Objective  To explore the inhibitory effect of thrombin inhibitors of tinzaparin on lung cancer angiogenesis and the molecular mechanism.

  Methods  Human umbilical vein endothelial cells(HUVCE) were treated with different concentrations of tinzaparin (10, 20, 50 and 100 U/L) and were divided into different groups. At the same time, cells with no addition of tinzaparin were assigned as control group. Cell counting kit-8(CCK-8) and Transwell experiments were used to detect cell viability and migration rate, respectively. The anti-angiogenesis effects of tinzaparin in vivo were further verified by in vitro test vessel formation experiment, network formation experiment and aortic loop experiment. Western blot was used to detect the protein content of thrombin, vascular endothelial-derived growth factor (VEGF), phosphorylated protein kinase B (p-AKT) and protein kinase B (AKT) in HUVEC cells and HCC827 cells of the control group and the 20 U/L tinzaparin group. The tumor quality and tumor volume of the control group and the 20 U/L timzapheparin group were measured. The anti-tumor and anti-angiogenic effects of tinzaparin were further verified by HCC827 xenograft; the tumor quality and volume of the control group and the 20 U/L timzapheparin group were measured; immunohistochemical method was used to detect expressions of Ki-67, CD31 and vascular endothelial cell A(VEGFA), proliferation and markers Ki-67 and CD31; apoptotic cells were detected in tumor tissues.

  Results  CCK-8 and Tranwell migration test found that timzapheparin (20 U/L) effectively inhibited the viability and migration of HUVEC cells. Compared with the control group, timzapheparin(20 U/L) effectively inhibited the formation of vessels of HUVEC cells(P < 0.05). This aortic ring measurement also showed that timzapheparin treatment at concentration of 20 U/L significantly reduced angiogenesis and inhibited the formation of new blood vessels in the CAM model (P < 0.01). Western blot detection revealed that the thrombin content in HUVEC cells and HCC827 cells of the 20 U/L timzapheparin group was significantly lower than that in control group(P < 0.01). The protein content of VEGF, p-AKT and Akt in HCC827 cells of 20 U/L timzapheparin group were lower than that of the control group(P < 0.05 or P < 0.01). HCC827 xenograft test showed that the 20 U/L timzapheparin group had lower tumor quality and volume than the control group(P < 0.01); compared with the control group, 20 U/L timzapheparin inhibited tumor proliferation, promoted tumor cell apoptosis, and inhibited angiogenesis.

  Conclusion  Timzapheparin reduces the expression of thrombin, inhibits cell proliferation and angiogenesis, and promotes apoptosis of lung cancer cell, and its anti-angiogenesis effect is related to the inactivation of Akt and its downstream signaling pathways.