The Effect Of A Human Disulfide-stabilized Diabody Against BFGF On The Growth Of Human Lung Cancer

Object: In order to investigate the effect of the Disulfide-stabilized diabody against bFGF(ds-Diabody) on the growth of human lung cancer A549 cells in vitro and in vivo and its antitumor mechanism.Methods: The engineered yeasts of ds-Diabody against bFGF were cultured in growth medium for 48 h. The medium was then chaged to induction medium and the ds-Diabody against bFGF was expressed in yeasts expression supernatant by inducing 1% methanol. The ds-Diabody against bFGF was purified from the yeast expression supernatant by ammonium sulfate precipitation and affinity chromatography of Ni-Seproase 6 FF and ion exchange chromatography of DEAE Sepharose FF. The purified ds-Diabody against bFGF was identified by SDS-PAGE and the antigen binding activity of the ds-Diabody against bFGF was tested by indirect ELISA assay. Human lung cancer A549 cells were cultured in vitro. The influence of the ds-Diabody against bFGF on human lung cancer A549 cells proliferation was analyzed by Cell Counting Kit-8. The effect of ds-Diabody against bFGF on the signal pathway of MAPK and Akt in A549 cells was assayed by western-blot. The influence of the ds-Diabody against bFGF on the migration and invasion of A549 cells was analyzed by scratch test and transwell assay. The subcutaneous xenograft tumor model was established to study the inhibitory effects of the ds-Diabody against bFGF on A549 tumor growth in vivo. One week later, when palpable tumors(≥5 mm in diameter) developed, the mice were randomly divided into three groups and administrated drugs every 3 days for a total of six injections. Weights of mice and tumor size were measured before every injection. After the first injection of antibodies, the blood samples(20 μl) of mice were collected from the caudal vein at different times(1 h, 71 h, 143 h, and 215 h). The residual anti-bFGF antibody activity in blood samples were detected by ELISA. The densities of micro-vessels and lymphatic vessels in tumor tissues were analyzed by immunohistochemistry.Results: The ds-Diabody against bFGF was successfully induced and expressed in the yeast supernatant. After purification by affinity chromatography of Ni-Seproase 6 FF and ion exchange chromatography of DEAE Sepharose FF, the purity of ds-Diabody against bFGF could reach more than 90%. The results of indirect ELISA assay indicated that the ds-Diabody against bFGF could specificly bind to bFGF. The ds-Diabody against bFGF could significantly inhibit the growth of A549 cells in a dose-dependent manner in vitro. The inhibition rate was about 40.94% when the concentration of the ds-Diabdoy against bFGF was 100 μg/m L. The ds-Diabody against bFGF could effectively inhibit the phosphorylation of Akt and MAPK in the bFGF/FGFR signal pathway. When the A549 cells were treated with 100 μg/m L ds-Diabody against bFGF for 30 min, the Akt and MAPK phosphorylation in the A549 cells decreased significantly. In the tumor cell scarification and invasion experiment, the ds-Diabody against bFGF could significantly inhibit the migration and invasion of A549 cells in vitro. The results of nude mice experiments showed that the ds-Diabody could significantly inhibit the growth of A549 cells in nude mice and the inhibition rate was 86.54% after administration six times. The residual anti-bFGF antibody activity of the ds-Diabody could steadily keep about 30-40% capacity than that of the injections; the results indicated that the ds-Diabody against bFGF could keep stable in mice. The immunohistochemistry results showed that the microvessels and lymphatic vessels were significantly reduced in the group of the ds-Diabody against bFGF, when compared to the PBS group.Conclusion: The human disulfide-stabilized diabody against bFGF could inhibit the proliferation, migration and invasion of human lung cancer A549 cells by blocking Akt and MAPK signaling pathways. The ds-Diabody against bFGF could reduce tumor microvessel density and lymphatic vessel density, thereby inhibiting the growth of mouse xenograft tumor of A549.