Targeted Therapy of Hepatocellular Carcinoma Using Gemcitabine-Incorporated GPC3 Aptamer

Abstract

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver, which can progress rapidly and has a poor prognosis. Glypican-3 (GPC3) has been proposed to be an important diagnostic biomarker and therapeutic target for HCC. Aptamers have emerged as promising drug delivery vehicles because of their high binding affinity for target molecules. Herein, we developed G12msi, a gemcitabine-incorporated DNA aptamer, targeting GPC3, and evaluated its binding specificity and anti-tumor efficacy in GPC3-overexpressing HCC cell lines and murine xenograft models. GPC3-targeted aptamers were selected by using the SELEX process and the chemotherapy drug gemcitabine was internally incorporated into the aptamer. To determine the binding affinity and internalization of the G12msi, flow cytometry and confocal microscopy were performed on GPC3-positive HepG2, Hep3B, and Huh7 cells, as well as a GPC3-negative A431 cell. The anti-tumor activities of G12msi were evaluated with in vitro and in vivo models. We found that G12msi binds to GPC3-overexpressing HCC tumor cells with high specificity and is effectively internalized. Moreover, G12msi treatment inhibited the cell proliferation of GPC3-positive HCC cell lines with minimal cytotoxicity in control A431 cells. In vivo systemic administration of G12msi significantly inhibited tumor growth of HCC HepG2 cells in xenograft models without causing toxicity. These results suggest that gemcitabine-incorporated GPC3 aptamer-based drug delivery may be a promising strategy for the treatment of HCC.

Keywords: GPC3; aptamer; aptamer-drug conjugate; gemcitabine; hepatocellular carcinoma.

Figure 1

Characterization of G12msi aptamers. (a) The secondary structures of G12msi generated by RNAstructure (http://rna.urmc.rochester.edu/RNAstructure.html). The sequence of G12msi is shown with modifications indicated (P, 5-[N-(1-naphthylmethyl)carboxamide]-2′-deoxyuridine; Z, 5-(N-benzylcarboxamide)-2′-deoxyuridine; N_Me, 2′-O-methoxy nucleotides; N_F, 2′-deoxy-2′-fluoro nucleotides; L, C3-spacer; D, gemcitabine). (b) Determination of binding affinity of G12m, G12ms and G12msi aptamers to GPC3 protein. Equilibrium dissociation constants (K_d) were determined by incubating GPC3 protein at varying concentrations of aptamer. (c) Nuclease resistance of G12m and G12msi aptamers in 50% serum. Aptamers were incubated with an equal volume of serum for 0, 3, 6, 12, 24, 48, and 72 h at 37 °C and analyzed by using polyacrylamide gel electrophoresis.

Figure 2

In vitro affinity and specificity of G12msi aptamers. (a) The specific binding capacity of G12msi to HCC cells was assessed by using flow cytometry. GPC3-positive HepG2, Hep3B, and Huh7 cell lines, and GPC3-negative A431 cells were stained with Cy5-labeled G12msi or scrambled G12msi. (b) Confocal microscopy analysis with Cy5-labeled G12msi or scrambled G12msi aptamers (Red) on GPC3 positive HepG2, Hep3B, Huh7 cells, and GPC3 negative A431 cells at 4 °C. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 20 μm.

Figure 3

Internalization and intracellular localization of G12msi aptamers. (a) Representative z-stack images with orthogonal side-views of HepG2, Hep3B, Huh7, and A431 cells treated with Cy5-labeled G12msi or scrambled G12msi aptamers (red) at 37 °C. Nuclei were stained with DAPI (blue). (b) The cellular trafficking of G12ms and G12msi aptamers in HepG2 cells. Representative images showing the intracellular colocalization of the Cy5-labeled G12ms and G12msi aptamers (red) with a lysosomal marker (LysoTracker™ Green DND-26; green) by using confocal microscopy. The nuclei were counterstained with DAPI (blue). Scale bars, 20 μm.

Figure 4

In vitro cytotoxicity of G12msi aptamer. (a) Cell viability analysis of GPC3-positive HepG2 and GPC3-negative A431 cells after treatment with increasing concentration of G12msi aptamer or gemcitabine. The relative amounts of viable cells were evaluated using the CCK-8 assay and cell viability (%) was normalized with untreated control cells. Data are shown as mean ± SD from three independent experiments. * p < 0.05, *** p < 0.0001, compared to control cells. (b) Representative confocal microscopic images of γ-H2AX nuclear foci (green) in response to G12msi aptamer. Nuclei were counterstained with DAPI (blue). Results represent the average of three independent experiments ± SD. * p < 0.05.

Figure 5

(a) Effect of nucleoside transporter inhibitor on the anti-proliferative activity of G12msi aptamer. Percentage of cell viability of G12msi aptamer and gemcitabine obtained after treatment of HepG2 cells with dipyridamole (DIP). Results are expressed as mean ± SD. * p < 0.05, ** p < 0.001, *** p < 0.0001. (b) Cell cycle analysis of HepG2 cells treated with G12msi and gemcitabine with or without dipyridamole. After staining with PI, cell cycle distribution was analyzed by using flow cytometry. (G1, gap1 phase; S, synthesis phase; G2/M, gap2/mitosis phase).

Figure 6

In vivo therapeutic efficacy of G12msi. (a) The growth curves of HepG2 tumor-bearing mice treated with intravenous injections of gemcitabine (18 mg/kg), G12msi (50 mg/kg, 100 mg/kg, 150 mg/kg, and 200 mg/kg), or vehicle (DPBS) alone. Representative image of tumors excised on day 15. * p < 0.05 (b) Changes in mean tumor weight and (c) the body weights of mice treated with gemcitabine, G12msi and vehicle. Red arrows indicate the time points of injection. Data are presented as the mean ± SD. * p < 0.05, compared to vehicle-treated group.