Docosahexaenoic Acid Inhibits Superoxide Dismutase 1 Gene Transcription in Human Cancer Cells: The Involvement of Peroxisome Proliferator-Activated Receptor α and Hypoxia-Inducible Factor-2α Signaling

Abstract

Docosahexaenoic acid (DHA; 22:6) is known to have anticancer activity, but its mechanisms of action remain to be further elucidated. We recently demonstrated that DHA downregulates superoxide dismutase (SOD) 1 gene expression, thereby weakening cellular antioxidant forces and enhancing cytotoxicity in various human cancer cells. The objective of this study was to investigate the mechanism of the inhibitory effect of DHA on SOD-1 gene expression in human cancer cells. A reporter gene assay indicated that DHA suppresses SOD-1 gene transcription in a time-and concentration-dependent manner in human cancer cells. Pretreatment with vitamin E did not block the inhibitory effect of DHA, indicating that this suppression does not depend on lipid peroxidation. The suppressive effect of DHA on SOD-1 gene transcription could be mimicked by the peroxisome proliferator-activator receptor (PPAR) ␣ ligand clofibrate but not the PPAR␥ ligand troglitazone, suggesting the involvement of PPAR␣ signaling. Deletion analysis of the key DNA binding elements in the SOD-1 gene promoter identified the distal hypoxia response element (HRE), but not the peroxisome proliferator response element or nuclear factor-B element, as essential for the suppressive effects of DHA. Coimmunoprecipitation confirmed that PPAR␣, but not PPAR␥, forms a complex with hypoxia-inducible factor (HIF)-2␣ in cancer cells. Chromatin immunoprecipitation analysis indicated that both DHA and clofibrate reduce HIF-2␣ binding to the HRE. Thus, we have identified the distal HRE in the SOD-1 gene promoter that mediates the suppression on the transcription of this gene by DHA, and we have demonstrated the involvement of PPAR␣ and HIF-2␣ signaling in this event.

MicroRNA-19 (miR-19) Regulates Tissue Factor Expression in Breast Cancer Cells

“…Western Blot Analysis-Western blot was performed as we described previously (24). In brief, cells were lysed with the lysis buffer, sonicated on ice, and centrifuged at 15,000 ϫ g for 15 min to remove insoluble material.…”

“…Co-immunoprecipitation-Co-immunoprecipitation using Ago2 antibody was performed as described previously (24). In short, cells growing in 100-mm dishes were washed with PBS and harvested by adding 150 l of IP buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM EDTA, 1 mM PMSF, and 1% Triton X-100).…”

Activation of Peroxisome Proliferator-activated Receptor α (PPARα) Suppresses Hypoxia-inducible Factor-1α (HIF-1α) Signaling in Cancer Cells

“…Recent studies demonstrated that these two drugs have anticancer properties in various model systems (9 -11), but the mechanisms behind their actions remain to be elucidated. We have reported previously that activation of PPAR␣ mediates the anticancer action of docosahexaenoic acid, likely through down-regulation of hypoxia signaling (12,13). We therefore hypothesized that the anticancer properties of PPAR␣ agonists may similarly be due to suppression of HIF-1␣ signaling in human cancer cells.…”

miR‐92 is a key oncogenic component of the miR‐17–92 cluster in colon cancer

“…Co-immunoprecipitation experiments were performed using an anti-Argonaute 2 (Ago2) antibody (Wako Pure Chemical Industries, Tokyo, Japan), as described previously. (12) The RNA was isolated from precipitates using ISOGEN reagent (Nippon Gene, Osaka, Japan) according to the manufacturer's instructions. Reverse transcriptase reactions were performed using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA).…”