利用小分子化合物研究肺腺癌细胞死亡及血管内皮细胞凋亡的分子机制
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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。
OBJECTIVE AND SIGNIFICANCE
Lung 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 CONTENTS
1.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.
METHODS
1.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 molecules
1.3 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 level
ROS level was analyzed by the fluorescent probe(DCHF).
8.Activity assay of SOD
SOD activity was performed according to the protocol of SOD detection kit.
9.Activity measurement of NADPH oxidase
Activity of NADPH oxidase was detected according to Li et al.[Li et al.,2002].
10.The models of angiogenesis in vitro and in vivo
10.1 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 derivatives
1.1 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 derivatives
At 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 DBO
3.1 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 2
4.1 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 angiogenesis
HUVECs 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.
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。
OBJECTIVE AND SIGNIFICANCE
Lung 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 CONTENTS
1.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.
METHODS
1.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 molecules
1.3 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 level
ROS level was analyzed by the fluorescent probe(DCHF).
8.Activity assay of SOD
SOD activity was performed according to the protocol of SOD detection kit.
9.Activity measurement of NADPH oxidase
Activity of NADPH oxidase was detected according to Li et al.[Li et al.,2002].
10.The models of angiogenesis in vitro and in vivo
10.1 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 derivatives
1.1 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 derivatives
At 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 DBO
3.1 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 2
4.1 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 angiogenesis
HUVECs 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.
引文
Abraham MC, Shaham S. Death without caspases, caspases without death. Trends Cell Biol, 2004, 14, 184-193.
Adams JM. Ways of dying multiple pathways to apoptosis. Genes Dev, 2003, 17. 2481-2495.
Allen LF, Sebolt-Leopold J, Meyer MB. CI-1040 (PD184352), a targeted signal transduction inhibitor of MEK (MAPKK). Semin Oncol, 2003, 30, 105-116.
Andriana BB, Tay TW, Maki I, Awal MA, Kanai Y, Kurohmaru M, Hayashi Y. An ultrastructural study on cytotoxic effects of mono (2-ethylhexyl) phthalate (MEHP) on testes in Shiba goat in vitro. J Vet Sci, 2004, 5, 235-240.
Artenie R, Artenie A, Cosovanu A. The cardiovascular significance of nitric oxide. Rev Med Chir Soc Med Nat Iasi, 1999,103,48-56.
Arthur CR, Gupton JT, Kellogg GE, Yeudall WA, Cabot MC, Newsham IF. Autophagic cell death, polyploidy and senescence induced in breast tumor cells by the substituted pyrrole JG-03-14, a novel microtubule poison. Biochem Pharmacol, 2007,74,981-991.
Auerbach R, Lewis R, Shinners B, Kubai L, Akhtar N. Angiogenesis Assays: A Critical Overview. Clin Chem. 2003,49, 32-40.
Bachelder RE, Marchetti A, Falcioni R. Activation of p53 function in carcinoma cells by the alpha(?)beta4 integrin. J Biol Chem, 1999, 274, 20733-20737.
Baehrecke EH. Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol, 2005,6,505-510.
Bampton Edward TW, Goemans CG, Niranjan D, Mizushima N, Tolkovsky AM. The dynamics of autophagy visualised in live cells: from autophagosome formation to fusion with endo/lysosomes. Autophagy, 2005,1, 023-036.
Banuelos A, Reyes E, Ocadiz R, Alvarez E, Moreno M, Monroy A, Gariglio P. Neocarzinostatin induces an effective p53-dependent response in human papillomavirus-positive cervical cancer cells. J Pharmacol Exp Ther, 2003, 306, 671-680.
Baril P, Gangeswaran R, Mahon PC, Caulee K. Kocher HM. Harada T. Periostin promotes invasiveness and resistance of pancreatic cancer cells to hypoxia-induced cell death: role of the beta4 integrin and the PI3k pathway. Oncogene, 2007, 26, 2082-2094.
Barradas M, Gonos ES, Zebedee Z. Koletta, E, Petropoulou C Delgado MD. Identification of a candidate tumor-suppressor gene specifically activated during Ras-induced senescence. Exp Cell Res, 2002, 273. 127-137.
Bellu AR, Kiel JA. Selective degradation of peroxisomes in yeasts. Microsc Res Tech. 2003,61,161-170.
Bertotti A, Comoglio PM, Trusolino L. β4 integrin activates a Shp2-Src signaling pathway that sustains HGF-induced anchorage-independent growth. J Cell Biol, 2006. 175.993-1003.
Boelens MC. van den Berg A, Vogelzang I, Wesseling J, Postma DS, Timens W. Differential expression and distribution of epithelial adhesion molecules in non-small cell lung cancer and normal bronchus. J Clin Pathol, 2007, 60, 608-614.
Brown KJ, Maynes SF, Bezos A. A novel in vitro assay for human angiogenesis. Lab Inves, 1996, 75, 539-555.
Butcher RA Schreiber SL. Using genome-wide transcriptional profiling to elucidate small-moiecule mechanism. Curr Opin Chem Biol, 2005, 9, 25-30.
Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature, 2000, 407, 249-257.
Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M, Wu Y, Bono F, Devy L, Beck H, Scholz D, Acker T, DiPalma T, Dewerchin M, Noel A, Stalmans I, Barra A, Blacher S, Vandendriessche T, Ponten A, Eriksson U, Plate KH, Foidart JM, Schaper W, Charnock-Jones DS, Hicklin DJ, Herbert JM, Collen D, Persico MG. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med, 2001, 7, 575-583.
Chen S, Hilcove S, Ding S. Exploring stem cell biology with small Molecules. Mol Biosys, 2006, 2,18-24.
Chu TS, Wu MS, Hsieh BS. Vasodilator agents inhibit opossum kidney proximal tubular cell growth. J Fomros Med Assoc, 2005,104, 374-377.
Coraux C, Delplanque A, Hinnrasky J, Peault B, Puchelle E, Gaillard D. Distribution of integrins during human fetal lung development. J Histochem Cytochem, 1998, 46,803-810.
Cremesti A, Paris F, Grassme H, Holler N, Tschopp J, Fuks Z, Gulbins E, Kolesnick R. Ceramide enables fas to cap and kill. J Biol Chem 2001, 276, 23954-23961.
Crotzer VL, Blum JS. Autophagy and intracellular surveillance: Modulating MHC class Ⅱ antigen presentation with stress. Proc Natl Acad Sci USA, 2005, 102, 7779-7780.
Cuervo AM, Dice JF. A receptor for the selective uptake and degradation of proteins by lysosomes. Science, 1996, 273, 501-503.
Daemi N, Thomasset N, Lissitzky JC, Dumortier J, Jacquier MF. Anti-β4 integrin antibodies enhance migratory and invasive abilities of human colon adenocarcinoma cells and their MMP-2 expression. Int J Cancer, 2000, 85, 850-856.
D'Ambra TE, Estep KG, Bell MR, Eissenstat MA, Josef KA, Ward SJ, Haycock DA. Conformationally restrained analogues of pravadoline: nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor. J Med Chem, 1992,35,124-135.
de Nigris F, Lerman A, Ignarro LJ. Oxidation-sensitive mechanisms, vascular apoptosis and atherosclerosis.Trends Mol Med, 2003, 9, 351-359. Dowling J, Yu Q C, Fuchs E. Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival. J Cell Biol, 1996, 134, 559-572.
Degenhardt K, Mathew R, Beaudoin B. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell, 2006, 10, 51-64.
Du AY, Zhao BX, Miao JY, Yin DL, Zhang SL. Safrole oxide induces apoptosis by up-regulating Fas and FasL instead of integrin beta4 in A549 human lung cancer cells. Bioorg Med Chem, 2006a. 14. 2438-2445.
Du AY. Zhao BX. Yin DL, Zhang SL. Miao JY. Safrole oxide induces apoptosis by activating caspase-3. -8, and -9 in A549 human lung cancer cells. Bioorg Med Chem Lett, 2006b, 16.81-83.
Du AY, Zhao BX, Yin DL, Zhang SL, Miao JY. Discovery of a novel small molecule. l-ethoxy-3-(3,4-methylenedioxyphenyl)-2-propanol, that induces apoptosis in A549 human lung cancer cells. Bioorg Med Chem, 2005, 13,4176-4183.
Du AY, Zhang SL, Miao JY. Zhao BX. Safrole oxide induces apoptosis in A549 human lung cancer cells. Exp Lung Res, 2004, 30, 419-429.
Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell, 2000, 102,33-42.
Du CQ, Zhao QT. Araki S, Zhang SL, Miao JY Apoptosis mediated by phosphatidylcholine-specific phospholipase C is associated with cAMP, p53 level, and cell-cycle distribution in vascular endothelial cells. Endothelium, 2003, 10, 141-147.
Edick MJ, Tesfay L, Lamb LE, Knudsen BS, Miranti CK. Inhibitionof integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Srcsignaling pathways in autophagic prostate epithelial cells induces caspase-independentdeath. Mol Biol Cell, 2007, 18, 2481-2490.
Eduardo O, Sandes A, licia G. Expression of inducible nitric oxide synthase in tumoral and non-tumoral epithelia from bladder cancer patients. Ntric oxide, 2005, 12, 39-45.
Falcioni R, Antonini A, Nistico P, Di Stefano S, Crescenzi M, Natali PG, Sacchi A. a6b4 and a6bl integrins associate with ErbB-2 in human carcinoma cell lines. Exp Cell Res, 1997,236, 76-85.
Falcioni R, Kennel SJ, Giacomini P, Zupi G, Sacchi A. Expression of tumor antigen correlated with metastatic potential of Levis lung carcinoma and B16 melanoma clones in mice. Cancer Res, 1986, 46, 5772-5778.
Fan CD, Wang WW, Zhao BX, Zhang SL, Miao JY. Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells. Bioorg Med Chem, 2006, 14, 3218-3222.
Farre JC, Subramani S. Peroxisome turnover by micropexophagy: an autophagy-related process. Trends Cell Biol, 2004,14, 515-523.
Feng C, Ye C, Liu X, Ma H, Li M. Beta4 integrin is involved in statin-induced endothelial cell death, Biochem Biophys Res Commun, 2004, 323, 858-864.
Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med, 2003, 9, 669-676.
Ferreira CG, Tolis C, Span SW, Peters GJ, van Lopik T, Kummer AJ, Pinedo HM, Giaccone G. Drug-induced apoptosis in lung cnacer cells is not mediated by the Fas/FasL (CD95/APO1) signaling pathway. Clin Cancer Res, 2000, 6,203-212.
Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med, 2003, 3, 643-651.
Folkman J . Anti-angiogenesis : new concept for therary of solid tumors. Ann Surg, 1972,175,409-416.
Foster KA, Oster CG, Mayer MM, Avery ML, Audus KL. Characterization of the A549 cell line as a type II pulmonary epithelial cell model for drug metabolism. Exp Cell Res, 1998, 243, 359-366.
Frederick L, Kiechle MD. Nitric oxide biochemisty pathophys and diction. Am J Clin pathlo, 2003,100, 567-575.
Fridman JS, Lowe SW. Control of apoptosis by p53. Oncogene, 2003, 33, 9030-9040.
Fujita T, Maruyama M, Araya J, Sassa K, Kawagishi Y, Hayashi R, Matsui S, Kashii T, Yamashita N, Sugiyama E, Kobayashi M. Hydrogen peroxide induces upregulation of Fas in human airway epithelial cells via the activation of PARP-p53 pathway. Am. J. Respir Cell Mol Biol, 2002, 27, 542-552.
Giancotti FG, Tarone G Positional control of cell fate through joint integrin/receptor protein kinase signaling. Annu Rev Cell Dev Biol, 2003, 19, 173-206.
Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst, 1973. 51. 1417-1423.
Giancotti FG. Targeting integrin β4 for cancer and anti-angiogenic therapy. Trends Pharmacol Sci, 2007, 28, 506-511.
Ginnan R. Guikema BJ, Halligan KE. Singer HA, Jourd'heuil D. Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases. Free Radic Biol Med, 2008; [Epub ahead of print]
Gleason B, Adley B, Rao MS, Diaz LK. Immunohistochemical detection of the b4 integrin subunit in pancreatic adenocarcinoma. J Histochem Cytochem, 2005, 53. 799-801.
Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol, 2000, 2, 156-162.
Grant DS, Kibbey MC, Kinsella JL, Cid MC, Kleinman HK. The role of basement membrane in angiogenesis and tumor growth. Pathol Res Pract, 1994, 190, 854-863.
Greenberger S, Shaish A, Varda-Bloom N, Levanon K, Breitbart E, Goldberg I, Barshack I, Hodish I, Yaacov N, Bangio L, Goncharov T, Wallach D, Harats D. Transcription-controlled gene therapy against tumor angiogenesis. J Clin Invest, 2004,113,1017-1024.
Grossman HB, Lee C, Bromberg J, Liebert M. Expression of the a6b4 integrin provides prognostic information in bladder cancer. Oncol Rep, 2000, 7, 13-16.
Guo W, Pylayeva Y, Pepe A, Yoshioka T, Muller WJ, Inghirami G, Giancotti FG. β4 integrin amplifies ErbB2 signaling to promote mammary tumorigenesis. Cell, 2006, 126, 489-502.
Hehnly H, Stamnes M. Regulating cy to skeleton-based vesicle motility. FEBS Lett, 2007,581,2112-2118.
Herrmann J, Lerman LO, Mukhopadhyay D, Napoli C, Lerman A. Angiogenesis in Atherogenesis. Arterioscler Thromb Vasc Biol, 2006, 26, 1948-1957.
Hiran TS, Mazurkiewicz JE, Kreienberg P, Rice FL, LaFlamme SE. Endothelial expression of the alpha6beta4 integrin is negatively regulated during angiogenesis. J Cell Sci, 2003, 116, 3771-3781.
Hsieh CC, Yen MH, Yen CH. Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells. Cardiovasc Res, 2001, 49, 135-145.
Iakovou K, Kazanis M, Vavayannis A, Bruni G, Romeo MR, Massarelli P, Teramoto S. Synthesis of oxypropanolamine derivatives of 3,4-dihydro-2H-l,4-benzoxazine, beta-adrenergic affinity, inotropic, chronotropic and coronary vasodilating activities. Eur J Med Chem, 1999, 34, 903-917.
Iglesias D, Fernandez-Peralta AM, Nejda N, Daimiel L, Azcoita MM, Oliart S. RIS1, a gene with trinucleotide repeats, is a target in the mutator pathway of colorectal carcinogenesis. Cancer Genet Cytogenet, 2006,167,138-144.
Schmitz I, Kirchhoff S, Krammer PH. Regulation of death receptor-mediated apoptosis pathways. Int J Biochem Cell Biol, 2000, 32:1123-1136.
Itoh N, Nagata S. A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J Biol Chem, 1993, 268, 10932-10937.
Iurlaro M, Vacca A, Minischetti M. Antiangiogensis by cyclosporine. Exp Hematol, 1998,26,1215-1222.
Jarvinen K, Pietarinen-Runtti P, Linnainmaa K, Raivio KO, Krejsa CM, Kavanagh T, Kinnula VL. Antioxidant defense mechanisms of human mesothelioma and lung adenocarcinoma cells. Am J Physiol Lung Cell Mol Physiol, 2000, 278, L696-L702.
Jiao PF, Zhao BX, Wang WW, He QX, Wan MS, Shin DS, Miao JY. Design, synthesis, and preliminary biological evaluation of 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine derivatives. Bioorg Med Chem Lett, 2006, 16, 2862-2867.
Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, Yoshimori T. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci, 2004,117,2805-2812.
Kabeya Y, Mizushima N, Ueno T. LC3. a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J, 2000, 19. 5720-5728.
Kamada Y, Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. Tor-mediated induction of autophagy via an Apgl protein kinase complex. J Cell Biol, 2000, 150. 1507-1513.
Kazmierski WM. Furfine E, Spaltenstein A, Wright LL. New, potent P1/P2-morpholinone-based HIV-protease inhibitors. Bioorg Med Chem Lett, 2006, 16,5226-5230.
Kettunen E, Nicholson AG, Nagy B, Wikman H, Seppanen JK, Stjernvall T. L1CAM, INP10, P-cadherin, tPA and ITGB4 over-expression in malignant pleural mesotheliomas revealed by combined use of cDNA and tissue microarray. Carcinogenesis, 2005, 26, 17-25.
Kimura T, Sakisaka T, Baba T, Yamada T, Takai Y. Involvement of the Ras-Ras-activated Rab5 guanine nucleotide exchange factor RIN2-Rab5 pathway in the hepatocyte growth factor-induced endocytosis of E-cadherin. J Biol Chem, 2006,281,10598-10609.
Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science, 2000, 290,1717-1721.
Klionsky DJ, Meijer AJ, Codogno P, Neufeld TP, Scott RC. Autophagy and P70S6 kinase. Autophagy, 2005, 1, 059-061.
Klionsky DJ . The molecular machinery of autophagy:unanswered questions. J Cell Sci, 2005,118, 7-18.
Koizumi K. Current surgical strategies for lung cancer with a focus on open thoracotomy and video-assisted thoracic surgery. J Nippon Med Sch, 2006, 73, 116-121.
Konig MF, Schlatter P, Burri PH. Microvascular growth in the chicken chorio-allantoic membrane. Adv Exp Med Biol, 1997, 411, 353-358.
Koukoulis G, Warren W, Virtanen I, Gould V. Immunolocalization of integrins in the normal lung and in pulmonary carcinomas. Human Pathol, 1997, 28, 1018-1025.
Krammer PH. CD95's deadly mission in the immune system. Nature, 2000, 407, 789-795.
Krneta J, Kroll J, Alves F, Prahst C. Sananbenesi F, Dullin C. Dissociation of angiogenesis and tumorigenesis in follistatin- and activin-expressing tumors. Cancer Res, 2006, 66, 5686-5695.
Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, Walsh K. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med, 2000, 6, 1004-1010.
Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell, 2004, 6, 463-477.
Li JM, Mullen AM, Yun S. Essential role of the NADPH oxidase subunit P47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor- alpha.CircRes, 2002, 90,143-150.
Liang SH, Clarke MF. Regulation of p53 localization. Eur J Biochem, 2001, 268, 2779-2783.
Liang XH, Jackson S, Seaman M. Induction of autophagy and inhibition of tumorigenesis by Beclin 1. Nature, 1999,402, 672-676.
Lipscomb EA, Mercurio AM. Mobilization and activation of a signaling competent alpha6beta4integrin underlies its contribution to carcinoma progression. Cancer Metastasis Rev, 2005,24, 413-423.
Litjens SH, de Pereda JM, Sonnenberg A. Current insights into the formation and breakdown of hemidesmosomes. Trends Cell Biol, 2006, 16, 376-383.
Liu ZZ, Chen HC, Cao SL, Li RT. A novel method for the synthesis of aryl glycidyl ethers. Synth Commun, 1994,24, 833.
Lohi J, Leivo I, Owaribe K, Burgeson RE, Franssila K, Virtanen I. Neoexpression of the epithelial adhesion complex antigens in thyroid tumours is associated with proliferation and squamous differentiation markers. J Pathol, 1998, 184, 191-196.
Lv X, Wang N, Su L, Zhang SL, Miao JY. Inhibition of PC-PLC blocked the survival of mouse neural cells by up-regulating the expression of integrin beta4 and Rb. Dev Neurosci. 2006. 28. 499-504.
Madri JA. Pratt BM. Tucker AM. Phenotypic modulation of endothelial cells by transforming growth factor-P depends upon the composition and organization of the extracellular matrix. J Cell Biol. 1988,106.1375-1384.
Maiuri MC, Zalckvar E, Kimchi A. Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol, 2007, 8, 741-752.
Mariotti A, Kedeshian PA, Dans M, Curatola AM. Gagnoux-Palacios L, Giancotti FG. EGF-R signaling through Fyn kinase disrupts the function of integrin a6b4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion. J Cell Biol, 2001, 155, 447-458.
Maxwell SA, Davis GE. Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines. Proc Natl Acad Sc. USA. 2000, 97, 3009-13014.
Mayer TU, Kapoor TM, Haggarty SJ. King RW, Schreiber SL, Mitchison TJ. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science, 1999, 286, 971-974.
Mercurio AM, Rabinovitz I. Towards a mechanistic understanding of tumor invasion - lessons from the a6b4 integrin. Semin. Cancer Biol, 2001, 11, 129-141.
Miao JY, Araki S, Kaji, K, Hayashi H. Integrin β4 is involved in apoptotic signal transduction in endothelial cells. Biochem Biophys Res Commun, 1997a, 233, 182-186.
Miao JY, Kaji K, Hayashi H, Araki S. Suppression of apoptosis by inhibition of phosphatidylcholine-specific phospholipase C in vascular endothelial cells, Endothelium, 1997b, 5, 231-239.
Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell, 2004, 15, 1101-1111.
Mortimore GE, Poso AR. Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu Rev Nutr, 1987, 7, 539-564.
Muta M, Matsumoto G, Hiruma K. Impact of vasculogenesis on solid tumor growth in a rat model. Oncol Rep, 2003, 10, 1213.
Nagata S, Golstein P. The Fas death factor. Science, 1995, 267,1449-1456.
Nguyen VA, Furhapter C, Romani N, Weber F, Sepp N. Infantile hemangioma is a proliferation of beta 4-negative endothelial cells adjacent to HLA-DR-positive cells with dendritic cell morphology. Hum Pathol, 2004, 35, 739-744.
Ni H, Dydensborg AB, Herring FE, Basora N, Gagne D, Vachon PH, Beaulieu JF. Upregulation of a functional form of the (34 integrin subunit in colorectal cancers correlates with c-Myc expression. Oncogene, 2005, 24, 6820-6829.
Nikolopoulos SN, Blaikie P, Yoshioka T, Guo W, Puri C, Tacchetti C, Giancotti FG. Targeted deletion of the integrin (34 signaling domain suppresses laminin-5-dependent nuclear entry of mitogen-activated protein kinases and NF-kB, causing defects in epidermal growth and migration. Mol Cell Biol, 2005, 25,6090-6102.
Nikolopoulos SN, Blaikie P, Yoshioka T, Guo W, Giancotti FG. Integrin β4 signaling promotes tumor angiogenesis. Cancer Cell, 2004, 6, 471-483.
Quinn L, Deveraux, John C Reed. IAP family proteins-suppressors of apoptosis. Genes Dev, 1999, 13, 239-252.
Pereira-Leal JB, Strom M, Godfrey RF, Seabra MC. Structural determinants of Rab and Rab Escort Protein interaction: Rab family motifs define a conserved binding surface. Biochem Biophys Res Commun, 2003, 301, 92-97.
Pfeffer SR. Rab GTPases: Specifying and deciphering organelle identity and function. Trends Cell Biol, 2001, 11,487-491.
Pipili-Synetos E, Kritikou S, Papadimitriou E, Athanassiadou A, Flordellis C, Maragoudakis ME. Nitric oxide synthase expression, enzyme activity and NO production during angiogenesis in the chick chorioallantoic membrane. Br J Pharmacol, 2000, 129, 207-213.
Ptasznik A, Nkaata Y, Kalota A, Emerson SG, GewirtzA. Short interfering RNA (sRiNA) target in the Lyn kinase induces apoptosis in primary , and durg-resistant, BCR-ABLI(+)leukemia cells. Nat Med, 2004. 10, 1187-1179.
RayChaudhury A, Frischer H, Malik AB. Inhibition of endothelial cell proliferation and bFGF-induced phenotypic modulation by nitric oxide. J Cell Biochem, 1996, 63. 125-134.
Reggiori F. Klionsky DJ. Autophagosomes: biogenesis from scratch? Curr Opin Cell Biol, 2005,17, 415-422.
Rivera M P. Multimodality therapy in the treatment of lung cancer. Semin Respir Cri. Care Med. 2004, 1,3-10.
Roberts P, Moshitch-Moshkovitz S, Kvam E, O'Toole E, Winey M, Goldfarb DS. Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae. Mol Biol Cell, 2003, 14, 129-141.
Rojas A, Figueroa H, Morales MA, Re L. Facing Up the ROS Labyrinth - Where To Go? Curr Vasc Pharmacol. 2006. 4, 277-289.
Rybczynski PJ, Zeck RE. Dudash J Jr. Combs DW. Burris TP, Yang M, Osborne MC, Chen X, Demarest KT. Benzoxazinones as PPARgamma agonists. 2. SAR of the amide substituent and in vivo results in a type 2 diabetes model. J Med Chem, 2004,47,196-209.
Sakkoula E, Pipili-Synetos E, Maragoudakis ME. Involvement of nitric oxide in the inhibition of angiogenesis by interleukin-2. Br J Pharmacol, 1997, 122, 793-795.
Sauer H, Wartenberg M. Reactive oxygen species as signaling molecules in cardiovascular differentiation of embryonic stem cells and tumor-induced angiogenesis. Antioxid Redox Signal, 2005, 7, 1423-34.
Sax JK, El-Deiry WS. p53 downstream targets and chemosensitivity. Cell Death Differ, 2003, 10,413-417.
Sehreiber SL. Chemical genetics resulting from a passion for synthetic organic chemistry. Bioogr Med Chem, 1998, 6, 1127-1152.
Segura I, Serrano A, De Buitrago GG, Gonzalez MA, Abad JL, Claveria C, Gomez L, Bernad A, Martinez-A C, Riese HH. Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis. FASEB J, 2002, 16,,833-841.
Sessa WC. The nitric oxide synthase family of proteins. J Vasc Res, 1994, 31, 131-143.
Shaw LM, Rabinovitz I, Wang HH, Toker A, Mercurio AM. Activation of phosphoinositide 3-OH kinase by the a6(34 integrin promotes carcinoma invasion. Cell, 1997,91,949-960.
Shekhar MPV, Werdell J, Tait L. Interaction with endothelial cell is prerequisite for branching ductal-alveolar morphogenesis and hyperplasia of preneoplastic human breast epithelial cell regulation by estrogen. Cancer Res, 2000, 60, 439-449.
Shi Y. Caspase activation: revisiting the induced proximity model. Cell, 2004, 117, 855-858.
Silva J, Silva JM, Barradas M, Garcia JM, Dominguez G, Garcia V. Analysis of the candidate tumor suppressor Ris-1 in primary human breast carcinomas. Mutat Res, 2006, 594, 78-85.
Smukste I, Stockwell BR. Advances in chemical genetics. Annu Rev Genomics Hum Genet, 2005, 6, 261-286.
Spring DR. Chemical genetics to chemical genomics: small molecules offer big insights. Chem Soc Rev, 2005, 34, 472-482.
Steiner L, Kroncke K, Fehsel K, Kolb-Bachofen V. Endothelial cells as cytotoxic effector cells: cytokine-activated rat islet endothelial cells lyse syngeneic islet cells via nitric oxide. Diabetologia, 1997,40, 150-155.
Stenmark H, Parton RG, Steele-Mortimer O, Liitcke A, Gruenberg J, Zerial M. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBOJ, 1994, 13, 1287-1296.
Stepp NT, Cornelius LA, Romani N. Polarized expression and basic fibroblast growth factor induced down-regulation of the a6|34 integrin complex on human microvascular endothelial cells. J Invest Dermatol, 1994,104, 266-270.
St Croix B, Rago C, Velculescu V, Traverso G, Romans KE, Montgomery E, Lal A, Riggins GJ. Lengauer C. Vogelstein B. Kinzler KW. Genes expressed in human tumor endothelium. Science. 2000. 289, 1197-1202.
Steven RH.Daniel JS.Hitoshi Y. Tumor growth enhancing effects of vacular endothelail growth factor are associated with increased nitric oxide synthase activity and inhibition of apoptosis in human breast carcinoma xenografts.Canc Lett, 2002.179, 95-101.
Su L. Zhao J, Zhao BX. Miao JY, Yin DL, Zhang SL. Safrole oxide induced vascular endothelial cell differentiation into neuron-like cells by depressing the reactive oxygen species level at the low concentration. Biochim Biophys Acta, 2006 Feb;1763(2):247-53.
Sulyok S. Wankell M, Alzheimer C, Werner S. Activin: an important regulator of wound repair, fibrosis. and neuroprotection. Mol Cell Endocrinol, 2004, 225, 127-132.
Sun SY, Yue P, Hong WK, Lotan R. Induction of Fas expression and augmentation of Fas/Fas ligand-mediated apoptosis by the synthetic retinoid CD437 in human lung cancer cells. Cancer Res, 2000, 60, 6537-6543.
Suryanto A, Herlambang K, Rachmatullah P. Comparison of Tumor Density by CT Scan Based on Histologic Type in Lung Cancer Patients. Acta Med Indones, 2005,37,195-198.
Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature, 1999, 397, 441-446.
Tagliabue E, Ghirelli C, Squicciarini P, Aiello P, Colnaghi MI, Menard S. Prognostic value of α6β4 integrin expression in breast carcinomas is affected by laminin production from tumor cells. Clin Cancer Res, 1998, 4, 407-410.
Tan W, Zhao B, Shi M, Sha L, Miao J. Shin D. Facile Synthesis and Evaluation of Cell A549 Growth Inhibitory Activity of 4-Aryl-6-aryloxymethylmorpholin-3-one Derivatives. Chinese J Chem, 2006, 24, 396-400.
Tani T, Karttunen T. Kiviluoto T, Kivilaakso E, Burgeson RE, Sipponen P, Virtanen I. α6β4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Continuous expression of laminins but not that of collagen Ⅶ is preserved in invasive parts of the carcinomas: implications for acquisition of the invading phenotype. Am J Pathol, 1996, 149, 781-793.
Thompson CB. Apoptosis in the pathogenesis and treat-ment of disease. Science, 1995,267,1456-1462.
Trusolino L, Bertotti A, Comoglio PM. A signaling adapter function for a6b4 integrin in the control of HGF-dependent invasive growth. Cell, 2001, 107, 643-654.
Tsuruta D, Hopkinson SB, Lane KD, Werner ME, Cryns VL, Jones JC. Crucial role of the specificity-determining loop of the integrin beta4 subunit in the binding of cells to laminin-5 and outside-in signal transduction. J Biol Chem, 2003, 278, 38707-38714.
Ushio-Fukai M. Redox signaling in angiogenesis: Role of NADPH oxidase. Cardiovasc Res, 2006, 71, 226-235.
Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ, Vaux DL. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell, 2000, 102, 43-53.
Wang WW, Liu X, Zhao J, Zhao BX, Zhang SL, Miao JY. A novel butyrolactone derivative inhibited apoptosis and depressed integrin β4 expression in vascular endothelial cells. Bioorg Med Chem Lett, 2007, 17, 482-485.
Waterman MJ, Stavridi ES, Waterman JL, Halazonetis TD. ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins. Nature Genetics, 1998, 19, 175-178.
Weaver VM, Lelievre S, Lakins JN, Chrenek MA, Jones JC, Giancotti F. β4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell. 2002, 2. 205-216.
Welt CK. The physiology and pathophysiology of inhibin, activin and follistatin in female reproduction. Curr Opin Obstet Gynecol, 2002. 14, 317-323.
Wilhelmsen K. Litjens SH. Sonnenberg A. Multiple functions of the integrin a6b4 in epidermal homeostasis and tumorigenesis. Mol Cell Biol, 2006, 26, 2877-2886.
Yang Y. Yang Y. Trent MB, He N. Lick SD. Zimniak P, Awasthi YC, Boor PJ. Glutathione-S-transferase A4-4 modulates oxidative stress in endothelium: possible role in human atherosclerosis. Atherosclerosis, 2004, 173, 211-221.
Yeh JR., Crews CM. Chemical genetics: adding to the developmental biology toolbox. Dev Cell, 2003, 5. 11-19.
Yokoi A, Kuormitsu J, Kawai T, Nagasu T, Sugi NH, Yoshimatsu K. Yoshino H, Owa T. Porfiling novel sulfonamide antitumor agents with cell-based phenotypic screens and array-based gene expression analysis. Mol Cancer Ther, 2002, 1, 275-286.
Xu ZX, Liang J, Haridas V, Gaikwad A, Connolly FP, Mills GB, Gutterman JU. A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated protein kinase. Cell Death Differ. 2007,14, 1948-1957.
Yu C, Wang Z, Dent P, Grant S. MEK1/2 inhibitors promote Ara-C-induced apoptosis but not loss of Deltapsi(m) in HL-60 cells. Biochem Biophys Res Commun, 2001, 286,1011-1018.
Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW, Vogelstein B. Identification and classification of p53-regulated genes. Proc Natl Acad Sci USA, 1999, 96, 14517-14522.
Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci USA, 2003, 100, 15077-15082.
Zerial M, McBride H. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol, 2001,2,107-117.
Zhao BX, Du AY, Miao JY, Zhao KW, Du CQ. Effects of novel safroleoxide derivatives, l-Methoxy-3-(3,4-methylenedioxy-benzyl)-2-propanol and 1-ethoxy-3-(3,4-methylenedioxybenzyl)-2-propanol,on apoptosis induced by deprivation of survival factors in vascular endothelial cells.Vas Pharm,2003,40,182-187.
Zhao J,Miao JY,Zhao BX,Zhang SL.Upregulating of Fas,integrin beta4 and P53and depressing of PC-PLC activity and ROS level in VEC apoptosis by safrole oxide.FEBS Lett,2005,579,5809-5813.
Zhao J,Miao JY,Zhao BX,Zhang SL,Yin DL.Suppressing Akt phosphorylation and activating Fas by safrole oxide inhibited angiogenesis and induced vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 and serum.Int J Biochem Cell Biol,2006,38,1603-1613.
Zhao J,Zhao BX,Du AY,Zhao QT,Miao JY.Effects of Novel Safrole Oxide Derivatives,1-Propyl-3-(3,4-methylenedioxyphenyl)-2-propanol and Isopropoxy-3-(3,4-methylenedioxyphenyl)-2-propanol,on Apoptosis Induced by Deprivation of Survival Factors in Vascular Endothelial Cells.Endothelium,2004a,11,267-273.
Zhao KW,Zhao QT,Zhang SL,Miao JY.Integrin beta4 mAb inhibited apoptosis induced by deprivation of growth factors in vascular endothelial cells,Acta Pharmacol Sin,2004b,25,733-737.
Zhou D,Harrison BL,Shah U,Andree TH,Hornby GA,Scerni R,Schechter LE,Smith DL.Studies toward the discovery of the next generation of antidepressants.Part 5:3,4-Dihydro-2H-benzo[1,4]oxazine derivatives with dual 5-HT1A receptor and serotonin transporter affinity.Bioorg Med Chem Lett,2006,16,1338-1341.
苗俊英,王洁,赵静等.蛋白质功能研究的有利工具-化学遗传学.生物医学工程研究,2004,23,63-66.
Adams JM. Ways of dying multiple pathways to apoptosis. Genes Dev, 2003, 17. 2481-2495.
Allen LF, Sebolt-Leopold J, Meyer MB. CI-1040 (PD184352), a targeted signal transduction inhibitor of MEK (MAPKK). Semin Oncol, 2003, 30, 105-116.
Andriana BB, Tay TW, Maki I, Awal MA, Kanai Y, Kurohmaru M, Hayashi Y. An ultrastructural study on cytotoxic effects of mono (2-ethylhexyl) phthalate (MEHP) on testes in Shiba goat in vitro. J Vet Sci, 2004, 5, 235-240.
Artenie R, Artenie A, Cosovanu A. The cardiovascular significance of nitric oxide. Rev Med Chir Soc Med Nat Iasi, 1999,103,48-56.
Arthur CR, Gupton JT, Kellogg GE, Yeudall WA, Cabot MC, Newsham IF. Autophagic cell death, polyploidy and senescence induced in breast tumor cells by the substituted pyrrole JG-03-14, a novel microtubule poison. Biochem Pharmacol, 2007,74,981-991.
Auerbach R, Lewis R, Shinners B, Kubai L, Akhtar N. Angiogenesis Assays: A Critical Overview. Clin Chem. 2003,49, 32-40.
Bachelder RE, Marchetti A, Falcioni R. Activation of p53 function in carcinoma cells by the alpha(?)beta4 integrin. J Biol Chem, 1999, 274, 20733-20737.
Baehrecke EH. Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol, 2005,6,505-510.
Bampton Edward TW, Goemans CG, Niranjan D, Mizushima N, Tolkovsky AM. The dynamics of autophagy visualised in live cells: from autophagosome formation to fusion with endo/lysosomes. Autophagy, 2005,1, 023-036.
Banuelos A, Reyes E, Ocadiz R, Alvarez E, Moreno M, Monroy A, Gariglio P. Neocarzinostatin induces an effective p53-dependent response in human papillomavirus-positive cervical cancer cells. J Pharmacol Exp Ther, 2003, 306, 671-680.
Baril P, Gangeswaran R, Mahon PC, Caulee K. Kocher HM. Harada T. Periostin promotes invasiveness and resistance of pancreatic cancer cells to hypoxia-induced cell death: role of the beta4 integrin and the PI3k pathway. Oncogene, 2007, 26, 2082-2094.
Barradas M, Gonos ES, Zebedee Z. Koletta, E, Petropoulou C Delgado MD. Identification of a candidate tumor-suppressor gene specifically activated during Ras-induced senescence. Exp Cell Res, 2002, 273. 127-137.
Bellu AR, Kiel JA. Selective degradation of peroxisomes in yeasts. Microsc Res Tech. 2003,61,161-170.
Bertotti A, Comoglio PM, Trusolino L. β4 integrin activates a Shp2-Src signaling pathway that sustains HGF-induced anchorage-independent growth. J Cell Biol, 2006. 175.993-1003.
Boelens MC. van den Berg A, Vogelzang I, Wesseling J, Postma DS, Timens W. Differential expression and distribution of epithelial adhesion molecules in non-small cell lung cancer and normal bronchus. J Clin Pathol, 2007, 60, 608-614.
Brown KJ, Maynes SF, Bezos A. A novel in vitro assay for human angiogenesis. Lab Inves, 1996, 75, 539-555.
Butcher RA Schreiber SL. Using genome-wide transcriptional profiling to elucidate small-moiecule mechanism. Curr Opin Chem Biol, 2005, 9, 25-30.
Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature, 2000, 407, 249-257.
Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M, Wu Y, Bono F, Devy L, Beck H, Scholz D, Acker T, DiPalma T, Dewerchin M, Noel A, Stalmans I, Barra A, Blacher S, Vandendriessche T, Ponten A, Eriksson U, Plate KH, Foidart JM, Schaper W, Charnock-Jones DS, Hicklin DJ, Herbert JM, Collen D, Persico MG. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med, 2001, 7, 575-583.
Chen S, Hilcove S, Ding S. Exploring stem cell biology with small Molecules. Mol Biosys, 2006, 2,18-24.
Chu TS, Wu MS, Hsieh BS. Vasodilator agents inhibit opossum kidney proximal tubular cell growth. J Fomros Med Assoc, 2005,104, 374-377.
Coraux C, Delplanque A, Hinnrasky J, Peault B, Puchelle E, Gaillard D. Distribution of integrins during human fetal lung development. J Histochem Cytochem, 1998, 46,803-810.
Cremesti A, Paris F, Grassme H, Holler N, Tschopp J, Fuks Z, Gulbins E, Kolesnick R. Ceramide enables fas to cap and kill. J Biol Chem 2001, 276, 23954-23961.
Crotzer VL, Blum JS. Autophagy and intracellular surveillance: Modulating MHC class Ⅱ antigen presentation with stress. Proc Natl Acad Sci USA, 2005, 102, 7779-7780.
Cuervo AM, Dice JF. A receptor for the selective uptake and degradation of proteins by lysosomes. Science, 1996, 273, 501-503.
Daemi N, Thomasset N, Lissitzky JC, Dumortier J, Jacquier MF. Anti-β4 integrin antibodies enhance migratory and invasive abilities of human colon adenocarcinoma cells and their MMP-2 expression. Int J Cancer, 2000, 85, 850-856.
D'Ambra TE, Estep KG, Bell MR, Eissenstat MA, Josef KA, Ward SJ, Haycock DA. Conformationally restrained analogues of pravadoline: nanomolar potent, enantioselective, (aminoalkyl)indole agonists of the cannabinoid receptor. J Med Chem, 1992,35,124-135.
de Nigris F, Lerman A, Ignarro LJ. Oxidation-sensitive mechanisms, vascular apoptosis and atherosclerosis.Trends Mol Med, 2003, 9, 351-359. Dowling J, Yu Q C, Fuchs E. Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival. J Cell Biol, 1996, 134, 559-572.
Degenhardt K, Mathew R, Beaudoin B. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell, 2006, 10, 51-64.
Du AY, Zhao BX, Miao JY, Yin DL, Zhang SL. Safrole oxide induces apoptosis by up-regulating Fas and FasL instead of integrin beta4 in A549 human lung cancer cells. Bioorg Med Chem, 2006a. 14. 2438-2445.
Du AY. Zhao BX. Yin DL, Zhang SL. Miao JY. Safrole oxide induces apoptosis by activating caspase-3. -8, and -9 in A549 human lung cancer cells. Bioorg Med Chem Lett, 2006b, 16.81-83.
Du AY, Zhao BX, Yin DL, Zhang SL, Miao JY. Discovery of a novel small molecule. l-ethoxy-3-(3,4-methylenedioxyphenyl)-2-propanol, that induces apoptosis in A549 human lung cancer cells. Bioorg Med Chem, 2005, 13,4176-4183.
Du AY, Zhang SL, Miao JY. Zhao BX. Safrole oxide induces apoptosis in A549 human lung cancer cells. Exp Lung Res, 2004, 30, 419-429.
Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell, 2000, 102,33-42.
Du CQ, Zhao QT. Araki S, Zhang SL, Miao JY Apoptosis mediated by phosphatidylcholine-specific phospholipase C is associated with cAMP, p53 level, and cell-cycle distribution in vascular endothelial cells. Endothelium, 2003, 10, 141-147.
Edick MJ, Tesfay L, Lamb LE, Knudsen BS, Miranti CK. Inhibitionof integrin-mediated crosstalk with epidermal growth factor receptor/Erk or Srcsignaling pathways in autophagic prostate epithelial cells induces caspase-independentdeath. Mol Biol Cell, 2007, 18, 2481-2490.
Eduardo O, Sandes A, licia G. Expression of inducible nitric oxide synthase in tumoral and non-tumoral epithelia from bladder cancer patients. Ntric oxide, 2005, 12, 39-45.
Falcioni R, Antonini A, Nistico P, Di Stefano S, Crescenzi M, Natali PG, Sacchi A. a6b4 and a6bl integrins associate with ErbB-2 in human carcinoma cell lines. Exp Cell Res, 1997,236, 76-85.
Falcioni R, Kennel SJ, Giacomini P, Zupi G, Sacchi A. Expression of tumor antigen correlated with metastatic potential of Levis lung carcinoma and B16 melanoma clones in mice. Cancer Res, 1986, 46, 5772-5778.
Fan CD, Wang WW, Zhao BX, Zhang SL, Miao JY. Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells. Bioorg Med Chem, 2006, 14, 3218-3222.
Farre JC, Subramani S. Peroxisome turnover by micropexophagy: an autophagy-related process. Trends Cell Biol, 2004,14, 515-523.
Feng C, Ye C, Liu X, Ma H, Li M. Beta4 integrin is involved in statin-induced endothelial cell death, Biochem Biophys Res Commun, 2004, 323, 858-864.
Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med, 2003, 9, 669-676.
Ferreira CG, Tolis C, Span SW, Peters GJ, van Lopik T, Kummer AJ, Pinedo HM, Giaccone G. Drug-induced apoptosis in lung cnacer cells is not mediated by the Fas/FasL (CD95/APO1) signaling pathway. Clin Cancer Res, 2000, 6,203-212.
Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med, 2003, 3, 643-651.
Folkman J . Anti-angiogenesis : new concept for therary of solid tumors. Ann Surg, 1972,175,409-416.
Foster KA, Oster CG, Mayer MM, Avery ML, Audus KL. Characterization of the A549 cell line as a type II pulmonary epithelial cell model for drug metabolism. Exp Cell Res, 1998, 243, 359-366.
Frederick L, Kiechle MD. Nitric oxide biochemisty pathophys and diction. Am J Clin pathlo, 2003,100, 567-575.
Fridman JS, Lowe SW. Control of apoptosis by p53. Oncogene, 2003, 33, 9030-9040.
Fujita T, Maruyama M, Araya J, Sassa K, Kawagishi Y, Hayashi R, Matsui S, Kashii T, Yamashita N, Sugiyama E, Kobayashi M. Hydrogen peroxide induces upregulation of Fas in human airway epithelial cells via the activation of PARP-p53 pathway. Am. J. Respir Cell Mol Biol, 2002, 27, 542-552.
Giancotti FG, Tarone G Positional control of cell fate through joint integrin/receptor protein kinase signaling. Annu Rev Cell Dev Biol, 2003, 19, 173-206.
Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst, 1973. 51. 1417-1423.
Giancotti FG. Targeting integrin β4 for cancer and anti-angiogenic therapy. Trends Pharmacol Sci, 2007, 28, 506-511.
Ginnan R. Guikema BJ, Halligan KE. Singer HA, Jourd'heuil D. Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases. Free Radic Biol Med, 2008; [Epub ahead of print]
Gleason B, Adley B, Rao MS, Diaz LK. Immunohistochemical detection of the b4 integrin subunit in pancreatic adenocarcinoma. J Histochem Cytochem, 2005, 53. 799-801.
Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol, 2000, 2, 156-162.
Grant DS, Kibbey MC, Kinsella JL, Cid MC, Kleinman HK. The role of basement membrane in angiogenesis and tumor growth. Pathol Res Pract, 1994, 190, 854-863.
Greenberger S, Shaish A, Varda-Bloom N, Levanon K, Breitbart E, Goldberg I, Barshack I, Hodish I, Yaacov N, Bangio L, Goncharov T, Wallach D, Harats D. Transcription-controlled gene therapy against tumor angiogenesis. J Clin Invest, 2004,113,1017-1024.
Grossman HB, Lee C, Bromberg J, Liebert M. Expression of the a6b4 integrin provides prognostic information in bladder cancer. Oncol Rep, 2000, 7, 13-16.
Guo W, Pylayeva Y, Pepe A, Yoshioka T, Muller WJ, Inghirami G, Giancotti FG. β4 integrin amplifies ErbB2 signaling to promote mammary tumorigenesis. Cell, 2006, 126, 489-502.
Hehnly H, Stamnes M. Regulating cy to skeleton-based vesicle motility. FEBS Lett, 2007,581,2112-2118.
Herrmann J, Lerman LO, Mukhopadhyay D, Napoli C, Lerman A. Angiogenesis in Atherogenesis. Arterioscler Thromb Vasc Biol, 2006, 26, 1948-1957.
Hiran TS, Mazurkiewicz JE, Kreienberg P, Rice FL, LaFlamme SE. Endothelial expression of the alpha6beta4 integrin is negatively regulated during angiogenesis. J Cell Sci, 2003, 116, 3771-3781.
Hsieh CC, Yen MH, Yen CH. Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells. Cardiovasc Res, 2001, 49, 135-145.
Iakovou K, Kazanis M, Vavayannis A, Bruni G, Romeo MR, Massarelli P, Teramoto S. Synthesis of oxypropanolamine derivatives of 3,4-dihydro-2H-l,4-benzoxazine, beta-adrenergic affinity, inotropic, chronotropic and coronary vasodilating activities. Eur J Med Chem, 1999, 34, 903-917.
Iglesias D, Fernandez-Peralta AM, Nejda N, Daimiel L, Azcoita MM, Oliart S. RIS1, a gene with trinucleotide repeats, is a target in the mutator pathway of colorectal carcinogenesis. Cancer Genet Cytogenet, 2006,167,138-144.
Schmitz I, Kirchhoff S, Krammer PH. Regulation of death receptor-mediated apoptosis pathways. Int J Biochem Cell Biol, 2000, 32:1123-1136.
Itoh N, Nagata S. A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J Biol Chem, 1993, 268, 10932-10937.
Iurlaro M, Vacca A, Minischetti M. Antiangiogensis by cyclosporine. Exp Hematol, 1998,26,1215-1222.
Jarvinen K, Pietarinen-Runtti P, Linnainmaa K, Raivio KO, Krejsa CM, Kavanagh T, Kinnula VL. Antioxidant defense mechanisms of human mesothelioma and lung adenocarcinoma cells. Am J Physiol Lung Cell Mol Physiol, 2000, 278, L696-L702.
Jiao PF, Zhao BX, Wang WW, He QX, Wan MS, Shin DS, Miao JY. Design, synthesis, and preliminary biological evaluation of 2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine derivatives. Bioorg Med Chem Lett, 2006, 16, 2862-2867.
Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, Yoshimori T. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci, 2004,117,2805-2812.
Kabeya Y, Mizushima N, Ueno T. LC3. a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J, 2000, 19. 5720-5728.
Kamada Y, Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. Tor-mediated induction of autophagy via an Apgl protein kinase complex. J Cell Biol, 2000, 150. 1507-1513.
Kazmierski WM. Furfine E, Spaltenstein A, Wright LL. New, potent P1/P2-morpholinone-based HIV-protease inhibitors. Bioorg Med Chem Lett, 2006, 16,5226-5230.
Kettunen E, Nicholson AG, Nagy B, Wikman H, Seppanen JK, Stjernvall T. L1CAM, INP10, P-cadherin, tPA and ITGB4 over-expression in malignant pleural mesotheliomas revealed by combined use of cDNA and tissue microarray. Carcinogenesis, 2005, 26, 17-25.
Kimura T, Sakisaka T, Baba T, Yamada T, Takai Y. Involvement of the Ras-Ras-activated Rab5 guanine nucleotide exchange factor RIN2-Rab5 pathway in the hepatocyte growth factor-induced endocytosis of E-cadherin. J Biol Chem, 2006,281,10598-10609.
Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science, 2000, 290,1717-1721.
Klionsky DJ, Meijer AJ, Codogno P, Neufeld TP, Scott RC. Autophagy and P70S6 kinase. Autophagy, 2005, 1, 059-061.
Klionsky DJ . The molecular machinery of autophagy:unanswered questions. J Cell Sci, 2005,118, 7-18.
Koizumi K. Current surgical strategies for lung cancer with a focus on open thoracotomy and video-assisted thoracic surgery. J Nippon Med Sch, 2006, 73, 116-121.
Konig MF, Schlatter P, Burri PH. Microvascular growth in the chicken chorio-allantoic membrane. Adv Exp Med Biol, 1997, 411, 353-358.
Koukoulis G, Warren W, Virtanen I, Gould V. Immunolocalization of integrins in the normal lung and in pulmonary carcinomas. Human Pathol, 1997, 28, 1018-1025.
Krammer PH. CD95's deadly mission in the immune system. Nature, 2000, 407, 789-795.
Krneta J, Kroll J, Alves F, Prahst C. Sananbenesi F, Dullin C. Dissociation of angiogenesis and tumorigenesis in follistatin- and activin-expressing tumors. Cancer Res, 2006, 66, 5686-5695.
Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, Walsh K. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med, 2000, 6, 1004-1010.
Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell, 2004, 6, 463-477.
Li JM, Mullen AM, Yun S. Essential role of the NADPH oxidase subunit P47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor- alpha.CircRes, 2002, 90,143-150.
Liang SH, Clarke MF. Regulation of p53 localization. Eur J Biochem, 2001, 268, 2779-2783.
Liang XH, Jackson S, Seaman M. Induction of autophagy and inhibition of tumorigenesis by Beclin 1. Nature, 1999,402, 672-676.
Lipscomb EA, Mercurio AM. Mobilization and activation of a signaling competent alpha6beta4integrin underlies its contribution to carcinoma progression. Cancer Metastasis Rev, 2005,24, 413-423.
Litjens SH, de Pereda JM, Sonnenberg A. Current insights into the formation and breakdown of hemidesmosomes. Trends Cell Biol, 2006, 16, 376-383.
Liu ZZ, Chen HC, Cao SL, Li RT. A novel method for the synthesis of aryl glycidyl ethers. Synth Commun, 1994,24, 833.
Lohi J, Leivo I, Owaribe K, Burgeson RE, Franssila K, Virtanen I. Neoexpression of the epithelial adhesion complex antigens in thyroid tumours is associated with proliferation and squamous differentiation markers. J Pathol, 1998, 184, 191-196.
Lv X, Wang N, Su L, Zhang SL, Miao JY. Inhibition of PC-PLC blocked the survival of mouse neural cells by up-regulating the expression of integrin beta4 and Rb. Dev Neurosci. 2006. 28. 499-504.
Madri JA. Pratt BM. Tucker AM. Phenotypic modulation of endothelial cells by transforming growth factor-P depends upon the composition and organization of the extracellular matrix. J Cell Biol. 1988,106.1375-1384.
Maiuri MC, Zalckvar E, Kimchi A. Kroemer G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol, 2007, 8, 741-752.
Mariotti A, Kedeshian PA, Dans M, Curatola AM. Gagnoux-Palacios L, Giancotti FG. EGF-R signaling through Fyn kinase disrupts the function of integrin a6b4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion. J Cell Biol, 2001, 155, 447-458.
Maxwell SA, Davis GE. Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines. Proc Natl Acad Sc. USA. 2000, 97, 3009-13014.
Mayer TU, Kapoor TM, Haggarty SJ. King RW, Schreiber SL, Mitchison TJ. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science, 1999, 286, 971-974.
Mercurio AM, Rabinovitz I. Towards a mechanistic understanding of tumor invasion - lessons from the a6b4 integrin. Semin. Cancer Biol, 2001, 11, 129-141.
Miao JY, Araki S, Kaji, K, Hayashi H. Integrin β4 is involved in apoptotic signal transduction in endothelial cells. Biochem Biophys Res Commun, 1997a, 233, 182-186.
Miao JY, Kaji K, Hayashi H, Araki S. Suppression of apoptosis by inhibition of phosphatidylcholine-specific phospholipase C in vascular endothelial cells, Endothelium, 1997b, 5, 231-239.
Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell, 2004, 15, 1101-1111.
Mortimore GE, Poso AR. Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu Rev Nutr, 1987, 7, 539-564.
Muta M, Matsumoto G, Hiruma K. Impact of vasculogenesis on solid tumor growth in a rat model. Oncol Rep, 2003, 10, 1213.
Nagata S, Golstein P. The Fas death factor. Science, 1995, 267,1449-1456.
Nguyen VA, Furhapter C, Romani N, Weber F, Sepp N. Infantile hemangioma is a proliferation of beta 4-negative endothelial cells adjacent to HLA-DR-positive cells with dendritic cell morphology. Hum Pathol, 2004, 35, 739-744.
Ni H, Dydensborg AB, Herring FE, Basora N, Gagne D, Vachon PH, Beaulieu JF. Upregulation of a functional form of the (34 integrin subunit in colorectal cancers correlates with c-Myc expression. Oncogene, 2005, 24, 6820-6829.
Nikolopoulos SN, Blaikie P, Yoshioka T, Guo W, Puri C, Tacchetti C, Giancotti FG. Targeted deletion of the integrin (34 signaling domain suppresses laminin-5-dependent nuclear entry of mitogen-activated protein kinases and NF-kB, causing defects in epidermal growth and migration. Mol Cell Biol, 2005, 25,6090-6102.
Nikolopoulos SN, Blaikie P, Yoshioka T, Guo W, Giancotti FG. Integrin β4 signaling promotes tumor angiogenesis. Cancer Cell, 2004, 6, 471-483.
Quinn L, Deveraux, John C Reed. IAP family proteins-suppressors of apoptosis. Genes Dev, 1999, 13, 239-252.
Pereira-Leal JB, Strom M, Godfrey RF, Seabra MC. Structural determinants of Rab and Rab Escort Protein interaction: Rab family motifs define a conserved binding surface. Biochem Biophys Res Commun, 2003, 301, 92-97.
Pfeffer SR. Rab GTPases: Specifying and deciphering organelle identity and function. Trends Cell Biol, 2001, 11,487-491.
Pipili-Synetos E, Kritikou S, Papadimitriou E, Athanassiadou A, Flordellis C, Maragoudakis ME. Nitric oxide synthase expression, enzyme activity and NO production during angiogenesis in the chick chorioallantoic membrane. Br J Pharmacol, 2000, 129, 207-213.
Ptasznik A, Nkaata Y, Kalota A, Emerson SG, GewirtzA. Short interfering RNA (sRiNA) target in the Lyn kinase induces apoptosis in primary , and durg-resistant, BCR-ABLI(+)leukemia cells. Nat Med, 2004. 10, 1187-1179.
RayChaudhury A, Frischer H, Malik AB. Inhibition of endothelial cell proliferation and bFGF-induced phenotypic modulation by nitric oxide. J Cell Biochem, 1996, 63. 125-134.
Reggiori F. Klionsky DJ. Autophagosomes: biogenesis from scratch? Curr Opin Cell Biol, 2005,17, 415-422.
Rivera M P. Multimodality therapy in the treatment of lung cancer. Semin Respir Cri. Care Med. 2004, 1,3-10.
Roberts P, Moshitch-Moshkovitz S, Kvam E, O'Toole E, Winey M, Goldfarb DS. Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae. Mol Biol Cell, 2003, 14, 129-141.
Rojas A, Figueroa H, Morales MA, Re L. Facing Up the ROS Labyrinth - Where To Go? Curr Vasc Pharmacol. 2006. 4, 277-289.
Rybczynski PJ, Zeck RE. Dudash J Jr. Combs DW. Burris TP, Yang M, Osborne MC, Chen X, Demarest KT. Benzoxazinones as PPARgamma agonists. 2. SAR of the amide substituent and in vivo results in a type 2 diabetes model. J Med Chem, 2004,47,196-209.
Sakkoula E, Pipili-Synetos E, Maragoudakis ME. Involvement of nitric oxide in the inhibition of angiogenesis by interleukin-2. Br J Pharmacol, 1997, 122, 793-795.
Sauer H, Wartenberg M. Reactive oxygen species as signaling molecules in cardiovascular differentiation of embryonic stem cells and tumor-induced angiogenesis. Antioxid Redox Signal, 2005, 7, 1423-34.
Sax JK, El-Deiry WS. p53 downstream targets and chemosensitivity. Cell Death Differ, 2003, 10,413-417.
Sehreiber SL. Chemical genetics resulting from a passion for synthetic organic chemistry. Bioogr Med Chem, 1998, 6, 1127-1152.
Segura I, Serrano A, De Buitrago GG, Gonzalez MA, Abad JL, Claveria C, Gomez L, Bernad A, Martinez-A C, Riese HH. Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis. FASEB J, 2002, 16,,833-841.
Sessa WC. The nitric oxide synthase family of proteins. J Vasc Res, 1994, 31, 131-143.
Shaw LM, Rabinovitz I, Wang HH, Toker A, Mercurio AM. Activation of phosphoinositide 3-OH kinase by the a6(34 integrin promotes carcinoma invasion. Cell, 1997,91,949-960.
Shekhar MPV, Werdell J, Tait L. Interaction with endothelial cell is prerequisite for branching ductal-alveolar morphogenesis and hyperplasia of preneoplastic human breast epithelial cell regulation by estrogen. Cancer Res, 2000, 60, 439-449.
Shi Y. Caspase activation: revisiting the induced proximity model. Cell, 2004, 117, 855-858.
Silva J, Silva JM, Barradas M, Garcia JM, Dominguez G, Garcia V. Analysis of the candidate tumor suppressor Ris-1 in primary human breast carcinomas. Mutat Res, 2006, 594, 78-85.
Smukste I, Stockwell BR. Advances in chemical genetics. Annu Rev Genomics Hum Genet, 2005, 6, 261-286.
Spring DR. Chemical genetics to chemical genomics: small molecules offer big insights. Chem Soc Rev, 2005, 34, 472-482.
Steiner L, Kroncke K, Fehsel K, Kolb-Bachofen V. Endothelial cells as cytotoxic effector cells: cytokine-activated rat islet endothelial cells lyse syngeneic islet cells via nitric oxide. Diabetologia, 1997,40, 150-155.
Stenmark H, Parton RG, Steele-Mortimer O, Liitcke A, Gruenberg J, Zerial M. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBOJ, 1994, 13, 1287-1296.
Stepp NT, Cornelius LA, Romani N. Polarized expression and basic fibroblast growth factor induced down-regulation of the a6|34 integrin complex on human microvascular endothelial cells. J Invest Dermatol, 1994,104, 266-270.
St Croix B, Rago C, Velculescu V, Traverso G, Romans KE, Montgomery E, Lal A, Riggins GJ. Lengauer C. Vogelstein B. Kinzler KW. Genes expressed in human tumor endothelium. Science. 2000. 289, 1197-1202.
Steven RH.Daniel JS.Hitoshi Y. Tumor growth enhancing effects of vacular endothelail growth factor are associated with increased nitric oxide synthase activity and inhibition of apoptosis in human breast carcinoma xenografts.Canc Lett, 2002.179, 95-101.
Su L. Zhao J, Zhao BX. Miao JY, Yin DL, Zhang SL. Safrole oxide induced vascular endothelial cell differentiation into neuron-like cells by depressing the reactive oxygen species level at the low concentration. Biochim Biophys Acta, 2006 Feb;1763(2):247-53.
Sulyok S. Wankell M, Alzheimer C, Werner S. Activin: an important regulator of wound repair, fibrosis. and neuroprotection. Mol Cell Endocrinol, 2004, 225, 127-132.
Sun SY, Yue P, Hong WK, Lotan R. Induction of Fas expression and augmentation of Fas/Fas ligand-mediated apoptosis by the synthetic retinoid CD437 in human lung cancer cells. Cancer Res, 2000, 60, 6537-6543.
Suryanto A, Herlambang K, Rachmatullah P. Comparison of Tumor Density by CT Scan Based on Histologic Type in Lung Cancer Patients. Acta Med Indones, 2005,37,195-198.
Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature, 1999, 397, 441-446.
Tagliabue E, Ghirelli C, Squicciarini P, Aiello P, Colnaghi MI, Menard S. Prognostic value of α6β4 integrin expression in breast carcinomas is affected by laminin production from tumor cells. Clin Cancer Res, 1998, 4, 407-410.
Tan W, Zhao B, Shi M, Sha L, Miao J. Shin D. Facile Synthesis and Evaluation of Cell A549 Growth Inhibitory Activity of 4-Aryl-6-aryloxymethylmorpholin-3-one Derivatives. Chinese J Chem, 2006, 24, 396-400.
Tani T, Karttunen T. Kiviluoto T, Kivilaakso E, Burgeson RE, Sipponen P, Virtanen I. α6β4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Continuous expression of laminins but not that of collagen Ⅶ is preserved in invasive parts of the carcinomas: implications for acquisition of the invading phenotype. Am J Pathol, 1996, 149, 781-793.
Thompson CB. Apoptosis in the pathogenesis and treat-ment of disease. Science, 1995,267,1456-1462.
Trusolino L, Bertotti A, Comoglio PM. A signaling adapter function for a6b4 integrin in the control of HGF-dependent invasive growth. Cell, 2001, 107, 643-654.
Tsuruta D, Hopkinson SB, Lane KD, Werner ME, Cryns VL, Jones JC. Crucial role of the specificity-determining loop of the integrin beta4 subunit in the binding of cells to laminin-5 and outside-in signal transduction. J Biol Chem, 2003, 278, 38707-38714.
Ushio-Fukai M. Redox signaling in angiogenesis: Role of NADPH oxidase. Cardiovasc Res, 2006, 71, 226-235.
Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ, Vaux DL. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell, 2000, 102, 43-53.
Wang WW, Liu X, Zhao J, Zhao BX, Zhang SL, Miao JY. A novel butyrolactone derivative inhibited apoptosis and depressed integrin β4 expression in vascular endothelial cells. Bioorg Med Chem Lett, 2007, 17, 482-485.
Waterman MJ, Stavridi ES, Waterman JL, Halazonetis TD. ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins. Nature Genetics, 1998, 19, 175-178.
Weaver VM, Lelievre S, Lakins JN, Chrenek MA, Jones JC, Giancotti F. β4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell. 2002, 2. 205-216.
Welt CK. The physiology and pathophysiology of inhibin, activin and follistatin in female reproduction. Curr Opin Obstet Gynecol, 2002. 14, 317-323.
Wilhelmsen K. Litjens SH. Sonnenberg A. Multiple functions of the integrin a6b4 in epidermal homeostasis and tumorigenesis. Mol Cell Biol, 2006, 26, 2877-2886.
Yang Y. Yang Y. Trent MB, He N. Lick SD. Zimniak P, Awasthi YC, Boor PJ. Glutathione-S-transferase A4-4 modulates oxidative stress in endothelium: possible role in human atherosclerosis. Atherosclerosis, 2004, 173, 211-221.
Yeh JR., Crews CM. Chemical genetics: adding to the developmental biology toolbox. Dev Cell, 2003, 5. 11-19.
Yokoi A, Kuormitsu J, Kawai T, Nagasu T, Sugi NH, Yoshimatsu K. Yoshino H, Owa T. Porfiling novel sulfonamide antitumor agents with cell-based phenotypic screens and array-based gene expression analysis. Mol Cancer Ther, 2002, 1, 275-286.
Xu ZX, Liang J, Haridas V, Gaikwad A, Connolly FP, Mills GB, Gutterman JU. A plant triterpenoid, avicin D, induces autophagy by activation of AMP-activated protein kinase. Cell Death Differ. 2007,14, 1948-1957.
Yu C, Wang Z, Dent P, Grant S. MEK1/2 inhibitors promote Ara-C-induced apoptosis but not loss of Deltapsi(m) in HL-60 cells. Biochem Biophys Res Commun, 2001, 286,1011-1018.
Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW, Vogelstein B. Identification and classification of p53-regulated genes. Proc Natl Acad Sci USA, 1999, 96, 14517-14522.
Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci USA, 2003, 100, 15077-15082.
Zerial M, McBride H. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol, 2001,2,107-117.
Zhao BX, Du AY, Miao JY, Zhao KW, Du CQ. Effects of novel safroleoxide derivatives, l-Methoxy-3-(3,4-methylenedioxy-benzyl)-2-propanol and 1-ethoxy-3-(3,4-methylenedioxybenzyl)-2-propanol,on apoptosis induced by deprivation of survival factors in vascular endothelial cells.Vas Pharm,2003,40,182-187.
Zhao J,Miao JY,Zhao BX,Zhang SL.Upregulating of Fas,integrin beta4 and P53and depressing of PC-PLC activity and ROS level in VEC apoptosis by safrole oxide.FEBS Lett,2005,579,5809-5813.
Zhao J,Miao JY,Zhao BX,Zhang SL,Yin DL.Suppressing Akt phosphorylation and activating Fas by safrole oxide inhibited angiogenesis and induced vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 and serum.Int J Biochem Cell Biol,2006,38,1603-1613.
Zhao J,Zhao BX,Du AY,Zhao QT,Miao JY.Effects of Novel Safrole Oxide Derivatives,1-Propyl-3-(3,4-methylenedioxyphenyl)-2-propanol and Isopropoxy-3-(3,4-methylenedioxyphenyl)-2-propanol,on Apoptosis Induced by Deprivation of Survival Factors in Vascular Endothelial Cells.Endothelium,2004a,11,267-273.
Zhao KW,Zhao QT,Zhang SL,Miao JY.Integrin beta4 mAb inhibited apoptosis induced by deprivation of growth factors in vascular endothelial cells,Acta Pharmacol Sin,2004b,25,733-737.
Zhou D,Harrison BL,Shah U,Andree TH,Hornby GA,Scerni R,Schechter LE,Smith DL.Studies toward the discovery of the next generation of antidepressants.Part 5:3,4-Dihydro-2H-benzo[1,4]oxazine derivatives with dual 5-HT1A receptor and serotonin transporter affinity.Bioorg Med Chem Lett,2006,16,1338-1341.
苗俊英,王洁,赵静等.蛋白质功能研究的有利工具-化学遗传学.生物医学工程研究,2004,23,63-66.
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