Date of Award

9-1-2020

Degree Name

Master of Science

Department

Molecular Cellular and Systemic Physiology

First Advisor

Hales, Dale

Abstract

Ovarian cancer is the fifth most lethal cancer in women (1) and the most lethal gynecological malignancy. In 2018, there were approximately 22,240 new diagnosed cases of ovarian cancer and 14,070 deaths in the United States alone (2). The lifetime risk for developing ovarian cancer in the United States is 1.3% or approximately 1 in 78 women. The five-year survival rate for women with ovarian cancer is a grim 47.6% (2) while the average five year survival rate for all cancers is about 68%. This dismal prognosis for ovarian cancer patients indicates the critical need for improved treatment options, efficient early detection methods and effective preventative measures for ovarian cancer (1). The objective of this study was to determine if DHA causes a reduction in cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) by blocking the activation of NF-κB regulated transcription in the ovary. DHA is a 22 carbon long-chain omega-3 polyunsaturated fatty acid that is biologically derived from Alpha-linolenic acid (ALA) found in flaxseed. COX-2 is an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. Prostaglandin E2 (PGE2) is a key regulator of inflammation which has been shown to be highly associated with ovarian cancer development and progression. Our laboratory studies ovarian cancer in the laying hen because it is the only known animal model to naturally develop ovarian cancer that both pathologically and histologically matches that of the human form of the disease. Dietary flaxseed is one of the richest vegetable sources of omega-3 polyunsaturated fatty acids. Our previous studies have shown that in laying hens, a long-term flaxseed supplemented diet reduces the incidence and severity of ovarian cancer and decreases COX-2 and PGE2. It was hypothesized that DHA, derived from ALA found in flaxseed, decreases inflammation in the ovaries by suppressing the activation of COX-2 and the production of PGE2 through inhibition of the NF-κB pathway. For this study, an NF-κB reporter plasmid was transfected into HEK293 cells. The reporter plasmid (“met-luc”) produces a secreted luciferase allowing sequential analysis of media from DHA and TNF-α treated cells to assess changes in NF-κB transcriptional activation. Tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB was used as a positive control. NF-κB activation was also assessed by measuring its nuclear translocation and cytoplasmic accumulation through immunocytochemistry (ICC) and western blot analysis. In a parallel study, immortalized ovarian surface epithelial (IOSE) cells were challenged with the same treatments of DHA and TNF-α. In these cells, COX-2 mRNA was assessed through RT-qPCR and COX-2 protein expression was analyzed through ICC and western blot.Our results indicate that DHA acts in a cell specific manner to reduce inflammation associated with cancer. We have found that in HEK293 cells DHA reduces TNFα induced NF-κB reporter activity. In contrast, ALA does not affect NF-κB reporter activity. HEK293 cells treated with TNFα alone indicated a dose-dependent increasing trend in nuclear translocation of the NF-κB p65 subunit and a decreasing trend in cytoplasmic p65, suggesting potential increased pathway activation. ICC suggests DHA treatment causes increased cytoplasmic sequestration of the NF-κB p65 subunits indicating inhibition of TNFα induced NF-κB activation. Western blot data also indicates a decreasing trend in nuclear NFκB p65 when cells are pretreated with DHA and subsequently challenged with TNF. The IOSE cells, were the only cells out of the cell lines tested (BG1, HEYC2, TOV112D, SKOV3, HEK293) to express COX-2. In these IOSE cells, TNFα alone showed a dose-dependent increasing trend in COX-2 protein (analyzed through ICC and western blot) and mRNA levels (analyzed through RT-qPCR). ICC analysis revealed that DHA reduces TNF induced COX-2 protein expression. However, the western blot did not further support this observation. Only a slight non-significant reduction with DHA treatment was observed. In addition, both DHA and TNFα, while also not significant, seemed to increase mRNA levels of COX-2 compared to control. This slight decreasing trend in COX-2 protein expression and increase in mRNA, could indicate a possible post-transcriptional mechanism of regulation of COX-2 by DHA independent of NF-κB in the IOSE cells. These data suggest that DHA could act via distinct mechanisms in a cell specific manner to potentially reduce COX-2 and subsequently PGE2 levels. DHA can act at the transcriptional level by reducing the nuclear translocation of NF-κB and transcriptional activation of NF-κB target genes such as COX-2 in some cell types. DHA also has the potential to work via a post-transcriptional mechanism to inhibit COX-2 and in turn reduce PGE2 levels. Both mechanisms ultimately have the potential to decrease the inflammation associated with ovarian cancer. This study describes the anti-inflammatory action of dietary flaxseed consumption, making flaxseed supplementation a promising preventive measure for reducing the risk of ovarian carcinogenesis.

Available for download on Wednesday, March 31, 2021

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