Fluorescence was measured at 520 em / 485 ex using a BMG Fluostar microplate

Medulloblastoma, a neuroectodermal tumor arising in the cerebellum, is the most common brain tumor found in children. We recently showed that nifurtimox induces production of reactive oxygen species (ROS) and subsequent apoptosis in neuroblastoma cells both in vitro and in vivo. Tetrathiomolybdate (TM) has been shown to decrease cell proliferation by inhibition of superoxide dismutase-1 (SOD1). Since both nifurtimox and TM increase ROS levels in cells, we investigated whether the combination of nifurtimox and TM would act synergistically in medulloblastoma cell lines (D283, DAOY). Genome-wide transcriptional analysis, by hybridizing RNA isolated from nifurtimox and TM alone or in combination treated and control cells (D283) on Affymetrix exon array gene chips was carried out to further confirm synergy. We show that nifurtimox and TM alone and in combination decreased cell viability and increased ROS levels synergistically. Examination of cell morphology following drug treatment (nifurtimox + TM) and detection of caspase-3 activation via Western blotting indicated that cell death was primarily due to apoptosis. Microarray data from cells treated with nifurtimox and TM validated the induction of oxidative stress, as many Nrf2 target genes (HMOX1, GCLM, SLC7A11 and SRXN1) (p<10-5) were upregulated. Other genes related to apoptosis, oxidative stress, DNA damage, protein folding and nucleosome formation were differentially involved in cells following treatment with nifurtimox + TM. Taken together, our results suggest nifurtimox and TM act synergistically in medulloblastoma cells in vitro, and that this combination warrants further studies as a new treatment for medulloblastoma. Introduction Medulloblastoma is the most common malignant brain tumor in children and accounts for 17% of all brain tumors in children 0-14 years old (1). Each year about five hundred children in the US are affected by the disease (2). Many of these children are less than 3 years old at diagnosis. These patients have a reduced 5-year survival rate or increased long-term morbidity. The overall 5-year survival rates range from 40 to 70%, with the lower rates corresponding to younger children. Current front-line treatments, which include intensive multiagent chemotherapy, surgical resection, and craniospinal radiation, often leave these patients with progressive neurocognitive and neuroendocrine defects (3-5). Despite these treatments, approximately one third of patients have recurrent disease. Therefore, novel, well-tolerated, effective treatments are needed to increase survival and prevent relapse without severe long-term toxicities. Reactive oxygen species (ROS) are products of normal metabolism, and can also be induced by xenobiotic exposure. Depending on their concentration, ROS can lead to different outcomes for the cell because of their ability to modify many intracellular signaling pathways, as well as cause non-specific damage to DNA, lipids, and proteins (6). Oxidative stress activates the Nrf2 pathway, which activates a battery of genes involved in detoxification and prevention of free radical formation to protect the cell and facilitate cell survival (7). Under conditions of normal redox homeostasis, the cell maintains a balance between ROS generation or oxidative stress and antioxidant and repair defense mechanisms (8). At low levels, ROS can cause cell proliferation, while at high levels, ROS cause cytotoxicity, inhibition of cell proliferation, and induction of apoptosis (9). In order to upset this balance so that an overproduction of ROS leads to apoptosis, it is necessary to deliver an excess amount of ROS to cells (9). This is the strategy employed when utilizing exogenous ROSgenerating drugs to treat tumor cells (10). In general, tumor cells are more active than normal cells in the production of O2and are under intrinsic oxidative stress, which makes them rely more heavily on cellular antioxidant enzymes (11). Because Antitumor activity of nifurtimox is enhanced with tetrathiomolybdate in medulloblastoma KAREN S. KOTO1, PAMELA LESCAULT3, LAURENT BRARD4, KYUKWANG KIM4, RAKESH K. SINGH4, JEFF BOND3, SHARON ILLENYE2, MARNI A. SLAvIK2, TAKAMARU ASHIKAGA1,5 and GISELLE L. SAULNIER SHOLLER1,2 1vermont Cancer Center, Departments of 2Pediatrics, 3Microbiology and Molecular Genetics, University of vermont College of Medicine, Burlington, vT; 4Department of Obstetrics and Gynecology, Women and Infants Hospital of RI, Alpert Medical School of Brown University, Providence, RI; 5Department of Medical Biostatistics, vermont Cancer Center, University of vermont, Burlington, vT, USA Received November 22, 2010; Accepted January 17, 2011 DOI: 10.3892/ijo.2011.971 Correspondence to: Dr Giselle L. Saulnier Sholler, Department of Pediatrics, FAHC-Smith 559, 111 Colchester Ave., Burlington, vT