Anti-Cancer Effects of 3, 3'-Diindolylmethane on Human Hepatocellular Carcinoma Cells Is Enhanced by Calcium Ionophore: The Role of Cytosolic Ca2+ and p38 MAPK.

Yuanyue Jiang,Michael Aschner,Yang Ye,Rongzhu Lu,Bingfang Wang,Jian Chen,Xinming Xu,Jie Gu,Yanfei Fang

doi:10.3389/fphar.2019.01167

Abstract

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 ([Ca2+]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. [Ca2+]i was measured with Fluo-3/AM by fluorescence microscopy. A23187, a calcium ionophore, was used to increase [Ca2+]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 [Ca2+]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 [Ca2+]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.

Highlights

Human hepatocellular carcinoma (HCC) is the sixth most common malignancy in the world and the third most common prevalent cause of cancer-related death worldwide (Forner et al, 2018)
The effects of DIM on liver cancer cell growth were evaluated with the CCK-8 assay
Western blotting analysis was consistent with the cell viability data, showing restoration of proliferation cell nuclear antigen (PCNA) levels in cells treated with the p38 inhibitor and DIM compared with DIM treatment alone (Figure 5C). These results suggest that DIM-induced inhibition of proliferation is dependent, at least in part, upon phospho-p38 activity and that the proliferation inhibition is highly correlated with the phospho-p38 MAPK pathway in both SMMC-77721 and HepG2 cells

Summary

Introduction

Human hepatocellular carcinoma (HCC) is the sixth most common malignancy in the world and the third most common prevalent cause of cancer-related death worldwide (Forner et al, 2018). As HCC is highly resistant to standard chemotherapy, surgical resection or other treatments, most current therapies have limited efficacy. Chemopreventive agents with low toxicity and high efficiency in inhibiting tumor growth are promising candidates for cancer therapy (Sun and Hai, 2006). 3,3’-diindolylmethane (DIM), derived from Brassica species of cruciferous vegetables (broccoli, cabbage, and cauliflower) has displayed antitumor activity in several human cancers, including colon (Kim et al, 2007), pancreatic (Abdelrahim et al, 2006), and prostate cancer (Beaver et al, 2012). High intake of cruciferous vegetables has been associated with lower incidence of lung and colorectal cancer (Higdon et al, 2007). DIM may offer potential prevention and therapy in HCC

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