索拉菲尼衍生物1118-20的抗肿瘤增殖和血管生成作用及机制研究

发布时间：2019-06-20 06:42

【摘要】：Sorafenib(索拉菲尼,Bay43-9006)是第一个口服的小分子多激酶抑制剂,广泛应用于晚期肾细胞癌、不可切除的肝癌患者。另外,在治疗乳腺癌、白血病、甲状腺癌等癌症类型中也具有很好的临床优势。到目前为止,索拉菲尼被推荐为治疗晚期肝癌的一线药物,晚期肾细胞癌的二线治疗药物和其它晚期或转移性甲状腺癌的推荐药物。索拉菲尼一方面通过直接靶向抑制丝氨酸／苏氨酸(Raf-1, B-Raf V600E, wild-type B-Raf and C-Raf),阻断肿瘤细胞增殖信号沿Raf/MEK/ERK通路传导；另一方面,通过抑制某些受体酪氨酸激酶(PDGFR-β, EGFR、VEGF、VEGFR、Flt-3、c-Kit等),抑制实体瘤血管内皮细胞形成血管,阻断肿瘤的血液营养供给,间接地抑制肿瘤的生长。然而在临床治疗中,sorafenib治疗导致的不良反应也不能忽视,使得治疗被迫中断或者降低剂量。常见的不良反应包括中性粒细胞减少、高血压、肝功能异常、肾功能损伤等,会影响患者的生活质量。同时,索拉菲尼是国外的专利保护药物,在中国售价昂贵,增加了中国患者治疗癌症的费用。因此,开发索拉菲尼衍生物中抗肿瘤活性高且低毒的多激酶抑制剂是临床急需的。鉴于Sorafenib在癌症治疗中较好的临床优势和在临床治疗中发生的不良反应等问题,李文保教授实验室在索拉菲尼结构的基础上,合成了一系列的索拉菲尼衍生物。在初期化合物活性筛选实验中,采用MTT实验评价了17个化合物对5种肿瘤细胞增殖的抑制作用。以抑制率和IC50值作为参考,我们筛选了6个具有研究潜力的化合物(1118-20、1124-21、1124-15、1125-67、1125-4、1118-41),都对5种肿瘤细胞的增殖具有较强的抑制作用。其中1118-20对上述5种肿瘤细胞抑制活性尤为明显,故选择1118-20作为研究对象,研究其抑制人肝癌细胞HepG2增殖的作用和机制。MTT和克隆形成实验表明,1118-20比sorafenib更能显著地抑制HepG2细胞的增殖；而1118-20对人正常肝细胞HL-7702的抑制作用较弱,与sorafenib无显著性差异；统计分析还发现,1118-20对肝癌细胞HepG2的细胞毒作用强于人正常肝细胞HL-7702。流式细胞仪检测细胞周期结果显示,1118-20可将HepG2细胞阻滞在S期。Hoechst 33258染色发现1118-20可诱导HepG2细胞发生细胞核浓缩、分裂、并边缘化等凋亡现象；Annexin-V/PI双染法发现1118-20可显著提高细胞凋亡的数量；western blotting检测发现1118-20可上调cleaved caspase-9、cleaved caspase-3及其底物裂解带cleaved PARP表达水平；JC-1染色和western blotting检测结果显示线粒体膜电势(△Ψm)下降,Mcl-1蛋白表达下调,Bax/Bcl-2比例上升,表明Bcl-2家族介导的线粒体凋亡途径被激活。进一步分析显示1118-20对HepG2细胞内增殖信号Wnt/β-catenin传导通路,EGFR/PI3K/Akt传导通路,Ras/ERK传导通路以及p53蛋白皆有影响。结果显示,1118-20不但上调抑癌蛋白p53的表达,还能抑制这三条信号通路,阻断增殖信号的传导,从而使肿瘤细胞的增殖阻滞。肿瘤细胞如果发生增殖或转移,甚至进一步恶化,肿瘤血管的形成是很有必要的。没有肿瘤血管,肿瘤细胞就不能获得足够的营养和氧气,也不能将无用的代谢物排泄出去,将极大地限制肿瘤的恶化和转移。MTT实验数据显示1118-20显著地抑制了人脐静脉血管内皮细胞HUVEC的增殖,且抑制效应比索拉菲尼更强。划痕试验表明1118-20可以减弱HUVECs的迁移能力；Western blotting结果显示1118-20可下调active-MMP-2、active-MMP-9的表达；体外管形成实验表明1118-20可抑制血管内皮细胞聚集形成血管管状结构;并且发现1118-20显著地降低血管形成相关蛋白(FGF-2、VEGF、VEGFR-2、EGFR)的表达,并能抑制VEGFR-2、EGFR的磷酸化,从而减弱形成血管的分子基础。综上所述,1118-20是一个抗肿瘤活性优于索拉菲尼,毒性较低的新化合物。在肝癌细胞中,1118-20不但激活Bcl-2家族介导的线粒体凋亡途径,还可调节Wnt/β-catenin通路、EGFR/PI3K/Akt通路、Ras/ERK通路抑制增殖信号的传导。1118-20还可通过下调蛋白抑制血管形成,间接限制肿瘤细胞的生长。这些数据表明1118-20可能是一个癌症治疗的潜在药物,具有较强的开发潜能。
[Abstract]:Sorafenib (Sorafenib, Bay43-9006) is the first oral micromolecule multi-kinase inhibitor, which is widely used in patients with advanced renal cell carcinoma and non-resectable liver cancer. In addition, the invention also has a good clinical advantage in the treatment of cancer types such as breast cancer, leukemia, thyroid cancer and the like. So far, Solani is recommended for the treatment of first-line drugs for advanced liver cancer, second-line treatment for advanced renal cell carcinoma and other advanced or metastatic thyroid cancer. so as to block the proliferation of tumor cells along the Raf/ MEK/ ERK pathway and, on the other hand, by inhibiting certain receptor tyrosine kinases (PDGFR-1, EGFR, VEGF, VEGFR, Flt-3, c-Kit, etc.), on the one hand, by direct targeting of a serine/ threonine (Raf-1, B-Raf V600E, a-type B-Raf and C-Raf); on the other hand, by inhibiting certain receptor tyrosine kinases (PDGFR-1, EGFR, VEGF, VEGFR, Flt-3, c-Kit, etc.), Inhibiting the formation of blood vessels of the vascular endothelial cells of the solid tumor, blocking the blood nutrient supply of the tumor, and indirectly inhibiting the growth of the tumor. However, in the clinical treatment, the adverse reaction caused by the treatment of the sorafenib cannot be ignored, so that the treatment is forced to be interrupted or the dose is reduced. Common adverse reactions include neutropenia, hypertension, liver function abnormality, renal function injury, etc., which may affect the quality of life of the patient. At the same time, Solani is a foreign patent protection drug, which is expensive in China and increases the cost of Chinese patients for cancer treatment. Therefore, the development of a multi-kinase inhibitor with high anti-tumor activity and low toxicity in the solani derivative is in urgent need. In view of the better clinical advantage of Sorafenib in the treatment of cancer and the adverse reactions that have occurred in clinical treatment, Professor Li Wenbao's laboratory has synthesized a series of solani derivatives on the basis of the structure of Sorafini. The inhibitory effect of 17 compounds on the proliferation of five tumor cells was evaluated by MTT assay in the initial compound activity screening experiment. As a reference to the inhibition and IC50 values, we screened six compounds (1118-20,1124-21,1124-15,1125-67,1125-4,1118-41) with the potential to study and have a strong inhibitory effect on the proliferation of 5 tumor cells. The inhibitory activity of 1118-20 on the above-mentioned five tumor cells was particularly obvious, and 1118-20 was selected as the research object to study the effect and mechanism of inhibiting the proliferation of human liver cancer cells HepG2. The results of MTT and clone formation showed that 1118-20 was more effective in inhibiting the proliferation of HepG2 cells than sorafenib, but the inhibitory effect of 1118-20 on human normal liver cells HL-7702 was weak and no significant difference with sorafenib. The statistical analysis also found that the cytotoxic effect of 1118-20 on HepG2 cells was stronger than that of human normal liver cells HL-7702. The cell cycle results were detected by flow cytometry, and the HepG2 cells were blocked in the S phase at 1118-20. Hoechst 33258 staining showed that 1118-20 could induce the nuclear concentration, division, and marginalization of HepG2 cells. The Annexin-V/ PI double-staining method found that 1118-20 could significantly increase the number of cell apoptosis. The western blotting assay found that 1118-20 could increase the level of clear PARP expression in clear caspase-9, clear caspase-3 and its substrate. The results of JC-1 staining and western blotting showed that the mitochondrial membrane potential decreased, the down-regulation of the Mcl-1 protein and the increase of the ratio of Bax/ Bcl-2, indicating that the Bcl-2 family-mediated mitochondrial apoptosis pathway was activated. Further analysis showed that 1118-20 had an effect on the proliferation signal Wnt/ P-cata conduction pathway, the EGFR/ PI3K/ Akt conduction pathway, the Ras/ ERK conduction pathway, and the p53 protein in HepG2 cells. The results show that 1118-20 not only upregulates the expression of the tumor suppressor protein p53, but also can inhibit the conduction of the proliferation signal, thereby blocking the proliferation of the tumor cells. The formation of a tumor vessel is necessary in the event of proliferation or metastasis or even further deterioration of the tumor cells. Without tumor blood vessels, the tumor cells can not obtain enough nutrients and oxygen, and the useless metabolites can not be discharged out, and the deterioration and metastasis of the tumor can be greatly limited. The MTT assay showed that 1118-20 significantly inhibited the proliferation of HUVEC in human umbilical vein endothelial cells, and the inhibitory effect was stronger. The scratch test shows that 1118-20 can reduce the migration ability of HUVECs; Western blotting results show that 1118-20 can downregulate the expression of active-MMP-2, active-MMP-9; in vitro tube formation experiments show that 1118-20 can inhibit the aggregation of vascular endothelial cells to form a vessel-like structure; and it has been found that 1118-20 significantly reduces angiogenesis-related proteins (FGF-2, VEGF, VEGFR-2, The expression of EGFR can inhibit the phosphorylation of VEGFR-2 and EGFR, thereby reducing the molecular basis for forming blood vessels. In conclusion,1118-20 is a new compound that has an anti-tumor activity that is superior to that of Solani and has a lower toxicity. In the liver cancer cell,1118-20 not only activates the Bcl-2 family-mediated mitochondrial apoptosis pathway, but also regulates the Wnt/ P-catenin pathway, the EGFR/ PI3K/ Akt pathway, and the Ras/ ERK pathway inhibits the conduction of the proliferation signal. These data suggest that 1118-20 may be a potential drug for cancer treatment and has a strong potential for development.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R96

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