control group. The data supported that HS20 could inhibit GPC3-positive liver tumor growth in vivo via signaling pathways other than the canonical Wnt/-catenin pathway. Discussion HSPGs play pivotal roles in tumorigenesis, tumor progression, and metastasis. These processes can be mediated by interactions with the HS chains of HSPGs. The HS chains serve as co-receptors for growth factors and facilitate ECM-growth factor interaction. In the present study, we found that the HS chains of GPC3 were involved in HCC cell migration via coordination with HGF signaling. Our findings suggest the role of HS in cell motility and provide evidence of the inhibition of tumor pathogenesis by targeting the HS domain of HSPGs. The emerging role of HSPG in tumor progression supports HS-based treatment for cancer therapies. One such strategy involves the heparanase inhibitor PI-88, which is a highly sulfated oligosaccharide mixture. PI-88 can inhibit angiogenesis and tumor growth by preventing FGF and VEGF receptor-HS interaction, and it is currently in a phase III clinical trial for HCC after surgical resection. PG545, an analog of PI-88, has been selected as the leading clinical candidate and is currently in a phase I clinical trial. Delteparin, a low molecular 9 / 13 Antibody Targeting the Heparan Sulfate Chains of Glypican-3 Fig 6. HS20 inhibited HGF-induced tumor spheroid formation. Representative photographs of Hep3B and Huh-7 spheroid. Hep3B and Huh-7 cells were treated with 50ng/ml HGF alone or co-cultured with 50 g/mL HS20 for 20 days. Human IgG was used as negative control. Scale bar indicates 50 m. The spheroid volume in each group described in. Each dot represents a spheroid. P<0.01 and P<0.001. Western blot to detect the expression of total c-Met and phosphorylated c-Met in spheroid. Hep3B cells and Huh-7 cells were co-cultured with 50ng/ml HGF and 50 g/mL HS20 for 20 days in a low attachment plate. Human IgG was used as a negative control. BALB/c nu/nu mice were subcutaneously inoculated with 10x106 Hep3B cells. When tumors reached an average volume of 100 mm3, mice were grouped and intravenously administered 25mg/kg HS20 twice a week. Values are mean SE from different mice. P<0.05. n = 4 for each group. PBS was used as vehicle. Arrows indicate antibody injection. BALB/c nu/nu mice were subcutaneously inoculated with 5x106 HepG2 cells. When tumors reached an average volume of 100 mm3, mice were grouped and intravenously administered 20mg/kg HS20 twice a week. Values are mean SE from different mice. P<0.05. n = 5 for each group. PBS was used as vehicle. Arrows indicate antibody injection. doi:10.1371/journal.pone.0137664.g006 weight non-anticoagulant heparin, also shows promising efficacy in the treatment of small cell lung cancer. These studies indicate that targeting PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19736622 HS may be a feasible option for cancer therapy. However, HS mimics alone may not provide effective A-83-01 chemical information anti-tumor treatment due to their limited specificity and potential side effects. Antibody therapy could represent a promising approach for HCC therapy given its high specificity to the tumor antigen. In addition to affecting HCC cells, HS20 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19734877 also blocks C-met activation in HepG2, a hepatoblastoma cell line with GPC3 expression. This provides the potential application of HS20 in different liver malignancies. GPC3 participates in HCC pathogenesis via multiple signaling mechanisms. Our previous study shows that HS20 blocks the interaction of GPC3 and Wnt3a, and subsequently inhibits th