| Purpose: Recent studies have showed that nanogold could inhibit the binding between VEGF165 (which has heparin binding domain) and its receptor VEGFR-2, conferring the inhibition of signal transduction induced by VEGF165. Here, we investigate whether nanogold could inhibit the proliferation of vascular endothelial cells and the angiogenesis of liver cancer. Previous reports have suggested that interleukin-12 (IL-12) could induce the expression of interferon-γ(INF-γ) and then allow INF-γto induce the expression of IP-10 (inducible protein-10) and MIG (monokine induced by interferon-gama), and inhibit the angiogenesis of tumor. Whereas the VEGF and Ang-2 are the main factors known by far, promoting the proliferation of vascular endothelial cells. We hypothesize that IL-12 present antiangiogenesis and anticancer effect by inhibiting the expression of Ang and VEGF gene in liver cancer tissue and inhibiting the proliferation of vascular endothelial cells, which needs to be confirmed by experiments.Methods: Immunoblotting was adopted to determine whether nanogold could specifically combine to heparin binding domain of bFGF. After nanogold acted on vascular endothelial cells, the changes of PLC—γ1 were examined. The effects of nanogold on the growth of vascular endothelial cells were assessed, while VEGF121 which has no heparin binding domain was set up as contral. Atomic force microscopy (AFM) was used to examine the sizes of nanogold acting with VEGF165 or VEGF121 and the changes of ultrastructure on cell surface treated with nanogold. The H22 liver cancer cells were transplanted into Balb/c nude mice, and mice were divided into two groups, experimental group treated with nanogold and control group treated with non nanogold. The microvascular density (MVD) was determined by imunohistochemistry method, and sizes and weights of liver cancer were examined after treatment with nanogold or not. 2. AdvmIL-12 was constructed and transfected into H22 cell. The transfection efficiency of AdvmIL-12 on H22 cell was detected. The expression of IL-12 protein in H22 cells was examined with ELISA and the effect of IL-12 on H22 cell growth was assessed. AdvmIL-12 was transfected into H22 cells in the experimental group, while the H22 cells were transfected with adenovirus in vector control group and the H22 cells were not transfected with anything as blank group. Then they were inoculated into the subrenal capsule or subcutaneously into right oxter of nude mouse, respectively. The expression of gene of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) in liver cancer tissue was detected by reverse transcription-polymerase chain reaction (RT-PCR). MVD was determined by imunohistochemistry method, and sizes and weights of liver cancer were measured. The results of P＜0.05 were considered to denote statistical significance.Results:1. Nanogold could bind to bEGF with heparin binding domain and inhibit the signal transduction of VEGF 165 and proliferation of vascular endothelial cells in vitro experiment. In contrast, no significant inhibition of nanogold on VEGF121 was observed.2. In vivo experiment suggested that MVD of liver cancer tissue treated by nanogold was 14.27±1.08, while control group had a MVD about 23.52±1.36. Therefore, the MVD in experimental group decreased significantly compared to that in control group (P＜0.01). In addition, compared to the control group, the group treated by nanogold had a significantly less tumor weights and volumes (P＜0.05).3. In nearby physiological conditions, AFM could probe the changes of ultrastructure on vascular endothelial cells surface before and aider the treatment with nanogold and VEGF165,VEGF121. When vascular endothelial cells were being in proliferation, there were many granules about hundreds nm in size on cell surface, the edges of cell membrane extended markedly, and cellular pseudopods prolonged obviously. In contrast, when cellular proliferations were inhibited by nanogold, the number of granules on cell surface decreased clearly, and cellular pseudopods shortened obviously.4. When multiplicity of infection (MOI) was 100 and time of infection (TOI) was 2 hours, the transfection efficiency of recombinant adenovirus was about 96.41%. The expressive quantity of IL-12 in experimental cell supematant was about (89.71±22.05) ng.48h-1.106cells-1. Data suggested that no significant inhibition effect of IL-12 against H22 cell growth was observed.5. In vivo experiment, data suggested that, compared to control groups, expression level of IL-12 in AdvmIL-12 group increased significantly. Compared to control group, the expression of VEGF and Ang-2 gene in experimental group decreased significantly. The ratioes of VEGF gene were 0.8867±0.1924 in experimental group, 1.1744±0.1189 in vector group, and 1.1874±0.1752 in blank group, respectively, and P＜0.05. The ratioes of Ang-2 gene were 0.7878±0.1471 in experimental group, 1.0319±0.1574 in vector group, and 1.0361±0.1536 in blank group, respectively, and P＜0.05. And the MVD, weights and volumes of tumor were significantly smaller than those in control groups, and P＜0.05.Conclusions:1. Nanogold could inhibit the signal transduction of VEGF165 and confer the significant inhibition effect against vascular endothelial cell proliferation induced by VEGF 165 in vitro experiment. In contrast, no significant inhibition effect of nanogold against VEGF121 was observed.2. Nanogold could inhibit the angiogenesis and the growth of H22 liver cancer by inhibiting the binding between VEGF165 and VEGFR-2 in vivo.3. AFM showed that nanogold inhibited the effects of VEGF165 on the proliferation of vascular endothelial cells.4. AdvmIL-12 could efficiently transfect into H22 cells and no significant inhibition effect oflL-12 against H22 cell growth was observed in vitro.5. In the nude mouse that absents T cells, IL-12 inhibited the angiogenesis and the growth of H22 liver cancer by inhibiting the expression of VEGF and Ang-2 gene in liver cancer tissue and the proliferation of vascular endothelial cells.These results in this experiment will be helpful for the antiangenesic treatment of liver cancer and other solid tumors.
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