OBJECTIVES: Recent studies have elucidated that diabetes mellitus (DM) patients exhibit an accelerated tumor progression, but the mechanism of its regulation is not yet fully understood. The following study seeks to examine the role of angiogenic factors in the growth of subcutaneously injected melanoma cancer using a diet-induced type II diabetic mouse model. METHODS: C57BL/6 mice were fed either a regular or high-fat, high-sucrose (HFHS) diet for 2 months (T2DM model; confirmed through a GTT) and subcutaneously injected with B16F0 melanoma cells. After a 1-week or 2-week incubation period, the tumor was extracted to examine its size, weight, vascularity, and gene/protein expression. In vitro studies were performed using endothelial cells to assess the effects of high-glucose on endothelial cell proliferation, migration, and tube formation. GLRX expression was examined in both tumor samples and endothelial cells. RESULTS: The results of the study showed that T2DM induced by a HFHS diet is able to promote tumor growth in both weight (2-week, p = 0.0070) and volume (1-week, p = 0.0351; 2-week, p = 0.0002). Tumors extracted from the HFHS diet group showed reduced expressions of angiogenic markers (ACTA2 (1-week, p = 0.0239; 2-week, p = 0.0123), KDR (1-week, p = 0.0091)) by western blot and a slightly reduced trend of angiogenesis (PECAM1) in histological analyses. GLRX expression was reduced in HFHS tumor samples (1-week, p = 0.0090) and, interestingly, lower amounts of GSH adducts (2-week, p = 0.0317) could be seen in 2-week tumors as well. In vitro studies of endothelial cells showed reduced trends of endothelial cell function (proliferation, migration, and tube formation) in high glucose medium. Also, it has been observed that high glucose may be able to stimulate GLRX expression in endothelial cells. CONCLUSION: The results of the following study have confirmed that B16F0 melanoma growth is, in fact, augmented in diet-induced diabetic mice; however, the vascularity and levels of angiogenic markers from the tumor tissues did not parallel the growth in its size. In vitro studies suggested that high glucose can impair EC function (i.e. proliferation, migration, and tube formation capabilities) as well as promote GLRX expression, which may be related to this discrepancy. Glutaredoxin-1 (GLRX), an enzyme which controls redox signaling, is upregulated in DM. Endothelial cell-specific GLRX overexpression in transgenic mice was found to stimulate subcutaneously injected melanoma (B16F0) growth, despite hindering limb revascularization after hind limb ischemia. The augmented tumor progression in DM may be associated with GLRX upregulation, alongside impaired ischemic limb revascularization and tumor angiogenesis; however, the mechanism of tumor growth in diabetes still lies inconclusive and further studies need to be examined to elucidate this phenomenon.

No description available.

The prognosis for multiple myeloma (MM) and the activated B-cell subtype of diffuse large B-cell lymphoma (ABC DLBCL) is poor. Gene expression profiling studies have identified that the transcription factor, nuclear factor kappa B (NF-κB) is overexpressed and confers a poor prognosis in MM and ABC DLBCL. NF-κB regulates the transcription of genes involved in cell proliferation and survival. Thus, several groups have tried to identify and/or develop agents that target NF-κB to improve therapy and patient prognosis for MM and ABC DLBCL. Our laboratory has shown that the manganese porphyrin MnTE-2-PyP5+ inhibits NF-κB in a murine lymphoma cell culture model and enhances tumor cell death in combination with dexamethasone and cyclophosphamide, two agents that are routinely used to treat these neoplasms. MnTE-2-PyP5+ inhibits NF-κB by glutathionylating p65, a member of the NF-κB family. The objective of the following studies was to determine whether MnTE-2-PyP5+ enhances the chemotherapeutic response in human MM and ABC DLBCL cells that overexpress and depend on NF-κB for survival. The following studies demonstrate that MnTE-2-PyP5+ glutathionylates and inhibits NF-κB in human MM and ABC DLBCL cells. MnTE-2-PyP5+ also synergizes with several MM and DLBCL chemotherapeutics, including dexamethasone, cyclophosphamide, vincristine and bortezomib to enhance cell death. The data from these human cell lines will provide the basis for future studies to test MnTE-2-PyP5+ in animal models and for translating MnTE-2-PyP5+ to the clinic.

New medical treatments, compounds that affect human health, nutritional supplements, and other substances, are introduced to society every day. The accurate determination of the potential toxicity from these substances is of critical importance to our society. Goals of the modern toxicologist not only involve the determination of the toxic potential of new substances but also: the elucidation of mechanisms; improving existing assays; and developing new assays to study toxicity. This thesis addresses these goals in two topics fundamental to toxicology. Re-evaluating the expression of dose and susceptibility of cells in culture The exposure of cells in culture to drugs, xenobiotics, and other compounds is one of the first tools used to determine the potential for toxicity. Problems can arise when results of these experiments are translated to next-level toxicity experiments (e.g. animals and humans). I hypothesized that "dose" in cell culture can be improved by designing and reporting experiments based on dose in moles per cell. When experiments were compared on an extracellular concentration basis, a large apparent variability in toxicity was observed. However, if these same exposures were expressed as moles per cell, all experiments yielded the same toxicity. In addition to the evaluation of mole per cell, I investigated the susceptibility of various cells to 1,4-benzoquinone. I hypothesized that upon exposure to toxins that bind covalently, larger cells would require more molecules per cell of toxin versus a smaller cell to achieve identical toxicities. I found a linear correlation between cell volume(pL) and ED50 (mole per cell where 50 % cell viability is lost), supporting my hypothesis. This work could improve current cell culture protocols and allow for better and less expensive determination of toxicities. Heritability of the red blood cell storage lesion Blood transfusions are an integral part of modern medicine with 5 million people receiving blood each year in the United States. There is growing evidence that red blood cells (RBCs) stored for longer periods are less therapeutically beneficial and could even be harmful to patients. This phenomenon of diminished RBC function with increased time in storage is called the storage lesion. However, there is great variation between different donors in the severity of the storage lesion in their donated RBCs. I hypothesized that part of this variability in the RBC storage lesion is determined by heritable genetic differences. To test this hypothesis, a study using mono- and di-zygotic twins was performed to determine the heritability of adenosine triphosphate (ATP), glutathione (GSH), glutathione disulfide (GSSG) and hemolysis in stored blood. Major discoveries in this study include: GSH, GSSG, and the half-cell reduction potential (Ehc) are heritable (57 %, 51 %, and 70 %, respectively) in non-stored RBCs. In addition, ATP was found to be heritable in two different storage solutions (62 % in AS-3, 71 % in CP2D); as well as GSH, GSSG, Ehc and hemolysis (59 %, 48 %, 64 %, and 53 %, respectively). These discoveries could eventually be used to develop new genetic tests that would predict the rate of deterioration in stored blood quality on an individual basis.

Selenium is a required trace element which was originally discovered by the Swedish chemist Jons Jacob Berzelius in 1817. It was initially believed to be a toxin as it was identified as being the cause of hoof maladies and excessive hair loss in horses that feed upon plants with high selenium content. It wasn’t until 1957 that the potential contributions of selenium to physiology were first demonstrated. Selenium is now known to play a critical role in the maintenance of human health. Interestingly, unlike other trace metals/semi-metals, selenium is directly incorporated into proteins in the form of the amino acid selenocysteine (Sec) in a very complicated and energetically costly fashion. Though rare, being found in only 25 human proteins, Sec proteins are involved in numerous vital biological processes including maintenance of redox homeostasis and anti-oxidant defense. Even though Sec is essential, the reason that Sec replaces its structural analog cysteine (Cys) in only 25 proteins is not widely agreed upon. A previous model suggests that the replacement of Cys with Sec provides enzymes with a type of catalytic advantage. The presence of Cys-containing orthologs of mammalian Sec-enzymes in other eukaryotes argues against this model. A newer model to explain the use of Sec is that the gain of function imparted to an enzyme by replacing Cys with Sec is the ability of Sec to impart chemical reversibility. Building on previous results from our lab demonstrating the ability of Sec to confer proteins with the ability to resist over oxidation we have elucidated the mechanism by which Sec containing thioredoxin reductase (TrxR) resists over oxidation. The ability of Sec-TrxR to resist oxidative inactivation is due to the greater electrophilicity of Sec relative to Cys. This allows for quicker resolution and prevents over oxidation. Based on these findings we also investigate the utility of the alkylating agent dimedone to probe the oxidation state of Sec. Interestingly, it was discovered that dimedone will react with seleneninic acid with the resulting adduct being labile. Additonally it was discovered that dimedone will also react with seleninic acid, resulting in the formation of a dimedone dimer. These results call into question the usefulness of dimedone in deteremining the oxidation state of Sec. Finally, we provide evidence that Sec-TrxR enzymes are able to catalyze single electron reductions. This is most likely due to the formation of a stable Sec radical intermediate. As a whole this project provides support for the theory that Sec was selected for due to its ability to convey chemical reversiablity to proteins.

The clinical potential of pharmacological ascorbate (P-AscH-; IV delivery achieving mM concentrations in blood) as an adjuvant in cancer therapy is being re-evaluated. At mM concentrations, P-AscH- is thought to exhibit anti-cancer activity via generation of a flux of H2O2 in tumors, which leads to oxidative distress. Here, we use cell culture models of pancreatic cancer, MIA PaCa-2, PANC-1, and 339 cells, to examine the effects of P-AscH- on DNA damage, and downstream consequences, including changes in bioenergetics. We have found that the high flux of H2O2 produced by P-AscH- induces both nuclear and mitochondrial DNA damage. In response to this DNA damage, we observed that poly (ADP-ribose) polymerase-1 (PARP-1) is hyperactivated, as determined by increased formation of poly (ADP-ribose) polymer. Using our unique absolute quantitation, we found that the P-AscH--mediated the overactivation of PARP-1, which results in consumption of NAD+, and subsequently depletion of ATP (potential energy crisis) leading to mitotic cell death. Time-course studies with MIA PaCa-2 cells showed that the level of NAD+ and ATP were reduced by 80% immediately after a 1-h exposure to P-AscH- (4 mM; 14 pmol cell-1); both species returned to near basal levels within 24 h. In parallel with these metabolic and energetic restorations, the lesions in nuclear DNA were removed within 3 h; however, even after 24 h, lesions in mitochondrial DNA were only partially repaired. We have also found that the Chk1 pathway has a major role in the maintenance of genomic integrity following treatment with P-AscH-. Hence, combinations of P-AscH- and Chk1 inhibitors could have the potential to improve outcomes of cancer treatment. Hyperactivation of PARP-1 and DNA repair are ATP-consuming processes. Using a Seahorse XF96 Analyzer, we observed no changes in OCR or ECAR/PPR following treatment with P-AscH-. OCR and ECAR/PPR together indicate the rate of production of intracellular ATP; therefore, the rate of production is unchanged after challenge with P-AscH-. Thus, the severe decrease in ATP is due solely to increased demand. Genetic deletion and pharmacological inhibition of PARP-1 preserved both NAD+ and ATP; however, the toxicity of P-AscH- remained. These data indicate that loss of NAD+ and ATP are secondary factors in the toxicity of P-AscH-, and damage to DNA is the primary factor. These preclinical findings can guide the best use of P-AscH- as an adjuvant in cancer therapy.