Inhibitory Effect of Embelin On Colon Carcinogenesis Is Peroxisome Proliferator-Activated Receptor γ (PPARγ)-Dependent

Abstract

BACKGROUND AND OBJECTIVES We have recently observed that down-regulation of X-linked inhibitor of apoptosis protein (XIAP) sensitizes colon cancer cells to the anticancer effect of PPARγ ligands in mice. In this study, we aimed to evaluate the effect of Embelin (EB), a reported antagonist of XIAP, on colon cancer cells In Vitro and In Vivo, with a particular focus on whether PPARγ is required for EB to exert its effect. MATERIALS AND METHODS Colon cancer cell line HCT116 cells were used for In Vitro study. A dominant negative PPARγ (dnPPARγ) was used to antagonize endogenous PPARγ in HCT116 cells. Cell proliferation and apoptosis were measured by Annexin V/PI staining and Cell Death ELISA. Effect of EB on NF-κB activity was determined by luciferase assay. For In Vivo studies, colon specific carcinogen 1, 2-Dimethylhydrazine dihydrochloride (DMH) was subcutaneously injected to induce colon cancer in PPARγ+/+ and PPARγ +/- mice. Mice were fed EB daily for 10 days before DMH injection, and continued for 30 more weeks. MATERIALS AND METHODS Colon cancer cell line HCT116 cells were used for In Vitro study. A dominant negative PPARγ (dnPPARγ) was used to antagonize endogenous PPARγ in HCT116 cells. Cell proliferation was measured by [3H]-thymidine incorporation assay, and apoptosis was determined by Annexin V/PI staining and Cell Death ELISA. Effect of EB on NF-κB activity was determined by a non-radioactive chemiluminescent activity assay and luciferase assay. For In Vivo studies, colon specific carcinogen 1,2-Dimethylhydrazine dihydrochloride (DMH) was subcutaneously injected to induce colon cancer in PPARγ+/+ and PPARγ +/- mice. Mice were fed EB daily for 10 days before DMH injection, and continued for 30 more weeks. RESULTS EB inhibited the proliferation and induced apoptosis in HCT116 cells with marked up-regulation of PPARγ. DnPPARγ markedly abrogated the effects of EB In Vitro. PPARγ+/- mice were more susceptible to DMH-induced colon carcinogenesis than PPARγ+/+ mice. EB significantly reduced the incidence of colon cancer in PPARγ+/+ but not in PPARγ+/- mice. EB inhibited NF-κB activity in PPARγ+/+ but marginally so in PPARγ+/- mice. In addition, EB significantly inhibited the expression of Survivin and cMyc, and this inhibition was PPARγ -dependent. CONCLUSION Reduced expression of PPARγ significantly sensitizes DMH-induced colon carcinogenesis. EB exerts an inhibitory effect on colon carcinogenesis but this effect was dependent on the presence of functional PPARγ.