Anti-Cancer Effects of 3, 3’-Diindolylmethane on Human Hepatocellular Carcinoma Cells Is Enhanced by Calcium Ionophore: The Role of Cytosolic Ca(2+) and p38 MAPK

Purpose: 3,3′-Diindolylmethane (DIM), derived from indole-3-carbinol (I3C) in the Brassica species of cruciferous vegetables, has anticancer effects, but its exact underlying mechanism of action is unknown. We explored the roles of cytosolic free calcium ([Ca(2+)](i)) and p38 MAPK in the anti-cancer effects of DIM in human hepatocellular carcinoma cells. Methods: Cell proliferation was measured with a Cell Counting Kit-8 (CCK-8) and the clonogenic formation assay. Cell apoptosis was examined by flow cytometric analysis and Hoechst dye staining. Cleaved-caspase3, cleaved-PARP, Bax, total, and phosphorylated p38 MAPK were assayed by western blotting. [Ca(2+)](i) was measured with Fluo-3/AM by fluorescence microscopy. A23187, a calcium ionophore, was used to increase [Ca(2+)](i) levels. Results: DIM inhibited cell proliferation in both SMMC-7721 and HepG2 cells in a concentration- and time-dependent manner. DIM also enhanced phosphorylation of p38 MAPK (p-p38), which was attenuated by SB203580. The proliferation inhibition and apoptosis induction by DIM were also blunted. In addition, DIM increased [Ca(2+)](i) in HCC cells, and this effect was inhibited by the calcium chelator, BAPTA-AM, resulting in reduced p-p38 MAPK activation and apoptosis in DIM-treated cells, though the proliferation inhibition by DIM was unchanged. However, the DIM-induced cell proliferation inhibition and apoptosis were significantly enhanced by A23187, a selective calcium ionophore, which was attributed to exaggerated p-p38 MAPK. Conclusions: The calcium ionophore enhanced DIM-induced anti-cancer effects in hepatocellular carcinoma cells, secondary to [Ca(2+)](i)-dependent activation of p38 MAPK. Treatment with a combination of DIM and calcium ionophore may offer a new approach to enhance the chemotherapeutic efficacy in liver cancer.