290 Alterations in Mitochondrial Dynamics Regulate Melanoma Tumor Progression Fulya Dal1, Sun-Hee Kim1, Yong Qin1, Yuuri Hashimoto1, and Suhendan Ekmekcioglu1 1University of Texas MD Anderson Cancer Center, USA The rate of mitochondrial biosynthesis and degradation can affect various aspects of tumor progression. Mitochondria are the primary producers of reactive oxygen species (ROS), which are involved in melanogenesis. However, the role of mitochondrial dynamics on melanogenesis remains controversial and requires mechanistic understanding to target the altered metabolism of cancer cells. Therefore, we hypothesized that aberrant mitochondrial biogenesis can promote melanoma progression by increasing metabolite and energy generation. In this study we showed that that mitochondrial fission protein, Drp-1, and fusion protein, Mfn-2, were highly expressed in MeWo, SK-Mel-28 and WM35 cells, compared to normal human keratinocytes. Moreover we found the loss of Drp-1 protein expression in vemurafenib (BRAF inhibitor)-resistant cell line, Mel-1617-BR, suggesting the change in mitochondrial dynamics may contribute to drug resistance. We also treated melanoma cells with mdivi-1, a mitochondrial fission inhibitor, to induce mitochondrial hyperfusion. However, at the concentration causing mitochondrial hyperfusion, mdivi-1 did not show any significant effect on melanoma cell viability. The effect of mdivi-1 on cell migration is currently under investigation to address further mechanistic outcomes. These results provide preliminary data to support the role of mitochondrial biogenesis in melanoma tumor progression.

291 The Role of Transition Metal Ions in Ascorbate-Induced Cytotoxicity in Pancreatic Cancer Juan Du1, Brett A Wagner1, Garry R Buettner1, and Joseph J Cullen1,2 1The University of Iowa, USA, 2Veterans Affairs Medical Center, USA Pharmacological ascorbate has been shown to induce toxicity in a various cancer cell lines both in vitro and in vivo. Pharmacological concentrations of ascorbate produce H2O2 via the formation of ascorbate radical (Asc ). The rate of oxidation of ascorbate is a function of the level of catalytically active iron and copper. Iron in cell culture media contributes significantly to the rate of H2O2 generation. In this study, we found that the presence of 0.5 or 10% FBS in DMEM decreased the rate of H2O2 formation and increased clonogenic survival of MIA PaCa-2 pancreatic cancer cells exposed to 0.5 mM ascorbate. Consistently, adding increasing amounts of apo-transferrin to DMEM media in the presence of 0.5 mM ascorbate decreased OCR (oxygen consumption rate) and the flux of H2O2, and increased clonogenic survival. To clarify how basal levels of intracellular labile iron affect ascorbate cytotoxicity, we compared intracellular labile iron levels of immortalized pancreatic ductal epithelial cells H6c7 with levels of labile iron in Kras cells (H6c7 that overexpress Kras) and several pancreatic cancer cell lines and the sensitivity of these cells to ascorbate (1 and 2 mM). Although Kras, MIA PaCa-2, Panc-1 and AsPC-1 cells were more sensitive to pharmacological ascorbate treatment than H6c7 cells as seen by clonogenic survival, only Panc-1 and AsPC-1 had higher basal levels of labile

iron than H6c7. There is no significant difference in intracellular labile iron content among H6c7, Kras and MIA PaCa-2 cells. Thus, intracellular labile iron levels may be only one of many factors that affect cellular sensitivity to ascorbate-induced cytotoxicity. Supported by VA Merit grant, NIH grants CA166800, CA137230, and CA169046.

292 High Glucose and Insulin Induce an Increase in Reactive Oxygen Species, Cell Migration and UPA Expression in Breast Cancer Cells MDA-MB-231 Luis Antonio Flores-López1, María Guadalupe Martínez-Hernández 1, and Luis Arturo Baiza-Gutman1 1Unidad de Morfofisiología, FES-Iztacala, UNAM, Mexico The mechanisms associated with the increased risk to develop cancer in patients with obesity or diabetes, are poorly understood. Cancer cell invasion involves extracellular proteases, like urokinase plasminogen activator (uPA), which participates starting a proteolytic cascade that contributes to the breakdown of extracellular matrix and its overexpression is correlated with aggressive tumors. The effect of high glucose and insulin on cell viability, migration and uPA expression in MDA-MB-231 breast cancer cells was evaluated. High glucose (30 mM) induced a significant increase of cell population, cell migration and reactive oxygen species production; these actions were potentiated by insulin treatment. The uPA mRNA was increased by high glucose (1.46±0.73 times), which was not further modified by simultaneous insulin treatment (1.83±0.47 times). The single chain, Pro-uPA and the two chain active uPA were increased in cells cultured with high glucose (25.68±6.43 % and 142.20±30.81 %). By casein-plasminogen zymography a higher activity of uPA in cell lysates was induced (7.75±1.17 times) by high glucose, High glucose plus insulin treatment also increases uPA activity but at lower magnitude (3.68±0.46 times). MDA-MB-231 cell proliferation and migration induced by high glucose probably was mediated by enhanced ROS and increased expression and activity of uPA, whereas, the additional increase of cell proliferation and migration induced by insulin in high glucose treated cells was associated with an additional increase in ROS production but not with further changes in uPA expression. In diabetic patients, hyperglycemia or early hyperinsulinemia can induce a proliferative and migratory phenotype in breast cancer cells. Supported by PAPIIT, DGAPA, UNAM, Project IN 223014, LA Flores is a postdoctoral fellow of DGAPA, UNAM.

293 Involvement of Hypoxia-Inducible Factor-1 in Resveratrol's Cell Growth Inhibitory Activity Joao Pedro Fonseca1, Bruna Ferreira da Silva1,2,3, Shawnnah Staples1, and Jeff A. Stuart1 1Brock University, Canada, 2University of Brasilia, Brazil, 3Scholarship from CNPq, Brazil One of the more robust effects attributed to resveratrol (RES) and related stilbenes is cell growth inhibitory activity that makes these molecules potentially attractive for anti-cancer therapies. Previously, we have shown in multiple cell lines that this effect

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