Reactive Oxygen Species Increase Mediated by Romo1 Expression Contributes to Tumor Progression

MitoSOX. Using Amplex Red, we observed a specific increase in H2O2 production in the rapamycin-treated cells. Co-treatment with N-acetyl cysteine attenuated the growth inhibitory effect of rapamycin and the observed increase in H2O2 levels. Immunoblots revealed increased MnSOD protein levels following rapamycin treatment. Nuclear run-on assays showed this to be due, at least in part, to increased transcription. Stable transfection of the Jurkat T lymphocytic leukemia cell line with SOD2 increased sensitivity to rapamycin. Exploring the mechanism underlying the increase MnSOD expression, we found that rapamycin treatment decreased serine 473 phosphorylation of Akt and determined that inhibition of the mTORC2 complex explained the increased SOD2 transcription. These results are the first to demonstrate that treatment of mantle cell lymphoma with rapamycin inhibits the mTORC2 complex, leading to increased ROS.

doi:10.1016/j.freeradbiomed.2012.10.101

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Gang Cheng , Jacek Zielonka1, Alexander R Dayton1, Joy Joseph1, and Balaraman Kalyanaraman1 1 Medical College of Wisconsin We reported that mitochondria-targeted nitroxide (Mito-CP) and 2deoxyglucose (2-DG) synergistically enhanced breast cancer cell death (Cheng G, et al. Cancer Res 72:2634-44, 2012). Ongoing studies in our laboratory also indicate that this combination therapy is lethal to other cancer cells. Mito-CP and 2-DG selectively inhibited ATP formed from glycolytic and oxidative phosphorylation in tumor cells. However, the effect of Mito-CP on intermediary metabolism has not been determined. To this end, we investigated the effect of Mito-CP on Kreb’s cycle metabolites in pancreatic cancer cell line, MiaPaCa-2.

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Ching-Fang Chang , Anne Diers1, and Neil Hogg1 1 Medical College of Wisconsin Most cancers have a metabolic phenotype which is characterized by a shift of energy metabolism from oxidative phosphorylation to glycolysis (the Warburg effect). Accumulating evidence has shown that reversing this metabolic alteration can sensitize cancer cells to death and metabolic modulators can increase the potency of anti-cancer drugs. L-CysNO is an efficient intracellular S-nitrosating agent. It has been shown that S-nitrosation of metabolic enzymes in glycolysis and mitochondria respiration alters their activities leading to the modulation of energy metabolism. Our previous studies showed that the administration of L-CysNO to bovine aortic endothelial cells increases cellular Snitrosation and inhibits the glycolytic enzyme GAPDH. Therefore, we hypothesize that L-CysNO can sensitize cancer cells to chemotherapeutic agents by inhibiting glycolysis. Human mammary adenocarcinoma MCF-7 cells were treated with LCysNO for 1 hour followed by 24 hours of increasing concentrations of doxorubicin, a widely used chemotherapeutic agent. L-CysNO enhanced the cytotoxicity of doxorubicin on MCF-7 cells in a concentration-dependent manner measured by clonogenic assay. Surprisingly, cellular GSH levels were almost doubled by CysNO treatment and this was unaffected by doxorubicin indicated cell death was not related to severe oxidative injury. The Seahorse extracellular flux (XF) analyzer was used to evaluate cellular glycolysis and mitochondria function, and adenine nucleotide pools were measured by HPLC. An increase of cellular ATP, NAD+ levels and mitochondrial ATPlinked oxygen consumption, and decrease of extracellular acidification rate after the treatments suggest that L-CysNO exposure not only resulted in an inhibition of cellular glycolysis but also enhanced mitochondrial ATP production, reversing the cancer metabolic phenotype. The detailed mechanisms of LCysNO-dependent sensitization are still under investigation; however, our study shows that L-CysNO can be a metabolic modulator with potential beneficial in cancer therapy in combination with traditional chemotherapeutics.

doi:10.1016/j.freeradbiomed.2012.10.102

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Results indicate the following: (1) At submicromolar concentrations, Mito-CP induces a dose- and time-dependent inhibition of proliferation in MiaPaCa-2 cells. (2) Using the XF24 extracellular flux analyzer, we observed that Mito-CP caused dose- and time-dependent inhibition in mitochondrial oxygen consumption rate (OCR) and mitochondrial functions. (3) Treatment with Mito-CP causes time- and concentrationdependent decrease in the components of Kreb’s cycle, including citric, cis-acotinic and iso-citric acids. (4) Mito-CP synergized with 2-DG in inhibiting the proliferation, inducing a significant decrease in intracellular ATP levels of MiaPaCa-2 cells. Collectively, these results suggest that the anticancer effects of mitochondriatargeted nitroxide, Mito-CP and 2-DG are related in part to inhibition of mitochondrial respiration, glycolysis and the Kreb’s cycle metabolism.

doi:10.1016/j.freeradbiomed.2012.10.103

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Jin Sil Chung , Sora Lee1, Gi Young Lee1, Jung Ar Shin2, Kee-Ho Lee3, and Young Do Yoo1 1 2 Korea University College of Medicine, Yonsei University College 3 of Medicine, Korea Korea Institute of Radiological and Medical Sciences Chronic ROS stress originated from the mitochondria contributes to a variety of pathological disorders including tumor progression. However, the factor involved in the production of mitochondrial ROS causing diseases has not been well identified. In the previous study, we identified a novel protein, Romo1, which produces ROS in the mitochondria. Romo1 was observed to be overexpressed in most cancer cell lines. Here, we showed that a higher level of Romo1 expression was detected in HCC tumors, compared with the corresponding normal liver tissue. Levels of Romo1 were increased, compared with normal liver tissues, in 63 of 95 HCC samples from patients. In HCC samples from patients, there was an inverse correlation between Romo1 overexpression and patient survival times. Increased levels of Romo1 also correlated with vascular invasion by the tumors, reduced

SFRBM 2012

differentiation, and larger tumor size. In addition to these results, we showed that Romo1 expression directly contribute to invasive activity of hepatoma cells. Exogenous expression of Romo1 in HCC cells increased their invasive activity, compared with control cells. Furthermore, Romo1 knockdown significantly suppressed the formation of visible lung metastatic nodules. We also investigated that Romo1-derived ROS are indispensable for proliferation of tumor cells by activating NF-țB. Romo1 knockdown decreased cellular ROS levels and NF-țB activity, resulting in cell growth inhibition. From these results, we suggest that Romo1 is a promising biomarker for hepatoma diagnosis and a new target for the development of anti-cancer drugs by inhibiting the origin of ROS production rather than elimination of intracellulary produced ROS.

doi:10.1016/j.freeradbiomed.2012.10.104

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Anne R. Diers , Matthew P. Fitzgerald2, Agnes Keszler1, Frederick E. Domann2, and Neil Hogg1 1 2 Medical College of Wisconsin, University of Iowa Inducible nitric oxide synthase (iNOS) produces high levels of the signaling molecule nitric oxide in vivo, and its expression is frequently deregulated in multiple tumor types including breast. Nitric oxide is a key regulator of several metabolic pathways such as glycolysis and oxidative phosphorylation. Direct links between central metabolism and epigenetic marks have been established and, in many cases, are based on the production of metabolic intermediates which serve as cofactors for epigenetic enzymes. Thus, we hypothesize that modulation of cellular bioenergetics by iNOS-derived nitric oxide will alter the epigenetic landscape of cancer cells. To test this, parental COH-BR1 human breast cancer cells which are iNOS-positive were engineered to stably express lentiviral shRNA to iNOS. Silencing was confirmed by measurement of iNOS protein levels and S-nitrosothiols, a nitric oxide-dependent protein modification. Using a Seahorse Bioscience XF24 Analyzer, basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR; an index of glycolytic flux) were compared in these cells, and parental COH-BR1 cells had lower basal OCR and higher basal ECAR than those with iNOS silenced indicating that iNOS-derived nitric oxide downregulates mitochondrial function and promotes a shift to glycolysis. No change in global DNA methylation was observed between parental and iNOS shRNA expressing cells. In addition, bisulfite genomic sequencing of the promoter of maspin, a tumor suppressor known to be epigenetically-silenced in breast cancer, revealed no local alterations in DNA methylation at this locus. In contrast, significant changes in modification of histones were observed between these cell lines, most notably diminished histone H3 trimethylation in cells lacking iNOS. These epigenetic changes also correlated with loss of maspin mRNA and protein expression. Together, these data indicate that iNOS-derived nitric oxide controls epigenetic events to regulate tumor suppressor expression and suggest a possible mechanistic link through central metabolism. (This work was supported, in part, by a SFRBM mini-fellowship)

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Rafal Nazarewicz1, Anna Dikalova1, Alfiya Bikineyeva1, Sergey Ivanov2, 1 and Sergey Dikalov 1 2 Vanderbilt University Medical Center, Yale Cancer Center Reactive oxygen species (ROS) play an important role in regulation of cell proliferation and cell survival. It has been previously reported that overexpression of mitochondrial SOD2 attenuates cancer. We hypothesize that scavenging of Ɣ mitochondrial O2 Ǧ in cancer cells selectively inhibits redox sensitive regulation of metabolic and cell survival pathways resulting in cell death. In order to test this hypothesis we have studied direct effect of mitochondria-targeted superoxide scavenger mitoTEMPO on B16-F0 mouse melanoma cells and tumor growth in nude mice model of human melanoma using A375 cells. In this work, for the first time, we show that mitochondriatargeted SOD mimetic mitoTEMPO inhibited cell growth, reduced viability and induced apoptosis in melanoma cells but did not affect nonmalignant skin fibroblasts. Treatment of melanoma cells with mitoTEMPO significantly diminished mitochondrial ROS, inhibited redox dependent Akt and Erk, restored activity of mitochondrial pyruvate dehydrogenase, reduced HIF1-Į and lactate dehydrogenase expression. These changes in signaling events caused metabolic switch from glycolysis to mitochondrial metabolism. Suppression of glycolysis in mitoTEMPO treated melanoma cells resulted in significant drop of cellular ATP and Ɣ induced cancer cell death. Scavenging of mitochondrial O2 Ǧ effectively suppresses growth of established tumor in the mouse model of human melanoma. Our data therefore support hypothesis that anticancer activity of mitochondria-targeted antioxidants is likely mediated by inhibition of ROS-sensitive cell signaling and metabolic changes leading to reduced cancer cell survival. We suggest that mitochondrial superoxide is a novel and promising pharmacological target to treat melanoma.

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doi:10.1016/j.freeradbiomed.2012.10.106

doi:10.1016/j.freeradbiomed.2012.10.105

SFRBM 2012

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