Synthesis of New Hydrophilic and Hydrophobic Cobinamides as NO-Independent sGC Activators

Herein, the synthesis of novel hydrophobic and hydrophilic cobinamides via aminolysis of vitamin B₁₂ derivatives that activate soluble guanyl cyclase (sGC) is presented. Unlike other sGC regulators, they target the catalytic domain of sGC and show higher activity than (CN)₂Cbi

PTBP1 response to H₂O₂-induced degradation varies in different breast cancer cell lines.

<p><b>A</b>: Western blot analysis examining PTBP1 expression in MDA231, MDA453, MDA468 and MCF7 cells treated with 1 mM H₂O₂ for 18 hours. Representative biological duplicates are shown. <b>B</b>: MDA453 cells are resistant to H₂O₂-induced PTBP1 degradation. <i>Top panel</i>: MDA453 cell were treated with different concentration of H₂O₂ and cell lysates were subjected to Western blot analysis with antibodies towards PTBP1 and β-actin. Shown blots are representative of four independent experiments with similar results. <i>Bottom panel</i>: densitometry analysis of PTBP1 protein levels normalized on β-actin levels. Averages for representative biological duplicates for each treatment are shown.</p

Insights into the mechanism of H₂O₂-induced PTBP1 down-regulation.

<p><b>A</b>: PTBP1 degradation occurs in time-dependent manner and is not prevented by proteasome inhibitor MG132. MDA468 cells were treated as in Fig. 1C with 1 mM H₂O₂ in the presence or absence of MG132 (10 µM). Western blot analysis was performed to visualize the expression of PTBP1. β-actin served as a loading control. Biological duplicates for each treatment are shown; blots are representative of three independent experiments with similar results. <b>B</b>: Densitometry analysis of PTBP1 protein levels normalized on β-actin. Averages for representative biological duplicates for each treatment are shown. <b>C</b>: H₂O₂ exposure does not affect PTBP1 mRNA levels. MDA468 cells were treated with indicated concentrations of H₂O₂ for 24 hours. Relative abundance of PTBP1 mRNA in samples was analyzed by RT-qPCR analysis. Average ΔCt ± SD for biological triplicates are shown. <b>D</b>: PTBP1 degradation depends on thiol oxidation and is not rescued by inhibition of <i>de novo</i> protein synthesis. Western blot analysis performed on MDA468 cell lysates treated for 24 hours with inhibitor of protein synthesis cycloxemide (2 µg/ml) and different factors inducing oxidative stress: 1 mM BSO (GSH depletion inducer); 1 mM HEDS (thiol oxidation inducer) and 0.01 units/ml of Glucose Oxidase (increases production of ROS). Shown Western blots are representative of three independent experiments with similar results.</p

H₂O₂ cytotoxicity analysis in MDA468 and MDA453 cells.

<p>Survival curve was generated in response to H₂O₂ dosage using trypan exclusion method and expressed as % of survival to untreated controls. Mean ± SD of three independent passages performed in triplicates are shown, *- p<0.05 by Student's t-test in comparison to control.</p

Hydrogen Peroxide Alters Splicing of Soluble Guanylyl Cyclase and Selectively Modulates Expression of Splicing Regulators in Human Cancer Cells

<div><h3>Background</h3><p>Soluble guanylyl cyclase (sGC) plays a central role in nitric oxide (NO)-mediated signal transduction in the cardiovascular, nervous and gastrointestinal systems. Alternative RNA splicing has emerged as a potential mechanism to modulate sGC expression and activity. C-α1 sGC is an alternative splice form that is resistant to oxidation-induced protein degradation and demonstrates preferential subcellular distribution to the oxidized environment of endoplasmic reticulum (ER).</p> <h3>Methodology/Principal Findings</h3><p>Here we report that splicing of C-α1 sGC can be modulated by H₂O₂ treatment in BE2 neuroblastoma and MDA-MD-468 adenocarcinoma human cells. In addition, we show that the H₂O₂ treatment of MDA-MD-468 cells selectively decreases protein levels of PTBP1 and hnRNP A2/B1 splice factors identified as potential α1 gene splicing regulators by <em>in silico</em> analysis. We further demonstrate that down-regulation of PTBP1 by H₂O₂ occurs at the protein level with variable regulation observed in different breast cancer cells.</p> <h3>Conclusions/Significance</h3><p>Our data demonstrate that H₂O₂ regulates RNA splicing to induce expression of the oxidation-resistant C-α1 sGC subunit. We also report that H₂O₂ treatment selectively alters the expression of key splicing regulators. This process might play an important role in regulation of cellular adaptation to conditions of oxidative stress.</p> </div

H₂O₂ exposure induces the expression of oxidation-resistant C-α1 sGC splice form in MDA468 and BE2 cells. A

<p>: RT-PCR detection of α1 (top) and C-α1 (bottom band) sGC transcripts following treatment with H₂O₂ (1 mM) or ODQ (20 µM), as indicated. RT-PCR products are separated on 3% agarose gel and stained with Ethidium Bromide. Upper band represents the message encoding canonical α1 protein (Transcripts 1–4, 270 bp); middle band is a non-specific product; bottom band indicates the C-α1 sGC transcript (Transcript 5, 94 bp). Biological triplicates representative of three independent experiments are shown. <b>B</b>: Ratio (mean ± SD) of the relative abundance of C-α1 and α1 transcripts quantified by densitometry. *p<0.05 by Student's t-test. <b>C</b>: Western blot detection of α1 (top) and C-α1 (bottom) proteins. MDA468 cells were treated with 1 mM H₂O₂ as indicated in presence or absence of MG132 (10 µM). Shown blots are representative of four independent experiments with similar results. <b>D</b>: Ratio of the relative abundance of C-α1 and α1 proteins quantified by densitometry. Data are shown as mean ± SD from four independent experiments.</p