木犀草素的抗胶质母细胞瘤作用及其机制研究

发布时间：2018-04-01 14:10

本文选题：木犀草素　切入点：胶质母细胞瘤　出处：《南京大学》2016年博士论文

【摘要】：胶质母细胞瘤是颅内肿瘤中最常见且恶性程度最高的恶性肿瘤,由于肿瘤高度增殖、侵袭以及对放化疗的耐受性,患者即使接受最积极的治疗手段,患者依然要面临手术疗效差、术后极易复发、生存期短的境地。因此,亟需我们开发新的治疗药物,寻找有效的治疗方法。木犀草素是一种天然黄酮类化合物,有文献报道其在体外、体内都有明确的抗肿瘤作用。研究表明,肺癌中木犀草素是Nrf2的有效的和选择性抑制剂,能有效抑制肺癌裸鼠种植瘤的生长并提高化疗药物的敏感性。胰岛素样生长因子(IGF-1)在多种恶性肿瘤组织中高表达,其介导的信号通路参与了肿瘤细胞凋亡调控。PI3K/AKT/mTOR信号通路的过度活化在肿瘤的发生发展过程中具有十分重要的作用,与肿瘤细胞的恶性生物学行为侵袭/迁移、凋亡和耐药等有着密不可分的关系。内质网应激反应是细胞应对外界刺激的一种保护性反应,但是研究表明长时间持续的内质网应激反应会诱导细胞发生凋亡。因此,本研究以胶质母细胞瘤细胞系U251MG和U87MG为体外模型,裸鼠皮下种植瘤为体内模型,详细研究木犀草素的抗胶质母细胞瘤作用,其对胶质母细胞瘤侵袭/迁移、凋亡的影响,以及对胶质母细胞瘤替莫唑胺耐药细胞的作用,并研究IGF-1R/PI3K/AKT/mTOR通路以及药物所引发的肿瘤细胞的内质网应激反应在这些过程中的作用。本课题共分为以下五个研究部分:第一部分木犀草素对胶质母细胞瘤细胞迁移的作用目的:研究木犀草素能否抑制胶质母细胞瘤细胞的迁移。方法:体外培养胶质母细胞瘤细胞系U251MG和U87MG,接种于96孔,利用CCK-8进行细胞增殖实验,将不同浓度梯度0,5,10,20,40,80μmol/L的木犀草素处理细胞24h后用CCK-8检测增殖,确定药物的给药浓度和时间(0,5,10,20μmol/L,24h)。之后再检测对U251MG和U87MG迁移的影响;利用western blot检测侵袭和迁移相关蛋白MMP-9,MMP-2,TIMP-1,TIMP-2的变化;利用 westernblot 检测 EMT 相关蛋白 Vimentin,β-catenin,N-cadherin 和 E-cadherin水平变化;利用罗丹明标记的鬼笔环肽染色细胞骨架蛋白,检测木犀草素对细胞形态和迁移表型的影响。结果:细胞增殖实验结果显示木犀草素浓度小于40μmol/L时对肿瘤细胞增殖没有明显影响;细胞划痕迁移实验表明木犀草素能显著抑制胶质母细胞瘤细胞的迁移;木犀草素能够明显下调侵袭和迁移相关蛋白MMP-2,MMP-9的表达而上调抑制蛋白TIMP-1,TIMP-2的表达;此外木犀草素能够阻止胶质母细胞瘤细胞的EMT过程。结论:木犀草素可以抑制胶质母细胞瘤细胞的迁移。第二部分木犀草素对胶质母细胞瘤细胞凋亡的影响目的:确定木犀草素对胶质母细胞瘤细胞的凋亡的作用。方法:利用胶质母细胞瘤细胞系U251MG和U87MG以及小鼠原代血管内皮细胞模型,用不同浓度的木犀草素处理细胞,用CCK-8检测细胞活力确定其对肿瘤细胞的杀伤作用;通过Annexin V和Propidium iodode(PI)染色利用流式细胞仪检测对胶质母细胞瘤细胞凋亡的影响。同时用TUNEL染色、免疫荧光和western blot检测cleaved-caspase-3的表达进一步确定其对肿瘤细胞凋亡的作用。利用裸鼠皮下种植模型,检测木犀草素在体内的抗肿瘤作用,并用western blot检测凋亡相关蛋白的表达,利用TUNEL检测肿瘤组织中的凋亡情况。结果:我们发现木犀草素在40,80μmol/L时在体外具有明显促进肿瘤细胞凋亡的作用;木犀草素能明显提高肿瘤细胞的TUNEL水平,cleaved-caspase3的蛋白水平明显升高;木犀草素能通过促进凋亡抑制胶质母细胞瘤裸鼠皮下种植瘤的生长。结论:木犀草素能促进胶质母细胞瘤细胞的凋亡并抑制体内肿瘤生长。第三部分内质网应激反应在木犀草素引发的胶质母细胞瘤细胞凋亡中的作用目的:明确内质网应激反应是否参与了木犀草素引起的肿瘤的凋亡。方法:利用荧光检测肿瘤细胞在药物刺激后ROS的水平情况,并利用western blot检测木犀草素(0,40,80μM)作用24h后氧化应激相关蛋白Nrf2、NQO-1、HO-1的水平变化:利用western blot检测40 μM木犀草素刺激不同时间(0,1,3,6,12,24h)及不同浓度(0,5,10,20，40,80μM)作用24h后内质网应激反应相关蛋白 p-PERK,p-eIF2α,ATF4,CHOP,Cleaved-caspase-12 的蛋白水平变化情况;利用JC-1染料检测40μM木犀草素处理24h后肿瘤细胞线粒体的膜电位变化情况,及免疫荧光检测处理后细胞色素c的表达情况,利用western blot检测线粒体凋亡相关蛋白Bcl-2,Bax的表达情况;利用western blot检测裸鼠皮下种植瘤中内质网应激相关蛋白ATF4,CHOP,Cleaved-caspase-12的表达情况。结果:用药物木犀草素刺激胶质母细胞瘤细胞后能够诱导其ROS水平的升高,降低氧化应激相关蛋白Nrf2、NQO-1和HO-1的水平。进而激活内质网应激反应,p-PERK、p-eIF2α蛋白在40μM木犀草素处理6h时最高,而CHOP,Cleaved-caspase-12和Cleaved-capase-3的水平在24h最高,而且在体外以剂量依赖形式促进促进内质网应激反应相关蛋白p-PERK、p-eIF2α、CHOP的表,在裸鼠体内同样具有促进ATF4、CHOP、Cleaved-caspase-12的水平。木犀草素的处理能导致肿瘤细胞内线粒体膜电位的下降,并抑制Bcl-2的表达而促进促凋亡蛋白Bax的表达。结论:木犀草素通过内质网应激反应诱导肿瘤细胞发生凋亡。第四部分p-IGF-1Rβ/PI3K/AKT/mTOR信号通路在木犀草素引起的胶质母细胞瘤细胞生物行为变化中的作用目的:研究木犀草素是否通过调控p-IGF-1Rβ/PI3K/AKT/mTOR信号通路来发挥抗胶质母细胞瘤作用。方法:用不同浓度(0,5,10,20,40,80μM)的木犀草素处理胶质母细胞瘤细胞U251MG 和 U87MG 24h 后,通过 western blot 检测细胞 p-IGF-1Rβ/PI3K/AKT/mTOR通路上各信号分子蛋白水平的变化;用p-IGF1R特异性激动剂IGF-1(100ng/ml)处理后,再次检测这些蛋白的变化,并利用western blot检测侵袭/迁移相关蛋白 MMP-2,MMP-9,TIMP-1,TIMP-2,Cleaved-caspase-3 的水平。结果:在木犀草素起到抑制胶质母细胞瘤细胞迁移的条件下,其能显著抑制细胞内IGF-1Rβ,AKT,mTOR的磷酸化水平,而对其总蛋白水平没有明显影响,说明木犀草素能抑制p-IGF-1Rβ/PI3K/AKT/mTOR信号通路;此外加入IGF-1后,升高的TIMP-1,TIMP-2，E-cadherin的水平出现下降,而被抑制的MMP-2,MMP-9,Vimentin的蛋白水平重新恢复。结论:木犀草素通过抑制p-IGF-1Rβ/PI3K/AKT/mTOR信号通路达到抑制胶质母细胞瘤细胞迁移和EMT过程的作用。第五部分木犀草素对胶质母细胞瘤替莫唑胺耐药细胞系的作用目的:通过建立的胶质母细胞瘤U251MG替莫唑胺耐药细胞系,验证其耐药表型,研究木犀草素对该耐药细胞系的促凋亡作用,及是否能逆转其对替莫唑胺的敏感性。方法:体外培养U251MG和U251MG-TR细胞,利用倒置显微镜观察期形态的变化,利用CCK-8检测其对替莫唑胺IC-50的变化,利用western blot和QRT-PCR检测多药耐药相关基因MRP-3的蛋白和mRNA表达;利用CCK-8检测不同浓度木犀草素(0,5,10,20,40,80μM)对耐药细胞系的杀伤作用,并与U251MG非耐药细胞比较,利用AV/PI染色在流式细胞仪上检测其对耐药细胞凋亡的作用,利用western blot检测凋亡相关蛋白Cleaved-caspase-3,Bcl-2,Bax的蛋白水平变化;利用western blot,QRT-PCR和免疫荧光检测耐药细胞系中Nrf2的水平,利用western blot,QRT-PCR检测耐药细胞系中NQO-1,HO-1的蛋白和mRNA水平;利用Nrf2慢病毒转染U251MG耐药细胞下调其Nrf2的表达,再利用CCK-8检测木犀草素对其的杀伤作用。将不同浓度的木犀草素和替莫唑胺联用,利用CCK-8检测其存活率,用AV/PI标记用流式细胞仪检测细胞凋亡,利用western blot检测相应Cleaved-caspase-3,Bax的蛋白水平。结果:我们实验室构建的U251MG替莫唑胺耐药细胞系(U251MG-TR)具有明显的耐药细胞特征,细胞变大而且成团块生长;其对替莫唑胺具有明显的耐药性,IC-50值上升9倍左右,多药耐药相关基因MRP-3的mRNA和蛋白水平明显升高;木犀草素能显著降低U251MG-TR的存活率,并且耐药细胞比非耐药细胞对木犀草素更加敏感,药物刺激后U251MG-TR细胞凋亡明显升高,凋亡相关蛋白Cleaved-caspase-3,Bax的水平明显升高,而抑制凋亡蛋白Bcl-2水平明显下降。U251MG-TR细胞中氧化应激相关蛋白Nrf2,NQO-1,HO-1的mRNA和蛋白水平都明显升高,用慢病毒将U251MG-TR细胞中Nrf2干扰后能降低耐药细胞对木犀草素的敏感性;木犀草素能够部分逆转U251MG-TR对替莫唑胺的耐药性。结论:木犀草素能诱导胶质母细胞瘤替莫唑胺耐药细胞的凋亡。总结本课题的研究结果从体外和体内两个方面均证实木犀草素可以通过p-IGF1R-β/PI3K/AKT/mTOR信号通路抑制胶质母细胞瘤细胞的迁移;并能通过激活持续的内质网应激反应而促进胶质母细胞瘤细胞发生凋亡:此外U251MG替莫唑胺耐药细胞对木犀草素十分敏感,这可能和耐药细胞高表达的Nrf2有关;而且,低剂量木犀草素就能提高耐药细胞对替莫唑胺的敏感性。因而,我们认为木犀草素具有多重抗胶质母细胞瘤作用,内质网应激反应诱导的凋亡在抗肿瘤过程中具有重要作用,可以为胶质母细胞瘤的治疗提供新的思路。
[Abstract]:Glioblastoma is the most malignant tumor in the most common intracranial tumors and malignant degree of tumor, because of high proliferation, invasion and tolerance to chemotherapy, even the patients accept the most active treatment, patients are still faced with poor operative effect, postoperative recurrence, short survival situation. Therefore, urgent we developed a new drug, to find effective treatment. Luteolin is a natural flavonoid, has been reported to the in vitro, in vivo have clear anti-tumor effect. The results show that lung cancer luteolin is potent and selective Nrf2 inhibitor, can effectively inhibit the lung cancer xenograft tumor growth and improve the sensitivity of chemotherapy drugs. Insulin like growth factor (IGF-1) is highly expressed in a variety of malignant tumors, the signal pathway mediated apoptosis of tumor cells is involved in the regulation of.PI3K/AKT/mTOR. The excessive activation of signaling pathway plays an important role in the process of tumor progression, invasion / migration and the biological behavior of malignant tumor cells, apoptosis and drug resistance are closely related. Endoplasmic reticulum stress is a protective response of cells respond to external stimuli, but studies have shown that endoplasmic reticulum stress in long time persistent will induce apoptosis. Therefore, the study on glioblastoma cell lines U251MG and U87MG as an in vitro model, nude mice subcutaneous tumor model in vivo, a detailed study of luteolin anti glioblastoma cell tumor and its invasion of glioblastoma cell migration, apoptosis, and the effect on glioma neuroblastoma temozolomide resistant cells, endoplasmic reticulum stress and study of IGF-1R/PI3K/AKT/mTOR induced drug pathway and tumor cells in these processes are used. This paper is divided into the following five parts: the first part of luteolin on migration of glioblastoma cells. Objective: To study whether luteolin inhibited the migration of glioblastoma cells. Methods: human glioblastoma cell lines U251MG and U87MG cultured in vitro were inoculated in 96 holes, cell proliferation experiments using CCK-8, will different 0,5,10,20,40,80 concentration gradient mol/L luteolin treated cells 24h after CCK-8 was used to detect the proliferation, determination of drug concentration and time (0,5,10,20 mol/L, 24h). After testing the effects on U251MG and U87MG migration; use of migration and invasion related protein MMP-9, Western blot, MMP-2 TIMP-1, TIMP-2 detection, using the change; Westernblot detection of EMT related protein Vimentin, beta -catenin, N-cadherin and E-cadherin level changes; stained cells skeleton protein using Luo Danming phalloidin,. Measuring the effect of luteolin on the morphology and migration of cell phenotype. Results: the cell proliferation assay showed that luteolin concentration lower than 40 mol/L has no obvious effect on tumor cell proliferation; cell scratch migration experiments show that luteolin can significantly inhibit the migration of glioblastoma cells; luteolin can significantly decrease the invasion and migration of MMP-2 protein, the expression of MMP-9 while regulation of suppressor protein TIMP-1, expression of TIMP-2; EMT process in addition luteolin can prevent glioblastoma cells. Conclusion: luteolin can inhibit the migration of glioblastoma cells. The second part: the effect of luteolin on glioblastoma cell apoptosis Objective: to determine luteolin on apoptosis of glioblastoma cells. Methods use: Glioblastoma cell lines U251MG and U87MG and primary murine vascular endothelial cell model with Cells treated with different concentrations of luteolin, determine its cytotoxic effect on tumor cells by CCK-8 detection of cell viability by Annexin V and Propidium iodode; (PI) staining using flow cytometry on glioblastoma cell apoptosis by TUNEL staining. At the same time, the expression of immune fluorescence and Western blot detection of cleaved-caspase-3 further confirmed the apoptosis of tumor cells. The nude mice model, detection of luteolin in vivo antitumor effect and expression of Western blot detection of apoptosis related proteins, apoptosis detected by TUNEL in tumor tissue. Results: we found that luteolin in 40,80 mol/L in vitro could obviously promote the apoptosis of tumor cells effect; luteolin can significantly improve tumor cell TUNEL level and protein level of cleaved-caspase3 increased significantly; can through the To promote the apoptosis of glioblastoma grown in nude mice subcutaneous tumor growth. Conclusion: luteolin can promote apoptosis of glioblastoma cells and inhibit tumor growth in vivo. Endoplasmic reticulum induced apoptosis of luteolin in third glioblastoma cells for apoptosis Objective: clear endoplasmic reticulum stress is involved in luteolin caused by the tumor. Methods: the tumor cells detected by fluorescence in the level of ROS after the stimulation of drugs, and the use of Western blot (0,40,80 M) detection of luteolin oxidative stress related protein Nrf2, 24h NQO-1, HO-1 levels by Western blot detection 40 M (0,1,3,6,12,24h) stimulation of luteolin in different time and different the concentration of (0,5,10,20,40,80 M) related proteins in the endoplasmic reticulum stress reaction after 24h p-PERK, ATF4, CHOP, p-eIF2 alpha, Cleaved-caspase-12 protein in water Flat changes; using membrane potential changes in JC-1 dye detection of tumor cells in mitochondrial M 24h 40 after luteolin treatment, the expression of cytochrome c and immunofluorescence assay after treated by Western blot detection of mitochondrial apoptosis related protein Bcl-2, Bax expression; the endoplasmic reticulum stress Western blot grown in nude mice subcutaneous tumor detection ATF4 protein, CHOP, expression of Cleaved-caspase-12. Results: the drug luteolin stimulation of glioblastoma cells to induced increase of ROS level, reduce the oxidative stress associated protein Nrf2, NQO-1 and HO-1 level. Then activation of endoplasmic reticulum stress, p-PERK, p-eIF2 protein in 40 M 6h treatment of luteolin the highest, while CHOP, Cleaved-caspase-12 and Cleaved-capase-3 levels in the highest 24h, and in vitro in a dose-dependent way to promote endoplasmic reticulum stress P-PERK protein, p-eIF2 alpha, CHOP, CHOP in nude mice with the same Cleaved-caspase-12, promoting ATF4 level. Luteolin treatment can lead to the decline of mitochondrial membrane potential in tumor cells, and inhibit and promote the expression of Pro apoptotic protein Bax Bcl-2 expression. Conclusion: luteolin induced apoptosis of tumor cells through endoplasmic reticulum changes in response to stress. The biological behavior of glioblastoma cells in fourth p-IGF-1R beta /PI3K/AKT/mTOR signaling pathway in the role of luteolin induced Objective: To study whether luteolin regulates p-IGF-1R beta /PI3K/AKT/mTOR signaling pathways play a role of anti glioma cells. Methods: different concentrations (0,5,10,20,40,80 M) of luteolin treatment of glioblastoma cells U251MG and U87MG 24h, by Western blot detection of cell p-IGF-1R beta /PI3K/AKT/mTOR pathway on the signal Molecular changes in protein levels; with p-IGF1R specific agonist IGF-1 (100ng/ml) after the treatment, again change detection of these proteins, and using Western blot to detect invasion / migration related proteins MMP-2, MMP-9, TIMP-1, TIMP-2, Cleaved-caspase-3 level. Results: in the luteolin to inhibit glioblastoma cell migration under the condition that it can significantly inhibit the intracellular IGF-1R beta, AKT, phosphorylation of mTOR, but has no effect on the total protein level, luteolin can inhibit p-IGF-1R beta /PI3K/AKT/mTOR signaling pathway; plus IGF-1, elevated TIMP-1, TIMP-2, E-cadherin levels declined, but was inhibited by MMP-2 MMP-9, the protein level of Vimentin recovered. Conclusion: luteolin can inhibit glioblastoma cell migration and EMT process by inhibiting the p-IGF-1R beta /PI3K/AKT/mTOR signal pathway. Fifth Effect of luteolin on points of glioblastoma temozolomide resistant cell line Objective: through the establishment of U251MG glioma cells of temozolomide resistant cell line, verify the resistance phenotype of luteolin induced apoptosis of the resistant cell line, and whether it can reverse its sensitivity to temozolomide. Methods: U251MG and U251MG-TR cells were cultured in vitro and observe the change of phase morphology by inverted microscope, to detect the changes of temozolomide IC-50, using CCK-8 western and QRT-PCR blot expression detection of multidrug resistance related gene MRP-3 and mRNA protein; using CCK-8 to detect different concentrations of luteolin (0,5,10,20,40,80 M) killing effect on drug resistance cell lines, and compared with non U251MG resistant cells, AV/PI staining was used to detect the drug resistance of cell apoptosis by flow cytometer, using Western blot to detect apoptosis related protein White Cleaved-caspase-3, Bcl-2, Bax protein level changes; using Western blot, Nrf2 QRT-PCR and immunofluorescence detection of drug resistance cell line level, using Western blot, NQO-1 QRT-PCR detection of drug-resistant cell lines, HO-1 and mRNA protein level; down regulated the expression of Nrf2 by lentivirus transfected Nrf2 resistant U251MG cells, using CCK-8 the killing effect of luteolin on detection. Different concentrations of luteolin and Temozolomide Combined with the use of CCK-8 to detect the survival rate of apoptosis was assayed by flow cytometry with AV/PI markers detection using western corresponding Cleaved-caspase-3 blot, the protein level of Bax. Results: We constructed U251MG temozolomide resistance cell line (U251MG-TR) has the obvious characteristics of the resistant cells, the cells became larger and mass growth; the temozolomide has obvious resistance, IC-50 value increased 9 times more. Drug resistance related gene MRP-3 and mRNA protein levels were significantly increased; luteolin can significantly reduce the survival rate of U251MG-TR, and resistant cells than non resistant cells more sensitive to luteolin, apoptosis of U251MG-TR cells after stimulation with drugs significantly increased the expression of apoptosis related proteins Cleaved-caspase-3, Bax level increased significantly, and the inhibition of apoptosis protein Bcl-2 decreased oxidative stress.U251MG-TR cell associated protein Nrf2, NQO-1, HO-1, mRNA and protein levels were significantly increased by lentiviral Nrf2 interference in U251MG-TR cells can decrease the sensitivity of resistant cells to luteolin; luteolin can partially reverse the U251MG-TR resistance to temozolomide. Conclusion: luteolin can induce the apoptosis of glioma cells to temozolomide drug resistant cells. Summarize the results of this research from two aspects of in vitro and in vivo showed that luteolin Can inhibit the migration of glioblastoma cells by p-IGF1R- beta /PI3K/AKT/mTOR signaling pathway; and through the activation of endoplasmic reticulum stress sustained and promote glioma cell apoptosis in U251MG cells to temozolomide resistance of luteolin is very sensitive, high expression and this may be related to Nrf2 resistant cells and low dose; luteolin can improve the resistance sensitivity of cells to temozolomide. Therefore, we believe that luteolin has the effect of multiple anti glioblastoma, apoptosis induced by endoplasmic reticulum stress plays an important role in the anti-tumor process, to provide new ideas for the treatment of glioblastoma.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.41

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