Suppression of Glypican 3 Inhibits Growth of Hepatocellular Carcinoma Cells through Up-Regulation of TGF-β2

Abstract

Glypican 3 (GPC3) is a valuable diagnostic marker and a potential therapeutic target in hepatocellular carcinoma (HCC). To evaluate the efficacy of targeting GPC3 at the translational level, we used RNA interference to examine the biologic and molecular effects of GPC3 suppression in HCC cells in vitro and in vivo. Transfection of Huh7 and HepG2 cells with GPC3-specific small interfering RNA (siRNA) inhibited cell proliferation (P < .001) together with cell cycle arrest at the G(1) phase, down-regulation of antiapoptotic protein (Bcl-2, Bcl-xL, and Mcl-1), and replicative senescence. Gene expression analysis revealed that GPC3 suppression significantly correlated with transforming growth factor beta receptor (TGFBR) pathway (P = 4.57e-5) and upregulated TGF-β2 at both RNA and protein levels. The effects of GPC3 suppression by siRNA can be recapitulated by addition of human recombinant TGF-β2 to HCC cells in culture, suggesting the possible involvement of TGF-β2 in growth inhibition of HCC cells. Cotransfection of siRNA-GPC3 with siRNA-TGF-β2 partially attenuated the effects of GPC3 suppression on cell proliferation, cell cycle progression, apoptosis, and replicative senescence, confirming the involvement of TGF-β2 in siRNA-GPC3-mediated growth suppression. In vivo, GPC3 suppression significantly inhibited the growth of orthotopic xenografts of Huh7 and HepG2 cells (P < .05), accompanied by increased TGF-β2 expression, reduced cell proliferation (observed by proliferating cell nuclear antigen staining), and enhanced apoptosis (by TUNEL staining). In conclusion, molecular targeting of GPC3 at the translational level offers an effective option for the clinical management of GPC3-positive HCC patients.

Antitumor Effect of SIRT1 Inhibition in Human HCC Tumor Models In Vitro and In Vivo

“…GPC3 promotes growth of HCC cells through stimulation of the canonical Wnt signaling pathway that regulates the expression of many downstream oncogenic proteins. A recent study has shown that molecular targeting of GPC3 enhanced TGF-b2 expression and signaling, which in turn inhibited HCC cell proliferation and cell-cycle progression, and induced replicative senescence (37). Loss of expression of AFP and GPC3 together with an increase of CDH1 suggests that cells are gaining a more differentiated phenotype.…”

Glypican-3 promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells through ERK signaling pathway

“…Depending on different cellular environments, GPC3 may either promote or inhibit cell growth. For example, transfection of Huh7 and HepG2 cells with GPC3-specific siRNA effectively inhibited cell proliferation (25). Cheng et al (26) reported that NIH3T3 cells transfected with a GPC3 expression vector revealed overexpression of GPC3 and excessive cell proliferation.…”

Anti-cancer Effects of Glypican-3 on Huh-7 Hepatocellular Carcinoma Cells

“…Of note, silencing GPC3 in hepatocellular carcinoma cells has been shown to inhibit cell proliferation and increase cell apoptosis [21]. Moreover, a study has shown that suppression of GPC3 inhibits cell proliferation and cell cycle progression in HepG2 cells but no significant effect in the Huh-7 cells [9]. Such a discrepancy could exist because of different cell lines as well as experimental conditions applied for the studies.…”

“…Glypicans regulate several signaling pathways such as Wnts, hedgehogs, fibroblast growth factors which could exert stimulatory or inhibitory effects on cells. In cells predominately controlled by hedgehogs signaling, GPC3 inhibits cell proliferation, whereas in cell predominately controlled by Wnt signaling pathway, GPC3 stimulates cell proliferation [9][10][11]. GPC-3 have shown to be overexpressed in hepatocellular carcinoma [12].…”

“…Silencing GPC3 in hepatocellular carcinoma cells inhibited cell proliferation and increased cell apoptosis. Moreover, another study shows that suppression of GPC3 inhibits cell proliferation and cell cycle progression in HepG2 cells but no significant effect in the Huh-7 cells [9]. However, the potential effect of overexpression of GPC3 in hepatocellular carcinoma remains unclear.…”