13, 14 To adjust for the transfection efficiency, 10 ng of the pRL-CMV vector (Promega) was cotransfected. Twenty-four hours later, cell lysates were prepared, and firefly and Renilla luciferase activities were quantitated with a dual-luciferase reporter assay system (Promega).
BALB/c nu/nu nude mouse xenografts were derived from SULF2-negative Hep3B vector and SULF2-positive Hep3B SULF2-5 cells.11 Immunohistochemistry was performed with an antibody against SULF2, GPC3, Wnt3a, or β-catenin.11 The primary antibody was replaced with 1% BSA/trishydroxymethylaminomethane-buffered saline for negative controls. The institutional animal care and use committee approved the protocols. Tissue sections were stained with antibodies against Ki-67 (Dako; 1:100) and caspase-3 (Cell Signaling; Selinexor 1:800) with a Dako Autostainer Plus and were counterstained with hematoxylin. Liver sections were TUNEL-stained with a peroxidase in situ cell death detection Tyrosine Kinase Inhibitor Library kit (Roche Diagnosis GmbH, Mannheim, Germany). The number of TUNEL-positive cells per 6 high-power fields (HPFs) was quantified. All data represent at least three independent experiments and are expressed as means and standard errors of the mean. Differences between groups were compared with an unpaired, two-tailed t test. Wnt3a is an important regulator of HCC growth.5 Desulfation of cell surface HSPGs by quail sulfatase 1 has been proposed to release sequestered Wnt ligands
bound to HSPGs at the cell surface and thus enhance the binding MCE of released Wnts to their Frizzled receptors.15 We investigated (1) the effects of SULF2 on Wnt signaling in HCC cells upon exposure to exogenous Wnt3a and (2) whether SULF2 activation of Wnt signaling is dependent on HS. Hep3B vector and Hep3B-SULF2-H cells were treated with the Wnt3a ligand (0, 2, or 10 ng/mL) for 24 hours and washed extensively. Wnt3a levels in cell lysates
were then compared by western immunoblotting. In Hep3B vector cells, there was a small increase in Wnt3a when cells were treated with 2 ng/mL Wnt3a, but there was no further increase at 10 ng/mL. In Hep3B-SULF2-H cells, the basal level of Wnt3a was higher. Treatment with 2 ng/mL Wnt3a did not increase Wnt3a; however, 10 ng/mL Wnt3a led to a substantial increase in Wnt3a, and this suggested that SULF2 increased endogenous Wnt3a levels (Fig. 1A). Moreover, the TOPFLASH luciferase reporter assay showed that Wnt3a stimulation of transiently transfected Hep3B-SULF2 cells induced significant Wnt/β-catenin pathway activity (P < 0.0002) as early as 6 hours after transfection and was sustained over 24 hours (Fig. 1B,C). Similar SULF2 enhancement of Wnt3a-induced TOPFLASH expression occurred in PLC/PRF/5 cells, which also have low SULF2 expression (P < 0.03; Supporting Fig. 1). Next, we determined whether Wnt3a binding to HCC cells is HS-dependent. Wnt3a binding was inhibited by HS in a dose-dependent manner (Fig. 2A-C).