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Transcriptomic effect of Anethole trithione (ATT) on human BPH-1 cells

ABSTRACT: The goal of this experiment was to evaluate the effect of antioxidant treatment on the transcriptome of epithelial cells from human benign prostate hyperplasia (BPH). The BPH-1 cell line has been immortalized from a 68-year-old BPH patient using SV-40 T-antigen (Hayward et al 1995, In Vitro Cell Dev Biol Anim 31, 14-24). BPH-1 cells were purchased in 2019 at DSMZ-German collection (ACC 143, reference 1402) and maintained in DMEM medium (Gibco, GlutamaxTM, 4.5g/L D-Glucose + Pyruvate, reference 31966-0.21) with 10% FBS (Eurobio, CVFSVF00-01). Anethole trithione (ATT) is an antioxidant molecule acting as a specific inhibitor of reactive oxygen species (ROS) production at the Iq site of the mitochondrial respiratory chain (Detaille et al, PLoS One 14, e0216385). ATT was initially resuspended at 10 mM in Dimethyl Sulfoxide (DMSO, Sigma D4540-500ML) then diluted in culture medium. For this experiment, 2 million BPH-1 cells were seeded in 100mm Petri dish. Media was replaced 24h after seeding, then fresh medium containing 10 µM ATT or vehicle (same dilution of DMSO without ATT) was added for 72h. Cells were trypsinized and centrifuged to dry pellets were stored at -20°C. RNA was extracted using NucleoSpin (Macherey Nagel) according to the manufacturer’s protocol and quantified using Nanodrop 2000 (Thermo Scientific). RNA quality was checked with Bioanalyser 2100 (Agilent), then 5µL RNA at a concentration of 120ng/µl were used for library generation. Clariom_S_Human arrays were used on the GeneChip 3000 7G scanner (Thermo Fisher Scientific). Results were processed with Transcriptome Analysis Console TAC4.0 (Thermo Fisher Scientific) and normalized with summarization methods RMA.

ORGANISM(S): Homo sapiens (human)

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PROVIDER: S-BSST1129 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:BackgroundBenign prostatic hyperplasia (BPH) is a common, chronic progressive disease. Inflammation is associated with prostatic enlargement and resistance to 5α-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-κB) pathway is linked to both inflammation and ligand-independent prostate cancer progression.MethodsNF-κB activation and androgen receptor variant (AR-V) expression were quantified in transition zone tissue samples from patients with a wide range of AUASS from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-κB activity. The effects on AR full length (AR-FL) and androgen-independent AR-V expression as well as cellular growth and differentiation were assessed.ResultsCanonical NF-κB signaling was found to be upregulated in late versus early stage BPH, and to be strongly associated with non-insulin dependent diabetes mellitus. Elevated expression of AR-variant 7 (AR-V7), but not other AR variants, was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS and TRUS volume. Forced activation of canonical NF-κB in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-κB and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment.ConclusionActivation of NF-κB and AR-V7 in the prostate is associated with increased disease severity. AR-V7 expression is inducible in human prostate cells by forced activation of NF-κB resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy.

Project description:Transcriptomic studies hold great potential towards understanding the human aging process. Previous transcriptomic studies have identified many genes with age-associated expression levels; however, small samples sizes and mixed cell types often make these results difficult to interpret.Using transcriptomic profiles in CD14+ monocytes from 1,264 participants of the Multi-Ethnic Study of Atherosclerosis (aged 55-94 years), we identified 2,704 genes differentially expressed with chronological age (false discovery rate, FDR???0.001). We further identified six networks of co-expressed genes that included prominent genes from three pathways: protein synthesis (particularly mitochondrial ribosomal genes), oxidative phosphorylation, and autophagy, with expression patterns suggesting these pathways decline with age. Expression of several chromatin remodeler and transcriptional modifier genes strongly correlated with expression of oxidative phosphorylation and ribosomal protein synthesis genes. 17% of genes with age-associated expression harbored CpG sites whose degree of methylation significantly mediated the relationship between age and gene expression (p?<?0.05). Lastly, 15 genes with age-associated expression were also associated (FDR???0.01) with pulse pressure independent of chronological age. Comparing transcriptomic profiles of CD14+ monocytes to CD4+ T cells from a subset (n?=?423) of the population, we identified 30 age-associated (FDR?<?0.01) genes in common, while larger sets of differentially expressed genes were unique to either T cells (188 genes) or monocytes (383 genes). At the pathway level, a decline in ribosomal protein synthesis machinery gene expression with age was detectable in both cell types.An overall decline in expression of ribosomal protein synthesis genes with age was detected in CD14+ monocytes and CD4+ T cells, demonstrating that some patterns of aging are likely shared between different cell types. Our findings also support cell-specific effects of age on gene expression, illustrating the importance of using purified cell samples for future transcriptomic studies. Longitudinal work is required to establish the relationship between identified age-associated genes/pathways and aging-related diseases.

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Transcriptomic effect of Anethole trithione (ATT) on human BPH-1 cells

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