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. 2012 Nov;19(11):1065-74.
doi: 10.1038/gt.2011.180. Epub 2012 Jan 5.

Antitumor effects of bladder cancer-specific adenovirus carrying E1A-androgen receptor in bladder cancer

Z Zhai  1 , Z Wang, S Fu, J Lu, F Wang, R Li, H Zhang, S Li, Z Hou, H Wang, R Rodriguez
Affiliations

Antitumor effects of bladder cancer-specific adenovirus carrying E1A-androgen receptor in bladder cancer

Z Zhai et al. Gene Ther. 2012 Nov.

Abstract

The high frequency of recurrence and poor survival rate of bladder cancer demand exploration of novel strategies. Gene therapy via adenovirus has shown promising potential for the treatment of tumors. We constructed a bladder cancer-specific adenovirus carrying E1A-androgen receptor (AR) under the control of UPII promoter and prostate stem cell antigen enhancer (PSCAE), designated as Ad/PSCAE/UPII/E1A-AR, and investigated its antitumor effects in vitro and in vivo. We demonstrated that Ad/PSCAE/UPII/E1A-AR could be selectively replicated in bladder tumor cell lines (5637, BIU87, EJ and T24) when compared with control adenovirus Ad/PSCAE/UPII/Luc. However, there was no evidence of cytotoxicity for normal human bladder cell line SV-HUC-1 and hepatoma cell line SMMC7721. AR agonist R1881 could strengthen the oncolytic effect of Ad/PSCAE/UPII/E1A-AR in bladder cancer cells. In addition, we demonstrated that intratumoral injection of Ad/PSCAE/UPII/E1A-AR into established subcutaneous human EJ tumors in nude mice could significantly regress the growth of tumor and markedly prolong survival for tumor-bearing mice; on the other hand, saline-treated tumors continued to grow rapidly. Our studies indicate that Ad/PSCAE/UPII/E1A-AR could effectively treat bladder cancer in vitro and in vivo. Furthermore, our findings provide a promising therapeutic modality for the treatment of bladder cancer.

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Figures

Figure 1
Figure 1
Construction and expression of Ad/PSCAE/UPII/E1A-AR. (a) Schematic diagram of the organization elements in the recombinant adenoviruses. Ad/PSCAE/UPII/E1A-AR is a conditionally replicative Ad5 virus, in which the human UPII promoter controls E1A gene expression. (b) RT-PCR analysis of AR, E1A, PSCAE and UPII expression. Human bladder cancer cell line EJ was treated with Ad/PSCAE/UPII/E1A-AR, Ad/PSCAE/UPII/E1A or Ad/PSCAE/UPII/Luc at an MOI of 20 for 72 h. mRNA was extracted from cells for RT-PCR analysis. The primers used are described in Materials and Methods. Glyceraldehyde 3-phosphate dehydrogenase was used as the internal standard.
Figure 2
Figure 2
The detection of luciferase activity. Bladder cancer cell lines and normal bladder cell line SV-HUC-1 were infected with Ad/PSCAE/UPII/Luc at an MOI of 20 for 24 h, and luciferase activity was analyzed and represented as mean±s.d. **P<0.01 versus SV-HUC-1; *P<0.05 versus SV-HUC-1.
Figure 3
Figure 3
Ad/PSCAE/UPII/E1A-AR inhibited the proliferation of bladder cancer cells and induced apoptosis in vitro. (A) Cell viability was examined after being treated with Ad/PSCAE/UPII/E1A-AR (◻), Ad/PSCAE/UPII/E1A (○) or Ad/PSCAE/UPII/Luc (▼) at an MOI of 0.1, 1, 10 and 100. Ad/CAE/UPII/E1A-AR suppressed the growth of bladder cancer cells, whereas it showed no inhibitory effect in normal bladder cells and non-bladder cancer cells. (B) Cell cycle was analyzed after treatment with Ad/PSCAE/UPII/E1A-AR or Ad/PSCAE/UPII/E1A using propidium iodide staining, and bladder cancer cells were infected with Ad/PSCAE/UPII/Luc as a control. (c) Quantitative assessment of the percentage of EJ, 5637, BIU87 and T24 cells in the sub-G1 phase. (D, E) Apoptotic cells were detected by transferase-mediated dUTP nick-end labeling. The number of apoptotic EJ, 5637 and BIU87 cells in the Ad/PSCAE/UPII/E1A-AR-treated group was significantly higher than that of T24 cells, and the number of apoptotic EJ, 5637 and BIU87 cells in the Ad/PSCAE/UPII/E1A-AR-treated group was significantly higher than that infected with Ad/PSCAE/UPII/E1A. Bladder cancer cells were infected with Ad/PSCAE/UPII/E1A-AR or Ad/PSCAE/UPII/E1A (20 MOI) for 72 h (× 100). (A, C) Ad/PSCAE/UPII/E1A-AR, (b, d) Ad/PSCAE/UPII/E1A and (e–h) control. (F) Caspase 3 activity was detected as described in Materials and methods, and the results are presented as mean±s.d. (G) Relative E1A gene expression levels of bladder cancer cells (BIU87, T24, 5637 and EJ) and normal bladder cells (SV-HUC-1) treated with Ad/PSCAE/UPII/E1A-AR were examined by semiquantitative RT-PCR with E1A-specific primers. The ΔΔCt method was used and glyceraldehyde 3-phosphate dehydrogenase was used for normalization. Results are presented as mean±s.d. **P<0.01 versus SV-HUC-1; *P<0.05 versus SV-HUC-1. (H) Virus particles in bladder cancer cells infected with Ad/PSCAE/UPII/E1A-AR or Ad/PSCAE/UPII/E1A (MOI = 10) for 72 h by TEM. (a) Bladder cancer cells without virus infection and (b) bladder cancer cells after infection with Ad/PSCAE/UPII/E1A-AR.
Figure 3
Figure 3
Ad/PSCAE/UPII/E1A-AR inhibited the proliferation of bladder cancer cells and induced apoptosis in vitro. (A) Cell viability was examined after being treated with Ad/PSCAE/UPII/E1A-AR (◻), Ad/PSCAE/UPII/E1A (○) or Ad/PSCAE/UPII/Luc (▼) at an MOI of 0.1, 1, 10 and 100. Ad/CAE/UPII/E1A-AR suppressed the growth of bladder cancer cells, whereas it showed no inhibitory effect in normal bladder cells and non-bladder cancer cells. (B) Cell cycle was analyzed after treatment with Ad/PSCAE/UPII/E1A-AR or Ad/PSCAE/UPII/E1A using propidium iodide staining, and bladder cancer cells were infected with Ad/PSCAE/UPII/Luc as a control. (c) Quantitative assessment of the percentage of EJ, 5637, BIU87 and T24 cells in the sub-G1 phase. (D, E) Apoptotic cells were detected by transferase-mediated dUTP nick-end labeling. The number of apoptotic EJ, 5637 and BIU87 cells in the Ad/PSCAE/UPII/E1A-AR-treated group was significantly higher than that of T24 cells, and the number of apoptotic EJ, 5637 and BIU87 cells in the Ad/PSCAE/UPII/E1A-AR-treated group was significantly higher than that infected with Ad/PSCAE/UPII/E1A. Bladder cancer cells were infected with Ad/PSCAE/UPII/E1A-AR or Ad/PSCAE/UPII/E1A (20 MOI) for 72 h (× 100). (A, C) Ad/PSCAE/UPII/E1A-AR, (b, d) Ad/PSCAE/UPII/E1A and (e–h) control. (F) Caspase 3 activity was detected as described in Materials and methods, and the results are presented as mean±s.d. (G) Relative E1A gene expression levels of bladder cancer cells (BIU87, T24, 5637 and EJ) and normal bladder cells (SV-HUC-1) treated with Ad/PSCAE/UPII/E1A-AR were examined by semiquantitative RT-PCR with E1A-specific primers. The ΔΔCt method was used and glyceraldehyde 3-phosphate dehydrogenase was used for normalization. Results are presented as mean±s.d. **P<0.01 versus SV-HUC-1; *P<0.05 versus SV-HUC-1. (H) Virus particles in bladder cancer cells infected with Ad/PSCAE/UPII/E1A-AR or Ad/PSCAE/UPII/E1A (MOI = 10) for 72 h by TEM. (a) Bladder cancer cells without virus infection and (b) bladder cancer cells after infection with Ad/PSCAE/UPII/E1A-AR.
Figure 4
Figure 4
CAR (a) and AR (b) protein expression levels in bladder cancer cells were determined by western blot. (a) The expression of CAR was detected in human bladder cancer cell lines 5637, T24, BIU-87 and EJ and in normal bladder cell line SV-HUC-1. Extracts from HEK293 were used as positive control, and β-actin was used as the internal standard. (b) The expression of AR was detected in human bladder cancer cell lines 5637, BIU-87, EJ and T24 and in normal bladder cell line SV-HUC-1. Extracts from LNCap, prostate cancer cell line, was used as positive control, and β-actin was used as the internal standard.
Figure 5
Figure 5
Expression of E1A protein and the effect of R1881 and flutamide on the activity of Ad/PSCAE/UPII/E1A-AR in bladder cancer cells. (a) R1881 significantly augmented the activity of Ad/PSCAE/UPII/E1A-AR in 5637, EJ and BIU87 cells, but not in T24 and SV-HUC-1 cells. Flutamide had no influence on the activity. Results are presented as mean±s.d. **P<0.01 versus the flutamide-treated group. (b) Expression of E1A protein by western blot analysis. Bladder cancer cells were treated with Ad/PSCAE/UPII/E1A or Ad/PSCAE/UPII/E1A-AR or Ad/PSCAE/UPII/Luc (20 MOI) for 72 h. Extracts from HEK293 were used as positive control, and β-actin was used as the internal standard. (c) Intensity of the optical density of E1A protein was analyzed.
Figure 6
Figure 6
Ad/PSCAE/UPII/E1A-AR suppressed bladder tumor growth in vivo. (A) EJ cells were injected subcutaneously into nude mice, and 2 weeks later the tumor-bearing mice were treated with an intratumoral injection of Ad/PSCAE/UPII/E1A-AR (◻), Ad/PSCAE/UPII/E1A (○), Ad/PSCAE/UPII/Luc (▼) or saline (▽) every other day five times. At 28 days after virus injection, tumor-bearing mice were killed. Bars represent mean±s.d. (B) Survival curves for mice treated with Ad/PSCAE/UPII/E1A-AR (–), Ad/PSCAE/UPII/E1A (…), Ad/PSCAE/UPII/Luc (—) or saline (…). (C) Images of xenografts treated with recombinant adenovirus. (a) Ad/PSCAE/UPII/E1A-AR, (b) Ad/PSCAE/UPII/E1A, (c) Ad/PSCAE/UPII/Luc and (d) saline. Scale bar = 10 mm. (D) Hematoxylin and eosin staining of sections of tumor of the brain, liver and lung harvested from animals receiving intratumoral injection of Ad/PSCAE/UPII/E1A-AR, Ad/PSCAE/UPII/E1A or saline. Arrows indicate the necrosis foci. (E) Particles arranged in the shape of a crystal lattice in tumor cells treated with Ad/PSCAE/UPII/E1A-AR under TEM ( × 30 000).
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