hGC33-Modified and Sorafenib-Loaded Nanoparticles have a Synergistic Anti-Hepatoma Effect by Inhibiting Wnt Signaling Pathway

Abstract

Delivery of tumor-specific inhibitors is a challenge in cancer treatment. Antibody-modified nanoparticles can deliver their loaded drugs to tumor cells that overexpress specific tumor-associated antigens. Here, we constructed sorafenib-loaded polyethylene glycol-b-PLGA polymer nanoparticles modified with antibody hGC33 to glypican-3 (GPC3 +), a membrane protein overexpressed in hepatocellular carcinoma. We found that hGC33-modified NPs (hGC33-SFB-NP) targeted GPC3⁺ hepatocellular carcinoma (HCC) cells by specifically binding to GPC3 on the surface of HCC cells, inhibited Wnt-induced signal transduction, and inhibited HCC cells in G0/1 by down-regulating cyclin D1 expression, thus attenuating HCC cell migration by inhibiting epithelial-mesenchymal transition. hGC33-SFB-NP inhibited the migration, cycle progression, and proliferation of HCC cells by inhibiting the Ras/Raf/MAPK pathway and the Wnt pathway in tandem with GPC3 molecules, respectively. hGC33-SFB-NP inhibited the growth of liver cancer in vivo and improved the survival rate of tumor-bearing mice. We conclude that hGC33 increases the targeting of SFB-NP to HCC cells. hGC33-SFB-NP synergistically inhibits the progression of HCC by blocking the Wnt pathway and the Ras/Raf/MAPK pathway.

Keywords: Glypican-3; Hepatocellular carcinoma; Targeted therapy; Wnt signal.

Fig. 1

Characterization of NPs. a TEM characterization of NPs, the scale bar indicates 100 nM; b the ¹H NMR spectra of synthetic hGC33-PLGA-b-PEG in CDCl3; c FTIR spectra of hGC33-PEG-b-PLGA and PEG-b- PLGA; d cumulative release profiles of SFB-NP and hGC33-SFB-NP in PBS (pH = 7.4) at 37 °C; e size changes of NPs incubated in DMEM medium containing 10% FBS over 14 d; f size changes of NPs incubated in PBS over 14 d. SFB, sorafenib; TEM, transmission electron microscopy; NPs, nanoparticles; ¹H NMR, ¹H-nuclear magnetic resonance spectroscopy; FTIR, Fourier transform infrared spectroscopy

Fig. 2

Expression of GPC3 and uptake of hGC33-coumarin6-NP in Li-7 and HepG2 cells. The cells, inoculated in the culture plate, were washed with PBS and incubated with 100 μg/ml hGC33-coumarin6-NP in DMEM for 2, 4, and 8 h. Nuclei were stained with DAPI, and the cells were fixed and detected by confocal laser scanning microscopy. GPC3 was not detected in Li-7 cells, but was highly expressed in HepG2 cells as detected by immunocytochemistry. The scale bar indicates 50 μM

Fig. 3

The proliferation of HepG2 and Li-7 cells was inhibited by hGC33, null-NP, and hGC33-null-NP. a Cell proliferation test was performed on GPC3-positive HepG2 cells treated with hGC33, null-NP or hGC33-null-NP; b cell proliferation tests were conducted on GPC3 negative Li-7 cells treated with hGC33, null-NP, and hGC33-null-NP. The cells were incubated with 0–2.5 μM hGC33, null-NP, or hGC33-null-NP for 1 day. The proliferation of the cells was measured with MTT method and standardized as untreated cells. All values represent the mean ± SD. Compared with the control group (0 μM) without antibody treatment, *P < 0.01; c the representative results of response of HepG2 to hGC33, null-NP, and hGC33-null-NP in hGC33 treatment (1.0 μM)

Fig. 4

hGC33-null-NP and hGC33-induced cell cycle arrest in G1 phase and inhibited cyclinD1 expression in HepG2 cells. a Representative cell–cycle diagram of various groups treated with hGC33-null-NP and hGC33. b Cell cycle analysis of various groups treated with hGC33-null-NP and hGC33. The HCC cells were incubated with 0.5 μm hGC33 or hGC33-null-NP (with the molar concentration of hGC33 as reference). *P < 0.05, the G1 phase of hGC33-null-NP or hGC33 cells was compared with that of 0 h cells. c, d After hGC33-null-NP or hGC33 treatment, cyclinD1 was significantly down regulated in HepG2 cells compared with that in the control group

Fig. 5

Expression of Wnt3a, FZD1, and FZD3 in HCC cell lines

Fig. 6

GPC3-activated Wnt signal transduction in HCC. Fifty percent Wnt3a DMEM medium was added with anti-wnt3a antibody, hGC33, or hGC33-null-NP. HepG2 (GPC3⁺⁺), Huh-7 (GPC3⁺) and Li-7 (GPC3⁻) cells were co-incubated for 48 h, and cell proliferation was measured by MTT assay. The data were expressed as mean ± SD (*P < 0.01)

Fig. 7

Inhibition of Wnt3a-induced β-catenin signaling by hGC33-null-NP or hGC33. a Compared with the control group, Wnt/β-catenin signaling pathway in HepG2 and Huh-7 cells treated with hGC33-null-NP or hGC33 was inhibited, and the levels of β-catenin and YAP were decreased, while the increased phosphorylated β-catenin and phosphorylated YAP molecules were unstable, and degraded in cytoplasm. The decreased β-catenin was difficult to maintain in the nucleus and drive the expression of CyclinD1, CD44, VEGF, and c-MYC, which resulted in the decrease of cyclinD1, CD44, VEGF, and c-myc protein levels. b The mechanism pattern of Wnt/β-catenin signaling pathway inhibited by hGC33-null-NP or hGC33

Fig. 8

Effect of hGC33-SFB-NP on EMT inhibition. a Compared with the control group, the hGC33-SFB-NP treatment group had less cell migration. Photographs were taken under an optical microscope (magnification, × 200). The error value represents the standard deviation of three independent experiments. *Compared with the control group, p < 0.01. b Compared with the control group, the EMT-related proteins snail, vimentin, and MMP-2 in HCCs treated with hGC33-SFB-NP decreased, whereas E-cadherin increased. c Molecular mechanism of EMT. EMT, epithelial–mesenchymal transition; MMP-2, matrix metalloproteinase-2; SFB, sorafenib; NP, nanoparticle

Fig. 9

The effect of hGC33-SFB-NP on xenotransplantation of HCC in nude mice and the changes of mice weight. Liver cancer cells were inoculated subcutaneously on the back of each nude mouse (n = 10). After 10 days, the tumor bearing mice were treated with PBS (control), free hGC33, free SFB, hGC33-null-NP, SFB-NP, and hGC33-SFB-NP. Tumor size (a, b) and body weight (c, d) of mice were monitored at designated time points