【作者】 何秋霞；

【导师】 苗俊英；

【作者基本信息】 山东大学 ， 细胞生物学， 2008， 博士

【摘要】 研究的目的和意义肺癌死亡率居于各种恶性肿瘤之首,而在肺癌中,肺腺癌是最常见的、发病率最高的一种。由于肺腺癌转移后禁忌手术,且对放疗不敏感,所以通常用化疗来治疗肺腺癌。目前,在肺腺癌治疗方面面临的主要问题包括开发新的化疗药物和阐明肺腺癌细胞生长、增殖、分化、转移和死亡的分子机制。然而,有关肺癌细胞自噬性死亡的分子机制方面的研究仅有很少报道,人们对肺腺癌细胞自噬性死亡的分子机制仍了解甚少。因此,揭示肺腺癌细胞自噬性死亡的分子机理具有重要的理论意义和应用价值。本研究选用A549细胞为材料研究肺腺癌生长的分子机制的原因在于:(1)A549细胞属于肺腺癌细胞系;(2)在肺腺癌研究领域,A549细胞通常作为一种标准模式细胞进行细胞学水平的检测;(3)目前对A549细胞自噬性死亡的分子机制了解甚少。化学遗传学是研究信号网络、蛋白质功能、代谢通路、开发创新性药物的有力工具。利用这种方法可以鉴定出一些在生物学过程中起重要调节作用的蛋白或基因,进而揭示一些复杂的细胞内过程,这为研究细胞死亡的分子机理提供了新思路。因此,本研究从化学遗传学角度出发,以小分子化合物作为干涉细胞生长的有效工具,对A549细胞自噬性死亡的分子机制进行了较为系统的研究。这些研究结果不仅有助于了解A549细胞自噬性死亡的分子机制,而且为肺腺癌治疗药物的有效开发提供一定的理论依据。血管形成不仅在肿瘤的生长过程中发挥关键作用,而且参与了肿瘤的转化过程,通过抑制血管形成来阻止肿瘤细胞的增殖已成为治疗肿瘤的有效策略之一。研究发现,血管内皮细胞的活化与增殖在血管形成中发挥关键作用,诱导血管内皮细胞凋亡是导致肿瘤血管退化的关键步骤。体外培养的人脐静脉血管内皮细胞是研究内皮细胞凋亡机制的良好材料。但目前有关人脐静脉血管内皮细胞凋亡的研究中诱导其凋亡的方法主要采用去除生长因子或去除生长因子并加入内皮细胞凋亡诱导剂(如manolide,MIBX等)的方法。有关生长因子存在条件下,人脐静脉血管内皮细胞凋亡的分子机制仍不清楚。本研究在加入生长因子的条件下,以小分子化合物作为凋亡诱导剂,对人脐静脉血管内皮细胞的凋亡机理进行了初步探讨。这些研究可加深对血管内皮细胞凋亡的分子机理的认识,为研究肿瘤血管退化的机制提供重要参考资料,有望为肺腺癌的有效治疗提供重要启迪。研究内容1.以吗啉酮衍生物作为诱导A549细胞凋亡的工具,研究A549细胞凋亡与细胞周期分布、P53和Fas蛋白累积量之间的关系。2.观察分析苯并噁嗪衍生物对A549细胞生长的影响,筛选能够有效抑制A549细胞生长的生物活性小分子。3.进一步分析2中筛选出的苯并噁嗪衍生物3f(6,8-二氯-2,3-二氢-3-羟甲基-1,4-苯并噁嗪,DBO)对A549细胞自噬性死亡的诱导作用。4.在上述实验中,我们发现DBO处理导致A549细胞中的膜整连蛋白β4显著下调,本实验进一步研究了膜整连蛋白β4在A549细胞自噬性死亡的作用。5.生长因子FGF-2存在的条件下,DBO诱导人脐静脉血管内皮细胞凋亡的研究及其对血管形成的影响研究方法1.细胞死亡方式的检测1.1利用倒置相差显微镜观察细胞形态的变化1.2 MTT法检测细胞存活率,由此对小分子化合物进行初步筛选。1.3吖啶橙(AO)染色后,通过荧光显微镜观察细胞核形态的变化以及酸性膜泡的数量和分布,从而判断细胞是否凋亡或自噬性死亡。1.4用免疫细胞化学法结合激光扫描共聚焦显微镜技术检测MAP1 LC3-Ⅱ(LC3-Ⅱ)的含量及其在细胞内分布的变化,以鉴定细胞是否发生自噬性死亡。1.5通过检测细胞培养液中乳酸脱氢酶的活性以鉴定细胞是否发生坏死。1.6 TUNEL法检测细胞凋亡率。1.7台盼蓝染色法检测细胞存活能力。1.8利用流式细胞仪检测细胞周期分布情况。2.细胞内蛋白水平及分布的检测:2.1免疫细胞化学法结合激光扫描共聚焦显微镜技术,检测P53、Fas、膜整连蛋白β4和MAP1 LC3-Ⅱ蛋白含量及其分布的变化。2.2 Western blot法检测P53、膜整连蛋白β4蛋白累积量的变化。3.DBO对A549细胞基因表达的影响利用人类全基因组cDNA芯片检测小分子化合物DBO对A549细胞基因表达的影响,具体参考Patterson的方法[Patterson et al.,2006]4.RIS1,FST,RIN2,RAB5B和膜整连蛋白β4的转录表达分析利用反转录PCR(RT-PCR)和琼脂糖凝胶电泳相结合,检测Ras诱导衰老基因(RIS1),滤泡素抑制素(FST),Ras与Rab相互作用因子2(RIN2),RAB5B和膜整连蛋白β4在mRNA水平上的变化。5.RNAi干扰蛋白表达:利用RNAi技术下调膜整连蛋白β4在A549细胞中的表达后,检测细胞存活率的变化,并采用TUNEL,LDH活性检测,AO染色和MAP1 LC3-Ⅱ免疫细胞化学检测等方法鉴别细胞的死亡方式。6.血管内皮细胞的提取和培养:参考Jaffe等的方法[Jaffe et al.,1973]7.ROS检测:利用荧光探针(DCHF)结合激光扫描共聚焦显微镜技术进行检测8.SOD活性检测:利用SOD检测试剂盒进行检测,具体操作按说明书进行。9.NADPH氧化酶活性检测:参考Li等的方法[Li et al,2002]10.体内外血管形成模型:10.1 Matrigel血管形成模型:参考Kureishi等的方法[Kureishi et al.,2000]10.2鸡胚尿囊膜(CAM)模型:参考Zhao等的方法[Zhao et al,2005]研究结果:1.吗啉酮衍生物诱导A549细胞凋亡及其分子机制1.1在48 h内,三种吗啉酮衍生物(化合物1,2和3,图1)以浓度依赖的方式显著降低A549细胞存活率(图2)。1.2流式细胞仪的分析结果表明,40μg/ml吗啉酮衍生物化合物1处理A549细胞48h后,与对照组相比,S期细胞所占比例下降,而G0/G1和G2/M期细胞比例上升。化合物2和3处理后,S期和G2/M期细胞所占比例均下降,而G0/G1期细胞比例明显上升(图3)。1.3倒置相差显微镜观察结果显示,40μg/ml的吗啉酮衍生物1,2和3分别处理A549细胞48 h后,细胞表现出明显的凋亡形态:胞体皱缩,胞浆内空泡明显,核固缩,折光性减弱,凋亡小体样结构形成(图4)。其中,化合物3的作用效果尤为明显。1.4 AO染色后利用荧光显微镜检测发现,40μg/ml的吗啉酮衍生物1,2和3分别处理A549细胞48 h后,细胞也呈现出典型的凋亡形态学特征:细胞核固缩、染色质凝集且边缘化明显、核碎裂,凋亡小体清晰可见(图5)。其中化合物3是三种吗啉酮衍生物中诱导A549细胞凋亡的最有效分子。1.5 40μg/ml的吗啉酮衍生物1,2和3分别处理A549细胞48 h后,细胞内P53蛋白含量明显增加,并向细胞核中聚集(图6)。细胞内Fas蛋白水平显著升高,在细胞膜上的分布也发生明显改变。在对照组,Fas弥散在整个细胞表面;而在化合物处理组,Fas蛋白在细胞膜上聚集成帽状,并有向细胞的一端迁移的趋势(图7)。吗啉酮衍生物通过Fas通路诱导A549细胞凋亡的机制与本实验室以前研究的黄樟素氧化物诱导A549细胞凋亡的机制类似,故对其诱导A549细胞凋亡的分子机理未做更深入的研究。2.苯并噁嗪衍生物初步筛选结果(MTT法检测细胞存活率)在24-48 h,200和400μM苯并噁嗪衍生物化合物3c,3d和3f都能够显著地抑制A549细胞生长(p＜0.05,图9),而化合物3a,3b,3e和3g对A549细胞的生长无显著影响(图10)。其中化合物3f(6,8-二氯-2,3-二氢-3-羟甲基-1,4-苯并噁嗪,DBO)的抑制效果最为明显。3.DBO诱导A549细胞自噬性死亡及其分子机制3.1 200μM DBO处理A549细胞48 h时,与对照组相比,G0/G1和G2/M期细胞所占比例明显上升(图12),P53蛋白含量显著增加(p＜0.05,图13),而膜整连蛋白β4的含量明显减少(p＜0.05,图14)。说明DBO极有可能通过上调P53蛋白、下调膜整连蛋白β4的含量,阻滞细胞于G1期,从而抑制A549细胞的生长。3.2 TUNEL和LDH检测发现,200μM DBO处理A549细胞48 h时,细胞凋亡率和培养液中LDH活性均无明显变化(图16和17),AO染色阳性率明显升高(图18)。免疫细胞化学检测的结果表明,在DBO处理组LC3-Ⅱ在细胞质中块状聚集,而在对照组中无此现象出现(图19)。这些结果说明DBO既没有诱导A549细胞凋亡也没有诱导其坏死,而极有可能是引起了A549细胞的自噬性死亡。3.3基因芯片的结果显示,200μM DBO处理A549细胞48 h时,共有77个基因发生了2倍以上量的显著变化,其中64(0.3%)个基因显著上调,13(0.06%)个基因明显下调(图20)。基于细胞生长率的变化以及酸性膜泡的增多,我们选择了Ras诱导的衰老相关基因1(RIS1),滤泡素抑制素(FST),RAB5B和RIN2等4个基因进行了进一步转录表达分析。RT-PCR结果显示,随着DBO处理时间的延长,A549细胞中RIS1,FST和RIN2的表达水平逐渐上升,RAB5B的表达水平逐渐下降(图21)。与其它基因不同的是,膜整连蛋白p4在处理24 h恢复到与对照组相当的水平,说明膜整连蛋白β4的表达可能随细胞周期的变化而变化。为阐明膜整连蛋白β4在调控A549肺腺癌细胞生长和增殖中的作用,我们利用膜整连蛋白β4特异性siRNA干扰其表达。膜整连蛋白β4下调后,虽然A549细胞的凋亡率和培养液中LDH活性均无明显变化(图23),但A549细胞存活率却明显降低(图22)。进一步分析表明,A549细胞内酸性膜泡数量明显增多,LC3-Ⅱ的表达显著增强、且呈斑块状聚集(图24),RIS1,FST和RIN2基因的表达明显增强,而RAB5B的表达明显减弱(图25)。意味着膜整连蛋白β4,RIS1,FST,RAB5B和RIN2可能参与调控A549细胞的生长和自噬性死亡过程。4.生长因子FGF2存在的条件下,DBO诱导人脐静脉血管内皮细胞凋亡及其分子机制4.1 50μM DBO处理人脐静脉血管内皮细胞24 h后,经检测发现膜整连蛋白β4的含量显著增加(图26),细胞凋亡率从21.3%上升到68.5%(p＜0.01,图27)。4.2 50μM DBO处理人脐静脉血管内皮细胞12 h时,细胞内的ROS和NO水平显著提高(图28和30A)。酶活分析表明细胞内NADPH氧化酶和iNOS的活性显著升高,而SOD和eNOS的活性无显著变化(图29和30B)。这意味着极有可能是由于NADPH氧化酶活性升高导致了细胞内ROS含量的增加,而iNOS活性的升高导致了NO含量增加。但在DBO处理人脐静脉血管内皮细胞前,若先用NADPH氧化酶的特异性抑制剂DPI处理细胞30分钟,则细胞内ROS和NO的含量均不能显著提高(图31),预示着在血管内皮凋亡中,NO的产生可能通过NADPH氧化酶与ROS代谢有关联。5.DBO对体内外血管形成的影响50μM DBO处理人脐静脉血管内皮细胞24 h时,分化形成的毛细血管样血管的数量最多。随着培养时间的延长,血管形成被抑制。到72h的时候,在DBO处理组几乎看不到毛细血管样结构存在(图32)。DBO(10-40 nmol)处理鸡胚尿囊膜3天后,与对照组相比,虽然一些大的血管仍存在,但是大量毛细血管消失(图33)。说明DBO能有效抑制血管形成。结论:1.吗啉酮衍生物是有效的肺腺癌细胞凋亡诱导剂,其中化合物3的作用最明显。2.膜整连蛋白β4可能是调节肺腺癌细胞存活与自噬性死亡的关键因子。3.苯并嗯嗪衍生物DBO可能通过诱导内皮细胞凋亡而抑制血管形成。更多还原

【Abstract】 OBJECTIVE AND SIGNIFICANCELung cancer mortality stands the first within all kinds of cancer.Among all types of lung cancer,lung adenocarcinoma occurs most commonly and its incident rate is the highest.Lung adenocarcinoma is insensitive to radiation therapy,and it is taboo against operation after metastasis.Therefore,chemotherapy is usually selected to cure lung adenocarcinoma.Current challenges in lung adenocarcinoma treatment include the search of the most promising new agents,which can be integrated into current methods of treatment,and clarification of the mechanism by which lung adenocarcinoma cells undergo growth,proliferation,differentiation,metastasis and death.However,few researches focused on lung adenocarcinoma cell autophagic death and the mechanism of it is poorly understood.Therefore,the further study on the mechanism of autophagic death in lung adenocarcinoma cells is of important theoretical significance and practical value.Why A549 cells were used to explore the molecular mechanisms of autophagy for lung adenocarcinoma in this study? First,A549 cells belong to lung adenocarcinoma cell lines.Second,in the field of anti-lung carcinoma drug discovery,A549 cells are often used as a model for cell-based assays.Third,at present,only a few fragmentary works has been done to explore the molecules involved in A549 cell autophagy and the mechanism of its autophagic death is still unclear.Chemical genetics is a powerful tool for the researches on signal transduction,protein function and discovering new drugs.Several proteins/genes, which play key roles in some biological process,can be identified from the perspective of chemical genetics.It is helpful to illuminate the mechanism of some complex intracellular phenomena.Therefore,chemical genetics provides a new strategy for studying the underlying molecule mechanism of cell death.Based on these background,small chemical molecules were used as a powerful tools to interfere with intracellular signal transduction networks related to cell proliferation, and to systematically investigated the mechanisms of autophagic death in A549 cells. The results of this study not only promote our understanding on the molecular mechanisms of autophagic death of A549 cells,but also provide theoretical bases for developing anti-lung adenocarcinoma agents to a certain extent.Angiogenesis is very important for tumor cell growth and progression. Therefore,inhibiting the proliferation of cancer cell by preventing angiogenesis is an effective way for cancer therapy.Some studies showed that activation and proliferation of vascular endothelial cells(VEC)play important roles in angiogenesis. Apoptosis of vascular endothelial cells would result in angiogenesis degeneration for tumor.Human umbilical vascular endothelial cell(HUVEC)is an excellent model for research on VEC apoptosis.At present,in the studies of HUVEC apoptosis,cell apoptosis generally was induced by depriving of growth factor with or without apoptosis inducer.However,in the presence of growth factor,the molecular mechanism of HUVEC apoptosis is still unclear.In this study,we used small chemical molecules as HUVEC apoptosis inducer to investigate the molecule mechanism of apoptosis in the presence of FGF 2.These data would promote our understanding on HUVEC apoptosis and offered the valuable information for further study in the degeneration of tumor vascular,which might provide ideas and objects for lung adenocarcinoma therapy.STUDY CONTENTS1.The apoptosis of A549 cell were induced by three novel morpholin-3-one derivatives and the potential roles of P53 and Fas proteins in A549 cells apoptosis were studied.2.The effects of benzoxazine derivatives on A549 cell viability were measured by MTT assay,and chemical molecules,which significantly inhibited the growth of A549 cells,were acquired including benzoxazine derivative compound 3f (DBO).3.As a most effective growth inhibitor of A549 cells among benzoxazine derivatives,the effect of DBO on autophagic death ofA549 cells was analyzed.4.In our study above,we found that integrinβ4 was obviously down regulated in A549 cells treated with DBO.Therefore.We further investigated the action of integrinβ4 in autophagic death of A549 cells.5.In the presence of FGF 2.the molecular mechanism of HUVEC apoptosis induced by DBO and the effect of DBO on angiogenesis were analyzed.METHODS1.Measurement of cell death mode:1.1 The changes of cell morphology were observed under phase contrast microscope.1.2 The cell viability was determined by MTT assay to screen small molecules1.3 Nuclear fragmentation,and the quantity and intracellular distribution of acidic vesicle were analyzed by acridine orange(AO)staining under fluorescent microscope.Accordingly,we estimated cell death mode:apoptosis or autophagic death?1.4 To estimate whether autophagic cell death was induced,the content and intracellualr distribution of MAP1 LC3-Ⅱ(LC3-Ⅱ)protein were detected by immunocytochemistry.1.5 To estimate whether necrosis happened,the activity of lactate dehydrogenase in culture medium was measured.1.6 Cell apoptotic rate was detected by TUNEL assay.1.7 The viability of A549 cell was examined by trypan blue exclusion.1.8 The distribution of cell cycle was anlyzed by flow cytometry.2.Analyses of protein contents and distribution:2.1 The contents and intracellular distribution of P53,Fas,integrinβ4 and MAP1 LC3-Ⅱproteins were examinated by immunocytochemistry.2.2 The contents of P53 and integrinβ4 proteins were analyzed by western blot assay.3.Effects of DBO on genes expression in A549 cells:The analysis of gene expression was performed by 22K human genome cDNA microarray according to Patterson et al[Patterson et al.,2006].4.RNA expression analyses of RIS1,FST,RIN2,RAB5B and integrinβ4: The mRNA abundances of RIS1(ras induced senescence 1),FST(Follistatin), RIN2(Ras and Rab interactor 2),RAB5B and integrinβ4 were analyzed by semi-quantitative RT-PCR and agarose gel electrophoresis.5.RNAi:When the contents of integrinβ4 in cells was down-regulated by RNAi,the cell viability was examined by MTT assay and cell death mode was identified by TUNEL assay,LDH activity assay,AO staining and LC3-Ⅱimmunocytochemistry.6.HUVEC culture:The extraction and culture of HUVEC were carried out according to Jaffe et al. [Jaffe et al.,1973].7.Measurement of ROS levelROS level was analyzed by the fluorescent probe(DCHF).8.Activity assay of SODSOD activity was performed according to the protocol of SOD detection kit.9.Activity measurement of NADPH oxidaseActivity of NADPH oxidase was detected according to Li et al.[Li et al.,2002].10.The models of angiogenesis in vitro and in vivo10.1 Capillary-like tube formation assay:The formation of vascular-like structures in HUVECs was analyzed by growth-factor-reduced Matrigel according to Kureishi et al.[Kureishi et al.,2000].10.2 Chick embryo chorioallantoic membrane(CAM)assay:CAM assay was performed as described previously[Zhao et al,2005].RESULTS:1.The molecular mechanisms of A549 cell apoptosis induced by novel morpholin-3-one derivatives1.1 Three novel morpholin-3-one derivatives(compound 1,2 and 3,figure 1) significantly inhibited A549 cell viability in a dose-dependent manner at 48 h (figure 2).1.2 After the cells were treated with 40μg/ml compound 1 for 48 h,the percentage of the cells in S phase obviously decreased and that in G1 and G2/M phases increased,compared to control.After the cells were incubated with 40μg/ml compound 2 and 3 for 48 h,the percentage of the cells in S and G2/M phases obviously decreased,and that in G1 phase obviously increased compared to control(figure 3).1.3 The changes of morphology associated with apoptosis occurred when the cells were treated with 40μg/ml morpholin-3-one derivatives for 48 h(figure 4),for example cell shrink,increasing of intracellular vacuoles,nuclear condensation, weaker refraction and apoptotic body formation.1.4 Treated with 40μg/ml three morpholin-3-one derivatives for 48 h,the cells presented chromatin condensation and DNA fragmentation by AO staining. Among the morpholin-3-one derivatives,compound 3 was the most effective apoptosis inducer(figure 5).1.5 When the cells were separately treated with 40μg/ml morpholin-3-one derivatives(compound 1,2 and 3)for 48 h,P53 protein was accumulated in nucleus and its relative content was significantly increased compared to control (figure 6).The relative content of Fas protein was also remarkably increased and its distribution on cell membrane was altered in the cells treated with the morpholin-3-one derivatives compared to control(figure 7).In the control group, Fas was diffusely distributed on the cell surface.However,in the cells treated with morpholin-3-one derivatives,Fas was assembled into patches and some patches were found to migrate toward one pole of the cell(figure 7).The results are similar with these of our previous report that safrole oxide could induce A549 cell apoptosis through Fas signal pathway.Therefore,we did not further explore the molecular mechanisms of A549 cell apoptosis induced by morpholin-3-one derivatives.2.The primary screen of benzoxazine derivativesAt 24-48 h,200 or 400μM compounds 3c,3d and 3f obviously inhibited A549 cell growth(figure 9),but the compounds 3a.3b,3e and 3g had no obvious effects on it(figure 10).Among compounds 3c,3d and 3f,compound 3f (6,8-dichioro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine,DBO)was the most effective one.3.The mechanism of A549 cell autophagic death induced by DBO3.1 Incubation with 200μM DBO for 48 h resulted in a substantial G0/G1 and G2/M1 phase block in A549 cells(figure 12).The level of P53 protein in cells treated with DBO was higher than that in control group(p＜0.05,figure 13).The relative content of integrinβ4 decreased obviously(p＜0.05,figure 14).These data suggested that DBO inhibited A549 cell viability probably through decreasing the level of integrinβ4,elevating the level of P53 and blocking A549 cells at G1 phase.3.2 TUNEL assay and LDH release analysis showed that there was no significant difference in cell apoptotic rate and LDH activity present in the medium of both control and test groups(figure 16 and 17).A549 cells showed quite extensive acridine orange staining,which is one of the indicatives of autophagic cell death (figure 18).DBO elevated the level of LC3-Ⅱ,which is now widely used as a specific marker of autopgagic cell death,and fluorescent dots were observed in DBO-treated A549 cells,while LC3-Ⅱwas diffusely distributed in the cytoplasm in control group(figure 19).These data suggested that DBO induced autophagic cell death instead of apoptosis and necrosis in A549 cells.3.3 The results from gene microarray showed that 77 genes were differentlv expressed more than two folds in A549 cells treated with 200μM DBO for 48 h (figure 20),including 64(0.3%)up-regulated genes and 13(0.06%) down-regulated genes.Based on the changes of cell growth and the increment of acidic vesicle,we selected RIS1,FST,RAB5B and RIN2 as candidates to be further investigated.DBO gradually increased the expressions of FST,RIS1 and RIN2 and decreased the expression of RAB5B with prolonged treatment(figure 21).It is interesting that the expression of integrinβ4 was recovered at 24 h, compared to control,indicating that this integrin subunit might change with cell cycle.To demonstrate the role of integrinβ4 in controlling A549 cell growth,we used integrinβ4 specific siRNA to down-regulate it.When the expression of integrinβ4 was blocked by the 40 nM siRNA treatment for 48 h.the cell viability decreased significantly(figure 22),while neither apoptosis nor necrosis happened(figure 23).Interestingly,in the cells treated with integrinβ4 specific siRNA,acidic vesicles accumulated and dispersed in cytoplasm(figure 24).In line with the results of AO staining,the LC3-Ⅱaggregated in A549 cells treated with integrinβ4 specific siRNA and the level of LC3-Ⅱalso increased compared to control(figure 24).Moreover,A549 cells treated with integrinβ4 specific siRNA showed significantly increased expressions of RIS1,FST and RIN2,and decreased expression of RAB5B(figure 25).These data indicated integrinβ4, FST,RIS1.RAB5B and RIN2 might were involved in regulating A549 cell growth and autophagic cell death.4.The apoptosis mechanism of HUVEC induced by DBO in the presence of FGF 24.1 In the HUVECs treated with 50μM DBO for 24 h,the cell apoptotic rate increased from 21.3%to 68.5%and the content of integrinβ4 was also obviously increased(figure 26 and 27).4.2 DBO(50μM treatment for 12 h)significantly increased the level of ROS and NO in HUVECs(figure 28 and 30A).Enzyme activity assay showed that the activity of NADPH oxidase and iNOS obviously increased,but that of SOD and eNOS did not obviously changed(figure 29 and 30B).The data indicated that DBO elevated the level of ROS and NO though increasing the activity of NADPH oxidase and iNOS.When NADPH oxidase was inhibited by its specific inhibitor dibenziodolium chloride(DPI),DBO could not elevate the levels of ROS and NO in HUVECs(figure 31).The results indicated that NO metabolism might be related to ROS metabolism in the process of VEC apoptosis.5.The effects of DBO on angiogenesisHUVECs treated with 50μM DBO for 24 h could differentiate towards capillary-like tubes to most extent on matrigel.But,with the prolonged culture, the formation of capillary-like tubes was inhibited by DBO.At 72 h,almost all capillary-like tubes were disrupted in DBO treatment group(figure 32).When the filter discs were soaked with DBO(10-40 nmol),some allantoic vessels existed while many capillary vessels disappeared on incubation for 3 days(figure 33).The data indicated that DBO effectively inhibited angiogenesis.Conclusions:1.The data suggested that the morpholin-3-one derivatives were new apoptosis inducers.Morpholin-3-one derivative compound 3 was the most effective one.2.Integrinβ4 might be a key factor in regulating lung adenocarcinoma cell survival and autophagic cell death.3.Our data suggested that DBO might inhibit angiogenesis through inducing VEC apoptosis.更多还原