The Role Of Oxidative Stress In The Establishment Of Resistance To Cisplatin In Epithelial Ovarian Cancer Cells

Epithelial ovarian cancer is the deadliest of all gynecologic cancers with an estimated 22,280 new cases and 14,240 deaths expected in 2016 in the US alone. This high mortality rate can be partially attributed to a lack of universal screening and the development of resistance to the recommended chemotherapeutics. Typically, the treatment of ovarian cancer requires both cytoreductive surgery (CRS) and platinum/taxane combination chemotherapy. Initially, 50–80% of patients with advanced disease will achieve complete clinical response. Unfortunately, most will relapse within 18 months with chemoresistant disease. Thus, understanding the mechanisms of platinum resistance is critical in order to improve the care of ovarian cancer patients. Several theories to explain cisplatin resistance have been proposed but failed to translate into clinical practice. Typically drug resistance mechanisms are multifactorial but can be broadly categorized as follows: 1) pharmacokinetic, resulting in inadequate intratumor cisplatin concentration, 2) tumor micro-environment, involving membrane transporters by reducing cisplatin uptake or increasing efflux, 3) increased inactivation and sequestration of cisplatin, 4) activation of DNA repair and antiapoptotic mechanisms, 5) decreased autophagy and, 6) cancer-cell specific mechanisms such as: acquired somatic mutations and epigenetic changes and persistence of slow growing cancer stem cells that maintain the cancer phenotype. We have recently characterized EOC tissues and cells to manifest a persistent pro-oxidant state with the upregulation of several key oxidant enzymes including: myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), and nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase with concurrent decrease in apoptosis compared to normal human ovarian tissues and cells. More importantly, shutting down the expression of one or more of these key oxidant enzymes reduced the pro-oxidant state, and significantly induced apoptosis. Single-nucleotide polymorphisms (SNPs) are point mutations that are selectively maintained in populations and are distributed throughout the human genome at an estimated overall frequency of at least one in every 1000 base pairs. Several SNPs in key oxidants and antioxidants enzymes have been associated with various cancers including ovarian. Therefore, we are proposing the following hypothesis: cisplatin treatment induces mutations in key oxidant and antioxidant enzymes resulting in further enhancement of oxidative stress and the acquisition of resistance in EOC cells. To test this hypothesis, we are proposing then following specific aims: Specific Aim 1: To determine the association of specific single nucleotide polymorphisms (SNPs) in key oxidant and antioxidant enzymes with EOC risk and survival in patients. The rationale of this aim is based on the fact that oxidative stress is strongly associated with several cancers, including ovarian. Known specific SNPs in oxidant and antioxidant genes may alter their expression profile and enzymatic functions. These SNPs have been reported to be associated not only with cancer risks, but also patient response to treatment and survival. The hypothesis of this aim is that specific SNPs in key oxidant and antioxidant enzymes are associated with overall patient survival. To achieve this aim, we will perform a case-control study using stored blood samples of research participants from the Karmanos Cancer Center. Individuals (n=143) recruited were divided into controls (n=94),) and ovarian cancer cases (n=49). Samples will undergo DNA extraction followed by TaqMan® SNP genotype analysis for rs4880 manganese superoxide dismutase (MnSOD), rs4673 (NAD(P)H oxidase (CYBA), rs3448 glutathione peroxidase (GPX1), rs2297518 inducible nitric oxide synthase (iNOS), rs1002149 glutathione reductase (GSR), and rs1001179 catalase (CAT). We will perform a multivariate analysis for identification of confounding variables and potential predictors of risk. Additionally, to study the impact of the SNPs on overall survival, Cox regression and Kaplan-Meier survival analyses will be used. Specific Aim 2: To determine the association of key oxidant and antioxidant enzymes as well as specific SNPs in these enzymes with the development of cisplatin resistance in EOC cells. The rationale of this aim is based on previous findings showing an association between the altered redox enzymes and EOC, in both patients and human cell lines. The hypothesis of this aim is that the acquisition of resistance to cisplatin in EOC cells is associated with enhanced pro-oxidant profile, as well as specific SNPs in key oxidant and antioxidant enzymes. To achieve this aim, we will utilize two human EOC cell lines, MDAH-2774 and SKOV-3 and their cisplatin resistant counterparts. We will perform TaqMan PCR genotyping, real-time RT-PCR, ELISA, and Griess assay to study the expression profile of the following genes: CYBA/NOX4, iNOS, CAT, SOD3, GSR and GPX1. To analyze the difference in the expression profiles of these genes for sensitive compared to resistant cells, we will use a Student’s t-test. Specific Aim 3: To determine whether specific SNP(s) in key oxidant and antioxidant enzymes cause the acquisition of cisplatin resistance in EOC cells. The rationale of this aim is based on the established fact that cisplatin treatment causes DNA damage, and the observation that specific SNPs in the redox enzymes were found to be associated with poor survival in patients. The hypothesis of this aim is: specific SNPs in key oxidant and antioxidant enzymes cause cisplatin resistance. To achieve this aim, we will utilize the CRISPR/Cas9 system to generate point mutations in sensitive EOC cells corresponding to the SNP genotype of the chemoresistant MDAH-2774 and SKOV-3 EOC cells. The cells will then be tested for cisplatin resistance using the MTT viability assay using the IC50 method. Results will be analyzed with regression analysis and student t-tests

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

<div><p>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 <i>BRCA1/2</i> negative (n = 53), high-risk <i>BRCA1/2</i> 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.</p></div

Cox regression analysis for selected SNPs in key oxidants and antioxidants genes in ovarian cancer.

<p>Adjusted Hazard Ratio (HR) for the variables included in the model.</p><p>* p<0.05, degrees of freedom = 1 for all analyses. CYBA; NAD(P)H oxidase subunit (NOX4), GPX; glutathione peroxidase, GSR; glutathione reductase, MnSOD; manganese superoxide dismutase, MPO; myeloperoxidase, NOS2; inducible nitric oxide synthase. For this analysis, several “Method”simulations were performed such as: forced entry (ENTER), forward LR (likelihood ratio), etc. The forward LR was chosen for the final analysis. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135739#pone.0135739.t004" target="_blank">Table 4</a> includes the strongest predictors kept by the model as well as those rejected by the model. The Cox regression model generated the scores in the table. The P-values are noted in the column significance; *p<0.05, is considered statistically significant.</p><p>Cox regression analysis for selected SNPs in key oxidants and antioxidants genes in ovarian cancer.</p

Characteristics of single nucleotide polymorphisms examined for genotyping.

<p>AA; amino acid, Ala; alanine, CAT; catalase, CYBA; NAD(P)H oxidase subunit (NOX4), GSR; glutathione reductase, GPX; glutathione peroxidase, His; histidine, Leu; leucine, MAF; minor allele frequency, MnSOD; manganese superoxide dismutase, MPO; myeloperoxidase, NCBI; National Center for Biotechnology Information, NOS2; nitric oxide synthase, Ser; serine, SNP; single nucleotide polymorphism, Tyr; tyrosine, Val; valine.</p><p>Characteristics of single nucleotide polymorphisms examined for genotyping.</p

Kaplan-Meier overall survival curves for in ovarian cancer utilizing a specific catalase SNP.

<p>The solid curve represents cases with (CC) homozygous wild-type genotype as compared to the dashed curve, which represents cases with homozygous mutant plus heterozygous mutant (CT+TT) genotypes. The X-axis represents patient survival in months; the Y-axis represents cumulative survival percentage. Chi-square p-value 0<0.05 is considered statistically significant.</p

Comparison of cases and controls based on demographic, personal or family history of cancer, and genotypic characteristics.

<p><i>*p< 0</i>.<i>05</i>, <i>CAT; catalase</i>, <i>CYBA; NAD(P)H oxidase subunit (NOX4)</i>, <i>GPX; glutathione peroxidase</i>, <i>GSR; glutathione reductase</i>, <i>MnSOD; manganese superoxide dismutase</i>, <i>MPO; myeloperoxidase</i>, <i>NOS2; inducible nitric oxide synthase</i>.</p><p>Comparison of cases and controls based on demographic, personal or family history of cancer, and genotypic characteristics.</p