory capacity. Intravenously injected GFP transgene product labeled Smad3-/- (MSCs) seeded 10-fold less effectively than ds-red transgene product labeled Smad3+/+ cells to the 80 days post 20 Gy irradiated lungs of C57BL/6J mice and proliferated less significantly for 60 days after cell injection. Female mice chimeric for male Smad3-/- compared to Smad3+/+ marrow showed decreased irradiation pulmonary fibrosis, Y+ stromal cell migration to the lungs, and improved survival. The data show that the reduced in vitro and in vivo migratory capacity of Smad3-/- bone marrow stromal cells correlates with decreased radiation pulmonary fibrosis observed in mice chimeric for Smad3-/- marrow.

Increased radioresistance, g(2)/m checkpoint inhibition, and impaired migration of bone marrow stromal cell lines derived from Smad3(-/-) mice.

Smad3 protein is a prominent member of the Tgfb receptor signaling pathway. Smad3(-/-) mice display decreased radiation-induced skin fibrosis, suggesting a defect in both Tgfb-mediated fibroblast proliferation and migration. We established bone marrow stromal cell lines from Smad3(-/-) mice and homozygous littermate(+/+) mice. Smad3(-/-) cells displayed a significant increase in radiation resistance with a D(0)=2.25+/- 0.14 Gy compared to Smad3(+/+) cells with a D(0)=1.75+/- 0.03 (P=0.023). Radioresistance was abrogated by reinsertion of the human SMAD3 transgene, resulting in a D(0)=1.49 0.10 (P=0.028) for Smad3(-/-)(3) cells. More Smad3(-/-) cells than Smad3(+/+) cells were in the G(2)/M phase; Smad3(-/-)(3) cells were similar to Smad3(+/+) cells. Smad3(+/+) cells exhibited increased apoptosis 24 h after 5 Gy (15%) or 8 Gy (43%) compared to less than 1% in Smad3(-/-) cells exposed to either dose. The movement of Smad3(-/-) cells, measured in an automated cell tracking system, was slower than that of Smad3(+/+) cells. Smad3(-/-)(3) cells resembled Smad3(+/+) cells. These studies establish concordance of a defective Tgfb signal transduction pathway, an increased proportion of G(2)/M cells, and radioresistance. The decreased migratory capacity of Smad3(-/-) cells in vitro correlates with decreased radiation fibrosis in vivo in mice deficient in Tgfb signaling.

Effect of EGFR antagonists gefitinib (Iressa) and C225 (Cetuximab) on MnSOD-plasmid liposome transgene radiosensitization of a murine squamous cell carcinoma cell line.

Radiation therapy of tumors of the head and neck region is compromised by dose limiting toxicity of normal tissues including the oral cavity and oropharyngeal mucosa. MnSOD-Plasmid Liposome (MnSOD-PL) intraoral gene therapy has been demonstrated to decrease normal tissue toxicity and also improve survival in mice with orthotopic SCC-VII squamous cell tumors on the floor of the mouth. Furthermore, intravenous administration of MnSOD-PL in mice with orthotopic tumors, or addition of MnSOD-PL to tumor cell lines in vitro produces a radiosensitizing effect attributable to differences in antioxidant pool responses of tumor cells compared to normal tissues following irradiation. To determine whether EGF receptor (EGFR) antagonists Iressa, or Cetuximab provided further improvement of radiation killing of squamous cell tumors, MnSOD-PL transfected or control SCCVII tumor cells were irradiated in vitro, and then the effect of EGFR receptor antagonists was tested. Cells transfected with MnSOD-PL were relatively radiosensitive D0 = 1.244 +/- 0.126 Gy compared to control D0 = 3.246 +/- 0.087 (p < 0.0001). Clonogenic radiation survival curves of SCCVII cells demonstrated radiosensitization by Iressa D0 = 2.770 +/- 0.134 Gy (p = 0.0264), but no significant radiosensitizing effect of Cetuximab D0 = 3.193 +/- 0.309 (p = 0.7338). The combination of MnSOD-PL plus Iressa further increased radiosensitivity of SCC-VII cells in vitro D0 = 0.785 +/- 0.01064 (p < 0.0001). The results suggest some synergy of the effectiveness of the EGFR antagonist Iressa on increasing the radiation killing of SCC-VII cells that supplements MnSOD-PL tumor radiosensitization.

Protection of esophageal multi-lineage progenitors of squamous epithelium (stem cells) from ionizing irradiation by manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) gene therapy.

BACKGROUND: Intraesophageal manganese superoxide dismutase plasmid liposome (MnSOD-PL) gene therapy protects against irradiation damage. MATERIALS AND METHODS: To determine whether esophageal side population (SP) stem cells were protected, epitope-tagged (hemagglutinin) (HA) MnSOD-PL was administered to C57BL/6J mice 24 hours prior to 30 Gy esophageal irradiation. SP cells were isolated, and apoptosis and multi-lineage vimentin/endothelin/F4/80 (macrophage) colonies in vitro were quantitated. RESULTS: The number and percent of SP cells, apoptotic cells, or numbers of multi-lineage vimentin/endothelin/F4/80-positive in vitro colonies isolated from non-irradiated HA-MnSOD-PL-treated or 30 Gy-irradiated esophagus did not differ between groups. Irradiation in vitro significantly increased apoptosis in explanted non-SP cells from control (p = 0.021) compared to MnSOD-PL-treated mice. Irradiation-induced cell division was significantly increased in SP cells from control-irradiated mice (p = 0.001), but not MnSOD-PL-treated mice. Irradiation-induced apoptosis detected in vivo at 5 days was decreased by MnSOD-PL. CONCLUSION: MnSOD-PL gene therapy protects esophageal SP cells from irradiation in vitro and in vivo.

MnSOD-plasmid liposome gene therapy decreases ionizing irradiation-induced lipid peroxidation of the esophagus.

BACKGROUND: Ionizing irradiation-induced cellular and tissue damage is mediated in part by resultant radiochemical reactions and resultant oxidative stress. Irradiation-induced reactive oxygen and nitrogen species include: superoxide, nitric oxide, hydroxyl radical and hydrogen peroxide. The biochemical combination of superoxide and nitric oxide radicals forms peroxynitrite, a potent oxidant known to induce lipid peroxidation. MATERIALS AND METHODS: The antioxidant capacity and lipid peroxidation of the esophagus were determined following irradiation. RESULTS: In the present studies, measurements of total antioxidant capacity did not change in the esophagus of control irradiated or control plasmid pNGVL3-PL intraesophageally-injected mice. In contrast, manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) intraesophageally-treated mice showed a significant increase in antioxidant capacity persisting for seven days. Lipid peroxidative changes induced in the control irradiated mouse esophagus decreased over seven days after irradiation of C3H/HeNHsd mice exposed to 37 Gy in a single fraction. MnSOD-PL radioprotective gene therapy administered intraorally 24 hours prior to irradiation did not significantly reduce the kinetics of induction of total peroxidated lipids over the first seven days after irradiation but did decrease lipid peroxidation at days 14 and 21. CONCLUSION: These studies demonstrate the antioxidant function of MnSOD-PL gene therapy to the esophagus, which is detectable as a reduction in irradiation-induced lipid peroxidation.

Adipocyte differentiation in Sod2(-/-) and Sod2(+/+) murine bone marrow stromal cells is associated with low antioxidant pools.

OBJECTIVE: Adipocytogenesis in bone marrow stromal cells (BMSCs) from manganese-superoxide dismutase-deficient (Sod2(-/-)) and wild-type (Sod2(+/+)) mice and the effect of antioxidant pool size were determined. METHODS: BMSCs from Sod2(-/-) or Sod2(+/+) mice were cultured with and without adipocytogenic supplements including: 10 mug/mL insulin, 1 muM dexamethasone, and 100 muM indomethacin. Oil Red-O-positive cells and reverse-transcriptase polymerase chain reaction measurement of peroxisome proliferator-activated receptor-gamma (PPARgamma) and lipoprotein lipase (LPL) were measured. Antioxidant glutathione levels (GSH) and glutathione peroxidase activity (GPX) were determined. RESULTS: Sod2(-/-) cells demonstrated constitutive adipocytogenesis in basal medium and generated 34% more adipocytes in adipocytogenic media. Growth of cells in the free radical scavenger antioxidant, amifostine (WR2721; 4 mM) decreased numbers of adipocytes in Sod2(-/-) BMSCs in both basal (38.0%, p = 0.037) and adipocytogenic (37.5%, p = 0.021) media and reduced to undetectable the levels of expression of PPARgamma and LPL. In contrast, Sod2(+/+) cells showed no detectable constitutive adipocytogenesis but formed adipocytes in adipocytogenic medium, with a decrease (43.7%, p = 0.001) by addition of WR2721. In basal conditions, Sod2(-/-) cells had lower GSH (78.6%; p = 0.0089) and GPX (52.7%; p < 0.001) levels than did Sod2(+/+) cells, which were increased in either medium by WR2721 treatment of Sod2(-/-) or Sod2(+/+) cells (all p < 0.001). Differentiation of BMSCs to adipocytes was inversely correlated with the level of GSH (r = -0.9427, p = 0.0167). Sod2(-/-) long-term bone marrow cultures had decreased hematopoiesis compared to those from Sod2(+/-) or Sod2(+/+) mice. CONCLUSION: The cellular redox pathway has a role in adipocyte differentiation of cells of the hematopoietic microenvironment.

Intraoral manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) radioprotective gene therapy decreases ionizing irradiation-induced murine mucosal cell cycling and apoptosis.

BACKGROUND: Single or multiple intraoral administrations of manganese superoxide dismutase-plasmid/liposomes (MnSOD-PL) to C3H/HeNHsd mice receiving single fraction or fractionated ionizing irradiation to the head and neck region have been shown to significantly decrease mucosal ulceration, weight loss and to improve survival. MATERIALS AND METHODS: To elucidate the mechanism of irradiation protection by MnSOD-PL and explore possible additive or synergistic protective effects with Amifostine (WR2721), mice received a single fraction of 19, 22.5, 25 or 30 Gy, or 24 fractions of 3 Gy irradiation to the oral cavity and oropharynx. Multiple parameters of irradiation-induced toxicity were quantitated in subgroups of each irradiated group of mice treated with single or multiple administrations of intraoral MnSOD-PL and/or intravenous WR2721. RESULTS: In 19 Gy single fraction irradiated mice, MnSOD-PL treatment the day before irradiation alone or in combination with intravenous WR2721 significantly decreased the irradiation induction of mucosal cell cycling as measured by 5-bromo-2-deoxyuridine (BuDR) uptake in oral cavity mucosal cells at 48 hours and decreased ulceration of the tongue at nine days after irradiation compared to control, irradiated or irradiated, WR2721-treated mice. Mice treated in single fractions of 22.5, 25 or 30 Gy showed MnSOD-PL protection against irradiation-induced oral mucosal apoptosis and xerostomia measured in decreased saliva output. In fractionated irradiated mice, twice weekly hemagglutinin (HA) epitope-tagged MnSOD uptake in oral cavity and tongue mucosal cells was not detectably altered by daily WR2721 intravenous administration. Mice treated with both radioprotective agents (MnSOD-PL and WR2721) demonstrated a significant decrease in irradiation-induced xerostomia (measured as reduced salivary gland output volume), mucosal ulceration and improved survival. CONCLUSION: Enhanced salivary gland function in WR2721-treated mice in the absence of detectable mucosal protection, coupled with relatively low uptake of HA-MnSOD in the salivary glands of intraorally-treated mice, suggests that a combination of both radioprotective agents may prove optimally effective for the prevention of the acute and late normal tissue toxicities of fractionated radiotherapy for head and neck cancer.

Mitochondrial localization of superoxide dismutase is required for decreasing radiation-induced cellular damage.

We investigated the importance of mitochondrial localization of the SOD2 (MnSOD) transgene product for protection of 32D cl 3 hematopoietic cells from radiation-induced killing. Four plasmids containing (1) the native human copper/zinc superoxide dismutase (Cu/ZnSOD, SOD1) transgene, (2) the native SOD2 transgene, (3), the SOD2 transgene minus the mitochondrial localization leader sequence (MnSOD-ML), and (4) the SOD2 mitochondrial leader sequence attached to the active portion of the SOD1 transgene (ML-Cu/ZnSOD) were transfected into 32D cl 3 cells and subclonal lines selected by kanamycin resistance. Clonogenic in vitro radiation survival curves derived for each cell clone showed that Cu/ZnSOD- and MnSOD-ML-expressing clones had no increase in cellular radiation resistance (D0=0.89 +/- 0.01 and 1.08 +/- 0.02 Gy, respectively) compared to parent line 32D cl 3 (D0=1.15 +/- 0.11 Gy). In contrast, cell clones expressing either SOD2 or ML-Cu/ZnSOD were significantly radioresistant (D0=2.1 +/- 0.1 and 1.97 +/- 0.17 Gy, respectively). Mice injected intraesophageally with SOD2-plasmid/liposome (MnSOD-PL) complex demonstrated significantly less esophagitis after 35 Gy compared to control irradiated mice or mice injected intraesophageally with Cu/ZnSOD-PL or MnSOD-ML-PL. Mice injected with intraesophageal ML-Cu/ZnSOD-PL showed significant radioprotection in one experiment. The data demonstrate the importance of mitochondrial localization of SOD in the in vitro and in vivo protection of cells from radiation-induced cellular damage.

Overexpression of the transgene for manganese superoxide dismutase (MnSOD) in 32D cl 3 cells prevents apoptosis induction by TNF-alpha, IL-3 withdrawal, and ionizing radiation.

OBJECTIVE: Stabilization of the mitochondria in IL-3-dependent hematopoietic progenitor cell line 32D cl 3 by overexpression of the transgene for manganese superoxide dismutase (MnSOD) prior to ionizing radiation prevents apoptosis. We now demonstrate that overexpression of the MnSOD transgene also protects 32D cl 3 cells from apoptosis caused by exposure to tumor necrosis factor-alpha (TNF-alpha) or withdrawal of interleukin (IL)-3. MATERIALS AND METHODS: The hematopoietic progenitor cell line, 32D cl 3, and subclones overexpressing the human MnSOD transgene, 1F2 or 2C6, were radiated to 1000 cGy or were exposed to TNF-alpha (0 to 100 etag/mL) or were subjected to IL-3 withdrawal. The cells were then examined at several time points for DNA strand breaks using a comet assay, depolarization of the mitochondrial membrane, activation of caspase-3, PARP cleavage, and apoptosis, and also for changes in cell cycle distribution. RESULTS: Overexpression of the transgene for MnSOD resulted in increased survival following exposure to radiation, exposure to TNF-alpha, or IL-3 withdrawal. The cell lines overexpressing MnSOD (1F2 or 2C6) displayed decreased radiation-induced, TNF-alpha-induced, or IL-3 withdrawal-induced mitochondrial membrane permeability, caspase-3 and PARP activation, and apoptosis. CONCLUSIONS: Overexpression of the human MnSOD transgene in 32D cl 3 cells results in stabilization of the mitochondria and reduction in radiation-, TNF-alpha-, or IL-3 withdrawal-induced damage. Thus, MnSOD stabilization of the mitochondrial membrane is relevant to reduction of apoptosis by several classes of oxidative stress inducers.

Manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) intratracheal gene therapy reduction of irradiation-induced inflammatory cytokines does not protect orthotopic Lewis lung carcinomas.

BACKGROUND: Intratracheal injection of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) prior to single fraction or fractionated irradiation of C57BL/6J mouse lung has been demonstrated to protect the lung from irradiation-induced damage. MATERIALS AND METHODS: To determine whether irradiation-induced inflammatory cytokine levels influenced the recovery of tumors following single fraction irradiation, mice with orthotopic Lewis Lung Carcinoma (3LL) tumors received MnSOD-PL treatment 24 hours after tumor implantation and 24 hours prior to irradiation. Subgroups were implanted with Alzet pumps continuously replacing levels of inflammatory cytokines over 7 days. RESULTS: In cytokine-treated mice, there was no detectable significant alteration in radiotherapy-mediated improved survival (tumor regrowth delay) compared to irradiated control mice. Each group of mice that received MnSOD-PL had increased survival compared to irradiated controls. CONCLUSION: These results support the anticipated safety of intrapulmonary MnSOD-PL gene therapy in lung cancer patients for protection of normal lung tissue from irradiation while allowing effective irradiation-mediated tumor control.

Potentiation of nitric oxide-induced apoptosis in p53-/- vascular smooth muscle cells.

The functional role of p53 in nitric oxide (NO)-mediated vascular smooth muscle cell (VSMC) apoptosis remains unknown. In this study, VSMC from p53-/- and p53+/+ murine aortas were exposed to exogenous or endogenous sources of NO. Unexpectedly, p53-/- VSMC were much more sensitive to the proapoptotic effects of NO than were p53+/+ VSMC. Furthermore, this paradox appeared to be specific to NO, because other proapoptotic agents did not demonstrate this differential effect on p53-/- cells. NO-induced apoptosis in p53-/- VSMC occurred independently of cGMP generation. However, mitogen-activated protein kinase (MAPK) pathways appeared to play a significant role. Treatment of the p53-/- VSMC with S-nitroso-N-acetylpenicillamine resulted in a marked activation of p38 MAPK and, to a lesser extent, of c-Jun NH(2)-terminal kinase, mitogen-activated protein kinase kinase (MEK) 1/2, and p42/44 (extracellular signal-regulated kinase, ERK). Furthermore, basal activity of the MEK-p42/44 (ERK) pathway was increased in the p53+/+ VSMC. Inhibition of p38 MAPK with SB-203580 or of MEK1/2 with PD-98059 blocked NO-induced apoptosis. Therefore, p53 may protect VSMC against NO-mediated apoptosis, in part, through differential regulation of MAPK pathways.