Antitumoral effects of hyaluronic acid inhibition by 4-methylumbelliferone (4MU) in an orthotopic hepatocellular carcinoma model in mice

LAURA ALANIZ; FLAVIA PICCIONI; MANGLIO RIZZO; MARIANA G. GARCIA; ANDRES RODRIGUEZ; MARIANA MALVICINI; JORGE B. AQUINO; CATALINA ATORRASAGASTI; JUAN BAYO; MARCELO SILVA; GUILLERMO MAZZOLINI

Congreso; The Liver Meeting 2010--AASLD's 61st Annual Meeting; 2010

American Association for the study of liver diseases

Introduction: Liver fibrosis is characterized by an excessive accumulation of extracellular matrix (ECM) components including hyaluronic acid (HA) that leads to progressive liver failure. In addition, cirrhosis is considered a pre-neoplastic disease for hepatocellular carcinoma (HCC). Altered HA deposition and composition has the ability to modulates tumor aggressiveness but its role in HCC is unknown. 4-Methylumbelliferone (4-MU) is a HA synthesis inhibitor with reported anticancer properties. Aim: To analyze the effects of HA inhibition by 4MU on HCC growth and fibrogenesis. Methods: We used an orthotopic in vivo model of HCC (Hepa129 cell line syngeneic with C3H/He mice) established in fibrotic livers. Fibrosis was induced by i.p. administration of thioacetamide (TAA) for 4 weeks. 4MU treatment was started 2 days before HCC cell implantation and continued up to 2 weeks. Fibrosis degree was quantified by applying Ishak score system. Hepatic HA levels were analyzed by histological staining using a HA-binding protein and systemic HA by ELISA. Surface markers (CD11c, CMH-II, 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 mass and its satellites. Proliferation (3H-thymidine) and apoptosis (anexin V) assays were performed to analyze a direct effect of 4MU on tumor cells. Results: Hepa129 cells expressed CD44 and have the ability to both bind and to produce HA. In vitro results showed that treatment of tumor cells with 4MU (1mM, 5mM) significantly reduced HCC cell proliferation and induced apoptosis (26.2% for 5mM) while nontumoral cells were unaffected. 4MU therapy reduced hepatic and systemic levels of HA. Macroscopic tumor size was similar in mice treated or not with 4MU but microscopic analysis showed important areas of necrosis only in treated animals. In addition, the number of tumor satellites was found 2-3-folds reduced in 4MU-treated mice. No signs of toxicity were observed and aminotransferases were not modified after 4MU treatment. We observed that untreated animals developed a F4-F5 fibrosis degree while mice treated with 4MU showed a reduced amount (F3). In addition, 4MU therapy improved maturation of intra-hepatic DCs (MHC-II and CD86), decreased IL-10  production (218 vs 86 pg/ml) and significantly enhanced MLR activity. In conclusion: These results suggest a role for 4-MU as anticancer agent in HCC. The potential antitumoral mechanisms of 4MU in this model could involve: i) inhibition of fibrogenesis, ii) stimulation of the immune system by dendritic cells activation, and iii) a direct antitumoral effect.