Midkine promoter-based conditionally replicative adenovirus for targeting midkine-expressing human bladder cancer model.
OBJECTIVE
To develop a novel therapeutic strategy against human bladder cancer using Ad-MK-E1a-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-E1a virus by measuring the E1a 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-E1a.


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-E1a infection, the E1a 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-E1a significantly inhibited KK47 and 5637 cell growth. In vivo, Ad-MK-E1a injection markedly inhibited KK47 tumor growth.


CONCLUSIONS
We have demonstrated the antitumor effect of Ad-MK-E1a in a human bladder cancer model overexpressing MK mRNA.
Terao
Shuji
S
Laboratory of Cell and Gene Therapy Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya-shi, Hyogo, Japan.
Shirakawa
Toshiro
T
Kubo
Shuji
S
Bishunu
Acharya
A
Lee
Sang-Jin
SJ
Goda
Kazumasa
K
Tsukuda
Mamoru
M
Hamada
Katsuyuki
K
Tagawa
Masatoshi
M
Takenaka
Atsushi
A
Fujisawa
Masato
M
Gotoh
Akinobu
A
D000256:Adenoviridae / Q000502:physiology; D000818:Animals; D005240:Feasibility Studies; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D000078792:Midkine; D009363:Neoplasm Proteins; D009414:Nerve Growth Factors; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000628:therapy*; D014779:Virus Replication
C066254:MDK protein, human; D009363:Neoplasm Proteins; D009414:Nerve Growth Factors; D000078792:Midkine