Adenovirus-mediated anti-AEG-1 ScFv expression driven by stathmin promoter inhibits tumor growth in cervical cancer

Abstract

Background: Astrocyte-elevated gene-1 (AEG-1) is over-expressed in many cancer cells and has multiple key functions in tumor initiation and progression. Currently, targeted-AEG-1 siRNA is one of the most common techniques to down-regulate AEG-1 expression, but the lack of tumor specificity and available delivery system make it difficult to enter clinical trials.

Methods: In this study, we creatively developed an adenovirus-mediated anti-AEG-1 single-chain antibody fragment (ScFv) expression system driven by a tumor specific promoter, and experimented with it in human cervical carcinoma cells to investigate the effect on tumor's proliferation and apoptosis.

Results: The results showed that of HeLa and SiHa cells treated with this recombinant anti-AEG-1 ScFv adenovirus not only inhibited cell growth, but induced apoptosis both in vitro and in vivo. Furthermore, we also observed that the expressions of several apoptosis-related genes like Akt 1 and c-Myc decreased, while NF-κB (p65) and cleaved caspase 3 increased on protein levels in vivo.

Conclusion: We concluded that stathmin promoter-driving anti-AEG-1 ScFv adenoviral system may be a breakthrough for its dual-specificity, and serve as an adjuvant tumor specific therapy method in the treatment for human cervical cancers.

Keywords: AEG-1; Adenovirus; Cervical cancer; ScFv; Stathmin promoter.

Fig. 1

AEG-1 ScFv expression detection. a Strong positive bands of AEG-1 ScFv gene in HeLa and SiHa cell lines transfected with pSFv vector were detected by RT-PCR, while negative results were observed in the cells with control vector. b AEG-1 ScFv expression detection by western blot, and the results were consistent with above RT-PCR

Fig. 2

Stathmin mRNA expression and stathmin promoter activity detection. a Strong positive bands of stathmin gene in human cervical carcinoma cells HeLa and SiHa were detected by RT-PCR, while negative result was observed in normal human cervical epithelial cells NCECS. b Stathmin promoter activity detection by luciferase reporter vectors. Cells were transfected with different luciferase reporter vectors of pGL3-sta-luc (with 451 bp stathmin promoter inserted), pGL3-CMV-luc (positive control) and pGL3-basic (negative control). pGL3-sta-luc showed high activity only in two cervical carcinoma cells compared with pGL3-CMV-luc and pGL3-basic. The histograms represent the average of three independent experiments

Fig. 3

The expressions of anti-AEG-1 ScFv in the cells that infected with recombinant adenovirus. a Strong positive bands of anti-AEG-1 ScFv gene were detected in HeLa and SiHa cell lines infected with SFv recombinant adenovirus by RT-PCR, while negative result was observed in cells infected with control viruses. b Anti-AEG-1 ScFv expression detection by western blot, and the results were as the same as above RT-PCR

Fig. 4

Anti-AEG-1 ScFv inhibited cell growth and induced cervical cancer cell apoptosis. a Cell viability detected by MTT assay. Blocking AEG-1 functions by anti-AEG-1 ScFv in HeLa and SiHa cells followed by a subsequent decrease in cell proliferation by 34.99% and 37.46%, respectively, at 72 h compared with the control cells (P < 0.05). b Colony formation assay. The number of colonies in the HeLa/Ad-sta-SFV and SiHa/Ad-sta-SFv cell groups (averaged colony number 265 and 239, respectively) was significantly reduced compared with uninfected HeLa or SiHa cells. The experiments were repeated thrice. **P < 0.05. c Caspase-3 activities detection. The results showed that the presence of cleaved caspase was markedly detected in Hela/Ad-sta-SFv and SiHa/Ad-sta-SFv compared with control groups, respectively, **P < 0.05, ***P < 0.01. d Western blot analysis for the indicated proteins. e Apoptosis was determined by by Flow Cytometry assay

Fig. 5

Xenograft tumor growth inhibition results. a The expressions of indicated proteins. Apoptosis-related oncogenes like Akt1, p-Akt (ser473) and p-Akt (Thr308) c-Myc were decreased while NF-κB (p65), cleaved caspase 3 increased on protein levels. b The tumor growth inhibition experiments results: The inhibition effect was demonstrated by comparing the size/weight of dissected tumors from each group after 4 weeks treatment, respectively, and each bar represents the tumor size/weight of mean ± SD of 10 animals per group. Significant difference from PBS group and Ad-sta group were represented