This study investigated the involvement of PPARgamma and PPARalpha signaling in the synergistic anticancer activity of clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and docosahexaenoic acid (DHA) in human cancer cells. The synergistic cytotoxicity of DHA and clioquinol was demonstrated in nine human cancer cell lines representing different tissues of origin. A2780, a well-established ovarian cancer model system, was chosen for further characterization because of its sensitivity to DHA and clioquinol. Both PPARalpha and PPARgamma were expressed in A2780 cells when analyzed with western blotting and reporter gene technique. Treatment of the cells with clofibrate (a PPARalpha agonist) and clioquinol for three days mimicked the synergy of DHA and clioquinol, whereas this synergy could not be seen with the use of troglitazone (a PPARgamma agonist) and clioquinol, suggesting that PPARalpha signaling is involved in the synergistic action. When used alone, the IC50 of clofibrate was 513 microM in A2780 cells. However, the addition of 5 microM clioquinol to clofibrate-treated cells led to a dramatic reduction of its IC50 value (148 microM). The combination effects index (CI) analysis confirmed the synergy of clioquinol and clofibrate on inhibiting cancer cell viability. Using inhibitors to block PPARalpha signaling diminished the synergistic cytotoxicity of clioquinol and DHA. These results provide pharmacological evidence that the synergistic anticancer action of clioquinol and DHA is mediated by PPARalpha signaling in human cancer cells.