Ascorbic Acid Attenuates Hyperoxia-Compromised Host Defense against Pulmonary Bacterial Infection.

Supraphysiological concentrations of oxygen (hyperoxia) can compromise host defense and increase susceptibility to bacterial infections, causing ventilator-associated pneumonia. The phagocytic activity of macrophages is impaired by hyperoxia-induced increases in the levels of reactive oxygen species (ROS) and extracellular high-mobility group box protein B1 (HMGB1). Ascorbic acid (AA), an essential nutrient and antioxidant, has been shown to be beneficial in various animal models of ROS-mediated diseases. The aim of this study was to determine whether AA could attenuate hyperoxia-compromised host defense and improve macrophage functions against bacterial infections. C57BL/6 male mice were exposed to hyperoxia (≥98% O2, 48 h), followed by intratracheal inoculation with Pseudomonas aeruginosa, and simultaneous intraperitoneal administration of AA. AA (50 mg/kg) significantly improved bacterial clearance in the lungs and airways, and significantly reduced HMGB1 accumulation in the airways. The incubation of RAW 264.7 cells (a macrophage-like cell line) with AA (0-1,000 µM) before hyperoxic exposure (95% O2) stabilized the phagocytic activity of macrophages in a concentration-dependent manner. The AA-enhanced macrophage function was associated with significantly decreased production of intracellular ROS and accumulation of extracellular HMGB1. These data suggest that AA supplementation can prevent or attenuate the development of ventilator-associated pneumonia in patients receiving oxygen support.

Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells.

Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.

Pharmacological characterization of 1-nitrosocyclohexyl acetate, a long-acting nitroxyl donor that shows vasorelaxant and antiaggregatory effects.

Nitroxyl (HNO) donors have potential benefit in the treatment of heart failure and other cardiovascular diseases. 1-Nitrosocyclohexyl acetate (NCA), a new HNO donor, in contrast to the classic HNO donors Angeli's salt and isopropylamine NONOate, predominantly releases HNO and has a longer half-life. This study investigated the vasodilatative properties of NCA in isolated aortic rings and human platelets and its mechanism of action. NCA was applied on aortic rings isolated from wild-type mice and apolipoprotein E-deficient mice and in endothelial-denuded aortae. The mechanism of action of HNO was examined by applying NCA in the absence and presence of the HNO scavenger glutathione (GSH) and inhibitors of soluble guanylyl cyclase (sGC), adenylyl cyclase (AC), calcitonin gene-related peptide receptor (CGRP), and K(+) channels. NCA induced a concentration-dependent relaxation (EC(50), 4.4 µM). This response did not differ between all groups, indicating an endothelium-independent relaxation effect. The concentration-response was markedly decreased in the presence of excess GSH; the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide had no effect. Inhibitors of sGC, CGRP, and voltage-dependent K(+) channels each significantly impaired the vasodilator response to NCA. In contrast, inhibitors of AC, ATP-sensitive K(+) channels, or high-conductance Ca(2+)-activated K(+) channels did not change the effects of NCA. NCA significantly reduced contractile response and platelet aggregation mediated by the thromboxane A(2) mimetic 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F(2)(α) in a cGMP-dependent manner. In summary, NCA shows vasoprotective effects and may have a promising profile as a therapeutic agent in vascular dysfunction, warranting further evaluation.

Role of nitric oxide in the chemistry and anticancer activity of etoposide (VP-16,213).

Originally identified as an innate cytotoxin, nitric oxide ((·)NO) formation in tumors can influence chemotherapy and exacerbate cancer progression. Here, we examined the hypothesis that (·)NO generation contributes to cancer cell drug resistance toward the widely used anticancer drug Etoposide (VP-16). The UV-vis spectrum of VP-16 was not changed by exposure of VP-16 to (·)NO in aqueous buffer. In contrast, reddish-orange compound(s) characteristic of o-quinone- and nitroso-VP-16 were readily generated in a hydrophobic medium (chloroform) in an oxygen-dependent manner. Similar products were also formed when the VP-16 radical, generated from VP-16 and horseradish peroxidase/H2O2, was exposed directly to (·)NO in chloroform in the presence of oxygen. Separation and spectral analysis of VP-16 reaction extracts by electron spin resonance and UV-vis indicated the generation of the phenoxy radical and the o-quinone of VP-16, as well as putative nitroxide, iminoxyl, and other nitrogen oxide intermediates. Nitric oxide products of VP-16 displayed significantly diminished topoisomerase II-dependent cleavage of DNA and cytotoxicity to human HL-60 leukemia cells. LPS-mediated induction of nitric oxide synthase in murine macrophages resulted in VP-16 resistance compared to Raw cells. Furthermore, (·)NO products derived from iNOS rapidly reacted with VP-16 leading to decreased DNA damage and cytotoxicity. Together, these observations suggest that the formation of (·)NO in tumors (associated macrophages) can contribute to VP-16 resistance via the detoxification of VP-16.

Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice.

Ascorbic acid is an essential nutrient commonly regarded as an antioxidant. In this study, we showed that ascorbate at pharmacologic concentrations was a prooxidant, generating hydrogen-peroxide-dependent cytotoxicity toward a variety of cancer cells in vitro without adversely affecting normal cells. To test this action in vivo, normal oral tight control was bypassed by parenteral ascorbate administration. Real-time microdialysis sampling in mice bearing glioblastoma xenografts showed that a single pharmacologic dose of ascorbate produced sustained ascorbate radical and hydrogen peroxide formation selectively within interstitial fluids of tumors but not in blood. Moreover, a regimen of daily pharmacologic ascorbate treatment significantly decreased growth rates of ovarian (P < 0.005), pancreatic (P < 0.05), and glioblastoma (P < 0.001) tumors established in mice. Similar pharmacologic concentrations were readily achieved in humans given ascorbate intravenously. These data suggest that ascorbate as a prodrug may have benefits in cancers with poor prognosis and limited therapeutic options.

Differential effects of reactive nitrogen species on DNA base excision repair initiated by the alkyladenine DNA glycosylase.

Chronic generation of reactive nitrogen species (RNS) can cause DNA damage and may also directly modify DNA repair proteins. RNS-modified DNA is repaired predominantly by the base excision repair (BER) pathway, which includes the alkyladenine DNA glycosylase (AAG). The AAG active site contains several tyrosines and cysteines that are potential sites for modification by RNS. In vitro, we demonstrate that RNS differentially alter AAG activity depending on the site and type of modification. Nitration of tyrosine 162 impaired 1,N(6)-ethenoadenine (epsilonA)-excision activity, whereas nitrosation of cysteine 167 increased epsilonA excision. To understand the effects of RNS on BER in vivo, we examined intestinal adenomas for levels of inducible nitric oxide synthase (iNOS) and AAG. A striking correlation between AAG and iNOS expression was observed (r = 0.76, P = 0.00002). Interestingly, there was no correlation between changes in AAG levels and enzymatic activity. We found AAG to be nitrated in human adenomas, suggesting that this RNS modification is relevant in the human disease. Expression of key downstream components of BER, apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase beta (POLbeta), was also examined. POLbeta protein was increased in nearly all adenomas compared with adjacent non-tumor tissues, whereas APE1 expression was only increased in approximately half of the adenomas and also was relocalized to the cytoplasm in adenomas. Collectively, the results suggest that BER is dysregulated in colon adenomas. RNS-induced posttranslational modification of AAG is one mechanism of BER dysregulation, and the type of modification may define the role of AAG during carcinogenesis.

Nitroxyl exacerbates ischemic cerebral injury and oxidative neurotoxicity.

Nitroxyl (HNO) donor compounds function as potent vasorelaxants, improve myocardial contractility and reduce ischemia-reperfusion injury in the cardiovascular system. With respect to the nervous system, HNO donors have been shown to attenuate NMDA receptor activity and neuronal injury, suggesting that its production may be protective against cerebral ischemic damage. Hence, we studied the effect of the classical HNO-donor, Angeli's salt (AS), on a cerebral ischemia/reperfusion injury in a mouse model of experimental stroke and on related in vitro paradigms of neurotoxicity. I.p. injection of AS (40 mumol/kg) in mice prior to middle cerebral artery occlusion exacerbated cortical infarct size and worsened the persistent neurological deficit. AS not only decreased systolic blood pressure, but also induced systemic oxidative stress in vivo indicated by increased isoprostane levels in urine and serum. In vitro, neuronal damage induced by oxygen-glucose-deprivation of mature neuronal cultures was exacerbated by AS, although there was no direct effect on glutamate excitotoxicity. Finally, AS exacerbated oxidative glutamate toxicity - that is, cell death propagated via oxidative stress in immature neurons devoid of ionotropic glutamate receptors. Taken together, our data indicate that HNO might worsen cerebral ischemia-reperfusion injury by increasing oxidative stress and decreasing brain perfusion at concentrations shown to be cardioprotective in vivo.

Generation of nitroxyl by heme protein-mediated peroxidation of hydroxylamine but not N-hydroxy-L-arginine.

The chemical reactivity, toxicology, and pharmacological responses to nitroxyl (HNO) are often distinctly different from those of nitric oxide (NO). The discovery that HNO donors may have pharmacological utility for treatment of cardiovascular disorders such as heart failure and ischemia reperfusion has led to increased speculation of potential endogenous pathways for HNO biosynthesis. Here, the ability of heme proteins to utilize H2O2 to oxidize hydroxylamine (NH2OH) or N-hydroxy-L-arginine (NOHA) to HNO was examined. Formation of HNO was evaluated with a recently developed selective assay in which the reaction products in the presence of reduced glutathione (GSH) were quantified by HPLC. Release of HNO from the heme pocket was indicated by formation of sulfinamide (GS(O)NH2), while the yields of nitrite and nitrate signified the degree of intramolecular recombination of HNO with the heme. Formation of GS(O)NH2 was observed upon oxidation of NH2OH, whereas NOHA, the primary intermediate in oxidation of L-arginine by NO synthase, was apparently resistant to oxidation by the heme proteins utilized. In the presence of NH2OH, the highest yields of GS(O)NH2 were observed with proteins in which the heme was coordinated to a histidine (horseradish peroxidase, lactoperoxidase, myeloperoxidase, myoglobin, and hemoglobin) in contrast to a tyrosine (catalase) or cysteine (cytochrome P450). That peroxidation of NH2OH by horseradish peroxidase produced free HNO, which was able to affect intracellular targets, was verified by conversion of 4,5-diaminofluorescein to the corresponding fluorophore within intact cells.

Nutritional Interventions for Mitochondrial OXPHOS Deficiencies: Mechanisms and Model Systems.

Multisystem metabolic disorders caused by defects in oxidative phosphorylation (OXPHOS) are severe, often lethal, conditions. Inborn errors of OXPHOS function are termed primary mitochondrial disorders (PMDs), and the use of nutritional interventions is routine in their supportive management. However, detailed mechanistic understanding and evidence for efficacy and safety of these interventions are limited. Preclinical cellular and animal model systems are important tools to investigate PMD metabolic mechanisms and therapeutic strategies. This review assesses the mechanistic rationale and experimental evidence for nutritional interventions commonly used in PMDs, including micronutrients, metabolic agents, signaling modifiers, and dietary regulation, while highlighting important knowledge gaps and impediments for randomized controlled trials. Cellular and animal model systems that recapitulate mutations and clinical manifestations of specific PMDs are evaluated for their potential in determining pathological mechanisms, elucidating therapeutic health outcomes, and investigating the value of nutritional interventions for mitochondrial disease conditions.

Workshop Report: Modulation of Antitumor Immune Responses by Dietary and Microbial Metabolites.

The human microbiota maintains an enormous and diverse capacity to produce a diet-dependent metabolome that impacts both host tissue and microbial community homeostasis. Recent discoveries support a growing appreciation that microbial metabolites derived from bioactive foods are also important regulators of host immune and metabolic functions. To gain a better understanding of the current evidence for the roles of dietary and microbial metabolites in tumor immunity, the Division of Cancer Biology and the Division of Cancer Prevention, National Cancer Institute, cosponsored a workshop on August 31 and September 1, 2016, in Bethesda, Maryland. Workshop participants examined several lines of converging science that link nutrition, microbiology, and tumor immunology and identified key concepts and research opportunities that will accelerate our understanding of these interactions. In addition, the participants identified some of the critical gaps and research challenges that could be addressed through interdisciplinary collaborations, including future opportunities for translating new information into novel cancer prevention and treatment strategies based on targeting host immune functions that are altered by metabolite sensing pathways.

Tumor macrophage redox and effector mechanisms associated with hypoxia.

Monocytes are recruited from the circulation into solid tumors where they differentiate into macrophages with unique phenotypes. While macrophages utilize oxygen in a broad range of immune effector functions, the generation of reactive oxygen and nitrogen oxide species is less clear in the setting of hypoxia, which can be a prominent feature of solid tumors. The relationships among innate immunity, redox systems, and the plasticity of phenotypic changes tumor-associated macrophages undergo in conjunction with tumor hypoxia will be examined.

Guanylyl cyclase-dependent chemotaxis of endothelial cells in response to nitric oxide gradients.

Nitric oxide (NO) is an important regulator of angiogenesis and neovascularization. The nature of endothelial cell motility responses to NO was examined using a Boyden chamber method. NO generated via decomposition of either DEA/NO or DETA/NO produced increases in human umbilical vein endothelial cell (HUVEC) chemotaxis, which were completely abrogated by ODQ, a soluble guanylyl cyclase inhibitor. Measurements of NO either directly by chemiluminescence or its chemistry with diaminofluorescein revealed that chemotaxis was driven by subtle NO gradients between the lower and the upper wells in this system. In addition to diffusion and volatilization from the upper chambers, the data showed that HUVEC consumption of NO contributed to these sustained gradients. Comparison of DEA/NO- and DETA/NO-mediated responses suggested that the persistence of spatial NO gradients is as significant as the absolute magnitude of NO exposure per unit time. The findings suggest that subnanomolar NO gradients are sufficient to mobilize endothelial cell migration into hypoxic tissue during neovascularization events, such as in wound healing and cancer.

Discriminating formation of HNO from other reactive nitrogen oxide species.

Nitroxyl (HNO) exhibits unique pharmacological properties that often oppose those of nitric oxide (NO), in part due to differences in reactivity toward thiols. Prior investigations suggested that the end products arising from the association of HNO with thiols were condition-dependent, but were inconclusive as to product identity. We therefore used HPLC techniques to examine the chemistry of HNO with glutathione (GSH) in detail. Under biological conditions, exposure to HNO donors converted GSH to both the sulfinamide [GSONH2] and the oxidized thiol (GSSG). Higher thiol concentrations generally favored a higher GSSG ratio, suggesting that the products resulted from competitive consumption of a single intermediate (GSNHOH). Formation of GSONH2 was not observed with other nitrogen oxides (NO, N2O3, NO2, or ONOO(-)),indicating that it is a unique product of the reaction of HNO with thiols. The HPLC assay was able to detect submicromolar concentrations of GSONH2. Detection of GSONH2 was then used as a marker for HNO production from several proposed biological pathways, including thiol-mediated decomposition of S-nitrosothiols and peroxidase-driven oxidation of hydroxylamine (an end product of the reaction between GSH and HNO) and NG-hydroxy-l-arginine (an NO synthase intermediate). These data indicate that free HNO can be biosynthesized and thus may function as an endogenous signaling agent that is regulated by GSH content.

The activation of metabolites of nitric oxide synthase by metals is both redox and oxygen dependent: a new feature of nitrogen oxide signaling.

Nitrite (NO(2)-), N (G)-hydroxy-L-arginine (NOHA), and hydroxylamine (NH(2)OH) are products of nitric oxide synthase (NOS) activity and can also be formed by secondary reactions of nitric oxide (NO). These compounds are commonly considered to be rather stable and as such to be dosimeters of NO biosynthesis. However, each can be converted via metal-catalyzed reactions into either NO or other reactive nitrogen oxide species (RNOS), such as nitrogen dioxide (NO(2)) and nitroxyl (HNO), which have biologic activities distinct from those of the parent molecules. Consequently, certain aspects of tissue regulation controlled by RNOS may be dictated to a significant extent by metal-dependent reactions, thereby offering unique advantages for cellular and tissue regulation. For instance, because many metal-catalyzed reactions depend on the redox and oxygen status of the cellular environment, such reactions could serve as redox indicators. Formation of RNOS by metal-mediated pathways would confine the chemistry of these species to specific cellular sites. Additionally, such mechanisms would be independent both of NO and NOS, thus increasing the lifetime of RNOS that react with NO. Thus metal-mediated conversion of nitrite, NOHA, and NH(2)OH into biologically active agents may provide a unique signaling mechanism. In this review, we discuss the biochemistry of such reactions in the context of their pharmacologic and biologic implications.

Meeting Report: The Role of the Mobilome in Cancer.

Approximately half of the human genome consists of repetitive sequence attributed to the activities of mobile DNAs, including DNA transposons, RNA transposons, and endogenous retroviruses. Of these, only long interspersed elements (LINE-1 or L1) and sequences copied by LINE-1 remain mobile in our species today. Although cells restrict L1 activity by both transcriptional and posttranscriptional mechanisms, L1 derepression occurs in developmental and pathologic contexts, including many types of cancers. However, we have limited knowledge of the extent and consequences of L1 expression in premalignancies and cancer. Participants in this NIH strategic workshop considered key questions to enhance our understanding of mechanisms and roles the mobilome may play in cancer biology. Cancer Res; 76(15); 4316-9. ©2016 AACR.

Ingress and reactive chemistry of nitroxyl-derived species within human cells.

The mechanisms that control the biological signaling and toxicological properties of the nitrogen oxide species nitroxyl (HNO) are largely unknown. The ingress and intracellular reactivity of nitroxyl-derived species were examined using Angeli's salt (AS), which decomposes initially to HNO and nitrite at physiologic pH. Exposure of 4,5-diaminofluorescein (DAF) to AS resulted in fluorescent product formation only in the presence of molecular oxygen. Kinetic analysis and the lack of signal from a nitric oxide (NO)-sensitive electrode suggested that these processes did not involve conversion of HNO to NO. On an equimolar basis, bolus peroxynitrite (ONOO(-)) exposure generated only 15% of fluorescent product formation observed from AS decomposition. Moreover, infusion of synthetic ONOO(-) at a rate comparable to AS decomposition resulted in only 4% of the signal. Quenching of AS-mediated product formation within intact human MCF-7 breast carcinoma cells containing DAF by addition of urate to buffer suggested involvement of an oxidized intermediate formed from reaction between HNO and oxygen. Conversely, intact cells competitively sequestered the HNO-derived species from reaction with DAF in solution. These data show this intermediate to be a long-lived diffusible species. Relative product yield from intracellular DAF was decreased 5- to 8-fold when cells were lysed immediately prior to AS addition, consistent with the partitioning of HNO and/or derived species into the cellular membrane, thereby shielding these reactive intermediates from either hydrolysis or cytoplasmic scavenger pools. These findings establish that oxygen-derived species of nitroxyl can readily penetrate and engage the intracellular milieu of cells and suggest this process to be independent of NO and ONOO(-) intermediacy. The substantial facilitation of oxygen-dependent nitroxyl chemistry by intact lipid bilayers supports a focusing role for the membrane in modulation of cellular constituents proteins by this unique species.

Inhibition of poly(ADP-RIBOSE) polymerase (PARP) by nitric oxide and reactive nitrogen oxide species.

The poly(ADP-ribose) polymerase (PARP) family of nuclear enzymes is involved in the detection and signaling of single strand breaks induced either directly by ionizing radiation or indirectly by the sequential action of various DNA repair proteins. Therefore, PARP plays an important role in maintaining genome stability. Because PARP proteins contain two zinc finger motifs, these enzymes can be targets for reactive nitrogen oxide intermediates (RNOS) generated as a result of nitric oxide (NO) biosynthesis in an aerobic environment. The effects of RNOS on the activity of purified PARP were examined using donor compounds. Both NO and nitroxyl (HNO) donors were found to be inhibitory in a similar time and concentration manner, indicating that PARP activity can be modified under both nitrosative and oxidative conditions. Moreover, these RNOS donors elicited comparable PARP inhibition in Sf21 insect cell extract and intact human MCF-7 cancer cells. The concentrations of donor required for 90% inhibition of PARP activity produce RNOS at a similar magnitude to those generated in the cellular microenvironment of activated leukocytes, suggesting that cellular scavenging of RNOS may not be protective against PARP modification and that inhibition of PARP may be significant under inflammatory conditions.

Heme proteins and nitric oxide (NO): the neglected, eloquent chemistry in NO redox signaling and regulation.

The role of nitric oxide (NO) in cellular physiology and signaling has been an important aspect in biomedical science over the last decade. As NO is a small uncharged radical, the chemistry of NO within the redox environment of the cell dictates the majority of its biological effects. The mechanisms that have received the most attention from a biological perspective involve reactions with oxygen and superoxide, despite the rich literature of metal-NO chemistry. However, NO and its related species participate in important chemistry with metalloproteins. In addition to the well known direct interactions of NO with heme proteins such as soluble guanylate cyclase and oxyhemoglobin, there is much important, but often underappreciated, chemistry between other nitrogen oxides and heme/metal proteins. Here the basic chemistry of nitrosylation and the interactions of NO and other nitrogen oxides with metal-oxo species such as found in peroxidases and monoxygenases are discussed.

Role of oxygen gradients in shaping redox relationships between the human intestine and its microbiota.

The unique anatomy and physiology of the intestine in conjunction with its microbial content create the steepest oxygen gradients in the body, which plunge to near anoxia at the luminal midpoint. Far from static, intestinal oxygen gradients ebb and flow with every meal. This in turn governs the redox effectors nitric oxide, hydrogen sulfide, and reactive oxygen species of both host and bacterial origin. This review illustrates how the intestine and microbes utilize oxygen gradients as a backdrop for mechanistically shaping redox relationships and a functional coexistence.

Ascorbic acid kills Epstein-Barr virus positive Burkitt lymphoma cells and Epstein-Barr virus transformed B-cells in vitro, but not in vivo.

Ascorbic acid has been shown to kill various cancer cell lines at pharmacologic concentrations. We found that Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) cells were more susceptible to ascorbic acid-induced cell killing than EBV-negative BL cells or EBV-transformed lymphoblastoid cells (LCLs). Ascorbic acid did not induce apoptosis in any of the tested cells but did induce the production of reactive oxygen species and cell death. Previously, we showed that bortezomib, a proteasome inhibitor, induces cell death in LCLs and EBV-positive BL cells. We found that ascorbic acid is strongly antagonistic for bortezomib-induced cell death in LCLs and EBV-positive BL cells. Finally, ascorbic acid did not prolong survival of severe combined immunodefiency mice inoculated with LCLs either intraperitoneally or subcutaneously. Thus, while ascorbic acid was highly effective at killing EBV-positive BL cells and LCLs in vitro, it antagonized cell killing by bortezomib and was ineffective in an animal model.