Fucoidan-induced ID-1 suppression inhibits the in vitro and in vivo invasion of hepatocellular carcinoma cells

Abstract

Introduction: Hepatocellular carcinoma (HCC) is a fast growing tumor associated with a high tendency for vascular invasion and distant metastasis. Fucoidan displays inhibitory effect on proliferation of cancer cells and protective effects on hepatocyte. In this study, we investigated the anti-metastatic effect of fucoidan on HCC cells, the key signal that modulates metastasis and the hepatoprotective effect over bile acid (BA)-induced apoptosis. Methods: The anti-metastatic effect of fucoidan was evaluated in vitro using an invasion assay with human HCC cells (Huh-7, SNU-761, and SNU-3085) under both normoxic (20% O2 and 5% CO2, at 37 ºC) and hypoxic (1% O2, 5% CO2, and 94% N2, at 37 ºC) conditions. Complementary DNA (cDNA) microarray analysis was performed to find the molecule which is significantly suppressed by fucoidan. In vivo study using a distant metastasis model by injecting HCC cells into spleen via portal vein was performed to confirm the inhibitory effect by fucoidan or small interfering RNA (siRNA) transfection. Immunoblot analyses were used to investigate the signaling pathway. Primary hepatocyte culture was performed to evaluate fucoidans hepatoprotective effect. Results: Fucoidan significantly suppressed the invasion of human HCC cells (Huh-7, SNU-761, and SNU-3085). Using cDNA microarray analysis, we found the molecule, ID-1, which was significantly suppressed by fucoidan treatment. Downregulation of ID-1 by siRNA significantly decreased invasion of HCC cells, both in vitro and in vivo (both P < 0.05) in a NDRG/CAP43-dependent manner. In immunoblot assay, downregulation of ID-1 by siRNA decreased the expressions of epithelial-mesenchymal transition markers including CK19, vimentin, MMP2, and fibronectin. Immunofluorescence study also revealed that actin rearrangement was inhibited when ID-1 was down-regulated in HCC cells. Interestingly, in SNU-761 cells, the ID-1 expressions under hypoxic conditions were lower as compared to those under normoxic conditions. Under hypoxic conditions, HIF-1α up-regulated NDRG-1/CAP43, while HIF-2α down-regulated ID-1, which might be a compensatory phenomenon against hypoxia-induced HCC invasion. Furthermore, fucoidan decreased BA-induced hepatocyte apoptosis as shown by the attenuation of caspase-8, and -7 cleavages, and suppressed the mobilization of caspase-8 and FADD into the death-inducing signaling complex. Conclusions: In conclusion, NDRG/CAP43-dependent down-regulation of ID-1 suppressed HCC invasion both in vitro and in vivo, which was modulated by fucoidan treatment, suggesting ID-1 is a novel therapeutic target for the treatment of metastatic HCC. Moreover, the compensatory down-regulation of ID-1 against hypoxia-induced HCC invasion was observed. Fucoidan protected hepatocytes from BA-induced apoptosis and preferentially inhibited invasion of HCC cells suggesting it may be a potent suppressor of tumor invasion with hepatoprotective effects.