Study on effect of thrombin inhibitor of tinzaparin in inhibiting angiogenesis of lung cancer and its molecular mechanism
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摘要:目的 探讨凝血酶抑制剂亭扎肝素对肺癌血管生成的抑制作用及分子机制。方法 使用不同浓度(10、20、50、100 U/L)亭扎肝素处理人脐静脉内皮细胞(HUVEC)并分组，同时设置对照组(不加亭扎肝素)，采用细胞计数试剂盒(CCK-8)法和Transwell实验分别检测各组细胞活力和细胞迁移情况。通过体外试管形成实验、网络形成试验和主动脉环试验进一步验证亭扎肝素的体外、体内抗血管生成作用，并采用蛋白质印迹法(Western blot)检测HUVEC和HCC827细胞中对照组与20 U/L亭扎肝素组的凝血酶含量和血管内皮生长因子(VEGF)、磷酸化蛋白激酶B(p-Akt)、蛋白激酶B(Akt)蛋白含量。通过HCC827异种移植物进一步验证亭扎肝素的抗肿瘤和抗血管生成作用，检测对照组与20 U/L亭扎肝素组的肿瘤质量、肿瘤体积。采用免疫组化方法检测肿瘤组织中Ki-67、CD31和血管内皮生长因子A(VEGFA)表达情况，并检测肿瘤组织中的凋亡细胞。结果 CCK-8法和Tranwell实验发现, 20 U/L亭扎肝素可有效抑制HUVEC的细胞活力和细胞迁移。网络形成试验显示，相较于对照组, 20 U/L亭扎肝素可有效抑制HUVEC的管形成，差异有统计学意义(P < 0.05)。主动脉环测定结果显示，相较于对照组, 20 U/L亭扎肝素减少了血管生成，抑制了CAM模型中的新血管形成，差异有统计学意义(P < 0.01)。Western blot检测结果显示, HUVEC和HCC827细胞中, 20 U/L亭扎肝素组的凝血酶含量均低于对照组，差异有统计学意义(P < 0.01); HCC827细胞中, 20 U/L亭扎肝素组的VEGF、p-Akt和Akt蛋白含量均低于对照组，差异有统计学意义(P < 0.05或P < 0.01)。HCC827异种移植物验证结果显示, 20 U/L亭扎肝素组的肿瘤质量、肿瘤体积均小于对照组，差异有统计学意义(P < 0.01); 与对照组比较, 20 U/L亭扎肝素可抑制肿瘤增殖，促进肿瘤细胞凋亡，并抑制血管生成。结论 亭扎肝素可降低凝血酶的表达，抑制细胞增殖和血管生成，促进肺癌细胞凋亡，其抗血管生成作用与Akt及其下游信号通路的失活有关。Abstract: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.
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Keywords:
- lung cancer /
- angiogenesis /
- thrombin inhibitors /
- timzapheparin /
- cell proliferation /
- apoptosis /
- protein kinase B
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图 1 亭扎肝素对HUVEC增殖和迁移的影响
A: 各组HUVEC细胞迁移距离比较; B: 各组HUVEC细胞活力比较。与对照组比较, *P < 0.05; 与20 U/L亭扎肝素组比较, #P < 0.05; 与50 U/L亭扎肝素组比较, △P < 0.05。
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图 2 亭扎肝素对体外和体内血管生成的影响
A: 2组HUVEC管形成的网络形成试验结果(放大倍数400倍); B: 2组相对管型长度比较; C: 主动脉环测定HUVEC血管生成结果(放大倍数400倍); D: 2组HUVEC微血管数量比较; E: CAM模型中的新血管形成结果(放大倍数400倍); F: 2组CAM模型中微血管数量比较。与对照组比较, *P < 0.05, **P < 0.01。
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图 3 亭扎肝素抑制VEGF和AKT的激活
A、B: HUVEC凝血酶含量的Western blot检测结果; C~G: HCC827细胞中VEGF、凝血酶、p-Akt、Akt蛋白含量的Western blot检测结果。与对照组比较, *P < 0.05, **P < 0.01。
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