Inhibition of Hyaluronic acid synthesis by 4-methylumbelliferone (4MU) upregulates dendritic cell activation in murine hepatocellular carcinoma (HCC) associated to liver fibrosis.

ALANIZ L; PICCIONI F; GARCÍA MG; RIZZO M; MAZZOLINI G

Viña del Mar, Chile

Congreso; 9º Congreso Latinoamericano de Inmunología; 2009

Liver fibrosis is characterized by an excessive deposition of extracellular matrix (ECM) components such as hyaluronan (HA), leading to progressive liver failure. Cirrhosis is considered a preneoplastic disease being hepatocellular carcinoma (HCC) one of the major complications. Abnormal HA accumulation modulates apoptosis, tumor progression and even the immune response. The aim of this work is to analyze whether inhibition of HA synthesis (by an specific inhibitor 4MU) during liver fibrosis might attenuate HCC development as well as its effects on antitumoral immune response. Methods: We used an in vivo model of HCC (Hepa129) with liver fibrosis. Fibrosis was induced by i.p. administration of thioacetamide for 4 weeks. 4MU (5mM) treatment was started 5 days before tumor cell implantation (performed by laparotomy) and injected for 2 weeks. Fibrosis degree was histologically quantified using Ishak score system by Masson's trichrome staining. Hepatic HA levels were analyzed by histological staining using a HA-binding protein. Costimulatory molecules (CD11c, CMHII, CD40, CD80, CD86) were analyzed in hepatic dendritic cells (DCs) by flow cytometry. Antitumoral effects of 4MU were analyzed by measuring the main hepatic tumor and its satellites. Proliferation and apoptosis assays were performed in order to analyze direct effect of 4MU on tumor cells. Results: We observed that untreated animals developed a F4-F5 fibrosis level while in mice treated with 4MU this level was reduced (F3). Tumor size was similar in mice treated or not with 4MU (163-189 mm3), however the number of satellites was found 2-folds reduced in treated mice. Mice that received 4MU therapy showed a significant activation of hepatic DCs compared with untreated mice (4,8% vs 2,6%).. In vitro results shown that treatment of tumor cells with 4MU (1mM, 5mM) significantly reduced cell proliferation and induced apoptosis (26,2% for 5mM). These results suggest an important role for hepatic HA during liver fibrogenesis and decreased HA synthesis would ameliorates HCC development. The potential antitumoral mechanisms of 4MU in this model could be inhibition fibrogenesis, stimulation of the immune system by dendritic cells activation and a direct antitumoral effect.in vivo model of HCC (Hepa129) with liver fibrosis. Fibrosis was induced by i.p. administration of thioacetamide for 4 weeks. 4MU (5mM) treatment was started 5 days before tumor cell implantation (performed by laparotomy) and injected for 2 weeks. Fibrosis degree was histologically quantified using Ishak score system by Masson's trichrome staining. Hepatic HA levels were analyzed by histological staining using a HA-binding protein. Costimulatory molecules (CD11c, CMHII, CD40, CD80, CD86) were analyzed in hepatic dendritic cells (DCs) by flow cytometry. Antitumoral effects of 4MU were analyzed by measuring the main hepatic tumor and its satellites. Proliferation and apoptosis assays were performed in order to analyze direct effect of 4MU on tumor cells. Results: We observed that untreated animals developed a F4-F5 fibrosis level while in mice treated with 4MU this level was reduced (F3). Tumor size was similar in mice treated or not with 4MU (163-189 mm3), however the number of satellites was found 2-folds reduced in treated mice. Mice that received 4MU therapy showed a significant activation of hepatic DCs compared with untreated mice (4,8% vs 2,6%).. In vitro results shown that treatment of tumor cells with 4MU (1mM, 5mM) significantly reduced cell proliferation and induced apoptosis (26,2% for 5mM). These results suggest an important role for hepatic HA during liver fibrogenesis and decreased HA synthesis would ameliorates HCC development. The potential antitumoral mechanisms of 4MU in this model could be inhibition fibrogenesis, stimulation of the immune system by dendritic cells activation and a direct antitumoral effect.