Bordetella源性胆固醇氧化酶诱导肺腺癌细胞凋亡的机制研究

Recent researchs have demonstrated that depletion of membrane cholesterol could induce apoptosis of tumor cells, which provided novel target for tumor therapy.Cholesterol oxidase (COD-B),an enzyme catalysing oxidation of cholesterol,was first applied in the field of tumor therapy. The results suggested that COD-B could induce apoptosis of human lung cancer A549 cells when it was incubated directly with A549.Expression of COD-B gene in A549 could also inhibit the growth of A549, and the inhibition effect of COD-B on A549 was enhanced by addition of cholesterol，which was opposite to previous reports.The functional mechanism of COD-B was unclear.In this project,the influence of COD-B on lung adenocarcinoma cells cultured in serum-starved or cholesterol-rich medium would be brought to light by incubating COD-B with cells and expression of COD-B in lung adenocarcinoma cells, which was beneficail to confirm the mechanism of apoptosis of lung adenocarcinoma cells induced by oxidation of membrane cholesterol and exogenous cholesterol.The mechanism of apoptosis of lung adenocarcinoma cells induced by COD-B would be revealed clearly,which possessed great theoretical significance and application value.By the virus-mediated expression of COD-B gene in animal model of tumor,the effect of COD-B enzymatic properties on the selective inhibiton on tumor and normal tissues would be confirmed, which determined whether COD-B would be promising candidates as therapeutic agents targeting tumors.Furthermore, It should be helpful to provide theoretical basis for screening tumor-killing protein at enzymatic point of view.

近期研究结果表明，膜胆固醇的清除能够诱导肿瘤细胞凋亡，为肿瘤治疗提供了新的靶点。我们首次将能够氧化胆固醇的胆固醇氧化酶（COD-B）应用到肿瘤治疗领域，结果显示，COD-B与肺癌细胞A549共孵育（胞外作用）及COD-B基因导入（胞内作用）均能够诱导A549凋亡，且添加外源胆固醇促进了A549的凋亡，其机制尚不清楚。本项目拟通过COD-B与肺腺癌细胞共孵育及COD-B基因导入肺腺癌细胞两种方式，分别在血清饥饿或高胆固醇条件下，研究COD-B对肺腺癌细胞的影响，明确膜胆固醇及外源胆固醇氧化在诱导细胞凋亡中的作用机制，确定COD-B诱导肺腺癌细胞凋亡的机理，具有重要的理论意义及应用价值；通过病毒介导COD-B基因在肿瘤动物模型中表达，确定COD-B的酶学性质对其在肿瘤及正常组织中选择性作用的影响，探讨COD-B作为肿瘤靶向药物的可能性，同时从酶学角度为肿瘤杀伤蛋白的筛选提供一定的理论依据。

胆固醇在肿瘤发生发展中起着关键作用，肿瘤细胞中胆固醇含量的改变可能影响着肿瘤进程。胆固醇氧化酶（COD）是来自微生物的一种黄素蛋白，能够催化胆固醇的氧化反应，但COD能否影响肿瘤细胞的生长与凋亡仍未可知。我们的结果证实，来源于Bordetella species的胆固醇氧化酶 (COD-B)在体内和体外都能够诱导肺腺癌细胞凋亡。COD-B抑制Akt和ERK1/2磷酸化具有时间、剂量依赖性，胆固醇补充并不能逆转COD-B的作用甚至进一步加强其作用。COD-B促进氧自由基的产生，补充外源胆固醇进一步提高氧自由基的水平。此外，CDO-B诱导JNK和p38的磷酸化，下调Bcl-2，上调Bax，激活caspase-3，促进细胞色素C的释放。COD-B的作用很可能是由于COD-B在氧化胆固醇的同时还产生过氧化氢所致。过氧化氢酶预处理只能部分阻断COD-B的作用，说明过氧化氢酶只能抑制过氧化氢的作用，对COD-B诱导的胆固醇缺失却无能为力。我们的结果显示COD-B通过降低胆固醇含量、提高氧自由基水平导致不可逆的细胞凋亡。由于COD-B能够氧化胆固醇为胆甾酮，我们进一步检测了胆甾酮对肺腺癌的影响。结果显示，高剂量的胆甾酮能够抑制肺腺癌细胞的生长，但低剂量的胆甾酮在不影响肺腺癌细胞生长的情况下却明显抑制肺腺癌细胞的迁移和侵袭。胆甾酮处理提升了ROS水平，瞬时激活了AMPKα1，上调HIF1，下调Bcl-2，诱导自噬。AMPKα1敲除部分抑制了胆甾酮诱导的自噬，但并不影响胆甾酮对HIF1和Bcl-2的作用。胆甾酮诱导的自噬促进HMGB1从胞核向胞浆的释放，阻止了HIF1的核迁移，抑制了MMP9和MMP2的激活。此外，胆甾酮能够促进时间依赖性的caveolin-1磷酸化、caveolin-1内化，抑制了Akt和NF-κB磷酸化，降低了snail和twist的表达。体内实验结果显示，胆甾酮抑制了肺腺癌脑转移。我们的结果显示，COD-B能够氧化肺腺癌细胞膜胆固醇，促进肺腺癌细胞的不可逆凋亡；低剂量的胆固醇氧化产物胆甾酮能够抑制肺腺癌细胞迁移和侵袭，在体内抑制肺腺癌脑转移。