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. 2013 Oct 14;11:257.
doi: 10.1186/1479-5876-11-257.

Antitumor Efficacy of a Recombinant Adenovirus Encoding Endostatin Combined With an E1B55KD-deficient Adenovirus in Gastric Cancer Cells

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Free PMC article

Antitumor Efficacy of a Recombinant Adenovirus Encoding Endostatin Combined With an E1B55KD-deficient Adenovirus in Gastric Cancer Cells

Li-xia Li et al. J Transl Med. .
Free PMC article

Abstract

Background: Gene therapy using a recombinant adenovirus (Ad) encoding secretory human endostatin (Ad-Endo) has been demonstrated to be a promising antiangiogenesis and antitumor strategy of in animal models and clinical trials. The E1B55KD-deficient Ad dl1520 was also found to replicate selectively in and destroy cancer cells. In this study, we aimed to investigate the antitumor effects of antiangiogenic agent Ad-Endo combined with the oncolytic Ad dl1520 on gastric cancer (GC) in vitro and in vivo and determine the mechanisms of these effects.

Methods: The Ad DNA copy number was determined by real-time PCR, and gene expression was assessed by ELISA, Western blotting or immunohistochemistry. The anti-proliferation effect (cytotoxicity) of Ad was assessed using the colorimetry-based MTT cell viability assay. The antitumor effects were evaluated in BALB/c nude mice carrying SGC-7901 GC xenografts. The microvessel density and Ad replication in tumor tissue were evaluated by checking the expression of CD34 and hexon proteins, respectively.

Results: dl1520 replicated selectively in GC cells harboring an abnormal p53 pathway, including p53 mutation and the loss of p14(ARF) expression, but did not in normal epithelial cells. In cultured GC cells, dl1520 rescued Ad-Endo replication, and dramatically promoted endostatin expression by Ad-Endo in a dose- and time-dependent manner. In turn, the addition of Ad-Endo enhanced the inhibitory effect of dl1520 on the proliferation of GC cells. The transgenic expression of Ad5 E1A and E1B19K simulated the rescue effect of dl1520 supporting Ad-Endo replication in GC cells. In the nude mouse xenograft model, the combined treatment with dl1520 and Ad-Endo significantly inhibited tumor angiogenesis and the growth of GC xenografts through the increased endostatin expression and oncolytic effects.

Conclusions: Ad-Endo combined with dl1520 has more antitumor efficacy against GC than Ad-Endo or dl1520 alone. These findings indicate that the combination of Ad-mediated antiangiogenic gene therapy and oncolytic Ad therapeutics could be one of promising comprehensive treatment strategies for GC.

Figures

Figure 1
Figure 1
dl1520 inhibited the proliferation of GC cells by selectively replicating in and destroying the cancer cells. A, B) The efficiencies of infection and replication of dl1520 in GC and normal cells. The infection efficiency (A) of dl1520 was shown as the dl1520 DNA copy number relative to β-actin at 0 hours after infection. The replication efficiency (B) of dl1520 was presented as the fold of the dl1520 DNA copy number at indicated time relative to that at 0 hours post-infection (One-way ANOVA, *p<0.05, **p<0.01 compared to that at 0 hours post-infection). C~E) The cytopathic effect (CPE) of dl1520 on GC cells. MTT cell proliferation assays were used to analyze the CPE of dl1520 on AGS (C), MGc80-3 (D) and SGC-7901 (E) GC cells. The results are presented as the percentages of viable cells related to the negative control (one-way ANOVA, *p<0.05, **p<0.01 compared to that at 0 MOIs). F) Western blotting analysis of protein levels of p14ARF in GC cells (Actin was used as the internal control). G) Quantitative RT-PCR analysis of the relative mRNA levels of p14ARF and p53 (normalized to that of GAPDH). H) The replication of dl1520 after modifying the p14ARF levels by knockdown or overexpression. AGS cells were transfected with pCD-p14ARF plasmid (pcDNA3.1(+) as a negative control), and MGc80-3 cells were transfected with p14ARF siRNA (si-p14ARF) (scrambled siRNA as a negative control). The cells were analyzed p14ARF expression by Western blotting after 48 hours post-transfection (H upper). Or the cells were infected with 10 MOIs of dl1520 after 24 hours post-transfection, and dl1520 DNA copy numbers were analyzed in GC cells after 48 hours post-infection (normalized against that at 0 hours) (H lower). (Student’s t test, *p< 0.05 compared with their respective control).
Figure 2
Figure 2
dl1520 rescued the replication of Ad-Endo in GC cells by supplying the E1A and E1B19k gene products. The replication of Ad-Endo is presented as the increase in the Ad-Endo DNA copy number, which was determined by real-time PCR. The results are shown here as the fold change in the Ad-Endo DNA copy number at the indicated time points relative to that at 0 hours post-infection. A) Ad-Endo DNA copies in GC cells at different time points after infection with 10 MOIs of Ad-Endo alone or in combination with 10 MOIs of dl1520 (two-way ANOVA, *p<0.05, **p<0.01 compared to that at 0 hours post-infection). B) The Ad-Endo DNA copy number in GC cells at 48 hours after infection with 10 MOIs of Ad-Endo alone or in combination with increasing MOIs of dl1520 (one-way ANOVA, *p<0.05, **p<0.01 compared to that of dl1520 at 0 MOIs). C) The Ad-Endo DNA copy number in MGc80-3 cells transiently transfected with the E1A or/and E1B19k genes 48 hours after infection with 10 MOIs of Ad-Endo (one-way ANOVA, **p<0.01 compared to the control pCD-EGFP). The ectopic expression of the E1A and E1B19k genes in MGc80-3 cells was analyzed by Western blotting (actin was used as the internal control) (C, Left).
Figure 3
Figure 3
dl1520 promoted endostatin expression by Ad-Endo in GC cells. GC cells were infected with Ad-Endo alone or combination with dl1520, and then the culture supernatants were collected at different time points. The endostatin concentrations were measured using a human endostatin ELISA kit (Shanghai ExCell Biology, Inc., Shanghai, China). The minimum detectable dose using this kit is 30 pg/mL. A) Endostatin concentrations in the culture supernatants of GC cells at different time points after infection with 10 MOIs of Ad-Endo alone or in combination with 10 MOIs of dl1520 (two-way ANOVA, *p<0.05, **p<0.01 compared to Ad-Endo alone). B) Endostatin concentrations in the culture supernatants at 48 hours after infection with 10 MOIs of Ad-Endo alone or in combination with increasing MOIs of dl1520 (one-way ANOVA, *p<0.05, **p<0.01 compared to that of dl1520 at 0 MOIs).
Figure 4
Figure 4
Ad-Endo enhanced the cytotoxic effects of dl1520 in GC cells. The in vitro cytotoxic effects were analyzed using the MTT cell proliferation assay. The proliferation activities of GC cells are represented as OD values at 570 nm (630 nm was used as the reference wavelength). The cytotoxic effects are represented as the inhibition rates (or inhibitory effects), and were calculated as follows: [(ODnegative control - ODexperiment)/ODnegative control]  ×  100%. A) The cytotoxic effects of 10 MOIs of Ad-Endo alone, 10 MOIs of dl1520 alone or the combination of the two on GC cells (one-way ANOVA, *p<0.05 compared with dl1520 alone and p<0.01 compared with Ad-Endo alone). B~D) The cytotoxic effects of 10 MOIs of dose dl1520 alone or in combination with increasing doses of Ad-Endo on AGS (B), MGc80-3 (C) and SGC-7901(D) GC cells (two-way ANOVA, *p<0.05, **p<0.01 compared to that of Ad-Endo at 0 MOIs). E) Ad replication in GC cells. Cells were infected 10 MOIs of dl1520 alone or in combination with 10 MOIs of Ad-Endo, and then the total Ad (Ad-Endo and dl1520) DNA copy number was detected by real-time PCR. Ad replication is presented as the increase in the total Ad DNA copy number. The results are shown here as the fold change in the total Ad DNA copy number at the indicated time points relative to that at 0 hours post-infection with dl1520 alone (two-way ANOVA, *p < 0.05, **p < 0.01 compared with dl1520 alone).
Figure 5
Figure 5
Antitumor effects of Ad-Endo combined with dl1520 on GC SGC-7901 xenografts in nude mice. A) The plasma concentration of endostatin in mice treated with a single intratumoral injection of Ad-Endo (5×108 pfu) alone or in combination with dl1520 (5×108 pfu). Blood plasma was sampled at the indicated time points, and the endostatin concentration was detected by ELISA (n=3) (two-way ANOVA, **p<0.01 compared to Ad-Endo alone). B, C) The growth inhibition of SGC-7901 xenografts by Ad-Endo or/and dl1520. B) The growth curves of SGC-7901 xenografts (two-way ANOVA, *p<0.01 compared to the control group; **p<0.01 compared to other three groups). C) Tumor weights (one-way ANOVA, *p< 0.01 compared with control group; **p<0.01 compared with the Ad-Endo+dl1520 group). D) Immunohistochemical assays of SGC-7901 xenografts to detect the expression of endostatin (upper), CD34 (middle) and the Ad hexon protein (lower) (×200). Brown staining indicates gene expression, and blue staining represents the cell nuclei. CD34, a marker of vascular endothelial cells, is used to assess the microvessel density in the tumor. Hexon staining indicates Ad replication.

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