Gene therapy for cancer through adenovirus vector‑mediated expression of the Ad5 early region gene 1A based on loss of IGF2 imprinting

Abstract

Loss of (genomic) imprinting (LOI) of the insulin-like growth factor 2 gene (IGF2) is a common epigenetic abnormality in many human cancers. IGF2 imprinting is regulated by differentially methylated domains (DMD) in the imprinting control region that is located between IGF2 and H19 on human chromosome 11. In the present study, combined expression of adenoviral vectors (Ad-EGFP and Ad-E1A) driven by H19 enhancer-DMD-H19 promoter complex was investigated and their effects on the tumor growth were assessed in vitro and in vivo. When infected with Ad-EGFP, the cancer cell lines with the LOI, such as HRT-18 and HT-29 cells, had the expression of the EGFP protein, whereas three cancer cell lines with the maintenance of imprinting (MOI) (HCT-116, MCF-7 and GES-1) had weak expression of EGFP. Furthermore, the expressed Ad-E1A significantly decreased cell viability and induced cell apoptosis only in HRT-18 and HT-29 cells in vitro, and effectively suppressed tumor development in HRT-18 and HT-29 xenograft in nude mice. It is concluded that this gene therapy vector is effective in the suppression of the growth of human colon cancer cells in vitro and in vivo, and that cancer gene therapy based on loss of IGF2 imprinting may prove to be a novel therapeutic option.

Figure 1

EGFP expression in the five cell lines infected with recombinant adenoviral vectors. (A) Infection with Ad-EGFP (10 PFU/cell) 24 h induced expression of EGFP in HRT-18 and HT-29 cells. (B) Microscope images show that there is only weak expression of EGFP in HCT-116, MCF-7 and GES-1 cells infected with Ad-EGFP (10 PFU/cell) at 48 h.

Figure 2

Analysis of the expression of hexon mRNA in infected cell lines. Real-time RT-PCR for hexon mRNA or β-actin (as an endogenous control for normalization) was performed and comparative quantification of hexon mRNA expression levels was carried out. The bars show the expression in relationship to HRT-18 cells after infecting with Ad-E1A 6 h (set to 1) as means ± SD of 3 separate experiments. Graphs are representative of 3 separate experiments. ^*P<0.05, ^**P<0.05 vs. expression level in MOI cells (HCT-116, MCF-7 and GES-1) at 12 h. ^#P<0.01, ^##P<0.01 vs. expression level in MOI cells at 24 h. ^▼P<0.01, ^▼▼P<0.01 vs. expression level in MOI cells at 48 h. ^▲P<0.01, ^▲▲P<0.01 vs. expression level in MOI cells at 72 h.

Figure 3

Analysis of the expression of E1A mRNA and protein in infected cell lines. (A) The expression of E1A and an internal control β-actin was evaluated by RT-qPCR after infecting with Ad-E1A (10 PFU/cell) 24 h. The bars show the expression in relationship to the GES-1 group (set to 1) as means ± SD of 3 separate experiments. E1A mRNA expression increased 7.36-fold in the HRT-18 group (^*P<0.01), 7.94-fold in the HT-29 group (^*P<0.01), 1.34-fold in the HCT-116 group (P>0.05) and 1.25-fold in the MCF-7 group (P>0.05). Graphs are representative of 3 separate experiments. (B) The expression of E1A and an internal control GAPDH was evaluated by western blot analysis after infecting with Ad-E1A (10 PFU/cell) 48 h. DT-A protein was positive in the HRT-18 and HT-29 cell lines, and negative in the HCT-116, MCF-7 and GES-1 cell lines.

Figure 4

In vitro cytotoxic effect of adenoviral vectors carrying the E1A gene. (A–C) Cell viability was determined by MTT assay 48, 72 and 96 h after infecting with increased MOI from 0 to 100 PFU/cell in five cell lines. The cell viability in HCT-116, MCF-7 and GES-1 cells infected with Ad-E1A (0–10 PFU/cell) showed no significant increase compared with the control group (P>0.05), but minimal cytotoxicity was seen when infected with Ad-E1A (50–100 PFU/cell) at 48, 72 and 96 h (P>0.05). The cell viability in LOI cells (HRT-18 and HT-29) infected with Ad-E1A (100 PFU/cell) showed a significant decrease compared with the control group at 48 h (^*P<0.05; ^#P<0.05). The cell viability in LOI cells infected with Ad-E1A (10–100 PFU/cell) showed a significant decrease compared with the control group at 72 h (^*P<0.05, ^**P<0.05, ^***P<0.01; ^#P<0.05, ^##P<0.05, ^###P<0.01). The cell viability in LOI cells infected with Ad-E1A (10–100 PFU/cell) showed a significant decrease compared with the control group at 96 h (^*P<0.01, ^**P<0.01, ^***P<0.01; ^#P<0.01, ^##P<0.01, ^###P<0.01). Graphs are representative of 3 separate experiments. (D) The apoptosis of cells was investigated using flow cytometry analysis 72 h after infecting with Ad-E1A or Ad-EGFP (10 PFU/cell). The apoptotic ratio in MOI cell lines (HCT-116, MCF-7 and GES-1) infected with Ad-E1A showed no significant increase compared with the control group (P>0.05), but the apoptotic ratio in LOI cell lines (HRT-18 and HT-29) infected with Ad-E1A showed a significant increase compared with the control group (^*P<0.01, ^#P<0.01). Graphs are representative of 3 separate experiments.

Figure 5

In vivo antitumor efficacy of adenoviral vectors carrying the E1A gene. (A and B) BALB/c mice were given subcutaneous implantation to establish tumor xenografts (HRT-18 and HT-29). Following the intratumoral injections with either the indicated adenoviruses (a total dosage of 10⁹ PFU/mouse), the tumor volume was observed for 28 days. The antitumor efficacy was marked in the Ad-E1A groups (^*P<0.01 for Ad-E1A vs. control group after 14 days, ^#P<0.01 for Ad-E1A vs. control group after 14 days).

Figure 6

Tumor apoptosis was confirmed after therapy with recombinant adenoviral vectors by TUNEL assay. (A) Immunohistochemical staining for tumor apoptosis (TUNEL) after treatment with recombinant adenoviral vectors. Representative images at ×400 magnification. (B) Quantification of apoptotic index after therapy with recombinant adenoviral vectors. The number of TUNEL-positive cells counted from the randomly selected fields of each tumor section. (^*P<0.01 vs. the PBS control, ^#P<0.01 vs. the PBS control).

Figure 6

Tumor apoptosis was confirmed after therapy with recombinant adenoviral vectors by TUNEL assay. (A) Immunohistochemical staining for tumor apoptosis (TUNEL) after treatment with recombinant adenoviral vectors. Representative images at ×400 magnification. (B) Quantification of apoptotic index after therapy with recombinant adenoviral vectors. The number of TUNEL-positive cells counted from the randomly selected fields of each tumor section. (^*P<0.01 vs. the PBS control, ^#P<0.01 vs. the PBS control).