线粒体ROS在低密度脂蛋白-胆固醇促结肠癌转移中的作用机制研究

The mortality of colon cancer mainly attributes to metastases, not the primary tumors from which these malignant lesions arise. Thus, the capacity to interdict the process of metastasis largely restricts our ability to effectively treat cancer. Retrospective studies showed that the abnormal cholesterol metabolism was involved in the colon adenocarcinoma initiation, yet the role of cholesterol metabolism in colon cancer metastasis has remained obscure because the blood cholesterol levels in colon cancer patients are generally not increased. However, numerous studies have shown increased levels of cholesterol in tumors as compared to normal tissues, and increased intracellular cholesterol levels are also frequently found in cancer cells. We demonstrated that increased low density lipoprotein - cholesterol(LDL-C) levels by injecting LDL-C locally into colon tumor significantly promoted colon cancer metastasis, and for the first time found that LDL-C induced metastasis may be related to mitochondrial ROS, the fundamental basis is essential but lacking. While the initial studies focused on the damaging effects of ROS, a recent paradigm shift has shown that mitochondrial ROS can act as signaling molecules to active pro-growth response. We hypothesize that mitochondrial ROS is one of the important causes of LDL-C induced colon cancer metastasis. In this study, ROS specific probes, ROS generation inhibitors and Matrix-roGFP will be used to demonstrate that ROS generation may be induced mainly in mitochondria-dependent manner by LDL-C and to clarify the underlying mechanism. Further, the oxysterols related to colon cancer metastasis will be separate and identify by ultra performance liquid chromatography with tandem mass spectrometry(UPLC-MS). Finally, we will clarify the role of ROS dependent metastasis signalings and the metastasis genes in colon cancer by real-time PCR, western blot and immunofluorescence assays. These analyses may provide a novel perspective about the link between LDL-C metabolism and colon cancer metastasis. A greater understanding of the role of mitochondrial ROS in colon cancer metastasis might lead eventually to a novel and/or targeted method for colon cancer treatment.

转移是导致结肠癌患者治疗失败和死亡的最主要原因。胆固醇代谢异常与结肠癌发生密切相关，但因结肠癌患者血胆固醇水平一般不高，导致其在结肠癌转移中的作用未被充分重视。现大量研究证实肿瘤组织内普遍存在胆固醇的蓄积。我们前期研究发现升高肿瘤组织内低密度脂蛋白-胆固醇（LDL-C）可显著促进结肠癌转移，并推测该现象可能与LDL-C升高线粒体ROS有关，但具体机制不详。本课题拟利用ROS特异性探针、ROS生成酶抑制剂和roGFP等证实LDL-C诱导的ROS来源于线粒体并明确其产生机制；利用UPLC-MS分离鉴定氧化甾醇的类型及含量，探讨其与结肠癌转移的相关性；利用RT-PCR、Western blot等研究线粒体ROS对转移相关信号通路及基因的影响，以期阐明LDL-C促进结肠癌转移的分子机制，为在血脂正常、系统降脂治疗受限时，治疗结肠癌转移提供一种新思路，具有重要的科学意义及潜在临床应用价值。

胆固醇代谢异常与结肠癌发生密切相关，但因复发性或转移性结肠癌患者一般体重正常、血胆固醇水平不高，导致其在结肠癌转移中的作用未被充分重视。 现大量研究证实肿瘤组织内普遍存在胆固醇的蓄积。本研究发现BMI正常的结肠癌患者中血清低密度脂蛋白-胆固醇(LDL-C)水平较健康者显著升高，LDL-C可显著促进结肠癌线粒体ROS水平及氧化甾醇升高（以27-OHC和7a-OHC升高为主）、可显著促进结肠癌转移和无糖耐受且该作用可被抗氧化剂逆转、可促进结肠癌细胞局部细胞因子CCL5上调和CCL11下调且CCL5升高和CCL11降低与结肠癌患者（尤其是TNM II期患者）5年生存负相关。我们发现低密度脂蛋白-胆固醇可调节结肠细胞内氧化还原状态、调节结肠癌细胞局部细胞因子，其中高CCL5水平和低CCL11水平可能为TNM II期结肠癌患者的5年生存的一种新预后指标，但目前仍未被重视。