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.

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Ching-Fang Chang1, Anne Diers1, and Neil Hogg1 1Medical 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 S-nitrosation 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 L-CysNO 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 ATP-linked 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 L-CysNO-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.

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Gang Cheng1, Jacek Zielonka1, Alexander R Dayton1, Joy Joseph1, and Balaraman Kalyanaraman1 1Medical College of Wisconsin We reported that mitochondria-targeted nitroxide (Mito-CP) and 2-deoxyglucose (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.

O

OP

9

Mito-CP

NO• OHOHO

O

OH

H

2-DG 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 concentration- dependent 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 mitochondria-targeted nitroxide, Mito-CP and 2-DG are related in part to inhibition of mitochondrial respiration, glycolysis and the Kreb’s cycle metabolism.

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Jin Sil Chung1, Sora Lee1, Gi Young Lee1, Jung Ar Shin2, Kee-Ho Lee3, and Young Do Yoo1 1Korea University College of Medicine, 2Yonsei University College of Medicine, Korea 3Korea 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

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doi:10.1016/j.freeradbiomed.2012.10.102

doi:10.1016/j.freeradbiomed.2012.10.103