Eukaryotic gene expression is a complex process and can be controlled at the level of transcription, post-transcription or translation and post-translation. In recent years, there is a growing interest in understanding the role of 3'-untranslated region (UTR) in regulating mRNA turnover and translation. The 3'-UTR harbors the poly(A) signal and post-transcriptional regulatory sequences like miRNA and AU-rich elements (AREs). The presence of multiple poly(A) sites often results in multiple transcripts; shorter transcripts correlating with more protein abundance. Manganese superoxide dismutase (MnSOD) is a nuclear encoded and mitochondrial matrix localized antioxidant enzyme that converts mitochondrial generated superoxide to hydrogen peroxide. Human MnSOD has two poly(A) sites resulting in two transcripts: 1.5 and 4.2 kb. We hypothesize that the 3'-UTR of MnSOD regulates its mRNA and protein levels as well as activity in response to growth states and environmental stress. Results from a Q-RT-PCR assay showed a preferential accumulation of the shorter MnSOD transcript during quiescence, which correlated with an increase in MnSOD activity. The accumulation of the longer MnSOD transcript during proliferation was associated with a decrease in MnSOD activity. Log transformed expression ratio of the longer to shorter transcript was also higher in proliferating epithelial non-cancerous (mammary: MCF-10A) and cancer cells (mammary: MB-231, SUM 159; oral squamous: SQ20B, FaDu, Cal27; and lung: A549, H292), suggesting that the abundance of the longer transcript is independent of cellular transformation status, instead it is dependent on cellular growth state. Interestingly, the abundance of the longer transcript directly correlated with percent S-phase (R2=0.86). The shorter transcript was enriched in irradiated MB-231 cells. MCF-10A cells exposed to 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of the environmental pollutant polychlorinated biphenyl 3, showed a significant decrease in the abundance of the 4.2 kb transcript due to a faster mRNA turnover, 14 h compared to 20 h in untreated control cells. The decrease in the 4.2 kb transcript levels was associated with a corresponding decrease in MnSOD protein levels and activity, which resulted in a significant inhibition of quiescent cells entry into the proliferative cycle. Deletion and reporter assays showed: (a) a significant decrease in reporter activity in constructs carrying multiple AREs that are present in the 3'-UTR of the longer MnSOD transcript; (b) irradiation increased the reporter activity of the constructs carrying the 3'-UTR sequence of the shorter MnSOD transcript and (c) N-acetyl-cysteine increased the reporter activity of constructs carrying multiple AREs. Because the longer transcript carries AREs, our results identified redox sensitive AREs as novel regulators of MnSOD transcript levels. We conclude that MnSOD 3'-UTR is a novel molecular sensor regulating MnSOD mRNA levels in response to different growth states and environmental stress. A better understanding of the 3'-UTR regulating gene expression could lead to the development of new molecular biology-based redox therapy designed to treat proliferative disorders.

Mitochondrial superoxide production during the early and late radiation response is increasingly recognized as a critical driver of oxidative damage and injury processes in mammalian cells. The role of Sirtuin 3, a key mitochondrial regulatory deacetylase, in preventing mitochondrial superoxide generation in conditions of nutrient and oxidative stress may be critical during the radiation response in mammalian liver. Because several tumor types express lower than normal levels of Sirtuin 3, the involvement of Sirtuin 3 in the radiation response may also provide clues to improving cancer radiation therapy and understanding the process of carcinogenesis. Studies of how the SIRT3 loss impacts the hepatic radiation response may also provide insight into the role of superoxide in normal liver physiology as well as in conditions of pathology. Increased superoxide production has largely been associated with disease, but oftentimes without clear demonstration of mechanism or even clear descriptions of pathogenesis. Here we identify a target of Sirtuin 3, the mitochondrial antioxidant enzyme manganese superoxide dismutase, and delineate the role that Sirtuin 3-mediated increases in manganese superoxide dismutase may be playing in the prevention of injury following biologically relevant doses of low linear energy transfer and high linear energy transfer radiation types including Cs-137 and Fe and Si particle radiation. Loss of Sirtuin 3 appears to correlate with decreases in hepatocellular carcinoma 16 months after 0.1 and 1 Gy doses of particle radiation known to increase hepatocellular carcinoma rates. These results indicate that Sirtuin 3 is a critical regulator of superoxide metabolism in the liver following whole body irradiation.

Multiple myeloma (MM) is a prevalent B-cell neoplasm that remains incurable with currently available chemotherapeutic drugs. Existing drug regimens result in initial disease remission but MM often relapses with an aggressive, drug resistant phenotype with uniform mortality. Bortezomib (BTZ, proteasome inhibitor) is a frontline anti-MM drug that is used for treatment of newly diagnosed and relapsed MM. However both intrinsic and acquired BTZ resistance is observed. Hence, gaining a mechanistic understanding of BTZ-resistance can provide novel targets to increase and restore BTZ cytotoxicity in MM. Studies show that BTZ-mediated proteasome inhibition generates oxidative stress therefore, BTZ resistance can be caused by an increase in cellular antioxidant capacity of MM cells. Antioxidants like superoxide dismutases (SODs), glutathione (GSH), and glutathione peroxidases (GPxs) can maintain cellular redox homeostasis and confer resistance to oxidative stress. Additionally, an increased glucose metabolism can assist in maintaining low reactive oxygen species (ROS) levels formed as by-products of endogenous or therapy induced oxidative stress. This led us to test the hypothesis that BTZ resistance in MM is linked to redox regulation via the antioxidant network and generation of reducing equivalents. Retrospective analysis of clinically annotated MM dataset shows a correlation between SOD1 gene expression, MM progression, and poor overall and event free survival. In a MM cell line model with intrinsic or acquired BTZ resistance, our results show a correlation between half maximal inhibitory concentration (IC50) of BTZ and CuZnSOD activity. Upon inhibition of CuZnSOD activity with a clinically approved drug, disulfiram (DSF, Antabuse), BTZ cytotoxicity was increased. Furthermore, enforced overexpression of CuZnSOD conferred BTZ resistance in an otherwise BTZ sensitive MM cell line. MM cell lines with differential intrinsic BTZ cytotoxicity displayed a correlation between BTZ IC50 and GSH levels as well as GPx-1 activity. Gene expression profiling data from patients showed that poor prognosis associates with increased glycolytic gene expression in MM. Also, MM cell lines with intrinsic resistance toward BTZ exhibited increased glucose uptake, increased mRNA expression and activity of glucose-6-phosphate dehydrogenase (G6PD) with increased cytotoxicity with glucose deprivation or 2-deoxyglucose (2-DG) treatment. In conclusion, our results provide a rationale for utilizing redox-based combination protocols of clinically approved drugs (i.e. DSF and 2-DG) with BTZ to improve MM therapy responses.

Recent advances in early detection and treatment of breast cancer have led to increasing numbers of long term survivors of breast cancer. There is a growing concern about the potential adverse effects of chemotherapy on cognitive functioning. The current study examines the neuroanatomical correlates of late neurocognitive effects of chemotherapy in elderly breast cancer survivors who have survived more than ten years and were exposed to chemotherapy at the time of their cancer treatment. The participants in this study are 30 women breast cancer survivors in the age range of 65-81 years. In this cross sectional design, regional brain volumes measured using magnetic resonance imaging were correlated with cognitive test scores using multiple regression analyses. The test scores from Wisconsin Card Sorting Test and Trail Making Test B are used as measures of executive function. The test scores from the Letter Number Sequencing subset of the Wechsler Adult Intelligence Scale (WAIS) are used for measures of working memory. We found support for the hypothesized association between reduction in performance on specific neuropsychological tests and reduced volumes predominantly in the frontal, temporal and subcortical white matter regions. These results suggest that the frontal, temporal and subcortical white matter region are a neuroanatomical correlate of cognitive impairment seen in our study population. Future research will be needed to discern whether the structural correlates of cognitive impairment seen in long term cancer survivors is likely to be developed as an imaging marker for cognitive evaluation and rehabilitation.

Oxidative protein surface mapping has gained popularity over recent years within the mass spectrometry (MS) community for gleaning information about the solvent accessibility of folded protein structures. The hydroxyl radical targets a wide breadth of reactive amino acids with a stable mass tag that withstands subsequent MS analysis. A variety of techniques exist for generating hydroxyl radicals, with most requiring sources of radiation or caustic oxidizing reagents. The purpose of this research was to evaluate the novel use of electrochemistry for accomplishing a comparable probe of protein structure with a more accessible tool. Two different working electrode types were tested across a range of experimental parameters, including voltage, flow rate, and solution electrolyte composition, to affect the extent of oxidation on intact proteins. Results indicated that the boron-doped diamond electrode was most valuable for protein research due to its capacity to produce hydroxyl radicals and its relatively low adsorption profile. Oxidized proteins were collected from the electrochemical cell for intact protein and peptide level MS analysis. Peptide mass spectral data were searched by two different “hybrid” software packages that incorporate de novo elements into a database search to accommodate the challenge of searching for more than forty possible oxidative mass shifts. Preliminary data showed reasonable agreement between amino acid solvent accessibility and the resulting oxidation status of these residues in aqueous solution, while more buried residues were found to be oxidized in “non-native” solution. Later experiments utilized higher flow rates to reduce protein residence time inside the electrochemical flow chamber, along with a different cell activation approach to improve controllability of the intact protein oxidation yield. A multidimensional chromatographic strategy was employed to improve dynamic range for detecting oxidation of lower reactivity residues. Along with increased levels of oxidation around “reactive hotspot” sites, the enhanced sensitivity of these measurements uncovered a significant level of background oxidation in control proteins. While further work is needed to determine the full utility that BDD electrochemistry can lend protein structural studies, the experimental refinements reported here pave the way for improvements that could lead to a high-throughput structural pipeline complementary to predictive modeling efforts.

With new insights on mechanism, there is renewed interest in the use of pharmacological ascorbate (AscH-) in cancer therapy. The generation of H2O2 with AscH- acting as an electron donor to O2 is central to AscH- -induced cytotoxicity. We hypothesized that catalytic manganoporphyrins (MnPs) would increase the rate of oxidation of AscH- thereby increasing the flux of H2O2, resulting in increased cytotoxicity. We tested three different MnPs: MnTBAP, MnT2EPyP, and MnT4MPyP, which represent a range of physicochemical and thermodynamic properties. Of the MnPs tested, MnT4MPyP had the greatest effect on increasing the rate of oxidation of AscH-, as seen by the concentration of ascorbate radical [Asc*-] and rate of oxygen consumption. MnPs and AscH-, when combined at concentrations that had minimal effects alone, synergistically increased the cytotoxicity as seen by decreased clonogenic survival in human pancreatic cancer cell lines. Catalase, but not superoxide dismutase, reversed the cytotoxicity of AscH- and MnP, consistent with an H2O2-mediated mechanism. In addition, there was a marked increase in the steady-state concentration of ascorbate radical upon the addition of MnPs to whole blood ex vivo from mice infused with ascorbate as well as from patients treated with pharmacologic ascorbate. The combination of MnT4MPyP with ascorbate inhibited in vivo tumor growth. We conclude that MnPs can increase the rate of oxidation of AscH-, leading to an increased flux of H2O2 resulting in increased ascorbate-induced cytotoxicity

Radiation is a common mode of cancer therapy that is well known to generate reactive oxygen species leading to cell damage and death. However, there are many limitations to radiation therapy including normal tissue toxicity and the presence of quiescent cancer cells that are radio-resistant. There are many factors that regulate normal and cancer cell radiation response including the cellular redox environment which includes a complex network of antioxidants. In this study, two specific objectives will be explored: (A) SEPP1 regulation of normal cell toxicity; and (B) FoxM1 regulation of quiescence-associated radiation resistance in human oral squamous carcinoma cells. Results from DHE-oxidation analysis show that in irradiated proliferating normal cells there is a late ROS accumulation that occurs independent of cell cycle checkpoint activation and precedes cell death. Additionally, Q-RT-PCR and immunoblot analysis show an increase in Selenoprotein P (SEPP1) expression following radiation. SEPP1 is an extracellular glycoprotein with proposed selenium transport and antioxidant functions. However, pretreatment of normal cells with sodium selenite or overexpression of sepp1 is able to mitigate radiation-induced normal cell toxicity. It is well-accepted that quiescent populations exist in most solid tumors and are often the reason for tumor recurrence. In this study, we see that quiescent head and neck cancer cells that are resistant to radiation have low basal expression of Forkhead box M1 (FoxM1) compared to proliferating cancer cells. FoxM1 is a transcription factor that has recently been implicated in the cellular response to oxidative stress. Results indicate that although basal expression is low in quiescent cells, following irradiation FoxM1 is increased in quiescent cancer cells but not in proliferating cancer cells. Additionally, pharmacological and genetic knockdown of FoxM1 led to sensitization of quiescent cancer cells indicating that FoxM1 inhibitors could be useful radiation sensitizers. Together, these objectives will help to identify possible treatment options to use in addition to radiation therapy to better target quiescence-associated resistant tumors and induce less normal cell toxicity.

High-dose pharmacologic ascorbate has promise as an adjuvant to traditional therapies for cancer. It is hypothesized that the peak plasma concentration is a key determinant in treatment efficacy. From the Phase I clinical trails on the use of pharmacological ascorbate as an adjuvant to Gemcitabine in the treatment of stage IV pancreatic cancer at the University of Iowa Hospitals and Clinics, we found that monitoring plasma ascorbate concentration [AscH-]pl with each infusion is both very time consuming and expensive for large scale implementation. A method to determine the amount and protocol to infuse ascorbate to achieve a desired patient [AscH-]pl would be of great benefit. Current models lack flexibility for various infusion proto- cols. Additionally, constructing a model of ascorbate pharmacokinetics would allow investigation of an optimal dosing regime to maintain constant plasma ascorbate levels. A mechanistic model and an empirical model were developed and validated. The mechanistic model suitably replicated the results obtained in the clinical trial but contained too many variables to be useful in a clinical setting. The empirical model showed good results in replicating the trial results and requires only a few easily measured variables to generate predictions High dose ascorbate has been shown to produce hydrogen peroxide. In furthering the studies of how ascorbate affects tumor cells, the action of glucose-6-phosphate dehydrogenase (G6PD) is considered because it supplies NADPH to several peroxide removal pathways. To this end, the kinetics of G6PD were studied using kinetic simulations. G6PD exhibits a reserve capacity, which is the difference between the activity when all intracellular NADP is oxidized to the rate at which is operates when intracellular NADP is at the physiologic 90 % reduced to 10 % oxidized ratio. These simulations yielded an interesting pattern which is also seen by evolutionary biologists. G6PD exhibits a response capacity, which is the difference between the maximum G6PD activity exhibited when there is no demand for NADPH greater than normal cell functions and the activity exhibited when all cellular NADP is oxidized.

Fabry disease is an X-linked lysosomal storage disease that results from a deficiency in the enzyme α-galactosidase (α-GAL). α-GAL hydrolyzes α-galactosides, and patients with Fabry disease suffer from an accumulation of these undegraded substrates. Human α-GAL naturally occurs as a homodimer, as determined through SEC and crystallographic analysis. This means its quaternary structure consists of two identical α-GAL subunits that are associated together into a single unit. Other species, such as rice, produce a monomeric form of α-GAL, consisting of only a single subunit. If α-GAL is functional as both a homodimer and monomer, then how does homodimerization affect the activity of human α-GAL? This can be answered through two model systems. First, a monomeric form of human α-GAL can be produced, testing the activity of human α-GAL in a monomeric state. A variant of α-GAL was engineered (called α-GALF273G/W277G) that appeared promising. Secondly, another system can be produced capable of stabilizing one active site of the dimer and testing the other active site for activity. Another lysosomal enzyme, α-N-acetylgalactosaminidase (α-NAGAL), shares 46% amino acid sequence identity and share 11 of 13 active site residues. Previously, an α-GAL variant (called α-GALE203S/L206A) was produced, that maintained the antigenicity of α-GAL, but had acquired the enzymatic specificity of α-N-acetylgalactosaminidase (α-NAGAL). A heterodimeric form of α-GAL can be produced combining one subunit of α-GAL with the engineered variant. The engineered site can be stabilized, while the wild-type site can be tested for activity. SEC analysis suggests α-GALF273G/W277G is a monomer, and its kinetic properties are reported. Evidence shows monomeric α-GAL could be useful as an improved enzyme replacement therapy. Western blotting and activity assays suggest the presence of the α-GAL/ α-GALE203S/L206A heterodimer.

Pharmacological ascorbate, intravenous administration of high-dose vitamin C aimed at peak plasma concentrations ~ 20 mM, has recently re-emerged, after a controversial history, as a potential anti-cancer agent in combination with standard-of-care radiation and chemotherapy-based regimens. The anti-cancer effects of ascorbate are hypothesized to involve the auto-oxidation or metal-catalyzed oxidation of ascorbate to generate H2O2, and preclinical in vitro and in vivo studies in a variety of disease sites demonstrate the efficacy of adjuvant ascorbate. Furthermore, phase I clinical trials in pancreatic and ovarian cancer have demonstrated safety and tolerability in combination with chemotherapy and preliminary results suggest therapeutic efficacy. Both preclinical in vitro and in vivo studies as well as phase I clinical trials suggest a cell-intrinsic mechanism of selective toxicity of cancer cells as compared to normal cells; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study aims to investigate the preclinical therapeutic efficacy of pharmacological ascorbate in combination with standard cancer therapies in three novel disease sites: non-small cell lung cancer (NSCLC), glioblastoma multiforme (GBM), and some histological subtypes of sarcoma. In vitro experiments demonstrate cancer cell-selective susceptibility to pharmacological ascorbate as compared to normal cells of identical cell lineages. Furthermore, in vivo murine xenograft models of NSCLC, GBM, and fibrosarcoma demonstrate therapeutic efficacy of pharmacological ascorbate in combination with chemotherapy and/or radiation as compared to chemotherapy and/or radiation alone without any additional therapeutic toxicity. Additionally, a phase I clinical trial in GBM subjects demonstrates the safety and tolerability of ascorbate in combination with radiation and temozolomide therapy. Although not powered for efficacy, preliminary results suggest that ascorbate may be efficacious in these subjects (median survival 18.2 months vs. 14.6 months in historical controls), and, importantly, that ascorbate therapy may be independent of MGMT promoter methylation status (median survival 23.0 months vs. 12.7 months in historical controls with absent MGMT promoter methylation). Preliminary results from a phase II clinical trial of ascorbate in combination with carboplatin/paclitaxel chemotherapy in advanced stage NSCLC subjects also demonstrate promising preliminary results related to efficacy (objective response rate (ORR) 29% and disease control rate (DCR) 93% vs. historical control ORR 15-19% and DCR 40%). In addition to demonstrating the potential efficacy of pharmacological ascorbate in combination with standard anti-cancer therapies, this work demonstrates that the selective toxicity of ascorbate may be mediated by perturbations in cancer cell oxidative metabolism. Increased mitochondrial-derived O2- and H2O2 disrupts cellular iron metabolism, resulting in increased iron uptake via Transferrin Receptor and a larger intracellular labile iron pool. The larger pool of labile iron in cancer cells underlies the selective sensitivity of cancer cells to ascorbate toxicity through pro-oxidant chemistry with ascorbate-produced H2O2. This mechanism is further supported by the finding of increased levels of O2- and labile iron in patient lobectomy-derived NSCLC tissue as compared to adjacent normal fresh frozen tissue. Together, these studies demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in NSCLC, GBM, and sarcoma therapy and propose that further investigations of tumor and systemic iron metabolism are required to determine if these alterations can be exploited to enhance therapeutic efficacy or serve as therapeutic biomarkers.