Proteasome‐mediated regulation of GATA2 expression and androgen receptor transcription in benign prostate epithelial cells

GATA2 has been shown to be an important transcription factor together with androgen receptor (AR) in prostate cancer cells. Less is known about GATA2 in benign prostate epithelial cells. We have investigated if GATA2 exogenous expression in prostate epithelial basal-like cells could induce AR transcription or luminal differentiation. Prostate epithelial basal-like (transit amplifying) cells were transduced with lentiviral vector expressing GATA2. Luminal differentiation markers were assessed by RT-qPCR, Western blot and global gene expression microarrays. We utilized our previously established AR and androgen-dependent fluorescence reporter assay to investigate AR activity at the single-cell level. Exogenous GATA2 protein was rapidly and proteasome-dependently degraded. GATA2 protein expression was rescued by the proteasome inhibitor MG132 and partly by mutating the target site of the E3 ligase FBXW7. Moreover, MG132-mediated proteasome inhibition induced AR mRNA and additional luminal marker gene transcription in the prostate transit amplifying cells. Different types of intrinsic mechanisms restricted GATA2 expression in the transit amplifying cells. The appearance of AR mRNA and additional luminal marker gene expression changes following proteasome inhibition suggests control of essential cofactor(s) of AR mRNA expression and luminal differentiation at this proteolytic level.publishedVersio

Tanshinones induce tumor cell apoptosis via directly targeting FHIT

Abstract The liposoluble tanshinones are bioactive components in Salvia miltiorrhiza and are widely investigated as anti-cancer agents, while the molecular mechanism is to be clarified. In the present study, we identified that the human fragile histidine triad (FHIT) protein is a direct binding protein of sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of Tanshinone IIA (TSA), with a Kd value of 268.4 ± 42.59 nM. We also found that STS inhibited the diadenosine triphosphate (Ap3A) hydrolase activity of FHIT through competing for the substrate-binding site with an IC50 value of 2.2 ± 0.05 µM. Notably, near 100 times lower binding affinities were determined between STS and other HIT proteins, including GALT, DCPS, and phosphodiesterase ENPP1, while no direct binding was detected with HINT1. Moreover, TSA, Tanshinone I (TanI), and Cryptotanshinone (CST) exhibited similar inhibitory activity as STS. Finally, we demonstrated that depletion of FHIT significantly blocked TSA’s pro-apoptotic function in colorectal cancer HCT116 cells. Taken together, our study sheds new light on the molecular basis of the anti-cancer effects of the tanshinone compounds

Proteasome‐mediated regulation of GATA2 expression and androgen receptor transcription in benign prostate epithelial cells

GATA2 has been shown to be an important transcription factor together with androgen receptor (AR) in prostate cancer cells. Less is known about GATA2 in benign prostate epithelial cells. We have investigated if GATA2 exogenous expression in prostate epithelial basal-like cells could induce AR transcription or luminal differentiation. Prostate epithelial basal-like (transit amplifying) cells were transduced with lentiviral vector expressing GATA2. Luminal differentiation markers were assessed by RT-qPCR, Western blot and global gene expression microarrays. We utilized our previously established AR and androgen-dependent fluorescence reporter assay to investigate AR activity at the single-cell level. Exogenous GATA2 protein was rapidly and proteasome-dependently degraded. GATA2 protein expression was rescued by the proteasome inhibitor MG132 and partly by mutating the target site of the E3 ligase FBXW7. Moreover, MG132-mediated proteasome inhibition induced AR mRNA and additional luminal marker gene transcription in the prostate transit amplifying cells. Different types of intrinsic mechanisms restricted GATA2 expression in the transit amplifying cells. The appearance of AR mRNA and additional luminal marker gene expression changes following proteasome inhibition suggests control of essential cofactor(s) of AR mRNA expression and luminal differentiation at this proteolytic level

An integrated meta-omics approach reveals the different response mechanisms of two anammox bacteria towards fluoroquinolone antibiotics

The emerging fluoroquinolone antibiotics (FQs) are highly influential in nitrogen removal from livestock wastewater. However, beyond the capability of nitrogen removal, little is known about the molecular mechanisms (e.g., shift of core metabolism and energy allocation) of different anaerobic ammonium-oxidizing bacteria (AnAOB) under continuous FQ stress. This study investigated the effects of ciprofloxacin, ofloxacin and their mixture at concentrations detected in livestock wastewater on two key anammox species in membrane bioreactors. It was found 20 μg/L FQs promoted nitrogen removal efficiency and community stability, and42–51 % of FQs were removed simultaneously. Integrated meta-omics analysis revealed varied gene expression patterns between the two dominant AnAOB, Candidatus Brocadia sapporoensis (B AnAOB) and Candidatus Kuenenia stuttgartiensis (K AnAOB). The nitrogen metabolic processes were bolstered in B AnAOB, while those involved in anammox pathway of K AnAOB were inhibited. This difference was tentatively attributed to the up-regulation of reactive oxygen species scavenger genes (ccp and dxf) and FQ resistance gene (qnrB72) in B AnAOB. Importantly, most enhanced core biosynthesis/metabolism of AnAOB and close cross-feeding with accompanying bacteria were also likely to contribute to their higher levels of biomass yield and metabolism activity under FQ stress. This finding suggests that B AnAOB has the advantage of higher nitrogen metabolism capacity over K AnAOB in livestock wastewater containing FQs, which is helpful for efficient and stable nitrogen removal by the functional anammox species

Novel STAT3 Inhibitors Targeting STAT3 Dimerization by Binding to the STAT3 SH2 Domain

Our drug discovery model has identified two novel STAT3 SH2 domain inhibitors 323–1 and 323–2 (delavatine A stereoisomers) in a series of experiments. In silico computational modeling, drug affinity responsive target stability (DARTS), and fluorescence polarization (FP) assays altogether determined that 323–1 and 323–2 directly target the STAT3 SH2 domain and inhibited both phosphorylated and non-phosphorylated STAT3 dimerization. Computational docking predicted that compound 323s bind to three subpockets of the STAT3 SH2 domain. The 323s inhibition of STAT3 dimerization was more potent than the commercial STAT3 SH2 domain inhibitor S3I-201 in the co-immunoprecipitation assay, correlating with computational docking data. The fluorescence polarization assay further confirmed that the compound 323s target the STAT3 SH2 domain by competitively abrogating the interaction between STAT3 and the SH2-binding peptide GpYLPQTV. Compared with S3I-201, the 323 compounds exhibited stronger inhibition of STAT3 and reduced the level of IL-6-stimulated phosphorylation of STAT3 (Tyr705) in LNCaP cells over the phosphorylation of STAT1 (Tyr701) induced by IFN-ɣ in PC3 cells or the phosphorylation of STAT1 (Ser727) in DU145 cells. Both compounds downregulated STAT3 target genes MCL1 and cyclin D1. Thus, the two compounds are promising lead compounds for the treatment of cancers with hyper-activated STAT3.publishedVersio

An androgen response element driven reporter assay for the detection of androgen receptor activity in prostate cells

<div><p>The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer, as is evident from the efficacy of androgen-deprivation therapy, AR is also the most frequently mutated gene, in castration resistant prostate cancer (CRPC). AR has therefore become an even more attractive therapeutic target in aggressive and disseminated prostate cancer. To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, a toolkit of AR expressor and androgen response element (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence assays. Interestingly, the background reporter activity in androgen-depleted medium was significantly higher in LNCaP cells compared to the prostate transit amplifying epithelial cell lines, EP156T-AR and 957E/hTERT-AR with exogenous AR. The androgen-induced signal to background was much higher in the latter benign prostate cells than in LNCaP cells. Androgen-independent nuclear localization of AR was seen in LNCaP cells and reduced ARE-signaling was seen following treatment with abiraterone, an androgen synthesis inhibitor. The ARE reporter activity was significantly stronger when stimulated by androgens than by β-estradiol, progesterone and dexamethasone in all tested cell types. Finally, no androgen-induced ARE reporter activity was observed in tumorigenic mesenchymal progeny cells of EP156T cells following epithelial to mesenchymal transition. This underscores the observation that expression of the classical luminal differentiation transcriptome is restricted in mesenchymal type cells with or without AR expression, and presence of androgen.</p></div

ARE promoter response and sensitivity to AR activity in 293FT cells.

<p>(A) Three different ARE promoter sequences, 241B, 248B and 249B inserted in a Gaussia luciferase reporter vector, were co-transfected with AR expression vector pLenti6.3/AR-GC-E2325 into 293FT cells. Cells were treated with 1 nM R1881 for 24 hours and Gaussia luciferase values were detected. These values were normalized by the MTS assay. (B) The 241B ARE promoter mCherry reporter vector was co-transfected with AR expression vector pLenti6.3/AR-GC-E2325 in 293FT cells. Fluorescence microscopy was performed after stimulating the cells with 1 nM R1881 for 24 hours. The error bars show the standard error of the mean (SEM) from three independent experiments. Significance was confirmed by using unpaired two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.</p

Comparison of different AR expression vectors in 293FT cells.

<p>293FT cells were co-transfected with ARE reporter CCS-1019L and three separate AR expression vectors. The cells were grown with or without synthetic androgen 1 nM R1881 for 24 hours. The AR expression was detected by measuring Firefly luminescent signals. These values were normalized by Renilla luminescence values. The error bars show the standard error of the mean (SEM) from three independent experiments. Significance was confirmed by using unpaired two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.</p

Internal normalization of ARE reporter signals in prostate cells.

<p>(A) FACS analysis of mCherry fluorescent signals in LNCaP-207-01 and LNCaP-207-02 cells grown in androgen free medium. Cells were treated with different concentrations of androgen R1881, <i>i</i>.<i>e</i>. 0.1 nM, 1 nM and 10 nM, and 10 μM enzalutamide (enza) with 1 nM R1881 for 24 hours. The values were normalized by constitutively expressed GFP fluorescent signals. (B) Multi-well mCherry RFU measurements of LNCaP-207-01 and LNCaP-207-02 cells grown in androgen free medium. The cells were treated with different concentrations of androgen R1881, <i>i</i>.<i>e</i>. 0.1 nM, 1 nM and 10 nM, and 10 μM enzalutamide with 1 nM R1881 for 24 hours. The mCherry RFU values were normalized by GFP RFU. (C) Fluorescence microscopy of LNCaP-207-01 cells treated with ± 1 nM R1881 and different concentrations of enzalutamide, <i>i</i>.<i>e</i>. 5 μM, 10 μM and 20 μM for 24 hours. The error bars show the standard error of the mean (SEM) from three independent experiments. Significance was confirmed by using unpaired two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.</p