[Construction of a DNA vaccine against extracellular domain 1-3 of Flk1 and its inhibitory effect on growth of liver cancer cell line H22].

BACKGROUND & OBJECTIVE: Angiogenesis plays an important role in the growth,invasion,and metastasis of most solid tumors. Vascular endothelial growth factor (VEGF) and its receptor flk-1 play a key role in tumor angiogenesis. Blocking VEGF-flk1 pathway may inhibit tumor growth. This study was to construct a DNA vaccine against extracellular domain 1-3 of flk1,and test its inhibitory effect on growth of liver cancer cell line H22.

METHODS: Extracellular domain 1-3 of flk1 was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR),and inserted into plasmid pcDNA3.1(+) to construct vaccine pcDNA3.1(+)-flk1-domain 1-3. The vaccine was transfected into COS7 cells,and protein expression of flk1-domain 1-3 was detected by Western blot. Standard 4-h (51)Cr releasing test was used to detect specific cytotoxic T lymphocyte (CTL) activity in vaccine-inoculated mice. To test vaccine's preventive effect,mice were divided into V,P, and S groups,treated with vaccine,pcDNA3.1(+), and saline, respectively. H22 cells were inoculated into mice 10 days later. The tumor size, tumor weight, mice survival time, tumor latent period, and microvessel density were recorded and analyzed.

RESULTS: Extracellular domain 1-3 of flk1 was cloned,and vaccine against it was constructed,both have been proved by DNA sequencing and comparing with data in GenBank. A protein of 44 kDa,which is consisted with flk1-domain 1-3 protein,expressed in COS7 cells inoculated with the DNA vaccine,specific CTL activity in these cells raised. After inoculated with H22 cells,tumor latent time of V group was (5.2+/-0.9) d,of P group was (4.0+/-0.7) d,of S group was (3.8+/-0.6) d; survival time of V group was (24.5+/-3.2) d,of P group was (14.7+/-2.6) d,of S group was (14.3+/-2.0) d; microvessel density of V group was 10.1+/-1.7,of P group was 27.3+/-3.3,of S group was 25.3+/-4.6; tumor weight of V group was (1.4+/-0.1) g,of P group was (1.8+/-0.2) g,of S group was (1.8+/-0.2)g. In comparison among groups,all data in V group were significantly different from P group and S group (P< 0.05),no significant difference existed between P group and S group (P >0.05).

CONCLUSION: The DNA vaccine against flk-1 may stimulate potent specific CTL activity, and inhibit growth of H22 cells by its anti-endothelial cell property.