Mohammed G. Saed

The Defensive Role of Cumulus Cells Against Reactive Oxygen Species Insult in Metaphase II Mouse Oocytes.

We investigated the ability of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radical (·OH), and hypochlorous acid (HOCl), to overcome the defensive capacity of cumulus cells and elucidate the mechanism through which ROS differentially deteriorate oocyte quality. Metaphase II mouse oocytes with (n = 1634) and without cumulus cells (n = 1633) were treated with increasing concentration of ROS, and the deterioration in oocyte quality was assessed by the changes in the microtubule morphology and chromosomal alignment. Oocyte and cumulus cell viability and cumulus cell number were assessed by indirect immunofluorescence, staining of gap junction protein, and trypan blue staining. The treated oocytes showed decreased quality as a function of increasing concentrations of ROS when compared to controls. Cumulus cells show protection against H2O2 and ·OH insult at lower concentrations, but this protection was lost at higher concentrations (>50 μmol/L). At higher H2O2 concentrations, treatment dramatically influenced the cumulus cell number and viability with resulting reduction in the antioxidant capacity making the oocyte more susceptible to oxidative damage. However, cumulus cells offered no significant protection against HOCl at any concentration used. In all circumstances in which cumulus cells did not offer protection to the oocyte, both cumulus cell number and viability were decreased. Therefore, the deterioration in oocyte quality may be caused by one or more of the following: a decrease in the antioxidant machinery by the loss of cumulus cells, the lack of scavengers for specific ROS, and/or the ability of the ROS to overcome these defenses.

The Role of Oxidative Stress in the Development of Cisplatin Resistance in Epithelial Ovarian Cancer

Objective: To investigate the role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer (EOC). Methods: Two parent EOC cell lines (MDAH-2774 and SKOV-3) and their chemoresistant counterparts (cisplatin, 50 µmol/L) were used. Total RNA was extracted and subjected to real-time reverse transcriptase polymerase chain reaction to evaluate the expression of glutathione reductase (GSR) and inducible nitric oxide synthase (iNOS), as well as nitrate/nitrite levels. Analysis of variance was used for main effects and Tukey for post hoc analysis at P < .05 for statistical significance. Results: Both cisplatin resistant cell lines displayed a significant decrease in GSR messenger RNA (mRNA) levels and activity (P < .01). As compared to sensitive controls, nitrate/nitrite levels were significantly higher in SKOV-3 cisplatin resistant cells while iNOS mRNA levels were significantly higher in MDAH-2774 cisplatin resistant cells (P < .05). Conclusion: Our data suggest that the development of cisplatin resistance tilts the balance toward a pro-oxidant state in EOC.

Nicotinamide Adenine Dinucleotide Phosphate Oxidase Is Differentially Regulated in Normal Myometrium Versus Leiomyoma

Uterine fibroids are the most common benign tumor in women. The goal of this study was to investigate whether nicotinamide adenine dinucleotide phosphate oxidase (NOX), a major source of superoxide and subsequent oxidative stress, was differentially regulated in myometrium versus leiomyoma. Expression levels of NOXs1-5, dual oxidase (DUOX), DUOX2, NOX organizer (NOXO) 1, NOX activator 1, p47phox, p67phox, and p22phox were determined in cells treated with hypoxia by real-time reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry in tissues. Expression of NOX4 increased in fibroid compared to myometrial tissues and cells. The NOX2, DUOX1, and p67phox were higher while p22phox was lower in fibroid than that in myometrial cells. Hypoxia increased NOX4, DUOX1, and NOXO1 and decreased p22phox in myometrial and reduced DUOX1 in fibroid cells. The NOX1, NOX3, NOX5, and DUOX2 were undetectable. Fibroid cells are characterized by a unique NOX profile, which promotes a severe prooxidant state that may be responsible for their development. Targeting these subunits may be beneficial for future therapeutic interventions.

A Single Nucleotide Polymorphism in Catalase Is Strongly Associated with Ovarian Cancer Survival

Ovarian cancer is the deadliest of all gynecologic cancers. Recent evidence demonstrates an association between enzymatic activity altering single nucleotide polymorphisms (SNP) with human cancer susceptibility. We sought to evaluate the association of SNPs in key oxidant and antioxidant enzymes with increased risk and survival in epithelial ovarian cancer. Individuals (n = 143) recruited were divided into controls, (n = 94): healthy volunteers, (n = 18), high-risk BRCA1/2 negative (n = 53), high-risk BRCA1/2 positive (n = 23) and ovarian cancer cases (n = 49). DNA was subjected to TaqMan SNP genotype analysis for selected oxidant and antioxidant enzymes. Of the seven selected SNP studied, no association with ovarian cancer risk (Pearson Chi-square) was found. However, a catalase SNP was identified as a predictor of ovarian cancer survival by the Cox regression model. The presence of this SNP was associated with a higher likelihood of death (hazard ratio (HR) of 3.68 (95% confidence interval (CI): 1.149–11.836)) for ovarian cancer patients. Kaplan-Meier survival analysis demonstrated a significant median overall survival difference (108 versus 60 months, p<0.05) for those without the catalase SNP as compared to those with the SNP. Additionally, age at diagnosis greater than the median was found to be a significant predictor of death (HR of 2.78 (95% CI: 1.022–7.578)). This study indicates a strong association with the catalase SNP and survival of ovarian cancer patients, and thus may serve as a prognosticator.

Lycopene, a powerful antioxidant, significantly reduces the development of the adhesion phenotype

Abstract Postoperative adhesions are a common medical complication of gynecologic and other pelvic surgeries resulting in persistent pelvic pain, obstruction of the intestines, and even infertility. The molecular mechanisms of postoperative adhesion development remain to be elucidated. We have recently described a role for reactive oxygen species, specifically superoxide, in the development of postoperative adhesions. In this study, we sought to determine whether lycopene, a potent antioxidant, reduces markers characteristic of the adhesion phenotype. Primary fibroblast cultures from normal peritoneum and adhesion tissues were utilized to determine mRNA levels of adhesion phenotype markers type I collagen, transforming growth factor-beta 1 (TGF-β1), and vascular endothelial growth factor (VEGF) in response to lycopene (24 hours, 10 μM) treatment. There was a 2 (p < 0.003), 4.7 (p < 0.004), and 1.6 fold (p < 0.004) increase in mRNA levels of type I collagen, TGF-β1, and VEGF, respectively, in adhesion as compared to normal peritoneal fibroblasts. Lycopene treatment led to a 6.8 and a 12.4 fold decrease in type I collagen mRNA levels, in normal peritoneal and adhesion fibroblasts, respectively (p < 0.005). Lycopene treatment led to a 4.2 (p < 0.03) and a 4.6 (p < 0.05) fold decrease in VEGF mRNA levels, in normal peritoneal and adhesion fibroblasts, respectively. Lycopene treatment led to a 7.0 fold decrease in TGF-β1 mRNA levels, in adhesion fibroblasts (p < 0.03). A 1.9 fold decrease in TGF-β1 mRNA was observed in normal peritoneal fibroblasts in response to treatment, although it was not significant. Lycopene substantially reduced levels of adhesion phenotype markers in normal peritoneal and adhesion fibroblasts and whether it will reduce postoperative adhesions needs to be further investigated.

Specific point mutations in key redox enzymes are associated with chemoresistance in epithelial ovarian cancer.

Abstract Oxidative stress plays an important role in the pathophysiology of ovarian cancer. Resistance to chemotherapy presents a significant challenge for ovarian cancer treatment. Specific single nucleotide polymorphisms (SNPs) in key redox enzymes have been associated with ovarian cancer survival and progression. The objective of this study was to determine whether chemotherapy induces point mutations in key redox enzymes that lead to the acquisition of chemoresistance in epithelial ovarian cancer (EOC). Human EOC cell lines and their chemoresistant counterpart were utilized for this study. Specific SNPs in key redox enzymes were analyzed by TaqMan SNP Genotyping. Activities and levels of key redox enzymes were determined by real‐time RT‐PCR, ELISA and a greiss assay. Point mutations in key redox enzymes were introduced into sensitive EOC cells via the CRISPR/Cas9 system. Cell viability and IC50 for cisplatin were determined by the MTT Cell Proliferation Assay. Data was analyzed with SPSS using Students two‐tailed t‐tests and One‐way ANOVA followed by Dunnetts or Tukeys post hoc tests, p<0.05. Here, we demonstrate that chemoresistant EOC cells are characterized by a further enhancement in oxidative stress as compared to sensitive counterparts. Additionally, chemoresistant EOC cells manifested specific point mutations, which are associated with altered enzymatic activity, in key redox enzymes that are not detected in sensitive counterparts. Supplementation of an antioxidant was able to successfully sensitize EOC cells to chemotherapeutics. Causality was established by the induction of these point mutations in sensitive EOC cells, which resulted in a significant increase in the level of chemoresistance. These findings indicate that chemotherapy induces specific point mutations in key redox enzymes that contribute to the acquisition of chemoresistance in EOC cells, highlighting a potential novel mechanism. Identification of targets for chemoresistance with either biomarker and/or screening potential will have a significant impact for the treatment of this disease. HighlightsChemoresistant ovarian cancer cells manifest an altered redox balance.Chemoresistant ovarian cancer cells manifest point mutations in key redox enzymes.Altered redox balance in chemoresistance is caused by mutations in redox enzymes.Point mutations in redox enzymes may be a potential mechanism of chemoresistance.Antioxidant supplementation with chemotherapeutics sensitizes ovarian cancer cells.

Shifting anaerobic to aerobic metabolism stimulates apoptosis through modulation of redox balance: Potential intervention in the pathogenesis of postoperative adhesions

OBJECTIVE To compare the effect of shifting anaerobic to aerobic metabolism on key regulators of oxidative stress, including extracellular superoxide dismutase (SOD3), inducible nitric oxide synthase (iNOS), and its product, nitric oxide (NO), as well as mitochondrial potential (Δψm) and apoptosis in fibroblasts established from normal peritoneum and adhesion tissues. DESIGN Prospective, experimental study. SETTING University medical center. PATIENT(S) Fibroblasts established from normal peritoneum and adhesion tissues from the same patients. INTERVENTION(S) Treatment with dichloroacetate (0, 20, 40, and 80 μg/mL, 24 hours). MAIN OUTCOME MEASURE(S) The expression of SOD3, iNOS, and NO levels were measured utilizing real-time reverse transcription-polymerase chain reaction and Greiss assay. The Δψm was evaluated by the JC-1 Mitochondrial Membrane Potential Assay. Apoptosis was determined by caspase-3 activity and TUNEL assays. Data were analyzed using SPSS 19.0. Mixed model repeated-measures analysis of variance was used with a Bonferroni correction. Significant interactions were analyzed with independent sample t tests. RESULT(S) Dichloroacetate increased apoptosis, SOD3 messenger RNA, iNOS messenger RNA, and NO levels in fibroblasts from peritoneum and adhesions. There was enhanced Δψm adhesion as compared with normal peritoneal fibroblasts. Creating oxidative stress by exposure by hypoxia markedly increased Δψm in fibroblasts from normal peritoneum to levels observed in adhesions; dichloroacetate protected against the effects of hypoxia. CONCLUSION(S) Anaerobic metabolism and oxidative stress are associated with the development of the adhesion phenotype, which manifests decreased apoptosis. Dichloroacetate induces adhesion fibroblasts to undergo apoptosis via modulation of redox homeostasis. These findings may provide targets for therapeutic treatment for reduction of profibrotic disorders, including postoperative adhesions.

The Role of Angiogenesis in the Persistence of Chemoresistance in Epithelial Ovarian Cancer

Objective: Chemoresistance remains a major challenge in the treatment of ovarian cancer. As part of a survival mechanism, tumor cells have been shown to release proangiogenic factors, such as vascular endothelial growth factor (VEGF), through a mechanism that involves the upregulation of hypoxia-induced factor (HIF)-1α. The objective of this study was to compare the expression of VEGF and its receptors (R1 and R2) as well as HIF-1α in chemoresistant epithelial ovarian cancer (EOC) cells to their chemosensitive counterparts and determine their impact on angiogenesis. Methods: Two human EOC cell lines, MDAH-2774 and SKOV-3, and their cisplatin- or taxotere-resistant counterparts were used. Total RNA and protein were subjected to real-time reverse transcriptase–polymerase chain reaction, immunoprecipitation/Western blot and enzyme-linked immunosorbent assay to evaluate the expression of VEGF, VEGF receptors (R1 and R2), and HIF-1α. Angiogenesis was assessed with an in vitro angiogenesis assay. Data were analyzed using independent Student t tests and chi-square. Results: Both taxotere- and cisplatin-resistant MDAH-2774 and SKOV-3 EOC cell lines manifested a significant decrease in VEGF, VEGF receptors, HIF-1α messenger RNA, and protein levels as compared to their chemosensitive counterparts. There was a significant decrease in the number and thickness of polygon blood vessel formation in chemoresistant EOC cells compared to chemosensitive counterparts. Conclusion: Cisplatin- and taxotere-resistant EOC cells are characterized by lower VEGF, VEGF receptors, and HIF-1α, and decreased angiogenesis. These findings may indicate a decrease in drug delivery at the tumor site, hence allowing the persistence of chemoresistant EOC cells.

Nicotinamide Adenine Dinucleotide Phosphate Oxidase Expression Is Differentially Regulated to Favor a Pro-oxidant State That Contributes to Postoperative Adhesion Development:

We have previously reported that superoxide (O2 •–) contributes to the development of postoperative adhesions. In this study, we determined whether O2 •– generating nicotinamide adenine dinucleotide phosphate oxidase (NOX) is differentially expressed in normal peritoneal and adhesion fibroblasts and tissues. The NOX isoforms were measured utilizing Western blot, immunohistochemistry, high-performance liquid chromatography, and real-time reverse transcription polymerase chain reaction. Expression and activity of NOX were found to be significantly higher in adhesion tissues and cells than that in normal peritoneal tissues and cells (P < .05). Levels of NOX2, NOX4, NOX activating protein 1, DUOX1, p47phox, and p22phox messenger RNA increased in adhesion fibroblasts when compared to normal peritoneal and increased in response to hypoxia in normal peritoneal fibroblasts. Thus, adhesion fibroblasts are characterized by a unique NOX expression profile, which maintains a pro-oxidant state that may be responsible for the persistence of the adhesion phenotype. Decreasing the activity of NOX by targeting these isoforms may be beneficial for future therapeutic interventions of postoperative adhesions.

Abstract B49: The role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer

Objective: To investigate the role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer (EOC). Methods: Two parent EOC cell lines (MDAH-2774 and SKOV-3) and their chemoresistant counterparts were used. Total RNA was extracted and subjected to real-time RT-PCR to evaluate the expression of inducible nitric oxide synthase (iNOS) and glutathione reductase (GSR) in all cells. Nitrate/nitrite levels and GSR activity were also measured using colorimetric and ELISA assays respectively. Cell proliferation was assessed using Ki-67 immunostaining in all cells. Unpaired t-tests were used for statistical analysis at p-value <0.05 for significance. Results: Both EOC cisplatin sensitive cells manifested significant increase in the expression and activity of GSR as compared to their resistant counterparts (p<0.05). On the other hand, EOC sensitive cells manifested a decrease in iNOS expression as compared to their resistant counterparts (p<0.05). Nitrate/nitrite levels were significantly decreased in EOC sensitive cells as compared to their resistant counterparts (p<0.05). Ki-67 immunofluorescence staining revealed increased proliferation in cisplatin resistant cells compared to controls for the MDAH-277a and SKOV-3 cell lines. Conclusion: Our data suggests that the development of cisplatin resistance heighten the prooxidant state in EOC leading to a further increase in cell proliferation. Citation Format: Jimmy Belotte, Nicole Fletcher, Awoniyi Awonuga, Mitchell Alexis, Husam Abu-soud, Michael Diamond, Mohammed Saed, Ghassan Saed. The role of oxidative stress in the development of cisplatin resistance in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B49.