3,3′-Diindolylmethane (DIM), a metabolic product of indole-3-carbinol extracted from cruciferous vegetables exhibits anti-inflammatory and anti-cancer properties. Earlier, the product has been demonstrated to possess anti-fibrotic properties; however, its protective effects on liver injury have not been clearly elucidated. In this study, we postulated the effects and molecular mechanisms of action of DIM on carbon tetrachloride (CCl4)-induced liver injury in mice. Acute liver injury was induced by a single intraperitoneal administration of CCl4 (1 ml/kg) into mice. DIM was injected via subcutaneous route for three days at various doses (2.5, 5 and 10 mg/kg) before CCl4 injection. Mice were sacrificed and serum was collected for quantification of serum transaminases. The liver was collected and weighed. Treatment with DIM significantly reduced serum transaminases levels (AST and ALT), tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS). CCl4- induced apoptosis was inhibited by DIM treatment by the reduction in the levels of cleaved caspase-3 and Bcl2 associated X protein (Bax). DIM treated mice significantly restored Cytochrome P450 2E1, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in CCl4 treated mice. In addition, DIM downregulated overexpression of hepatic nuclear factor kappa B (NF-κB) and inhibited CCl4 mediated apoptosis. Our results suggest that the protective effects of DIM against CCl4- induced liver injury are due to the inhibition of ROS, reduction of pro-inflammatory mediators and apoptosis.

Poncirus fructus (PF) is a phytochemical compound extracted from the dry, immature fruits of Poncirus trifoliate . PF is traditionally used to treat gastrointestinal disorders, allergies, and inflammatory disease. In East Asia, PF is also known for its anticancer properties. There are numerous reports on the anticancer and anti-inflammatory effects of PF in a wide range of cancers and gastrointestinal diseases, respectively. However, the role of PF in inducing apoptosis and suppressing the invasiveness of hepatocellular carcinoma (HCC) remains unclear. This study investigated the ability of PF to induce apoptosis and inhibit the invasiveness and migratory ability of HCC cell lines (Hep3B and Huh7). Wound healing, Transwell migration and invasion, and colony-formation assays, as well as flow cytometry, were used to analyze cell proliferation, migration, invasion, and apoptosis. Epithelial-mesenchymal transition (EMT)-related and apoptotic proteins were assessed by western blotting. The mitochondrial membrane potential of the Hep3B and Huh7 cells was observed with tetramethylrhodamine ethyl ester. The reactive oxygen species (ROS) level was determined by dihydroethidium (DHE) staining. PF treatment significantly decreased the proliferation of Hep3B and Huh7 cells in a dose-dependent manner, reduced the mitochondrial membrane potential, increased ROS levels, decreased the protein levels of Bcl-2, and increased the protein levels of Bax and cleaved caspase-3 and 9, suggesting that PF mediated HCC apoptosis via a mitochondrial pathway. Our findings showed that PF prevented HCC cell migration and invasion by inhibiting the EMT process and downregulating MMP-2 and MMP-9 activities. The results suggest the potential anticancer effects of PF by inhibiting proliferation, inducing apoptosis, and reducing the invasion and migration of HCC cells.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide with limited treatment options. Biomarker-based active phenolic flavonoids isolated from medicinal plants might shed some light on potential therapeutics for treating HCC. 3,3′-diindolylmethane (DIM) is a unique biologically active dimer of indole-3-carbinol (I3C), a phytochemical compound derived from Brassica species of cruciferous vegetables—such as broccoli, kale, cabbage, and cauliflower. It has anti-cancer effects on various cancers such as breast cancer, prostate cancer, endometrial cancer, and colon cancer. However, the molecular mechanism of DIM involved in reducing cancer risk and/or enhancing therapy remains unknown. The aim of the present study was to evaluate anti-cancer and therapeutic effects of DIM in human hepatoma cell lines Hep3B and HuhCell proliferation was measured with MTT and trypan blue colony formation assays. Migration, invasion, and apoptosis were measured with Transwell assays and flow cytometry analyses. Reactive oxygen species (ROS) intensity and the loss in mitochondrial membrane potential of Hep3B and Huh7 cells were determined using dihydroethidium (DHE) staining and tetramethylrhodamine ethyl ester dye. Results showed that DIM significantly suppressed HCC cell growth, proliferation, migration, and invasion in a concentration-dependent manner. Furthermore, DIM treatment activated caspase-dependent apoptotic pathway and suppressed epithelial–mesenchymal transition (EMT) via ER stress and unfolded protein response (UPR). Taken together, our results suggest that DIM is a potential anticancer drug for HCC therapy by targeting ER-stress/UPR.

The ErbB3 binding protein 1 (Ebp1) has been reported in several cancers, in which it can act as either a pro‐oncogenic regulator or a tumor suppressor. However, the biological function and molecular mechanism of Ebp1 p48 in hepatocellular carcinoma (HCC) remain unclear. Here, we report that the long isoform of Ebp1, p48, is highly expressed in HCC tissues compared with normal tissues. Ebp1 p48 expression was correlated with the tumor size in HCC patients. Silencing Ebp1 p48 by transduction with lentiviral shEbp1 dramatically reduced the proliferation rate, soft agar colony generation, and tumor formation in vivo. We further demonstrated that Ebp1 p48 knockdown resulted in decreased p38 phosphorylation, which subsequently reduced hypoxia‐inducible factor 1α (HIF1α) expression. Moreover, Ebp1 p48 knockdown led to an upregulation of p53 expression through MDM2 downregulation. Taken together, these results suggest that the Ebp1/p38/HIF1α signaling pathway and the Ebp1‐mediated downregulation of p53 are involved in hepatocarcinogenesis. Therefore, Ebp1 and its downstream signaling pathways may be promising therapeutic targets of HCC.