Matching Items (4)
- All Subjects: Bexarotene
- All Subjects: SIRT1
- Creators: Hackney Price, Jennifer
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
- Member of: Theses and Dissertations
Vitamin D, a bioactive lipid and essential nutrient, is obtained by humans through either endogenous synthesis in response to UV light exposure or via nutritional intake. Once activated to its hormonal form, vitamin D binds to and activates the nuclear vitamin D receptor (VDR). Activation of VDR is known to modulate gene transcription in vitamin D target tissues such as kidney, colon, and bone; however, less is known about the ability of VDR to respond to "nutritional modulators". One such potential VDR modulator is resveratrol, a plant-derived polyphenol and potent antioxidant nutrient that also functions as a chemopreventative. Resveratrol is known to activate sirtuin-1, a deacetylase enzyme with potential anti-aging properties. This study explores the potential for resveratrol, an anticancer nutraceutical, to upregulate VDR activity through its effector protein, sirtuin-1. Furthermore, due to its putative interactions with several intracellular signaling pathways, klotho has been proposed as an anti-aging protein and tumor suppressor gene, while the Wnt/β-catenin signaling pathway drives enhanced cellular proliferation leading to numerous types of cancers, especially colorectal neoplasia. Thus, the ability of klotho to cooperate with vitamin D to inhibit oncogenic β-catenin signaling was also analyzed. The experiments and resultant data presented in this thesis explore the potential role of VDR as a physiologically relevant nutritional sensor in human cells. This novel study reveals the importance of nutrient modulation of the VDR system by vitamin D and resveratrol and how this might represent a molecular mechanism that is responsible for the putative anti-cancer actions of vitamin D. Furthermore, this study enhances our understanding of how vitamin D/VDR and resveratrol interact with klotho and how this interaction affects β-catenin signaling to mitigate oncogenic growth and differentiation. This works demonstrates that the vitamin D hormone serves as a likely chemopreventive agent for various types of cancers through control of anti-oxidation and cellular proliferation pathways via its nuclear receptor. Our results also indicate the potential for resveratrol, an anticancer nutraceutical, to upregulate VDR activity through SIRT1. Furthermore, the novel data presented in this work illustrate that klotho, an anti-aging protein, cooperates with vitamin D to synergistically inhibit oncogenic β-catenin signaling. Ultimately, this study enhances our understating of the molecular pathways that underpin nutritional chemoprevention, and how modulation of these pathways via dietary intervention may lead to advances in public health strategies to eventually curb carcinogenesis.
Bexarotene is a commercially produced drug commonly known as Targetin presecribed to treat cutaneous T-cell lymphoma (CTCL). Bex mimics the actions of natural 9-cis retinoic acid in the body, which are derived from Vitamin A in the diet and boost the immune system. Bex has been shown to be effective in the treatment of multiple types of cancer, including lung cancer. However, the disadvantages of using Bex include increased instances of hypothyroidism and excessive concentrations of blood triglycerides. If an analog of Bex can be developed which retains high affinity RXR binding similar to the 9-cis retinoic acid while exhibiting less interference for heterodimerization pathways, it would be of great clinical significance in improving the quality of life for patients with CTCL. This thesis will detail the biological profiling of additional novel (Generation Two) analogs, which are currently in submission for publication, as well as that of Generation Three analogs. The results from these studies reveal that specific alterations in the core structure of the Bex "parent" compound structure can have dramatic effects in modifying the biological activity of RXR agonists.
Bexarotene (Bex) is a FDA-approved drug used to treat cutaneous T-cell lymphoma (CTCL). It binds with high affinity to the retinoid-X-receptor (RXR), a nuclear receptor implicated in numerous biological pathways. Bex may have the potential to attenuate estrogenic activity by acting as an estrogen receptor alpha (ERα) signaling antagonist, and can therefore be used to treat ERα-positive cancers, such as breast cancer. Using dual luciferase reporter assays, real-time qRT-PCR, and metabolic proliferation assays, the anti-estrogenic properties of Bex were ascertained. However, since Bex produces numerous contraindications, select novel RXR drug analogs were also evaluated. Results revealed that, in luciferase assays, Bex could significantly (P < 0.01) inhibit the transcriptional activity of ERα, so much so that it rivaled ER pan-antagonist ZK164015 in potency. Bex was also able to suppress the proliferation of two breast cancer cell models, MCF-7 and T-47D, and downregulate the expression of an estrogen receptor target gene (A-myb), which is responsible for cell proliferation. In addition, novel analogs A30, A33, A35, and A38 were evaluated as being more potent at inhibiting ERE-mediated transcription than Bex at lower concentrations. Analogs A34 and A35 were able to suppress MCF-7 cell proliferation to a degree comparable to that of Bex. Inhibition of T-47D cell proliferation, by contrast, was best achieved by analogs A34 and A36. For those with ERα – positive breast cancer who are refractory to current chemotherapeutics used to treat breast cancer, Bex and its analogs may prove to be useful alternative options.
The significance of hormonal vitamin D in the numerous facets of health stresses the importance of elucidating the molecular mechanism(s) associated with 1,25D-VDR signaling modulators (e.g., resveratrol and sirtuin-1). Resveratrol (Res), a natural antioxidant, is a potent activator of NAD-dependent deacetylase sirtuin-1 (SIRT-1), an enzyme associated with longevity in animal models. This present study employed mammalian 2-hybrid (M2H) and vitamin D responsive element (VDRE)-based transcriptional assays to investigate the potential effects of Res and SIRT-1 on VDR signal transduction. Results from VDRE-based assays indicate that Res and SIRT-1 potentiate 1,25D-VDR activity via cell-and-promoter-specific pathways. In addition, 1,25D displacement experiments revealed an increase in VDR-bound radiolabeled 1,25D in the presence of Res, suggesting that Res may potentiate VDR transactivation by stimulating 1,25D binding. M2H assays in HEK293 cells were then utilized to assess levels of interaction between VDR and VDR comodulators, including RXR, SRC-1, and DRIP-205. Both Res and SIRT-1 increased the ability of VDR to associate with RXR; however, SRC-1 and DRIP-205 interactions were not enhanced. The activity of a novel, non-acetylatable VDR mutant, K413R, was probed revealing that K413R possesses amplified transactivation capacity over wild-type VDR. A SIRT-1 inhibitor, EX-527, was used to suppress endogenous SIRT-1, resulting in significantly decreased VDR transactivation. Finally, qPCR results in HEK293 cells revealed that the 1,25D-mediated induction of CYP24A1, an endogenous VDR target gene, was enhanced (85%) by SIRT-1 while Res increased CYP24A1 expression by 294%. The combination of 1,25D, SIRT-1, and Res amplified CYP24A1 expression by 326% over 1,25D, although this effect did not reach statistical significance when compared to the Res only treated group. We conclude that acetylation of VDR comprises a negative feedback loop that attenuates 1,25D-VDR signaling. This loop is suppressed by resveratrol/SIRT-1-catalyzed deacetylation of VDR, restoring VDR activity. The two compounds, 1,25-dihydroxyvitamin D (1,25D, vitamin D) and 5-hydroxytryptamine (5-HT, serotonin), have been proposed to play a significant role in abnormal social behavior associated with psychological conditions including autism spectrum disorders (ASDs) and depression; however, the mechanism underlying these associations has yet to be elucidated. Deficiencies in 1,25D or 5-HT have been linked to the increased incidence of ASDs. Thus, examining the modulation of genes involved in 5-HT biosynthesis, reuptake, and degradation is fundamental in linking low 1,25D levels to the increased incidence of psychiatric disorders. We propose that 1,25D regulates tryptophan hydroxylase-2 (TPH2), the initial and rate-limiting enzyme in the biosynthetic pathway of 5-HT. In order to evaluate the regulation of TPH2 in neuronal cells, three formulations of media were examined to optimize the cell culture conditions necessary for growth and morphology of embryonic rat medullary raphe (B14) serotonergic neurons. Next, quantitative real time-PCR (qPCR) was utilized to examine TPH2 expression in cultured human glioblastoma (U-87) cells and rat serotonergic neurons (B-14). Human TPH2 mRNA in U-87 cells was induced dose-dependently resulting in a 2.4-fold increase at 10 nM 1,25D. Strikingly, TPH2 mRNA in B-14 cells was observed to be 26- to 86-fold upregulated at 10 nM 1,25D; however, 1 nM and 100 nM 1,25D elicited significantly smaller inductions (8-fold and 1.2-fold, respectively).