Detection of STAT3 as S-nitrosylating protein by biotin switch method.

<p><b>A</b>. Detection of biotinylated proteins after biotin-switch method in the presence or absence of S-nitrosoglutathione (GSNO; 0.5 mM). Omission of biotin-HPRT indicates the specificity of S-nitrosylation by biotin-switch method. <b>B</b>. Detection of nitrosylation of various proteins including EGFR, p65, Akt and STAT3 in ovarian cancer cells upon GSNO treatment. <b>C</b>. STAT3 was immunoprecipitated from nitrosylated protein lysate after biotin switch assay and its biotinylation was detected by anti-biotin-HRP using western blot analysis. <b>D</b>. A2780, C200 and SKOV3 cell lines were treated with GSNO (0.5 mM) or oxidized GSNO (0.5 mM) as control for 2 hours followed by immunoblot analysis for detection of tyrosine phosphorylation of STAT3 at 705 residue and total STAT3. <b>E</b>. Under similar experimental conditions as “D”, A2780, C200 and SKOV3 cells were processed for biotin switch method for detection of STAT3. Sample in lane 4 was treated with GSNO as lane 2, except during processing for biotin switch assay; the addition of HPDP was omitted. Upper panel shows the biotinylated proteins after biotin-switch assay. Nitrosylated STAT3 was detected by pulling down biotinylated proteins by streptavidin agarose followed by immunoblot analysis using anti-STAT3 antibody. Lower bands show the total levels of input STAT3 processed for biotin switch assay.</p

Preclinical Therapeutic Potential of a Nitrosylating Agent in the Treatment of Ovarian Cancer

<div><p>This study examines the role of s-nitrosylation in the growth of ovarian cancer using cell culture based and <i>in vivo</i> approaches. Using the nitrosylating agent, S-nitrosoglutathione (GSNO), a physiological nitric oxide molecule, we show that GSNO treatment inhibited proliferation of chemoresponsive and chemoresistant ovarian cancer cell lines (A2780, C200, SKVO3, ID8, OVCAR3, OVCAR4, OVCAR5, OVCAR7, OVCAR8, OVCAR10, PE01 and PE04) in a dose dependent manner. GSNO treatment abrogated growth factor (HB-EGF) induced signal transduction including phosphorylation of Akt, p42/44 and STAT3, which are known to play critical roles in ovarian cancer growth and progression. To examine the therapeutic potential of GSNO <i>in vivo</i>, nude mice bearing intra-peritoneal xenografts of human A2780 ovarian carcinoma cell line (2×10⁶) were orally administered GSNO at the dose of 1 mg/kg body weight. Daily oral administration of GSNO significantly attenuated tumor mass (p<0.001) in the peritoneal cavity compared to vehicle (phosphate buffered saline) treated group at 4 weeks. GSNO also potentiated cisplatin mediated tumor toxicity in an A2780 ovarian carcinoma nude mouse model. GSNO’s nitrosylating ability was reflected in the induced nitrosylation of various known proteins including NFκB p65, Akt and EGFR. As a novel finding, we observed that GSNO also induced nitrosylation with inverse relationship at tyrosine 705 phosphorylation of STAT3, an established player in chemoresistance and cell proliferation in ovarian cancer and in cancer in general. Overall, our study underlines the significance of S-nitrosylation of key cancer promoting proteins in modulating ovarian cancer and proposes the therapeutic potential of nitrosylating agents (like GSNO) for the treatment of ovarian cancer alone or in combination with chemotherapeutic drugs.</p></div

Oral administration of GSNO abrogates tumor growth in A2780 bearing nude mice.

<p><b>A</b>. Schematic diagram of experimental design. In brief, A2780 ovarian cancer cell line was injected interperitoneally in nude mice and S-nitrosoglutathione (GSNO) was given orally daily from day 3 at the dose of 1 mg/kg of body weight till the end of the study. Phosphate buffered saline (PBS) was given as vehicle. <b>B</b>. Decreased abdominal circumference of GSNO treated mice compared to vehicle treated groups measured at week 4 (**p<0.01 treated compared to vehicle). <b>C.</b> Decreased excised tumor weights of GSNO treated mice compared to vehicle treated at week 4 (***p<0.001 treated compared to vehicle). Results are shown as mean ± SD of 10–14 individual animals. <b>D</b>. Representative gross morphological picture of tumor mass and tumor associated with ovary of vehicle and GSNO treated mice. <b>E</b>. Measurements of viable tumor size of GSNO vs PBS treated mice, as described in methods (***p<0.001 treated compared to vehicle). <b>F.</b> Count of mitotic cells and <b>G.</b> count of CD31 positive vessels per HPF (x400) in GSNO vs PBS treated mice, as described in methods), counts were performed from 5 fields of 3 different tumors from each group. NS; non-significant treated compared to vehicle.</p

Detection of STAT3 as S-nitrosylating protein by biotin switch method.

<p><b>A</b>. Detection of biotinylated proteins after biotin-switch method in the presence or absence of S-nitrosoglutathione (GSNO; 0.5 mM). Omission of biotin-HPRT indicates the specificity of S-nitrosylation by biotin-switch method. <b>B</b>. Detection of nitrosylation of various proteins including EGFR, p65, Akt and STAT3 in ovarian cancer cells upon GSNO treatment. <b>C</b>. STAT3 was immunoprecipitated from nitrosylated protein lysate after biotin switch assay and its biotinylation was detected by anti-biotin-HRP using western blot analysis. <b>D</b>. A2780, C200 and SKOV3 cell lines were treated with GSNO (0.5 mM) or oxidized GSNO (0.5 mM) as control for 2 hours followed by immunoblot analysis for detection of tyrosine phosphorylation of STAT3 at 705 residue and total STAT3. <b>E</b>. Under similar experimental conditions as “D”, A2780, C200 and SKOV3 cells were processed for biotin switch method for detection of STAT3. Sample in lane 4 was treated with GSNO as lane 2, except during processing for biotin switch assay; the addition of HPDP was omitted. Upper panel shows the biotinylated proteins after biotin-switch assay. Nitrosylated STAT3 was detected by pulling down biotinylated proteins by streptavidin agarose followed by immunoblot analysis using anti-STAT3 antibody. Lower bands show the total levels of input STAT3 processed for biotin switch assay.</p

GSNO treatment attenuates STAT3 activation and proliferative signaling in ovarian cancer cell lines.

<p>A2780 (<b>A</b>) and SKOV3 (<b>B</b>) cells were plated, serum starved overnight and treated with S-nitrosoglutathione (GSNO; 0.5 mM) or inactive control (oxidized GSNO; 0.5 mM) in the presence or absence of HB-EGF (50 ng/ml) for various time periods (5–20 min). Cells were harvested at indicated time points and processed for the detection of various signaling molecules including pSTAT3 (Tyr705), pAkt (Ser473) and p-p42/44 (Thr202/Tyr204) using their specific antibodies from Cell Signaling (Danvers, MA). Total STAT3, Akt, p42/44 and β-actin was used for equal loading. Blots are representative of two independently run experiments.</p

GSNO attenuates cell proliferation in ovarian cancer cell lines.

<p>Percentage viability of A2780, C200, PE01, PE04, SKOV3 and OV202 treated with indicated doses of S-nitrosoglutathione (GSNO; 0.1–1 mM) was determined by MTT assay. Inactive GSNO (oxidized, last bar) was used as a control. The data represents 3 individual experiments done in triplicate. ***p<0.001; **p<0.01; *p<0.05 and NS; not significant compared to untreated cells using Student’s t-test (Prism).</p

<i>In vitro</i> nitrosylation of STAT3 affects its DNA binding ability.

<p><b>A</b>. Nuclear extract (NE) from SKOV3 was isolated and 20 µg was incubated with S-nitrosoglutathione (GSNO) or oxidized GSNO (100 µM) at 4°C in the presence or absence of DTT (1 mM). After 4 hours of incubation, DNA binding ability of nuclear STAT3 was examined by incubating nitrosylated NE with STAT3 gel shift oligonucleotides conjugated with agarose followed by immunoblot analysis with anti-STAT3 antibody. Since STAT3 recruits coactivator, including p300, we also examined the recruitment of p300 under similar experimental conditions. STAT3 and pSTAT3 (Y705) were examined before pulling down STAT3 with gel shift oligonucleotides conjugated with agarose. Nitrosylation of STAT3 was also examined under similar experimental conditions using biotin switch method. Total STAT3 level was examined in the input of NE from SKOV3, which was used to show that an equal protein amount was used in this experiment. <b>B</b>. Recombinant STAT3 (1 µg) was incubated with GSNO or oxidized GSNO (0.1 mM) at 4°C in the presence or absence of DTT (1 mM). After 2 hours of incubation, DNA binding ability of recombinant STAT3 is examined by pulling down Stat3 by incubating with gel shift oligonucleotides conjugated with agarose.</p

GSNO inhibits colony formation in chemosensitive and chemoresistant ovarian cancer cell line.

<p>Cells (2×10³)/well (A2780, C200, PEO1 and PEO4) were plated in 6-well plates and treated with indicated concentrations of S-nitrosoglutathione (GSNO) once. Oxidized GSNO was used as a negative control (last bar). After 2 weeks, colonies were stained with MTT and counted. Results are shown as mean ± SD of triplicates. *p<0.05, **p<0.01, ***p<0.001 and NS; not significant compared to untreated cells using Student’s t-test (Prism).</p

GSNO potentiates cisplatin induced cytotoxicity in A2780 bearing nude mice.

<p><b>A</b>. Schematic diagram of experimental design. <b>B</b>. Cumulative excised tumor weight from individual mice at 4 weeks with S-nitrosoglutathione (GSNO; 1 mg/kg of body weight) (panel 2), cisplatin (4 mg/kg of body weight) (panel 3) and GSNO (1 mg/kg of body weight) and cisplatin (4 mg/kg of body weight) combination (panel 4). Cisplatin was given 3 times by intraperitoneal route at day 7, 14 and 21 post-tumor injections. Results are shown as mean ± SD of 7 individual animals. ***p<0.001 combination of GSNO + cisplatin treated group compared to untreated or cisplatin alone treated group; **p<0.01 GSNO treated group compared to untreated group; *p<0.05 cisplatin treated group compared to untreated group; #p<0.05 combination of GSNO + cisplatin group compared to GSNO alone group.</p

Mitochondrial respiration indices.

<p>Mitochondrial oxygen consumption rate (OCR) of control and DM rats: A) Mitochondrial basal OCR (Δ OCR from basal minus of Antimycin-A). B) ADP OCR (ADP minus Oligomycin). C) Maximum OCR (FCCP minus Antimycin-A). D) FCCP-induced mitochondrial respiratory reserve capacity. (Area under curve of FCCP minus area under curve oligomycin) was plotted as a graph. E) The ratio between state 3 and 4 respirations was depicted as the respiratory control rate (RCR). The data expressed are mean ± SEM. N = 6–11. ** <i>p</i> < 0.01 <i>vs</i> control;.***P<0.0001 <i>vs</i> control.</p