A novel approach to glioma therapy using an oncolytic adenovirus with two specific promoters

Abstract

Gliomas are the most common type of primary brain tumor in adults, where more than half of the cases are malignant, and the prognosis is poor. The early viral 1A (E1A) protein has been widely recognized to be essential for adenoviral replication and production of progeny virions in human cells, a process that is regulated by human telomerase reverse transcriptase. The p53 gene, as a tumor suppressor, regulates diverse cellular processes, including cell cycle arrest, cell autophagy, senescence and apoptosis. Dysfunction of the p53 pathways is common in malignant gliomas. Exogenous expression of p53 during adenovirus replication in human cancer cells may accelerate cell death and improve the release of early virus progeny. In the present study, a conditionally replicative adenovirus (CRAd) Ad-Tp-E1A-Gp-p53, which expressed functional p53 protein when replicating in cancer cells, was constructed. Next, the level of p53 expression in U251 cells was determined by western blot analysis, and the inhibitory effect of Ad-Tp-E1A-Gp-p53 on U251 cells was detected via an MTT assay. The results indicated that p53 expression was upregulated with an increase in the multiplicity of infection (MOI) of Ad-Tp-E1A-Gp-p53. Additionally, the inhibitory effects of Ad-Tp-E1A-Gp-p53 in different groups were significantly different (P<0.05), with the inhibition ratio of the experimental groups being higher, compared with the control group (P<0.05). Furthermore, the inhibition ratio increased with increases in the MOI of Ad-Tp-E1A-Gp-p53. Therefore, the expression of functional p53 and that of E1A may increase the potency of CRAd, and overexpression of p53 through CRAd is a promising approach to more effective treatments in a number of human cancer types.

Keywords: conditionally replicative adenovirus; early viral 1A; glial fibrillary acidic protein; human telomerase reverse transcriptase; p53.

Figure 1.

Activity of pGL3-hTERTp and pGL3-GFAP in different cells. (A) The plasmids (pGL3-hTERTp and pGL3-GFAP) were transfected into the cultured MRC-5 (11), U251 and T98 G cells (magnification, ×100). (B) The relative luciferase activity of pGL3-hTERTp and pGL3-GFAP was detected using a dual-luciferase reporter assay. Data are presented as the mean ± standard deviation (n=3). **P<0.01. NS, not significant. hTERT, human telomerase reverse transcriptase; GFAP, glial fibrillary acidic protein.

Figure 2.

Verification of pGL3-ENGFAP and PCA19-ENGFAP plasmids by gel electrophoresis, according to the size (bp) of excised fragments. (A) Excised fragments of pGL3-ENGFAP were detected by gel electrophoresis. Lanes 1–6, double digestion with MluI and XhoI; lane 7, GeneRuler™ DNA Ladder mix (10,000 bp); and lanes 8–13, double digestion with PvuI and PvuII. (B) Excised fragments of PCA19-ENGFAP were detected by gel electrophoresis. Lanes 1–6, double digestion with EcoRI and XbaI; lane 7, GeneRuler™ DNA Ladder Mix; and lanes 8–13, digestion with PstI. GFAP, glial fibrillary acidic protein.

Figure 3.

Verification of PCA19-Gp53 and p74-Tp-Gp53 plasmids by gel electrophoresis, according to the size (bp) of excised fragments. (A) Excised fragments of PCA19-Gp53 detected by gel electrophoresis. Lanes 1–6, double digestion with EcoRI and SalI; lane 7, GeneRuler™ DNA Ladder mix (10,000 bp); lanes 8–13, digestion with PvuII. (B) Excised fragments of P74-Tp-Gp53 detected by gel electrophoresis. Lanes 1–6, digestion of PCA19-Gp53 by BglII, SacI and XbaI, NcoI and SalI, BamHI, PstI and NcoI, respectively; and lane 7, GeneRuler™ DNA Ladder Mix (10,000 bp). (C) DNA fragments of Ad-Tp-E1A-Gp-p53 were detected by gel electrophoresis. Lanes 1–3, amplification of Ad-Tp-E1A-Gp-p53 using the GT154+GT156 primer; lanes 4, 10 and 16, negative control; lane 5, amplification of plasmid P74-TP using the GT154+GT156 primer; lanes 6 and 12, GeneRuler™ DNA Ladder Mix; lanes 7–9, amplification of Ad-Tp-E1A-Gp-p53 using the W267+W268 primer; lane 11, amplification of plasmid PENTER-p53 using the W267+W268 primer; lanes 13–15, amplification of Ad-Tp-E1A-Gp-p53 using the W331+W332 primer; and lane 17, amplification of plasmidpGL3-GFAP by using the W331+W332 primer. E1A, early viral 1A; GFAP, glial fibrillary acidic protein.

Figure 4.

Adenovirus Ad-Tp-E1A-Gp-p53 suppresses the growth of U251 cells. (A) Quantitative analysis of p53 expression via western blot analysis. β-actin gene was used as an internal control. (B) Inhibitory effect of Ad-Tp-E1A-Gp-p53 on the growth of U251 cells as detected by MTT. Data are presented as the mean ± standard deviation (n=3). *P<0.05. Group 1, 1 MOI; group 2, 10 MOI; group 3, 100 MOI; group 4, 1,000 MOI. MOI, multiplicity of infection. E1A, early viral 1A.