median life expectancy of 12 months from diagnosis. GBM is characterized by a heterogeneous population of cells that are genetically unstable, and resistant to chemotherapy. Additionally, GBMs display high activity of system xc which is a sodium-independent electroneutral transporter containing a catalytic subunit: xCT (SLC7A11). System xc is capable of contributing to the antioxidant capacity by importing cystine into a cell, where it is then reduced to cysteine and used to synthesize the antioxidant glutathione (GSH). It was previously shown that inhibition of system xc slows tumor growth in vitro and extends survival in vivo. We performed in silico analysis that revealed GBM patients with high expression of SLC7A11 had a worse prognosis compared to patients with lower expression of SLC7A11. Furthermore, high intracellular GSH levels in cancer cells were associated with drug resistance and resistance to alkylating agents such as temozolomide (TMZ). Therefore, we generated stable xCT over-expressing, and xCT knock-down, U251 GBM cell lines to determine whether increased system xc expression protects GBM from both oxidative- and genotoxic-stress. As expected, xCT knock-down cells exhibited decreased GSH levels and increased levels of intracellular ROS while the xCT over-expressing cells were more resistant to oxidative stress. Additionally, over-expression of system xc resulted in decreased sensitivity to TMZ which was not solely mediated by GSH. The elevated xCT expression correlated with a cancer stem cell (CSC) phenotype as shown by increased expression of CSC-associated markers and neurosphere formation. It has been established that CSCs possess enhanced mechanisms of protection from ROS that render them resistant to chemo- and radiotherapy. Therefore, we demonstrate that system xc in GBM not only promotes survival under oxidative stress but may also modulate sensitivity to chemotherapy treatment partially mediated by an increased CSC phenotype. These findings indicate that therapeutic manipulation of system xc either alone, or in combination with other treatments, may improve clinical outcome in GBM patients.

309 Chrysin Mitigates Chemically Induced Renal Carcinogenesis by Attenuation of Oxidative Stress, Hyper Proliferation and Apoptosis at Preclinical Stage Summya Rashid1, Sana Nafees1, Nemat Ali1, and Sarwat Sultana1 1Jamia Hamdard, India The present study was designed to investigate the

carcinogenesis in Wistar rats. Ferric nitrilotriacetate (Fe-NTA) is a potent nephrotoxin and known renal carcinogen. CH is a natural flavonoid found in many plant extracts, propolis, blue passion flower. It has anti-oxidative, anti-inflammatory, anti-apoptotic properties. Renal cancer was initiated by single intraperitoneal injection of N-nitrosodiethylamine and promoted by twice weekly administration of Fe-NTA for 16 weeks. CH was administered at two doses daily. The possible mechanism could be induction of oxidative stress, cellular proliferation via up regulation of PCNA and ki-67 along with diminution of apoptosis by down regulating p53, bax, caspase 3, cytochrome c and upregulating Bcl-2. Prophylactic treatment of CH mitigated serum toxicity markers, reduced tumor incidences, regulated proliferation and induced apoptosis. These results provide a powerful evidence for the chemo preventive efficacy of CH at pre-clinical stage.

310 Activation of Nrf2 Pathway in the Cancer Stem Cell-Enriched Mammosphere System and Its Involvement in Sphere Growth and Stress Resistance In-geun Ryoo1, Bo-hyun Choi1, and Mi-Kyoung Kwak1 1The Catholic University of Korea, Republic of Korea Cancer stem cells (CSCs) are believed to be responsible for cancer recurrence due to high levels of expressions of drug efflux transporters and antioxidant genes. Nonadherent mammospheres of breast cancer cells are known to be CSC-enriched populations and have been used to investigate CSC properties. Here we investigated the role of NF-E2-related factor 2 (Nrf2) in mammosphere growth and stress resistance using the breast carcinoma cell line MCF7. In mammospheres, levels of Nrf2 protein and its target gene expression were significantly higher than monolayer cultured MCF7. Mammosphere Nrf2 protein maintained its high level under cycloheximide incubation and ubiquitinated Nrf2 protein accumulated, indicating that Nrf2 increase is resulted from protein stabilization. As an underlying mechanism, we demonstrated that enzymatic function and expression of the proteasome are decreased in mammospheres. Moreover, mammospheres retained a higher level of p62 and the silencing of p62 attenuated Nrf2 activation in mammospheres. As a functional implication, Nrf2-silenced mammospheres showed high levels of reactive oxygen species and cell death, and thereby the knockdown sphere growth was retarded compared to the control cells. Furthermore, the increase of drug efflux transporters and detoxifying enzymes were repressed in Nrf2-silenced mammospheres; therefore these mammospheres did not develop anticancer drug resistance unlike the control mammospheres. Collectively, these results indicate that decreased proteasome function and p62 increase led to Nrf2 activation in CSC-enriched mammospheres, and further suggest that mammosphere Nrf2 may play a role in CSCs survival and anticancer drug resistance. Key words: Nrf2, mammospheres, cancer stem cells, proteasome, p62, reactive oxygen species, breast cancer

311 Copper-Zinc Superoxide Dismutase as a Target for Enhancing Bortezomib Cytotoxicity in Multiple Myeloma Kelley Salem1, Michael L McCormick1, Erik Wendlandt1, Fenghuang Zhan1, and Apollina Goel1 1University of Iowa, USA Multiple myeloma (MM) remains an incurable B-cell malignancy that frequently exhibits intrinsic and acquired chemo-resistance. As MM patients show increased systemic oxidative stress, we queried if adaptation to oxidative stress may be linked to therapeutic resistance to the anti-MM drug bortezomib (BTZ, Velcade). Retrospective clinical data was analyzed for gene expression of copper-zinc superoxide dismutase (CuZnSOD, SOD1) where increased SOD1 expression correlated with MM progression, high-risk disease, and adverse survival outcomes. In vitro studies utilized drug naïve MM cell lines with intrinsic differential apoptotic sensitivities towards BTZ (MM.1S, 8226, U266) and the BTZ resistant (BR) lines (MM.1SBR, 8226BR). Enzymatic activities and mRNA expression of key antioxidant enzymes (CuZnSOD, catalase, glutathione peroxidase-1) and glutathione (GSH) levels were determined. In the cell line model, up-regulation of antioxidant enzymes (CuZnSOD and GPx-1) and GSH levels associated with intrinsic- and adaptive-BTZ resistance

S130 SFRBM 2014

doi: 10.1016/j.freeradbiomed.2014.10.205

doi: 10.1016/j.freeradbiomed.2014.10.206

doi: 10.1016/j.freeradbiomed.2014.10.207

chemopreventive potential of Chrysin (CH) against two stage renal