Induction of apoptosis by Dae-Hwang-Mok-Dan-Tang in HCT-116 colon cancer cells through activation of caspases and inactivation of the phosphatidylinositol 3-kinase/Akt signaling

Integr Med Res. 2017 Jun;6(2):179-189. doi: 10.1016/j.imr.2017.03.003. Epub 2017 May 26. Park C1, Hong SH2, Choi YH2,3. Author information 1 Department of Molecular Biology, College of Natural Sciences, Dongeui University, Busan, Korea. 2 Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, Korea. 3 Anti-Aging Research Center & Blue-Bio Industry Regional Innovation Center, Dongeui University, Busan, Korea. Abstract BACKGROUND: Dae-Hwang-Mok-Dan-Tang (DHMDT), a traditional Korean medicine, contains five species of medicinal plants and has been used to treat patients with digestive tract cancer for hundreds of years; however, its anticancer mechanism is poorly understood. In the present study, we investigated the proapoptotic effects of DHMDT in human colon cancer HCT-116 cells. METHODS: Cytotoxicity was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was detected using 4,6-diamidino-2-phenyllindile staining, agarose gel electrophoresis, and flow cytometry. The protein levels were determined using Western blot analysis. Caspase activity was measured using a colorimetric assay. RESULTS: Treatment with DHMDT resulted in a growth inhibition coupled with apoptosis induction, which was associated with the downregulation of members of IAP (inhibitor of apoptosis protein) family, including XIAP and survivin, and the activation of caspase-9 and -3 accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase and phospholipase C-γ1. DHMDT treatment also showed a correlation with the translocation of proapoptotic Bax to mitochondria, the loss of mitochondrial membrane permeabilization, and the cytochrome c release from the mitochondria to the cytosol. Moreover, DHMDT increased the levels of death receptor-associated ligands and enhanced activation of caspase-8 and cleavage of its substrate, Bid. However, the pan-caspase inhibitor could reverse DHMDT-induced apoptosis. In addition, DHMDT suppressed the phosphoinositide 3-kinase (PI3K)/Akt pathway, and treatment with a potent inhibitor of PI3K further increased the apoptotic activity of DHMDT. CONCLUSION: Our data showed that DHMDT induces HCT-116 cell apoptosis by activating intrinsic and extrinsic apoptosis pathways and by suppressing the PI3K/Akt signal pathway; however, further studies are needed to identify the active compounds. KEYWORDS: Dae-Hwang-Mok-Dan-Tang; HCT-116 cells; PI3K/Akt; apoptosis; caspase PMID: 28664141 PMCID: PMC5478258 DOI: 10.1016/j.imr.2017.03.003 Free PMC Article Share on FacebookShare on TwitterShare on Google+