53 research outputs found
Inhibition of Inflammatory Gene Expression in Keratinocytes Using a Composition Containing Carnitine, Thioctic Acid and Saw Palmetto Extract
Chronic inflammation of the hair follicle (HF) is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA). Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr) and its glycoside, β-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid) could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS) provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4) associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities
Histone deacetylase inhibitors: A new mode for inhibition of cholesterol metabolism
<p>Abstract</p> <p>Background</p> <p>Eukaryotic gene expression is a complex process involving multiple cis and trans activating molecules to either facilitate or inhibit transcription. In recent years, many studies have focused on the role of acetylation of histone proteins in modulating transcription, whereas deacetylation of these same proteins is associated with inactivation or repression of gene expression. This study explores gene expression in HepG2 and F9 cell lines treated with Trichostatin A (TSA), a potent histone deacetylase inhibitor.</p> <p>Results</p> <p>These experiments show that TSA treatment results in clear repression of genes involved in the cholesterol biosynthetic pathway as well as other associated pathways including fatty acid biosynthesis and glycolysis. TSA down regulates 9 of 15 genes in this pathway in the F9 embryonal carcinoma model and 11 of 15 pathway genes in the HepG2 cell line. A time course study on the effect of TSA on gene expression of various enzymes and transcription factors involved in these pathways suggests that down regulation of <it>Srebf2 </it>may be the triggering factor for down regulation of the cholesterol biosynthesis pathway.</p> <p>Conclusion</p> <p>Our results provide new insights in the effects of histone deacetylases on genes involved in primary metabolism. This observation suggests that TSA, and other related histone deacetylase inhibitors, may be useful as potential therapeutic entities for the control of cholesterol levels in humans.</p
Effects of 1α,25 dihydroxyvitamin D3 and testosterone on miRNA and mRNA expression in LNCaP cells
<p>Abstract</p> <p>Background</p> <p>There is evidence from epidemiological and <it>in vitro </it>studies that the biological effects of testosterone (T) on cell cycle and survival are modulated by 1,25-dihydroxyvitamin D<sub>3 </sub>(1,25(OH)<sub>2</sub>D<sub>3</sub>) in prostate cancer. To investigate the cross talk between androgen- and vitamin D-mediated intracellular signaling pathways, the individual and combined effects of T and 1,25(OH)<sub>2</sub>D<sub>3 </sub>on global gene expression in LNCaP prostate cancer cells were assessed.</p> <p>Results</p> <p>Stringent statistical analysis identifies a cohort of genes that lack one or both androgen response elements (AREs) or vitamin D response elements (VDREs) in their promoters, which are nevertheless differentially regulated by both steroids (either additively or synergistically). This suggests that mechanisms in addition to VDR- and AR-mediated transcription are responsible for the modulation of gene expression. Microarray analysis shows that fifteen miRNAs are also differentially regulated by 1,25(OH)<sub>2</sub>D<sub>3 </sub>and T. Among these miR-22, miR-29ab, miR-134, miR-1207-5p and miR-371-5p are up regulated, while miR-17 and miR-20a, members of the miR-17/92 cluster are down regulated. A number of genes implicated in cell cycle progression, lipid synthesis and accumulation and calcium homeostasis are among the mRNA targets of these miRNAs. Thus, in addition to their well characterized effects on transcription, mediated by either or both cognate nuclear receptors, 1,25(OH)<sub>2</sub>D<sub>3 </sub>and T regulate the steady state mRNA levels by modulating miRNA-mediated mRNA degradation, generating attenuation feedback loops that result in global changes in mRNA and protein levels. Changes in genes involved in calcium homeostasis may have specific clinical importance since the second messenger Ca<sup>2+ </sup>is known to modulate various cellular processes, including cell proliferation, cell death and cell motility, which affects prostate cancer tumor progression and responsiveness to therapy.</p> <p>Conclusions</p> <p>These data indicate that these two hormones combine to drive a differentiated phenotype, and reinforce the idea that the age dependent decline in both hormones results in the de-differentiation of prostate tumor cells, which results in increased proliferation, motility and invasion common to aggressive tumors. These studies also reinforce the potential importance of miRNAs in prostate cancer progression and therapeutic outcomes.</p
Thrombospondin-1 Type 1 Repeats in a Model of Inflammatory Bowel Disease: Transcript Profile and Therapeutic Effects
Thrombospondin-1 (TSP-1) is a matricellular protein with regulatory functions in inflammation and cancer. The type 1 repeats (TSR) domains of TSP-1 have been shown to interact with a wide range of proteins that result in the anti-angiogenic and anti-tumor properties of TSP-1. To ascertain possible functions and evaluate potential therapeutic effects of TSRs in inflammatory bowel disease, we conducted clinical, histological and microarray analyses on a mouse model of induced colitis. We used dextran sulfate sodium (DSS) to induce colitis in wild-type (WT) mice for 7 days. Simultaneously, mice were injected with either saline or one form of TSP-1 derived recombinant proteins, containing either (1) the three type 1 repeats of the TSP-1 (3TSR), (2) the second type 1 repeat (TSR2), or (3) TSR2 with the RFK sequence (TSR2+RFK). Total RNA isolated from the mice colons were processed and hybridized to mouse arrays. Array data were validated by real-time qPCR and immunohistochemistry. Histological and disease indices reveal that the mice treated with the TSRs show different patterns of leukocytic infiltration and that 3TSR treatment was the most effective in decreasing inflammation in DSS-induced colitis. Transcriptional profiling revealed differentially expressed (DE) genes, with the 3TSR-treated mice showing the least deviation from the WT-water controls. In conclusion, this study shows that 3TSR treatment is effective in attenuating the inflammatory response to DSS injury. In addition, the transcriptomics work unveils novel genetic data that suggest beneficial application of the TSR domains in inflammatory bowel disease
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Thrombospondin-1 in a Murine Model of Colorectal Carcinogenesis
Colorectal Cancer (CRC) is one of the late complications observed in patients suffering from inflammatory bowel diseases (IBD). Carcinogenesis is promoted by persistent chronic inflammation occurring in IBD. Understanding the mechanisms involved is essential in order to ameliorate inflammation and prevent CRC. Thrombospondin 1 (TSP-1) is a multidomain glycoprotein with important roles in angiogenesis. The effects of TSP-1 in colonic tumor formation and growth were analyzed in a model of inflammation-induced carcinogenesis. WT and TSP-1 deficient mice (TSP-1-/-) of the C57BL/6 strain received a single injection of azoxymethane (AOM) and multiple cycles of dextran sodium sulfate (DSS) to induce chronic inflammation-related cancers. Proliferation and angiogenesis were histologically analyzed in tumors. The intestinal transcriptome was also analyzed using a gene microarray approach. When the area containing tumors was compared with the entire colonic area of each mouse, the tumor burden was decreased in AOM/DSS-treated TSP-1-/- versus wild type (WT) mice. However, these lesions displayed more angiogenesis and proliferation rates when compared with the WT tumors. AOM-DSS treatment of TSP-1-/- mice resulted in significant deregulation of genes involved in transcription, canonical Wnt signaling, transport, defense response, regulation of epithelial cell proliferation and metabolism. Microarray analyses of these tumors showed down-regulation of 18 microRNAs in TSP-1-/- tumors. These results contribute new insights on the controversial role of TSP-1 in cancer and offer a better understanding of the genetics and pathogenesis of CRC
Microarray analysis in B cells among siblings with/without MS - role for transcription factor TCF2
Gene expression during normal and FSHD myogenesis
<p>Abstract</p> <p>Background</p> <p>Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, <it>DUX4</it>, that can encode a protein containing two homeodomains. A <it>DUX4 </it>transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how.</p> <p>Methods</p> <p>Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods.</p> <p>Results</p> <p>Many of the ~17,000 examined genes were differentially expressed (> 2-fold, <it>p </it>< 0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis-specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked <it>DUX4 </it>RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types.</p> <p>Conclusions</p> <p><it>DUX4</it>'s pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated <it>DUX4 </it>expression at the myoblast or myotube stages. Our model could explain why <it>DUX4</it>'s inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.</p
Multisubstrate analog inhibitors of glucosamine synthase as potential antifungal agents.
Inhibition of the chitin biosynthetic enzyme, glucosamine synthase (EC.2.6.1.16, L-glutamine:D-Fructose-6-phosphate amidotransferase) has been shown to be a promising approach to the development of antifungal agents targeting the cell wall. The best known inhibitors of glucosamine synthase possess epoxy-ketones and {dollar}\\alpha,\\beta{dollar} unsaturated carbonyls. In vivo, these inherently reactive moieties are easily deactivated by nucleophiles like glutathione. Hence we decided to develop inhibitors lacking such highly reactive groups. Development of multisubstrate analogs is a logical approach to inhibition of multisubstrate enzymes. The compounds investigated in our study possess structural modifications of both glutamine and fructose-6-phosphate so as to bind to both binding sites on the enzyme leading to a potent inhibition of the enzyme. IC{dollar}\\sb{lcub}50{rcub}{dollar} data indicated AP3 to be a potent inhibitor of glucosamine synthase from Candida albicans. The correlation between the distance from the {dollar}\\alpha{dollar}-carbon of the amino acid to the phosphonate phosphorus indicates that shorter chain lengths provide better inhibitors. CGS19755, a structural analog of AP5 showed a higher potency than AP4, indicating that the rigidity and hydrophobicity enhances binding to this site. Our data indicates that the distance between the phosphate binding site and the amino acid binding site is {dollar}\\rm{lcub}\\approx{rcub}3\\ A\\sp\\circ{dollar}. However AP3 does not inhibit the C. albicans enzyme when a protease inhibitor like PMSF was used in the isolation procedure. One explanation for this observation could be that proteases released during previous isolation procedure cleave some portion on the enzyme allowing AP3 to access the active site and inhibit the enzyme. In the presence of PMSF, this is prevented and AP3 cannot reach the active site. AP3 did not inhibit glucosamine synthase isolated from S. cereviseae with or without using PMSF. This reinforces existing evidence that significant differences exist between the enzyme protein from S. cereviseae and C. albicans. The unstable nature of the partially purified enzyme extract however did not allow us to determine a K{dollar}\\rm\\sb{lcub}i\\ app{rcub}{dollar} for these compounds. Hence we cannot state whether the inhibition is multisubstrate or selectively competitive with respect to either substrate. Repeating this study with the homogeneously purified enzyme obtainable from recently developed clones would clarify these areas
Microarray analysis in B cells among siblings with/without MS - role for transcription factor TCF2
Abstract Background We investigated if global gene expression and transcription networks in B-lymphocytes of siblings with multiple sclerosis (MS) were different from healthy siblings. Results Using virus-transformed immortalized B cells and human whole genome bioarrays with validation using RT-qPCR, we found that in siblings with MS, genes for CXCL10, serpin B1 and FUT4 were up regulated whereas CDC5L, TNFRSF19 and HLA-DR were down regulated, among others; transcription analysis showed two intersecting clusters of transcriptional factors - the larger, governed by the upregulated transcription factor 2 (TCF2) and the smaller network regulated by the downregulated CDC5L. Conclusion No study has linked TCF2 to MS and to better understand the role of TCF2 in MS, studies in larger cohorts are required.</p
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