A Far-Red-Emitting Fluorescence Probe for Sensitive and Selective Detection of Peroxynitrite in Live Cells and Tissues

In this study, the far-red-emitting fluorescence probe <b>1</b>, containing a rhodamine derivative and a hydrazide reactive group, was developed for peroxynitrite detection and imaging. This probe, which is cell permeable and shows high sensitivity and selectivity in fluorometric detection of peroxynitrite over other ROS/RNS, was successfully utilized to detect exogenous and endogenous peroxynitrite in HeLa and RAW 264.7 cells, respectively. More importantly, <b>1</b> can also be used to detect endogenous peroxynitrite generated in <i>Pseudomonas aeruginosa</i> (PAO1)-infected mouse bone marrow-derived neutrophils. We anticipate that the new probe will serve as a powerful molecular imaging tool in investigations of the role(s) played by peroxynitrite in a variety of physiological and pathological contexts

Insulin Protects Cardiac Myocytes from Doxorubicin Toxicity by Sp1-Mediated Transactivation of Survivin

<div><p>Insulin inhibits ischemia/reperfusion-induced myocardial apoptosis through the PI3K/Akt/mTOR pathway. Survivin is a key regulator of anti-apoptosis against doxorubicin-induced cardiotoxicity. Insulin increases survivin expression in cardiac myocytes to mediate cytoprotection. However, the mechanism by which survivin mediates the protective effect of insulin against doxorubicin-associated injury remains to be determined. In this study, we demonstrated that pretreatment of H9c2 cardiac myocytes with insulin resulted in a significant decrease in doxorubicin-induced apoptotic cell death by reducing cytochrome c release and caspase-3 activation. Doxorubicin-induced reduction of survivin mRNA and protein levels was also significantly perturbed by insulin pretreatment. Reducing survivin expression with survivin siRNA abrogated insulin-mediated inhibition of caspase-3 activation, suggesting that insulin signals to survivin inhibited caspase-3 activation. Interestingly, pretreatment of H9c2 cells with insulin or MG132, a proteasome inhibitor, inhibited doxorubicin-induced degradation of the transcription factor Sp1. ChIP assay showed that pretreatment with insulin inhibited doxorubicin-stimulated Sp1 dissociation from the <i>survivin</i> promoter. Finally using pharmacological inhibitors of the PI3K pathway, we showed that insulin-mediated activation of the PI3K/Akt/mTORC1 pathway prevented doxorubicin-induced proteasome-mediated degradation of Sp1. Taken together, insulin pretreatment confers a protective effect against doxorubicin-induced cardiotoxicity by promoting Sp1-mediated transactivation of survivin to inhibit apoptosis. Our study is the first to define a role for survivin in cellular protection by insulin against doxorubicin-associated injury and show that Sp1 is a critical factor in the transcriptional regulation of survivin.</p></div

Effect of insulin on the doxorubicin-induced changes in Sp1 and p53.

<p>(A, D) H9c2 cardiac myocytes were left untreated or treated with the indicated concentration of doxorubicin (<i>Doxo</i>) for 12 h, and (B, C, E) serum-deprived cells were left untreated or pretreated with insulin (200 nM) for 1 h prior to treatment with doxorubicin (1 μM) for 12 h. Whole cell lysates were analyzed by immunoblotting with anti-Sp1, anti-phospho-p53 (Ser¹⁵), anti-p53 and anti-GAPDH antibodies. Graphs represent the mean ±S.D. of the normalized densitometric analyses of Sp1 protein levels (A, **<i>p</i> < 0.01, n = 3; B, ***<i>p</i> < 0.001, n = 5). (D, E) Sp1 mRNA amount was determined by RT-PCR (28 cycles). Note that the blots in <i>panel C</i> represent one of three independent experiments.</p

Doxorubicin-induced Sp1 degradation by proteasome-mediated proteolysis.

<p>(A–C) H9c2 cardiac myocytes were pretreated with proteasome inhibitor MG132 (50 nM) for 1 h and treated with 1 μM doxorubicin (<i>Doxo</i>) for either 12 h (A) or 24 h (B, C). (A, B) Whole cell lysates were analyzed by immunoblotting using anti-Sp1 and anti-survivin antibodies. Graphs represent the mean ±S.D. of the normalized densitometric analyses of Sp1 (A, n = 5) or survivin (B, n = 5) protein levels (***<i>p</i> < 0.001). (C) Total RNA was analyzed by RT-PCR (28 cycles) with specific primers to <i>survivin</i> gene. Graph represents the mean ±S.D. of the normalized densitometric analysis of <i>survivin</i> mRNA levels (**<i>p</i> < 0.01; ***<i>p</i> < 0.001, n = 3).</p

Inhibitory effect of insulin on the doxorubicin-induced Sp1 degradation via PI3K/Akt/mTORC1 pathway in H9c2 cardiac myocytes.

<p>(A) Serum-deprived H9c2 cardiac myocytes were left untreated or pretreated with 200 nM insulin (<i>Ins</i>) for 1 h and treated with 1 μM doxorubicin (<i>Doxo</i>) for 12 h. (B–D) Cells were pretreated with 2 μM PI3K inhibitor LY294002 (<i>LY</i>) (B), 1 μM mTORC1 inhibitor rapamycin (<i>Rapa</i>) (C), or 5 μM p70S6K inhibitor PF4708671 (<i>PF</i>). (D) for 1 h, followed by treatment with insulin (200 nM) for 1 h and then with doxorubicin (1 μM) for 12 h. Whole cell lysates were analyzed by immunoblotting for protein levels or phosphorylation status of Akt, mTORC1 and p70S6K and for protein levels of Sp1 using antibodies listed in Materials and Methods. Note that these blots represent one of three independent experiments. Graphs represent the mean ±S.D. of the normalized densitometric analyses of Sp1 protein levels (*<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001, n = 3).</p

Effect of doxorubicin and/or insulin on the transcriptional activity of Sp1 and p53.

<p>(A) Multiple sequence alignment (ClustalW2) of the proximal promoter regions of the rat, mouse and human <i>survivin</i> genes. Black arrow indicates the transcriptional start site and +1 points out the translation start codon. Canonical p53, Sp1, Sp1-like sites of the mouse and human <i>survivin</i> genes are boxed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref012" target="_blank">12</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref038" target="_blank">38</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref040" target="_blank">40</a>]. Two gray bars indicate the primers for ChIP analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref031" target="_blank">31</a>]; a red bar corresponds to human CHR sequences; two blue bars highlight human CDE regions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135438#pone.0135438.ref050" target="_blank">50</a>]. (B) Serum-deprived cells were left untreated or pretreated with 200 nM insulin (<i>Ins</i>) for 1 h and treated with 1 μM doxorubicin (<i>Doxo</i>) for 12 h. Cross-linked cell lysates were subjected to ChIP analysis with anti-Sp1 or anti p53-antibody. RT-PCR (36 cycles) was performed with ChIP primers as listed in Materials and Methods (n = 5). (C–E) One day after transfection with Sp1 siRNA (20 nM), cells were left untreated or pretreated with insulin (200 nM) for 1 h and treated with doxorubicin (1 μM) for 24 h. Whole cell lysates were immunoblotted with anti-Sp1, anti-survivin and anti-caspase-3 (active form) antibodies (C, D), and total RNA was analyzed by RT-PCR (28 cycles) with specific primers to <i>survivin</i> gene (E). Note that these results represent one of three independent experiments.</p

Inhibitory effect of insulin on the doxorubicin-induced survivin down-regulation.

<p>(A, B) Serum-deprived H9c2 cardiac myocytes were left untreated or pretreated with 200 nM insulin (<i>Ins</i>) for 1 h and treated with 1 μM doxorubicin (<i>Doxo</i>) for 24 h. (A) Whole cell lysates were separated by SDS-PAGE gel and analyzed by immunoblotting with anti-survivin antibody. Graph represents the mean ±S.D. of the normalized densitometric analysis of survivin protein levels from 8 independent experiments (***<i>p</i> < 0.001, n = 8). (B) Total RNA was analyzed by RT-PCR (28 cycles) using primers specific to <i>survivin</i> gene. Graph represents the mean ±S.D. of the normalized densitometric analysis of <i>survivin</i> mRNA levels from 3 independent experiments (***<i>p</i> < 0.001, n = 3). (C) Immunofluorescence microscopy images were obtained using anti-survivin antibody followed by staining with DAPI (200×). (D, E) One day after transfection with either scrambled RNA (scRNA) or survivin siRNA (20 nM), H9c2 cardiac myocytes were left untreated or pretreated with insulin (200 nM) for 1 h and treated with doxorubicin (1 μM) for 24 h. Whole cell lysates were blotted with antibodies against survivin and cleaved-caspase-3 (active form). Note that the blots in <i>panel D</i> and <i>E</i> represent one of three independent experiments. <i>n</i>.<i>s</i>. not significant.</p

Protective effect of insulin on the doxorubicin-induced cell death in H9c2 cardiac myocytes.

<p>(A) H9c2 cardiac myocytes were left untreated or treated with 1 μM doxorubicin (<i>Doxo</i>) for 24 h, 48 h, and 72 h. (B) Serum-deprived H9c2 cardiac myocytes were left untreated or pretreated with the indicated concentration of insulin (<i>Ins</i>) for 1 h prior to treatment with 1 μM doxorubicin (<i>Doxo</i>) for 24 h. Cell viability was assessed by the MTT assay (**<i>p</i> < 0.01; ***<i>p</i> < 0.001, n = 3 performed in triplicates). (C–F) Serum-deprived cells were left untreated or pretreated with insulin (200 nM) for 1 h prior to treatment with doxorubicin (1 μM) for 24 h. (C) Represented images of the TUNEL assay (100×). Treated cells were incubated with TUNEL reaction mixture, followed by staining with DAPI. (D) The TUNEL-stained cells were counted under fluorescence microscopy and presented as a bar graph (***<i>p</i> < 0.001, n = 3 performed in triplicates). (E) Caspase-3 activity was determined by immunoblot analysis of active form of caspase-3. (F) Mitochondrial (<i>Mito</i>) and cytosolic (<i>Cyto</i>) fractions were separated by SDS-PAGE gel and analyzed by immunoblotting with anti-cytochrome C (<i>Cyt C</i>) antibodies. GAPDH or β-actin bands show that equal amounts of sample were loaded and VDAC1 is used as a loading control for mitochondrial fraction. Note that blots represent one of three independent experiments. Values are mean ±S.D.</p