Gene transfer of a soluble receptor of VEGF inhibits the growth of experimental eyelid malignant melanoma.
OBJECTIVE
To determine the effect of adenovirus-mediated gene transfer of a soluble receptor of vascular endothelial growth factor (VEGF) on the growth of experimental eyelid malignant melanoma.


METHODS
An adenovirus vector encoding a soluble VEGF receptor/flt-1 (Adflt-ExR) was constructed. The bovine retinal endothelial cells (ECs) were incubated in a culture medium of 293E1 cells infected by means of an adenovirus vector or uninfected (control), which contained human recombinant VEGF, and the [3H]thymidine uptake was tested. The experimental eyelid malignant melanoma was induced by the injection of B16 melanoma cells (4 x 10(6) cells) into the right upper eyelid of BALB/c nu/nu mice, and the size of the tumor was recorded for 3 weeks after tumor cell injection. The effect of Adflt-ExR was examined in three ways. Model 1: B16 cells were infected by Adflt-ExR beforehand (at a multiplicity of infection [MOI] of 10) and injected into the eyelid. Model 2: Adflt-ExR was injected into pre-established B16 cell-induced eyelid malignant melanoma. Model 3: Adflt-ExR was injected into the femoral muscle of mice before B16 cell injection into the eyelid, and the remote effect was evaluated. An adenovirus vector bearing the LacZ gene (AdLacZ) or phosphate-buffered saline was used as a control. The amount of VEGF and the flt-ExR protein was measured by sandwich enzyme-linked immunosorbent assay (ELISA). Vascularization was evaluated by counting the number and the size of the vessels.


RESULTS
The supernatant of Adflt-ExR-transfected cells clearly inhibited VEGF-induced bovine retinal EC proliferation in vitro. In models 1 and 2, the tumor growth in Adflt-ExR-treated mice was significantly lower than that of controls (P < 0.05). In model 3, no significant difference was found (P = 0.14). The molar ratio of VEGF/flt-ExR protein was clearly low in the tumors of Adflt-ExR-treated mice in models 1 and 2 (P < 0.01) but not in model 3 (P > 0.05). In vessel density, the tumors in Adflt-ExR-treated mice had fewer vessels than tumors in control animals in models 1 and 2 (P < 0.05).


CONCLUSIONS
Adenovirus-mediated gene transfer of a soluble form of VEGF receptor (flt-1) gene inhibited the growth of the experimental eyelid malignant melanoma. This method may be useful as an antiangiogenic therapy for eyelid malignant melanoma.
Shiose
S
S
Department of Ophthalmology, Research Institute of Angiocardiology and Cardiovascular Clinic, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Sakamoto
T
T
Yoshikawa
H
H
Hata
Y
Y
Kawano
Y
Y
Ishibashi
T
T
Inomata
H
H
Takayama
K
K
Ueno
H
H
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002417:Cattle; D016228:Endothelial Growth Factors / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005142:Eyelid Neoplasms / Q000098:blood supply / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon / Q000235:genetics; D008222:Lymphokines / Q000378:metabolism; D008297:Male; D008545:Melanoma / Q000098:blood supply / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D051379:Mice; D008807:Mice, Inbred BALB C; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000235:genetics* / Q000378:metabolism; D012171:Retinal Vessels / Q000378:metabolism; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D011518:Proto-Oncogene Proteins; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1