[Preparation and functional study of an adenovirus vector expressing human plasminogen kringle 5 gene].
Tumor angiogenesis plays a pivotal role in the progress of tumor. Among the various endogenous angiogenic inhibitors discovered, the human plasminogen kringle 5 (K5) has been demonstrated to be a potential inhibitor of the proliferation and migration of vascular endothelial cells in vitro. The replication-incompetent adenovirus (Ad) vector Adeno-X-CMV-K5 (Ad-K5) (where CMV is cytomegalovirus) was constructed and its antiangiogenic effect was tested on vascular endothelial cell and tumor cell. For the construction, the K5 cDNA was fused in-frame with human plasminogen signal sequence and inserted into the eukaryotic expression vector pcDNA3 to form pcDNA3K5. The recombinant plasmid was subcloned into the shuttle plasmid pShuttle under the control of the constitutive CMV immediate-early promoter. The plasmid carrying the cDNA for K5 (pShuttleKS) was then recombined with the Adeno-X viral DNA and transformed into E. coli DH5alpha. The resultant recombinant plasmid pAd-K5 was transfected into human embryonic kidney (HEK) 293 cells with liposome. The adenovirus expressing human plasminogen kringle 5 (Ad-K5) was successfully packaged and propagated in 293 cells, as detected by the cytopathic effect (CPE) on the cells, and the viral titer in the supernatant was 5 x 10(8) pfu/mL by plaque assay. Both human umbilical vein endothelial cell line ECV304 and human breast carcinoma cell line MDA-MB-231 were infected with Ad-K5 and Ad-LacZ, which was used the negative control, and assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compared with uninfected control and Ad-LacZ infected control, Ad-K5 infected group at 80 MOI (multiplicity of infection) significantly inhibited ECV304 proliferation; the difference between uninfected control and Ad-LacZ infected control was not significant. In contrast, there was no significant difference in the proliferation of MDA-MB-231 among all the treatments. In addition, the Ad-K5 at 100 MOI inhibited the differentiation and tube formation of ECV304 on ECMatrix gel. These results suggested that the recombinant replication-defective Adenovirus expressing human plasminogen kringle 5 inhibited the proliferation, differentiation and tube formation of ECV304 and had no effect on the proliferation of MDA-MB-231. Adenovirus mediated human plasminogen kringle 5 gene therapy may be a potential treatment of cancer through angiogenesis inhibition.
Yang
Jian
J
The Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai 200032, China. hysong@shmu.edu.cn
Wang
Yue-Xiang
YX
Guan
Xiao-Qun
XQ
Ma
Chun-Gu
CG
Wang
Long-Sheng
LS
Song
Hou-Yan
HY
D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D018919:Neovascularization, Physiologic / Q000235:genetics / Q000502:physiology; D010446:Peptide Fragments / Q000235:genetics* / Q000502:physiology; D010958:Plasminogen / Q000235:genetics* / Q000502:physiology; D016133:Polymerase Chain Reaction
D010446:Peptide Fragments; C491499:plasminogen kringle 5; D010958:Plasminogen