Docosahexaenoic acid inhibits superoxide dismutase 1 gene transcription in human cancer cells: the involvement of peroxisome proliferator-activated receptor alpha and hypoxia-inducible factor-2alpha signaling.

Docosahexaenoic acid (DHA; n-3, 22:6) is known to have anticancer activity, but its mechanisms of action remain to be further elucidated. We recently demonstrated that DHA down-regulates 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) alpha ligand clofibrate but not the PPARgamma ligand troglitazone, suggesting the involvement of PPARalpha 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-kappaB element, as essential for the suppressive effects of DHA. Coimmunoprecipitation confirmed that PPARalpha, but not PPARgamma, forms a complex with hypoxia-inducible factor (HIF)-2alpha in cancer cells. Chromatin immunoprecipitation analysis indicated that both DHA and clofibrate reduce HIF-2alpha 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 PPARalpha and HIF-2alpha signaling in this event.