Anti-metastatic Effect Of Disintegrin From Gloydius Brevlcaudus Venom On JEG-3Cells In Vitro And ItsRelevant Signaling Pathways

Disintegrin is a small molecule polypeptide which is mainly separated from the venom of Gloydius Brevlcaudus，it can inhibit the binding of integrin and its ligand.Disintegrin contains arginine-glycine-aspartic acid (Arg-Gluy-Asp, RGD) or lysine-glycine-aspartic acid(Lys-Gluy-Asp, KGD). The RGD or KGD sequences are the binding sites of disintegrin and integrin ligands which including extracellular matrixc(ECM)、fibrinogen(Fg）and fibronectin(FN）. Integrin ligands and disintegrin which have RGD or KGD can compete with each other for the same binding site in the integrin and disintegrin can competitively inhibit the action of integrin ligands. because of blocking cellular interactions between tumor cells and the ECM/other cells, thereby it can inhibit platelet aggregation formation of tumor thrombus, tumor angiogenesis, and reducing the ability of tumor cell invasion, metastasis.Disintegrin was isolated and purified from Gloydius brevicaudus venom (GBV). We investigate the effects of disintegrin on growing, adhesion, migration, invasion, and apoptosis in human choriocarcinoma cell line JEG-3. Investegated the effects of disintegrin on the expression of Id1、MMP9and CD73in JEG-3cell.1Effect of disintegrin on JEG-3cells, proliferation in vitro Culture JEG-3cell in vitro as the research object, treated with differentconcentration gradient (0、5、10、15、20�'�25μg/ml) disintegrin in24h, MTT method was used to detect absorbance at490nm groups(A490). We can found that the disintegrin groups can inhibit the proliferation of JEG-3cells in vitro significantly (P<0.01) and the effect is dose dependent. The median inhibitory rate (IC50) was18.00μ g/ml, when the concentration of25μ g/ml, the inhibition rate can reach69.47%. 2Effect of disintegrin on JEG-3cells, adhesion in vitroWe use cell matrix adhesion method to detect the effect of disintegrin on JEG-3cell adhesion in vitro. Observate samples under inverted microscope, cell adhesion we can find that high dose group can inhibit cell adhesion more than low dose group and the control group. The JEG-3cells were treated with disintegrin (0、5、10、20、35、40μg/ml) groups in2h first, then inoculated into96holes plate which have been covered with Fn in7h, MTT assay was used to detect the absorbance at490nm groups. The results showed that each concentration group corresponding to the inhibition rates were20.26、29.31、40.41、49.63and52.62%， the results were statistically significant.3Effect of disintegrin on JEG-3cells, migration in vitro The scratch test was used to examine the impact of disintegrin on JEG-3cells,migration in vitro. Set up the control group and18μ g/ml group. Observe and record the growth state of the cells under inverted microscope after0、24、48h.After24h we can find that the control group cells gradually moved to the scratch area while the experimental group has no cell migration but arc shrinking; after48h,the control group growing dense but the experimental group, scratch area was significantly larger, big range appeared round shrinkage, display disintegrin can lower the movement ability of JEG-3cell.4Effect of disintegrin on JEG-3cells, invasion in vitro Application of Transwell cell reconstituted basement membrane invasion assaywas used to analyze the effect of disintegrin on invasion of JEG-3cells，after treated by drugs in24h, Observe and record the growth state of the cells under inverted microscope.We can find that transmembrane cell number of the experimental group was significantly less than the control group, disintegrin can significantly reduce the invasion ability of JEG-3cells.5Effect of disintegrin on JEG-3cells, apoptosis in vitro The Annexin-V FITC/PI double staining of flow cytometry was used to detectcell apoptosis rate. JEG-3cells were treated by disintegrin after24h, Annexin-V FITC/PI flow cytometry can distinguish normal、early apoptosis、 late apoptosis and necrosis cells from the the sample. The four quadrants were counted the proportion of cells, we can found that early apoptosis rate、the survival rate and the apoptosis rate of the experimental group were11.8%±2.24%,75.1%±6.63%,11.8%±3.54%while corresponding to the control group，the data were6.0%±5.89%,85.4%±8.78%and7.8%±4.31%. The results show that disintegrin can promote the apoptosis of JEG-3cells, the difference has statistical significance (P<0.05).6Effect of disintegrin on JEG-3cells, expression of Id1, MMP9and CD73in mRNART-PCR method was used to detect the expression of Id1, MMP9and CD73of mRNA in JEG-3cells which treated by disintegrin in24h.After treated by0、4μg/ml disintegrin24h，JEG-3cells， total RNA was extracted for RT-PCR,to detect the expression of Id1, MMP9, CD73in mRNA.The experimental results show: disintegrin can significantly reduce the expression of mRNA of Id1、 MMP9and CD73in JEG-3cells (P＜0.01).Conclusion： GBVDI can significantly inhibit human choriocarcinoma cell line JEG-3proliferation, adhesion and migration, can reduce its ability of the invasion and metastasis, promote its apoptosis in vitro and can significantly reduce the expression of mRNA in Id1、MMP9and CD73which are interrelated with cell differentiation, invasion, adhesion, migration and survival.