The Influence Of Tumor-derived Von Willebrand Factor On Tumor Spreading Ability And The Effect Of Astragalin On The Process

Background and Objective: The interplay between cancer cells and platelets has been recognized as a "deadly ally" in cancer metastasis.The interaction through cell surface receptor and autocrine factor promoted the formation of tumor embolus and metastasis,von Willebrand factor(VWF)probably play a critical role among the interaction of cancer cells,endothelial cells and platelets,the effect and implication of VWF in the process was until unclear.The clinical study showed the serum VWF level of cancer patient increased,and it was closely correlated with tumor degree,metastasis and poor prognosis.These results suggested that cancer cell perhaps modified the biological behavior through generation of tumor-derived VWF to promote hematogenous spread and poor prognosis.The object of this study was to investigate the generation of tumor derived VWF in cancer tissue and cancer cell lines,and the influence of tumor derived VWF on biological behavior,we also studied the effect of astragalin on the VWF mediated cancer metastasis.Methods: 1.Collected the plasm of patient with gastric adenocarcinoma and measured the plasm VWF level by ELISA,detected the histological degree and stages,analyzed the relation between VWF expression level and histological feature by histology and immunohistochemical staining.2.Detected the VWF expression in gastric cancer cell lines by immunofluorescence,Immunohistostaining,transmission electron microscope,immunoelectron microscopy,western blotting and PCR.Detected the influence of exogenous VWF on aggregation and adhesion of gastric cancer cells.3.Constructed transgene gastric cancer cells which over expressed VWF,studied the effect of tumor derived VWF on cell adhesion of cancer cell to endothelial cell and platelets by microfluidics experiment,and analyzed the effect of the VWF receptor in the process.4.Studied the influence of tumor derived VWF on tumor blood spread and generation of metastasis by animal experiment,in vivo imaging and digital slicing technique.5.Treated the animal with astragalin and investigated the effect of astragalin on cancer spread and proliferation mediated by VWF.studied the influence of astragalin on proliferation,adhesion using in vitro experiments,approached the inhibition mechanisms of astragalin on tumor spread mediated by VWF.Results: The examinations of cancer tissue and blood samples for 105 patients with precancerous lesions and gastric adenocarcinoma showed the plasma VWF levels were significantly higher in patients with gastric adenocarcinoma compared with patients with intraepithelial neoplasia and health volunteer(P<0.05),The serum VWF level was also higher in patients with late stages(TNM ?/?)than patients with early stages(TNM ?/?)(P<0.05),the patients with poorly differentiated cancer had higher level of serum VWF concentration than the patients with moderately and well differentiated cancer(P<0.05).The immunohistochemical staining showed cancer tissue expressed VWF,the expression level negatively correlated with differentiated degree(r =-0.4256,P<0.05).The immunofluorescence,PCR and Western blotting detection indicated the gastric cancer cell lines such as BGC823,MKN45 expressed VWF,and the level of expression was influenced by thrombin and culture microenvironments.The detection of transmission electron microscope and immunoelectron microscopy displayed Weibel-Palade body like structure in BGC823 cells and could been labeled by gold conjugated VWF antibody,this was similar with endothelium.Aggregation experiment and adhesion experiment showed exogenous VWF could promote cancer cell aggregation and adhesion of cancer cells to endothelium and platelets.The promoting effect could been inhibited by antibody and si RNA of VWF.In the microfluidics experiment,the adhesion ability of BGC-VWF cell which transfected with a plasmid expressing the full-length VWF c DNA to platelets coated pipe significantly increased(P < 0.01).We analyzed on potential mechanism of cell adhesion mediated by VWF,find that the antibody of GPIb? could inhibit the adhesion of BGC823 cell to endothelium and platelets;The antibody of integrin could inhibit the adhesion between BGC-VWF and platelets.The inhibitor of PI3 K could inhibit the expression of VWF in gastric cancer cell,and inhibit the adhesion reaction to endothelium and platelets.In vivo experiment showed the NOD/SCID mice were injected with BGC-VWF cells from tail vein had a worse prognosis.In vivo imaging study for mice showed the fluorescence signal of animal which was injected with BGC-VWF cells significantly enhanced.Intraperitoneal injection of astragalin alternate day could inhibit the increase of fluorescence signal.Compared the data of lung fluorescence signal and digital slice information,we found the BGC-VWF group had higher lung fluorescence signal and bearing tumor area than the BGC-Sham group which transfected empty plasmid,the astragalin experiment treatment could decrease the lung fluorescence signal and bearing tumor area.The in vitro experiment showed astragalin couldn't influence the aggregation and adhesion of cancer cells based on platelets,but could inhibited the VWF synthesis and release in HUVECS,reduces the adhesive capacity between cancer cell and endotheliocyte,inhibited the cell proliferation and promote apoptosis,and also augment antitumor immune response.Conclusion: The human gastric adenocarcinoma tissue and cell lines could express the protein and m RNA of VWF,the tumor-derived VWF could promote cancer cell aggregation and adhesion to platelets and endothelium via GPIb? and Integrin,and promote the lung metastasis of cancer cell through bloodstream.The astragalin probably suppress VWF-overexpressing cancer cell development in lung metastases through reduce adhesive capacity between cancer cell and endotheliocyte,promoting apoptosis,antitumor immunity and inhibiting cell proliferation.