Nukhet Aykin-Burns

A Dynamic Pathway for Calcium-Independent Activation of CaMKII by Methionine Oxidation

Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.

SIRT3 Is a Mitochondria-Localized Tumor Suppressor Required for Maintenance of Mitochondrial Integrity and Metabolism during Stress

The sirtuin gene family (SIRT) is hypothesized to regulate the aging process and play a role in cellular repair. This work demonstrates that SIRT3(-/-) mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3(-/-) MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3(-/-) MEFs and reverses the tumor-permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3(-/-) mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondria-localized tumor suppressor.

Mitochondrial and H2O2 Mediate Glucose Deprivation-induced Stress in Human Cancer Cells

The hypothesis that glucose deprivation-induced cytotoxicity in transformed human cells is mediated by mitochondrial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and H2O2 was first tested by exposing glucose-deprived SV40-transformed human fibroblasts (GM00637G) to electron transport chain blockers (ETCBs) known to increase mitochondrial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and H2O2 production (antimycin A (AntA), myxothiazol (Myx), or rotenone (Rot)). Glucose deprivation (2–8 h) in the presence of ETCBs enhanced parameters indicative of oxidative stress (i.e. GSSG and steady-state levels of oxygen-centered radicals) as well as cytotoxicity. Glucose deprivation in the presence of AntA also significantly enhanced cytotoxicity and parameters indicative of oxidative stress in several different human cancer cell lines (PC-3, DU145, MDA-MB231, and HT-29). In addition, human osteosarcoma cells lacking functional mitochondrial electron transport chains (rho(0)) were resistant to glucose deprivation-induced cytotoxicity and oxidative stress in the presence of AntA. In the absence of ETCBs, aminotriazole-mediated inactivation of catalase in PC-3 cells demonstrated increases in intracellular steady-state levels of H2O2 during glucose deprivation. Finally, in the absence of ETCBs, overexpression of manganese containing superoxide dismutase and/or mitochondrial targeted catalase using adenoviral vectors significantly protected PC-3 cells from toxicity and oxidative stress induced by glucose deprivation with expression of both enzymes providing greater protection than was seen with either alone. Overall, these findings strongly support the hypothesis that mitochondrial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and H2O2 significantly contribute to glucose deprivation-induced cytotoxicity and metabolic oxidative stress in human cancer cells.

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.

Mitochondrial Complex II Dysfunction Can Contribute Significantly to Genomic Instability after Exposure to Ionizing Radiation

Abstract Dayal, D., Martin, S. M., Owens, K. M., Aykin-Burns, N., Zhu, Y., Boominathan, A., Pain, D., Limoli, C. L., Goswami, P. C., Domann, F. E. and Spitz, D. R. Mitochondrial Complex II Dysfunction Can Contribute Significantly to Genomic Instability after Exposure to Ionizing Radiation. Ionizing radiation induces chronic metabolic oxidative stress and a mutator phenotype in hamster fibroblasts that is mediated by H2O2, but the intracellular source of H2O2 is not well defined. To determine the role of mitochondria in the radiation-induced mutator phenotype, end points of mitochondrial function were determined in unstable (CS-9 and LS-12) and stable (114) hamster fibroblast cell lines derived from GM10115 cells exposed to 10 Gy X rays. Cell lines isolated after irradiation demonstrated a 20–40% loss of mitochondrial membrane potential and an increase in mitochondrial content compared to the parental cell line GM10115. Surprisingly, no differences were observed in steady-state levels of ATP (P > 0.05). Unstable clones demonstrated increased oxygen consumption (two- to threefold; CS-9) and/or increased mitochondrial electron transport chain (ETC) complex II activity (twofold; LS-12). Using Western blot analysis and Blue Native gel electrophoresis, a significant increase in complex II subunit B protein levels was observed in LS-12 cells. Furthermore, immunoprecipitation assays revealed evidence of abnormal complex II assembly in LS-12 cells. Treatment of LS-12 cells with an inhibitor of ETC complex II (thenoyltrifluoroacetone) resulted in significant decreases in the steady-state levels of H2O2 and a 50% reduction in mutation frequency as well as a 16% reduction in CAD gene amplification frequency. These data show that radiation-induced genomic instability was accompanied by evidence of mitochondrial dysfunction leading to increased steady-state levels of H2O2 that contributed to increased mutation frequency and gene amplification. These results support the hypothesis that mitochondrial dysfunction originating from complex II can contribute to radiation-induced genomic instability by increasing steady-state levels of reactive oxygen species.

Cigarette Smoke Induces Cellular Senescence via Werner's Syndrome Protein Down-regulation

RATIONALE Werners syndrome is a genetic disorder that causes premature aging due to loss-of-function mutations in a gene encoding a member of the RecQ helicase family. Both Werners syndrome and cigarette smoking accelerate aging. No studies have examined the effect of cigarette smoke on Werners syndrome protein. OBJECTIVES To investigate the role of Werners syndrome protein in cigarette smoke-induced cellular senescence. METHODS Cellular senescence and amounts of Werners syndrome protein were measured in fibroblasts isolated from patients with emphysema and compared with age-matched nonsmokers. The in vitro effects of cigarette smoke on amounts of Werners syndrome protein, function, and senescence were also evaluated in primary human lung fibroblasts and epithelial cells. MEASUREMENTS AND MAIN RESULTS Cultured lung fibroblasts isolated from patients with emphysema exhibited a senescent phenotype accompanied by a decrease in Werners syndrome protein. Cigarette smoke extract decreased Werners syndrome protein in cultured fibroblasts and epithelial cells. Werners syndrome protein-deficient fibroblasts were more susceptible to cigarette smoke-induced cellular senescence and cell migration impairment. In contrast, exogenous overexpression of Werners syndrome protein attenuated the cigarette smoke effects. CONCLUSIONS Cigarette smoke induces cellular senescence and cell migration impairment via Werners syndrome protein down-regulation. Rescue of Werners syndrome protein down-regulation may represent a potential therapeutic target for smoking-related diseases.

Sirt3, mitochondrial ROS, ageing, and carcinogenesis.

One fundamental observation in cancer etiology is that the rate of malignancies in any mammalian population increases exponentially as a function of age, suggesting a mechanistic link between the cellular processes governing longevity and carcinogenesis. In addition, it is well established that aberrations in mitochondrial metabolism, as measured by increased reactive oxygen species (ROS), are observed in both aging and cancer. In this regard, genes that impact upon longevity have recently been characterized in S. cerevisiae and C. elegans, and the human homologs include the Sirtuin family of protein deacetylases. Interestingly, three of the seven sirtuin proteins are localized into the mitochondria suggesting a connection between the mitochondrial sirtuins, the free radical theory of aging, and carcinogenesis. Based on these results it has been hypothesized that Sirt3 functions as a mitochondrial fidelity protein whose function governs both aging and carcinogenesis by modulating ROS metabolism. Sirt3 has also now been identified as a genomically expressed, mitochondrial localized tumor suppressor and this review will outline potential relationships between mitochondrial ROS/superoxide levels, aging, and cell phenotypes permissive for estrogen and progesterone receptor positive breast carcinogenesis.

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.

Mitochondrial electron transport chain blockers enhance 2-deoxy-D-glucose induced oxidative stress and cell killing in human colon carcinoma cells

Increasing evidence suggests that cancer cells (relative to normal cells) have altered mitochondrial electron transport chains (ETC) that are more likely to form reactive oxygen species (ROS; i.e. O2 •-and H2O2) resulting in a condition of chronic metabolic oxidative stress, that maybe compensated for by increasing glucose and hydroperoxide metabolism. In the current study, the ability of an inhibitor of glucose metabolism, 2-deoxy-D-glucose (2DG), combined with mitochondrial electron transport chain blockers (ETCBs) to enhance oxidative stress and cytotoxicity was determined in human colon cancer cells. Treatment of HT29 and HCT116 cancer cells with Antimycin A (Ant A) or rotenone (Rot) increased carboxy-dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidine (DHE) oxidation, caused the accumulation of glutathione disulfide and enhanced 2DG-induced cell killing. In contrast, Rot did not enhance the toxicity of 2DG in normal human fibroblasts supporting the hypotheses that cancer cells are more susceptible to inhibition of glucose metabolism in the presence of ETCBs. In addition, 2-methoxy-antimycin A (Meth A; an analog of Ant A that does not have ETCB activity) did not enhance 2DG-induced DHE oxidation or cytotoxicity in cancer cells. Finally, in HT29 tumor bearing mice treated with the combination of 2DG (500 mg/kg) + Rot (2 mg/kg) the average rate of tumor growth was significantly slower when compared to control or either drug alone. These results show that 2DG-induced cytotoxicity and oxidative stress can be significantly enhanced by ETCBs in human colon cancer cells both in vitro and in vivo.

Oxidative stress in a phenylketonuria animal model

Oxidative stress is seen in various metabolic disorders for unknown reasons. Oxidative stress is defined as an imbalance between pro-oxidant and antioxidant status in favor of the former. This study investigated whether oxidative stress exists in phenylketonuria (PKU) using the BTBR-Pah(enu2) animal model for PKU. Animals (14-24 weeks old) were sacrificed and brain and red blood cells (RBCs) were obtained aseptically. The lipid peroxidation by-product, evaluated as malondialdehyde (MDA), was significantly higher in the brains and RBCs of PKU animals (n = 6) than in controls (n = 6). Glutathione/glutathione disulfide, a good indicator for tissue thiol status, was significantly decreased both in the brains and RBCs. Some antioxidant enzymes were also analyzed in RBCs, including glucose-6-phosphate dehydrogenase (G6PD), which provides the RBCs main reducing power, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and catalase detoxifies H2O2 by catalyzing its reduction to O2 and H2O. Both catalase and G6PD were significantly increased in the RBCs of PKU animals.