Nanoceria: A Rare-Earth Nanoparticle as a Novel Anti-Angiogenic Therapeutic Agent in Ovarian Cancer

Ovarian cancer (OvCa) is the fifth most common cause of death from all cancers among women in United Sates and the leading cause of death from gynecological malignancies. While most OvCa patients initially respond to surgical debulking and chemotherapy, 75% of patients later succumb to the disease. Thus, there is an urgent need to test novel therapeutic agents to counteract the high mortality rate associated with OvCa. In this context, we have developed and engineered Nanoceria (NCe), nanoparticles of cerium oxide, possessing anti-oxidant properties, to be used as a therapeutic agent in OvCa. We show for the first time that NCe significantly inhibited production of reactive oxygen species (ROS) in A2780 cells, attenuated growth factor (SDF1, HB-EGF, VEGF(165) and HGF) mediated cell migration and invasion of SKOV3 cells, without affecting the cell proliferation. NCe treatment also inhibited VEGF165 induced proliferation, capillary tube formation, activation of VEGFR2 and MMP2 in human umbilical vascular endothelial cells (HUVEC). NCe (0.1 mg/kg body weigh) treatment of A2780 ovarian cancer cells injected intra-peritoneally in nude mice showed significant reduction (p \u3c 0.002) in tumor growth accompanied by decreased tumor cell proliferation as evident from reduced tumor size and Ki67 staining. Accumulation of NCe was found in tumors isolated from treated group using transmission electron microscopy (TEM) and inductively coupled plasma mass spectroscopy (ICP-MS). Reduction of the tumor mass was accompanied by attenuation of angiogenesis, as observed by reduced CD31 staining and specific apoptosis of vascular endothelial cells. Collectively, these results indicate that cerium oxide based NCe is a novel nanoparticle that can potentially be used as an anti-angiogenic therapeutic agent in ovarian cancer

Metformin Suppresses Ovarian Cancer Growth and Metastasis with Enhancement of Cisplatin Cytotoxicity In Vivo12

Ovarian cancer is the most lethal gynecologic cancer in women. Its high mortality rate (68%) reflects the fact that 75% of patients have extensive (>stage III) disease at diagnosis and also the limited efficacy of currently available therapies. Consequently, there is clearly a great need to develop improved upfront and salvage therapies for ovarian cancer. Here, we investigated the efficacy of metformin alone and in combination with cisplatin in vivo. A2780 ovarian cancer cells were injected intraperitoneally in nude mice; A2780-induced tumors in nude mice, when treated with metformin in drinking water, resulted in a significant reduction of tumor growth, accompanied by inhibition of tumor cell proliferation (as assessed by immunohistochemical staining of Ki-67, Cyclin D1) as well as decreased live tumor size and mitotic cell count. Metformin-induced activation of AMPK/mTOR pathway was accompanied by decreased microvessel density and vascular endothelial growth factor expression. More importantly, metformin treatment inhibited the growth of metastatic nodules in the lung and significantly potentiated cisplatin-induced cytotoxicity resulting in approximately 90% reduction in tumor growth compared with treatment by either of the drugs alone. Collectively, our data show for the first time that, in addition to inhibiting tumor cell proliferation, metformin treatment inhibits both angiogenesis and metastatic spread of ovarian cancer. Overall, our study provides a strong rationale for use of metformin in ovarian cancer treatment

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

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

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

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

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 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

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

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