Influence Of Adinbitor On The Immunity Function Of Mice, Tumor Growth And Metastasis

Background: Disintegrins are a family of small molecule proteins containing theArg-Gly-Asp （RGD） or Lys-Gly-Asp （KGD） motifs in rich of cysteines from snakevenoms. By competitively binding with the integrin receptors αⅡ b β3, αvβ3andα5β1,the RGD motif can prevent the adhesion of tumor cells with extracellular matrix（ECM） and induce the apoptosis of tumor cells. It can inhibit the adhesion, proliferation,metastasis and angiogenesis of tumor cells by blocking the integrin-focal adhesionkinase （FAK） pathway. Adinbitor was cloned from the venom gland of LvshunAgkistrodon halys brevicaudus stejneger. Adinbitor is composed by73amino acidresidues with an RGD three-peptide sequence featured for disintegrins and12cysteineresidues. It is a novel member of the disintegrin family with a molecular weight of9kDa. As an antagonist to a variety of integrin receptors, Adinbitor shows apparentanti-tumor activities.Objective:To study the effects of Adinbitor on the proliferation, migration andinvasion behaviors of hepatoma cells and on the immunity, tumor development andmetastasis of tumor-bearing mice.Methods:1.In vitro experiments:The inhibitory effects of Adinbitor on mousehepatoma H₂₂cells and highly lymphatic metastatic potential Hca-F cells weredetermined by using cell counting kit-8（CCK-8） assay. The effect of Adinbitor on theinvasion and migration capacities of Hca-F cells were assayed by using Transwellchamber method.2. In vivo experiments: Tumor-bearing mice model was established byimplanting hepatoma H₂₂cells into BALB/C mouse. And80mice were randomlydivided into Adinbitor oral administration and intraperitoneal injection administrationgroups. The mice from each group were then divided into4sub-groups with thetreatments of0mg/kg （designated as control group）,0.5mg/kg,1.25mg/kg and5mg/kg Adinbitor （as low, moderate, high dose group）. Then, the live status of the mice wasclosely monitored and recorded. On the15thday after Adinbitor administration, all themice were sacrificed following their fundus bloods were taken. The levels of IL-18inserum were determined. Pathological analysis was performed for the tumor tissues andneck tissues of the mice both from the experimental and control groups.20establishedimmunological unming mice were randomly divided into experimental and controlgroups. The experimental group mice were treated with Adinbitor （1.25mg/kg） daily byoral administration and the control group mice were treated with the same dose of saline.On the7thday after treatments, all the mice were sacrificed following their fundusbloods were taken. Then, the serum concentration of immunoglobulin IgG wasmeasured.Results:1. Adinbitor could effectively inhibit the proliferations of H₂₂and Hca-F cells,and the migration and invasion of Hca-F cells.2. Results from experimental mouse hepatoma indicated that Adinbitorsignificantly downregulated the protein expression levels of vascular endothelial growthfactor （VEGF） and CD34（P<0.05）. The mean optical density （MOD） and microvesseldensity （MVD） of control group mice was0.181±0.023and25.25±5.52, respectively.For the mice orally administrated with high-dose of Adinbitor, the MOD and MVDwere0.118±0.02and11.87±2.99. And0.119±0.013and13.25±3.24were measuredfor the MOD and MVD of the mice treated with moderate dose of Adinbitor.Correspondingly, the administrations of moderate and high doses of Adinbitor couldapparently improve the survival rate of tumor-bearing mice.3. The experimental results on the effects of Adinbitor on the immune function ofmice showed that the mouse serum level of IgG （P<0.05） was significantly increasedfollowing Adinbitor treatment. Except for the mice treated with low dose of Adinbitor,the administration of Adinbitor could increase the mouse serum level of IL-18（P<0.05）.Apparently, Adinbitor could enhance the immune function of tumor-bearing mice.Conclusion:1. Adinbitor apparently inhibits the proliferations of H₂₂and Hca-F cells as wellas reduces the invasion and migration capacities of Hca-F.2. Potentially by inhibiting the proliferation of endothelial cells, Adinbitor mightbe in help of inhibiting tumor angiogenesis, metastasis and malignancy and extendingthe survival duration of tumor-bearing mice. 3. We propose the primary inhibitory action mechanism of Adinbitor on tumordevelopment and metastasis in current work. Adinbitor might directly inhibit theproliferation, migration and invasion of tumor cells. It may also activate and stimulatethe proliferation of immunocytes by secreting inflammatory cytokines andimmunoglobulin, which results in enhanced body’s immune function and suppressedtumor angiogenesis and tumor development.