Access Type

Open Access Dissertation

Date of Award

January 2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Ghassan M. Saed

Abstract

Ovarian cancer is the leading cause of death from gynecologic malignancies yet the underlying pathophysiology is not clearly established. The disease is usually diagnosed in the advanced stage and carries a poor prognosis. The 5-year survival rate is greater than 75% if diagnosis of the cancer occurs at an early stage; however, this rate drops to 20% when the tumor has spread beyond its origin. Thus, a method for early detection is critically needed, which can help prolong, or even save lives. Currently, an effective screening test for ovarian cancer is lacking. Many tests have been evaluated but have been found to be lacking in sensitivity, specificity, or both. This becomes even more difficult as ovarian cancer is so rare in the general population, with an estimated prevalence of 0.04%. To date, 96% is the highest sensitivity recorded for the early detection of ovarian cancer. However, because of the low prevalence of ovarian cancer, a sensitivity and specificity of 96% would only detect 1 case of cancer per 100 oophorectomies done for positive screens due to the high false positive rate.

Clinical and epidemiological investigations have provided evidence supporting the role of reactive oxygen species (ROS) in the prognosis and metastasis of cancer due to exogenous factors that lead to chronic inflammation. In addition, cancer cells are frequently under persistent oxidative stress, which participates in cancer progression as well as in the selection of resistant cells that are unable to be eliminated by apoptosis. A recent novel finding from our laboratory has shown that myeloperoxidase (MPO) is expressed in ovarian cancer cell lines and tissues, with minimal or no expression detected in normal ovarian tissues. Myeloperoxidase is an abundant heme protein, which plays a central role in the formation of reactive oxidant and free radical species. Additionally, under conditions of oxidative stress, MPO serves as a source of free iron.

Another source of ROS is the superoxide producing nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase, which we observed to be upregulated in ovarian cancer as compared to normal ovarian tissues. It has been reported that mutations in specific regions of NAD(P)H oxidase subunits contribute to the enhancement of the enzyme activity with subsequent increase in superoxide (O2 ●−) production, contributing to enhanced levels of oxidative stress.

Therefore, there is compelling eveidence to support the role of oxidative stress in the pathogenesis of ovarian cancer, yet the mechanisms responsible for the dissemination of EOC are not fully understood. The objective of this work is to identify mechanisms of oxidative stress in the pathogenesis of ovarian cancer. The central hypothesis of this study is that ovarian cancer is controlled by mechanisms that emanate from an altered redox balance. To test this hypothesis, the following specific aims have been investigated:

Specific Aim I: Determine the oxidative stress profile in epithelial ovarian cancer and determine the effect of the modulation of specific key players of oxidative stress on the persistence of the oncogeneic phenotype. To determine the oxidative stress profile, the expression of the following markers will be evaluated: NAD(P)H oxidase, nitrate/nitrite, glutathione, glutathione reductase, glutathione peroxidase, glutathione s-transferase, inducible nitric oxide synthase, myeloperoxidase, catalase, and superoxide dismutase in epithelial ovarian cancer cells and tissues as compared to their normal counterparts. These markers were selected based on extensive preliminary and published results, utilizing a cell culture model and ovarian cancer tissues. Modulation of key players of oxidative stress will contribute to delineation of the mechanisms of development of ovarian cancer. To achieve this, a combination of knockdown or inhibition of specific pro-oxidants ulizing siRNA or direct inhibition will be performed followed by the assessment of the expression other pro-oxidants as well as the effect on apoptosis.

Specific Aim II: To assess the effectiveness of MPO and free iron levels in detection of early stage ovarian cancer. The mechanism by which MPO functions under oxidative stress is not well defined. MPO has been indicated to serve as a source of free iron under oxidative stress. The hypothesis of this aim is that serum MPO and free iron levels can be utilized to distinguish patients with stage I ovarian cancer from those with normal ovaries or benign gynecologic conditions as well as from late state ovarian cancer. MPO and free iron levels will be measured in the sera and tissues of women at the time of ovarian cancer diagnosis as compared to healthy women or those with benign gynecologic disorders respectively. The biological significance of the relationship between serum MPO and free iron, in conjunction with the poor prognosis of ovarian cancer has the potential for future clinical applications. .

Specific Aim III: To assess whether a SNP in NAD(P)H oxidase is associated with increased risk of development of ovarian cancer. Single nucleotide polymorphisms (SNPs) are associated with increased risk of several cancers. We evaluated the association of a SNP (rs4673) in the CYBA gene, encoding the p22phox subunit of NAD(P)H oxidase, substituting allele C with T at position 242 located on chromosome 16q24, in high-risk individuals with or without ovarian cancer, with or without a deleterious BRCA1/2 mutation. This SNP is known to be associated an increased activity of the enzyme, resulting in an increase in superoxide production. TaqMan® SNP Genotype analysis and the QuantStudio 12K Flex Real-Time PCR System were utilized to determine the frequency of this SNP in individuals with ovarian cancer as compared to noncancer individuals.

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