production and partial transformation. Taken together, our results suggest that a BLT2-Nox1-linked cascade is responsible for the elevated ROS generation in Ras-transformed cells. Our finding may contribute to clarifying the signaling events underlying the enhanced levels of ROS frequently observed in various transformed cells and possibly serve as a basis for developing new therapeutic strategies for human cancers. 92 6PDOO 0ROHFXOH 2[LGDWLYH 0HWDEROLVP ,QKLELWRUV IRU 0HWDVWDWLF 0HODQRPD 7KHUDS\ Kyle C. Kloepping 1 , Mitchell C. Coleman 1 , Brett A. Wagner 1 , James A. Jacobus 1 , Kranti A. Mapuskar 1 , Garry R. Buettner 1 , Douglas R. Spitz 1 , and Michael K. Schultz 1 1 The University of Iowa Metastatic melanoma incidence is increasing faster than any other form of cancer worldwide. Despite advances in chemotherapeutic, biological, and targeted therapies, no treatment affords durable benefit to melanoma patients. The current study explores a novel approach to melanoma therapy utilizing a small-molecule triphenylphosphonium (TPP) platform technology that targets fundamental differences in oxidative metabolism and response to oxidative stress in melanoma cells relative to normal cells. Based on the mitochondrial targeting properties of TPP, a small library of TPP derivatives were examined to determine if the molecular composition of TPP variants could be modified to affect melanoma cell oxidative metabolism and viability. In vitro measurements of cell viability, mitochondrial membrane potential, electron transport chain complex activity, oxidative stress, and oxygen consumption rates were conducted. In vivo studies were performed in mice bearing melanoma tumor xenografts to determine if TPP treatment can be tolerated and induce a reduction in melanoma tumor growth via increased oxidative stress. Results indicate that TPP derivatives can be designed to disrupt oxidative metabolism and lead to melanoma cell death via increased oxidative stress. Further, melanoma tumor bearing mice treated with TPP exhibited decreased melanoma tumor growth rates compared to untreated mice. Importantly, TPP treatment was well tolerated and tumor lysates showed increased oxidative stress markers, thus providing evidence that TPP derivatives can be designed to disrupt oxidative metabolism and selectively kill melanoma cells via increased oxidative stress. 93 P725 6ZLWFKHV $HURELF *O\FRO\VLV WR 2[LGDWLYH 3KRVSKU\ODWLRQ LQ &HOOXODU %LRHQHUJHWLFV 8QGHU 5DGLDWLRQ Chung-Ling Lu 1 and Jian Jian Li 1 1 University of California Davis In 1924 Otto Warburg discovered that cancer cells tended to convert glucose into lactate to produce energy even under the presence of oxygen. This primary finding in cancer cell metabolism is called aerobic glycolysis known as Warburg effect. Tumors thus were believed to use aerobic glycolysis to produce cellular energy and to use those intermediates as sources to support cell proliferation. Therefore, switching the aerobic glycolysis to the pathway of oxidative phosphorylation could be a potential approach to minimize the growth and invasion of tumors. One of the central proteins that involve in tumor proliferation is the mammalian target of rapamycin. The relation between mTOR signaling pathway and cell energy metabolism has been extensively studied, however, whether regulation of mTOR can change the way cells produce their energy is unknown. mTOR dysfunction can transform normal cells into tumor-like cells and switch the energy metabolism to aerobic glycolysis. Also mTOR were found on the mitochondria surface and inhibition of mTOR can decrease the function of mitochondria. Therefore, mTOR activity is highly related to the mitochondrial function but how mTOR controls the difference of bioenergetics still needs to be elucidated. Here we showed that in cancer cells mTOR expression is increased but mTOR level on the mitochondria decreased compared to normal cells. In addition, mitochondrial ATP generation was lowered and glycolysis was enhanced. However, mTOR was localized to mitochondrial outer membrane in 5 Gy-treated tumor cells to enhance mitochondrial bioenergetics. These results demonstrate a new role of mTOR that is able to switch the aerobic glycolysis to oxidative phosphorylation in cellular bioenergetics. These results support the notion that cellular metabolism can be dynamically reprogrammed by mTOR in tumor cells under different stress conditions. 94 1LWULF 2[LGH $FWLYDWHV S UDV E\ 61LWURV\ODWLRQ GXULQJ /RVV RI ,QWHJULQ0HGLDWHG &HOO0DWUL[ &RQWDFW /HDGLQJ WR ,QFUHDVHG 6XSHUR[LGH /HYHO DQG &HOO 6XUYLYDO Fabiana Henriques Melo 1 , Fernanda Molognoni 1 , Ana Moretti 2 , Heraldo Possolo de Souza 2 , and Miriam Galvonas Jasiulionis 1 1 UNIFESP,Brazil, 2 USP, Brazil A melanocyte malignant transformation model was developed after submitting a non-tumorigenic melanocyte lineage (melan-a cells) to sequential cycles of loss of integrin-mediated cell-matrix contact. Since anoikis resistance is one of the capabilities acquired by tumorigenic cells, our aim was to investigate which alterations lead melanocytes to survive and developed tumors. In the first 3 hours of inappropriate attachment to extracellular matrix, melan-a cells showed increased superoxide anion and nitric oxide levels. Uncoupled endothelial nitric oxide synthase was identified as one of the main sources of superoxide anion during anchorage impediment, since L-sepiapterin, precursor of eNOS cofactor BH4, decreased superoxide anion levels. Recently, NADPH oxidase was identified as another source of superoxide anion during anchorage blocade, since NSC, a RAC inhibitor, decreased superoxide levels. Moreover, NOX 1 and 4, but not NOX 2 and 3 expressions were increased during anchorage blockade. Rac, a small Rho GTPase, is a NADPH oxidase cytosolic component that is recruited to membrane after its activation by ras. Ras signaling pathway was activated after 30 min in melan-a cells submitted to anchorage blockade. In appropriate redox environments, NO has the potential to induce the nitrosylation of cellular targets, modulating its function. Ras activation during de-adhesion was abrogated by PTIO, a NO scavenger and nitrosylation of Ras was detected, suggesting the involvement of NO in the activation of Ras signaling pathway. The maintenance of a pro-oxidant environment seems to be important in cell survival, since PTIO rendered melan-a melanocytes more sensitive to anoikis in the same way as superoxide anion depletion. PTIO decreased superoxide levels, showing its involvement in superoxide regulation production. Our results suggest for the first time in malignant transformation, that SFRBM 2012 S46 doi:10.1016/j.freeradbiomed.2012.10.120 doi:10.1016/j.freeradbiomed.2012.10.121 doi:10.1016/j.freeradbiomed.2012.10.122