Pentabromopseudilin: a myosin V inhibitor suppresses TGF-<b>β</b> activity by recruiting the type II TGF-<b>β</b> receptor to lysosomal degradation

<p>Pentabromopseudilin (PBrP) is a marine antibiotic isolated from the marine bacteria <i>Pseudomonas bromoutilis</i> and <i>Alteromonas luteoviolaceus</i>. PBrP exhibits antimicrobial, anti-tumour, and phytotoxic activities. In mammalian cells, PBrP is known to act as a reversible and allosteric inhibitor of myosin Va (MyoVa). In this study, we report that PBrP is a potent inhibitor of transforming growth factor-β (TGF-β) activity. PBrP inhibits TGF-β-stimulated Smad2/3 phosphorylation, plasminogen activator inhibitor-1 (PAI-1) protein production and blocks TGF-β-induced epithelial–mesenchymal transition in epithelial cells. PBrP inhibits TGF-β signalling by reducing the cell-surface expression of type II TGF-β receptor (TβRII) and promotes receptor degradation. Gene silencing approaches suggest that MyoVa plays a crucial role in PBrP-induced TβRII turnover and the subsequent reduction of TGF-β signalling. Because, TGF-β signalling is crucial in the regulation of diverse pathophysiological processes such as tissue fibrosis and cancer development, PBrP should be further explored for its therapeutic role in treating fibrotic diseases and cancer.</p

Cholest-4-en-3-one attenuates TGF-β responsiveness by inducing TGF-β receptors degradation in Mv1Lu cells and colorectal adenocarcinoma cells

<p><b>Purpose:</b> The transforming growth factor-beta (TGF-β) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-β signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) <i>in vitro</i>.</p> <p><b>Methods:</b> The inhibitory effects of cholest-4-en-3-one on TGF-β-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-β receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation.</p> <p><b>Results:</b> Cholest-4-en-3-one attenuated TGF-β signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-β-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-β responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-β receptors and facilitating rapid degradation of TGF-β and thus suppressing TGF-β-induced signaling.</p> <p><b>Conclusions:</b> Our results suggest that cholest-4-en-3-one inhibits TGF-β signaling may be due, in part to the translocation of TGF-β receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-β deficiency.</p

Chemical structures of (A) cholesterol and (B) euphol.

<p>Chemical structures of (A) cholesterol and (B) euphol.</p

Euphol decreased the abundance of TβR-I and TβR-II in Mv1Lu (A) and MKN45 (B) cells after.

<p>Mv1Lu and MKN45 cells were treated with several concentrations of euphol at 37°C for 48 hours, after which the cell lysates were subjected to Western blot analysis using anti-TβR-I, anti-TβR-II, TfR, EEA-1, caveolin-1, and anti-β-actin antibodies (A and C), followed by quantification by densitometry (B and D). The ratio of the relative amounts of TβR-I, TβR-II, and β-actin in cells treated without euphol was taken as 100% TGF-β receptor expression. The data are representative of a total of four independent analyses; values are mean ± s.d. significantly lower than control cells: *<i>P</i><0.05 versus control.</p

Euphol inhibits TGF-β-induced fibronectin expression.

<p>(A) AGS cells were treated with TGF-β (100 pM) ± euphol (5–40μg/ml) for 24 hours. Total RNA were isolated and the expressions of fibronectin were determined by RT-PCR. GAPDH was used as a loading control. (B) AGS cells were treated with TGF-β (100 pM) ± euphol for 48 hours. Total protein extracts from treated cells were Western blotted with anti-fibronectin or anti-β-actin monoclonal antibody. β-actin was used as a loading control; results were quantified by densitometry showing in lower panel. Data represent the means ± s.d. of three independent experiments *<i>P</i><0.01 versus TGF-β-induced expression.</p

Immunofluorescent localization of TβR-II and caveolin-1(or flotillin-2) in Mv1Lu cells treated with and without euphol and TGF-β.

<p>Mv1Lu cells that transiently expressed TβR-II-HA were treated with or without 10 μg/ml euphol at 37°C for 1 hour, after which they were incubated with or without 100 pM TGF-β at 37°C for 30 minutes. The cells were then fixed with cold methanol and incubated with mouse anti-HA antibodies (Fig 5A and 5B, a-d), rabbit anti-caveolin-1 antibodies (Fig 5A, e-h), or rabbit anti-flotillin-2 antibodies (Fig 5B, e-h) followed by incubation with rhodamine-conjugated donkey anti-mouse antibodies or FITC-conjugated goat anti-rabbit antibodies. The fluorescence in the cells was measured using confocal fluorescence microscopy. Bar, 20 μm. The white arrows indicate colocalization of TβR-II-HA and caveolin-1 (or flotillin-2) at the cell surface (j) and in endocytic vesicles (l).</p

Inhibition of TGF-β-induced transcriptional activation by euphol.

<p>(A) Mv1Lu cells with stable expression of the PAI-1 luciferase promoter were treated with increasing concentration of euphol. The gray bars in (A) represent the cells without TGF-β treatment. The black bars represent the cells treated with 100 pM TGF-β. (B,C,D) Mv1Lu cells were transfected with CAGA₁₂-Luc, collagen, or a fibronectin luciferase plasmid, and AGS (E) and MKN45 (F) gastric cancer cells were transfected with CAGA₁₂-Luc, Mv1Lu, AGS, and MKN45 cells were treated with TGF-β (100 pM, +β), euphol (Eu), or MβCD (CD). Luciferase activity was measured as described in the methods section. Columns show mean of three independent experiments performed in triplicated; bars indicate s.d.; *<i>P</i><0.05 (compare with TGF-β treatment), **<i>P</i><0.01.</p

Magnesium oxide use and reduced risk of dementia: a retrospective, nationwide cohort study in Taiwan

<p><b>Objective:</b> Dietary magnesium may be associated with a lower risk of dementia; however, the impact of magnesium oxide (MgO), a common laxative, on dementia has yet to be elucidated. This study aimed to investigate the association between the usage of MgO and the risk of developing dementia.</p> <p><b>Methods:</b> We used a dataset from the National Health Research Institute Database (NHRID) of Taiwan containing one million randomly sampled subjects to identify patients aged ≥50 years with no history of MgO usage. A total of 1547 patients who had used MgO were enrolled, along with 4641 controls who had not used the MgO propensity score matched by age, gender and comorbidity, at a ratio of 1:3. After adjusting for confounding risk factors, a Cox proportional hazards model was used to compare the risk of developing dementia during a 10 year follow-up period.</p> <p><b>Results:</b> Of the enrolled patients, 44 (2.84%) developed dementia, when compared to 199 (4.28%) in the control group. The Cox proportional hazards regression analysis revealed that the patients who had used MgO were less likely to develop dementia with a crude hazard ratio of 0.617 (95% CI, 0.445–0.856, <i>p</i> = .004). After adjusting for age, gender, comorbidity, geographical area and urbanization level of residence, and monthly income, the adjusted hazard ratio was 0.517 (95% CI, 0.412–0.793, <i>p</i> = .001).</p> <p><b>Conclusions:</b> The patients who used MgO had a decreased risk of developing dementia. Further studies on the effects of MgO in reducing the risk of dementia are therefore warranted.</p