Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines

Abstract

Aim: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line.

Methods: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blotting, respectively. Apoptosis of HCC cells and normal liver cells was detected by cytometric assay with Hoechst33258 staining. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to investigate proliferation of HCC cells and normal liver cells, and cell cycle was assayed by flow cytometry.

Results: RT-PCR, ELISA and Western blotting showed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24. MTT indicated that SG600-IL24 could suppress the growth of HepG2, Hep3B, MHCC97L, with an inhibition rate of 75% ± 2.5%, 85% ± 2.0%, 72% ± 1.8%, respectively (P < 0.01), promote the apoptosis of HepG2, Hep3B, MHCC97L, with an apoptosis rate of 56.59% ± 4.0%, 78.36% ± 3.5%, 43.39% ± 2.5%, respectively (P < 0.01), and block the HCC cell lines in the G2/M phase with a blocking rate of 35.4% ± 4.2%, 47.3% ± 6.2%, 42% ± 5.0%, respectively (P < 0.01) but not the normal liver cell line in a p53-independent manner.

Conclusion: SG600-IL24 can selectively suppress the proliferation and apoptosis of HCC cell lines in vitro but not normal liver cell line L02 in a p53-independent manner. Compared with Ad.IL-24, SG600-IL24 can significantly enhance the antitumor activity in HCC cell lines.

Figure 1

Expression of adenovirus-mediated melanoma differentiation-associated-7/interleukin-24 mRNA in hepatocellular carcinoma cell lines of HepG2, Hep3B and MHCC97L and human normal liver cell line L02. Cells were infected with 10 multiplicity of infection of Ad.IL-24, SG600-EGFP, SG600-IL24,harvested at 24 h, treated as described in “Materials and Methods”. A: Lanes 1, 2, 3, 4: Ad.IL-24 group, SG600-EGFP group, control group and SG600-IL24 group; B: β-actin corresponsively.

Figure 2

Expression of melanoma differentiation-associated-7/interleukin-24 after infection with SG600-IL24 protein in hepatocellular carcinoma cells and normal liver cells. Cells infected with 10 multiplicity of infection of Ad.IL-24, SG600-EGFP and SG600-IL24 were collected 48 h after infection as described in “Materials and Methods”. A: Lane 1: Control group; Lane 2: Ad.IL-24 group; Lane 3: SG600-EGFP group; Lanes 4, 5, 6, 7: HepG2, Hep3B, MHCC97L and L02 SG600-IL24 groups; B: β-actin corresponsively.

Figure 3

Cell viability of different hepatocellular carcinoma cells and normal liver cells infected with oncolytic adenoviruses SG600-IL24 and replicant replication-deficient adenovirus Ad.IL-24 measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at 24, 48, 72 and 96 h after infection. Results are presented as means ± SD (n = 5).

Figure 4

Flow cytometry (A) and histography (B) showing SG600-IL24 induced G2/M arrest in hepatocellular carcinoma cells after SG600-IL24 infection.