Inhibitory effect of Survivin promoter-regulated oncolytic adenovirus carrying P53 gene against gallbladder cancer

Abstract

Gene therapy has become an important strategy for treatment of malignancies, but problems remains concerning the low gene transferring efficiency, poor transgene expression and limited targeting specific tumors, which have greatly hampered the clinical application of tumor gene therapy. Gallbladder cancer is characterized by rapid progress, poor prognosis, and aberrantly high expression of Survivin. In the present study, we used a human tumor-specific Survivin promoter-regulated oncolytic adenovirus vector carrying P53 gene, whose anti-cancer effect has been widely confirmed, to construct a wide spectrum, specific, safe, effective gene-viral therapy system, AdSurp-P53. Examining expression of enhanced green fluorecent protein (EGFP), E1A and the target gene P53 in the oncolytic adenovirus system validated that Survivin promoter-regulated oncolytic adenovirus had high proliferation activity and high P53 expression in Survivin-positive gallbladder cancer cells. Our in vitro cytotoxicity experiment demonstrated that AdSurp-P53 possessed a stronger cytotoxic effect against gallbladder cancer cells and hepatic cancer cells. The survival rate of EH-GB1 cells was lower than 40% after infection of AdSurp-P53 at multiplicity of infection (MOI) = 1 pfu/cell, while the rate was higher than 90% after infection of Ad-P53 at the same MOI, demonstrating that AdSurp-P53 has a potent cytotoxicity against EH-GB1 cells. The tumor growth was greatly inhibited in nude mice bearing EH-GB1 xenografts when the total dose of AdSurp-P53 was 1 × 10(9) pfu, and terminal dUTP nick end-labeling (TUNEL) revealed that the apoptotic rate of cancer cells was (33.4 ± 8.4)%. This oncolytic adenovirus system overcomes the long-standing shortcomings of gene therapy: poor transgene expression and targeting of only specific tumors, with its therapeutic effect better than the traditional Ad-P53 therapy regimen already on market; our system might be used for patients with advanced gallbladder cancer and other cancers, who are not sensitive to chemotherapy, radiotherapy, or who lost their chance for surgical treatment.

Figure 1

Schematic diagram of the recombinant adenoviruses. Compared with the wild type of adenovirus, the expression cassette of P53 or EGFP was inserted into adenoviral genome to replace E1 region and generated the recombinant adenoviruses Ad‐P53 or Ad‐EGFP. The Survivin promoter was used to regulate the E1a gene, and the expression cassette of P53 or EGFP was inserted into adenoviral E1 region upstream of E1a gene, then generated the recombinant oncolytic adenoviruses AdSurp‐P53 or AdSurp‐EGFP. ITR: inverted terminal repeats; ψ: adenovirus 5 packaging signal; E1a‐pro: the wild type of E1a promoter; E1b‐pro: the wild type of E1b promoter, including E1b‐55KD and E1b‐19KD; CMV: cytomegalovirus promoter; Surp: Survivin promoter.

Figure 2

Detection of Survivin expression and promoter activity. (A), Total RNA was extracted using TriZol reagent and Survivin mRNA was amplified by RT‐PCR. Survivin mRNA was positive in EH‐GB1 and BEL‐7404 cells but not in normal cell line MRC‐5 and BJ. (B), Luciferase reporter gene was used to detect the relative activity of Survivin promoter. The luciferase activities of pGL3‐basic and pGL3‐hSurp were normalized with that of pGL3‐Control in each cell line and shown as percentages. The Survivin promoter activities were high in EH‐GB1 and BEL‐7404 cells and low in MRC‐5 and BJ cells (**p = 0.0026).

Figure 3

Specific proliferation of Survivin promoter‐mediated oncolytic adenovirus. (A), When MOI = 5 pfu/cell, AdSurp‐EGFP proliferated in cancer cells and resulted in high EGFP expression; whereas EGFP expression was low in BJ and MRC‐5 cells. But AdSurp‐EGFP mediated higher level of EGFP expression in cancer cells EH‐GB1 and the same level of EGFP expression in normal cells MRC‐5 when infected at the same MOI of 5 pfu/cell, original magnification × 200. (B), The EGFP‐positive cell percentage of every tested cell line was counted within 5 medium‐power fields (original magnification × 200) under microscope, and showed in histograms, **p < 0.01. (C), Western blotting analysis detected AdSurp‐P53‐mediated E1A and P53 expression in cancer cells, which was significantly higher than that mediated by Ad‐P53, and both AdSurp‐P53 and Ad‐P53 resulted in almost no expression in normal cells at MOI = 5 pfu/cell.

Figure 4

Cytotoxic effect of tumor‐specific AdSurp‐P53 against cancer cells. Cells were seeded into 96‐well plate at 104 cell/well and were infected with AdSurp‐P53 and Ad‐P53 when MOI = 0.01 to 100 pfu/cell; the cytotixic effects of AdSurp‐P53 and Ad‐P53 were compared on cancer cells and normal cells, *p < 0.05; **p < 0.01.

Figure 5

Anti‐tumor effect and apoptosis‐inducing role of AdSurp‐P53 in nude mice. (A), Compared with the control group, both AdSurp‐P53 and Ad‐P53 achieved noticeable therapeutic effects in mice bearing gallbladder cancer EH‐GB1 xenografts, AdSurp‐P53 showed better anti‐tumor efficacy than Ad‐P53, *p < 0.05; **p < 0.01. (B), H&E staining, immunostaining, and TUNEL labeling showed cancer cell pathomorphological changes, P53 expression and apoptosis. (C), Apoptotic cell percentages were counted in 5 high‐power fields, *p < 0.05; **p < 0.01 compared with control group.