Inhibition of hepatocellular carcinoma growth by adenovirus-mediated expression of human telomerase reverse transcriptase COOH-27 terminal polypeptide in mice

Abstract

A 27-kDa C-terminal fragment of human telomerase reverse transcriptase, hTERTC27, has previously been reported to inhibit the growth and tumorigenicity of HeLa human cervical cancer cells and U87-MG human glioblastoma multiforme cells. However, the antitumor effects of hTERTC27 in hepatoma and its underlying mechanisms are unclear. In the current study, the therapeutic effect of hTERTC27, mediated by recombinant adenovirus, in hepatocellular carcinoma (HCC) was explored in vitro and in vivo to investigate the possible mechanisms. The results indicated that recombinant adenovirus carrying hTERTC27 (rAdv-hTERTC27) effectively inhibited the growth and induced apoptosis of the Hepa 1-6 HCC cells. Dendritic cells transduced with rAdv-hTERTC27 were highly effective at inducing antigen-specific T cell proliferation and increasing the activated cytotoxicity of T cells against Hepa 1-6 cells. HCC was inhibited significantly when a single dose of 5×10⁷ pfu rAdv-hTERTC27 was administered intravenously. In summary, the results of this study demonstrated that rAdv-hTERTC27 may serve as a reagent for intravenous administration when combined with telomerase-based gene therapy and immunotherapy for cancer.

Keywords: cytotoxic T lymphocytes; gene therapy; hTERTC27; hepatocellular carcinoma; immunotherapy.

Figure 1

rAdv-hTERTC27 inhibits growth and induces apoptosis of Hepa 1–6 hepatocellular carcinoma cells. Hepa 1–6 cells were infected with recombinant adenovirus or vehicle for 48 h. (A) Cell viability was assessed using a CCK-8 assay and the results are presented as the mean ± SD of three independent experiments. ^*P<0.05, vs. PBS and rAdv-EGFP. (B) Cell apoptosis was evaluated by a cell death detection kit and the results are presented as the mean ± SD of three independent experiments. ^*P<0.05, vs. PBS and rAdv-EGFP. (C) Apoptotic cells were determined by PI and Hochest 33258 staining. The red and blue colors represent apoptotic cells (magnification, ×200). rAdv, recombinant adenovirus; hTERTC27, 27-kDa C-terminal fragment of human telomerase reverse transcriptase; PI, propidium iodide.

Figure 2

DCs transfected with rAdv-hTERTC27 induce T lymphocyte proliferation and prime cytotoxic activity of CTLs. (A) Following transfection with or without recombinant adenovirus for 24 h, DCs were cocultured with lymphocytes for 72 h at ratios of DC:T of 1:5, 1:10, 1:20 and 1:40. Following stimulation, the lymphocyte proliferation activity was analyzed using CCK-8 colorimetric assays. Experiments were repeated three times and representative results are presented. ^*P<0.05, vs. PBS and rAdv-EGFP. (B) Allogeneic T cells were cocultured with rAdv-hTERTC27-DCs, rAdv-EGFP-DCs and PBS-DCs for 72 h. Hepa 1–6 cells, as target cells, were cocultured with effector cells (CTLs) at the indicated ratios (E:T = 5:1, 20:1 and 40:1) for 72 h. Cytotoxicity assay assessed by CCK-8 colorimetric assays. Experiments were repeated three times and results are presented. ^*P<0.05, vs. PBS and rAdv-EGFP. DCs, dendritic cells; rAdv, recombinant adenovirus; hTERTC27, 27-kDa C-terminal fragment of human telomerase reverse transcriptase; CTL, cytotoxic T lymphocyte; E, effector; T, target.

Figure 3

rAdv-hTERTC27 significantly reduces tumor size and prolongs survival rate of C57BL/6 mouse hepatic capsule injected with Hepa 1–6 cells. (A) Following 7-days post tumor cell injection, 5.0×10⁷ pfu of rAdv-hTERTC27 and rAdv-EGFP, and an equal volume of PBS and hTERTC27 polypeptide, were directly injected via the tail vein. Images of the mice were obtained following sacrifice. (B) rAdv-hTERTC27 markedly inhibits tumor volume. (C) rAdv-hTERTC27 significantly increases survival time. Mean life span of each mouse was observed daily. ^*P<0.05 vs. PBS, rAdv-EGFP and hTERTC27.