大花旋覆花内酯及其衍生物抑制细胞增殖的作用机制
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摘要
炎症反应在动脉粥样硬化、肿瘤等疾病的发生发展过程中具有重要作用。致炎因子和细胞因子可通过细胞膜受体激活胞内多条信号通路,进而诱导转录因子活化,调节下游靶基因的表达活性,最终导致细胞行为的改变,并且该过程贯穿动脉粥样硬化和肿瘤形成的诸多阶段。临床流行病学资料也证实,以抗炎为靶标的治疗措施能有效降低动脉粥样硬化等心血管疾病和多种肿瘤的发病风险。因此,寻找或者合成具有较强抗炎活性的先导化合物对于治疗心血管及肿瘤相关性疾病具有重要意义。
欧亚旋覆花属菊科植物,属辛温之药,入肺、大肠经,具有消痰、行水、降气、止呕、抗菌、消炎和镇痛之功效,主要用于治疗胸中痰结、胁下胀满、咳喘、呃逆、心下痞硬、大腹水肿等症。构—效分析表明,从欧亚旋覆花中分离提取的活性单体——大花旋覆花内酯(1-O-acetylbritannilactone,ABL),可通过阻断核因子-κB(nuclear factor-kappa B,NF-κB)信号通路抑制炎性因子诱导的血管平滑肌细胞(vascular smooth muscle cell, VSMC)的炎症应答,对损伤引起的血管内膜增生具有明显的预防作用;6位引入酯酰基的1,6-O2-ABL具有更强的抗炎作用。细胞的过度增殖是炎症诱发血管狭窄和肿瘤形成的主要病理机制,ABL除了抑制炎症应答外,是否能够直接抑制细胞的过度增殖尚不清楚。本研究从细胞和整体水平上系统观察了ABL及其修饰衍生物对VSMC和乳腺癌细胞增殖的抑制效应,进而探讨其作用机制。旨在挖掘ABL新的药理效应,为扩展药物研发提供依据。
1 ABL抑制血小板源性生长因子(platelet-derived growth factor,PDGF)诱导的VSMC增殖及其机制
动脉粥样硬化、冠状动脉血管成形术后再狭窄等慢性炎性病变主要涉及血管内膜的损伤并由此导致的VSMC的迁移和增殖。局部炎症引起血小板聚集,暴露于管腔的VSMC以及内皮细胞在各种因素的刺激下可释放出多种细胞因子。其中,PDGF可通过活化细胞内信号转导途径从而促进VSMC迁移和增殖,在血管新生内膜形成过程中起着重要作用。本研究证实ABL通过阻断PDGF激活的ERK1/2信号级联而抑制DNA的合成,抑制VSMC增殖,并且诱导细胞凋亡。
1.1 ABL抑制PDGF诱导的VSMC DNA合成和细胞增殖
采用BrdU掺入实验发现,给予PDGF(20 ng/ml)刺激VSMC 24 h后,细胞增殖活性明显增高。而ABL(5、10、20μM)与PDGF共同作用24 h后,VSMC增殖活性明显下降。细胞计数分析也观察的相似的实验结果。而单独应用10μM ABL处理VSMC 24 h,细胞的DNA合成和增殖活性未见明显改变。提示ABL可抑制PDGF诱导的VSMC增殖而对静止的细胞无影响。
1.2 ABL使PDGF诱导VSMC的细胞周期停滞于G1期
流式细胞术分析显示,ABL(10、20μM)与PDGF(20 ng/ml)共同作用VSMC 24 h后,处于G1期的细胞数较PDGF组明显增加,与此同时,S期细胞数目明显减少。提示ABL抑制PDGF诱导的VSMC增殖的作用与细胞周期进程被阻滞于G1期有关。
1.3 ABL通过上调p21而抑制细胞周期素(cyclins)和细胞周期素依赖激酶(CDKs)的表达
用不同浓度ABL(5、10、20μM)与PDGF(20 ng/ml)共同作用VSMC 24 h后,Western blot检测细胞周期相关蛋白的表达。结果显示,ABL可剂量依赖性地抑制cyclinD1、cyclinA、cyclinE、CDK2、CDK4和CDK6的表达。此外,免疫共沉淀分析表明,ABL(20μM)可明显抑制p21-CDKs复合物的形成。这说明ABL阻滞VSMC周期进程与抑制细胞周期蛋白表达与活性有关。
1.4 ABL可通过活化Caspases促进VSMC的凋亡
细胞凋亡分析结果表明,ABL(5、10、20μM)与PDGF(20 ng/ml)共同作用VSMC 24 h后,VSMC凋亡率较单纯PDGF(20 ng/ml)刺激组显著升高。随着ABL剂量的增加,促凋亡蛋白Bax的表达逐渐升高,而抑凋亡蛋白Bcl-xL和Bcl-2的表达逐渐下降。此外,还发现ABL可通过诱导Caspase-3与Caspase-9的活性而促进VSMC凋亡。
1.5 ABL抑制PDGF诱导的VSMC迁移
平面迁移实验结果显示,ABL(5、10、20μM)可剂量依赖性地抑制PDGF(20 ng/ml)诱导的VSMC迁移。跨膜迁移实验结果还表明,ABL不仅可以显著抑制PDGF诱导的VSMC跨膜迁移,而且对于静止的VSMC迁移同样具有抑制作用。
1.6 ABL抑制PDGF诱导的ERK1/2信号通路的激活
免疫共沉淀分析结果显示,ABL(5、10、20μM)可剂量依赖性地抑制PDGF(20 ng/ml)诱导的PDGF受体(PDGFR)的磷酸化,对PDGFR下游的MEK1/2、ERK1/2的激活分析显示,增加的MEK1/2和ERK1/2的磷酸化水平也随着ABL浓度的增加而逐渐降低。通过转染持续激活的MEK1/2表达质粒pCMV-MEK1,发现强制活化的ERK1/2可部分逆转ABL处理所致的VSMC增殖抑制,提示抑制ERK1/2信号通路的活化是ABL抗VSMC增殖的重要环节。
1.7 ABL抑制球囊损伤诱导的新生内膜形成
为了确定ABL在体内的抗VSMC增殖活性,建立大鼠颈总动脉内皮球囊损伤模型。形态学分析显示,给予ABL(26 mg/kg/day)的大鼠,球囊损伤诱导的血管新生内膜的增生程度较单纯球囊损伤组明显减轻,其面积分别为0.05±0.02 mm2和0.15±0.04 mm2,两者比较具有显著性差异(P < 0.01)。对血管组织切片进行TUNEL染色发现,ABL组新生内膜中阳性染色细胞数目较球囊损伤组明显升高,提示ABL可诱导新生内膜细胞发生凋亡。
综上所述,ABL可以阻断PDGF激活的ERK1/2信号通路,从而阻滞VSMC细胞周期的进程,抑制细胞DNA的合成,诱导细胞凋亡,并且可以抑制大鼠颈总动脉球囊损伤模型中血管新生内膜的形成。
2 ABL与Celecoxib协同抑制环加氧酶-2(COX-2)的表达和乳腺癌细胞的生长
以往研究证实,ABL具有抑制COX-2表达和PGE2释放的作用,但是ABL在肿瘤治疗方面是否具有成药性尚不清楚。COX-2特异抑制剂Celecoxib在乳腺癌的治疗和预防方面的有效性已得到临床证实,但是由于其副作用多的缺点而限制了该药的应用。基于ABL和Celecoxib具有相似的作用靶点,本研究探讨了二者联用对乳腺癌细胞的抑制作用,并对其抗肿瘤活性和药理机制进行了评价。
2.1 ABL和Celecoxib协同抑制乳腺癌细胞的增殖
将处于对数生长期的乳腺癌细胞(MDA-MB-231,MDA-MB-468和MCF-7)接种于96孔板中,分别加入不同浓度的ABL(5、10、25、50、100、150μM)和Celecoxib(2.5、5、10、20、40μM)处理细胞48 h后,用MTT法检测细胞增殖活力。结果显示,随着化合物浓度的增加,各种乳腺癌细胞增殖活力不断下降,呈剂量依赖性的特点。然而,在两种化合物共同作用于乳腺癌细胞后,ABL(50μM)与Celecoxib(5μM)就可以产生明显的抑制作用,且抑制强度均明显高于相同剂量的单一制剂处理组(P < 0.05)。用Calcusyn软件分析发现,ABL与Celecoxib联用CI值<1,表现为二者的协同抑制作用。此外,应用MDA-MB-231细胞裸鼠体内异种移植瘤模型证实,ABL(15 mg/kg/day)与Celecoxib(5 mg/kg/day)的联用对移植瘤生长的抑制作用较相同剂量的单一制剂的效果明显增强。
2.2 ABL和Celecoxib协同诱导乳腺癌细胞凋亡
通过流式细胞术、细胞凋亡ELISA及DAPI染色分析了两种化合物对乳腺癌细胞凋亡的影响。结果显示,在COX-2高表达的MDA-MB-231细胞中,ABL(50μM)与Celecoxib(5μM)联用对细胞凋亡的促进作用明显高于相同剂量的单化合物处理组(P < 0.05)。而在COX-2低表达的MDA-MB-468和MCF-7细胞中,两种化合物联用对凋亡的影响不明显。此外,通过Western blot检测发现,两种制剂联用能够显著激活MDA-MB-231细胞中的Caspase-3与Caspase-9,从而促进细胞凋亡的发生。
2.3 ABL和Celecoxib协同抑制乳腺癌细胞COX-2表达和活性
为了进一步探讨ABL与Celecoxib协同作用促进高表达COX-2的MDA-MB-231乳腺癌细胞凋亡的作用机制,进而检测了ABL和Celecoxib对COX-2的表达和活性的影响。RT-PCR、Western blot和免疫荧光染色结果表明,在高表达COX-2的MDA-MB-231细胞中,ABL(50μM)与Celecoxib(5μM)的联用明显抑制COX-2的转录和表达,减少PGE2的分泌,抑制作用明显高于相同剂量的单化合物处理组;在COX-2低表达的MCF-7细胞中,ABL(50μM)与Celecoxib(5μM)的联用同样可以抑制TPA(0.1μM)诱导的COX-2的表达。
2.4 ABL和Celecoxib协同抑制乳腺癌细胞COX-2基因转录的分子机制
报告基因分析结果表明,在转染COX-2启动子报告基因的MCF-7细胞中,ABL(50μM)与Celecoxib(5μM)可以协同抑制TPA(0.1μM)诱导的COX-2报告基因转录活性(P < 0.05)。染色质免疫沉淀(ChIP)和DNA蛋白亲和分析实验发现,ABL与Celecoxib的协同效应是通过抑制转录因子ATF-2、CREB-1和c-Fos的激活及其与COX-2基因启动子区的CRE和AP-1元件的结合从而下调COX-2基因的转录活性。
2.5 ABL和Celecoxib协同抑制乳腺癌细胞中Akt和p38信号通路的激活
Akt和MAPK信号通路在介导COX-2基因的转录激活中发挥重要作用。在MCF-7细胞中,ABL(50μM)与Celecoxib(5μM)可以协同抑制TPA(0.1μM)诱导的Akt和p38信号通路的激活,而对JNK和ERK途径的影响不明显。将Akt表达质粒Ca-Akt和p38表达质粒MKK6b分别转染MCF-7细胞,均可以部分消除ABL与Celecoxib对COX-2的报告基因活性的协同抑制效应,转录因子活性分析也得到相似的结果。
综上所述,在细胞和整体水平上,ABL可以通过抑制乳腺癌细胞COX-2的表达和激活从而协同增强Celecoxib的抗肿瘤效应,为两者的联用提供实验依据。
3 ABL衍生物ABL-N通过激活JNK信号通路促进乳腺癌细胞凋亡
以往研究表明,ABL抑制肿瘤生长和诱导细胞凋亡的作用较弱,为了提高ABL的效力和成药性,对ABL的6-OH进行修饰,筛选得到ABL-N。活性分析显示,ABL-N对多种肿瘤细胞株的增殖具有很强的抑制效应。在前期研究的基础上,本部分重点观察了ABL-N对体外培养的人乳腺癌细胞增殖、凋亡的影响,并且通过体内实验对ABL-N的抗瘤活性进行了评价。
3.1 ABL-N抑制肿瘤细胞的增殖
将多种组织来源的肿瘤细胞株( MCF-7、MDA-MB-468、MDA-MB-231、Du145、PC-3、LoVo和HT-29)接种于96孔板中,分别加入不同浓度ABL-N(5、10、20、40μM)处理细胞1、3、6、12、24 h后,用MTT法检测各种细胞的增殖活力。结果显示,随着ABL-N浓度的增加和作用时间的延长,各种肿瘤细胞株增殖活力逐渐下降,提示ABL-N具有较广的抗肿瘤谱,其抗肿瘤增殖作用具有剂量和时间依赖性。
3.2 ABL-N阻滞乳腺癌细胞周期进程
用流式细胞仪检测细胞周期分布结果显示,ABL-N(20μM)作用于乳腺癌细胞(MDA-MB-231,MDA-MB-468和MCF-7)6、12、24 h后,可使处于G2/M期的细胞数明显增加,提示ABL-N可能是通过抑制细胞周期进程,使细胞停滞于G2/M期从而抑制细胞的增殖。
3.3 ABL-N通过诱导Caspases活化而促进乳腺癌细胞凋亡
通过DAPI染色、细胞凋亡ELISA及流式细胞术分析了ABL-N对乳腺癌细胞凋亡的影响。结果显示,ABL-N(20μM)作用于乳腺癌细胞(MDA-MB-231,MDA-MB-468和MCF-7)6、12、24 h后,细胞凋亡率随着作用时间的延长而逐渐升高。Caspases活性分析发现,ABL-N(20μM)可使MDA-MB-231和MDA-MB-468细胞中Caspases-3/7的活性升高;而在雌激素受体阳性的MCF-7细胞中只有Caspases-8的活性明显升高。分别用Caspases抑制剂z-VAD-fmk和Caspases-3特异抑制剂z-DEVD-fmk预处理MDA-MB-231细胞,发现两者均可以部分逆转ABL-N诱导的MDA-MB-231细胞凋亡。结果表明Caspases介导了ABL-N促凋亡的作用。
3.4 ABL-N对乳腺癌细胞中Bcl-2家族成员表达的差异调节
ABL-N(20μM)分别作用MDA-MB-231细胞6、12、24 h后,用Western blot检测Bcl-2家族成员的表达情况。结果显示,随着ABL-N作用时间的延长,抗凋亡蛋白Bcl-2的表达水平逐渐下降,促凋亡蛋白Bax和Bad的表达逐渐升高,且Bax/Bcl-2比值增大。
3.5 ABL-N诱导乳腺癌细胞MAPK信号途径的激活
为了揭示ABL-N诱导乳腺癌细胞凋亡的关键信号通路,分别观察ABL-N对MAPK信号途径活化的影响。结果显示,ABL-N(20μM)分别处理MDA-MB-231细胞0.5、1、3、6、12、24 h,随着时间的延长,JNK和p38的磷酸化水平逐渐升高,而磷酸化的ERK变化不明显。JNK激酶活性分析显示,ABL-N可诱导MDA-MB-231中JNK下游底物c-Jun的磷酸化,但是,将GST-JNK1在体外与ABL-N共孵育时,并没有观察到c-Jun的磷酸化,提示ABL-N通过激活JNK上游的信号分子而发挥作用的。进而用JNK特异性抑制剂SP600125或p38特异性抑制剂SB203580处理细胞,发现只有SP600125能够逆转ABL-N诱导的细胞凋亡,由此证明JNK信号通路的激活介导了ABL-N引发的细胞凋亡。
3.6 ABL-N抑制MDA-MB-231细胞裸鼠体内异种移植瘤的生长
为了观察ABL-N在体内的抗肿瘤活性,进一步构建了MDA-MB-231细胞异种移植瘤模型,评价ABL-N对移植瘤生长的影响。结果显示,ABL-N(15 mg/kg/day)可明显抑制移植瘤的生长,而对裸鼠的一般状况无明显影响,表明ABL-N是一种低毒、高效的活性化合物。
总之,ABL-N是一种具有较广的抗肿瘤谱的化合物,可通过诱导Caspases活化、激活JNK信号通路促进体外培养乳腺癌细胞的凋亡,抑制体内肿瘤生长。
结论
1 ABL通过阻滞细胞周期进程和诱导凋亡而抑制VSMC增殖和损伤诱导的新生内膜增生。
2在细胞和整体水平上,ABL通过抑制乳腺癌细胞COX-2的表达和活性从而协同增强Celecoxib的抗肿瘤效应。
3在体内外,ABL衍生物ABL-N可通过激活JNK信号通路和上调Bax/Bcl-2比值而促进乳腺癌细胞凋亡,抑制肿瘤生长。
Neointimal formation, predominantly consisting of vascular smooth muscle cell (VSMC) growth and migration, is a major pathogenic process of hyperproliferative vascular disorders, such as atherosclerosis and restenosis after balloon angioplasty and stent placement. Although the mechanisms implicated in the process of restenosis have not been completely resolved, the accumulating evidences suggest that inflammation plays a key role in neointimal growth after angioplasty. Recently, abnormally elevated expression of cyclooxygenase-2 (COX-2) has been frequently observed in cancer tissues and some researches supports the concept that COX-2 could provide an early target for cancer prevention. The epidemiological studies have reported the inverse association between the regular use of non-steroidal anti-inflammatory drugs (NSAIDs) and the incidence of many cancers. Therefore, using the anti-inflammatory drugs and agents could suppress the production of inflammatory mediators and in turn block the initiation and progression of inflammation-associated diseases, including hyperproliferative vascular disorders and cancers.
Acetylbritannilactone (ABL), a new active extract isolated from a traditional Chinese medicinal herb Inula Britannica L, is a kind of sesquiterpenes and has been shown to possess anti-inflammatory and anticancer activities. In the previous work, it is demonstrated that that ABL inhibits the expression of inflammation-associated genes and it possesses anticancer properties. It is showed that the properties of ABL have been attributed, at least in part, to its ability to inhibit COX-2. Here, we evaluated the effects of ABL on VSMC and cancer cells and then investigated the intracellular signaling pathways as possible mechanisms.
1 Acetylbritannilactone induce G1 arrest and apoptosis in vascular smooth muscle cells
Neointimal formation is a major pathogenic process of hyperproliferative vascular disorders, such as atherosclerosis and restenosis after balloon angioplasty and stent placement. In response to arterial injury, varieties of inflammatory vasoactive and mitogenic factors are released. Among them, platelet-derived growth factor (PDGF) is one of the most potent mitogenic and chemotactic agents for SMCs and plays a pivotal role in the onset and development of various vascular proliferative diseases. Therefore, inhibition of VSMC growth, either by targeting cellular mediators of the proliferative response or by interfering with the cell cycle machinery, represents a potentially effective therapeutic approach to prevent against restenosis after revascularization therapies. ABL has been receiving strong attention as a preventing agent against cancer and inflammatory diseases. However, the effects of ABL on VSMC proliferation and apoptosis have not yet been clarified. Therefore, in the present study, we evaluated the regulation of ABL on VSMC cycle and apoptosis and then investigated the intracellular signaling pathways as possible mechanisms in vitro and in vivo.
1.1 ABL inhibits DNA synthesis and cell proliferation in VSMCs
Growth-arrested VSMCs were treated with PDGF (20 ng/ml) for 24 h in the presence of ABL, and DNA synthesis was measured by BrdU-incorporation assay. Pretreatment for 2 h with ABL efficiently inhibited PDGF-stimulated cell proliferation in a concentration-dependent manner. Similar results obtained under the same concentrations by cell counts.
1.2 ABL induces G1 cell cycle arrest
To characterize the contribution of cell cycle arrest to the reduction in VSMC proliferation, flow cytometric analysis was performed for DNA content. ABL treatment arrested the cell cycle at G1 phase, resulting in a decrease in the fraction of cells in the S phase. These data suggested that inhibition of VSMC proliferation by ABL might be associated with the induction of G1 arrest.
1.3 ABL up-regulates p21cip1 and inhibits cyclins and CDKs
As it has been shown that cyclins, CDKs, and CDKIs play crucial roles in the regulation of cell cycle progression, we analyzed the effects of ABL on the expression of these cell cycle regulatory proteins. Cyclin D1, A, and E were up-regulated in response to PDGF at 24 h and the effects could be blocked by ABL treatment. Similarly, a significant reduction in the expression of CDK2, CDK4, and CDK6 was observed. We also examined the effect of ABL on the induction of p21cip1 and showed that the decrease in p21cip1 expression stimulated by PDGF was markedly attenuated by ABL. Furthermore, we immunoprecipitated p21cip1 from total cell lysates, and studied it binding with CDK2, CDK4 and CDK6, which showed an increase in the bound levels of the proteins after ABL treatment. Thus, these results revealed that the ABL-induced enhancement of the p21cip1 played an important role in the ABL-induced G1 arrest of cell cycle progression in VSMCs, possibly through their inhibition of CDK kinase activity.
1.4 ABL induces apoptosis of VSMCs
To determine whether the ABL-induced loss of the proliferation in VSMCs was associated with the induction of apoptosis, nucleosome fragmentation in the cytoplasm was determined. The data showed that ABL dramatically enhanced apoptosis in VSMCs in a concentration-dependent manner. Moreover, cell lysates were prepared from VSMCs stimulated with PDGF and following treatment for 24 h with ABL and were subjected to western bolt for Bcl-xL, Bcl-2, and Bax. This revealed that ABL treatment induced a concentration-dependent reduction in the levels of the anti-apoptotic proteins Bcl-xL and Bcl-2 with a concomitant increase in the levels of pro-apoptotic protein Bax compared with the cells that were not treated with ABL. Based on the above results showing induction of apoptosis by ABL, we analyzed the levels of cleaved caspase-9 and caspase-3 treated with ABL for 24 h. Our data showed that ABL treatment significantly increased the cleaved caspase-9 and caspase-3 in VSMCs induced by PDGF.
1.5 ABL suppresses VSMC migration
Migration of VSMCs is regarded as the essential step leading to neointimal thickening in atherosclerosis and restenosis. The wounding assay showed that PDGF (20 ng/ml) enhanced the basal migration of VSMCs by≈5-fold. Pretreatment with ABL potently suppressed chemoattractant induced migration of VSMCs in a concentration-dependent manner. Similar results were obtained in the Boyden chamber assay. The results also showed that ABL inhibited the chemotaxis of VSMCs stimulated with chemoattractant in the lower chamber. Moreover, ABL also reduced the random motion induced by PDGF (20 ng/ml) in both the upper and lower chambers.
1.6 ABL inhibits PDGF-stimulated phosphorylation of ERK1/2
To investigate the molecular mechanisms of the antiproliferative and pro-apoptotic effects exerted by ABL, the phosphorylation of each mitogen-activated protein kinase (MAPK) pathways were examined. ABL treatment significantly inhibited the ERK1/2 activation stimulated with PDGF in a concentration-dependent manner. Likewise, ABL also effectively suppressed the capacity of PDGF to stimulate the phosphorylation of MEK1/2. In addition, we examined the influence of ABL on the ligand-induced tyrosine phosphorylation of theβPDGFR. The data showed that ABL significantly inhibited theβPDGFR phosphorylation stimulated with PDGF. Therefore, it is very likely that ABL interferes with the pathway from theβPDGFR, via MEK1/2, to ERK1/2.
1.7 ABL suppresses VSMC proliferation and induces apoptosis in vivo
Furthermore, we showed that ABL also resulted in a significantly reduction of neointimal formation in carotid arteries (neointimal area, 0.15±0.04 vs. 0.05±0.02 mm2, P < 0.01). To evaluate the effects of ABL on VSMC apoptosis in vivo, we performed TUNEL assay. At 14 days after injury, the ABL-treated group showed higher levels of apoptosis than the vehicle-treated group (ABL vs. vehicle-treated, 15.6±2.9% vs. 4.3±1.1%), confirming that the induction of apoptosis was one of mechanisms of ABL inhibiting neointimal formation.
In summary, the present study provides some important new insights into the molecular mechanisms of action of ABL in VSMCs. Our results suggest that ABL is capable of suppressing the abnormal VSMC proliferation, accompanied by the induction of apoptosis in vivo and in vitro. In this regard, it may be of interest to investigate the feasibility of ABL administration in patients with vascular restenosis.
2 Acetylbritannilactone synergistically potentiates the growth inhibitory effect of celecoxib on human breast cancer cells through suppressing cyclooxygenase-2 expression
Recently, the epidemiological studies have reported the inverse association between the regular use of non-steroidal anti-inflammatory drugs (NSAIDs) and the incidence of breast cancer. However, the long-term use of traditional NSAIDs may be limited due to undesired side effects among the users. Unlike traditional NSAIDs, several studies have indicated that celecoxib may provide effective approaches to prevent and treat breast cancer. ABL is a kind of sesquiterpenes and has been shown to possess anti-inflammatory and anticancer activities. It is showed that the properties of ABL have been attributed, at least in part, to its ability to inhibit COX-2. With the goal of enhancing the chemopreventive effects in breast cancer, we tested the effects of the celecoxib and ABL combination in vitro and in vivo.
2.1 Celecoxib and ABL synergistically inhibited human breast cancer cell growth
The growth-inhibitory effects of celecoxib (2.5-40μM) and ABL (5-150μM) on cell viability were first assessed by MTT assay and calculated as the percentage of viable cells relative to untreated cells, respectively. Celecoxib and ABL inhibited growth of these three cancer cell lines (MDA-MB-231, MDA-MB-468 and MCF-7) in a concentration dependent manner. Although single ABL (50μM) induced only weak growth inhibition, the addition of the dose of ABL to celecoxib (5μM) that was almost no effect on the growth of the breast cancer cells, induced a pronounced decrease in cell viability. Furthermore, MDA-MB-231 cells were treated with both celecoxib and ABL simultaneously at fixed 1: 5 and 1:10 dose ratio for 48 h. CI values were determined using the commercial software package Calcusyn. The CI values of the celecoxib and ABL were less than 1. These results showed a synergistic inhibition between celecoxib and ABL in the growth of MDA-MB-231 cells, suggesting that celecoxib and ABL may be an effective combination for the inhibition of MDA-MB-231 cell growth due to their synergistic efficacy. Because the combination of celecoxib and ABL was more effective at inhibiting cultured cancer cells, effect on tumor development was examined next in vivo. Treatment with single celecoxib (5 mg/kg) or ABL (15 mg/kg) had weaker inhibitory effect on MDA-MB-231 tumor growth, while the combination treatment significantly reduced tumor volume by 50% (P < 0.05) after 30 days of treatment.
2.2 Celecoxib and ABL synergistically induced apoptosis
To examine whether the synergistic growth inhibition by combined treatment with celecoxib and ABL may be explained by the induction of apoptosis, the extent of apoptosis was assessed following 48 h exposure of cells to the agents. The combination of celecoxib (5μM) and ABL (50μM) significantly increased the percentage of apoptotic cells compared with single agents in MDA-MB-231 cells. However, celecoxib, ABL, or combined agents showed weaker induction of apoptosis in MCF-7 and MDA-MB-468 cells that express low levels of COX-2. To confirm the induction of apoptosis by the combination, expression of apoptosis markers also analyzed in MDA-MB-231 cells by Western blot after treatments with the agents. The combination treatment, but not individual agents, resulted in marked increased degradation of caspase-3 and caspase-9 in MDA-MB-231 cells. These events were associated with increased apoptosis proportion of cancer cells.
2.3 Celecoxib and ABL synergistically suppressed COX-2 expression and activity
To determine the mechanisms in MDA-MB-231 cell apoptosis induced by the combination treatment of celecoxib and ABL, the expression of COX-2 mRNA and protein was examined. COX-2 was overexpressed at mRNA and protein levels in MDA-MB-231 cells, slightly decreased by the single treatments with celecoxib (5μM) or ABL (50μM), but markedly decreased by the combination of the two agents. Consistent with the Western blot results, in immunofluorescence staining analyses, the combination treatment of celecoxib (5μM) and ABL (50μM) on MDA-MB-231cells for 48 h led to a drastic decrease in cytoplasmic COX-2 expression was not affected by the two agents treatment. Moreover, the combination of celecoxib (5μM) and ABL (50μM) also markedly suppressed TPA (0.1μM)-induced COX-2 expression in MCF-7 cells. PGE2 is derived by COX-2 catalysis, which represents COX-2 activity. The combination of celecoxib and ABL almost totally (>90%) diminished PGE2 production.
2.4 Celecoxib and ABL synergistically suppressed COX-2 gene transcription
To investigate the mechanisms by which the agent combination inhibited COX-2 expression, the luciferase-reporter construct containing a fragment of the COX-2 promoter (?1432/+59) was transfected into MCF-7 cells that was induced by TPA to express COX-2. The combination treatment of celecoxib and ABL significantly reduced TPA-induced COX-2 promoter activity (P < 0.05). The inhibitory effect of the combination of the two agents was significantly stronger than the effects produced by either celecoxib or ABL at their low doses. Furthermore, the recruitments of transcription factors to the CRE and AP-1 elements were evaluated by ChIP assay. TPA increased the recruitment of the transcription factors ATF-2, CREB-1, c-Fos and c-Jun to the COX-2 promoter region containing CRE and AP-1 sites, respectively. However, the combination of celecoxib and ABL significantly reduced the binding of ATF-2, CREB-1 and c-Fos binding activity than each agent alone. The similar results were observed in DAPA experiments using the probe containing the CRE and AP-1 binding sites.
2.5 Synergistic inhibition of the transcription factors by celecoxib and ABL is associated with suppression of Akt and p38 signaling
The activation of specific transcription factors, which triggers COX-2 expression, is regulated by Akt and MAPKs cascades. For this, the roles of these intracellular signaling pathways in inhibition of ATF-2, CREB-1 and c-Fos activation by single and combination of celecoxib and ABL were examined using antibodies that identify the active (phosphorylated) forms of these kinases. Western blot analysis showed that exposure of MCF-7 cells to celecoxib or ABL alone for 2 h resulted in a slight decrease in Akt and p38 phosphorylation induced by TPA. However, the combination of agents reduced levels of active Akt and p38 to a greater degree versus either agent alone. To further determine whether the observed changes in signaling are responsible for reduction of transcription activation of COX-2 gene, MCF-7 cells were transfected with the COX-2-Luc along with a constitutively active Akt expression (Ca-Akt) or p38 expression (MKK6b) vectors. The results showed that transfection of cells with Ca-Akt or MKK6b significantly abolished the inhibition of TPA-induced COX-2 gene promoter activity by the combination of celecoxib and ABL. In addition, the Ca-Akt or MKK6b also protected cells from agents-induced apoptosis and supported the idea that the attenuation of Akt and p38 activation contributed to the enhanced induction of apoptosis by the combination of celecoxib and ABL.
In summary, celecoxib and ABL combination synergistically inhibit the growth of breast cancer cells in vitro and in vivo. In particular, ABL may synergistically enhance the activity of celecoxib on breast cancer growth inhibition via suppressing COX-2 transcriptional activation and expression.
3 ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation
In the previous work, it is demonstrated that that ABL inhibits the expression of inflammation-associated genes and it possesses anticancer properties. In the course of our continuing search for cytotoxic ABL analogues, we synthesized the compound 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-4-yl 2-(6-methoxynaphthalen-2-yl)propanoate (ABL-N), which in preliminary studies showed exceptional anti-proliferative activity against several human cancer cell types. Here, we showed that ABL-N was more potent than ABL in the ability to induce apoptosis, at a low concentration, of human breast cancer cells and investigated the therapeutic potential of the ABL-N and its underlying mechanism of action.
3.1 ABL-N reduces the viability of various carcinoma cell lines
The inhibitory effects of ABL and ABL-N on various carcinoma cell lines were estimated using the MTT cellular survival assay. The results showed that ABL-N treatment inhibited cell growth with similar IC50 (approximately 12-20μM) after a 24 h treatment. It indicated that ABL-N was a broad-spectrum inhibitory agent of the human carcinoma cells.
3.2 ABL-N arrests cells in G2/M phase of the cell cycle
Because ABL-N can effectively inhibit cell viability, we reasoned that this effect might be attributable to its ability to interfere with the cell cycle. MDA-MB-231 cells were incubated with 20μM ABL-N for 6, 12 and 24 h, and the cell cycle analysis was done by PI uptake. The results showed that the ratio of cells in G2/M phase and the cells with hypodiploid DNA contents (sub-G1) were significantly accumulated over the treatment periods. Moreover, we also analyzed cell cycle in MDA-MB-468 and MCF-7 cells and found the G2/M arrest induced by ABL-N as well.
3.3 ABL-N induces apoptosis in breast cancer cells
We next analyzed whether the ABL-N-induced cell viability reduction in human breast cancer cells involved apoptosis. MDA-MB-231 cells were treated with 20μM ABL-N and apoptosis was assayed by two different methods. DAPI staining showed that the condensed and fragmented nuclei increased with ABL-N treatment. Nucleosome fragmentation further determined by Cell Death Detection ELISAPLUS confirmed that cells treated for 6 h with 20μM ABL-N underwent apoptosis. Furthermore, caspase activities were measured with Caspase-Glo assays. Treatment with ABL-N induced the activation of caspases-3/7, -8 and -9 in MDA-MB-231 cells. However, we found that caspase-8 activity is significantly higher in MCF-7 cells treated with ABL-N. To define the role of caspase activation in ABL-N-induced apoptosis, we treated MDA-MB-231 cells with pan-caspase inhibitor z-VAD-fmk (50μM) and caspase-3-specific inhibitor (z-DEVD-fmk) (50μM) before challenge with ABL-N (20μM). z-VAD-fmk pretreatment for 1 h abrogated ABL-N-induced apoptosis as measured by the nucleosome fragmentation and the appearance of sub-G1 cells. The caspase-3-specific inhibitor z-DEVD-fmk also reduced ABL-N-induced apoptosis in MDA-MB-231 cells. These results suggested that activation of caspase cascade was essential for ABL-N-induced apoptosis in breast cancer cells.
3.4 ABL-N modulates the expression of Bcl-2 family proteins in breast cancer cells
The effects of ABL-N on the expression of anti-apoptotic protein Bcl-2 and the pro-apoptotic proteins Bax and Bad in breast cancer cells were evaluated. The data showed a marked increase in the level of Bax and Bad, which started at 6 h and peaked at 24 h of treatment with ABL-N in MDA-MB-231 cells. In contrast, reduced Bcl-2 protein appeared later at 12 h. Then, the ratio of Bax and Bcl-2 was measured by a densitometric analysis of the bands. The data showed that ABL-N treatment resulted in a time-dependent increase in Bax/Bcl-2 ratio in MDA-MB-231 cells that favors apoptosis.
3.5 ABL-N induces the activation of JNK and p38 signaling in breast cancer cells
Activation of MAPK is involved in many aspects of the control of cellular proliferation and apoptosis in response to a variety of extracellular stimulus. We therefore examined the effects of ABL-N on the activation of several MAPK pathways. The data showed that ABL-N treatment induced activation of JNK and p38 in a time-dependent manner. The activation of JNK by ABL-N was further confirmed by the analysis of phosphorylation of its downstream substrate c-Jun. It showed that c-Jun was phosphorylated following ABL-N treatment, which occurred over the same sustained period as JNK activation. To gain further insight into the mechanism by which ABL-N treatment affects JNK, we assayed JNK activity in ABL-N-treated cells as well as the direct effect of ABL-N on GST-JNK1 fusion proteins activity. The data showed that JNK activity began to increase after treatment with 20μM ABL-N for 3 h and maximum activation was achieved 12 h after treatment. In addition, using GST-JNK1 fusion proteins, we found that the JNK activity was unaffected by the presence of ABL-N, indicating ABL-N activated JNK indirectly by activating signaling molecules located upstream in the JNK cascades. To determine the role of the activation of JNK and p38 in ABL-N-induced apoptosis, MDA-MB-231 cells were treated with the JNK inhibitor SP600125 or the p38 inhibitor SB203580 and their effects on cell apoptosis were examined. The data showed that reduction of cell viability by ABL-N was effectively abolished by SP600125, but SB203580 only had a slight effect on the decreased viability by ABL-N. These results suggested that the activation of JNK signaling is responsible for the ABL-N-induced apoptosis.
3.6 ABL-N inhibits the growth of human breast cancer xenografts
Because ABL-N treatment showed the effective growth inhibition in cultured breast cancer cells, we subsequently carried out in vivo study using MDA-MB-231-derived cancer xenografts in nude mice. The data showed that the i.p. treatment with ABL-N (15 mg/kg) caused a significant inhibition of tumor growth as early as 20 days after treatment and persisted after 34 days. In summary, our studies suggest that ABL-N significantly induces apoptosis in breast cancer cells. This induction is associated with the activation of caspases and JNK signaling pathways. Moreover, ABL-N treatment caused a significant inhibition of tumor growth in vivo. Therefore, it is thought that ABL-N might be a potential drug for use in breast cancer prevention and intervention.
CONCLUSIONS
1 Acetylbritannilactone induce G1 arrest and apoptosis in vascular smooth muscle cells.
2 Acetylbritannilactone synergistically potentiates the growth inhibitory effect of celecoxib on human breast cancer cells through suppressing cyclooxygenase-2 expression.
3 ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation.
欧亚旋覆花属菊科植物,属辛温之药,入肺、大肠经,具有消痰、行水、降气、止呕、抗菌、消炎和镇痛之功效,主要用于治疗胸中痰结、胁下胀满、咳喘、呃逆、心下痞硬、大腹水肿等症。构—效分析表明,从欧亚旋覆花中分离提取的活性单体——大花旋覆花内酯(1-O-acetylbritannilactone,ABL),可通过阻断核因子-κB(nuclear factor-kappa B,NF-κB)信号通路抑制炎性因子诱导的血管平滑肌细胞(vascular smooth muscle cell, VSMC)的炎症应答,对损伤引起的血管内膜增生具有明显的预防作用;6位引入酯酰基的1,6-O2-ABL具有更强的抗炎作用。细胞的过度增殖是炎症诱发血管狭窄和肿瘤形成的主要病理机制,ABL除了抑制炎症应答外,是否能够直接抑制细胞的过度增殖尚不清楚。本研究从细胞和整体水平上系统观察了ABL及其修饰衍生物对VSMC和乳腺癌细胞增殖的抑制效应,进而探讨其作用机制。旨在挖掘ABL新的药理效应,为扩展药物研发提供依据。
1 ABL抑制血小板源性生长因子(platelet-derived growth factor,PDGF)诱导的VSMC增殖及其机制
动脉粥样硬化、冠状动脉血管成形术后再狭窄等慢性炎性病变主要涉及血管内膜的损伤并由此导致的VSMC的迁移和增殖。局部炎症引起血小板聚集,暴露于管腔的VSMC以及内皮细胞在各种因素的刺激下可释放出多种细胞因子。其中,PDGF可通过活化细胞内信号转导途径从而促进VSMC迁移和增殖,在血管新生内膜形成过程中起着重要作用。本研究证实ABL通过阻断PDGF激活的ERK1/2信号级联而抑制DNA的合成,抑制VSMC增殖,并且诱导细胞凋亡。
1.1 ABL抑制PDGF诱导的VSMC DNA合成和细胞增殖
采用BrdU掺入实验发现,给予PDGF(20 ng/ml)刺激VSMC 24 h后,细胞增殖活性明显增高。而ABL(5、10、20μM)与PDGF共同作用24 h后,VSMC增殖活性明显下降。细胞计数分析也观察的相似的实验结果。而单独应用10μM ABL处理VSMC 24 h,细胞的DNA合成和增殖活性未见明显改变。提示ABL可抑制PDGF诱导的VSMC增殖而对静止的细胞无影响。
1.2 ABL使PDGF诱导VSMC的细胞周期停滞于G1期
流式细胞术分析显示,ABL(10、20μM)与PDGF(20 ng/ml)共同作用VSMC 24 h后,处于G1期的细胞数较PDGF组明显增加,与此同时,S期细胞数目明显减少。提示ABL抑制PDGF诱导的VSMC增殖的作用与细胞周期进程被阻滞于G1期有关。
1.3 ABL通过上调p21而抑制细胞周期素(cyclins)和细胞周期素依赖激酶(CDKs)的表达
用不同浓度ABL(5、10、20μM)与PDGF(20 ng/ml)共同作用VSMC 24 h后,Western blot检测细胞周期相关蛋白的表达。结果显示,ABL可剂量依赖性地抑制cyclinD1、cyclinA、cyclinE、CDK2、CDK4和CDK6的表达。此外,免疫共沉淀分析表明,ABL(20μM)可明显抑制p21-CDKs复合物的形成。这说明ABL阻滞VSMC周期进程与抑制细胞周期蛋白表达与活性有关。
1.4 ABL可通过活化Caspases促进VSMC的凋亡
细胞凋亡分析结果表明,ABL(5、10、20μM)与PDGF(20 ng/ml)共同作用VSMC 24 h后,VSMC凋亡率较单纯PDGF(20 ng/ml)刺激组显著升高。随着ABL剂量的增加,促凋亡蛋白Bax的表达逐渐升高,而抑凋亡蛋白Bcl-xL和Bcl-2的表达逐渐下降。此外,还发现ABL可通过诱导Caspase-3与Caspase-9的活性而促进VSMC凋亡。
1.5 ABL抑制PDGF诱导的VSMC迁移
平面迁移实验结果显示,ABL(5、10、20μM)可剂量依赖性地抑制PDGF(20 ng/ml)诱导的VSMC迁移。跨膜迁移实验结果还表明,ABL不仅可以显著抑制PDGF诱导的VSMC跨膜迁移,而且对于静止的VSMC迁移同样具有抑制作用。
1.6 ABL抑制PDGF诱导的ERK1/2信号通路的激活
免疫共沉淀分析结果显示,ABL(5、10、20μM)可剂量依赖性地抑制PDGF(20 ng/ml)诱导的PDGF受体(PDGFR)的磷酸化,对PDGFR下游的MEK1/2、ERK1/2的激活分析显示,增加的MEK1/2和ERK1/2的磷酸化水平也随着ABL浓度的增加而逐渐降低。通过转染持续激活的MEK1/2表达质粒pCMV-MEK1,发现强制活化的ERK1/2可部分逆转ABL处理所致的VSMC增殖抑制,提示抑制ERK1/2信号通路的活化是ABL抗VSMC增殖的重要环节。
1.7 ABL抑制球囊损伤诱导的新生内膜形成
为了确定ABL在体内的抗VSMC增殖活性,建立大鼠颈总动脉内皮球囊损伤模型。形态学分析显示,给予ABL(26 mg/kg/day)的大鼠,球囊损伤诱导的血管新生内膜的增生程度较单纯球囊损伤组明显减轻,其面积分别为0.05±0.02 mm2和0.15±0.04 mm2,两者比较具有显著性差异(P < 0.01)。对血管组织切片进行TUNEL染色发现,ABL组新生内膜中阳性染色细胞数目较球囊损伤组明显升高,提示ABL可诱导新生内膜细胞发生凋亡。
综上所述,ABL可以阻断PDGF激活的ERK1/2信号通路,从而阻滞VSMC细胞周期的进程,抑制细胞DNA的合成,诱导细胞凋亡,并且可以抑制大鼠颈总动脉球囊损伤模型中血管新生内膜的形成。
2 ABL与Celecoxib协同抑制环加氧酶-2(COX-2)的表达和乳腺癌细胞的生长
以往研究证实,ABL具有抑制COX-2表达和PGE2释放的作用,但是ABL在肿瘤治疗方面是否具有成药性尚不清楚。COX-2特异抑制剂Celecoxib在乳腺癌的治疗和预防方面的有效性已得到临床证实,但是由于其副作用多的缺点而限制了该药的应用。基于ABL和Celecoxib具有相似的作用靶点,本研究探讨了二者联用对乳腺癌细胞的抑制作用,并对其抗肿瘤活性和药理机制进行了评价。
2.1 ABL和Celecoxib协同抑制乳腺癌细胞的增殖
将处于对数生长期的乳腺癌细胞(MDA-MB-231,MDA-MB-468和MCF-7)接种于96孔板中,分别加入不同浓度的ABL(5、10、25、50、100、150μM)和Celecoxib(2.5、5、10、20、40μM)处理细胞48 h后,用MTT法检测细胞增殖活力。结果显示,随着化合物浓度的增加,各种乳腺癌细胞增殖活力不断下降,呈剂量依赖性的特点。然而,在两种化合物共同作用于乳腺癌细胞后,ABL(50μM)与Celecoxib(5μM)就可以产生明显的抑制作用,且抑制强度均明显高于相同剂量的单一制剂处理组(P < 0.05)。用Calcusyn软件分析发现,ABL与Celecoxib联用CI值<1,表现为二者的协同抑制作用。此外,应用MDA-MB-231细胞裸鼠体内异种移植瘤模型证实,ABL(15 mg/kg/day)与Celecoxib(5 mg/kg/day)的联用对移植瘤生长的抑制作用较相同剂量的单一制剂的效果明显增强。
2.2 ABL和Celecoxib协同诱导乳腺癌细胞凋亡
通过流式细胞术、细胞凋亡ELISA及DAPI染色分析了两种化合物对乳腺癌细胞凋亡的影响。结果显示,在COX-2高表达的MDA-MB-231细胞中,ABL(50μM)与Celecoxib(5μM)联用对细胞凋亡的促进作用明显高于相同剂量的单化合物处理组(P < 0.05)。而在COX-2低表达的MDA-MB-468和MCF-7细胞中,两种化合物联用对凋亡的影响不明显。此外,通过Western blot检测发现,两种制剂联用能够显著激活MDA-MB-231细胞中的Caspase-3与Caspase-9,从而促进细胞凋亡的发生。
2.3 ABL和Celecoxib协同抑制乳腺癌细胞COX-2表达和活性
为了进一步探讨ABL与Celecoxib协同作用促进高表达COX-2的MDA-MB-231乳腺癌细胞凋亡的作用机制,进而检测了ABL和Celecoxib对COX-2的表达和活性的影响。RT-PCR、Western blot和免疫荧光染色结果表明,在高表达COX-2的MDA-MB-231细胞中,ABL(50μM)与Celecoxib(5μM)的联用明显抑制COX-2的转录和表达,减少PGE2的分泌,抑制作用明显高于相同剂量的单化合物处理组;在COX-2低表达的MCF-7细胞中,ABL(50μM)与Celecoxib(5μM)的联用同样可以抑制TPA(0.1μM)诱导的COX-2的表达。
2.4 ABL和Celecoxib协同抑制乳腺癌细胞COX-2基因转录的分子机制
报告基因分析结果表明,在转染COX-2启动子报告基因的MCF-7细胞中,ABL(50μM)与Celecoxib(5μM)可以协同抑制TPA(0.1μM)诱导的COX-2报告基因转录活性(P < 0.05)。染色质免疫沉淀(ChIP)和DNA蛋白亲和分析实验发现,ABL与Celecoxib的协同效应是通过抑制转录因子ATF-2、CREB-1和c-Fos的激活及其与COX-2基因启动子区的CRE和AP-1元件的结合从而下调COX-2基因的转录活性。
2.5 ABL和Celecoxib协同抑制乳腺癌细胞中Akt和p38信号通路的激活
Akt和MAPK信号通路在介导COX-2基因的转录激活中发挥重要作用。在MCF-7细胞中,ABL(50μM)与Celecoxib(5μM)可以协同抑制TPA(0.1μM)诱导的Akt和p38信号通路的激活,而对JNK和ERK途径的影响不明显。将Akt表达质粒Ca-Akt和p38表达质粒MKK6b分别转染MCF-7细胞,均可以部分消除ABL与Celecoxib对COX-2的报告基因活性的协同抑制效应,转录因子活性分析也得到相似的结果。
综上所述,在细胞和整体水平上,ABL可以通过抑制乳腺癌细胞COX-2的表达和激活从而协同增强Celecoxib的抗肿瘤效应,为两者的联用提供实验依据。
3 ABL衍生物ABL-N通过激活JNK信号通路促进乳腺癌细胞凋亡
以往研究表明,ABL抑制肿瘤生长和诱导细胞凋亡的作用较弱,为了提高ABL的效力和成药性,对ABL的6-OH进行修饰,筛选得到ABL-N。活性分析显示,ABL-N对多种肿瘤细胞株的增殖具有很强的抑制效应。在前期研究的基础上,本部分重点观察了ABL-N对体外培养的人乳腺癌细胞增殖、凋亡的影响,并且通过体内实验对ABL-N的抗瘤活性进行了评价。
3.1 ABL-N抑制肿瘤细胞的增殖
将多种组织来源的肿瘤细胞株( MCF-7、MDA-MB-468、MDA-MB-231、Du145、PC-3、LoVo和HT-29)接种于96孔板中,分别加入不同浓度ABL-N(5、10、20、40μM)处理细胞1、3、6、12、24 h后,用MTT法检测各种细胞的增殖活力。结果显示,随着ABL-N浓度的增加和作用时间的延长,各种肿瘤细胞株增殖活力逐渐下降,提示ABL-N具有较广的抗肿瘤谱,其抗肿瘤增殖作用具有剂量和时间依赖性。
3.2 ABL-N阻滞乳腺癌细胞周期进程
用流式细胞仪检测细胞周期分布结果显示,ABL-N(20μM)作用于乳腺癌细胞(MDA-MB-231,MDA-MB-468和MCF-7)6、12、24 h后,可使处于G2/M期的细胞数明显增加,提示ABL-N可能是通过抑制细胞周期进程,使细胞停滞于G2/M期从而抑制细胞的增殖。
3.3 ABL-N通过诱导Caspases活化而促进乳腺癌细胞凋亡
通过DAPI染色、细胞凋亡ELISA及流式细胞术分析了ABL-N对乳腺癌细胞凋亡的影响。结果显示,ABL-N(20μM)作用于乳腺癌细胞(MDA-MB-231,MDA-MB-468和MCF-7)6、12、24 h后,细胞凋亡率随着作用时间的延长而逐渐升高。Caspases活性分析发现,ABL-N(20μM)可使MDA-MB-231和MDA-MB-468细胞中Caspases-3/7的活性升高;而在雌激素受体阳性的MCF-7细胞中只有Caspases-8的活性明显升高。分别用Caspases抑制剂z-VAD-fmk和Caspases-3特异抑制剂z-DEVD-fmk预处理MDA-MB-231细胞,发现两者均可以部分逆转ABL-N诱导的MDA-MB-231细胞凋亡。结果表明Caspases介导了ABL-N促凋亡的作用。
3.4 ABL-N对乳腺癌细胞中Bcl-2家族成员表达的差异调节
ABL-N(20μM)分别作用MDA-MB-231细胞6、12、24 h后,用Western blot检测Bcl-2家族成员的表达情况。结果显示,随着ABL-N作用时间的延长,抗凋亡蛋白Bcl-2的表达水平逐渐下降,促凋亡蛋白Bax和Bad的表达逐渐升高,且Bax/Bcl-2比值增大。
3.5 ABL-N诱导乳腺癌细胞MAPK信号途径的激活
为了揭示ABL-N诱导乳腺癌细胞凋亡的关键信号通路,分别观察ABL-N对MAPK信号途径活化的影响。结果显示,ABL-N(20μM)分别处理MDA-MB-231细胞0.5、1、3、6、12、24 h,随着时间的延长,JNK和p38的磷酸化水平逐渐升高,而磷酸化的ERK变化不明显。JNK激酶活性分析显示,ABL-N可诱导MDA-MB-231中JNK下游底物c-Jun的磷酸化,但是,将GST-JNK1在体外与ABL-N共孵育时,并没有观察到c-Jun的磷酸化,提示ABL-N通过激活JNK上游的信号分子而发挥作用的。进而用JNK特异性抑制剂SP600125或p38特异性抑制剂SB203580处理细胞,发现只有SP600125能够逆转ABL-N诱导的细胞凋亡,由此证明JNK信号通路的激活介导了ABL-N引发的细胞凋亡。
3.6 ABL-N抑制MDA-MB-231细胞裸鼠体内异种移植瘤的生长
为了观察ABL-N在体内的抗肿瘤活性,进一步构建了MDA-MB-231细胞异种移植瘤模型,评价ABL-N对移植瘤生长的影响。结果显示,ABL-N(15 mg/kg/day)可明显抑制移植瘤的生长,而对裸鼠的一般状况无明显影响,表明ABL-N是一种低毒、高效的活性化合物。
总之,ABL-N是一种具有较广的抗肿瘤谱的化合物,可通过诱导Caspases活化、激活JNK信号通路促进体外培养乳腺癌细胞的凋亡,抑制体内肿瘤生长。
结论
1 ABL通过阻滞细胞周期进程和诱导凋亡而抑制VSMC增殖和损伤诱导的新生内膜增生。
2在细胞和整体水平上,ABL通过抑制乳腺癌细胞COX-2的表达和活性从而协同增强Celecoxib的抗肿瘤效应。
3在体内外,ABL衍生物ABL-N可通过激活JNK信号通路和上调Bax/Bcl-2比值而促进乳腺癌细胞凋亡,抑制肿瘤生长。
Neointimal formation, predominantly consisting of vascular smooth muscle cell (VSMC) growth and migration, is a major pathogenic process of hyperproliferative vascular disorders, such as atherosclerosis and restenosis after balloon angioplasty and stent placement. Although the mechanisms implicated in the process of restenosis have not been completely resolved, the accumulating evidences suggest that inflammation plays a key role in neointimal growth after angioplasty. Recently, abnormally elevated expression of cyclooxygenase-2 (COX-2) has been frequently observed in cancer tissues and some researches supports the concept that COX-2 could provide an early target for cancer prevention. The epidemiological studies have reported the inverse association between the regular use of non-steroidal anti-inflammatory drugs (NSAIDs) and the incidence of many cancers. Therefore, using the anti-inflammatory drugs and agents could suppress the production of inflammatory mediators and in turn block the initiation and progression of inflammation-associated diseases, including hyperproliferative vascular disorders and cancers.
Acetylbritannilactone (ABL), a new active extract isolated from a traditional Chinese medicinal herb Inula Britannica L, is a kind of sesquiterpenes and has been shown to possess anti-inflammatory and anticancer activities. In the previous work, it is demonstrated that that ABL inhibits the expression of inflammation-associated genes and it possesses anticancer properties. It is showed that the properties of ABL have been attributed, at least in part, to its ability to inhibit COX-2. Here, we evaluated the effects of ABL on VSMC and cancer cells and then investigated the intracellular signaling pathways as possible mechanisms.
1 Acetylbritannilactone induce G1 arrest and apoptosis in vascular smooth muscle cells
Neointimal formation is a major pathogenic process of hyperproliferative vascular disorders, such as atherosclerosis and restenosis after balloon angioplasty and stent placement. In response to arterial injury, varieties of inflammatory vasoactive and mitogenic factors are released. Among them, platelet-derived growth factor (PDGF) is one of the most potent mitogenic and chemotactic agents for SMCs and plays a pivotal role in the onset and development of various vascular proliferative diseases. Therefore, inhibition of VSMC growth, either by targeting cellular mediators of the proliferative response or by interfering with the cell cycle machinery, represents a potentially effective therapeutic approach to prevent against restenosis after revascularization therapies. ABL has been receiving strong attention as a preventing agent against cancer and inflammatory diseases. However, the effects of ABL on VSMC proliferation and apoptosis have not yet been clarified. Therefore, in the present study, we evaluated the regulation of ABL on VSMC cycle and apoptosis and then investigated the intracellular signaling pathways as possible mechanisms in vitro and in vivo.
1.1 ABL inhibits DNA synthesis and cell proliferation in VSMCs
Growth-arrested VSMCs were treated with PDGF (20 ng/ml) for 24 h in the presence of ABL, and DNA synthesis was measured by BrdU-incorporation assay. Pretreatment for 2 h with ABL efficiently inhibited PDGF-stimulated cell proliferation in a concentration-dependent manner. Similar results obtained under the same concentrations by cell counts.
1.2 ABL induces G1 cell cycle arrest
To characterize the contribution of cell cycle arrest to the reduction in VSMC proliferation, flow cytometric analysis was performed for DNA content. ABL treatment arrested the cell cycle at G1 phase, resulting in a decrease in the fraction of cells in the S phase. These data suggested that inhibition of VSMC proliferation by ABL might be associated with the induction of G1 arrest.
1.3 ABL up-regulates p21cip1 and inhibits cyclins and CDKs
As it has been shown that cyclins, CDKs, and CDKIs play crucial roles in the regulation of cell cycle progression, we analyzed the effects of ABL on the expression of these cell cycle regulatory proteins. Cyclin D1, A, and E were up-regulated in response to PDGF at 24 h and the effects could be blocked by ABL treatment. Similarly, a significant reduction in the expression of CDK2, CDK4, and CDK6 was observed. We also examined the effect of ABL on the induction of p21cip1 and showed that the decrease in p21cip1 expression stimulated by PDGF was markedly attenuated by ABL. Furthermore, we immunoprecipitated p21cip1 from total cell lysates, and studied it binding with CDK2, CDK4 and CDK6, which showed an increase in the bound levels of the proteins after ABL treatment. Thus, these results revealed that the ABL-induced enhancement of the p21cip1 played an important role in the ABL-induced G1 arrest of cell cycle progression in VSMCs, possibly through their inhibition of CDK kinase activity.
1.4 ABL induces apoptosis of VSMCs
To determine whether the ABL-induced loss of the proliferation in VSMCs was associated with the induction of apoptosis, nucleosome fragmentation in the cytoplasm was determined. The data showed that ABL dramatically enhanced apoptosis in VSMCs in a concentration-dependent manner. Moreover, cell lysates were prepared from VSMCs stimulated with PDGF and following treatment for 24 h with ABL and were subjected to western bolt for Bcl-xL, Bcl-2, and Bax. This revealed that ABL treatment induced a concentration-dependent reduction in the levels of the anti-apoptotic proteins Bcl-xL and Bcl-2 with a concomitant increase in the levels of pro-apoptotic protein Bax compared with the cells that were not treated with ABL. Based on the above results showing induction of apoptosis by ABL, we analyzed the levels of cleaved caspase-9 and caspase-3 treated with ABL for 24 h. Our data showed that ABL treatment significantly increased the cleaved caspase-9 and caspase-3 in VSMCs induced by PDGF.
1.5 ABL suppresses VSMC migration
Migration of VSMCs is regarded as the essential step leading to neointimal thickening in atherosclerosis and restenosis. The wounding assay showed that PDGF (20 ng/ml) enhanced the basal migration of VSMCs by≈5-fold. Pretreatment with ABL potently suppressed chemoattractant induced migration of VSMCs in a concentration-dependent manner. Similar results were obtained in the Boyden chamber assay. The results also showed that ABL inhibited the chemotaxis of VSMCs stimulated with chemoattractant in the lower chamber. Moreover, ABL also reduced the random motion induced by PDGF (20 ng/ml) in both the upper and lower chambers.
1.6 ABL inhibits PDGF-stimulated phosphorylation of ERK1/2
To investigate the molecular mechanisms of the antiproliferative and pro-apoptotic effects exerted by ABL, the phosphorylation of each mitogen-activated protein kinase (MAPK) pathways were examined. ABL treatment significantly inhibited the ERK1/2 activation stimulated with PDGF in a concentration-dependent manner. Likewise, ABL also effectively suppressed the capacity of PDGF to stimulate the phosphorylation of MEK1/2. In addition, we examined the influence of ABL on the ligand-induced tyrosine phosphorylation of theβPDGFR. The data showed that ABL significantly inhibited theβPDGFR phosphorylation stimulated with PDGF. Therefore, it is very likely that ABL interferes with the pathway from theβPDGFR, via MEK1/2, to ERK1/2.
1.7 ABL suppresses VSMC proliferation and induces apoptosis in vivo
Furthermore, we showed that ABL also resulted in a significantly reduction of neointimal formation in carotid arteries (neointimal area, 0.15±0.04 vs. 0.05±0.02 mm2, P < 0.01). To evaluate the effects of ABL on VSMC apoptosis in vivo, we performed TUNEL assay. At 14 days after injury, the ABL-treated group showed higher levels of apoptosis than the vehicle-treated group (ABL vs. vehicle-treated, 15.6±2.9% vs. 4.3±1.1%), confirming that the induction of apoptosis was one of mechanisms of ABL inhibiting neointimal formation.
In summary, the present study provides some important new insights into the molecular mechanisms of action of ABL in VSMCs. Our results suggest that ABL is capable of suppressing the abnormal VSMC proliferation, accompanied by the induction of apoptosis in vivo and in vitro. In this regard, it may be of interest to investigate the feasibility of ABL administration in patients with vascular restenosis.
2 Acetylbritannilactone synergistically potentiates the growth inhibitory effect of celecoxib on human breast cancer cells through suppressing cyclooxygenase-2 expression
Recently, the epidemiological studies have reported the inverse association between the regular use of non-steroidal anti-inflammatory drugs (NSAIDs) and the incidence of breast cancer. However, the long-term use of traditional NSAIDs may be limited due to undesired side effects among the users. Unlike traditional NSAIDs, several studies have indicated that celecoxib may provide effective approaches to prevent and treat breast cancer. ABL is a kind of sesquiterpenes and has been shown to possess anti-inflammatory and anticancer activities. It is showed that the properties of ABL have been attributed, at least in part, to its ability to inhibit COX-2. With the goal of enhancing the chemopreventive effects in breast cancer, we tested the effects of the celecoxib and ABL combination in vitro and in vivo.
2.1 Celecoxib and ABL synergistically inhibited human breast cancer cell growth
The growth-inhibitory effects of celecoxib (2.5-40μM) and ABL (5-150μM) on cell viability were first assessed by MTT assay and calculated as the percentage of viable cells relative to untreated cells, respectively. Celecoxib and ABL inhibited growth of these three cancer cell lines (MDA-MB-231, MDA-MB-468 and MCF-7) in a concentration dependent manner. Although single ABL (50μM) induced only weak growth inhibition, the addition of the dose of ABL to celecoxib (5μM) that was almost no effect on the growth of the breast cancer cells, induced a pronounced decrease in cell viability. Furthermore, MDA-MB-231 cells were treated with both celecoxib and ABL simultaneously at fixed 1: 5 and 1:10 dose ratio for 48 h. CI values were determined using the commercial software package Calcusyn. The CI values of the celecoxib and ABL were less than 1. These results showed a synergistic inhibition between celecoxib and ABL in the growth of MDA-MB-231 cells, suggesting that celecoxib and ABL may be an effective combination for the inhibition of MDA-MB-231 cell growth due to their synergistic efficacy. Because the combination of celecoxib and ABL was more effective at inhibiting cultured cancer cells, effect on tumor development was examined next in vivo. Treatment with single celecoxib (5 mg/kg) or ABL (15 mg/kg) had weaker inhibitory effect on MDA-MB-231 tumor growth, while the combination treatment significantly reduced tumor volume by 50% (P < 0.05) after 30 days of treatment.
2.2 Celecoxib and ABL synergistically induced apoptosis
To examine whether the synergistic growth inhibition by combined treatment with celecoxib and ABL may be explained by the induction of apoptosis, the extent of apoptosis was assessed following 48 h exposure of cells to the agents. The combination of celecoxib (5μM) and ABL (50μM) significantly increased the percentage of apoptotic cells compared with single agents in MDA-MB-231 cells. However, celecoxib, ABL, or combined agents showed weaker induction of apoptosis in MCF-7 and MDA-MB-468 cells that express low levels of COX-2. To confirm the induction of apoptosis by the combination, expression of apoptosis markers also analyzed in MDA-MB-231 cells by Western blot after treatments with the agents. The combination treatment, but not individual agents, resulted in marked increased degradation of caspase-3 and caspase-9 in MDA-MB-231 cells. These events were associated with increased apoptosis proportion of cancer cells.
2.3 Celecoxib and ABL synergistically suppressed COX-2 expression and activity
To determine the mechanisms in MDA-MB-231 cell apoptosis induced by the combination treatment of celecoxib and ABL, the expression of COX-2 mRNA and protein was examined. COX-2 was overexpressed at mRNA and protein levels in MDA-MB-231 cells, slightly decreased by the single treatments with celecoxib (5μM) or ABL (50μM), but markedly decreased by the combination of the two agents. Consistent with the Western blot results, in immunofluorescence staining analyses, the combination treatment of celecoxib (5μM) and ABL (50μM) on MDA-MB-231cells for 48 h led to a drastic decrease in cytoplasmic COX-2 expression was not affected by the two agents treatment. Moreover, the combination of celecoxib (5μM) and ABL (50μM) also markedly suppressed TPA (0.1μM)-induced COX-2 expression in MCF-7 cells. PGE2 is derived by COX-2 catalysis, which represents COX-2 activity. The combination of celecoxib and ABL almost totally (>90%) diminished PGE2 production.
2.4 Celecoxib and ABL synergistically suppressed COX-2 gene transcription
To investigate the mechanisms by which the agent combination inhibited COX-2 expression, the luciferase-reporter construct containing a fragment of the COX-2 promoter (?1432/+59) was transfected into MCF-7 cells that was induced by TPA to express COX-2. The combination treatment of celecoxib and ABL significantly reduced TPA-induced COX-2 promoter activity (P < 0.05). The inhibitory effect of the combination of the two agents was significantly stronger than the effects produced by either celecoxib or ABL at their low doses. Furthermore, the recruitments of transcription factors to the CRE and AP-1 elements were evaluated by ChIP assay. TPA increased the recruitment of the transcription factors ATF-2, CREB-1, c-Fos and c-Jun to the COX-2 promoter region containing CRE and AP-1 sites, respectively. However, the combination of celecoxib and ABL significantly reduced the binding of ATF-2, CREB-1 and c-Fos binding activity than each agent alone. The similar results were observed in DAPA experiments using the probe containing the CRE and AP-1 binding sites.
2.5 Synergistic inhibition of the transcription factors by celecoxib and ABL is associated with suppression of Akt and p38 signaling
The activation of specific transcription factors, which triggers COX-2 expression, is regulated by Akt and MAPKs cascades. For this, the roles of these intracellular signaling pathways in inhibition of ATF-2, CREB-1 and c-Fos activation by single and combination of celecoxib and ABL were examined using antibodies that identify the active (phosphorylated) forms of these kinases. Western blot analysis showed that exposure of MCF-7 cells to celecoxib or ABL alone for 2 h resulted in a slight decrease in Akt and p38 phosphorylation induced by TPA. However, the combination of agents reduced levels of active Akt and p38 to a greater degree versus either agent alone. To further determine whether the observed changes in signaling are responsible for reduction of transcription activation of COX-2 gene, MCF-7 cells were transfected with the COX-2-Luc along with a constitutively active Akt expression (Ca-Akt) or p38 expression (MKK6b) vectors. The results showed that transfection of cells with Ca-Akt or MKK6b significantly abolished the inhibition of TPA-induced COX-2 gene promoter activity by the combination of celecoxib and ABL. In addition, the Ca-Akt or MKK6b also protected cells from agents-induced apoptosis and supported the idea that the attenuation of Akt and p38 activation contributed to the enhanced induction of apoptosis by the combination of celecoxib and ABL.
In summary, celecoxib and ABL combination synergistically inhibit the growth of breast cancer cells in vitro and in vivo. In particular, ABL may synergistically enhance the activity of celecoxib on breast cancer growth inhibition via suppressing COX-2 transcriptional activation and expression.
3 ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation
In the previous work, it is demonstrated that that ABL inhibits the expression of inflammation-associated genes and it possesses anticancer properties. In the course of our continuing search for cytotoxic ABL analogues, we synthesized the compound 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-4-yl 2-(6-methoxynaphthalen-2-yl)propanoate (ABL-N), which in preliminary studies showed exceptional anti-proliferative activity against several human cancer cell types. Here, we showed that ABL-N was more potent than ABL in the ability to induce apoptosis, at a low concentration, of human breast cancer cells and investigated the therapeutic potential of the ABL-N and its underlying mechanism of action.
3.1 ABL-N reduces the viability of various carcinoma cell lines
The inhibitory effects of ABL and ABL-N on various carcinoma cell lines were estimated using the MTT cellular survival assay. The results showed that ABL-N treatment inhibited cell growth with similar IC50 (approximately 12-20μM) after a 24 h treatment. It indicated that ABL-N was a broad-spectrum inhibitory agent of the human carcinoma cells.
3.2 ABL-N arrests cells in G2/M phase of the cell cycle
Because ABL-N can effectively inhibit cell viability, we reasoned that this effect might be attributable to its ability to interfere with the cell cycle. MDA-MB-231 cells were incubated with 20μM ABL-N for 6, 12 and 24 h, and the cell cycle analysis was done by PI uptake. The results showed that the ratio of cells in G2/M phase and the cells with hypodiploid DNA contents (sub-G1) were significantly accumulated over the treatment periods. Moreover, we also analyzed cell cycle in MDA-MB-468 and MCF-7 cells and found the G2/M arrest induced by ABL-N as well.
3.3 ABL-N induces apoptosis in breast cancer cells
We next analyzed whether the ABL-N-induced cell viability reduction in human breast cancer cells involved apoptosis. MDA-MB-231 cells were treated with 20μM ABL-N and apoptosis was assayed by two different methods. DAPI staining showed that the condensed and fragmented nuclei increased with ABL-N treatment. Nucleosome fragmentation further determined by Cell Death Detection ELISAPLUS confirmed that cells treated for 6 h with 20μM ABL-N underwent apoptosis. Furthermore, caspase activities were measured with Caspase-Glo assays. Treatment with ABL-N induced the activation of caspases-3/7, -8 and -9 in MDA-MB-231 cells. However, we found that caspase-8 activity is significantly higher in MCF-7 cells treated with ABL-N. To define the role of caspase activation in ABL-N-induced apoptosis, we treated MDA-MB-231 cells with pan-caspase inhibitor z-VAD-fmk (50μM) and caspase-3-specific inhibitor (z-DEVD-fmk) (50μM) before challenge with ABL-N (20μM). z-VAD-fmk pretreatment for 1 h abrogated ABL-N-induced apoptosis as measured by the nucleosome fragmentation and the appearance of sub-G1 cells. The caspase-3-specific inhibitor z-DEVD-fmk also reduced ABL-N-induced apoptosis in MDA-MB-231 cells. These results suggested that activation of caspase cascade was essential for ABL-N-induced apoptosis in breast cancer cells.
3.4 ABL-N modulates the expression of Bcl-2 family proteins in breast cancer cells
The effects of ABL-N on the expression of anti-apoptotic protein Bcl-2 and the pro-apoptotic proteins Bax and Bad in breast cancer cells were evaluated. The data showed a marked increase in the level of Bax and Bad, which started at 6 h and peaked at 24 h of treatment with ABL-N in MDA-MB-231 cells. In contrast, reduced Bcl-2 protein appeared later at 12 h. Then, the ratio of Bax and Bcl-2 was measured by a densitometric analysis of the bands. The data showed that ABL-N treatment resulted in a time-dependent increase in Bax/Bcl-2 ratio in MDA-MB-231 cells that favors apoptosis.
3.5 ABL-N induces the activation of JNK and p38 signaling in breast cancer cells
Activation of MAPK is involved in many aspects of the control of cellular proliferation and apoptosis in response to a variety of extracellular stimulus. We therefore examined the effects of ABL-N on the activation of several MAPK pathways. The data showed that ABL-N treatment induced activation of JNK and p38 in a time-dependent manner. The activation of JNK by ABL-N was further confirmed by the analysis of phosphorylation of its downstream substrate c-Jun. It showed that c-Jun was phosphorylated following ABL-N treatment, which occurred over the same sustained period as JNK activation. To gain further insight into the mechanism by which ABL-N treatment affects JNK, we assayed JNK activity in ABL-N-treated cells as well as the direct effect of ABL-N on GST-JNK1 fusion proteins activity. The data showed that JNK activity began to increase after treatment with 20μM ABL-N for 3 h and maximum activation was achieved 12 h after treatment. In addition, using GST-JNK1 fusion proteins, we found that the JNK activity was unaffected by the presence of ABL-N, indicating ABL-N activated JNK indirectly by activating signaling molecules located upstream in the JNK cascades. To determine the role of the activation of JNK and p38 in ABL-N-induced apoptosis, MDA-MB-231 cells were treated with the JNK inhibitor SP600125 or the p38 inhibitor SB203580 and their effects on cell apoptosis were examined. The data showed that reduction of cell viability by ABL-N was effectively abolished by SP600125, but SB203580 only had a slight effect on the decreased viability by ABL-N. These results suggested that the activation of JNK signaling is responsible for the ABL-N-induced apoptosis.
3.6 ABL-N inhibits the growth of human breast cancer xenografts
Because ABL-N treatment showed the effective growth inhibition in cultured breast cancer cells, we subsequently carried out in vivo study using MDA-MB-231-derived cancer xenografts in nude mice. The data showed that the i.p. treatment with ABL-N (15 mg/kg) caused a significant inhibition of tumor growth as early as 20 days after treatment and persisted after 34 days. In summary, our studies suggest that ABL-N significantly induces apoptosis in breast cancer cells. This induction is associated with the activation of caspases and JNK signaling pathways. Moreover, ABL-N treatment caused a significant inhibition of tumor growth in vivo. Therefore, it is thought that ABL-N might be a potential drug for use in breast cancer prevention and intervention.
CONCLUSIONS
1 Acetylbritannilactone induce G1 arrest and apoptosis in vascular smooth muscle cells.
2 Acetylbritannilactone synergistically potentiates the growth inhibitory effect of celecoxib on human breast cancer cells through suppressing cyclooxygenase-2 expression.
3 ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation.
引文
[1] Ip JH, Fuster V, Badimon L, Badimon J, Taubman MB, Chesebro JH. Syndromes of accelerated atherosclerosis: role of vascular injury and smooth muscle cell proliferation. J Am Coll Cardiol 1990;15:1667-1687
[2] Tanner FC, Boehm M, Akyurek LM, San H, Yang ZY, Tashiro J, et al. Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation. Circulation 2000;101:2022-2025
[3] Ihling C, Menzel G, Wellens E, Monting JS, Schaefer HE, Zeiher AM. Topographical association between the cyclin-dependent kinases inhibitor P21, p53 accumulation, and cellular proliferation in human atherosclerotic tissue. Arterioscler Thromb Vasc Biol 1997;17:2218-2224
[4] Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 1995;9:1149-1163
[5] Lee B, Moon SK. Resveratrol inhibits TNF-alpha-induced proliferation and matrix metalloproteinase expression in human vascular smooth muscle cells. J Nutr 2005;135:2767-2773
[6] Isner JM, Kearney M, Bortman S, Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation 1995;91:2703-2711
[7] Sata M, Perlman H, Muruve DA, Silver M, Ikebe M, Libermann TA, et al. Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response. Proc Natl Acad Sci U S A 1998;95:1213-1217
[8] Bai N, Lai CS, He K, Zhou Z, Zhang L, Quan Z, et al. Sesquiterpene lactones from Inula britannica and their cytotoxic and apoptotic effects on human cancer cell lines. J Nat Prod 2006;69:531-535
[9] Park EJ, Kim J. Cytotoxic sesquiterpene lactones from Inula britannica. Planta Med 1998;64:752-754
[10] Rafi MM, Bai NS, Chi Tang H, Rosen RT, White E, Perez D, et al. A sesquiterpenelactone from Inula britannica induces anti-tumor effects dependent on Bcl-2 phosphorylation. Anticancer Res 2005;25:313-318
[11] Liu B, Han M, Wen JK. Acetylbritannilactone Inhibits Neointimal Hyperplasia after Balloon Injury of Rat Artery by Suppressing Nuclear Factor-{kappa}B Activation. J Pharmacol Exp Ther 2008;324:292-298
[12] Liu YP, Wen JK, Zheng B, Zhang DQ, Han M. Acetylbritannilactone suppresses lipopolysaccharide-induced vascular smooth muscle cell inflammatory response. Eur J Pharmacol 2007;577:28-34
[13] Han M, Wen JK, Zheng B, Cheng Y, Zhang C. Serum deprivation results in redifferentiation of human umbilical vascular smooth muscle cells. Am J Physiol Cell Physiol 2006;291:C50-58
[14] Iijima K, Yoshizumi M, Hashimoto M, Akishita M, Kozaki K, Ako J, et al. Red wine polyphenols inhibit vascular smooth muscle cell migration through two distinct signaling pathways. Circulation 2002;105:2404-2410
[15] Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest 1983;49:327-333
[16] Li JM, Brooks G. Cell cycle regulatory molecules (cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors) and the cardiovascular system; potential targets for therapy? Eur Heart J 1999;20:406-420
[17] Willis S, Day CL, Hinds MG, Huang DC. The Bcl-2-regulated apoptotic pathway. J Cell Sci 2003;116:4053-4056
[18] Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2:647-656
[19] Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485-495
[20] Jiang C, Wang Z, Ganther H, Lu J. Caspases as key executors of methyl selenium-induced apoptosis (anoikis) of DU-145 prostate cancer cells. Cancer Res 2001;61:3062-3070
[21] Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker ofchemotherapy-induced apoptosis. Cancer Res 1993;53:3976-3985
[22] Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes (1). N Engl J Med 1992;326:242-250
[23] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801-809
[24] Rosenkranz S, Knirel D, Dietrich H, Flesch M, Erdmann E, Bohm M. Inhibition of the PDGF receptor by red wine flavonoids provides a molecular explanation for the "French paradox". FASEB J 2002;16:1958-1960
[25] Jones SM, Kazlauskas A. Growth-factor-dependent mitogenesis requires two distinct phases of signalling. Nat Cell Biol 2001;3:165-172
[26] Hu Y, Zou Y, Dietrich H, Wick G, Xu Q. Inhibition of neointima hyperplasia of mouse vein grafts by locally applied suramin. Circulation 1999;100:861-868
[27] Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 1999;79:1283-1316
[28] Gallo R, Padurean A, Jayaraman T, Marx S, Roque M, Adelman S, et al. Inhibition of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle. Circulation 1999;99:2164-2170
[29] Ruef J, Meshel AS, Hu Z, Horaist C, Ballinger CA, Thompson LJ, et al. Flavopiridol inhibits smooth muscle cell proliferation in vitro and neointimal formation In vivo after carotid injury in the rat. Circulation 1999;100:659-665
[30] Yang HM, Kim HS, Park KW, You HJ, Jeon SI, Youn SW, et al. Celecoxib, a cyclooxygenase-2 inhibitor, reduces neointimal hyperplasia through inhibition of Akt signaling. Circulation 2004;110:301-308
[31] Chandrasekar B, Tanguay JF. Platelets and restenosis. J Am Coll Cardiol 2000;35:555-562
[32] Hoshi S, Goto M, Koyama N, Nomoto K, Tanaka H. Regulation of vascular smooth muscle cell proliferation by nuclear factor-kappaB and its inhibitor, I-kappaB. J Biol Chem 2000;275:883-889
[33] Nagata Y, Todokoro K. Requirement of activation of JNK and p38 for environmental stress-induced erythroid differentiation and apoptosis and of inhibition of ERK for apoptosis. Blood 1999;94:853-863
[34] Zelivianski S, Spellman M, Kellerman M, Kakitelashvilli V, Zhou XW, Lugo E, et al. ERK inhibitor PD98059 enhances docetaxel-induced apoptosis of androgen-independent human prostate cancer cells. Int J Cancer 2003;107:478-485
[35] Pullikuth AK, Catling AD. Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: a perspective. Cell Signal 2007;19:1621-1632
[36] Ray RM, Vaidya RJ, Johnson LR. MEK/ERK regulates adherens junctions and migration through Rac1. Cell Motil Cytoskeleton 2007;64:143-156
[1] Chang SH, Liu CH, Conway R, Han DK, Nithipatikom K, Trifan OC, et al. Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression. Proc Natl Acad Sci U S A 2004;101:591-596
[2] Howe LR, Subbaramaiah K, Brown AM, Dannenberg AJ. Cyclooxygenase-2: a target for the prevention and treatment of breast cancer. Endocr Relat Cancer 2001;8:97-114
[3] Harris RE, Chlebowski RT, Jackson RD, Frid DJ, Ascenseo JL, Anderson G, et al. Breast cancer and nonsteroidal anti-inflammatory drugs: prospective results from the Women's Health Initiative. Cancer Res 2003;63:6096-6101
[4] Schreinemachers DM, Everson RB. Aspirin use and lung, colon, and breast cancer incidence in a prospective study. Epidemiology 1994;5:138-146
[5] Hinz B, Brune K. Cyclooxygenase-2--10 years later. J Pharmacol Exp Ther 2002;300:367-375
[6] Harris RE, Alshafie GA, Abou-Issa H, Seibert K. Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase 2 inhibitor. Cancer Res 2000;60:2101-2103
[7] Basu GD, Pathangey LB, Tinder TL, Lagioia M, Gendler SJ, Mukherjee P. Cyclooxygenase-2 inhibitor induces apoptosis in breast cancer cells in an in vivo model of spontaneous metastatic breast cancer. Mol Cancer Res 2004;2:632-642
[8] Grosch S, Maier TJ, Schiffmann S, Geisslinger G. Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst 2006;98:736-747
[9] Kang HK, Lee E, Pyo H, Lim SJ. Cyclooxygenase-independent down-regulation of multidrug resistance-associated protein-1 expression by celecoxib in human lung cancer cells. Mol Cancer Ther 2005;4:1358-1363
[10] Shishodia S, Koul D, Aggarwal BB. Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis. J Immunol 2004;173:2011-2022
[11] Surh YJ. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003;3:768-780
[12] Liu B, Han M, Wen JK. Acetylbritannilactone Inhibits Neointimal Hyperplasia after Balloon Injury of Rat Artery by Suppressing Nuclear Factor-{kappa}B Activation. J Pharmacol Exp Ther 2008;324:292-298
[13] Liu YP, Wen JK, Zheng B, Zhang DQ, Han M. Acetylbritannilactone suppresses lipopolysaccharide-induced vascular smooth muscle cell inflammatory response. Eur J Pharmacol 2007;577:28-34
[14] Bai N, Lai CS, He K, Zhou Z, Zhang L, Quan Z, et al. Sesquiterpene lactones from Inula britannica and their cytotoxic and apoptotic effects on human cancer cell lines. J Nat Prod 2006;69:531-535
[15] Rafi MM, Bai NS, Chi Tang H, Rosen RT, White E, Perez D, et al. A sesquiterpenelactone from Inula britannica induces anti-tumor effects dependent on Bcl-2 phosphorylation. Anticancer Res 2005;25:313-318
[16] Park EJ, Kim J. Cytotoxic sesquiterpene lactones from Inula britannica. Planta Med 1998;64:752-754
[17] Han M, Wen JK, Zheng B, Zhang DQ. Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression. Life Sci 2004;75:675-684
[18] Subbaramaiah K, Dannenberg AJ. Cyclooxygenase 2: a molecular target for cancer prevention and treatment. Trends Pharmacol Sci 2003;24:96-102
[19] Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27-55
[20] Han M, Wen JK, Zheng B, Cheng Y, Zhang C. Serum deprivation results in redifferentiation of human umbilical vascular smooth muscle cells. Am J Physiol Cell Physiol 2006;291:C50-58
[21] Inoue H, Yokoyama C, Hara S, Tone Y, Tanabe T. Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element. J Biol Chem 1995;270:24965-24971
[22] Zheng B, Han M, Bernier M, Zhang XH, Meng F, Miao SB, et al. Kruppel-like factor 4 inhibits proliferation by platelet-derived growth factor receptor beta-mediated, not by retinoic acid receptor alpha-mediated, phosphatidylinositol 3-kinase and ERK signaling invascular smooth muscle cells. J Biol Chem 2009;284:22773-22785
[23] Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochem J 1994;302 ( Pt 3):723-727
[24] Chang YJ, Wu MS, Lin JT, Chen CC. Helicobacter pylori-Induced invasion and angiogenesis of gastric cells is mediated by cyclooxygenase-2 induction through TLR2/TLR9 and promoter regulation. J Immunol 2005;175:8242-8252
[25] Degner SC, Kemp MQ, Bowden GT, Romagnolo DF. Conjugated linoleic acid attenuates cyclooxygenase-2 transcriptional activity via an anti-AP-1 mechanism in MCF-7 breast cancer cells. J Nutr 2006;136:421-427
[26] Zhao X, Goswami M, Pokhriyal N, Ma H, Du H, Yao J, et al. Cyclooxygenase-2 expression during immortalization and breast cancer progression. Cancer Res 2008;68:467-475
[27] Eckert LB, Repasky GA, Ulku AS, McFall A, Zhou H, Sartor CI, et al. Involvement of Ras activation in human breast cancer cell signaling, invasion, and anoikis. Cancer Res 2004;64:4585-4592
[28] Liu XH, Rose DP. Differential expression and regulation of cyclooxygenase-1 and -2 in two human breast cancer cell lines. Cancer Res 1996;56:5125-5127
[29] Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 1997;9:240-246
[30] Tang Q, Chen W, Gonzales MS, Finch J, Inoue H, Bowden GT. Role of cyclic AMP responsive element in the UVB induction of cyclooxygenase-2 transcription in human keratinocytes. Oncogene 2001;20:5164-5172
[31] Kim EH, Na HK, Kim DH, Park SA, Kim HN, Song NY, et al. 15-Deoxy-Delta12,14-prostaglandin J2 induces COX-2 expression through Akt-driven AP-1 activation in human breast cancer cells: a potential role of ROS. Carcinogenesis 2008;29:688-695
[32] Falandry C, Canney PA, Freyer G, Dirix LY. Role of combination therapy with aromatase and cyclooxygenase-2 inhibitors in patients with metastatic breast cancer. Ann Oncol 2009;20:615-620
[33] Canney PA, Machin MA, Curto J. A feasibility study of the efficacy and tolerability of the combination of Exemestane with the COX-2 inhibitor Celecoxib in post-menopausalpatients with advanced breast cancer. Eur J Cancer 2006;42:2751-2756
[34] Mustafa A, Kruger WD. Suppression of tumor formation by a cyclooxygenase-2 inhibitor and a peroxisome proliferator-activated receptor gamma agonist in an in vivo mouse model of spontaneous breast cancer. Clin Cancer Res 2008;14:4935-4942
[35] Pockaj BA, Basu GD, Pathangey LB, Gray RJ, Hernandez JL, Gendler SJ, et al. Reduced T-cell and dendritic cell function is related to cyclooxygenase-2 overexpression and prostaglandin E2 secretion in patients with breast cancer. Ann Surg Oncol 2004;11:328-339
[36] Shao J, Sheng H, Inoue H, Morrow JD, DuBois RN. Regulation of constitutive cyclooxygenase-2 expression in colon carcinoma cells. J Biol Chem 2000;275:33951-33956
[37] Wu KK, Liou JY, Cieslik K. Transcriptional Control of COX-2 via C/EBPbeta. Arterioscler Thromb Vasc Biol 2005;25:679-685
[38] Schroer K, Zhu Y, Saunders MA, Deng WG, Xu XM, Meyer-Kirchrath J, et al. Obligatory role of cyclic adenosine monophosphate response element in cyclooxygenase-2 promoter induction and feedback regulation by inflammatory mediators. Circulation 2002;105:2760-2765
[39] Kang YJ, Wingerd BA, Arakawa T, Smith WL. Cyclooxygenase-2 gene transcription in a macrophage model of inflammation. J Immunol 2006;177:8111-8122
[40] Tang HY, Shih A, Cao HJ, Davis FB, Davis PJ, Lin HY. Resveratrol-induced cyclooxygenase-2 facilitates p53-dependent apoptosis in human breast cancer cells. Mol Cancer Ther 2006;5:2034-2042
[41] Han S, Inoue H, Flowers LC, Sidell N. Control of COX-2 gene expression through peroxisome proliferator-activated receptor gamma in human cervical cancer cells. Clin Cancer Res 2003;9:4627-4635
[42] Chun KS, Kim SH, Song YS, Surh YJ. Celecoxib inhibits phorbol ester-induced expression of COX-2 and activation of AP-1 and p38 MAP kinase in mouse skin. Carcinogenesis 2004;25:713-722
[43] Zhang J, Bui TN, Xiang J, Lin A. Cyclic AMP inhibits p38 activation via CREB-induced dynein light chain. Mol Cell Biol 2006;26:1223-1234
[44] Hsu AL, Ching TT, Wang DS, Song X, Rangnekar VM, Chen CS. Thecyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem 2000;275:11397-11403
[45] Tegeder I, Pfeilschifter J, Geisslinger G. Cyclooxygenase-independent actions of cyclooxygenase inhibitors. FASEB J 2001;15:2057-2072
[46] Maier TJ, Janssen A, Schmidt R, Geisslinger G, Grosch S. Targeting the beta-catenin/APC pathway: a novel mechanism to explain the cyclooxygenase-2-independent anticarcinogenic effects of celecoxib in human colon carcinoma cells. FASEB J 2005;19:1353-1355
[1] Mellstedt H. Cancer initiatives in developing countries. Ann Oncol 2006;17 Suppl 8:viii24-viii31
[2] Althuis MD, Dozier JM, Anderson WF, Devesa SS, Brinton LA. Global trends in breast cancer incidence and mortality 1973-1997. Int J Epidemiol 2005;34:405-412
[3] Wu CC, Chan ML, Chen WY, Tsai CY, Chang FR, Wu YC. Pristimerin induces caspase-dependent apoptosis in MDA-MB-231 cells via direct effects on mitochondria. Mol Cancer Ther 2005;4:1277-1285
[4] Garcia-Carbonero R, Supko JG. Current perspectives on the clinical experience, pharmacology, and continued development of the camptothecins. Clin Cancer Res 2002;8:641-661
[5] Rowinsky EK, Donehower RC. Paclitaxel (taxol). N Engl J Med 1995;332:1004-1014
[6] Zhu JY, Lavrik IN, Mahlknecht U, Giaisi M, Proksch P, Krammer PH, et al. The traditional Chinese herbal compound rocaglamide preferentially induces apoptosis in leukemia cells by modulation of mitogen-activated protein kinase activities. Int J Cancer 2007;121:1839-1846
[7] Huang M, Gao H, Chen Y, Zhu H, Cai Y, Zhang X, et al. Chimmitecan, a novel 9-substituted camptothecin, with improved anticancer pharmacologic profiles in vitro and in vivo. Clin Cancer Res 2007;13:1298-1307
[8] Tamvakopoulos C, Dimas K, Sofianos ZD, Hatziantoniou S, Han Z, Liu ZL, et al. Metabolism and anticancer activity of the curcumin analogue, dimethoxycurcumin. Clin Cancer Res 2007;13:1269-1277
[9] Liu J, Zhou W, Li SS, Sun Z, Lin B, Lang YY, et al. Modulation of orphan nuclear receptor Nur77-mediated apoptotic pathway by acetylshikonin and analogues. Cancer Res 2008;68:8871-8880
[10] Wang Z, Qiu J, Guo TB, Liu A, Wang Y, Li Y, et al. Anti-inflammatory properties and regulatory mechanism of a novel derivative of artemisinin in experimental autoimmune encephalomyelitis. J Immunol 2007;179:5958-5965
[11] Zhang M, Huang J, Xie X, Holman CD. Dietary intakes of mushrooms and green tea combine to reduce the risk of breast cancer in Chinese women. Int J Cancer2009;124:1404-1408
[12] Razis ED, Fountzilas G. Paclitaxel: epirubicin in metastatic breast cancer--a review. Ann Oncol 2001;12:593-598
[13] Hainsworth JD, Greco FA. Etoposide: twenty years later. Ann Oncol 1995;6:325-341
[14] Jordan MA, Thrower D, Wilson L. Mechanism of inhibition of cell proliferation by Vinca alkaloids. Cancer Res 1991;51:2212-2222
[15] Liu B, Han M, Wen JK. Acetylbritannilactone Inhibits Neointimal Hyperplasia after Balloon Injury of Rat Artery by Suppressing Nuclear Factor-{kappa}B Activation. J Pharmacol Exp Ther 2008;324:292-298
[16] Liu YP, Wen JK, Zheng B, Zhang DQ, Han M. Acetylbritannilactone suppresses lipopolysaccharide-induced vascular smooth muscle cell inflammatory response. Eur J Pharmacol 2007;577:28-34
[17] Bai N, Lai CS, He K, Zhou Z, Zhang L, Quan Z, et al. Sesquiterpene lactones from Inula britannica and their cytotoxic and apoptotic effects on human cancer cell lines. J Nat Prod 2006;69:531-535
[18] Rafi MM, Bai NS, Chi Tang H, Rosen RT, White E, Perez D, et al. A sesquiterpenelactone from Inula britannica induces anti-tumor effects dependent on Bcl-2 phosphorylation. Anticancer Res 2005;25:313-318
[19] Park EJ, Kim J. Cytotoxic sesquiterpene lactones from Inula britannica. Planta Med 1998;64:752-754
[20] Easty GC, Easty DM, Monaghan P, Ormerod MG, Neville AM. Preparation and identification of human breast epithelial cells in culture. Int J Cancer 1980;26:577-584
[21] Edwards PA, Brooks IM, Bunnage HJ, Foster AV, Ellison ML, O'Hare MJ. Clonal analysis of expression of epithelial antigens in cultures of normal human breast. J Cell Sci 1986;80:91-101
[22] Adhami VM, Aziz MH, Mukhtar H, Ahmad N. Activation of prodeath Bcl-2 family proteins and mitochondrial apoptosis pathway by sanguinarine in immortalized human HaCaT keratinocytes. Clin Cancer Res 2003;9:3176-3182
[23] Han M, Wen JK, Zheng B, Cheng Y, Zhang C. Serum deprivation results in redifferentiation of human umbilical vascular smooth muscle cells. Am J Physiol Cell Physiol 2006;291:C50-58
[24] Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485-495
[25] Jiang C, Wang Z, Ganther H, Lu J. Caspases as key executors of methyl selenium-induced apoptosis (anoikis) of DU-145 prostate cancer cells. Cancer Res 2001;61:3062-3070
[26] Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 1993;53:3976-3985
[27] Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol 2007;35:495-516
[28] Willis S, Day CL, Hinds MG, Huang DC. The Bcl-2-regulated apoptotic pathway. J Cell Sci 2003;116:4053-4056
[29] Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2:647-656
[30] Wada T, Penninger JM. Mitogen-activated protein kinases in apoptosis regulation. Oncogene 2004;23:2838-2849
[31] Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001;81:807-869
[32] Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995;270:1326-1331
[33] Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998;281:1322-1326
[34] Tafani M, Schneider TG, Pastorino JG, Farber JL. Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition. Am J Pathol 2000;156:2111-2121
[35] Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 1997;139:1281-1292
[36] Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2001;15:2922-2933
[37] Hunot S, Flavell RA. Apoptosis. Death of a monopoly? Science 2001;292:865-866
[38] Nicholson DW, Thornberry NA. Apoptosis. Life and death decisions. Science 2003;299:214-215
[39] Janicke RU, Sprengart ML, Wati MR, Porter AG. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 1998;273:9357-9360
[40] Piwocka K, Bielak-Mijewska A, Sikora E. Curcumin induces caspase-3-independent apoptosis in human multidrug-resistant cells. Ann N Y Acad Sci 2002;973:250-254
[41] Heerdt BG, Houston MA, Anthony GM, Augenlicht LH. Initiation of growth arrest and apoptosis of MCF-7 mammary carcinoma cells by tributyrin, a triglyceride analogue of the short-chain fatty acid butyrate, is associated with mitochondrial activity. Cancer Res 1999;59:1584-1591
[42] Nagata S. Fas ligand-induced apoptosis. Annu Rev Genet 1999;33:29-55
[43] Liu B, Han M, Wen JK, Wang L. Livin/ML-IAP as a new target for cancer treatment. Cancer Lett 2007;250:168-176
[44] Wang L, Zhang Q, Liu B, Han M, Shan B. Challenge and promise: roles for Livin in progression and therapy of cancer. Mol Cancer Ther 2008;7:3661-3669
[45] Woo JH, Kim YH, Choi YJ, Kim DG, Lee KS, Bae JH, et al. Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species, down-regulation of Bcl-XL and IAP, the release of cytochrome c and inhibition of Akt. Carcinogenesis 2003;24:1199-1208
[46] Qanungo S, Das M, Haldar S, Basu A. Epigallocatechin-3-gallate induces mitochondrial membrane depolarization and caspase-dependent apoptosis in pancreatic cancer cells. Carcinogenesis 2005;26:958-967
[47] Park C, Jin CY, Kim GY, Choi IW, Kwon TK, Choi BT, et al. Induction of apoptosis by esculetin in human leukemia U937 cells through activation of JNK and ERK. Toxicol Appl Pharmacol 2008;227:219-228
[48] Tsuiki H, Tnani M, Okamoto I, Kenyon LC, Emlet DR, Holgado-Madruga M, et al. Constitutively active forms of c-Jun NH2-terminal kinase are expressed in primary glial tumors. Cancer Res 2003;63:250-255
[49] Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 2000;288:870-874
[50] Antonyak MA, Moscatello DK, Wong AJ. Constitutive activation of c-Jun N-terminalkinase by a mutant epidermal growth factor receptor. J Biol Chem 1998;273:2817-2822
[51] Neumann-Haefelin E, Qi W, Finkbeiner E, Walz G, Baumeister R, Hertweck M. SHC-1/p52Shc targets the insulin/IGF-1 and JNK signaling pathways to modulate life span and stress response in C. elegans. Genes Dev 2008;22:2721-2735
[52] Kasper B, Brandt E, Ernst M, Petersen F. Neutrophil adhesion to endothelial cells induced by platelet factor 4 requires sequential activation of Ras, Syk, and JNK MAP kinases. Blood 2006;107:1768-1775
[1] Buday L, Downward J. Roles of cortactin in tumor pathogenesis. Biochim Biophys Acta 2007;1775:263-273
[2] DesMarais V, Ghosh M, Eddy R, Condeelis J. Cofilin takes the lead. J Cell Sci 2005;118:19-26
[3] Kruchten AE, McNiven MA. Dynamin as a mover and pincher during cell migration and invasion. J Cell Sci 2006;119:1683-1690
[4] Lambrechts A, Van Troys M, Ampe C. The actin cytoskeleton in normal and pathological cell motility. Int J Biochem Cell Biol 2004;36:1890-1909
[5] Watanabe T, Noritake J, Kaibuchi K. Regulation of microtubules in cell migration. Trends Cell Biol 2005;15:76-83
[6] Vicente-Manzanares M, Webb DJ, Horwitz AR. Cell migration at a glance. J Cell Sci 2005;118:4917-4919
[7] Gerthoffer WT. Mechanisms of vascular smooth muscle cell migration. Circ Res 2007;100:607-621
[8] Gerthoffer WT. Migration of airway smooth muscle cells. Proc Am Thorac Soc 2008;5:97-105
[9] Deakin NO, Turner CE. Paxillin comes of age. J Cell Sci 2008;121:2435-2444
[10] Palamidessi A, Frittoli E, Garre M, Faretta M, Mione M, Testa I, et al. Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration. Cell 2008;134:135-147
[11] Zech T, Machesky L. Rab5 and rac team up in cell motility. Cell 2008;134:18-20
[12] Papakonstanti EA, Stournaras C. Cell responses regulated by early reorganization of actin cytoskeleton. FEBS Lett 2008;582:2120-2127
[13] Le Clainche C, Carlier MF. Regulation of actin assembly associated with protrusion and adhesion in cell migration. Physiol Rev 2008;88:489-513
[14] Ahmed I, Ponery AS, Nur EKA, Kamal J, Meshel AS, Sheetz MP, et al. Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces. Mol Cell Biochem 2007;301:241-249
[15] Lammermann T, Bader BL, Monkley SJ, Worbs T, Wedlich-Soldner R, Hirsch K, et al.Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 2008;453:51-55
[16] Schmidt A, Brixius K, Bloch W. Endothelial precursor cell migration during vasculogenesis. Circ Res 2007;101:125-136
[17] Daley WP, Peters SB, Larsen M. Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci 2008;121:255-264
[18] Kumar S, Maxwell IZ, Heisterkamp A, Polte TR, Lele TP, Salanga M, et al. Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics. Biophys J 2006;90:3762-3773
[19] Zhou X, Rowe RG, Hiraoka N, George JP, Wirtz D, Mosher DF, et al. Fibronectin fibrillogenesis regulates three-dimensional neovessel formation. Genes Dev 2008;22:1231-1243
[20] Kim HD, Guo TW, Wu AP, Wells A, Gertler FB, Lauffenburger DA. Epidermal growth factor-induced enhancement of glioblastoma cell migration in 3D arises from an intrinsic increase in speed but an extrinsic matrix- and proteolysis-dependent increase in persistence. Mol Biol Cell 2008;19:4249-4259
[21] Wang JH, Lin JS. Cell traction force and measurement methods. Biomech Model Mechanobiol 2007;6:361-371
[22] Zaman MH, Kamm RD, Matsudaira P, Lauffenburger DA. Computational model for cell migration in three-dimensional matrices. Biophys J 2005;89:1389-1397
[23] Thibault MM, Buschmann MD. Migration of bone marrow stromal cells in 3D: 4 color methodology reveals spatially and temporally coordinated events. Cell Motil Cytoskeleton 2006;63:725-740
[24] Schindler M, Nur EKA, Ahmed I, Kamal J, Liu HY, Amor N, et al. Living in three dimensions: 3D nanostructured environments for cell culture and regenerative medicine. Cell Biochem Biophys 2006;45:215-227
[25] Seasholtz TM, Majumdar M, Brown JH. Rho as a mediator of G protein-coupled receptor signaling. Mol Pharmacol 1999;55:949-956
[26] Tzima E. Role of small GTPases in endothelial cytoskeletal dynamics and the shear stress response. Circ Res 2006;98:176-185
[27] Hall A. Rho GTPases and the actin cytoskeleton. Science 1998;279:509-514
[28] Burridge K, Wennerberg K. Rho and Rac take center stage. Cell 2004;116:167-179
[29] Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992;70:401-410
[30] Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992;70:389-399
[31] Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995;81:53-62
[32] Huang F, Chotiner JK, Steward O. Actin polymerization and ERK phosphorylation are required for Arc/Arg3.1 mRNA targeting to activated synaptic sites on dendrites. J Neurosci 2007;27:9054-9067
[33] Yip SC, El-Sibai M, Coniglio SJ, Mouneimne G, Eddy RJ, Drees BE, et al. The distinct roles of Ras and Rac in PI 3-kinase-dependent protrusion during EGF-stimulated cell migration. J Cell Sci 2007;120:3138-3146
[34] El-Sibai M, Nalbant P, Pang H, Flinn RJ, Sarmiento C, Macaluso F, et al. Cdc42 is required for EGF-stimulated protrusion and motility in MTLn3 carcinoma cells. J Cell Sci 2007;120:3465-3474
[35] Ridley AJ. Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol 2006;16:522-529
[36] Carlier MF, Pantaloni D. Control of actin assembly dynamics in cell motility. J Biol Chem 2007;282:23005-23009
[37] Pruyne D, Evangelista M, Yang C, Bi E, Zigmond S, Bretscher A, et al. Role of formins in actin assembly: nucleation and barbed-end association. Science 2002;297:612-615
[38] Chang F, Peter M. Cell biology. Formins set the record straight. Science 2002;297:531-532
[39] Kovar DR, Harris ES, Mahaffy R, Higgs HN, Pollard TD. Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell 2006;124:423-435
[40] Anton IM, Jones GE, Wandosell F, Geha R, Ramesh N. WASP-interacting protein (WIP): working in polymerisation and much more. Trends Cell Biol 2007;17:555-562
[41] Anton IM, Jones GE. WIP: a multifunctional protein involved in actin cytoskeletonregulation. Eur J Cell Biol 2006;85:295-304
[42] Paavilainen VO, Oksanen E, Goldman A, Lappalainen P. Structure of the actin-depolymerizing factor homology domain in complex with actin. J Cell Biol 2008;182:51-59
[43] Carlier MF, Laurent V, Santolini J, Melki R, Didry D, Xia GX, et al. Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility. J Cell Biol 1997;136:1307-1322
[44] van Rheenen J, Song X, van Roosmalen W, Cammer M, Chen X, Desmarais V, et al. EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells. J Cell Biol 2007;179:1247-1259
[45] Sidani M, Wessels D, Mouneimne G, Ghosh M, Goswami S, Sarmiento C, et al. Cofilin determines the migration behavior and turning frequency of metastatic cancer cells. J Cell Biol 2007;179:777-791
[46] Conti MA, Adelstein RS. Nonmuscle myosin II moves in new directions. J Cell Sci 2008;121:11-18
[47] Wu MH. Endothelial focal adhesions and barrier function. J Physiol 2005;569:359-366
[48] Broussard JA, Webb DJ, Kaverina I. Asymmetric focal adhesion disassembly in motile cells. Curr Opin Cell Biol 2008;20:85-90
[49] Turner CE. Paxillin interactions. J Cell Sci 2000;113 Pt 23:4139-4140
[50] Su WH, Chen HI, Jen CJ. Polymorphonuclear leukocyte transverse migration induces rapid alterations in endothelial focal contacts. J Leukoc Biol 2007;82:542-550
[51] Galbraith CG, Yamada KM, Galbraith JA. Polymerizing actin fibers position integrins primed to probe for adhesion sites. Science 2007;315:992-995
[52] Gupton SL, Eisenmann K, Alberts AS, Waterman-Storer CM. mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration. J Cell Sci 2007;120:3475-3487
[53] Chuang YY, Valster A, Coniglio SJ, Backer JM, Symons M. The atypical Rho family GTPase Wrch-1 regulates focal adhesion formation and cell migration. J Cell Sci 2007;120:1927-1934
[54] Lyle KS, Raaijmakers JH, Bruinsma W, Bos JL, de Rooij J. cAMP-induced Epac-Rap activation inhibits epithelial cell migration by modulating focal adhesion and leading edgedynamics. Cell Signal 2008;20:1104-1116
[55] Ziegler WH, Liddington RC, Critchley DR. The structure and regulation of vinculin. Trends Cell Biol 2006;16:453-460
[56] Guan JL. Cell biology. Integrins, rafts, Rac, and Rho. Science 2004;303:773-774
[57] Palazzo AF, Eng CH, Schlaepfer DD, Marcantonio EE, Gundersen GG. Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling. Science 2004;303:836-839
[58] Nakamura J, Shigematsu S, Yamauchi K, Takeda T, Yamazaki M, Kakizawa T, et al. Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens. Biochem Biophys Res Commun 2008;374:699-703
[59] Wei JF, Wei L, Zhou X, Lu ZY, Francis K, Hu XY, et al. Formation of Kv2.1-FAK complex as a mechanism of FAK activation, cell polarization and enhanced motility. J Cell Physiol 2008;217:544-557
[60] Kang SS, Kook JH, Hwang S, Park SH, Nam SC, Kim JK. Inhibition of matrix metalloproteinase-9 attenuated neural progenitor cell migration after photothrombotic ischemia. Brain Res 2008;1228:20-26
[61] Gong Y, Hart E, Shchurin A, Hoover-Plow J. Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice. J Clin Invest 2008;118:3012-3024
[62] Takino T, Saeki H, Miyamori H, Kudo T, Sato H. Inhibition of membrane-type 1 matrix metalloproteinase at cell-matrix adhesions. Cancer Res 2007;67:11621-11629
[63] dos Remedios CG, Chhabra D, Kekic M, Dedova IV, Tsubakihara M, Berry DA, et al. Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol Rev 2003;83:433-473
[64] Bellion A, Baudoin JP, Alvarez C, Bornens M, Metin C. Nucleokinesis in tangentially migrating neurons comprises two alternating phases: forward migration of the Golgi/centrosome associated with centrosome splitting and myosin contraction at the rear. J Neurosci 2005;25:5691-5699
[65] Komatsu S, Ikebe M. The phosphorylation of myosin II at the Ser1 and Ser2 is critical for normal platelet-derived growth factor induced reorganization of myosin filaments. Mol Biol Cell 2007;18:5081-5090
[66] Clark K, Langeslag M, Figdor CG, van Leeuwen FN. Myosin II andmechanotransduction: a balancing act. Trends Cell Biol 2007;17:178-186
[67] Buss F, Kendrick-Jones J. How are the cellular functions of myosin VI regulated within the cell? Biochem Biophys Res Commun 2008;369:165-175
[68] Ezratty EJ, Partridge MA, Gundersen GG. Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase. Nat Cell Biol 2005;7:581-590
[69] Ballestrem C, Wehrle-Haller B, Hinz B, Imhof BA. Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration. Mol Biol Cell 2000;11:2999-3012
[70] Krendel M, Zenke FT, Bokoch GM. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton. Nat Cell Biol 2002;4:294-301
[71] Basu R, Chang F. Shaping the actin cytoskeleton using microtubule tips. Curr Opin Cell Biol 2007;19:88-94
[72] Palazzo AF, Joseph HL, Chen YJ, Dujardin DL, Alberts AS, Pfister KK, et al. Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Curr Biol 2001;11:1536-1541
[73] Gomes ER, Jani S, Gundersen GG. Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell 2005;121:451-463
[74] Nomachi A, Nishita M, Inaba D, Enomoto M, Hamasaki M, Minami Y. Receptor tyrosine kinase Ror2 mediates Wnt5a-induced polarized cell migration by activating c-Jun N-terminal kinase via actin-binding protein filamin A. J Biol Chem 2008;283:27973-27981
[75] Rivero F, Koppel B, Peracino B, Bozzaro S, Siegert F, Weijer CJ, et al. The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development. J Cell Sci 1996;109 ( Pt 11):2679-2691
[76] Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, et al. Cell migration: integrating signals from front to back. Science 2003;302:1704-1709
[77] Faux CH, Parnavelas JG. The role of intracellular calcium and RhoA in neuronal migration. Sci STKE 2007;2007:pe62
[78] Sobieszek A. Calmodulin-dependent autophosphorylation of smooth muscle myosinlight chain kinase: intermolecular reaction mechanism via dimerization of the kinase and potentiation of the catalytic activity following activation. Biochemistry 1995;34:11855-11863
[79] San Martin A, Lee MY, Williams HC, Mizuno K, Lassegue B, Griendling KK. Dual regulation of cofilin activity by LIM kinase and Slingshot-1L phosphatase controls platelet-derived growth factor-induced migration of human aortic smooth muscle cells. Circ Res 2008;102:432-438
[80] Theisen CS, Wahl JK, 3rd, Johnson KR, Wheelock MJ. NHERF links the N-cadherin/catenin complex to the platelet-derived growth factor receptor to modulate the actin cytoskeleton and regulate cell motility. Mol Biol Cell 2007;18:1220-1232
[81] Sossey-Alaoui K, Li X, Ranalli TA, Cowell JK. WAVE3-mediated cell migration and lamellipodia formation are regulated downstream of phosphatidylinositol 3-kinase. J Biol Chem 2005;280:21748-21755
[82] Wang C, Navab R, Iakovlev V, Leng Y, Zhang J, Tsao MS, et al. Abelson interactor protein-1 positively regulates breast cancer cell proliferation, migration, and invasion. Mol Cancer Res 2007;5:1031-1039
[83] Enomoto A, Murakami H, Asai N, Morone N, Watanabe T, Kawai K, et al. Akt/PKB regulates actin organization and cell motility via Girdin/APE. Dev Cell 2005;9:389-402
[84] Enomoto A, Ping J, Takahashi M. Girdin, a novel actin-binding protein, and its family of proteins possess versatile functions in the Akt and Wnt signaling pathways. Ann N Y Acad Sci 2006;1086:169-184
[85] Ghosh P, Garcia-Marcos M, Bornheimer SJ, Farquhar MG. Activation of Galphai3 triggers cell migration via regulation of GIV. J Cell Biol 2008;182:381-393
[86] Kitamura T, Asai N, Enomoto A, Maeda K, Kato T, Ishida M, et al. Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin. Nat Cell Biol 2008;10:329-337
[87] Jiang P, Enomoto A, Jijiwa M, Kato T, Hasegawa T, Ishida M, et al. An actin-binding protein Girdin regulates the motility of breast cancer cells. Cancer Res 2008;68:1310-1318
[88] Huang C, Jacobson K, Schaller MD. MAP kinases and cell migration. J Cell Sci 2004;117:4619-4628
[89] Pullikuth AK, Catling AD. Scaffold mediated regulation of MAPK signaling andcytoskeletal dynamics: a perspective. Cell Signal 2007;19:1621-1632
[90] Han MY, Kosako H, Watanabe T, Hattori S. Extracellular signal-regulated kinase/mitogen-activated protein kinase regulates actin organization and cell motility by phosphorylating the actin cross-linking protein EPLIN. Mol Cell Biol 2007;27:8190-8204
[91] Szczur K, Xu H, Atkinson S, Zheng Y, Filippi MD. Rho GTPase CDC42 regulates directionality and random movement via distinct MAPK pathways in neutrophils. Blood 2006;108:4205-4213
[92] Jog NR, Jala VR, Ward RA, Rane MJ, Haribabu B, McLeish KR. Heat shock protein 27 regulates neutrophil chemotaxis and exocytosis through two independent mechanisms. J Immunol 2007;178:2421-2428
[93] Chan A, Akhtar M, Brenner M, Zheng Y, Gulko PS, Symons M. The GTPase Rac regulates the proliferation and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients. Mol Med 2007;13:297-304
[94] Feldner JC, Brandt BH. Cancer cell motility--on the road from c-erbB-2 receptor steered signaling to actin reorganization. Exp Cell Res 2002;272:93-108
[95] Lorenowicz MJ, Fernandez-Borja M, Hordijk PL. cAMP signaling in leukocyte transendothelial migration. Arterioscler Thromb Vasc Biol 2007;27:1014-1022
[96]Ichiki T. Role of cAMP response element binding protein in cardiovascular remodeling: good, bad, or both? Arterioscler Thromb Vasc Biol 2006;26:449-455
[97] Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell 1996;84:359-369
[98] Xiao F, Wang XF, Li JM, Xi ZQ, Lu Y, Wang L, et al. Overexpression of N-WASP in the brain of human epilepsy. Brain Res 2008;1233:168-175
[99] Liu L, Chen L, Chung J, Huang S. Rapamycin inhibits F-actin reorganization and phosphorylation of focal adhesion proteins. Oncogene 2008;27:4998-5010
[100] Mendoza-Naranjo A, Gonzalez-Billault C, Maccioni RB. Abeta1-42 stimulates actin polymerization in hippocampal neurons through Rac1 and Cdc42 Rho GTPases. J Cell Sci 2007;120:279-288
[2] Tanner FC, Boehm M, Akyurek LM, San H, Yang ZY, Tashiro J, et al. Differential effects of the cyclin-dependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation. Circulation 2000;101:2022-2025
[3] Ihling C, Menzel G, Wellens E, Monting JS, Schaefer HE, Zeiher AM. Topographical association between the cyclin-dependent kinases inhibitor P21, p53 accumulation, and cellular proliferation in human atherosclerotic tissue. Arterioscler Thromb Vasc Biol 1997;17:2218-2224
[4] Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 1995;9:1149-1163
[5] Lee B, Moon SK. Resveratrol inhibits TNF-alpha-induced proliferation and matrix metalloproteinase expression in human vascular smooth muscle cells. J Nutr 2005;135:2767-2773
[6] Isner JM, Kearney M, Bortman S, Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation 1995;91:2703-2711
[7] Sata M, Perlman H, Muruve DA, Silver M, Ikebe M, Libermann TA, et al. Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response. Proc Natl Acad Sci U S A 1998;95:1213-1217
[8] Bai N, Lai CS, He K, Zhou Z, Zhang L, Quan Z, et al. Sesquiterpene lactones from Inula britannica and their cytotoxic and apoptotic effects on human cancer cell lines. J Nat Prod 2006;69:531-535
[9] Park EJ, Kim J. Cytotoxic sesquiterpene lactones from Inula britannica. Planta Med 1998;64:752-754
[10] Rafi MM, Bai NS, Chi Tang H, Rosen RT, White E, Perez D, et al. A sesquiterpenelactone from Inula britannica induces anti-tumor effects dependent on Bcl-2 phosphorylation. Anticancer Res 2005;25:313-318
[11] Liu B, Han M, Wen JK. Acetylbritannilactone Inhibits Neointimal Hyperplasia after Balloon Injury of Rat Artery by Suppressing Nuclear Factor-{kappa}B Activation. J Pharmacol Exp Ther 2008;324:292-298
[12] Liu YP, Wen JK, Zheng B, Zhang DQ, Han M. Acetylbritannilactone suppresses lipopolysaccharide-induced vascular smooth muscle cell inflammatory response. Eur J Pharmacol 2007;577:28-34
[13] Han M, Wen JK, Zheng B, Cheng Y, Zhang C. Serum deprivation results in redifferentiation of human umbilical vascular smooth muscle cells. Am J Physiol Cell Physiol 2006;291:C50-58
[14] Iijima K, Yoshizumi M, Hashimoto M, Akishita M, Kozaki K, Ako J, et al. Red wine polyphenols inhibit vascular smooth muscle cell migration through two distinct signaling pathways. Circulation 2002;105:2404-2410
[15] Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest 1983;49:327-333
[16] Li JM, Brooks G. Cell cycle regulatory molecules (cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors) and the cardiovascular system; potential targets for therapy? Eur Heart J 1999;20:406-420
[17] Willis S, Day CL, Hinds MG, Huang DC. The Bcl-2-regulated apoptotic pathway. J Cell Sci 2003;116:4053-4056
[18] Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2:647-656
[19] Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485-495
[20] Jiang C, Wang Z, Ganther H, Lu J. Caspases as key executors of methyl selenium-induced apoptosis (anoikis) of DU-145 prostate cancer cells. Cancer Res 2001;61:3062-3070
[21] Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker ofchemotherapy-induced apoptosis. Cancer Res 1993;53:3976-3985
[22] Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes (1). N Engl J Med 1992;326:242-250
[23] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801-809
[24] Rosenkranz S, Knirel D, Dietrich H, Flesch M, Erdmann E, Bohm M. Inhibition of the PDGF receptor by red wine flavonoids provides a molecular explanation for the "French paradox". FASEB J 2002;16:1958-1960
[25] Jones SM, Kazlauskas A. Growth-factor-dependent mitogenesis requires two distinct phases of signalling. Nat Cell Biol 2001;3:165-172
[26] Hu Y, Zou Y, Dietrich H, Wick G, Xu Q. Inhibition of neointima hyperplasia of mouse vein grafts by locally applied suramin. Circulation 1999;100:861-868
[27] Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 1999;79:1283-1316
[28] Gallo R, Padurean A, Jayaraman T, Marx S, Roque M, Adelman S, et al. Inhibition of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle. Circulation 1999;99:2164-2170
[29] Ruef J, Meshel AS, Hu Z, Horaist C, Ballinger CA, Thompson LJ, et al. Flavopiridol inhibits smooth muscle cell proliferation in vitro and neointimal formation In vivo after carotid injury in the rat. Circulation 1999;100:659-665
[30] Yang HM, Kim HS, Park KW, You HJ, Jeon SI, Youn SW, et al. Celecoxib, a cyclooxygenase-2 inhibitor, reduces neointimal hyperplasia through inhibition of Akt signaling. Circulation 2004;110:301-308
[31] Chandrasekar B, Tanguay JF. Platelets and restenosis. J Am Coll Cardiol 2000;35:555-562
[32] Hoshi S, Goto M, Koyama N, Nomoto K, Tanaka H. Regulation of vascular smooth muscle cell proliferation by nuclear factor-kappaB and its inhibitor, I-kappaB. J Biol Chem 2000;275:883-889
[33] Nagata Y, Todokoro K. Requirement of activation of JNK and p38 for environmental stress-induced erythroid differentiation and apoptosis and of inhibition of ERK for apoptosis. Blood 1999;94:853-863
[34] Zelivianski S, Spellman M, Kellerman M, Kakitelashvilli V, Zhou XW, Lugo E, et al. ERK inhibitor PD98059 enhances docetaxel-induced apoptosis of androgen-independent human prostate cancer cells. Int J Cancer 2003;107:478-485
[35] Pullikuth AK, Catling AD. Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: a perspective. Cell Signal 2007;19:1621-1632
[36] Ray RM, Vaidya RJ, Johnson LR. MEK/ERK regulates adherens junctions and migration through Rac1. Cell Motil Cytoskeleton 2007;64:143-156
[1] Chang SH, Liu CH, Conway R, Han DK, Nithipatikom K, Trifan OC, et al. Role of prostaglandin E2-dependent angiogenic switch in cyclooxygenase 2-induced breast cancer progression. Proc Natl Acad Sci U S A 2004;101:591-596
[2] Howe LR, Subbaramaiah K, Brown AM, Dannenberg AJ. Cyclooxygenase-2: a target for the prevention and treatment of breast cancer. Endocr Relat Cancer 2001;8:97-114
[3] Harris RE, Chlebowski RT, Jackson RD, Frid DJ, Ascenseo JL, Anderson G, et al. Breast cancer and nonsteroidal anti-inflammatory drugs: prospective results from the Women's Health Initiative. Cancer Res 2003;63:6096-6101
[4] Schreinemachers DM, Everson RB. Aspirin use and lung, colon, and breast cancer incidence in a prospective study. Epidemiology 1994;5:138-146
[5] Hinz B, Brune K. Cyclooxygenase-2--10 years later. J Pharmacol Exp Ther 2002;300:367-375
[6] Harris RE, Alshafie GA, Abou-Issa H, Seibert K. Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase 2 inhibitor. Cancer Res 2000;60:2101-2103
[7] Basu GD, Pathangey LB, Tinder TL, Lagioia M, Gendler SJ, Mukherjee P. Cyclooxygenase-2 inhibitor induces apoptosis in breast cancer cells in an in vivo model of spontaneous metastatic breast cancer. Mol Cancer Res 2004;2:632-642
[8] Grosch S, Maier TJ, Schiffmann S, Geisslinger G. Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors. J Natl Cancer Inst 2006;98:736-747
[9] Kang HK, Lee E, Pyo H, Lim SJ. Cyclooxygenase-independent down-regulation of multidrug resistance-associated protein-1 expression by celecoxib in human lung cancer cells. Mol Cancer Ther 2005;4:1358-1363
[10] Shishodia S, Koul D, Aggarwal BB. Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis. J Immunol 2004;173:2011-2022
[11] Surh YJ. Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer 2003;3:768-780
[12] Liu B, Han M, Wen JK. Acetylbritannilactone Inhibits Neointimal Hyperplasia after Balloon Injury of Rat Artery by Suppressing Nuclear Factor-{kappa}B Activation. J Pharmacol Exp Ther 2008;324:292-298
[13] Liu YP, Wen JK, Zheng B, Zhang DQ, Han M. Acetylbritannilactone suppresses lipopolysaccharide-induced vascular smooth muscle cell inflammatory response. Eur J Pharmacol 2007;577:28-34
[14] Bai N, Lai CS, He K, Zhou Z, Zhang L, Quan Z, et al. Sesquiterpene lactones from Inula britannica and their cytotoxic and apoptotic effects on human cancer cell lines. J Nat Prod 2006;69:531-535
[15] Rafi MM, Bai NS, Chi Tang H, Rosen RT, White E, Perez D, et al. A sesquiterpenelactone from Inula britannica induces anti-tumor effects dependent on Bcl-2 phosphorylation. Anticancer Res 2005;25:313-318
[16] Park EJ, Kim J. Cytotoxic sesquiterpene lactones from Inula britannica. Planta Med 1998;64:752-754
[17] Han M, Wen JK, Zheng B, Zhang DQ. Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression. Life Sci 2004;75:675-684
[18] Subbaramaiah K, Dannenberg AJ. Cyclooxygenase 2: a molecular target for cancer prevention and treatment. Trends Pharmacol Sci 2003;24:96-102
[19] Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27-55
[20] Han M, Wen JK, Zheng B, Cheng Y, Zhang C. Serum deprivation results in redifferentiation of human umbilical vascular smooth muscle cells. Am J Physiol Cell Physiol 2006;291:C50-58
[21] Inoue H, Yokoyama C, Hara S, Tone Y, Tanabe T. Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. Involvement of both nuclear factor for interleukin-6 expression site and cAMP response element. J Biol Chem 1995;270:24965-24971
[22] Zheng B, Han M, Bernier M, Zhang XH, Meng F, Miao SB, et al. Kruppel-like factor 4 inhibits proliferation by platelet-derived growth factor receptor beta-mediated, not by retinoic acid receptor alpha-mediated, phosphatidylinositol 3-kinase and ERK signaling invascular smooth muscle cells. J Biol Chem 2009;284:22773-22785
[23] Appleby SB, Ristimaki A, Neilson K, Narko K, Hla T. Structure of the human cyclo-oxygenase-2 gene. Biochem J 1994;302 ( Pt 3):723-727
[24] Chang YJ, Wu MS, Lin JT, Chen CC. Helicobacter pylori-Induced invasion and angiogenesis of gastric cells is mediated by cyclooxygenase-2 induction through TLR2/TLR9 and promoter regulation. J Immunol 2005;175:8242-8252
[25] Degner SC, Kemp MQ, Bowden GT, Romagnolo DF. Conjugated linoleic acid attenuates cyclooxygenase-2 transcriptional activity via an anti-AP-1 mechanism in MCF-7 breast cancer cells. J Nutr 2006;136:421-427
[26] Zhao X, Goswami M, Pokhriyal N, Ma H, Du H, Yao J, et al. Cyclooxygenase-2 expression during immortalization and breast cancer progression. Cancer Res 2008;68:467-475
[27] Eckert LB, Repasky GA, Ulku AS, McFall A, Zhou H, Sartor CI, et al. Involvement of Ras activation in human breast cancer cell signaling, invasion, and anoikis. Cancer Res 2004;64:4585-4592
[28] Liu XH, Rose DP. Differential expression and regulation of cyclooxygenase-1 and -2 in two human breast cancer cell lines. Cancer Res 1996;56:5125-5127
[29] Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol 1997;9:240-246
[30] Tang Q, Chen W, Gonzales MS, Finch J, Inoue H, Bowden GT. Role of cyclic AMP responsive element in the UVB induction of cyclooxygenase-2 transcription in human keratinocytes. Oncogene 2001;20:5164-5172
[31] Kim EH, Na HK, Kim DH, Park SA, Kim HN, Song NY, et al. 15-Deoxy-Delta12,14-prostaglandin J2 induces COX-2 expression through Akt-driven AP-1 activation in human breast cancer cells: a potential role of ROS. Carcinogenesis 2008;29:688-695
[32] Falandry C, Canney PA, Freyer G, Dirix LY. Role of combination therapy with aromatase and cyclooxygenase-2 inhibitors in patients with metastatic breast cancer. Ann Oncol 2009;20:615-620
[33] Canney PA, Machin MA, Curto J. A feasibility study of the efficacy and tolerability of the combination of Exemestane with the COX-2 inhibitor Celecoxib in post-menopausalpatients with advanced breast cancer. Eur J Cancer 2006;42:2751-2756
[34] Mustafa A, Kruger WD. Suppression of tumor formation by a cyclooxygenase-2 inhibitor and a peroxisome proliferator-activated receptor gamma agonist in an in vivo mouse model of spontaneous breast cancer. Clin Cancer Res 2008;14:4935-4942
[35] Pockaj BA, Basu GD, Pathangey LB, Gray RJ, Hernandez JL, Gendler SJ, et al. Reduced T-cell and dendritic cell function is related to cyclooxygenase-2 overexpression and prostaglandin E2 secretion in patients with breast cancer. Ann Surg Oncol 2004;11:328-339
[36] Shao J, Sheng H, Inoue H, Morrow JD, DuBois RN. Regulation of constitutive cyclooxygenase-2 expression in colon carcinoma cells. J Biol Chem 2000;275:33951-33956
[37] Wu KK, Liou JY, Cieslik K. Transcriptional Control of COX-2 via C/EBPbeta. Arterioscler Thromb Vasc Biol 2005;25:679-685
[38] Schroer K, Zhu Y, Saunders MA, Deng WG, Xu XM, Meyer-Kirchrath J, et al. Obligatory role of cyclic adenosine monophosphate response element in cyclooxygenase-2 promoter induction and feedback regulation by inflammatory mediators. Circulation 2002;105:2760-2765
[39] Kang YJ, Wingerd BA, Arakawa T, Smith WL. Cyclooxygenase-2 gene transcription in a macrophage model of inflammation. J Immunol 2006;177:8111-8122
[40] Tang HY, Shih A, Cao HJ, Davis FB, Davis PJ, Lin HY. Resveratrol-induced cyclooxygenase-2 facilitates p53-dependent apoptosis in human breast cancer cells. Mol Cancer Ther 2006;5:2034-2042
[41] Han S, Inoue H, Flowers LC, Sidell N. Control of COX-2 gene expression through peroxisome proliferator-activated receptor gamma in human cervical cancer cells. Clin Cancer Res 2003;9:4627-4635
[42] Chun KS, Kim SH, Song YS, Surh YJ. Celecoxib inhibits phorbol ester-induced expression of COX-2 and activation of AP-1 and p38 MAP kinase in mouse skin. Carcinogenesis 2004;25:713-722
[43] Zhang J, Bui TN, Xiang J, Lin A. Cyclic AMP inhibits p38 activation via CREB-induced dynein light chain. Mol Cell Biol 2006;26:1223-1234
[44] Hsu AL, Ching TT, Wang DS, Song X, Rangnekar VM, Chen CS. Thecyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem 2000;275:11397-11403
[45] Tegeder I, Pfeilschifter J, Geisslinger G. Cyclooxygenase-independent actions of cyclooxygenase inhibitors. FASEB J 2001;15:2057-2072
[46] Maier TJ, Janssen A, Schmidt R, Geisslinger G, Grosch S. Targeting the beta-catenin/APC pathway: a novel mechanism to explain the cyclooxygenase-2-independent anticarcinogenic effects of celecoxib in human colon carcinoma cells. FASEB J 2005;19:1353-1355
[1] Mellstedt H. Cancer initiatives in developing countries. Ann Oncol 2006;17 Suppl 8:viii24-viii31
[2] Althuis MD, Dozier JM, Anderson WF, Devesa SS, Brinton LA. Global trends in breast cancer incidence and mortality 1973-1997. Int J Epidemiol 2005;34:405-412
[3] Wu CC, Chan ML, Chen WY, Tsai CY, Chang FR, Wu YC. Pristimerin induces caspase-dependent apoptosis in MDA-MB-231 cells via direct effects on mitochondria. Mol Cancer Ther 2005;4:1277-1285
[4] Garcia-Carbonero R, Supko JG. Current perspectives on the clinical experience, pharmacology, and continued development of the camptothecins. Clin Cancer Res 2002;8:641-661
[5] Rowinsky EK, Donehower RC. Paclitaxel (taxol). N Engl J Med 1995;332:1004-1014
[6] Zhu JY, Lavrik IN, Mahlknecht U, Giaisi M, Proksch P, Krammer PH, et al. The traditional Chinese herbal compound rocaglamide preferentially induces apoptosis in leukemia cells by modulation of mitogen-activated protein kinase activities. Int J Cancer 2007;121:1839-1846
[7] Huang M, Gao H, Chen Y, Zhu H, Cai Y, Zhang X, et al. Chimmitecan, a novel 9-substituted camptothecin, with improved anticancer pharmacologic profiles in vitro and in vivo. Clin Cancer Res 2007;13:1298-1307
[8] Tamvakopoulos C, Dimas K, Sofianos ZD, Hatziantoniou S, Han Z, Liu ZL, et al. Metabolism and anticancer activity of the curcumin analogue, dimethoxycurcumin. Clin Cancer Res 2007;13:1269-1277
[9] Liu J, Zhou W, Li SS, Sun Z, Lin B, Lang YY, et al. Modulation of orphan nuclear receptor Nur77-mediated apoptotic pathway by acetylshikonin and analogues. Cancer Res 2008;68:8871-8880
[10] Wang Z, Qiu J, Guo TB, Liu A, Wang Y, Li Y, et al. Anti-inflammatory properties and regulatory mechanism of a novel derivative of artemisinin in experimental autoimmune encephalomyelitis. J Immunol 2007;179:5958-5965
[11] Zhang M, Huang J, Xie X, Holman CD. Dietary intakes of mushrooms and green tea combine to reduce the risk of breast cancer in Chinese women. Int J Cancer2009;124:1404-1408
[12] Razis ED, Fountzilas G. Paclitaxel: epirubicin in metastatic breast cancer--a review. Ann Oncol 2001;12:593-598
[13] Hainsworth JD, Greco FA. Etoposide: twenty years later. Ann Oncol 1995;6:325-341
[14] Jordan MA, Thrower D, Wilson L. Mechanism of inhibition of cell proliferation by Vinca alkaloids. Cancer Res 1991;51:2212-2222
[15] Liu B, Han M, Wen JK. Acetylbritannilactone Inhibits Neointimal Hyperplasia after Balloon Injury of Rat Artery by Suppressing Nuclear Factor-{kappa}B Activation. J Pharmacol Exp Ther 2008;324:292-298
[16] Liu YP, Wen JK, Zheng B, Zhang DQ, Han M. Acetylbritannilactone suppresses lipopolysaccharide-induced vascular smooth muscle cell inflammatory response. Eur J Pharmacol 2007;577:28-34
[17] Bai N, Lai CS, He K, Zhou Z, Zhang L, Quan Z, et al. Sesquiterpene lactones from Inula britannica and their cytotoxic and apoptotic effects on human cancer cell lines. J Nat Prod 2006;69:531-535
[18] Rafi MM, Bai NS, Chi Tang H, Rosen RT, White E, Perez D, et al. A sesquiterpenelactone from Inula britannica induces anti-tumor effects dependent on Bcl-2 phosphorylation. Anticancer Res 2005;25:313-318
[19] Park EJ, Kim J. Cytotoxic sesquiterpene lactones from Inula britannica. Planta Med 1998;64:752-754
[20] Easty GC, Easty DM, Monaghan P, Ormerod MG, Neville AM. Preparation and identification of human breast epithelial cells in culture. Int J Cancer 1980;26:577-584
[21] Edwards PA, Brooks IM, Bunnage HJ, Foster AV, Ellison ML, O'Hare MJ. Clonal analysis of expression of epithelial antigens in cultures of normal human breast. J Cell Sci 1986;80:91-101
[22] Adhami VM, Aziz MH, Mukhtar H, Ahmad N. Activation of prodeath Bcl-2 family proteins and mitochondrial apoptosis pathway by sanguinarine in immortalized human HaCaT keratinocytes. Clin Cancer Res 2003;9:3176-3182
[23] Han M, Wen JK, Zheng B, Cheng Y, Zhang C. Serum deprivation results in redifferentiation of human umbilical vascular smooth muscle cells. Am J Physiol Cell Physiol 2006;291:C50-58
[24] Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis 2000;21:485-495
[25] Jiang C, Wang Z, Ganther H, Lu J. Caspases as key executors of methyl selenium-induced apoptosis (anoikis) of DU-145 prostate cancer cells. Cancer Res 2001;61:3062-3070
[26] Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 1993;53:3976-3985
[27] Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol 2007;35:495-516
[28] Willis S, Day CL, Hinds MG, Huang DC. The Bcl-2-regulated apoptotic pathway. J Cell Sci 2003;116:4053-4056
[29] Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2:647-656
[30] Wada T, Penninger JM. Mitogen-activated protein kinases in apoptosis regulation. Oncogene 2004;23:2838-2849
[31] Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001;81:807-869
[32] Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995;270:1326-1331
[33] Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998;281:1322-1326
[34] Tafani M, Schneider TG, Pastorino JG, Farber JL. Cytochrome c-dependent activation of caspase-3 by tumor necrosis factor requires induction of the mitochondrial permeability transition. Am J Pathol 2000;156:2111-2121
[35] Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 1997;139:1281-1292
[36] Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2001;15:2922-2933
[37] Hunot S, Flavell RA. Apoptosis. Death of a monopoly? Science 2001;292:865-866
[38] Nicholson DW, Thornberry NA. Apoptosis. Life and death decisions. Science 2003;299:214-215
[39] Janicke RU, Sprengart ML, Wati MR, Porter AG. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 1998;273:9357-9360
[40] Piwocka K, Bielak-Mijewska A, Sikora E. Curcumin induces caspase-3-independent apoptosis in human multidrug-resistant cells. Ann N Y Acad Sci 2002;973:250-254
[41] Heerdt BG, Houston MA, Anthony GM, Augenlicht LH. Initiation of growth arrest and apoptosis of MCF-7 mammary carcinoma cells by tributyrin, a triglyceride analogue of the short-chain fatty acid butyrate, is associated with mitochondrial activity. Cancer Res 1999;59:1584-1591
[42] Nagata S. Fas ligand-induced apoptosis. Annu Rev Genet 1999;33:29-55
[43] Liu B, Han M, Wen JK, Wang L. Livin/ML-IAP as a new target for cancer treatment. Cancer Lett 2007;250:168-176
[44] Wang L, Zhang Q, Liu B, Han M, Shan B. Challenge and promise: roles for Livin in progression and therapy of cancer. Mol Cancer Ther 2008;7:3661-3669
[45] Woo JH, Kim YH, Choi YJ, Kim DG, Lee KS, Bae JH, et al. Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species, down-regulation of Bcl-XL and IAP, the release of cytochrome c and inhibition of Akt. Carcinogenesis 2003;24:1199-1208
[46] Qanungo S, Das M, Haldar S, Basu A. Epigallocatechin-3-gallate induces mitochondrial membrane depolarization and caspase-dependent apoptosis in pancreatic cancer cells. Carcinogenesis 2005;26:958-967
[47] Park C, Jin CY, Kim GY, Choi IW, Kwon TK, Choi BT, et al. Induction of apoptosis by esculetin in human leukemia U937 cells through activation of JNK and ERK. Toxicol Appl Pharmacol 2008;227:219-228
[48] Tsuiki H, Tnani M, Okamoto I, Kenyon LC, Emlet DR, Holgado-Madruga M, et al. Constitutively active forms of c-Jun NH2-terminal kinase are expressed in primary glial tumors. Cancer Res 2003;63:250-255
[49] Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, et al. Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science 2000;288:870-874
[50] Antonyak MA, Moscatello DK, Wong AJ. Constitutive activation of c-Jun N-terminalkinase by a mutant epidermal growth factor receptor. J Biol Chem 1998;273:2817-2822
[51] Neumann-Haefelin E, Qi W, Finkbeiner E, Walz G, Baumeister R, Hertweck M. SHC-1/p52Shc targets the insulin/IGF-1 and JNK signaling pathways to modulate life span and stress response in C. elegans. Genes Dev 2008;22:2721-2735
[52] Kasper B, Brandt E, Ernst M, Petersen F. Neutrophil adhesion to endothelial cells induced by platelet factor 4 requires sequential activation of Ras, Syk, and JNK MAP kinases. Blood 2006;107:1768-1775
[1] Buday L, Downward J. Roles of cortactin in tumor pathogenesis. Biochim Biophys Acta 2007;1775:263-273
[2] DesMarais V, Ghosh M, Eddy R, Condeelis J. Cofilin takes the lead. J Cell Sci 2005;118:19-26
[3] Kruchten AE, McNiven MA. Dynamin as a mover and pincher during cell migration and invasion. J Cell Sci 2006;119:1683-1690
[4] Lambrechts A, Van Troys M, Ampe C. The actin cytoskeleton in normal and pathological cell motility. Int J Biochem Cell Biol 2004;36:1890-1909
[5] Watanabe T, Noritake J, Kaibuchi K. Regulation of microtubules in cell migration. Trends Cell Biol 2005;15:76-83
[6] Vicente-Manzanares M, Webb DJ, Horwitz AR. Cell migration at a glance. J Cell Sci 2005;118:4917-4919
[7] Gerthoffer WT. Mechanisms of vascular smooth muscle cell migration. Circ Res 2007;100:607-621
[8] Gerthoffer WT. Migration of airway smooth muscle cells. Proc Am Thorac Soc 2008;5:97-105
[9] Deakin NO, Turner CE. Paxillin comes of age. J Cell Sci 2008;121:2435-2444
[10] Palamidessi A, Frittoli E, Garre M, Faretta M, Mione M, Testa I, et al. Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration. Cell 2008;134:135-147
[11] Zech T, Machesky L. Rab5 and rac team up in cell motility. Cell 2008;134:18-20
[12] Papakonstanti EA, Stournaras C. Cell responses regulated by early reorganization of actin cytoskeleton. FEBS Lett 2008;582:2120-2127
[13] Le Clainche C, Carlier MF. Regulation of actin assembly associated with protrusion and adhesion in cell migration. Physiol Rev 2008;88:489-513
[14] Ahmed I, Ponery AS, Nur EKA, Kamal J, Meshel AS, Sheetz MP, et al. Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces. Mol Cell Biochem 2007;301:241-249
[15] Lammermann T, Bader BL, Monkley SJ, Worbs T, Wedlich-Soldner R, Hirsch K, et al.Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 2008;453:51-55
[16] Schmidt A, Brixius K, Bloch W. Endothelial precursor cell migration during vasculogenesis. Circ Res 2007;101:125-136
[17] Daley WP, Peters SB, Larsen M. Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci 2008;121:255-264
[18] Kumar S, Maxwell IZ, Heisterkamp A, Polte TR, Lele TP, Salanga M, et al. Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics. Biophys J 2006;90:3762-3773
[19] Zhou X, Rowe RG, Hiraoka N, George JP, Wirtz D, Mosher DF, et al. Fibronectin fibrillogenesis regulates three-dimensional neovessel formation. Genes Dev 2008;22:1231-1243
[20] Kim HD, Guo TW, Wu AP, Wells A, Gertler FB, Lauffenburger DA. Epidermal growth factor-induced enhancement of glioblastoma cell migration in 3D arises from an intrinsic increase in speed but an extrinsic matrix- and proteolysis-dependent increase in persistence. Mol Biol Cell 2008;19:4249-4259
[21] Wang JH, Lin JS. Cell traction force and measurement methods. Biomech Model Mechanobiol 2007;6:361-371
[22] Zaman MH, Kamm RD, Matsudaira P, Lauffenburger DA. Computational model for cell migration in three-dimensional matrices. Biophys J 2005;89:1389-1397
[23] Thibault MM, Buschmann MD. Migration of bone marrow stromal cells in 3D: 4 color methodology reveals spatially and temporally coordinated events. Cell Motil Cytoskeleton 2006;63:725-740
[24] Schindler M, Nur EKA, Ahmed I, Kamal J, Liu HY, Amor N, et al. Living in three dimensions: 3D nanostructured environments for cell culture and regenerative medicine. Cell Biochem Biophys 2006;45:215-227
[25] Seasholtz TM, Majumdar M, Brown JH. Rho as a mediator of G protein-coupled receptor signaling. Mol Pharmacol 1999;55:949-956
[26] Tzima E. Role of small GTPases in endothelial cytoskeletal dynamics and the shear stress response. Circ Res 2006;98:176-185
[27] Hall A. Rho GTPases and the actin cytoskeleton. Science 1998;279:509-514
[28] Burridge K, Wennerberg K. Rho and Rac take center stage. Cell 2004;116:167-179
[29] Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992;70:401-410
[30] Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992;70:389-399
[31] Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995;81:53-62
[32] Huang F, Chotiner JK, Steward O. Actin polymerization and ERK phosphorylation are required for Arc/Arg3.1 mRNA targeting to activated synaptic sites on dendrites. J Neurosci 2007;27:9054-9067
[33] Yip SC, El-Sibai M, Coniglio SJ, Mouneimne G, Eddy RJ, Drees BE, et al. The distinct roles of Ras and Rac in PI 3-kinase-dependent protrusion during EGF-stimulated cell migration. J Cell Sci 2007;120:3138-3146
[34] El-Sibai M, Nalbant P, Pang H, Flinn RJ, Sarmiento C, Macaluso F, et al. Cdc42 is required for EGF-stimulated protrusion and motility in MTLn3 carcinoma cells. J Cell Sci 2007;120:3465-3474
[35] Ridley AJ. Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol 2006;16:522-529
[36] Carlier MF, Pantaloni D. Control of actin assembly dynamics in cell motility. J Biol Chem 2007;282:23005-23009
[37] Pruyne D, Evangelista M, Yang C, Bi E, Zigmond S, Bretscher A, et al. Role of formins in actin assembly: nucleation and barbed-end association. Science 2002;297:612-615
[38] Chang F, Peter M. Cell biology. Formins set the record straight. Science 2002;297:531-532
[39] Kovar DR, Harris ES, Mahaffy R, Higgs HN, Pollard TD. Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell 2006;124:423-435
[40] Anton IM, Jones GE, Wandosell F, Geha R, Ramesh N. WASP-interacting protein (WIP): working in polymerisation and much more. Trends Cell Biol 2007;17:555-562
[41] Anton IM, Jones GE. WIP: a multifunctional protein involved in actin cytoskeletonregulation. Eur J Cell Biol 2006;85:295-304
[42] Paavilainen VO, Oksanen E, Goldman A, Lappalainen P. Structure of the actin-depolymerizing factor homology domain in complex with actin. J Cell Biol 2008;182:51-59
[43] Carlier MF, Laurent V, Santolini J, Melki R, Didry D, Xia GX, et al. Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility. J Cell Biol 1997;136:1307-1322
[44] van Rheenen J, Song X, van Roosmalen W, Cammer M, Chen X, Desmarais V, et al. EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells. J Cell Biol 2007;179:1247-1259
[45] Sidani M, Wessels D, Mouneimne G, Ghosh M, Goswami S, Sarmiento C, et al. Cofilin determines the migration behavior and turning frequency of metastatic cancer cells. J Cell Biol 2007;179:777-791
[46] Conti MA, Adelstein RS. Nonmuscle myosin II moves in new directions. J Cell Sci 2008;121:11-18
[47] Wu MH. Endothelial focal adhesions and barrier function. J Physiol 2005;569:359-366
[48] Broussard JA, Webb DJ, Kaverina I. Asymmetric focal adhesion disassembly in motile cells. Curr Opin Cell Biol 2008;20:85-90
[49] Turner CE. Paxillin interactions. J Cell Sci 2000;113 Pt 23:4139-4140
[50] Su WH, Chen HI, Jen CJ. Polymorphonuclear leukocyte transverse migration induces rapid alterations in endothelial focal contacts. J Leukoc Biol 2007;82:542-550
[51] Galbraith CG, Yamada KM, Galbraith JA. Polymerizing actin fibers position integrins primed to probe for adhesion sites. Science 2007;315:992-995
[52] Gupton SL, Eisenmann K, Alberts AS, Waterman-Storer CM. mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration. J Cell Sci 2007;120:3475-3487
[53] Chuang YY, Valster A, Coniglio SJ, Backer JM, Symons M. The atypical Rho family GTPase Wrch-1 regulates focal adhesion formation and cell migration. J Cell Sci 2007;120:1927-1934
[54] Lyle KS, Raaijmakers JH, Bruinsma W, Bos JL, de Rooij J. cAMP-induced Epac-Rap activation inhibits epithelial cell migration by modulating focal adhesion and leading edgedynamics. Cell Signal 2008;20:1104-1116
[55] Ziegler WH, Liddington RC, Critchley DR. The structure and regulation of vinculin. Trends Cell Biol 2006;16:453-460
[56] Guan JL. Cell biology. Integrins, rafts, Rac, and Rho. Science 2004;303:773-774
[57] Palazzo AF, Eng CH, Schlaepfer DD, Marcantonio EE, Gundersen GG. Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling. Science 2004;303:836-839
[58] Nakamura J, Shigematsu S, Yamauchi K, Takeda T, Yamazaki M, Kakizawa T, et al. Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens. Biochem Biophys Res Commun 2008;374:699-703
[59] Wei JF, Wei L, Zhou X, Lu ZY, Francis K, Hu XY, et al. Formation of Kv2.1-FAK complex as a mechanism of FAK activation, cell polarization and enhanced motility. J Cell Physiol 2008;217:544-557
[60] Kang SS, Kook JH, Hwang S, Park SH, Nam SC, Kim JK. Inhibition of matrix metalloproteinase-9 attenuated neural progenitor cell migration after photothrombotic ischemia. Brain Res 2008;1228:20-26
[61] Gong Y, Hart E, Shchurin A, Hoover-Plow J. Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice. J Clin Invest 2008;118:3012-3024
[62] Takino T, Saeki H, Miyamori H, Kudo T, Sato H. Inhibition of membrane-type 1 matrix metalloproteinase at cell-matrix adhesions. Cancer Res 2007;67:11621-11629
[63] dos Remedios CG, Chhabra D, Kekic M, Dedova IV, Tsubakihara M, Berry DA, et al. Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol Rev 2003;83:433-473
[64] Bellion A, Baudoin JP, Alvarez C, Bornens M, Metin C. Nucleokinesis in tangentially migrating neurons comprises two alternating phases: forward migration of the Golgi/centrosome associated with centrosome splitting and myosin contraction at the rear. J Neurosci 2005;25:5691-5699
[65] Komatsu S, Ikebe M. The phosphorylation of myosin II at the Ser1 and Ser2 is critical for normal platelet-derived growth factor induced reorganization of myosin filaments. Mol Biol Cell 2007;18:5081-5090
[66] Clark K, Langeslag M, Figdor CG, van Leeuwen FN. Myosin II andmechanotransduction: a balancing act. Trends Cell Biol 2007;17:178-186
[67] Buss F, Kendrick-Jones J. How are the cellular functions of myosin VI regulated within the cell? Biochem Biophys Res Commun 2008;369:165-175
[68] Ezratty EJ, Partridge MA, Gundersen GG. Microtubule-induced focal adhesion disassembly is mediated by dynamin and focal adhesion kinase. Nat Cell Biol 2005;7:581-590
[69] Ballestrem C, Wehrle-Haller B, Hinz B, Imhof BA. Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration. Mol Biol Cell 2000;11:2999-3012
[70] Krendel M, Zenke FT, Bokoch GM. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton. Nat Cell Biol 2002;4:294-301
[71] Basu R, Chang F. Shaping the actin cytoskeleton using microtubule tips. Curr Opin Cell Biol 2007;19:88-94
[72] Palazzo AF, Joseph HL, Chen YJ, Dujardin DL, Alberts AS, Pfister KK, et al. Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Curr Biol 2001;11:1536-1541
[73] Gomes ER, Jani S, Gundersen GG. Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell 2005;121:451-463
[74] Nomachi A, Nishita M, Inaba D, Enomoto M, Hamasaki M, Minami Y. Receptor tyrosine kinase Ror2 mediates Wnt5a-induced polarized cell migration by activating c-Jun N-terminal kinase via actin-binding protein filamin A. J Biol Chem 2008;283:27973-27981
[75] Rivero F, Koppel B, Peracino B, Bozzaro S, Siegert F, Weijer CJ, et al. The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development. J Cell Sci 1996;109 ( Pt 11):2679-2691
[76] Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, et al. Cell migration: integrating signals from front to back. Science 2003;302:1704-1709
[77] Faux CH, Parnavelas JG. The role of intracellular calcium and RhoA in neuronal migration. Sci STKE 2007;2007:pe62
[78] Sobieszek A. Calmodulin-dependent autophosphorylation of smooth muscle myosinlight chain kinase: intermolecular reaction mechanism via dimerization of the kinase and potentiation of the catalytic activity following activation. Biochemistry 1995;34:11855-11863
[79] San Martin A, Lee MY, Williams HC, Mizuno K, Lassegue B, Griendling KK. Dual regulation of cofilin activity by LIM kinase and Slingshot-1L phosphatase controls platelet-derived growth factor-induced migration of human aortic smooth muscle cells. Circ Res 2008;102:432-438
[80] Theisen CS, Wahl JK, 3rd, Johnson KR, Wheelock MJ. NHERF links the N-cadherin/catenin complex to the platelet-derived growth factor receptor to modulate the actin cytoskeleton and regulate cell motility. Mol Biol Cell 2007;18:1220-1232
[81] Sossey-Alaoui K, Li X, Ranalli TA, Cowell JK. WAVE3-mediated cell migration and lamellipodia formation are regulated downstream of phosphatidylinositol 3-kinase. J Biol Chem 2005;280:21748-21755
[82] Wang C, Navab R, Iakovlev V, Leng Y, Zhang J, Tsao MS, et al. Abelson interactor protein-1 positively regulates breast cancer cell proliferation, migration, and invasion. Mol Cancer Res 2007;5:1031-1039
[83] Enomoto A, Murakami H, Asai N, Morone N, Watanabe T, Kawai K, et al. Akt/PKB regulates actin organization and cell motility via Girdin/APE. Dev Cell 2005;9:389-402
[84] Enomoto A, Ping J, Takahashi M. Girdin, a novel actin-binding protein, and its family of proteins possess versatile functions in the Akt and Wnt signaling pathways. Ann N Y Acad Sci 2006;1086:169-184
[85] Ghosh P, Garcia-Marcos M, Bornheimer SJ, Farquhar MG. Activation of Galphai3 triggers cell migration via regulation of GIV. J Cell Biol 2008;182:381-393
[86] Kitamura T, Asai N, Enomoto A, Maeda K, Kato T, Ishida M, et al. Regulation of VEGF-mediated angiogenesis by the Akt/PKB substrate Girdin. Nat Cell Biol 2008;10:329-337
[87] Jiang P, Enomoto A, Jijiwa M, Kato T, Hasegawa T, Ishida M, et al. An actin-binding protein Girdin regulates the motility of breast cancer cells. Cancer Res 2008;68:1310-1318
[88] Huang C, Jacobson K, Schaller MD. MAP kinases and cell migration. J Cell Sci 2004;117:4619-4628
[89] Pullikuth AK, Catling AD. Scaffold mediated regulation of MAPK signaling andcytoskeletal dynamics: a perspective. Cell Signal 2007;19:1621-1632
[90] Han MY, Kosako H, Watanabe T, Hattori S. Extracellular signal-regulated kinase/mitogen-activated protein kinase regulates actin organization and cell motility by phosphorylating the actin cross-linking protein EPLIN. Mol Cell Biol 2007;27:8190-8204
[91] Szczur K, Xu H, Atkinson S, Zheng Y, Filippi MD. Rho GTPase CDC42 regulates directionality and random movement via distinct MAPK pathways in neutrophils. Blood 2006;108:4205-4213
[92] Jog NR, Jala VR, Ward RA, Rane MJ, Haribabu B, McLeish KR. Heat shock protein 27 regulates neutrophil chemotaxis and exocytosis through two independent mechanisms. J Immunol 2007;178:2421-2428
[93] Chan A, Akhtar M, Brenner M, Zheng Y, Gulko PS, Symons M. The GTPase Rac regulates the proliferation and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients. Mol Med 2007;13:297-304
[94] Feldner JC, Brandt BH. Cancer cell motility--on the road from c-erbB-2 receptor steered signaling to actin reorganization. Exp Cell Res 2002;272:93-108
[95] Lorenowicz MJ, Fernandez-Borja M, Hordijk PL. cAMP signaling in leukocyte transendothelial migration. Arterioscler Thromb Vasc Biol 2007;27:1014-1022
[96]Ichiki T. Role of cAMP response element binding protein in cardiovascular remodeling: good, bad, or both? Arterioscler Thromb Vasc Biol 2006;26:449-455
[97] Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell 1996;84:359-369
[98] Xiao F, Wang XF, Li JM, Xi ZQ, Lu Y, Wang L, et al. Overexpression of N-WASP in the brain of human epilepsy. Brain Res 2008;1233:168-175
[99] Liu L, Chen L, Chung J, Huang S. Rapamycin inhibits F-actin reorganization and phosphorylation of focal adhesion proteins. Oncogene 2008;27:4998-5010
[100] Mendoza-Naranjo A, Gonzalez-Billault C, Maccioni RB. Abeta1-42 stimulates actin polymerization in hippocampal neurons through Rac1 and Cdc42 Rho GTPases. J Cell Sci 2007;120:279-288