br Materials and Methods br Results br

Materials and Methods
Results
Discussion High-dose vitamin C has been studied in multiple cancers and has shown controversial clinical effects (Cameron and Pauling, 1978, 1976; Creagan et al., 1979; Moertel et al., 1985). The contradictory clinical results can be at least partially explained by different routes of vitamin C administration applied, i.e., either orally or intravenously. Recent reports indicate that a certain ROS concentration is required for high-dose vitamin C to induce cytotoxicity in cancer cells. The generation of ascorbyl- and H2O2 radicals by PAA increases ROS stress in cancer cells (Du et al., 2012). These studies including preclinical and clinical were performed in solid tumors, such as glioblastoma (Herst et al., 2012), pancreatic cancer (Du et al., 2015), ovarian cancer (Ma et al., 2014), prostate cancer (Chen et al., 2012; Pollard et al., 2010), hepatoma (Verrax and Calderon, 2009), colon cancer (Pires et al., 2016), mesothelioma (Ranzato et al., 2011), breast cancer (Yun et al., 2015), bladder cancer (Gilloteaux et al., 2010), and neuroblastoma (Deubzer et al., 2010). Reports are lacking to show that PAA can be used as a pro-oxidant drug in the treatment of “liquid” tumors, where tumor cells are surrounded by blood. This environmental difference between solid tumor and blood cancer has the potential to influence the PAA efficacy on cancer cell death even when given at high doses, because ascorbic RO4929097 Supplier generated ROS are much easier permeabilized in liquid tumor than in solid tumor. In this study, we report that PAA is efficacious in killing MM cells in vitro and in vivo models, which generated levels of 20–40mM ascorbate and 500nM ascorbyl radicals after intraperitoneal administration of 4g ascorbate per kilogram of body weight (Chen et al., 2008), in xenograft MM mice. These data suggest that PAA may show a therapeutic advantage to blood cancers vs solid tumors because of the communication between tumor cells and blood plasma. We have shown that Fpn1 regulates iron export in MM cells and LIP in vitro and in vivo (Gu et al., 2015). In addition, ferritin also regulates LIP by sequestering free iron in an oxidized form to prevent formation of free radicals (Pantopoulos et al., 2012). Our preliminary data show that overexpression of Fpn1 in MM cell line OCI-MY5 results in increased viability compared to wild type cells after PAA treatment. We hypothesize that Fpn1 expressing MM cells are less sensitive to PAA because their cytosolic iron content is reduced by Fpn1. To test if resistance to PAA is indeed due to low cytosolic iron content, we depleted cytosolic iron by pre-incubating cells with an iron chelator, deferoxamine (DFO). DFO is poorly membrane permeable. However, it has been used to chelate intracellular iron in multiple studies including cells expressing Fpn1 (Delaby et al., 2005; Knutson et al., 2003). ARP1 MM cells pre-treated with DFO (200μM, 3h) followed by PAA treatment showed a higher viability than cells not pre-treated with DFO. These results strongly suggest that the mechanism of PAA killing of MM cells is indeed iron-dependent. In addition, Fpn1 is significantly down-regulated in CD138+ primary MM cells, while the iron importer, transferrin receptor 1, is significantly upregulated in CD138+ MM cells compared to normal plasma cells, further supporting that MM cells have higher iron content than non-tumor cells. PAA showed increased killing of MM cells derived from almost all primary MM patients and smoldering MM, but not from MGUS patients. These results suggest that PAA administration in SMM may be able to prevent progression to symtomatic MM. Though ROS and H2O2 are well known factors mediating PAA-induced cancer cell death (Espey et al., 2011; Levine et al., 2011), a single molecular mechanism cannot explain these observations because multiple pathways are involved in the downstream effects of ROS and H2O2 (Venturelli et al., 2015). Necorosis, caspase-dependent and caspase-independent apoptosis, and autophagy were reported in ascorbate-induced cell death in different types of cancer (Chen et al., 2015). A recent study by Yun and colleagues demonstrated that vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH, but spares normal cells (Yun et al., 2015). Recently the deep-sequencing data have identified that RAS family genes show the most frequent mutations in MM. KRAS, NRAS and BRAF are mutated in 22%, 20% and 7% of MM samples respectively from the analysis of 733 patients, and these two RAS mutations occur exclusively in MM patients (Manier et al., 2017; Lohr et al., 2014). Although we did not specifically test if PAA was more sensitive to RAS-mutated MM samples, we found PAA was sensitive to all 9 MMs and 2 SMMs, in whom the RAS mutation is unknown, and 3 MM cell lines (without any RAS mutations) but insensitive to 2 MGUS samples, suggesting that the disease stage rather than the mutation of RAS and/or BRAF is the major predictive factor for PAA sensitivity in MM treatment. Other molecular mechanisms including ATP depletion and ATM-AMPK signaling have been reported to explain PAA-induced cell death (Cullen, 2010; Du et al., 2010; Chen et al., 2012; Ma et al., 2014). In this study, TEM data indicate that mitochondrial morphology and structure are significantly altered after PAA treatment. Furthermore, AIF1 was originally discovered as an intermembrane space (IMS) component of mitochondria and characterized as a pro-apoptotic gene (Susin et al., 1999; Joza et al., 2001). Therefore, we focused on AIF1 as one of the pathways by which PAA-induced MM cell death. The pro-apoptotic AIF1 or truncated AIF1 (tAIF) is cleaved from the full-length AIF1 by calpains and/or cathepsins after a caspase-independent cell death insult (Joza et al., 2009; Modjtahedi et al., 2006; Sevrioukova, 2011; Artus et al., 2010). tAIF moves from the mitochondria to the cytosol and nucleus, where it initiates chromatolysis and caspase-dependent and caspase-independent cell death (Nikoletopoulou et al., 2013; Artus et al., 2010). Our data show that PAA increases AIF1 cleavage and translocation from mitochondria to cytoplasm and nucleus. Overexpression of AIF1 in MM cells increases while knock-down of AIF1 prevents PAA-induced MM cell death, indicating that AIF1 plays a critical role in mediating PAA-induced MM cell death. Because the mitochondrial apoptogenic factors such as cytochrome c and Bcl-2 family proteins are also important for the activation of caspases, future work will have to determine if AIF1 is the major pathway related to PAA activity in cancer cells as well as the exact relationship with other mitochondrial apotogenetic factors. In addition, the necrosis and apoptosis markers, such as RIP1/3 and caspases 3/8/9, are cleaved after PAA administration. It is therefore possible that PAA activates caspase 8 resutling in RIP1 cleavage and necrosis (Rajput et al., 2011) evidenced by strong caspase 8 cleavage after a short-term treatment with PAA.