缺氧诱导因子-1α对肺癌脑转移的作用及其机制的研究
|
摘要
目的探讨缺氧与肺癌细胞释放缺氧诱导因子-1α(HIF-1α)的关系,揭示肺癌脑转移的可能机制。方法建立A549肺癌细胞缺氧模型,缺氧培养A549细胞0.5、2、4、8、12和24 h(设同时点常氧培养为对照组)后,ELISA法测定A549细胞培养液内HIF-1α质量浓度。Transwell小室构建体外血脑屏障模型,A549细胞培养液缺氧不同时间后,检测体外血脑屏障模型的跨内皮阻抗(TER)变化,以反映体外血脑屏障通透性的改变;计数Transwell下室培养液中的A549细胞数。将缺氧不同时间的A549细胞培养液经尾静脉注入Wistar大鼠,伊文思兰检测动物血脑屏障通透性改变; Western blot法测定紧密连接相关蛋白Claudin-5在大鼠脑组织中的表达变化。结果与对照组比较,A549细胞缺氧培养2 h后,其细胞培养液内HIF-1α质量浓度增高,体外血脑屏障模型TER减小,穿过Transwell进入下室的缺氧A549细胞数增多(P均<0.05),以缺氧8 h时上述作用最明显(P<0.01), 24 h恢复至对照组水平。在大鼠体内实验中,与同时点对照组比较,尾静脉注入缺氧2 h的A549细胞培养液后,大鼠脑组织中伊文思兰质量百分比增加,大鼠脑组织中Claudin-5蛋白表达降低(P均<0.05),并且以注入缺氧8 h的A549细胞培养液作用最明显(P<0.01),注入缺氧24 h的A549细胞培养液则与同时点对照组水平相当。结论缺氧可引起肺癌细胞释放HIF-1α增多。增多的HIF-1α导致血脑屏障紧密连接蛋白Claudin-5表达减少,血脑屏障通透性增大,肺癌细胞转移入脑。
Objective To study the relationship between hypoxia and the hypoxia inducible factor-1α(HIF-1α) from lung cancer cells, to reveal the possible mechanism of brain metastases of lung cancer. Methods The hypoxia model of A549 lung cancer cells was established. After hypoxia culture of A549 cells for 0.5, 2, 4, 8, 12 and 24 h(normal oxygen culture at the same time point was set as the control group), the mass concentration of HIF-1α in A549 lung cancer cell culture medium were determined by ELISA. Transwell chamber was used to construct an in vitro blood brain barrier model, was treated with A549 lung cancer cell culture medium after different time points of hypoxia, Tran endothelial resistance(TER) change of blood-brain barrier model in instrument, to reflect the changes of blood-brain barrier permeability in vitro; A549 lung cancer cells in the culture medium were counted under Transwell room. A549 lung cancer cells with hypoxia at different time points injected into Wistar rats via tail vein, Western blot method was used to menstruate expression of tight junction associated protein Claudin-5 in the brain tissues, Evans blue to detect the change of blood brain barrier permeability in rats. Results Compared with the control group, the HIF-1α mass concentration in the cell culture solution of A549 increased, the in vitro blood-brain barrier model TER decreased, and the cell number of A549 that passed through transwell into the lower chamber increased(all P<0.05) after hypoxia 2 h, the above effect was most obvious when hypoxia 8 h(all P<0.01). After hypoxia 24 h, it was restored to the control group level. In the in vivo experiment of rats, compared with the control group, the mass percent of Evans blue in rat brain tissues increased after A549 cell culture solution with hypoxia 2 h was injected via caudal vein, meaning increased the permeability of rat blood brain barrier, while the expression of Claudin-5 protein in rat brain tissues decreased(all P<0.05). The effect was most obvious when A549 cell culture solution with hypoxia 8 h was injected into rat tail vein(P<0.01). Ejection *国家自然科学基金(No.81101912)、河北省高等学校科学技术研究项目(No.ZD20160082)、河北省卫生厅科学研究基金项目(No. 20170901) 和华北理工大学研究生创新项目(No.2019 S32)资助△ 通信作者, E-mail: qinlj20012003@163.com of hypoxia 24 h A549 cell culture solution yielded the same effects as those in the control group. Conclusion Hypoxia can induce the increase of HIF-1α in lung cancer cells. The increase of HIF-1α results in the decrease of Claudin-5 expression and increase of blood-brain barrier permeability, leading to lung cancer cells metastasis into the brain.
Objective To study the relationship between hypoxia and the hypoxia inducible factor-1α(HIF-1α) from lung cancer cells, to reveal the possible mechanism of brain metastases of lung cancer. Methods The hypoxia model of A549 lung cancer cells was established. After hypoxia culture of A549 cells for 0.5, 2, 4, 8, 12 and 24 h(normal oxygen culture at the same time point was set as the control group), the mass concentration of HIF-1α in A549 lung cancer cell culture medium were determined by ELISA. Transwell chamber was used to construct an in vitro blood brain barrier model, was treated with A549 lung cancer cell culture medium after different time points of hypoxia, Tran endothelial resistance(TER) change of blood-brain barrier model in instrument, to reflect the changes of blood-brain barrier permeability in vitro; A549 lung cancer cells in the culture medium were counted under Transwell room. A549 lung cancer cells with hypoxia at different time points injected into Wistar rats via tail vein, Western blot method was used to menstruate expression of tight junction associated protein Claudin-5 in the brain tissues, Evans blue to detect the change of blood brain barrier permeability in rats. Results Compared with the control group, the HIF-1α mass concentration in the cell culture solution of A549 increased, the in vitro blood-brain barrier model TER decreased, and the cell number of A549 that passed through transwell into the lower chamber increased(all P<0.05) after hypoxia 2 h, the above effect was most obvious when hypoxia 8 h(all P<0.01). After hypoxia 24 h, it was restored to the control group level. In the in vivo experiment of rats, compared with the control group, the mass percent of Evans blue in rat brain tissues increased after A549 cell culture solution with hypoxia 2 h was injected via caudal vein, meaning increased the permeability of rat blood brain barrier, while the expression of Claudin-5 protein in rat brain tissues decreased(all P<0.05). The effect was most obvious when A549 cell culture solution with hypoxia 8 h was injected into rat tail vein(P<0.01). Ejection *国家自然科学基金(No.81101912)、河北省高等学校科学技术研究项目(No.ZD20160082)、河北省卫生厅科学研究基金项目(No. 20170901) 和华北理工大学研究生创新项目(No.2019 S32)资助△ 通信作者, E-mail: qinlj20012003@163.com of hypoxia 24 h A549 cell culture solution yielded the same effects as those in the control group. Conclusion Hypoxia can induce the increase of HIF-1α in lung cancer cells. The increase of HIF-1α results in the decrease of Claudin-5 expression and increase of blood-brain barrier permeability, leading to lung cancer cells metastasis into the brain.
引文
[1] SATOH H, ISHIKAWA H, YAMASHITA YT, et al. Patterns of brain metatasis in lung cancer. Oncol Rep,2001, 8(4):781-783.
[2] LEE HW, LEE JI, LEE SJ, et al. Patient-derived xenografts from non-small cell lung cancer brain metastases are valuable translational platforms for the development of personalized targeted therapy. Clin Cancer Res,2015,21(5):1172-1182.
[3] 米东辉, 王静, 李立, 等.HIF-1α 表达与肺癌生物学行为关系的研究.中国老年学杂志,2007,27(9):880-881.
[4] 桑洁, 刘萍, 赵宝全.血脑屏障分子组成研究进展.国际药学研究杂志,2011,38(3):201-205.
[5] HARFORD-WRIGHT E, LEWIS KM, VINK R. Towards drug discovery for brain tumours: interaction of kinins and tumours at the blood brain barrier interface. Recent Pat CNS Drug Discov,2011,6(1):31-40.
[6] THOMPSON EM, FRENKEL EP, NEUWELT EA. The paradoxical effect of bevacizumab in the therapy of malignant gliomas. Neurology,2011,76(1):87-93.
[7] LEMASSON B, SERDUC R, MAISIN C, et al. Monitoring blood-brain barrier status in a rat model of glioma receiving therapy: dual injection of low-molecular-weight and macromolecular MR contrast media. Radiology,2010,257(2):342-352.
[8] PANG Z, FENG L, HUA R, et al. Lactoferrin-conjugated biodegradable polymersome holding doxorubicin and tetrandrine for chemotherapy of glioma rats. Mol Pharm,2010,7(6):1995-2005.
[9] BLACK KL,YIN D,ONG JM,et al. PDE5 inhibitors enhance tumor permeability and efficacy of chemotherapy in a rat brain tumor model. Brain Res,2008,1230:290-302[2018-02-05]. https://doi.org/10.1016/j.brainres.
[10] ZHANG Z, YANJ, SHI H. Role of hypoxia inducible factor 1 in hyperglycemia-exacerbated blood-brain barrier disruption in ischemic stroke. Neurobiol Dis,2016,95:82-92[2018-02-05]. https://doi.org/10.1016 /j.nbd.2016.07.012.
[11] GALDEANO C, GADD MS, SOARES P, et al. Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von Hippel-Lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities. J Med Chem,2014,57(20):8657-8663.
[12] CHEN W, JADHAV V, TANG J,et al.HIF-1 alpha inhibition ameliorates neonatal brain damage after hypoxic- ischemic injury. Acta Neurochir Suppl,2008,102:395-399.
[13] CHENC, HU Q, YAN J, et al. Early inhibition of HIF-1alpha with small interfering RNA reduces ischemic- reperfused brain injury in rats. Neurobiol Dis,2009,33(3):509-517.
[2] LEE HW, LEE JI, LEE SJ, et al. Patient-derived xenografts from non-small cell lung cancer brain metastases are valuable translational platforms for the development of personalized targeted therapy. Clin Cancer Res,2015,21(5):1172-1182.
[3] 米东辉, 王静, 李立, 等.HIF-1α 表达与肺癌生物学行为关系的研究.中国老年学杂志,2007,27(9):880-881.
[4] 桑洁, 刘萍, 赵宝全.血脑屏障分子组成研究进展.国际药学研究杂志,2011,38(3):201-205.
[5] HARFORD-WRIGHT E, LEWIS KM, VINK R. Towards drug discovery for brain tumours: interaction of kinins and tumours at the blood brain barrier interface. Recent Pat CNS Drug Discov,2011,6(1):31-40.
[6] THOMPSON EM, FRENKEL EP, NEUWELT EA. The paradoxical effect of bevacizumab in the therapy of malignant gliomas. Neurology,2011,76(1):87-93.
[7] LEMASSON B, SERDUC R, MAISIN C, et al. Monitoring blood-brain barrier status in a rat model of glioma receiving therapy: dual injection of low-molecular-weight and macromolecular MR contrast media. Radiology,2010,257(2):342-352.
[8] PANG Z, FENG L, HUA R, et al. Lactoferrin-conjugated biodegradable polymersome holding doxorubicin and tetrandrine for chemotherapy of glioma rats. Mol Pharm,2010,7(6):1995-2005.
[9] BLACK KL,YIN D,ONG JM,et al. PDE5 inhibitors enhance tumor permeability and efficacy of chemotherapy in a rat brain tumor model. Brain Res,2008,1230:290-302[2018-02-05]. https://doi.org/10.1016/j.brainres.
[10] ZHANG Z, YANJ, SHI H. Role of hypoxia inducible factor 1 in hyperglycemia-exacerbated blood-brain barrier disruption in ischemic stroke. Neurobiol Dis,2016,95:82-92[2018-02-05]. https://doi.org/10.1016 /j.nbd.2016.07.012.
[11] GALDEANO C, GADD MS, SOARES P, et al. Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von Hippel-Lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities. J Med Chem,2014,57(20):8657-8663.
[12] CHEN W, JADHAV V, TANG J,et al.HIF-1 alpha inhibition ameliorates neonatal brain damage after hypoxic- ischemic injury. Acta Neurochir Suppl,2008,102:395-399.
[13] CHENC, HU Q, YAN J, et al. Early inhibition of HIF-1alpha with small interfering RNA reduces ischemic- reperfused brain injury in rats. Neurobiol Dis,2009,33(3):509-517.