In the present study, the anti-tumor efficacy of α-TEA, a derivative of RRR-α-tocopherol, was investigated in LNCaP and PC-3-GFP human prostate cancer cells. Data show that α-TEA induced apoptosis in both cell lines in a time- and dose-dependent manner. Data show that α-TEA induces apoptosis through the activation of pro-apoptotic Fas signaling and inhibition of pro-survival Akt signaling pathways. The role of FADD and Daxx in α-TEA-induced apoptosis was determined. Data show that α-TEA promotes the association of FADD with Fas. FADD siRNA significantly reduced α-TEA-induced apoptosis in LNCaP cells. However, in PC-3-GFP cells, FADD siRNA caused apoptosis in the absence of α-TEA, and in the presence of FADD siRNA, α-TEA-induced apoptosis was significantly enhanced, indicating pro-survival activity of FADD in PC-3-GFP cells. Although α-TEA did not change the total protein levels of Daxx, it did promote the association of Daxx with Fas. α-TEA-induced apoptosis was significantly reduced by Daxx siRNA, and enhanced by overexpression of wild type Daxx, showing the pro-apoptotic role of Daxx in α-TEA-induced apoptosis. α-TEA inhibited phosphorylation of all three Akt isoforms; namely, Akt1, Akt2, and Akt3, thereby removed the phosphorylation inhibition on FOXO1 and FOXO1-mediated upregulation of FasL enhanced apoptosis through Fas signaling pathway. Studies have shown that selenium is of value in prostate cancer prevention. Here we document that methylseleninic acid (MSA) acts synergistically with α-TEA to induce apoptosis in LNCaP and PC-3-GFP human prostate cancer celld in culture. Western blot analyses indicate the involvement of caspases-8, -9, and -3, as well as Akt, in the synergistic effect of α-TEA and MSA. In a preclinical PC-3-GFP xenograft mouse model, α-TEA and MSC separately and together significantly reduced tumor burden and metastatic lesions in lungs and lymph nodes. However, synergism with the combination that in cell culture were not obtained in the animal study. α-TEA alone was as effective as, perhaps better than, the combination treatment in ruducing tumor burden and inhibiting metastases. Thus, data support α-TEA alone, rather than α-TEA plus selenium, as a treatment for human prostate cancer. / text

Anti-inflammatory specific cytokines and chemokines are elevated in many advanced tumors and correlate with poor prognosis. However, the differential expression of anti-inflammatory cytokines and chemokines in prostate cancer is not known. We investigated the hypotheses that androgen unresponsive DU145 and PC3 prostate cancer cells and androgen responsive LNCaP prostate cancer cells, differentially expressed selected anti-inflammatory and pro-inflammatory cytokines and chemokines and that, dendritic cells pulsed with prostate tumor antigens will induce mainly pro-inflammatory cytokines and chemokines in T cells using mouse models. Our results indicated that anti-inflammatory specific cytokines IL-1 0, IL-4, and anti-inflammatory specific chemokine CCL- 17 (TARC) and cognate receptor CCR4 are expressed in prostate cancer cell lines. Quantitative real-time PCR (qRT-PCR) revealed an almost five-fold increase in chemokine CCL17 and its cognate receptor CCR4 mRNA in androgen unresponsive DU145 and PC3 prostate cancer cell lines compared to androgen responsive prostate tumor LNCaP. Protein analysis indicated significantly increased secretion of anti-inflammatory cytokine IL- 10 by DU145 and PC3 compared to LNCaP. Furthermore, pro-inflammatory cytokine IFN-y and pro-inflammatory chemokine IP- 10 secretion were significantly less in these prostate cancer cells, when compared to immortalized normal prostate epithelial cells. Our in- vivo analysis revealed that T cells were activated by pulsed dendritic cells shown in the increase mRNA expression of pro-inflammatory cytokine IFN-y and pro-inflammatory chemokine IP- 10, and cognate receptor CXCR3. However, a predominant pro-inflammatory response was not observed as anti-inflammatory cytokines and chemokines were also seen. The production of anti-inflammatory cytokines and chemokines suggests a possible mechanism for prostate cancer to evade host immune responses by negatively modulating immune responses that are necessary for destroying cancers cells.. Cytokine and chemokine profiles could be used as potential prognostic markers for disease progression. Additionally, an effacious vaccine will depend on its ability to inhibit the recruitment of known distinct functional anti-inflammatory effector molecules, implicated in prostate cancer progression.

published_or_final_version / Pathology / Master / Master of Philosophy

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Cancer cells have the ability to survive when oxygen is limiting, and upregulate the pathway of fatty acid synthesis, owing in part to alterations in their metabolism. I compared the metabolic phenotypes of the prostate cancer cell lines LNCaP, DU145, and PC3 assessing energy metabolism, and metabolic gene expression. I also explored the plasticity of the metabolic phenotype following passage, selection and in vivo growth. Finally, I explored the sensitivity of the fatty acid synthesis pathway to low oxygen. LNCaP cells had a more oxidative phenotype based on oxygen consumption, lactate production, enzyme assays, and mRNA levels. While DU145 and PC3 cells were more glycolytic, they were unresponsive to dichloroacetate (DCA), and dinitrophenol (DNP), stimulators of oxygen consumption. Mitochondrial dysfunction in the PC3 and DU145 cells may explain this phenomenon, for they possessed normal cardiolipin levels but lower mitochondrial enzyme activities (cytochrome oxidase (COX), citrate synthase (CS)). When LNCaP cells were subjected to high passage, with and without clonal selection, the derived lines acquired a dysfunctional oxidative phenotype, becoming more glycolytic. Clonal selection appeared to have the most dramatic effect on cellular metabolism. This finding is supported by decreased oxygen consumption, increased lactate production, and a reduction in the activity of the oxidative enzymes CS and COX in the clonally selected LNCaP-luc cell line. Similar to the DU145 and PC3 cells, NAO fluorescence indicates that the oxidative impairment in these LNCaP-derived lines may be due to a reduction in mitochondrial activity. The pattern of metabolic gene expression iii seen in vitro was unaffected when LNCaP cells were grown as subcaspular and muscle xenografts in immunodeficient mice, though xenografts did exhibit indications of an hypoxic response (elevated VEGF mRNA). Oxygen deprivation in vitro increased mRNA for HIF and responsive genes but not SREBP responsive genes. Similarly, oxygen deprivation had no influence on triglyceride levels in any of the lines suggesting that the SREBP axis may not be directly modulated by oxygen levels. Collectively these studies demonstrate differences in the metabolism of these prostate cancer models, with important ramifications of therapeutic strategies involving metabolic targets. / Thesis (Master, Biology) -- Queen's University, 2008-09-25 17:28:13.418

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