Inhibition of Hyaluronic acid synthesis by 4-methylumbelliferone (4MU) reduces fibrosis in a murine model of orthotopic hepatocellular carcinoma (HCC) associated to liver fibrosis.

FLAVIA PICCIONI; MARIANA MALVICINI; MIGUEL RIZZO; NESTOR KIPPES; CATALINA ATORRASAGASTI; ANDREZ RODRIGUEZ; MARCELO AMANTE; ESTEBAN FIORE; JUAN BAYO; JORGE AQUINO; MARIANA GARCIA; LAURA ALANIZ; GUILLERMO MAZZOLINI

Petersberg

Conferencia; European association for the study of the liver; 2011

European association for the study of the liver

Backgrounds & Aims: Liver fibrogenesis is characterized by an excessive production and deposition of extracellular matrix (ECM) components such as hyaluronic acid (HA), mainly produced by hepatic stellate cells (HSCs) that had transdifferentiated to myofibroblasts. These cells also increase alpha-smooth muscle actin expression levels according to degree of their activation state. Cirrhosis is the endstage consequence of liver fibrosis, and is considered a preneoplastic disease being hepatocellular carcinoma (HCC) one of the major complications. The aim of this work was to analyze if inhibition of HA synthesis by the specific inhibitor 4-methylumbelliferone (4-MU) reduced fibrosis development, and as a consequence, tumoral progression of HCC. Methods: We used an orthotopic Hepa129 HCC model established in fibrotic livers induced by hioacetamide. We evaluated 4-MU effects on HSCs (LX2, CFSC-2G) in vitro by proliferation and apoptosis,and on primary cultured hepatocytes by apoptosis and cytotoxicity assays. Fibrogenesis was also analyzed in vivo by Masson s trichrome and Sirious Red staining for collagen fibres, and immunohistochemistry for alpha-smooth muscle actin. Collagen and alpha-smooth muscle actin quantification was performed by morphometric analysis. Fibrosis stage was assessed according to Metavir score (no fibrosis F0, cirrhosis F4). Results: 4-MU treatment 2-3-folds reduced number of tumor satellites, reduced proliferation (64% and 24% for CFSC-2G and LX2, respectively for 4MU 0,25 mM) and induced apoptosis (Apoptosis index=28 and 24 for CFSC-2G and LX2, respectively, for 4MU 5mM) of HSCs in vitro while primary cultured hepatocytes remained unaffected (5% cytotoxicity). Animals treated with 4-methylumbelliferone developed a reduced fibrosis degree in comparison to controls (F1-2 vs F2-3, respectively). Importantly, treatment decreased significantly the amount of collagen compared to non-treated mice (0.65±0.08% vs 1.48 ±0.11%, respectively), the same as for alpha-smooth muscle actin+ (0.17±0.02% vs 0.40±0.04%, respectively). Conclusions: Our results suggest a role for 4-methylumbelliferone as antitumoral, antifibrotic and non-toxic agent in hepatocellular carcinoma with underlying fibrosis.