Adenovirus-mediated herpes simplex virus thymidine kinase gene therapy combined with ganciclovir induces hepatoma cell apoptosis

Abstract

The present study aimed to examine the apoptotic effects of adenovirus (ADV)-mediated herpes simplex virus thymidine kinase (ADV-TK) combined with ganciclovir (GCV) in tissues obtained from patients with hepatocellular carcinoma in order to provide a theoretical basis for the development of this gene therapy program. Apoptosis detection was conducted using the terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling assay and the apoptosis index was compared between the experimental; and control groups. Furthermore, the protein expression levels of caspase-3, B-cell lymphoma-2 (Bcl-2), Bcl-2-assoicated protein X (Bax) and nuclear factor (NF)-κB were examined in pathological specimens using immunohistochemical staining. The Bax/Bcl-2 ratio and the release of cytochrome c were examined using western blot analysis. Results indicated that combined ADV-TK and GCV treatment significantly increased the number of apoptotic cells compared with the control group (P<0.05). Immunohistological analysis revealed that ADV-TK and GCV treatment significantly increased the number of caspase-3-positive cells, reduced the Bax/Bcl-2 ratio and NF-κB expression levels and promoted the release of cytochrome c compared with the control group (P<0.01). In conclusion, the present results suggest that combined ADV-TK and GCV treatment exerts its effect through the apoptotic signaling pathway.

Keywords: apoptosis mechanism; gene therapy; hepatocellular carcinoma.

Figure 1.

Combined adenovirus-mediated herpes simplex virus thymidine kinase and ganciclovir treatment induces hepatic cell apoptosis. (A) A TUNEL assay revealed apoptotic-positive hepatic cells marked by green staining. The blue DAPI staining marked intact DNA (magnification, ×400). (B) The apoptotic index was calculated. Data are presented as the mean ± standard deviation. TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling; DAPI, 4′6-diamidino-2-phenylindole.

Figure 2.

Expression of apoptosis-associated proteins in tissues of patients with hepatocellular carcinoma detected by immunohistochemical staining and western blot analysis. (A) Image of positively stained cells and their localization in the combined ADV-TK and GCV treatment group and control groups. Analyses of (B) caspase-3, (C) Bax/Bcl-2 ratio and (D) NF-κB were performed in the combined ADV-TK and GCV treatment group and control group. (E) Representative images of western blot analysis. (F) Histogram of relative protein expression analysis. Data are presented as the mean ± standard deviation. **P<0.01 vs. control. ADV-TK, adenovirus-mediated herpes simplex virus thymidine kinase; GCV, ganciclovir; NF-κB, nuclear factor-κB; Bcl-2, B-cell lymphoma; Bax, B-cell lymphoma-associated protein X; Cyt C, cytochrome c; Cyto, cytosol; Mito, mitochondria.