We read with great interest an article by Zittermann et al.1 showing that hepatocellular carcinoma (HCC) cells infected with lentivirus for soluble glypican 3 (sGPC3) expression had a lower proliferation rate. The authors indicated that the growth inhibition might be due to the sGPC3 protein secreted by infected HCC cells. Here, we provide direct evidence showing that the sGPC3 protein does in fact inhibit the growth of HCC cells in vitro.
Our experiments are summarized in Figure 1. Consisting of 580 amino acids, GPC3 (also called MXR7, OCI-5, or GTR2-2) is a heparan sulfate proteoglycan that is attached to the cell surface via a glycosyl-phosphatidylinositol (GPI) anchor. We constructed an expression plasmid (called pMH133) for producing sGPC3 (GPC3ΔGPI, amino acid residues Q25-H559) that lacks the COOH-terminal hydrophobic GPI-anchoring domain2 (Fig. 1a). The recombinant sGPC3 protein with a His tag at the COOH-terminal was purified on a 1-mL Ni Sepharose column (HisTrap FF; GE Healthcare, Piscataway, NJ) from the serum-free media of human HEK293F cells (Invitrogen, Carlsbad, CA) transfected with pMH133. Fractions of the single dominant peak were collected using the ÄKTAexplorer FPLC system (GE Healthcare), run on a SDS-PAGE gradient gel (4–20%) (Invitrogen), pooled, and concentrated. Final protein concentration was measured using Coomassie Plus Protein Assay Reagent (Thermo Scientific/Pierce, Rockford, IL). The recombinant sGPC3 protein detected by the 1G12 antibody specific for the COOH-terminal of GPC3 (Santa Cruz Biotechnology, Santa Cruz, CA) was heavily glycosylated and had a high molecular weight consistent with those of native GPC3 proteins found in HCC cells1–5 (Figs. 1b and 1c). To examine whether or not recombinant sGPC3 protein can inhibit the growth of HCC cells, we screened 6 HCC lines (SK-Hep-1, HepG2, Hep3B, Huh-1 Huh-4, and Huh-7) by flow cytometry and found that the HepG2 cell line had the highest protein expression level of GPC3 on the cell surface (Fig. 1d). We then incubated HepG2 cells with various concentrations (1–100 μg/mL) of sGPC3. Figure 1e shows a dose-dependent inhibition of cell proliferation measured by WST-8 cell proliferation assays (Dojindo, Rockville, MD). Using the Prism software (GraphPad Software, San Diego, CA), we estimated EC50 was about 15 μg/mL (0.2 μM). We then treated HepG2 cells with sGPC3 and evaluated the cells daily in the course of 6 days by live cell counting using Countess Automated Cell Counter (Invitrogen) (Fig. 1f) and WST-8 cell progression assays (Fig. 1g). Cell proliferation was significantly inhibited as early as 72 h (day 3) after treatment.
In summary, our data show that recombinant sGPC3 protein can inhibit the proliferation of HCC in vitro. The precise mechanisms require further investigation.