Prostate cancer is the most frequently diagnosed non-skin cancer and the third leading cause of cancer mortality among men in the US. Androgens are functionally required for the normal growth of the prostate gland and play a critical role in prostate tumor development and progression. Epithelial-mesenchymal-transition (EMT) is an important process during normal development, and cancer cell metastasis. This study examined the ability of androgens to influence EMT of prostate cancer epithelial cells and evaluate the effect of taxol chemotherapy on androgen signaling in prostate cancer cells in prostate cancer. The EMT pattern was evaluated on the basis of expression of the epithelial markers as well as cytoskeleton reorganization in respond to DHT (1nM) and/or TGFβ (5ng/ml). Overexpressing and silencing approaches to regulate androgen receptor (AR) expression were conducted to determine the involvement of AR in EMT in the presence or absence of an AR antagonist. The AR transcriptional activity was determined on the basis of prostate specific antigen (PSA) mRNA expression and the androgen-response element (ARE) luciferase reporter assay. The interaction of AR and tubulin was investigated using immunoprecipitation, immunofluorescence as well as introduction of a truncated AR in human prostate cancer cells. Our results demonstrate that androgens induce the EMT pattern in prostate tumor epithelial cell with Snail activation and led to significant changes in prostate cancer cell migration and invasion potential. Expression levels of AR inversely correlated with androgen-mediated EMT in prostate tumor epithelial cells, pointing to a low AR content required for the EMT phenotype. Our study also reveals that treatment of prostate cancer cells with Paclitaxel or Nocodaxol inhibits androgen-dependent, as well as androgen-independent AR nuclear translocation and activation potentially via targeting the interaction of AR and microtubule cytoskeletal structures. Our findings on multiple aspects of AR function in prostate cancer development and progression may enhance the understanding of AR targeting therapy being a double-sided sword in the context of tumor microenvironment. These studies provide new insights into the mechanism of action of chemotherapy agents and the development of therapeutic resistance within tubulin/microtubule repertoire in prostate cancer cells.

The purpose of this study was to determine whether androgen promotes AT1aR expression on smooth muscle to confer high prevalence of AngII-induced AAAs in hyperlipidemic mice. In addition, we also investigate the role of androgen in the progression of established AngII-induced AAAs. First, we sought to examine the role of endogenous androgen in the growth of established AngII-induced AAAs. By castrating male mice, we demonstrated that removal of endogenous androgen significantly decreased the progressive lumen dilation of established AngII-induced AAAs in male ApoE-/- mice, but had no effect on external AAA diameters. These results suggest that androgen contributes to the progression of established AAAs through distinct mechanisms that differentially influence aortic lumen and wall diameters. We also investigate whether androgen regulates aortic AT1aR expression to promote AngII-induced AAA formation. Our data demonstrated that in male and female mice, both endogenous and exogenous androgen stimulate AT1aR level particularly in abdominal aortas. This androgen-dependent enhanced expression of abdominal aortic AT1aR was correlated with increased AngIIinduced AAA formation in male and female mice. Smooth muscle AT1aR deficiency significantly reduced luminal and external diameters of abdominal aortas as well as the incidence of AngII-induced AAAs in adult female mice administered exogenous androgen. Collectively, these results indicate that in adult mice androgen stimulate smooth muscle AT1aR expression to promote AngII-induced AAA formation. To determine the role of androgen during development on AT1aR expression on SMC and AngII-induced vascular pathologies, we exposed neonatal female mice to one single dose of testosterone. Our data demonstrated that neonatal testosterone administration dramatically increased AngII-induced AAA, atherosclerosis and ascending aortic aneurysms in adult female mice. In addition, smooth muscle AT1aR deficiency reduced effects of neonatal testosterone to promote AAAs, but had no effect on the other two AngII-induced vascular pathologies. In summary, our findings demonstrated that androgen, both in adult life and during development, stimulate smooth muscle AT1aR expression and promote AngII-induced AAA in female hyperlipidemic mice.

A previous thoracic aortic aneurysm time course study from our laboratory determined that ascending aortic dilation was significantly increased by day 5, and reached a plateau by day 28 of angiotensin II (AngII) infusion. We also found that mice had hemorrhage localized to the ascending aortas by day 5 of AngII infusion. The purpose of these studies was to provide mechanistic insight into the development of AngII-induced ascending aortic hemorrhage. Male C57BL/6 mice fed normal diet were subcutaneously infused with either AngII (1000 ng/kg/min) or saline for 5 days. To examine cellular heterogeneity, hemorrhaged ascending aortas were collected and sectioned serially for histological staining and immunostaining. I was unable to identify an entry point for blood into the media of the aortic root and ascending aorta. However, I found incomplete intimo-medial dissection near the hemorrhaged regions that may potentially be contiguous with the blood. To investigate infiltration of immune cells during AngII infusion, immunohistochemistry of hemorrhaged ascending aortas was performed. The numbers of macrophages and neutrophils in AngII-infused aortas were increased in both medial and adventitial areas when compared with saline-infused aortas. Therefore, infiltration of immune cells at the point of dissection is associated with aortic hemorrhage during AngII infusion.

T-2 toxin (T-2), produced by the genus Fusarium, is a cytotoxic trichothecene mycotoxin, a feed contaminant, and has been shown to be immunomodulatory. It is suspected that T-2-associated immunomodulation is mediated partly through the hypothalamic-pituitary-adrenal axis. The presence of endotoxin, a bacterial product capable of activating the hypothalamic-pituitary-adrenal axis as well as the levels of several hormones, also associated with activation of the hypothalamic-pituitary-adrenal axis, were determined in both vehicle- and toxin-treated animals. Endotoxemia was evident twenty-four hours after a single oral exposure to T-2. Blood levels of adrenocorticotropic hormone and corticosterone, parameters of the stress response, also increased twenty-four hours after T-2 exposure. Hypothalamic norepinephrine and serum corticosterone levels increased in a dose-related manner after two weeks of T-2 exposure. An increased corticosteroid level was associated with thymic involution leading potentially to decreased T-dependent antibody response, a known effect of T-2. The effects of exposure to T-2 on the development of both T-dependent and T-independent antibody response were determined in nonoperated, sham-operated and adrenalectomized mice. T-2 decreased the antibody response to a T-dependent antigen and increased a T-independent response. The effects of T-2 were partially nullified by adrenalectomy. These results provide a further confirmation of the postulate that the hypothalamic-pituitary-adrenal axis plays an important role in T-2 toxin-immunomodulation. In vitro studies were undertaken to investigate the direct effects of T-2 on various populations of lymphatic cells. Exposure to T-2 after twenty-four hours caused an increase in the uptake of 3H-thymidine by mouse splenic cells. Pokeweed mitogen stimulation also increased in this system; the response to lipopolysaccharide increased to a lesser extent. However, T-cell responses to phytohaemagglutinin and concanavalin A (Con A) decreased. Thymic cells were also sensitive to T-2. The possibility of pharmacological activity of T-2 with thymocytes was investigated. Both specific and nonspecific cell associations were observed. The association of T-2 with thymocytes was altered in the presence of dexamethasone, a synthetic corticosteroid. T-2 was shown to have both indirect as well as direct activities on the immune system. Endocrine dysfunction resulting from chronic stress and possible pharmacologic activity of T-2 provide the impetus for further investigations.

Small ubiquitin-like modifier (SUMO) regulates numerous biological functions. In a previous study we found that sumoylation of HSF2 is involved in regulating HSF2 bookmarking function, but the mechanism that mediates this regulation was unknown. The results in my work support the intriguing hypothesis that polycomb protein, Mel-18, actually functions as an anti-SUMO E3 protein, interacting both with HSF2 and the SUMO E2 Ubc9, but acting to inhibit Ubc9 activity and thereby decrease sumoylation of the HSF2. This study also suggested that Mel-18 negatively regulates the sumoylation of other cellular proteins, and we extend its targets to RanGAP1 protein. The results also show that RanGAP1 sumoylation is decreased during mitosis, and that this is associated with increased interaction between RanGAP1 and Mel-18. Previous studies showed little evidence of anti-SUMO E3 proteins, however, my study, taken together, found Mel-18 actually functions as a novel anti-SUMO E3 protein, interacting both with substrates and the SUMO E2 Ubc9 but acting to inhibit Ubc9 activity to decrease sumoylation of target proteins and also provide an explanation for how mitotic HSF2/RanGAP1 sumoylation is regulated. This finding also gives a clue for a future study direction in Mel-18 as a tumor suppressor: the anti-SUMO E3 function. Additionally, we identify a single-nucleotide polymorphism in another human PcG protein, Bmi-1, that changes a cysteine residue within its RING domain, cysteine 18, to a tyrosine. This C18Y polymorphism is associated with a significant decrease in levels of the Bmi-1 protein. Furthermore, the C18Y Bmi-1 protein exhibits a very high level of ubiquitination compared to wild-type Bmi-1, suggesting that that the low levels of this form of Bmi-1 are due to its destruction by the ubiquitin-proteasome system. Consistent with this hypothesis, treatment of cells with the proteasome inhibitor MG-132 results in a significant increase in levels of C18Y Bmi-1. This is the first example of a polymorphism in human Bmi- 1 that reduces levels of this important protein.

The blood-brain barrier (BBB) is a dynamic interface, mainly consisting of highly specialized brain microvascular endothelial cells (BMECs) that segregate the central nervous system (CNS) from the peripheral circulation. Impairment of the BBB, due to disruption of tight junction (TJ) proteins and inflammatory responses, may initiate and/or contribute to the progress of CNS disorders, including stroke. Stroke is the second leading cause of death worldwide. It has been shown that aging and environmental pollutants can induce brain endothelium dysfunction, and are considered as risk factors for stroke. Deficiency of telomerase is highly linked with aging-associated vascular diseases. Evidence indicates that patients with shorter telomere length are at higher risk of heart disease or stroke. Results in this dissertation address the influence of telomerase reverse transcriptase (TERT), a key component of telomerase, on the BBB integrity in the context of ischemic stroke induced brain injury. Our results indicate that aging-related BBB alterations aggregate the stroke outcomes by inducing oxidative stress and stimulating proinflammatory responses on the brain microvessels. The ability of the BBB to protect the brain from harmful compounds indicates that the BBB may be targeted by chemical toxicants in the peripheral circulation. Polychlorinated biphenyls (PCBs) are persistent organic pollutants that frequently bind to nanoparticles (NPs) in the environment. Our results demonstrate that binding PCB153, one of the most abundant PCB congeners in the environment, to silica nanoparticles (PCB153-NPs) potentiates cerebrovascular toxicity and stroke outcomes via stimulation of inflammatory responses and disruption of BBB integrity. These events are mediated by activation of toll-like receptor 4 (TLR4), which subsequently recruits tumor necrosis factor-associated factor 6 (TRAF6) and initiates the production of multiple inflammatory mediators. Research presented in this dissertation demonstrates that aging and environmental pollutants play crucial roles in modifying the function of the BBB through alterations of inflammatory responses and TJ protein expression, which further contribute to the progression of stroke-induced cerebral ischemic injury.

Hexavalent chromium, Cr(VI), is an established human carcinogen that is a worldwide environmental health concern. It is well understood that reactive oxygen species, genomic instability, and DNA damage repair deficiency are important contributors to Cr(VI)-induced carcinogenesis. After decades of research some cancer hallmarks remain understudied for the mechanism of Cr(VI) carcinogenesis. Dysregulated cellular energetics have been established as a hallmark of cancer. Energy pathways that become dysregulated in cancer include mitochondrial respiration, lipogenesis, pentose phosphate pathway, one carbon metabolism, and increased anaerobic glycolysis in the presence of oxygen or ‘Warburg effect’. To investigate metabolism changes in Cr(VI) carcinogenesis, we exposed human lung epithelial cells (BEAS-2B cells) to Cr(VI) for six months and isolated a colony from soft agar. To confirm the results in the BEAS-2B cells, we used two other sets of Cr(VI)-transformed cells, human lung epithelial cells (BEP2D cells) and human lung fibroblasts (WTHBF-6 cells). We found increased lipogenesis related protein expressions including: ATP citrate lyase (ACLY), acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FASN) in Cr(VI)-transformed cells as compared to passage-matched control cells. We also observed increased palmitic acid levels, confirming that Cr(VI)-transformed cells were making more lipids. Cr(VI)-transformed BEAS-2B cells had decreased colony formation in soft agar and decreased cell growth when treated with a FASN inhibitor (C75). ACLY, ACC1, and FASN protein expressions were also increased in chromate-induced lung tumors in human tissue samples. We also observed that Cr(VI)-transformed human lung cells (BEAS-2B, BEP2D, and WTHBF-6 cells) had no major changes in their mitochondrial respiration as measured by the Seahorse Analyzer when compared to their passage-matched control cells. Conversely, xenograft tumor-derived cells had mitochondrial respiratory dysfunction. Interestingly, we also found that Cr(VI)-transformed human lung cells (BEAS-2B, BEP2D, and WTHBF-6 cells) had no major changes in their glycolytic function as measured by the Seahorse Analyzer when compared to their passage-matched control cells. Similarly, these cells did not have changes in glycolytic enzymes or extracellular L-lactate levels. Moreover, xenograft tumor-derived cells showed no changes in glycolytic endpoints or L-lactate levels. This indicates these cells did not undergo the ‘Warburg effect’. These data demonstrate that increased lipogenesis is important to Cr(VI)-induced lung carcinogenesis and are consistent with the cancer literature which reports that increased lipogenesis proteins occur during carcinogenesis. Additionally, our results indicate mitochondrial respiratory dysfunction is likely a result of the tumor microenvironment and a later step during Cr(VI) carcinogenesis. Lastly, we observed the ‘Warburg effect’ is not required for Cr(VI)-induced carcinogenesis in vitro. However, it remains to be shown if the ‘Warburg effect’ is still a consequence or contributing factor for tumorigenesis. Future studies are needed to investigate other metabolic pathways in Cr(VI)-induced carcinogenesis. In conclusion, some metabolism pathways are important to Cr(VI)-induced carcinogenesis, while others appear not to be.

The rate of cancer patients is increasing as the development of science and technology. Twenty million cancer survivors are estimated living in the United States by 2025. However, many cancer survivors show cognitive dysfunction, negatively affecting the quality of life. These cognitive impairments are recognized as chemotherapy-induced cognitive impairment (CICI), also called "chemo brain" by cancer survivors, including the diminished ability of memory and learning, hard to concentrate and focus, as well as diminution of executive function and processing speed. The etiologies and pathologies of CICI are complicated, especially in most cases the anti-cancer drug cannot cross the blood-brain barrier (BBB). One of the significant candidate mechanisms underlying CICI is chemotherapy-induced, oxidative damage-mediated tumor necrosis factor-alpha (TNF-a) elevation. One of the prototypes of reactive oxygen species (ROS)-generating chemotherapeutic agents is Doxorubicin, normally used as part of multi-drug chemotherapeutic regimens to treat solid tumors and lymphomas. In this dissertation, TNF-a null (TNFKO) mice were used to investigate the role of TNF-a in Dox-induced, oxidative damage-mediated alterations in brain. Dox-induced oxidative damage in brain is ameliorated and brain mitochondrial function is preserved in brains of TNFKO mice. Both Dox-decreased levels of hippocampal choline-containing compounds and activities of brain phospholipases are partially protected in the TNFKO group. It is shown in this dissertation that Dox-targeted mitochondrial damage and levels of brain choline-containing metabolites, as well as changes in the activity of phospholipases, including both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD), are decreased in the CNS and associated with oxidative damage mediated by TNF-a. The results are discussed with respect to identifying a potential therapeutic target to protect against cognitive problems after chemotherapy and thereby improve the quality of life of cancer survivors. We also tested the effect of a chemotherapy drug adjuvant, 2-mercaptoethane sulfonate sodium (MESNA), on CICI in this dissertation research. MESNA ameliorated Dox-induced oxidative protein damage in plasma and led to decreased oxidative damage in brain. MESNA was demonstrated to rescue the memory deficits caused by Dox in the novel object recognition test. The activity of PC-PLC was preserved when MESNA was co-administered with Dox. This study is the first evidence for demonstrating the protective effects of MESNA on Dox-related protein oxidation, cognitive decline, phosphocholine levels, and PC-PLC activity in brain and suggests novel potential therapeutic targets and strategies to mitigate CICI. Parkinson Disease (PD) is considered as the second most neurodegenerative disease, associated with aging and gender. Although the detailed mechanisms remain unknown, inflammation and oxidative damage are two main etiological factors of PD. Certain genetic factors have been discovered related to this disease. Thus, using rodent models with relative gene mutations are the main strategies to investigate PD. However, few rodent models showed same clinical and biochemical features of PD. PTEN-induced putative kinase -1 (PINK1) knockout (KO) rat is the rodent model used in this dissertation research. The oxidative damage in the brain of PINK1 KO rats, the ventricle sizes, and neurochemical metabolite profiles in these rats as a function of age and gender were measured. Distinct gender- and age-related alterations were found, many consistent with those in PD. The proteome of brain of PINK1 KO rat as a function of age and gender also was studied. Based on the collected data, the suitability of this unique rat as a faithful model of known characteristics of PD with our results is discussed.

Background Herbal marijuana alternatives (HMA), legal plant products adulterated with synthetic cannabinoid receptor agonists, represent a growing public health concern. Only a few case reports describe HMA and synthetic cannabinoid’s clinical toxicity. We describe an outbreak of HMA abuse primarily in the Midwest, the clinical presentation of HMA toxicity, and clinical and forensic testing. Methods During the course of ongoing surveillance for emerging drugs of abuse between November 2009 and August 2010, we retrospectively and prospectively identified a convenience sample comprising 81 cases of abuse of HMA products. Subject demographics, vital signs, lab results and urine were obtained (when available) and tested via gas chromatography mass spectrometry (GCMS) analysis. Samples of HMAs and synthetic cannabinoids were also analyzed via GCMS. Results HMA users were predominantly young males who inhaled HMAs. Analysis of their urine detected synthetic cannabinoid parent compound in one subject. GCMS analysis of synthetic cannabinoids established a reference library that confirmed the presence of synthetic cannabinoids in sampled HMA products. Conclusion HMA products were available in head shops, gas stations, and via the Internet. We have confirmed the presence of synthetic cannabinoids in these HMA products. The tachycardia, hypertension, agitation, anxiety, vomiting and hallucinations observed in this convenience sample are not readily explained by the presence of synthetic cannabinoids acting on CB1 and CB2 receptors. Further research must be done on HMA products and their abusers.

Sulfiredoxin (Srx) is an antioxidant enzyme that can be induced by oxidative stress. It promotes oncogenic phenotypes of cell proliferation, colony formation, migration, and metastasis in lung, skin and colon cancers. Srx reduces the overoxidation of 2-cysteine peroxiredoxins in cells, in addition to its role of removing glutathione modification from several proteins. In this study, I explored additional physiological functions of Srx in lung cancer through studying its interacting proteins. Protein disulfide isomerase (PDI) family members, thioredoxin domain containing protein 5 (TXNDC5) and protein disulfide isomerase family A member 6 (PDIA6), were detected to interact with Srx. Therefore, I proposed that TXNDC5 and PDIA6 are important for the oncogenic phenotypes of Srx in lung cancer. In chapter one, I presented background information about the role of Srx as an antioxidant enzyme in cancer. I also explained the functional significance of PDIs as oxidoreductase and chaperones in cells. In chapter two, I verified the Srx-TXNDC5/PDIA6 interaction in HEK293T and A549 cells by co-immunoprecipitation and other assays. In TXNDC5 and PDIA6, the N-terminal thioredoxin-like domain (D1) is determined to be the main platform for interaction with Srx. The Srx-TXNDC5 interaction was enhanced by H2O2 treatment in A549 cells. Srx was determined to localize in the endoplasmic reticulum (ER) of A549 cells along with TXNDC5 and PDIA6. This localization was confirmed by both subcellular fractionation and immunofluorescence imaging experiments. In chapter three I focused on studying the physiological function of Srx interacting proteins in the ER. A549 subcellular fractionation results showed that TXNDC5 facilitates Srx retention in the ER. Moreover, TXNDC5 and Srx were found to participate in chaperone activities in lung cancer. Both proteins contributed in the refolding of heat-shock induced protein aggregates. In addition, TXNDC5 and PDIA6 were found to enhance the protein refolding in response to H2O2 treatment. Conversely, Srx appeared to have an inhibitory effect on protein folding under same treatment conditions. Downregulation of Srx, TXNDC5, or PDIA6 significantly reduced cell viability in response to tunicamycin treatment. TXNDC5 knockdown decreased the time required for the splicing of X-box binding protein-1 (XBP-1). In either knockdown Srx or TXNDC5 cells, there was an observable decrease in the expression of GRP78 and the splicing of spliced XBP-1. These results suggest a possible role of Srx in unfolded protein response signaling. TXNDC5 and PDIA6, similar to Srx, contribute to the proliferation, anchorage independent colony formation and migration of lung cancer cells. In this dissertation I concluded that Srx TXNDC5, and PDIA6 proteins participate in oxidative protein folding in lung cancer. Srx and TXNDC5 can modulate unfolded protein response (UPR) sensor activation and growth inhibition. Furthermore, TXNDC5 and PDIA6 can promote tumorigenesis of lung cancer cells. Therefore, the molecular interaction of Srx with TXNDC5/PDIA6 has the potential to be used as novel therapeutic targets for lung cancer treatment.