Adenovirus-mediated human endostatin gene delivery in the treatment of mouse melanoma

Abstract

Objective To observe the bioactivity of adenovirus-mediated human endostatin gene in vivo and in vitro.Methods B16F10 melanoma cells and human endothelial cells(ECV 304)were both transfected with recombinant adenovirus containing green fluorescent protein(Ad-GFP)or human endostatin gene (Ad-mES) at various multiplicity of infection(MOI).Then,the expression of endostatin gene was detected by RT-PCR,and the growth of cells by MTT assay.B16F10 cells were inoculated into the back of mice to establish melanoma models,which were classified into treated groups intratumorally injected with Ad-mES once (single Ad-mES group) or repeatedly(repetitive Ad-mES group)with an interval of 7 days,and control groups intratumorally injected with Ad-GFP (Ad-GFP group)or phosphate buffred solution (PBS group).Subsequently,the growth of tumors was observed at an interval of 4 days;tumor tissue samples were obtained from killed mice and subjected to the detection of endostatin expression with immunohistochemistry on day 7 after the first intratumoral injection;transmission electron microscopy was used to observe the apoptosis of tumor cells and endothelial cells on day 14.Results Adenovirus could efficiently infect targeted cells.When the MOI of Ad-GFP was 10,20,50,100,200 and 500,B16F10 cells were infected at a rate of 15.6%,35%,73%,88%,95.2%and 97%,respectively,and ECV304 cells at a rate of 19%,35%,80%,90%,97%and 98.5%,respectively.The endosmtin gene was proved to be expressed in targeted cells.Ad-mES had no obvious effect on B16F10 cells,but inhibited the growth of ECV 304 ceils,and the inhibitive effect was enhanced with the concentration of Ad-mES.The rate of tumor formation was 100% on day 8 after injection of B16F10 cells,and the earliest death of mice was observed on dav 16 in PBS group,day 18 in Ad-GFP group,day 20 in single Ad-mES group and repetive Ad-mES group.Conclusions The recombinant Ad-mES could affect the bioactivity of targeted cells in vivo and in vitro.Intratumoral injection of Ad-mES prolonged the survival of mice bearing melanoma and deccelarated the growth of melanoma. Key words: Melanoma; Endostatins; Gene therapy