Soo-Mi Lee

Wnt signaling enhances the activation and survival of human hepatic stellate cells

Wnt signaling was implicated in pulmonary and renal fibrosis. Since Wnt activity is enhanced in liver cirrhosis, Wnt signaling may also participate in hepatic fibrogenesis. Thus, we determined if Wnt signaling modulates hepatic stellate cell (HSC) activation and survival. Wnt3A treatment significantly activated human HSCs, while this was inhibited in secreted frizzled‐related protein 1 (sFRP1) overexpressing cells. Wnt3A treatment significantly suppressed TRAIL‐induced apoptosis in control HSCs versus sFRP1 over‐expressing cells. Particularly, caspase 3 was more activated in sFRP1 over‐expressing cells following TRAIL and Wnt3A treatment. These observations imply that Wnt signaling promotes hepatic fibrosis by enhancing HSC activation and survival.

Eupatilin attenuates bile acid-induced hepatocyte apoptosis.

BackgroundIn cases of cholestasis, bile acids induce hepatocyte apoptosis by activating death receptor-mediated apoptotic signaling cascades. Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is a pharmacologically active ingredient found in Artemisia asiatica and exhibits cytoprotective effects against experimentally induced gastrointestinal, pancreatic, and hepatic damage. This study was undertaken to examine if eupatilin modulates bile acid-induced hepatocyte apoptosis.MethodsHuh-BAT cells, a human hepatocellular carcinoma cell line stably transfected with a bile acid transporter, were used in this study. Apoptosis was quantified using 4′,6-diamidino-2-phenylindole dihydrochloride staining, and its signaling cascades were explored by immunoblot analysis. Kinase signaling was evaluated by immunoblotting and by using selective inhibitors. Eupatilins in vivo effect on bile acid-induced hepatocyte apoptosis was explored in bile duct-ligated rats.ResultsEupatilin significantly reduced bile acid-mediated hepatocyte apoptosis by attenuating bile acid-induced caspase 8 cleavage. Eupatilin diminished the bile acid-induced activation of mitogen-activated protein kinases, including p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. In particular, the eupatilin-mediated inhibition of bile acid-induced c-Jun N-terminal kinase activation was found to be responsible for attenuating caspase 8 cleavage. Moreover, eupatilin diminished hepatocyte apoptosis in bile duct-ligated rats.ConclusionsEupatilin attenuates bile acid-induced hepatocyte apoptosis by suppressing bile acid-induced kinase activation. Therefore, eupatilin might be therapeutically efficacious in a variety of human liver diseases associated with cholestasis.

Hypoxia-inducible adrenomedullin accelerates hepatocellular carcinoma cell growth

Adrenomedullin is implicated in tumor progression and induced by hypoxia. We evaluated if adrenomedullin signaling is active in hepatocellular carcinoma (HCC), especially under hypoxic conditions, and to analyze its prognostic implication in HCC patients. HCC cells expressed adrenomedullin and its receptor, and hypoxia induced adrenomedullin expression. Adrenomedullin stimulated HCC cell growth via Akt activation, which was prevented by adrenomedullin peptide inhibitor. Clinico-pathological analysis revealed adrenomedullin extent was related to vascular invasion and N-cadherin intensity, which were reported to indicate a poor prognosis. In conclusion, adrenomedullin signaling is hypoxia-inducible and functionally active in HCCs, and its expression may be a prognostic factor.

Ursodeoxycholic acid-induced inhibition of DLC1 protein degradation leads to suppression of hepatocellular carcinoma cell growth

Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, has been shown to inhibit mitogenic signaling and suppressing cell proliferation in colonic tumorigenesis. The transcription of DLC1 (deleted in liver cancer), a tumor suppressor gene, is frequently silenced in various types of human cancer. In this study, we postulated that UDCA may inhibit DLC1 protein degradation in hepatocellular carcinoma (HCC) cells, and increased DLC1 expression may suppress HCC cell growth. Human HCC cell lines were used in this study. The methylation status was measured by methylation-specific PCR following sodium bisulfite treatment. Cell proliferation was assessed using an MTS assay. Kinase signaling cascades were evaluated by immunoblot analysis. For assessing ubiquitination, immunoprecipitation analysis was used. To inhibit cellular protein, specific small interfering RNAs (siRNAs) were transfected into cells. DLC1 protein levels increased over time following UDCA treatment. Specifically, UDCA increased the half-life of the DLC1 protein by inhibiting proteasomal degradation of DLC1 without affecting ubiquitination of the DLC1 protein. In addition, HCC cell growth was suppressed following UDCA treatment and this growth suppression was significantly reversed following transfection with DLC1-siRNA. Inhibition of DLC1 increased cellular proliferation; this was reduced after Rho-inhibitor treatment. Finally, RhoA activity was reduced following UDCA treatment; this result was reversed and thus increased following DLC1-siRNA transfection. In conclusion, these results demonstrate that UDCA induces DLC1 protein expression by inhibiting proteasomal DLC1 degradation in a ubiquitin-independent manner, and that DLC1 induction participates in UDCA-induced suppression of HCC cell growth. These observations implicate UDCA as an anti-proliferative agent in HCC.

Sodium taurocholate cotransporting polypeptide mediates dual actions of deoxycholic acid in human hepatocellular carcinoma cells: enhanced apoptosis versus growth stimulation

PurposeThe hydrophobic bile acid, deoxycholic acid (DC), can induce apoptosis in hepatocytes. The roles of DC and its transporter are not yet established in hepatocellular carcinoma (HCC) cells. We investigated DC-induced alterations in HCC cell growth, with a particular focus on the effect of the expression of bile acid (BA)-transporting Na+-dependent taurocholic cotransporting polypeptides (NTCPs).MethodsWe determined NTCP expression in four human HCC cell lines: Huh-BAT, Huh-7, SNU-761, and SNU-475. NTCP expression and apoptotic signaling cascades were examined by immunoblot analyses. Cell viability was assessed using the 3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assay. Wound healing and invasion assays were performed to evaluate cell migration and invasion abilities. Real-time polymerase chain reaction was performed to measure IL-8 expression levels. Nuclear factor kappa B (NF-κB) activity was evaluated by enzyme-linked immunosorbent assay.ResultsThe HCC cell lines revealed varying NTCP expression levels, and DC treatment had dual effects, depending on NTCP expression. DC induced apoptosis in NTCP-positive HCC cells, especially under hypoxic conditions. In NTCP-negative HCC cells, simultaneous treatment with DC and cyclooxygenase inhibitor markedly decreased aggressive cellular behaviors via the inhibition of NF-κB/COX-2/IL-8 pathways.ConclusionHydrophobic bile acid offers therapeutic potential for patients with advanced HCC via different mechanisms depending on NTCP expression levels within the tumor.

Enhanced interleukin-2 diphtheria toxin conjugate-induced growth suppression in retinoic acid-treated hypoxic hepatocellular carcinoma cells

Hypoxia induces survival signals in hepatocellular carcinoma (HCC). Hypoxia and retinoic acid (RA) may also induce interleukin-2 (IL-2) receptor expression, and thus we evaluated if RA and IL-2 receptor-targeted therapy are indicated in hypoxic HCCs. RA induced IL-2 receptor expression (R alpha, R beta, R gamma) in HCC cells, whereas hypoxia specifically induced IL-2 R gamma expression. IL-2 stimulated hypoxic HCC cell growth via p42/44 MAPK activation. Combination of denileukin diftitox and RA significantly suppressed hypoxic HCC cell growth compared to single agent-treated or normoxic cells. Therefore, denileukin diftitox and RA may be therapeutically implicated in infiltrative HCCs which are exposed to hypoxic environments.