【作者】 刘彬；

【导师】 温进坤； 韩梅；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2010， 博士

【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。更多还原

【Abstract】 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 cellsNeointimal 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 VSMCsGrowth-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 arrestTo 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 VSMCsTo 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 migrationMigration 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 ERK1/2To 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 vivoFurthermore, 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 expressionRecently, 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 growthThe 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 apoptosisTo 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 activityTo 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 transcriptionTo 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 signalingThe 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 activationIn 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 linesThe 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 cycleBecause 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 cellsWe 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 cellsThe 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 cellsActivation 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 xenograftsBecause 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.CONCLUSIONS1 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.更多还原