【作者】 张倩；

【导师】 马望；

【作者基本信息】 郑州大学 ， 临床医学博士（肿瘤学）（专业学位）， 2023， 博士

【摘要】 研究背景肺癌的死亡率和发病率在世界所有恶性肿瘤中均位居首位。其中约85%的肺癌患者病理类型为非小细胞肺癌(NSCLC)。在过去的几十年,靶向和免疫治疗在肺癌领域取得了长足的进步,但NSCLC患者整体预后仍然较差。因此,寻找治疗NSCLC的潜在靶点和新的治疗手段尤为重要。姜黄素,是一种从传统药物姜黄中提取的天然多酚,具有抗病毒、抗菌、抗炎症、抗氧化和抗增殖活性。姜黄素可能通过作用于多种信号通路和潜在的分子靶点来延缓NSCLC的发生和进展,在NSCLC的临床治疗过程中可能是一种十分有前景的候选药物。GBA(Galbanic acid),加蓬酸,是一种倍半萜香豆素,具有多种抗肿瘤活性。然而,姜黄素是否通过激活自噬诱导对NSCLC的凋亡及机制未见报道,以及姜黄素能否作为协同GBA治疗NSCLC的理想制剂及其机制仍不明确。因此,本研究旨在探讨姜黄素及其与GBA联合对非小细胞肺癌细胞的治疗作用并阐明其潜在机制。第一部分 姜黄素诱导非小细胞肺癌自噬介导的细胞凋亡目的:研究姜黄素对NSCLC细胞的自噬、凋亡的影响,并探索姜黄素作用于NSCLC的分子机制。方法:1.利用递增浓度的姜黄素分别处理NSCLC细胞系(A549和PC-9),MTT法检测姜黄素对细胞增值和活力的影响。2.A549和PC-9细胞分别暴露于40 μM和20 μM姜黄素48h后,用Hoechst 33258染色细胞核并在荧光显微镜下观察,对细胞的表型进行鉴定。3.利用western blot法检测各组细胞凋亡途径的下游标志性蛋白:BAX、cleaved-caspase-3、cleaved-caspase-8、cleaved-caspase-9,Bcl-2。4.利用mRFP-GFP-LC3双荧光腺病毒自噬指示系统转染A549细胞,再用姜黄素处理细胞24 h,在共聚焦显微镜下对各组具有mRFP和GFP斑点的细胞进行定量,以标记和监测LC3和自噬流的变化。5.western blot法检测各组自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ的表达及自噬关键底物beclin-1的表达。6.使用ROS检测试剂盒及通过流式细胞术分析各组细胞内ROS(活性氧)的产生,研究ROS在姜黄素诱导的NSCLC细胞凋亡过程中的作用。7.使用ROS抑制剂N-乙酰半胱氨酸(NAC)孵育细胞,并用western blot检测自噬、凋亡相关蛋白的表达,以验证ROS是否介导姜黄素诱导NSCLC细胞凋亡和自噬的作用。8.利用western blot检测MAPK信号通路中的关键蛋白ERK、JNK和p38,以进一步探索姜黄素在NSCLC中分子作用机制。9.在姜黄素存在或不存在的情况下,分别采用JNK抑制剂SP600125、p38抑制剂SB203580预处理细胞,再用40 μM姜黄素孵育A549细胞24 h,利用western blot检测自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ,凋亡相关蛋白cleaved caspase-3以及p-JNK/JNK,p-p38/P38的水平,验证MAPK信号通路是否参与姜黄素诱导的细胞凋亡和自噬。10.采用NAC(ROS抑制剂)对细胞进行预处理,Western blot法检测p-JNK/JNK,p-p38/P38的水平,以进一步阐明ROS与姜黄素诱导的MAPK信号通路之间的关系。结果:1.姜黄素以浓度依赖性和时间依赖性的方式降低细胞活性;40μM的姜黄素对A549细胞48 h的活力抑制高达50%,20 μM的姜黄素对PC-9细胞株显示出约50%的抑制能力。2.40 μM和20 μM姜黄素分别处理后的A549和PC细胞中的细胞核皱缩呈现散点状,并且A549和PC-9的凋亡细胞缩小。对缩小的细胞核和总的细胞核比例进行统计学分析表明姜黄素处理后细胞凋亡显著增加。3.A549和PC-9细胞用40 μM和20 μM姜黄素处理48h后,WB结果显示BAX、cleaved-caspase-3(c-cas-3)、cleaved-caspase8(c-cas-8)和 cleaved-caspase-9(c-cas-9)的表达显著性增加,而Bcl-2以剂量依赖性方式降低。这些数据表明姜黄素可能通过诱导的细胞凋亡抑制非小细胞肺癌细胞的细胞生长。4.mRFP-GFP-LC3腺病毒感染实验发现,姜黄素在A549细胞中诱导的GFP-LC3和mRFP-LC3斑点形成显著增强,表明姜黄素可以促进细胞自噬。5.随着姜黄素浓度的增加,LC3-Ⅱ/LC3-Ⅰ的比例显著上调,表明姜黄素处理细胞过程中激活了自噬的反应。此外,beclin-1的表达在两种非小细胞肺癌细胞中显著增强。6.经姜黄素处理的A549细胞和PC细胞的ROS显著上调。7.用NAC抑制ROS的产生后,LC3-Ⅱ/LC3-Ⅰ、cleaved caspase-3 和Cleaved PARP的表达显著下调,表明抑制ROS的产生能有效的下调细胞凋亡的自噬。8.A549和PC-9细胞分别用40 μM和20 μM姜黄素处理48 h后,WB结果显示MAPK通路p-ERK/ERK、p-JNK/JNK和p-p38/p38 的表达上调。9.与单独的姜黄素处理组相比,p38抑制SB203580和JNK抑制剂SP600125干预后的细胞中,自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ和细胞凋亡相关蛋白Cleaved caspase-3显著下调,并降低p-JNK和p-p3 8蛋白水平。10.与单独的姜黄素处理组相比,NAC干预后的细胞中MAPK信号通路相关蛋白p-JNK/JNK,p-p38/P38显著下调,即NAC阻止了姜黄素在A549和PC-9细胞中诱导的p-JNK和p-p38的活化。第二部分 姜黄素增强GBA诱导的抗NSCLC作用目的:研究姜黄素联合GBA抗NSCLC的作用及机制探索。方法:1.MTT法分别测定递增浓度GBA(0、20、40、80 μM)、递增浓度的姜黄素(0、15、30、45 μM),以及固定浓度的GBA分别与低浓度姜黄素(10、15、20 μM)结合使用处理后的A549细胞活性,评估其对NSCLC细胞A549的细胞增值的影响。2.Annexin V-FITC和PI凋亡检测试剂盒对姜黄素(15 μM)和GBA(40 μM)单独处理和联合处理进行直接染色,利用流式细胞术进行分析,以探究姜黄素和GBA在A549中的毒性作用的介导路径。3.通过免疫印迹分析检测凋亡信号通路的下游蛋白:Bax、cleaved-caspase-3、-9、Bcl-2。4.进行Transwell试验以观察GBA和/或姜黄素处理后A549细胞的迁移能力,利用westernblot检测E-cadherin、vimentin和snail的表达水平。5.用基质凝胶侵袭试验观察各组处理后A549细胞的侵袭能力,利用westernblot检测细胞侵袭的标志物uPA和MMP2的表达6.利用GFP-LC3荧光自噬腺病毒指示系统感染A549细胞并监测LC3和自噬流的变化,利用westernblot检测自噬标记蛋白的水平,包括LC3-Ⅱ、LC3-Ⅰ和Beclin-1。7.用雷帕霉素(mTOR抑制剂)或胰岛素(AKT激活剂)预处理细胞,然后分组培养用药,MTT法验证姜黄素和GBA的联用是否通过Akt/mTOR信号通路介导NSCLC细胞死亡;westernblot分析潜在的蛋白质,包括AKT(Ser473)、mTOR(Ser2448)及其下游靶标p70s6k(Ser371)的磷酸化表达。8.用雷帕霉素阻断Akt/mTOR信号通路,利用qRT-PCR检测侵袭相关基因MMP2和uPA的mRNA表达,WesternBlotting测定评细胞凋亡相关蛋白Cleaved caspase 3、自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比例的表达。结果1.GBA或者姜黄素单独处理以浓度依赖性方式抑制A549细胞生长,并且40 μM的GBA表现出约60%的生长抑制。15 μM低浓度的姜黄素和40 μM GBA对A549细胞具有更好的协同抑制效果,比单独使用姜黄素或GBA更有效。这种特殊的联合治疗被用于后续进一步的研究。2.与空白对照组相比,姜黄素(15 μM)处理对细胞凋亡的诱导作用不明显,GBA(40 μM)单独处理A549细胞后,处于凋亡阶段的细胞比例显著增多,而低浓度姜黄素(15μM)和GBA(40μM)联合处理A549细胞后,处于凋亡阶段的细胞比例相较于GBA(40 μM)单独处理组显著增多,这种现象表明联合治疗可能导致A549细胞进一步凋亡。3.与单独使用GBA或空白对照组相比,姜黄素和GBA的联合使用增强了凋亡信号通路的下游蛋白cleaved-caspase-3、-9和Bax的表达,并减少Bcl-2的活化。4.Transwell结果显示,15 μM姜黄素不影响细胞迁移,40 μM GBA可显著减少迁移细胞的数量;与GBA处理相比,15 μM姜黄素与GBA结合使用可进一步显著减少迁移细胞,WB结果显示低浓度的姜黄素和GBA的组合可以有效增强A549细胞中E-cadherin的表达,降低vimentin和snail的表达水平。5.姜黄素和GBA对细胞侵袭的协同抑制作用较单用GBA显著增强,联合处理组能显著降低介导细胞侵袭的标志物uPA和MMP2的表达。6.姜黄素与GBA联合处理对比单独的GBA处理可显著增加A549细胞中以LC3绿色荧光强度为标志的自噬的斑点形成。与GBA组相比,暴露于15μM姜黄素和GBA显著增加了LC3-Ⅱ/LC3-Ⅰ和Beclin-1的表达水平。以上结果表明低浓度姜黄素在A549细胞中增强GBA诱导的细胞自噬。7.40 μM雷帕霉素显著抑制A549细胞的增殖,并在与GBA和姜黄素合用时进一步降低。当暴露于用姜黄素、GBA合用组后,用100 nM胰岛素预处理时,细胞活力显著上调。姜黄素或GBA处理后AKT、mTOR和p70s6k的磷酸化显著下调,40 μM雷帕霉素预处理可加重姜黄素或/和GBA处理引起的抑制作用,而胰岛素预处理可逆转或至少减轻这种抑制作用。研究结果表明Akt/mTOR通路可能介导GBA和姜黄素对A549细胞的抑制作用。8.与姜黄素和GBA联合处理相比,暴露于雷帕霉素加姜黄素和GBA的细胞中MMP2 和uPA的mRNA表达明显下调,Cleaved caspase 3、LC3-Ⅱ/LC3-Ⅰ显著增加。这些结果证实阻断Akt/mTOR信号通路参与调节姜黄素和GBA诱导的A549细胞抗肿瘤作用(侵袭、自噬、凋亡)的事实。结论(1)姜黄素以剂量和时间依赖性方式抑制细胞活性,并促进NSCLC细胞凋亡和自噬;诱导ROS的产生显著增加,诱导的ROS通过激活caspase和自噬相关蛋白导致细胞凋亡和自噬;ROS-MAPK通路介导了姜黄素诱导非小细胞肺癌细胞自噬和细胞凋亡的作用;(2)姜黄素可显著增强GBA诱导的NSCLC细胞凋亡和自噬,并能进一步增强GBA诱导的细胞迁移和侵袭能力;姜黄素通过抑制非小细胞肺癌细胞中的Akt/mTOR信号通路增强了 GBA诱导的抗肿瘤作用。更多还原

【Abstract】 Background:Lung cancer is the cancer with the highest mortality rate among all men.The mortality and morbidity of lung cancer rank first among all malignant tumors in the world.About 85%of lung cancer patients belong to non-small cell lung cancer(NSCLC).Over the past few decades,great strides have been made in new treatments such as targeted drugs and immunotherapy,but the prognosis for patients with NSCLC remains poor.Therefore,finding potential targets and new therapeutic options for the treatment of NSCLC is crucial and urgent.Curcumin,a natural polyphenol extracted from the traditional medicine turmeric,has antiviral,antibacterial,anti-inflammatory,antioxidant and antiproliferative activities.Curcumin can delay the occurrence and progression of NSCLC by acting on multiple signaling pathways and potential molecular targets.GBA(Coumarin)is a sesquiterpene coumarin with various antitumor activities.However,Whether curcumin can induce apoptosis of NSCLC by activating autophagy and its mechanism have not been reported,and whether curcumin can be used as an ideal preparation for the treatment of NSCLC with GBA and its mechanism are still unclear.Here,this study aimed to investigate the therapeutic effect of curcumin and its combination with GBA on non-small cell lung cancer cells and to clarify the underlying mechanism.Part Ⅰ:Curcumin induces autophagy-mediated apoptosis in non-small cell lung cancerObjective:To study the effect of curcumin on autophagy and apoptosis of NSCLC cells,and to analyze the molecular mechanism of curcumin in NSCLC.Methods:1.NSCLC cell lines(A549 and PC-9)were treated with increasing concentration of curcumin.The cell proliferation and viability was assessed by MTT assay.2.Treated with 40μM curcumin in A549 cells and 20 μM curcumin in PC-9 cells respectively After 48 hours,the nucleus of cells were stained with Hoechst 33258 and observed under the fluorescence microscope to identify the cell phenotype.3.The expression of marker proteins involved in apoptosis pathway:BAX,cleaved-capase-3,cleaved-capase-8,cleaved-capase-9,Bcl-2 in each group was estimated by Western blot after treatment.4.Autophagy was determined by confocal microscopy after infection with mRFP-GFP-LC3 dual-fluorescent adenovirus.Cells which with mRFP and GFP double spots in each group were quantified under a confocal microscope to label and monitor the changes of LC3 and autophagy flow.5.The expression levels of autophagy-related protein LC3-II/LC3-I and the expression levels of the key substrates of autophagy,beclin-1 were assessed by Western blot.6.Study of reactive oxygen species(ROS)by flow cytometry,and to explore the role of ROS in the process of curcumin-induced apoptosis of NSCLC cells.7.The cells were incubated with ROS inhibitor N-acetylcysteine(NAC)and the expression of autophagy and apoptosis-related proteins were detected by western blot to verify whether ROS mediated the effect of curcumin on inducing apoptosis and autophagy of NSCLC cells.8.The key proteins of ERK,JNK and p38 in MAPK signal pathway were detected by Western blot to further explore the molecular mechanism of curcumin in NSCLC.9.In the presence or absence of curcumin,cells were pretreated with JNK inhibitor SP600125 and p38 inhibitor SB203580 respectively,and then treated with 40 μM curcumin for 24 hours,and the expression of marker proteins involved in autophagyand apoptosis was estimated by Western Blotting assays to verify whether MAPK signal pathway was involved in curcumin-induced apoptosis and autophagy.10.Cells were pretreated with ROS inhibitor NAC,then the expression of p-JNK/JNK,p-p38/P38 were detected by Western blot to further clarify the relationship between ROS and curcumin-induced MAPK signal pathway.Results:1.Curcumin significantly reduced cell the viability in a concentration-dependent and time-dependent manner.40 μM curcumin inhibited the activity on A549 cells by up to 50%for 48 hours,while 20 μM curcumin showed about 50%inhibition on PC9 cells.2.The nuclei of cells treated with 40 μM and 20 μM curcumin were scattered,and the size of apoptotic cells were smaller.The ratio of the reduced nucleus to the total nucleus showed that the apoptosis of cells increased significantly after curcumin treatment.3.The expression of BAX,cleaved-cacase-3(c-cas-3),cleaved-cacase-8(c-cas-8)and cleaved-cacase-9(c-cas-9)increased significantly After 48h treatment with 40μM curcumin in A549 and 20 μM curcumin in PC-9 cells,while the expression of Bcl-2 decreased in a dose-dependent manner.These data showed that curcumin may inhibit the growth of non-small cell lung cancer cells by inducing apoptosis way.4.Curcumin enhanced the formation of GFP-LC3 and mRFP-LC3 spots significantly in A549 cells,indicating that curcumin can promote cell autophagy which was showed in mRFP-GFP-LC3 infection experiment.5.The proportion of LC3-Ⅱ/LC3-Ⅰ was significantly increased,indicating that curcumin activated autophagy during the treatment of non-small cell lung cancer with the increase of curcumin concentration for 48h.In addition,the expression of beclin-1 was also significantly enhanced in both types of non-small cell lung cancer cells.6.ROS of A549 cells and PC cells was significantly up-regulated during curcumin treatment.7.The expression of LC3-Ⅱ/LC3-Ⅰ,cleared caspase-3 and cleared PARP was significantly down-regulated with NAC,indicating that inhibition of ROS production can effectively reduce autophagy of cell apoptosis.8.The expression of p-ERK/ERK,p-JNK/JNK and p-p38/p38 in MAPK pathway was up-regulated after 48 h treatment with 40μM curcumin and 20 μM curcumin.9.Compared with the curcumin treatment group alone,in the cells after the intervention of p38 inhibitor SB203580 and JNK inhibitor SP600125,the autophagy-related protein LC3-Ⅱ/LC3-Ⅰ and apoptosis-related protein Cleared caspase-3 was significantly down-regulated,and the expression of p-JNK and p-p38 protein was decreased.10.Compared with the curcumin treatment group alone,the MAPK signal pathway related proteins p-JNK/JNK,p-p38/P38 in the cells after NAC intervention were significantly down-regulated.NAC prevented the activation of p-JNK and p-p38 induced by curcumin in A549 and PC-9 cells,and reversed the apoptosis and autophagy reaction induced by curcumin treatment.Part Ⅱ Curcumin enhances GBA-induced anti-NSCLC effectObjective:To study the effect and mechanism of curcumin combined with GBA on NSCLC Methods:1.Use MTT to measure the effect of each group on the proliferation of NSCLC A549 cells with the increasing concentration of GBA(0,20,40,80μM),increasing concentration of curcumin(0,15,30,45 μM)And 40 μM GBA combined with low concentration of curcumin(10,15,20 μM)of A549 cells.2.Annexin V-FITC and PI apoptosis detection kit were used to detect curcumin(15 μM)And GBA(40μM),and flow cytometry was used for analysis to explore the pathway of the toxicity of curcumin and GBA in A549.3.The downstream protein of apoptosis signal pathway was detected by westernblot to further explore the potential mechanism of synergistic apoptotic effect on A549 cells.4.Transwell test was conducted to observe the migration of A549 cells after GBA and/or curcumin treatment.Western blot was used to detect the expression level of E-cadherin,vimentin and snail.5.The invasion of A549 cells treated by each group was observed by matrix gel invasion test.Western blot was used to detect the expression of uPA and MMP2,markers of cell invasion.6.A549 cells were infected with GFP-LC3 fluorescent autophagic adenovirus indicator system to monitor the changes of LC3 and autophagic flow.The autophagic marker proteins,including LC3-Ⅱ,LC3-Ⅰ and Beclin-1,were detected by western blot.7.Cells were pretreated with rapamycin(mTOR inhibitor)or insulin(AKT activator),and then cultured in each group.MTT method was used to verify whether the combination of curcumin and GBA mediated NSCLC cell death through Akt/mTOR signal pathway.Westernblot of potential proteins,including the phosphorylation expression of AKT(Ser473),mTOR(Ser2448)and its downstream target p70s6k(Ser371).8.Using rapamycin to block the Akt/mTOR signaling pathway,qRT PCR was used to detect the mRNA expression of invasion related genes of MMP2 and uPA.Western blotting was used to evaluate the expression of cell apoptosis related protein of Cleared caspase 3 and autophagy related protein of LC3-Ⅱ/LC3-Ⅰ ratio.Results:1.GBA or curcumin alone significantly inhibited the growth of A549 cells in a concentration-dependent manner,and 40 μM GBA showed about 60%growth inhibition.15μM curcumin and 40μM GBA had a better synergistic inhibitory effect on A549 cells,which was more effective than curcumin or GBA alone.This special combination was used for further research.2.Compared with the control group,cell apoptosis was not obvious with 15μM curcumin.After A549 cells were treated alone.The proportion of cell apoptosis increased in 40 μM GBA group significantly.The proportion of apoptotic cells in 15μM curcumin combined with 40 μM GBA group was higher than that of 40 μM GBA group,which indicated that the combined treatment might cause significant apoptosis of A549 cells.3.Compared with GBA alone or the control group,the combination of curcumin and GBA increased the expression of the downstream proteins of the apoptotic signal pathway,cleaved-capase-3,-9 and Bax,and decreased the activation of Bcl-2.4.The Transwell test showed that cell migration was not affected with 15 μM Curcumin,40 μM GBA could significantly reduce the number of migrating cells;Compared with GBA treatment,the combination of curcumin and GBA could further significantly reduce the number of migrating cells.WB results showed that the combination of curcumin at low concentration and GBA can effectively enhance the expression of E-cadherin in A549 cells and reduce the expression level of vimentin and snail.5.The synergistic inhibition of curcumin and GBA on cell invasion was significantly stronger than that of GBA alone.The combined treatment group could significantly reduce the expression of the markers of cell invasion uPA and MMP2.6.Compared with GBA alone,the combination of curcumin and GBA could significantly increase the formation of autophagic spots marked by LC3 green fluorescence intensity in A549 cells.Compared with GBA group,15 μM curcumin combined with 40μM GBA group significantly increased the expression level of LC3-II/LC3-I and Beclin-1.These results showed that low concentration of curcumin enhanced GBA-induced autophagy in A549 cells.7.40 μM rapamycin significantly inhibited the proliferation of A549 cells and further decreased when combined with GBA and curcumin.When exposed to the combination of curcumin and GBA and pretreated with 100 nM insulin,compared with GBA and curcumin,the cell viability was significantly increased.The phosphorylation of AKT,mTOR and p70s6k was significantly decreased after curcumin or GBA treatment,40 μM Rapamycin pretreatment could aggravate the inhibition caused by curcumin or/and GBA treatment,while insulin pretreatment could reverse or at least alleviate this inhibition.The results showed that Akt/mTOR pathway may mediate the inhibition of GBA and curcumin on A549 cells.8.Compared with the combination group of curcumin and GBA,the mRNA expression of MMP2 and uPA in the cells exposed to rapamycin plus curcumin and GBA was significantly down-regulated,and the levels of cleared caspase 3,LC3-Ⅱ/LC3-Ⅰ were significantly increased.These results confirmed the fact that blocking Akt/mTOR signal pathway was involved in regulating the antitumor effect(invasion,autophagy,apoptosis)of A549 cells induced by curcumin and GBA.Conclusion:(1)Curcumin treatment inhibits cell activity in a dose-and time-dependent manner,and promotes apoptosis and autophagy of NSCLC cells;Curcumin induces a significant increase in ROS production,and the induced ROS leads to apoptosis and autophagy by activating caspase and autophagy-related proteins;ROS-MAPK pathway mediates the role of curcumin in inducing autophagy and apoptosis of NSCLC cells;(2)Curcumin can significantly enhance GBA-induced NSCLC cell apoptosis and autophagy of NSCLC cells,and further enhance GBA-induced cell migration and invasion;Curcumin enhanced the antitumor effect induced by GBA by inhibiting Akt/mTOR signal pathway in NSCLC cells.更多还原