Cells were incubated with 25 μM of NCX-4040 for 6 h, then with cisplatin for 1 h (ICdose for A2780 WT) with a 24 h follow up time

Cell viability was evaluated using MTT assay. Data represent mean ± SD obtained from 4 independent experiments and expressed as a percentage of the control cells treated with vehicle (1% DMSO). *p < 0.01 Control group. **p < 0.05 cDDP or NCX-4040 group.<p><b>Copyright information:</b></p><p>Taken from "NCX-4040, a nitric oxide-releasing aspirin, sensitizes drug-resistant human ovarian xenograft tumors to cisplatin by depletion of cellular thiols"</p><p>http://www.translational-medicine.com/content/6/1/9</p><p>Journal of Translational Medicine 2008;6():9-9.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2267444.</p><p></p

Representative EPR spectra obtained from control (NCX-4040, A2780 cDDP cells) and NCX-4040 treatment groups

The cells were incubated with NCX-4040 in the presence of spin trap (MGD)Fe. Also shown is an EPR spectrum of an authentic complex of NO with Fe(MGD)obtained by adding SNAP (an NO donor) to spin trap. () Time-course of nitric oxide release by NCX-4040 in A2780 WT, A2780 cDDP, and A2780 cDDP cells treated with 1 mM buthionine sulfoximine (BSO) for 24 h. () Representative DAF-FM DA fluorescence images of untreated (control) and treated (1, 2 or 4 hours) with NCX-4040 (100 μM). () Quantitative results on the DAF-FM DA fluorescence data. *p < 0.01 Control group.<p><b>Copyright information:</b></p><p>Taken from "NCX-4040, a nitric oxide-releasing aspirin, sensitizes drug-resistant human ovarian xenograft tumors to cisplatin by depletion of cellular thiols"</p><p>http://www.translational-medicine.com/content/6/1/9</p><p>Journal of Translational Medicine 2008;6():9-9.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2267444.</p><p></p

Groups of mice (4–6 per group) were inoculated with the A2780 cDDP human ovarian cancer cells in the upper portion of hind leg

Seven days after inoculation, two groups were injected daily (i.p.) with NCX-4040 (5 mg/kg), of which one group received a single i.p. injection of cisplatin on day 11 (8 mg/kg). The third group received single dose of cisplatin on day 11 (8 mg/kg). One group received aspirin (5 mg/kg, daily). Control group received vehicle. () Tumor growth curve for control, cisplatin, and combination of treatment (NCX-4040 and cisplatin). () Tumor growth volume data (mean ± SE, expressed as percent of control group) on the 19day after injection of cancer cells. *p < 0.05 compared to control group. **p < 0.05 versus cDDP group. NCX-4040 and aspirin (ASA), a metabolic product of NCX-4040, showed no significant effect. The results show that pretreatment with NCX-4040 was effective in enhancing the efficacy of cisplatin in inhibiting the growth of cisplatin-resistant ovarian cancer xenografts in mice.<p><b>Copyright information:</b></p><p>Taken from "NCX-4040, a nitric oxide-releasing aspirin, sensitizes drug-resistant human ovarian xenograft tumors to cisplatin by depletion of cellular thiols"</p><p>http://www.translational-medicine.com/content/6/1/9</p><p>Journal of Translational Medicine 2008;6():9-9.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2267444.</p><p></p

A redox-sensitive probe (RSSR) was injected intratumorally (100 mM; 15 μl), then sequential images were obtained for 30 min

The EPR images show the probe distribution in the tumor, while the tumor itself is not visible. () Representative images of time-course of redox probe distribution in untreated A2780 WT and A2780 cDDP tumors, as well as in A2780 cDDP tumor treated with NCX-4040 (5 mg/kg, daily). () Total redox levels expressed as a percent of A2780 WT level. () Glutathione (GSH) levels in the tumors excised on day 19 post-inoculation. Data represent mean ± SD obtained from 4 mice/group. *p < 0.05 A2780 WT group. **p < 0.05 A2780 cDDP group. The results demonstrate that NCX-4040 depleted thiol levels in the cisplatin-resistant tumor.<p><b>Copyright information:</b></p><p>Taken from "NCX-4040, a nitric oxide-releasing aspirin, sensitizes drug-resistant human ovarian xenograft tumors to cisplatin by depletion of cellular thiols"</p><p>http://www.translational-medicine.com/content/6/1/9</p><p>Journal of Translational Medicine 2008;6():9-9.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2267444.</p><p></p

Xenograft endometrial tumor weight and expression of STAT3 in mice treated with metformin.

<p>A xenograft study was done in which nude mice were injected with 1 x 10⁶ Ishikawa endometrial cancer cells subcutaneously in the right flank. After tumors were at least 3–5 mm in diameter, treatment with control, metformin 100 mg/kg, or metformin 200 mg/kg was started. Mice were sacrificed after 4 weeks of treatment. <b>A</b>. Tumor weight (in grams) from the mice with the 3 largest tumors in each group. <b>B</b>. Average body weight (g) of the mice in each group at conclusion of the study. <b>C</b>. Western blot results of STAT3 and associated proteins after treatment with control, 100 mg/kg, or 200 mg/kg of metformin.</p

High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer

<div><p>Objectives</p><p>STAT3 is over-expressed in endometrial cancer, and diabetes is a risk factor for the development of type 1 endometrial cancer. We therefore investigated whether glucose concentrations influence STAT3 expression in type 1 endometrial cancer, and whether such STAT3 expression might be inhibited by metformin.</p><p>Methods</p><p>In Ishikawa (grade 1) endometrial cancer cells subjected to media with low, normal, or high concentrations of glucose, expression of STAT3 and its target proteins was evaluated by real-time quantitative PCR (qPCR). Ishikawa cells were treated with metformin and assessed with cell proliferation, survival, migration, and ubiquitin assays, as well as Western blot and qPCR. Expression of apoptosis proteins was evaluated with Western blot in Ishikawa cells transfected with a STAT3 overexpression plasmid and treated with metformin. A xenograft tumor model was used for studying the <i>in vivo</i> efficacy of metformin.</p><p>Results</p><p>Expression of STAT3 and its target proteins was increased in Ishikawa cells cultured in high glucose media. <i>In vitro</i>, metformin inhibited cell proliferation, survival and migration but induced apoptosis. Metformin reduced expression levels of pSTAT3 ser727, total STAT3, and its associated cell survival and anti-apoptotic proteins. Additionally, metformin treatment was associated with increased degradation of pSTAT3 ser727. No change in apoptotic protein expression was noticed with STAT3 overexpression in Ishikawa cells. <i>In vivo</i>, metformin treatment led to a decrease in tumor weight as well as reductions of STAT3, pSTAT3 ser727, its target proteins.</p><p>Conclusions</p><p>These results suggest that STAT3 expression in type 1 endometrial cancer is stimulated by a high glucose environment and inhibited by metformin.</p></div

Degradation of pSTAT3 ser727 in grade 1 endometrial cancer cells treated with meformin.

<p>After Ishikawa cells were treated with control, 10 mM, or 20 mM metformin in high glucose media for 48h, samples were subjected to ubiquitin assay. The ubiquitinated proteins were subjected to immunoblot for pSTAT3 Ser727 and blotted with ubiquitin antibody. A ubiquitination smear of pSTAT3 Ser727 is seen in the metformin treated ishikawa cells under proteasomal inhibition using MG-132.</p

Metformin inhibits STAT3, its regulatory proteins and upregulated apoptosis-related proteins, in grade 1 endometrial cancer cells.

<p><b>A</b>. Western blot of STAT3 and its regulatory proteins in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h in high-glucose conditions. <b>B</b>. qPCR of STAT3 and some of its regulatory genes in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h. Groups significantly different than control (p<0.05) are indicated with an asterisk (*). <b>C</b>. Metformin did not affects pSTAT3 and STAT3 in normal glucose conditions.</p

Metformin inhibits grade 1 endometrial cancer cell proliferation, survival, migration, and induces apoptosis.

<p><b>A</b>. Sulforhodamine B (SRB) assay measured Ishikawa cell proliferation with increasing concentrations of metformin after 24h or 48h, in high-glucose media. <b>B</b>. Proportion of Ishikawa cells surviving (compared to control) after treatment with 10 mM or 20 mM of metformin. <b>C</b>. Cell migration assay, with Ishikawa cells subjected to control, 10 mM, or 20 mM metformin for 24h. Control (0h) is also shown. <b>D</b>. Quantification of % wound closure; the 10 mM and 20 mM treatment groups were each significantly different than control (p<0.05; noted with asterisk [*]). <b>E</b>. Western blot of apoptosis-related protein expression in Ishikawa cells after treatment with control, 10 mM, or 20 mM for 48h.</p

Expression of apoptosis and cell proliferation-related proteins in metformin-treated grade 1 endometrial cancer cells overexpressing STAT3.

<p>Ishikawa endometrial cancer cells were transfected with a STAT3-overexpressing plasmid. <b>A</b>. Western blot confirming overexpression of pSTAT3 ser727 and total STAT3. <b>B</b>. Western blot of proteins involved in apoptosis or cell proliferation in control or STAT3-overexpressing Ishikawa cells treated with control or 20 mM metformin in high-glucose medium for 48h. (C: control, TR: transfection reagent only, OE: transfected with STAT3-overexpressing plasmid, Ctrl: control, Met: metformin).</p