Abstract B45: Anti-inflammatory actions of DHA via inhibition of the NF-κB pathway

Abstract

Abstract The objective of this study is to determine if docosahexaenoic acid (DHA), a 22 carbon long-chain omega-3 fatty acid found in flaxseed, causes a reduction in cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) by blocking the activation of Nuclear Factor Kappa light chain enhancer of activated B cells (NF-κB). NF-κB is a transcription factor that regulates expression of many stress response genes. Many cancers, including ovarian cancer, have a dysregulated NF-κB pathway. The NF-κB transcriptional complex is sequestered in an inactivate state in the cytoplasm. I Kappa Beta Kinase (IKK) phosphorylation of I Kappa Beta (IKB) permits the translocation of the NF-κB complex into the nucleus and activation of transcription. COX-2, an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins, such as PGE2, is regulated by NF-κB. PGE2 helps regulate and promote inflammation associated with increased incidence and severity of ovarian cancer. Our laboratory studies the laying hen because it is the only known animal model to spontaneously develop ovarian cancer. Flaxseed, the richest vegetable source of omega-3 fatty acids, may be effective in the prevention of ovarian cancer. Our previous studies have shown that in hens, a long-term flaxseed supplemented diet decreases the incidence and severity of ovarian cancer and decreases COX-2 and PGE2 levels. It is hypothesized that DHA will decrease inflammation 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 HEK293T cells using polyethylenimine (PEI). The reporter plasmid (“met-luc” with metridia luciferase driven by the NF-κB promoter) produces a secreted luciferase allowing sequential analysis of media from treated cells to assess changes in NF-κB activation. Tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB is a positive control. NF-κB activation is assessed by immunocytochemistry (ICC) and Western blot by measuring its translocation into the nucleus. Data show that DHA reduces TNFα-induced NF-κB reporter activity. HEK293T cells treated with TNFα alone showed an increase in nuclear NF-κB p65 subunits, suggesting pathway activation. ICC suggests DHA treatment causes cytoplasmic sequestration of the NF-κB p105 and p65 subunits, indicating inhibition of TNFα-induced NF-κB activation. Western blot data indicate an increase in cytoplasmic NF-κB p65 subunit after TNFα and DHA treatment compared to cells treated with only TNFα. These data suggest that DHA could prevent the release of the NF-κB subunits for nuclear translocation, therefore decreasing COX-2 and PGE2 levels associated with inflammation seen in ovarian cancer. This could be a possible mechanism of action for the chemosuppressive role of long-term flaxseed consumption in ovarian cancer in laying hens. (Funding: NIH RO1AT005295). Citation Format: Kara Starkweather, Karen Hales, Dale Hales. Anti-inflammatory actions of DHA via inhibition of the NF-κB pathway [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B45.