Mitchell C. Coleman

Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress.

Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3⁻/⁻ livers at 3 months of age. Livers of Sirt3⁻/⁻ mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null Sirt3 into Sirt3⁻/⁻ MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSOD(K122-R)), increased MnSOD activity when expressed in MnSOD⁻/⁻ MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3⁻/⁻ MEFs with lenti-MnSOD(K122-R) inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3⁻/⁻ livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.

2-deoxy-D-glucose causes cytotoxicity, oxidative stress, and radiosensitization in pancreatic cancer.

Glucose metabolism as assessed by (18)FDG PET imaging provides prognostic information in patients with pancreatic cancer but the implications of manipulating glucose metabolism for therapeutic purposes are unknown. Based on previous results with other cancer cell types, we hypothesized that inhibition of glucose metabolism in pancreatic cancer cells would cause cell killing via oxidative stress resulting from disruptions in thiol metabolism. 2-Deoxy-D-glucose (2DG), a chemical inhibitor of glucose metabolism, and glucose deprivation induced cytotoxicity in human pancreatic cancer cells in a time-and dose-dependent manner as well as causing significant increases in metabolic oxidative stress as measured by increased glutathione disulfide accumulation and NADP(+)/NADPH ratios. Simultaneous administration of the thiol antioxidant N-acetylcysteine protected pancreatic cancer cells against the c-ytotoxic effects of 2DG as well as reversing 2DG-induced glutathione disulfide accumulation and augmenting intracellular cysteine pools. In nude mice with heterotopic pancreatic tumors, the combination of 2DG and ionizing radiation resulted in greater inhibition of tumor growth and increased survival, relative to either agent alone. These results support the hypothesis that inhibiting glucose metabolism causes cytotoxicity in human pancreatic cancer cells via metabolic oxidative stress and disruptions in thiol metabolism. These results also support the speculation that inhibitors of glucose metabolism can be used in combination with classical oxidative stress-inducing agents (such as ionizing radiation) to enhance therapeutic responses in pancreatic cancer.

Nuclear factor-κB and manganese superoxide dismutase mediate adaptive radioresistance in low-dose irradiated mouse skin epithelial cells

Mechanisms governing inducible resistance to ionizing radiation in untransformed epithelial cells pre-exposed to low-dose ionizing radiation (LDIR; </=10 cGy) are not well understood. The present study provides evidence that pre-exposure to 10 cGy X-rays increases clonogenic survival of mouse skin JB6P+ epithelial cells subsequently exposed to 2 Gy doses of gamma-rays. To elucidate the molecular pathways of LDIR-induced adaptive radioresistance, the transcription factor nuclear factor-kappaB (NF-kappaB) and a group of NF-kappaB-related proteins [i.e., p65, manganese superoxide dismutase (MnSOD), phosphorylated extracellular signal-regulated kinase, cyclin B1, and 14-3-3zeta] were identified to be activated as early as 15 min after LDIR. Further analysis revealed that a substantial amount of both 14-3-3zeta and cyclin B1 accumulated in the cytoplasm at 4 to 8 h when cell survival was enhanced. The nuclear 14-3-3zeta and cyclin B1 were reduced and increased at 4 and 24 h, respectively, after LDIR. Using YFP-fusion gene expression vectors, interaction between 14-3-3zeta and cyclin B1 was visualized in living cells, and LDIR enhanced the nuclear translocation of the 14-3-3zeta/cyclin B1 complex. Treatment of JB6P+ cells with the NF-kappaB inhibitor IMD-0354 suppressed LDIR-induced expression of MnSOD, 14-3-3zeta, and cyclin B1 and diminished the adaptive radioresistance. In addition, treatment with small interfering RNA against mouse MnSOD was shown to inhibit the development of LDIR-induced radioresistance. Together, these results show that NF-kappaB, MnSOD, 14-3-3zeta, and cyclin B1 contribute to LDIR-induced adaptive radioresistance in mouse skin epithelial cells.

Paclitaxel combined with inhibitors of glucose and hydroperoxide metabolism enhances breast cancer cell killing via H2O2-mediated oxidative stress

Cancer cells (relative to normal cells) demonstrate alterations in oxidative metabolism characterized by increased steady-state levels of reactive oxygen species (i.e., hydrogen peroxide, H(2)O(2)) that may be compensated for by increased glucose metabolism, but the therapeutic significance of these observations is unknown. In this study, inhibitors of glucose (i.e., 2-deoxy-d-glucose, 2DG) and hydroperoxide (i.e., l-buthionine-S,R-sulfoximine, BSO) metabolism were utilized in combination with a chemotherapeutic agent, paclitaxel (PTX), thought to induce oxidative stress, to treat breast cancer cells. 2DG + PTX was more toxic than either agent alone in T47D and MDA-MB231 human breast cancer cells, but not in normal human fibroblasts or normal human mammary epithelial cells. Increases in parameters indicative of oxidative stress, including steady-state levels of H(2)O(2), total glutathione, and glutathione disulfide, accompanied the enhanced toxicity of 2DG + PTX in cancer cells. Antioxidants, including N-acetylcysteine and polyethylene glycol-conjugated catalase and superoxide dismutase, inhibited the toxicity of 2DG + PTX and suppressed parameters indicative of oxidative stress in cancer cells, whereas inhibition of glutathione synthesis using BSO further sensitized breast cancer cells to 2DG + PTX. These results show that combining inhibitors of glucose (2DG) and hydroperoxide (BSO) metabolism with PTX selectively (relative to normal cells) enhances breast cancer cell killing via H(2)O(2)-induced metabolic oxidative stress, and suggest that this biochemical rationale may be effectively utilized to treat breast cancers.

All-trans-retinoic acid induces manganese superoxide dismutase in human neuroblastoma through NF-κB

Retinoids are signaling molecules that are involved in proliferation, differentiation, and apoptosis during development. Retinoids exert their effects, in part, by binding to nuclear receptors, thereby altering gene expression. Clinical use of retinoids in the treatment of neuroblastoma is of interest due to their success in management of acute promyelocytic leukemia. Using the SK-N-SH human neuroblastoma cell line we investigated the effects of the differentiation agent all-trans-retinoic acid (ATRA) on the expression of manganese superoxide dismutase (MnSOD), an enzyme previously shown to enhance differentiation in vitro. Manganese superoxide dismutase mRNA, protein, and activity levels increased in a time-dependent manner upon treatment with ATRA. Nuclear levels of the NF-kappaB proteins p50 and p65 increased within 24 h of ATRA administration. This increase paralleled the degradation of the cytoplasmic inhibitor IkappaB-beta. Furthermore an increase in DNA binding to a NF-kappaB element occurred within a 342-bp enhancer (I2E) of the SOD2 gene with 10 microM ATRA treatment. Reporter analysis showed that ATRA-mediated I2E-dependent luciferase expression was attenuated upon mutation of the NF-kappaB element, suggesting a contribution of this transcription factor to retinoid-mediated upregulation of MnSOD. This study identifies SOD2 as a retinoid-responsive gene and demonstrates activation of the NF-kappaB pathway in response to ATRA treatment of SK-N-SH cells. These results suggest that signaling events involving NF-kappaB and SOD2 may contribute to the effects of retinoids used in cancer therapy.

Inhibition of Glutamate Cysteine Ligase Activity Sensitizes Human Breast Cancer Cells to the Toxicity of 2-Deoxy-d-Glucose

It has been hypothesized that cancer cells increase glucose metabolism to protect against metabolic fluxes of hydroperoxides via glutathione-dependent peroxidases. 2-Deoxy-D-glucose, inhibits glucose metabolism and has been shown to cause cytotoxicity in cancer cells that is partially mediated by disruptions in thiol metabolism. In the current study, human breast cancer cells were continuously treated (24 hours) with 2-deoxy-D-glucose, and total glutathione content as well as the expression of the first enzyme in the glutathione synthetic pathway [glutamate cysteine ligase (GCL)] were found to be induced 2.0-fold. Inhibiting GCL activity during 2-deoxy-D-glucose exposure using l-buthionine-[S,R]-sulfoximine (BSO) significantly enhanced the cytotoxic effects of 2-deoxy-D-glucose and caused increases in endpoints indicative of oxidative stress, including % oxidized glutathione and steady-state levels of pro-oxidants as assayed using an oxidation-sensitive fluorescent probe. These results show that treatment of human breast cancer cells with 2-deoxy-d-glucose causes metabolic oxidative stress that is accompanied by increases in steady-state levels of GCL mRNA, GCL activity, and glutathione content. Furthermore, inhibition of 2-deoxy-D-glucose-mediated induction of GCL activity with BSO increases endpoints indicative of oxidative stress and sensitizes cancer cells to 2-deoxy-D-glucose-induced cytotoxicity. These results support the hypothesis that drug combinations capable of inhibiting both glucose and hydroperoxide metabolism may provide an effective biochemical strategy for sensitizing human cancer cells to metabolic oxidative stress.

Manganese Superoxide Dismutase gene dosage affects chromosomal instability and tumor onset in a mouse model of T cell lymphoma

Increased reactive oxygen species (ROS) such as superoxide have been implicated as causal elements of oncogenesis. A variety of cancers have displayed changes in steady-state levels of key antioxidant enzymes, with the mitochondrial form of superoxide dismutase (MnSOD) being commonly implicated. Increasing MnSOD expression suppresses the malignant phenotype in various cancer cell lines and suppresses tumor formation in xenograft and transgenic mouse models. We examined the impact of MnSOD expression in the development of T cell lymphoma in mice expressing proapoptotic Bax. Lck-Bax38/1 transgenic mice were crossed to mice overexpressing MnSOD (Lck-MnSOD) as well as MnSOD+/- mice. The effects of MnSOD on apoptosis, cell cycle, chromosomal instability (CIN), and lymphoma development were determined. The apoptotic and cell cycle phenotypes observed in thymocytes from control and Bax transgenic mice were unaffected by variations in MnSOD levels. Remarkably, increased gene dosage of MnSOD significantly decreased aneuploidy in premalignant thymocytes as well as the onset of tumor formation in Lck-Bax38/1 mice. The observed effects of MnSOD support a role for ROS in CIN and tumor formation in this mouse model of T cell lymphoma.

Amifostine Induces Antioxidant Enzymatic Activities in Normal Tissues and a Transplantable Tumor That Can Affect Radiation Response

PURPOSE To determine whether amifostine can induce elevated manganese superoxide dismutase (SOD2) in murine tissues and a transplantable SA-NH tumor, resulting in a delayed tumor cell radioprotective effect. METHODS AND MATERIALS SA-NH tumor-bearing C3H mice were treated with a single 400 mg/kg or three daily 50 mg/kg doses of amifostine administered intraperitoneally. At selected time intervals after the last injection, the heart, liver, lung, pancreas, small intestine, spleen, and SA-NH tumor were removed and analyzed for SOD2, catalase, and glutathione peroxidase (GPx) enzymatic activity. The effect of elevated SOD2 enzymatic activity on the radiation response of SA-NH cells was determined. RESULTS SOD2 activity was significantly elevated in selected tissues and a tumor 24 h after amifostine treatment. Catalase and GPx activities remained unchanged except for significant elevations in the spleen. GPx was also elevated in the pancreas. SA-NH tumor cells exhibited a twofold elevation in SOD2 activity and a 27% elevation in radiation resistance. Amifostine administered in three daily fractions of 50 mg/kg each also resulted in significant elevations of these antioxidant enzymes. CONCLUSIONS Amifostine can induce a delayed radioprotective effect that correlates with elevated levels of SOD2 activity in SA-NH tumor. If limited to normal tissues, this delayed radioprotective effect offers an additional potential for overall radiation protection. However, amifostine-induced elevation of SOD2 activity in tumors could have an unanticipated deleterious effect on tumor responses to fractionated radiation therapy, given that the radioprotector is administered daily just before each 2-Gy fractionated dose.

WR-1065, the active metabolite of amifostine, mitigates radiation-induced delayed genomic instability.

Compounds that can protect cells from the effects of radiation are important for clinical use, in the event of an accidental or terrorist-generated radiation event, and for astronauts traveling in space. One of the major concerns regarding the use of radio-protective agents is that they may protect cells initially, but predispose surviving cells to increased genomic instability later. In this study we used WR-1065, the active metabolite of amifostine, to determine how protection from direct effects of high- and low-LET radiation exposure influences genomic stability. When added 30 min before irradiation and in high concentrations, WR-1065 protected cells from immediate radiation-induced effects as well as from delayed genomic instability. Lower, nontoxic concentrations of WR-1065 did not protect cells from death; however, it was effective in significantly decreasing delayed genomic instability in the progeny of irradiated cells. The observed increase in manganese superoxide dismutase protein levels and activity may provide an explanation for this effect. These results confirm that WR-1065 is protective against both low- and high-LET radiation-induced genomic instability in surviving cells.

Genomic instability induced by mutant succinate dehydrogenase subunit D (SDHD) is mediated by O2−• and H2O2

SDHD mutations are associated with human cancers but the mechanisms that may contribute to transformation are unknown. The hypothesis that mutations in SDHD increase levels of superoxide leading to genomic instability was tested using site-directed mutagenesis to generate a truncated SDHD cDNA that was expressed in Chinese hamster fibroblasts. Stable expression of mutant SDHD resulted in 2-fold increases in steady-state levels of superoxide that were accompanied by a significantly increased mutation rate as well as a 70-fold increase in mutation frequency at the hprt locus. Overexpression of MnSOD or treatment with polyethylene glycol conjugated (PEG)-catalase suppressed mutation frequency in SDHD mutant cells by 50% (P<0.05). Simultaneous treatment with PEG-catalase and PEG-SOD suppressed mutation frequency in SDHD mutant cells by 90% (P<0.0005). Finally, 95% depletion of glutathione using l-buthionine-[S,R]-sulfoximine (BSO) in SDHD mutant cells caused a 4-fold increase in mutation frequency (P<0.05). These results demonstrate that mutations in SDHD cause increased steady-state levels of superoxide which significantly contributed to increases in mutation rates and frequency mediated by superoxide and hydrogen peroxide. These results support the hypothesis that mutations in SDHD may contribute to carcinogenesis by increasing genomic instability mediated by increased steady-state levels of reactive oxygen species.