Date of Award

5-1-2016

Degree Name

Doctor of Philosophy

Department

Molecular, Cellular, and Systemic Physiology

First Advisor

Hales, Dale

Abstract

Ovarian cancer (OvCa) is the most fatal gynecological malignancy, with over 21,290 new cases diagnosed in 2015. The 5 year survival rate for patients diagnosed at Stage IV is 17% in the United States. It has been reported that estrogen (E2) can promote metastasis of ovarian tumor cells through its nuclear receptor (ER). E2 is a potent mitogen and can stimulate the growth and invasion of ovarian cancer cells. We have previously shown that flaxseed diet decreases the severity and incidence of OvCa in hens, the only animal model that develops OvCa spontaneously and the disease is pathologically and histologically similar to the human disease.. While it is well established that the n-3 polyunsaturated fatty acids from flaxseed are anti-inflammatory the phytoestrogenic properties of its lignan, secoisolaricirescinol diglucoside (SDG) have not been fully explored. SDG metabolites, enterolactone (ED) and enterodiol (EL) can decrease ER signaling by competing for binding sites with E2, potentially decreasing expression of E2 target genes involved in proliferation and survival. Our goal was to analyze the effect of flaxseed diet on E2 signaling and metabolism in the normal and cancerous ovarian tissues. We hypothesized that due to the phytoestrogenic properties of the flax lignan, flaxseed diet can affect the downstream signaling of ER there by altering the expression of its target genes. In the 3 year old pre-neoplastic hen ovaries, 15% flaxseed supplemented diet was most effective in decreasing the expression of ER and its target genes like IGFBP4, IGFBP5, IRS1 that are a part of the IGF signaling pathway and are also implicated in a significant number of malignancies. Whole flaxseed diet also decreased the expression of the anti-apoptotic protein, BCL2L1 possibly by reducing the activation of the NFkB pathway. Ovarian tumors from 4 year old hens appeared to over-express estrogen receptor along the glandular area of the tumor but not the stroma. In the 3 year old pre-neoplastic hen ovaries, whole flaxseed and its components, defatted flax meal and flax oil decreased AKT2 mRNA expression but did not affect its activation. Another major regulator of intracellular signaling, ERK 1/2 MAPK appeared to be upregulated in tumors from flaxseed fed hens. Whole flaxseed diet was also significant in altering the metabolism of E2. This was suggested by the increased 2-hydroxyestrone/16-hydroxyestrone (2OHE/16OHE) ratio (a marker for reduced risk of cancer), in the serum from normal hens that were fed 15% flaxseed. It was also demonstrated that whole flaxseed and its components led to a significant decrease in CYP1B1 expression, an enzyme frequently upregulated in cancers, in the hen ovarian tumor tissues. Levels of the pro-apoptotic and anti-proliferative metabolite, 2-methoxyestradiol were significantly increased with whole flaxseed diet in the serum from 3 year old (pre-neoplastic hens) as well as 4 year old hens (cancerous and normal hens). These results demonstrated that whole flaxseed had a more significant effect in decreasing the expression of the cancer implicated end-points and increasing the levels of protective metabolites in comparison to either of its components, individually. Mechanistic studies with the in vitro model using the BG1FR ovarian cancer cells indicated that that 2-methoxyestradiol could induced apoptosis with a parallel increase in p38 activation. Since we observe a similar correlation between 2-methoxyestradiol and p38 in vivo, we believe that flaxseed diet maintains an anti-proliferative, anti-inflammatory and pro-apoptotic ovarian microenvironment by increasing 2-methoxyestradiol levels.

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