Effect Of Bevacizumab On Tight Junction Proteins Of Vascular Endothelial Cells

Background Platinum-based chemotherapy can significantly prolong the survival period of non-squamous non-small cell lung cancer(NSCLC)patients,but drug resistance will finally inevitably occur,which reveals increased tumor invasion and metastasis.Claudin-5(CLDN5)is a protein member in tight junction(TJ)structure expressed in both endothelial and epithelial cells,which is confirmed to be involved in proliferation and leakage of endothelial cells(ECs)and malignancies’ metastases.This study is to investigate how dose bevacizumab,a VEGF antibody applied in clinic to improve chemotherapeutic efficacy,affect tight junction protein CLDN5 and subsequently influence tumor cells’ invasion and potential metastasis.Methods Human umbilical vein ECs(HUVECs)and mouse retinal microvascular ECs(MRMECs)were introduced in this study.Western-blot,quantitative-PCR,immunofluorescence and immunohistochemistry were used to observe the regulation of bevacizumab on CLDN5.The functional experiments in cell biology were applied to study the effect of bevacizumab and CLDN5 on the migration,invasion and permeability of ECs.The si RNAs of CLDN5 were used to explore the effect of CLDN5 on endothelial cells of blood vessels.The inhibitor and si RNA of TGFβ1,JNK or PI3 K were used to determine the up-stream molecules of CLDN5.The immunoprecipitation assay was used to explore the relationship between VEGFA,JNK and PI3 K.Results data was analyzed using a Students two sample t-test.Results Low concentration of bevacizumab up-regulated the expression of CLDN5,while high concentration down-regulated it.CLDN5 was mediated by JNK,PI3 K and TGFβ1 signal molecules,which confirmed by inhibitor or si RNA of JNK,PI3 K and TGFβ1.High concentration of bevacizumab enhanced the abilities of migration,invasion and permeation of HUVECs.The abilities of migration and permeation of HUVECs were also enhanced and the proliferation ability was decreased by silencing the expression of CLDN5.The aforementioned mechanisms were also validated in MRMECs.Conclusions High concentration of bevacizumab was more likely to increase tumor’s ability of invasion and potential metastasis via down-regulating the expression of tight junction protein CLDN5,which was down regulated by TGFβ1.Low concentration of bevacizumab augmented the expression of CLDN5 through inhibiting VEGF and upregulated the expression of PI3 K and JNK.