Midkine promoter-based conditionally replicative adenovirus for targeting midkine-expressing human bladder cancer model
Midkine promoter-based conditionally replicative adenovirus for targeting midkine-expressing human bladder cancer model
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DOI：
10.1016/j.urology.2007.07.003
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发表时间：
2007-11-01
期刊：
影响因子：
2.1
通讯作者：
Gotoh, Akinobu
中科院分区：
文献类型：
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作者：
Terao, Shuji;Shirakawa, Toshiro;Gotoh, Akinobu
目的：利用Ad-MK-Ela-a midkine （MK）启动子调控的、有条件复制的腺病毒，开发一种新的治疗人类膀胱癌的策略。方法体外检测多种人类肿瘤细胞系，包括膀胱癌（KK47、5637和T24）、肺癌（A549）和头颈癌（H891）。在每个细胞系中，我们用TaqMan实时定量聚合酶链反应检测MK mRNA的表达，用荧光素酶测定质粒转染后的MK启动子活性，以及用巨细胞病毒- β -半乳糖质粒共转染的转导效率。在这些细胞中，我们通过实时聚合酶链反应（real-time polymerase chain reaction）测量Ela DNA拷贝数来评估Ad-MK-Ela病毒的细胞类型特异性复制，并使用Alamar blue试验评估该病毒对细胞生长的抑制作用。在动物实验中，裸鼠皮下接种KK47细胞，然后瘤内注射磷酸盐缓冲盐水或Ad5-CMV-LacZ或Ad-MK-Ela。结果5637、A549和KK47细胞的MK mRNA表达量和MK启动子驱动的荧光素酶活性均明显高于T24和H891细胞。Ad-MK-EIa感染后，KK47、5637和A549细胞的Ela DNA拷贝数明显高于T24和H891细胞。在感染倍数为0.01时，Ad-MK-Ela显著抑制KK47和5637细胞的生长。在体内，注射Ad-MK-Ela可明显抑制KK47肿瘤的生长。结论：我们在过表达MK mRNA的人膀胱癌模型中证实了Ad-MK-Ela的抗肿瘤作用。
OBJECTIVES To develop a novel therapeutic strategy against human bladder cancer using Ad-MK-Ela-a midkine (MK) promoter-regulated, conditionally replicating, adenovirus.METHODS We tested several human cancer cell lines in vitro, including those of bladder cancer (KK47, 5637, and T24), lung cancer (A549), and head and neck cancer (H891). In each cell line, we examined MK mRNA expression by TaqMan real-time quantitative polymerase chain reaction, MK promoter activity, after plasmid transfection, using a luciferase assay, and the transduction efficiency by co-transfection with the cytomegalovirus-beta-gal plasmid. In these cells, we assessed the cell type-specific replication of Ad-MK-Ela virus by measuring the Ela DNA copy number by real-time polymerase chain reaction and the cell growth inhibition due to this virus using the Alamar blue assay. In animal studies, nude mice were subcutaneously inoculated with KK47 cells and later intratumorally injected with phosphate-buffered saline or Ad5-CMV-LacZ or Ad-MK-Ela.RESULTS The MK mRNA expression level and MK promoter-driven luciferase activity were relatively greater and markedly increased, respectively, in the 5637, A549, and KK47 cells than in the T24 and H891 cells. After Ad-MK-EIa infection, the Ela DNA copy number increased more significantly in the KK47, 5637, and A549 cells than in the T24 and H891 cells. At a multiplicity of infection of 0.01, Ad-MK-Ela significantly inhibited KK47 and 5637 cell growth. In vivo, Ad-MK-Ela injection markedly inhibited KK47 tumor growth.CONCLUSIONS We have demonstrated the antitumor effect of Ad-MK-Ela in a human bladder cancer model overexpressing MK mRNA.