Involvement of annexin A8 in the properties of pancreatic cancer
TSPAN8参与小鼠非酒精性脂肪性肝病的脂质代谢
非酒精性脂肪性肝病（NAFLD ）是与脂代谢异常有关的世界范围内最常见的慢性肝病［1］。
据估计全球NAFLD 患病率约为25%，近几十年来其患病率日益增长［2,3］。
NAFLD 疾病谱广泛，从轻度脂肪变性（即肝细胞内的脂滴沉积）到以脂肪变性、炎症和肝细胞气球样变性为特征的非酒精性脂肪性肝炎（NASH ）［4］。
单纯性肝脂肪变性是良性和可逆的；然而，NASH 是一种潜在的严重疾病，可进一步发展为肝硬化和肝细胞癌（HCC ）［5,6］。
目前该病的发病机制还未完全阐明，多种理论的提出如：二次打击学说、多次打击学说、脂质异位沉积、肠道菌群失调等都只是冰山一角［7］。
尽管其发病机制复杂，但NAFLD/NASH 的一个不变特征是它总是在肝脂肪变性的背景下发展，即肝细胞中异常脂滴的积累。
当各种细胞内和细胞外应激事件与脂肪变性相结合时，肝细胞会经历应激/损伤反应，最终导致细胞损伤和死亡，这是NAFLD 的关键特征［8］。
因此，探索脂质代谢的相关机制对于了解NAFLD 的病理生理学至关重要。
四跨膜蛋白8（TSPAN8）是一种具有四次跨膜结构的小相对分子质量的膜蛋白，其通过与自身和其他各种细胞信号分子相互作用形成TSPAN8介导的蛋白质复合物。
这些蛋白质复合物有助于构建富含四跨膜蛋白TSPAN8is involved in lipid metabolism in non-alcoholic fatty liver disease in miceZHANG Jia,XUE Wei,ZHANG Shujun,ZHU Yali,YANG Cheng,GAO Yue,SHI Lingfeng,HUANG WenxiangDepartment of Infectious Diseases,First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China摘要：目的探讨四跨膜蛋白8（TSPAN8）在非酒精性脂肪性肝病（NAFLD ）发生发展中的变化及其在脂质代谢中的作用。
下调Pannexin2通道能增强顺铂诱导睾丸癌(I-10)细胞凋亡
睾丸癌是年轻男性最常见的恶性实体肿瘤，其中生殖细胞肿瘤占所有睾丸肿瘤的大多数［1］。
顺铂是治疗不同类型实体肿瘤的一线化疗药物［2］，与其他药物联合使用对睾丸癌具有很高的治愈率。
但治疗时产生的耐药性和肝肾毒性等不良反应，限制了顺铂等铂类药物在临床上的应用。
因此，寻找特异性较高，对铂类化疗药物具有特殊敏感性的新靶点对临床上睾丸癌的治疗策略具有重要的指导意义。
泛连接蛋白（Panxs ）是Panchin 等在无脊椎动物体内找到的一类缝隙连接蛋白［3］，包括3种Panx 亚型，主要包括：Panx-1、Panx-2和Panx-3［4-5］。
其结构由6个Panx 蛋白形成一个六聚体半通道，但是Panx 蛋白糖基Down-regulation of pannexin 2channel enhances cisplatin-induced apoptosis in testicular cancer I-10cellsYAO Yanxue,DONG Shuying,ZHU Chenlu,HU Miao,DU Baolong,TONG Xuhui School of Pharmacy,Bengbu Medical College,Bengbu 233030,China摘要：目的探讨Pannexin2（Panx-2）蛋白在睾丸癌细胞中的表达水平以及干扰Panx-2表达对顺铂诱导睾丸癌（I-10）细胞凋亡的影响。
方法免疫印迹法Panx-2蛋白在睾丸癌细胞中的表达水平；以睾丸癌I-10细胞株为研究对象，实验分为转染试剂对照组（mork 组）、阴性对照质粒组（NC 组）、干扰Panx-2组（shRNA1质粒组和shRNA2质粒组），采用免疫印迹法验证Panx-2的表达；在转染细胞中添加16μmol/L 的顺铂诱导细胞死亡，通过MTT 法分别检测细胞在24、48、72h 的存活率，集落克隆法检测细胞的集落形成能力；在顺铂（16μmol/L ）处理8h 后，采用AnnexinV/PI 双染法检测细胞的早期凋亡；在顺铂（16μmol/L ）作用24h 后，免疫印迹法检测细胞凋亡相关蛋白caspase-3、Bcl-2和Bax 的表达水平。
S100A8-A9在炎症和肿瘤中的应用及作用机制研究进展
S100A8-A9在炎症和肿瘤中的应用及作用机制研究进展中国现代医药杂志2011年9月第13卷第9期MMJC,Sep2011,Vn113,No.9S100A8/A9在炎症和肿瘤中的应用及作用机制研究进展蒋秋林孙晓红$100A8蛋白fCalgranulinA蛋白,MRP8蛋白)与$100A9蛋白(CalgranulinB蛋白,MRP14蛋白)均属于钙结合蛋白S100蛋白家族成员,两者常以钙离子依赖性方式形成异源二聚体S100A8/A9蛋白复合物【1(以下简称SIOOA8/A9),在循环中性粒细胞,单核巨噬细胞中表达,而在正常巨噬细胞和淋巴细胞中无表达,在慢性炎症环境下,两蛋白也表达于上皮中:可参与炎症反应,调节细胞生长分化,增长抑制,诱导细胞凋亡[21等.随着近年来对其研究的不断深入,发现St00A8/A9与多种疾病密切相关,特别是其对于肿瘤性疾病增殖和凋亡的可能作用.使其成为研究热点.1S1o0A8和S1o0A9皆为S100蛋白家族成员S1oo蛋白家族是一个由小分子量蛋白组成的钙结合蛋白家族,共有22名家族成员.SIO0蛋白两端为氨基末端EF一1和羧基末端EF一2手型结构,两端分别包含一个由l4和12个氨基酸残基组成的钙粘环,中间为铰链区,组成4个Or.一螺旋.形成螺旋一环一螺旋(helix—loop—helix)i~结构.SIO0蛋白家族各个成员的结构不同体现在铰链区和羧基末端的氨基酸序列不同,从而具有不同的生物学活性[31.另外,SIO0蛋白对钙离子的亲和力较经典的钙离子感受器(如钙调蛋白)要弱,其KD大约是200～500uMt".$100蛋白家族中多个成员的表达在多种肿瘤形成过程中出现异常.同时该家族中有16名成员染色体定位均位于1号染色体长臂2区1带(1q21区带),该区稳定性差,易发生染色体的缺失,易位,重叠等改变,与肿瘤的发生关系密切;且表皮分化复合体(epidermaldifferentiationeomplex,EDC)也定位在染色体lq21区带.与人类上皮终末分化密切相关.S100蛋白参与信号转导,细胞增殖和移动,细胞周期调整,运输和分化等多种分子生物过程.1.1S100A8,S1OOA9结构特征$100A8蛋白为一个由267个碱基对编码的88个氨基酸残基构成的低分子蛋白(14kDa),S100A9蛋白为相对分子质量较d~(13kDa)的蛋白, 两者均属于$100蛋白家族成员.具有特征性SIO0蛋白家族4一螺旋环形和EF手型结构.S100A8和S100A9有强烈的形成异源二聚体的趋势,而异源二聚体的形成导致铰链区3一螺旋和2一钙粘环结构改变.钙粘环疏水表面暴露,结合各种靶蛋白,可传递Ca信号以及调节胞质中的Caz+浓度[41.在机体中发挥着重要的生物学作用.作者单位:421002衡阳.南华大学附属南华医院通讯作者:孙晓红l25?正常生理状态下.SIOOA8/A9存在于上皮细胞,角质化细胞等分化良好的组织中,主要定位于胞浆,也可转移到细胞骨架和质膜上,以利于细胞内钙离子的聚集;另外,在髓系细胞分化过程中也有表达,在粒细胞和单核细胞中高表达,约占中性粒细胞胞质蛋白45%t6j.而炎症病变早期渗出的炎性细胞中也表达S100A8/A9.它们是中性粒细胞化学趋化性和黏附性的强有力的诱导剂.在炎症反应过程中发挥重要作用m.在某些病理条件下.例如:系统性红斑狼疮,牛皮癣,皮肤恶性肿瘤等,上皮细胞也表达Sl00A8/A9tSJ.1.2S100A8,Sl0oA9表达特点S100A8,S100A9在感染性疾病恢复期是低下的.而在感染性疾病和非感染性炎症时显着升高.有文献报道在艾滋病,口腔念珠菌病和分枝杆菌病中存在SIOOA8/A9的高表达q.Kane等【1lI发现同一风湿性关节炎患者的关节腔液标本中S100A8/A9的表达量约是血清标本的10倍.另外,S100A8/A9表达水平在系统性红斑狼疮,系统性硬化病进展期,牛皮癣和慢性肺病中明显升高;在炎症性肠病粪便中明显高于肠易激综合征和健康人,且S100A8/A9复合物浓度与一些特殊的炎症性疾病f囊性纤维化,类风湿关节炎,慢性支气管炎1的活动呈特征性的正相关㈣.最近研究发现.在人类多种原发和浸润性肿瘤中检测到SIOOA8和$100A9的高表达.S1OOA8和S100A9在胃癌,肺腺癌,乳腺癌组织中表达增高㈣.Kimt等发现在大肠腺瘤和早期大肠癌血清中S100A8和S100A9显着表达,且在大肠癌中的肿瘤浸润的免疫细胞中也有高表达.Hermani等[.63发现SIOOA8和S100A9在前列腺上皮内瘤变和前列腺腺癌中的表达明显高于良性的前列腺增生组织.尤其在高分化前列腺腺癌中表达最强.Petersson等分析卵巢肿瘤患者血清和卵巢积液,发现只有在恶性卵巢肿瘤患者的血清中及卵巢积液中能够检测到S100A8/A9.而良性卵巢囊肿患者的血清及卵巢积液中未发现SIOOA8/A9的表达.另外,在某些上皮来源的肿瘤性疾病中,发现S100A8/A9表达下调,例如.在食管鳞状细胞癌和子宫鳞状细胞癌等多种上皮来源的肿瘤中检测到表达S100A8/A9明显下调;Roesch等I删采用cDNA芯片及基质辅助激光解吸电离飞行时间质谱的方法均检测到S100A8,A9在头颈部鳞状细胞癌中表达明显下调.2S100A8/A9的活性,应用及作用机制2.1S100A8/A9的胞内外活性在细胞内环境中,作为Ca感受器的S100蛋白家族不断改变自身的构象以适应Ca2+内流并且通过粘附胞内其它蛋白调整Ca"信号.Kerkhoff等研究表明.胞内S100A8/A9与烟酰胺腺嘌吟二核苷酸磷酸126?中国现代医药杂志2011年9月第13卷第9期MMJC,Sep2011,V ol13,No.9 fNADPH)氧化酶复合物相互作用.藉此提高NADPH氧化酶的活性,NADPH氧化过程异常可以产生细胞内活性氧(ROS1,导致细胞凋亡.当S100A8/A9与胞质氧化酶活化因子交互作用时将花生四烯酸转移至膜结合gp91phox中,而花生四烯酸与gp91phox的结合是NADPH氧化酶活化的必需因素.S100A8/A9一旦从活化的吞噬细胞中释放人细胞外,就通过掠夺病原微生物生长所必需的微量金属离子,如Ca",Zn和Mn,而发挥抗微生物作用l2ll.S100A8/A9还显示出细胞因子类似物的作用,包括活化糖基化终产物受体(RAGE),促进白细胞在炎症区的聚集和将花生四烯酸输送到靶细胞.研究发现胞外SIOOA8/A9表现出对多种细胞f包括肿瘤细胞1有抑制生长和诱导凋亡的作用[231,例如:人胃癌细胞系,MCF一7乳腺癌细胞系和KELLY人神经母细胞瘤等.2.2SIOOA8/A9的应用及作用机制在炎症性疾病中.SIOOAS/A9作为嗜中性粒细胞的一个炎性因子.对炎症灶周围的白细胞具有很强的趋化作用.在急性炎症多形核白细胞中,胞膜联合型S100A8/A9优先表达,且这些细胞释放大量TNF—ot和IL一1.表明膜表面表达的SIOOA8/A9可以修复有宿主防御功能的巨噬细胞_lI.V ogl等[241近来发现在单核细胞迁移过程中,S100A8/A9与细胞膜上微管蛋白有关,并且介导内皮细胞转移.有研究发现它与伤口愈合密切相关,其可能机制就是参与伤口上皮角蛋白细胞骨架重建和f或1对炎症细胞的趋化作用1251.S100A8和S100A9还可诱导HIV一1长末端重复启动子活化,另外,SIOOA9能与喹啉一3一甲酰胺小分子特异性结合,在控制自身免疫性疾病过程中起重要作用,被认为是一个新的用于治疗人类自身免疫性疾病的药物靶标1271.Haigh[2s] 等在对牙周炎治疗前后唾液中蛋白组成的研究中发现,S100A8/A9含量在治疗后显着增加.S100A8/A9用于炎症反应标志物甚至优于CRP和ERS["】.鉴于其在炎症反应中的多种表现,有望成为炎症预测和治疗的新选择.近来研究表明,S100A8/A9还具有细胞毒性,可诱导某些细胞的凋亡以及抑制细胞增殖.Ghavami1291发现SIOOA8/A9通过线粒体路径发挥促凋亡活性:一是快速降低线粒体膜电位;二是通过阻止细胞色素C的释放而抑制线粒体的分裂;三是诱导线粒体中Bax(凋亡活化基因)迁移及二聚体的形成;四是使BCL2家族(凋亡抑制基因)在凋亡和抑制凋亡的平衡中趋向于凋亡:还发现[3o1.S100A8/A9可能通过两条途径导致结肠癌细胞的凋亡.一是通过经典的线粒体一细胞色素C旁路途径;另一个是通过作为Caspase一3稳定剂的2n,S100A8/A9与Zn结合,降低细胞内zn+浓度.激活Caspase一3,导致凋亡.另外,S100A8/A9下调人类宫颈癌CasKi细胞系中MMP一2的表达,并且可通过诱导CasKi细胞凋亡抑制其增殖和侵袭转移_3I】.有研究发现.肿瘤组织中S100蛋白异常表达与肿瘤分期和预后具有相关性.SIOOA9在宫颈癌同步放,化疗高敏感组中的表达强度和表达率高于低敏感组;在肝癌,乳腺癌中的表达与分化程度呈负相关[321.3展望S100A8/A9是免疫细胞产生的有效的炎性因子,也是有力的促凋亡因子,其作用机制十分复杂,虽已取得部分进展,还有待进一步深入研究.上述研究结果表明S100A8/A9在肿瘤性疾病中存在差异表达且功能多样,使其具有广阔的应用前景,有可能成为恶性肿瘤预防,治疗和预后评定的新指标.参考文献1KorndorferIP,BruecknerF,SkerraA,eta1.Thecrystalstructureofthehuman(S100A8/S100A9)2heterotetramer,ealprotectin,illus- trateshoweonformationalchangesofinteractingalpha—helices candeterminespecificassociationoftwoEF—handproteins[J].J MolBiol,2007370(5):8872NackenW,RothJ,SorgC,eta1.S1OOA9,S100A8:Myel0idrepresen- tativesofS100proteinfamilyasprominentplayersininnateim—munity[J].MicroscResTech,2003,60(6):569-5803RavasiT,HsuK,GoyetteJ,eta1.ProbingtheSIO0proteinfamily throughgenomicandfunctionalanalysis叫.Genomics,2004,84(1): 10-224ZimmerDB,CornwallEH,LandarA,eta1.The$100proteinfami—ly:history,function,andexpression.BrainResBull,1995,37(4):4175KongJP,DingF,ZhouCN,eta1.Lossofmyeloid—relatedProteins8 andmyeloid—relatedproteins14expressioninhumanesophageal squamouscellcarcinomacorrelateswithpoordifferentiation[J1. 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生物信息学分析胰腺癌组织关键基因表达及其意义
基金项目：国家自然科学基金资助项目（８１９６０５１６）作者单位：７３００００ 兰州大学第一临床医学院、兰州大学第一医院通讯作者：朱克祥，电子信箱：ｆｌｅｘｚｈｕ６９１０＠ｓｉｎａ．ｃｏｍ生物信息学分析胰腺癌组织关键基因表达及其意义王乾合 赵立然 朱克祥摘 要 目的 利用生物信息学方法对ＧＥＯ数据库进行探索，获取胰腺癌发生、发展的核心基因，分析胰腺癌发生、发展的潜在机制。
方法 使用ＧＥＯｑｕｅｒｙ包从ＧＥＯ数据库获取胰腺癌相关芯片数据（ＧＳＥ６２４５２），利用Ｌｉｍｍａ包进行差异分析，结合ｃｌｕｓｔｒｅＰｒｏｆｉｌｅｒ包对上调基因和下调基因分别进行ＧＯ功能和ＫＥＧＧ通路分析，利用Ｓｔｒｉｎｇ在线网站对差异基因进行蛋白互作网络分析，利用Ｃｙｓｔｏｓｃｏｐｅｓ软件筛选出核心基因，最后借助ＴＣＧＡ数据库和芯片数据（ＧＳＥ２８７３５）对核心基因进行再次验证。
结果利用生物信息学方法共获得２８６个差异基因，其中包含上调基因１８９个，下调基因９７个。
富集分析结果显示上调基因涉及细胞组织黏附、细胞外基质组织构成等功能以及蛋白消化与吸收、ＰＩ３Ｋ－Ａｋｔ等信号通路相关。
下调基因与脂类消化、细胞壁破裂等功能和胰腺分泌等信号通路密切相关。
通过蛋白互作网络筛选出２０个关键基因，利用ＧＥＰＩＡ数据库对关键基因进行生存分析验证，发现３个基因（ＭＭＰ１１、ＬＡＭＢ３、ＰＬＡＵ）与胰腺癌预后明显相关。
结论 通过生物信息学方法最终筛选出３个核心基因，为胰腺癌的诊断和预后提供了新的思路。
关键词 胰腺癌 靶向治疗 ＧＥＯ中图分类号 Ｒ７３ 文献标识码 Ａ ＤＯＩ １０．１１９６９／ｊ．ｉｓｓｎ．１６７３ ５４８Ｘ．２０２１．０５．００９ＥｘｐｒｅｓｓｉｏｎａｎｄＳｉｇｎｉｆｉｃａｎｃｅｏｆＫｅｙＧｅｎｅｓｉｎＰａｎｃｒｅａｔｉｃＣａｎｃｅｒＴｉｓｓｕｅｓ：ＡＢｉｏｉｎｆｏｒｍａｔｉｃｓＡｎａｌｙｓｉｓ． ＷａｎｇＱｉａｎｈｅ，ＺｈａｏＬｉｒａｎ，ＺｈｕＫｅｘ ｉａｎｇ．ＴｈｅＦｉｒｓｔＣｌｉｎｉｃａｌＭｅｄｉｃａｌＣｏｌｌｅｇｅ，ＬａｎｚｈｏｕＵｎｉｖｅｒｓｉｔｙ，ＴｈｅＦｉｒｓｔＨｏｓｐｉｔａｌｏｆＬａｎｚｈｏｕＵｎｉｖｅｒｓｉｔｙ，Ｇａｎｓｕ７３００００，ＣｈｉｎａＡｂｓｔｒａｃｔ Ｏｂｊｅｃｔｉｖｅ Ｇｅｎｅｅｘｐｒｅｓｓｉｏｎｏｍｎｉｂｕｓ（ＧＥＯ）ｄａｔａｂａｓｅｓｗａｓａｎａｌｙｚｅｄｂｙｕｓｉｎｇｂｉｏｉｎｆｏｒｍａｔｉｃｓｔｏｓｃｒｅｅｎｔｈｅｋｅｙｇｅｎｅｓ，ａｎｄｔｏａｎａｌｙｚｅｔｈｅｐｏｔｅｎｔｉａｌｍｅｃｈａｎｉｓｍｓｏｆｐａｎｃｒｅａｔｉｃｃａｎｃｅｒ．Ｍｅｔｈｏｄｓ ＭｉｃｒｏａｒｒａｙｄａｔａｏｆＧＳＥ６２４５２ｗｅｒｅｏｂｔａｉｎｅｄｆｒｏｍＧＥＯｄａｔａｂａｓｅｓｕｓｉｎｇＧＥＯｑｕｅｒｙｐａｃｋａｇｅａｎｄｄｉｆｆｅｒｅｎｔｉａｌｌｙｅｘｐｒｅｓｓｅｄｇｅｎｅｓｗｅｒｅｓｃｒｅｅｎｅｄｕｓｉｎｇＬｉｍｍａｐａｃｋａｇｅ．ＷｉｔｈｔｈｅｃｌｕｓｔｒｅＰｒｏｆｉｌｅｒｐａｃｋａｇｅ，ＧＯｆｕｎｃｔｉｏｎａｎｄＫＥＧＧｐａｔｈｗａｙａｎａｌｙｓｉｓｗｅｒｅｃａｒｒｉｅｄｏｕｔｆｏｒｕｐ－ｒｅｇｕｌａｔｅｄｇｅｎｅｓａｎｄｄｏｗｎ－ｒｅｇｕｌａｔｅｄｇｅｎｅｓｒｅｓｐｅｃｔｉｖｅｌｙ．ＰｒｏｔｅｉｎｉｎｔｅｒａｃｔｉｏｎｎｅｔｗｏｒｋａｎａｌｙｓｉｓｏｆｄｉｆｆｅｒｅｎｔｉａｌｌｙｅｘｐｒｅｓｓｅｄｇｅｎｅｓｗａｓａｎａｌｙｚｅｄｕｓｉｎｇＳｔｒｉｎｇｏｎｌｉｎｅｗｅｂｓｉｔｅｓ，ａｎｄＣｙｓｔｏｓｃｏｐｅｓｓｏｆｔｗａｒｅｗａｓｕｓｅｄｔｏｓｃｒｅｅｎｃｏｒｅｇｅｎｅｓ．Ｆｉｎａｌｌｙ，ｔｈｅｃｏｒｅｇｅｎｅｓｗｅｒｅｖｅｒｉｆｉｅｄｗｉｔｈＴＣＧＡｄａｔａｂａｓｅａｎｄｃｈｉｐｄａｔａ（ＧＳＥ２８７３５）．Ｒｅｓｕｌｔｓ Ａｔｏｔａｌｏｆ２８６ｄｉｆｆｅｒｅｎｔｉａｌｌｙｅｘｐｒｅｓｓｅｄｇｅｎｅｓｗｅｒｅｅｘｔｒａｃｔｅｄｂｙｂｉｏｉｎｆｏｒｍａｔｉｃｓ，ｉｎｃｌｕｄｉｎｇ１８９ｕｐ－ｒｅｇｕｌａｔｅｄｇｅｎｅｓａｎｄ９７ｄｏｗｎ－ｒｅｇｕｌａｔｅｄｇｅｎｅｓ．Ｅｎｒｉｃｈｍｅｎｔａｎａｌｙｓｉｓｒｅｖｅａｌｅｄｔｈａｔｕｐｒｅｇｕｌａｔｅｄｇｅｎｅｓｗｅｒｅｃｌｏｓｅｌｙａｓｓｏｃｉａｔｅｄｗｉｔｈｔｈｅｆｕｎｃｔｉｏｎａｎｄｐａｔｈｗａｙｅｎｒｉｃｈｍｅｎｔｓｕｃｈａｓｃｅｌｌｕｌａｒｔｉｓｓｕｅａｄｈｅｓｉｏｎ，ｅｘｔｒａ ｃｅｌｌｕｌａｒｍａｔｒｉｘｔｉｓｓｕｅｃｏｍｐｏｓｉｔｉｏｎ，ｐｒｏｔｅｉｎｄｉｇｅｓｔｉｏｎａｎｄａｂｓｏｒｐｔｉｏｎ，ａｎｄＰＩ３Ｋ－Ａｋｔｓｉｇｎａｌｉｎｇｐａｔｈｗａｙｓ．Ｄｏｗｎ－ｒｅｇｕｌａｔｅｄｇｅｎｅｓａｒｅｃｌｏｓｅ ｌｙｒｅｌａｔｅｄｔｏｌｉｐｉｄｄｉｇｅｓｔｉｏｎ，ｃｅｌｌｗａｌｌｒｕｐｔｕｒｅａｎｄｐａｎｃｒｅａｔｉｃｓｅｃｒｅｔｉｏｎｓｉｇｎａｌｉｎｇｐａｔｈｗａｙｓ．Ｔｗｅｎｔｙｃｏｒｅｇｅｎｅｓｗｅｒｅｓｃｒｅｅｎｅｄｕｓｉｎｇｔｈｅｐｒｏ ｔｅｉｎｉｎｔｅｒａｃｔｉｏｎｎｅｔｗｏｒｋ，ａｎｄｓｕｒｖｉｖａｌａｎａｌｙｓｉｓｏｆｋｅｙｇｅｎｅｓｗｅｒｅｐｅｒｆｏｒｍｅｄｕｓｉｎｇｔｈｅＧＥＰＩＡｄａｔａｂａｓｅ．Ｗｅｆｏｕｎｄｔｈａｔｔｈｒｅｅｇｅｎｅｓ（ＭＭＰ１１，ＬＡＭＢ３，ＰＬＡＵ）ｗｅｒｅｓｉｇｎｉｆｉｃａｎｔｌｙｃｏｒｒｅｌａｔｅｄｗｉｔｈｔｈｅｐｒｏｇｎｏｓｉｓｏｆｐａｎｃｒｅａｔｉｃｃａｎｃｅｒ．Ｃｏｎｃｌｕｓｉｏｎ Ｔｈｒｅｅｃｏｒｅｇｅｎｅｓｗｅｒｅｓｅ ｌｅｃｔｅｄｂｙｂｉｏｉｎｆｏｒｍａｔｉｃｓｍｅｔｈｏｄ，ｗｈｉｃｈｐｒｏｖｉｄｅｄａｎｅｗｉｄｅａｆｏｒｔｈｅｄｉａｇｎｏｓｉｓａｎｄｐｒｏｇｎｏｓｉｓｏｆｐａｎｃｒｅａｔｉｃｃａｎｃｅｒ．Ｋｅｙｗｏｒｄｓ Ｐａｎｃｒｅａｔｉｃｃａｎｃｅｒ；Ｔａｒｇｅｔｅｄｔｈｅｒａｐｙ；ＧＥＯ 胰腺癌（ｐａｎｃｒｅａｔｉｃｃａｒｃｉｎｏｍａ）是一种恶性程度极高的消化道肿瘤，在恶性肿瘤中是导致癌症相关死亡的第３大原因。
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Enhanced effect of gemcitabine by emodin agaist pancreatic cancer in vivo via Cytochrome C-regulated apoptotic pathwayAbstractBachground:With high toxicity,gemcitabine is currently the best treatment for pancreatic cancer.Agents that can enhance the effects of gemcitabine with no or low toxicity are needed for treating pancreatic cancer. Emodin, a natural anthraquinone derivative, is one such agent that has been shown to induce apoptosis in other tumor cells via downregulating Bcl-2/Bax ratio and promoting release of Cytochrome C (CytC),but with very low toxicity. The aim of this study was to evaluate whether emodin can enhance the effect of gemcitabine on pancreatic cancer in vivo and investigate the possible mechanisms of the enhancement.Methods: In vitro, proliferation suppression of SW1990 pancreatic cancer cells induced by 48 hours of therapy with emodin(40μM/L)、gemcitabine(20μM/）or emodin(40μM/L) combined with gemcitabine(20μM/L）was detected by MTT assay. Furthermore,cell apoptosis after drug therapy was determined by Flow Cytometry（FCM）and caspase 3 activation was detected by western blotting.In vivo，a total of 48 nude mice inoculated with SW1990 cells were randomly assigned in 4 experimental groups. Animals in each group were received intravenous injection of emodin(40mg/kg), gemcitabine(120mg/kg), emodin(40mg/kg) plus gemcitabine(80mg/kg), or 0.9% sodium chloride, respectively. Tumor size during therapy was measured every six days. Cell proliferation was evaluated by immunohistochemical analysis of Ki-67 and apoptotic cell death was determined by TUNEL-staining analysis. The expressions of Bcl-2, Bax, active caspase-3 and Cytochrome C were assessed by immunohistochemistry and western blotting.Results:In vitro,emodin inhibited the proliferation of SW1990 cells and potentiated cell apoptosis induced by gemcitabine, which was evidenced by caspase 3 activation in combination group .In vivo, tumors treated with combination of emodin(40mg/kg) and gemcitabine(80mg/kg) showed significant reductions in volume,Ki-67 proliferation index and Bcl-2/Bax ratio (compared with other groups),which induced increasing release of Cytochrome C from mitochondria to cytoplasm and triggered Caspase 3 activation leading to apoptosis. Conclusion:Our results suggest that emodin improve the antitumor effect of gemcitabine,even at a lower dose of gemcitabine,which could decrease chemotherapeutic toxicity,on transplanted pancreatic cancer through enhancing apoptosis induced by gemcitabine, the mechanism of which might be through downregulating Bcl-2/Bax ratio and promoting release of Cytochrome C from mitochondria into cytoplasm.Abstract(备用版)Gemcitabine is currently the best treatment available for pancreatic cancer，but with high toxicity. Agents that can enhance the effects of gemcitabine with no or low toxicity are needed for thetreatment of pancreatic cancer. Emodin, a natural anthraquinone derivative, is one such agent that has been shown to induce apoptosis in other tumor cells via downregulating Bcl-2/Bax and promoting release of Cytochrome C,but with very low toxicity. The aim of this study was to evaluate whether emodin can enhance the effect of gemcitabine on pancreatic cancer in vitro and in vivo and investigate the possible mechanisms of the enhancement.In vitro,emodin inhibited the proliferation of SW1990 cell line and potentiated the apoptosis induced by gemcitabine, which was evidenced by activation of caspase 3 in combination group .In vivo, tumors from nude mice subcutaneously injected with SW1990 cells and treated with a combination of emodin (40mg/kg) and gemcitabine(80mg/kg) showed significant reductions in volume,Ki-67 proliferation index and expression of Bcl-2/Bax ratio (compared with tumors from mice treated with sodium chloride,emodin alone(40mg/kg),or gemcitabine alone(120mg/kg)),which induced increasing release of Cytochrome C from mitochondria to cytoplasm and triggered Caspase 3 activation leading to apoptosis.Taken together,our results suggest that emodin improved the antitumor effect of gemcitabine,even at a lower dose of gemcitabine which could decrease the toxicity of chemotherapy, on transplanted tumor of SW1990 cell line through enhancing apoptosis induced by gemcitabine, the mechanism of which might be through downregulating Bcl-2/Bax ratio and promoting release of Cytochrome C from mitochondria into cytoplasm.Key words:Emodin;Gemcitabine;Apoptosis;Bcl-2;Bax;Cytochrome C1.IntroductionsPancreatic cancer is a highly malignant tumor in alimentary system with very poor prognosis.1-year survival rate of patients with advanced pancreatic cancer was 8%,and 5-year survival rate of them was 3%(Jemal et al. 2002)，with the median overall survival of only 6 months[2]. Complete resection of the tumor is currently the only curative option but only 10-15% of patients present with localized, potentially resectable disease at the time of diagnosis(Hilbig and Oettle 2010).Previous researches show that gemcitabine,as a DNA nucleotide analogue,has a good effect on many solid tumors including pancreatic cancer,and as a adjuvant drug after curative-intent resection,can prolong disease-free survival to at least 6 months[4].Therefore,gemcitabine was used to treat advanced pancreatic cancer as a standard drug[5; 6].However,the effect of gemcitabine was limited with a low remission rate of about 10%[7; 8],and median overall survival by adjuvant medication with gemcitabine after operation was only 22.1 months[4].Most patient would develop resistance to gemcitabine ,which contains strong toxic side effect and is very expensive.Previous studies discovered many kinds of medicinescan strengthen the therapeutic effect of gemcitabine on pancreatic cancer[9; 10; 11; 12]. Accordingto the above reasons,it is valuable to find a drug which can potentiate the effect of gemcitabine but with low toxic effect and price.Emodin（1,3,8-trihydroxy-6-methylanthraquinone）is a natural anthraquinone derivative isolated from Rheum palmatum L.Previous studies have demonstrated that emodin can inhibit cell growth and induce apoptosis in several types of tumor cells.Emodin,with low toxicity to normal cells,can induce tumor cells to produce a lot of reactive oxygen species(ROS) ,which r esults in downregulation of Bcl-2 gene and upregulation of Bax,leading to release of Cytochrome c frommitochondria,finally resulting in more tumor cells apoptosis[13; 14].In this study,based on in vitro cell culture and transplanted tumor in athymic nu/nu mice,we investigated our hypothesis that emodin could significantly enhance antitumor effect of gemcitabine on pancreatic cancer in vivo via Cytochrome C-regulated apoptotic pathway.Materials and methodsCell lines and animalsThe human pancreatic cancer cell line SW1990 was purchased from American Type Culture Collection.6~7week female athymic BALB/c nu/nu mice were purchased from Shang Hai Cancer Institute for tumor implantation.All animals were maintained in a sterile enviroment and cared for within animal experiment center of Wenzhou Medical College.Drug and agentsEmodin was purchased from Sigma-Aldrich (St. Louise, MO, USA), dissolved in dimethylsulfoxide(DMSO) .The final concentration of DMSO was <0.1%.Gemcitabine was purchased from Ely Lilly,dissolved in 0.9% sodium chloride. Rabbit anti-human anti-Bax,anti-bcl-2 polyclonal antibodies, and anti-Cytochrome C (CytC), anti-active caspase-3,anti-NF-κB (p65) monoclonal antibodies were bought from Abcam.In Situ Cell Death Detection Kit was purchased from Roche, Germany.Cell cultureThe human pancreatic cancer cell lines SW1990 was maintained in RPMI1640 supplemented with 10% fetal bovine serum,100 units/ml penicillin,and 100mg/ml streptomycin in a humidified incubator containing 5% CO2 in air at 37 ℃.Cell growth assayTo assess the viability of cells, cell numbers were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) (Sigma, St. Louis,MO, USA), performed according to the method of Gerlier and Thomasset [22]. Briefly, cells were plated at a density of 5 ×103 cells/well in 96-well microtiter plates. After treatment, 20 ul of MTT solution (5 mg/mL in phosphate-buffered saline (PBS)) was added to each well and the plates were incubated. The supernatant was aspirated and the MTT formazan was dissolved in 150 ul of DMSO. The plates were mixed for 10 min on a gyratory shaker, and absorbance was measured with an ELISA reader (BIO-Tek ELx800, Winooski, VT,USA) at a wavelength of 490 nm. Experiment was repeated thrice.Cell apoptosis assayAfter cells have been exposed to gemcitabine(20 μmol/L)for 48h,emodin(40 μmol/L)for 48h,or their combinations（40 μmol/L emodin coincubat ed with 20 μmol/L gemcitabine）for 48h at six-well plates,SW1990 cells were washed,harvested,and counted.1×105 cells were re-suspended in 100 µL binding buffer,before 10 µL of AnnexinV and 5 µL of PI were added,and incubated in the dark for 15 min at room temperature,according to the manufacturer’s instruction (Biosea,China).The apoptosis rate(%) was determined with a cytometer (Epics AltraII,Beckman Coulter,USA).Cells were also viewed under an inverse fluorescent microscopy.Experiment was repeated thrice.Caspase 3 activity assayCaspase-3 activities were assayed using a Colorimetric Protease Assay Kit according to the manufa cturer’s instructions (Keygen Biotech, Nanjing, China). Briefly, 1×106cells were harvested after treatment,washed three times with PBS and resuspended in 50 μL of the lysis buffer, kept on ice for 60 min, and then centrifuged (10,000×g, 1 min at 4 ℃). Supe rnatants containing 100 μg of protein were incubated with 5 μL of enzyme specific colorimetric substrates Ac-DEVD-pNA (2 mM), at 37 ℃ for 4 h. The colorimetric release of p-nitroaniline from the Ac-DEVD-pNA substrate was measured using a light wave of 405 nm with an ELISA reader (BIO-Tek ELx800, Winooski, VT, USA).Pancreatic cancer xenograft tumor modelSW1990 cells in log-phase were digested with trypsin, and were finally resuspended withserum free culture medium. Tumor xenografts were established by subcutaneous inoculation of 2 × 106 SW1990 pancreatic cancer cells into the right abdominal flanks of BALB/c female mice (4 to 6 weeks old, 18 to 20 g in weight, purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences, Shanghai, China). A total of 48 animals were randomly divided into four groups, 12 mice for each group, and were housed in Specific Pathogen Free (SPF) level laboratory. Three weeks after inoculation, when tumor grew to desired size (largest diameter ≥ 5 mm), mice were received different treatments every three days. Animals in the emodin, gemcitabine, combination treatment group or control were received intraperitoneal injection of 40 mg/kg emodin, 125 mg/kg gemcitabine, 40 mg/kg emodin plus 80 mg/kg gemcitabine, or 0.9% sodium chloride, respectively. Mice body weight and tumor size was measured every six days after drug treatments. Tumor size was calculated using the following formula: Volume = (4/3) π [(length + width)/4]3. The inhibition ratio was calculated by the following formula: Inhibition rate (%) = [1 − (mean tumor volume of drug-treated mice before experiment − mean tumor volume of drug-treated mice at the end of the experiment / mean tumor volume of control mice before experiment − mean tumor volume of control mice at the end of the experiment)] × 100%. One week after the final injection (day 37), the animals were weighed and sacrificed. The subcutaneous tumors were excised and the tumor volume and tumor weight was measured.A part of the tumor tissue was formalin-fixed and paraffin-embedded for immunohistochemistry. The rest part was frozen in liquid nitrogen for EMSA and western blot analysis.Ethical approval for this study was given by Ethics Committee at Wenzhou Medical College.Ki-67 immunohistochemistry.Formalin-fixed, paraffin-embedded sections (4μm) were stained with anti-Ki-67 (rabbit monoclonal clone SP6;NeoMarkers, Fremont, CA) antibody as described previously (30). Results were expressed as percentage of Ki-67+ cells±SE per×400 magnification. A total of ten ×400 fields was examined and counted from three tumors of each of the treatment groups.Tunel assay detect tumor apoptosisTo assess the degree of apoptosis,we used the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling(Tunel)method after two weeks of therapy.Tunel staining was performed by stantard methods with 4μm tissue section. Operated as shown in tunel kit steps,tissue section was add tunel reaction solution on the sections at 37 ℃ for 1 hour,.Laser scanning confocal microscope under 400 folds observation camera was used, with excitation wavelength 488nm and emission wavelength 568nm. We observed 10 field vision of strongest fluorescence on each slice .Immunohistochemistry detected apoptosis relative protein:Bax,Bcl-2,Cytochrome c,Active caspase 3Sections were cut from paraffin embedded pancreatic cancer tissues.Immunostaining was performed using primary antibodies specific for Bcl-2,Bax and Cytochrome c with appropriate dilutions ,followed by staining with appropriate HRP-conjugated secondary antibodies,The slides were developed in diaminobenzidine and counterstained with a weak solution of haematoxylin solution stain.The stained slides were dehydrated and mounted in permount and visualized on a microscope.Images were captured with an attached camera linked to a computer.Mitochondria and cytoplasm ingredient of Tumor tissue were isolatedFresh tumor tissue were homogenated and then transfered to centrifuge tube, 800 ×g centrifugation for 5 min at 4 º C. Supernatant were collected and transferred to a new centrifuge tube. After 800 × g centrifugation for 5 min at 4 º C, we discarded the precipitate. The supernatant was transferred to a new centrifuge tube. After 12,000 ×g centrifugation 10 min at 4ºC,the supernatant containing cytoplasmic components and the sediment containing mitochondrial were separated and stored in -80 o C until use for detecting Cytochrome c in them by western blot analysis.Western blot detected apoptosis relative protein in tumor tissue:Bax ,Bcl-2,Cytochrome CProteins were routinely extracted from tumor tissues using radioimmunoprecipitation assay (RIPA) buffer .The protein concentration was detected by bicinchoninic acid (BCA) assay.The proteins were then fractionated by SDS-PAGE,electrotransferred to PVDF membranes, blocked with 5% non-fat milk, and then probed with primary antibodies and HRP-conjugated anti-rabbit secondary antibody. After washing, the bound antibody complexes were detected using an ECL chemiluminescence reagent (Amersham).Statistical analysisDate are expressed as means±SD.Statistical analysis was performed using SPSS software.Differences between group mean values were determined by one-way analysis of variance followed by two-tailed Student′s t-test for unpaired samples,assuming equalvariances.p<0.05 was considerer statistically significant. ResultsEmodin potentiates growth inhibition induced by gemcitabine in pancreatic cancer cellsWe investigated the effect of a combination of emodin and gemcitabine on cell viability by MTT assay. For these studies,SW1990 cells were treated with gemcitabine(20μM) for 48h,emodin(40μM) for 48h,or their combination(emodin (40μM) coincubat ed with gemcitabine(20μM) for 48h). Viable cells were evaluated by MTT assay. We found that combination treatment with emodin plus gemcitabine resulted in more loss of cell viability than either emodin or gemcitabine alone in pancreatic cancer cells (Fig. 1).20406080100120Co n t r o lE m o d i nG e mc i t ab i n eG e mc i t a b i n e+E m o d i nC e l l v i a b i l i t y （% o f c o n t r o l )****Fig.1Cells were treated with gemcitabine(20μM) for 48h,emodin(40μM) for 48h,or theircombination(emodin (40μM) coincubat ed with gemcitabine(20μM ) for 48h ) and analyzed for viable cells by MTT assay as described in Section 2. Data are presented as mean ± S.E. of nine replicates from three independent experiments. *P < 0.01 compared with sodium chloride treatment; **P < 0.01 compared with sodium chloride, emodin or gemcitabine treatment.Emodin sensitizes pancreatic cancer cells to gemcitabine-induced apoptosis in vitroSW1990 cells were treated with gemcitabine (20μM) for 48h,emodin(40μM )for 48h,or their combination (emodin (40μM) combined with gemcitabine(20μM) for 48h), before stained with AnnexinV/PI for flow cytometry determination of the apoptoticrate,and observation under fluorescent microscopy . We found that single emodin increased the apoptotic rate from 4.6% to 21.6%,while single gemcitabine increased the apoptotic rate from 4.6% to 15.2% ,all with statistical significance (P <0.05).Combination of gemcitabine and emodin produced significantly more pronounced apoptosis compared with single-agent treatment (29.6%,P <0.05) (Fig.2A and B). To further examine the ability of the combination therapy to induce apoptosis, caspase-3 activities in SW1990 cell line tested were evaluated. The combination of emodin and gemcitabine caused an additional increase in caspase-3 activity (Fig. 2C) by comparison with either agent alone. Next, we evaluated caspase-3 activation in both cell lines tested by Western blotting. The combined treatments also induced an additional activation of caspase-3 in SW1990 cells (Fig. 2D). These in vitro studies prompted us to conduct in vivo testing of our hypothesis, and the results are presented below5101520253035C o nt r o lE m o di nG e mc i t a b in eE m o di n+G e m c i t ab i n eA p o p t o t i c c e l l s (%)****(A)(B)50100150200250300350400450500C o nt r o lEm o di nG e mc i t a b in eG e mc i t a b in e +E mo di nC a s p a s e 3 a c t i v i t y (% o f c o n t r o l )****(C)Active caspase 317 KD beta-actin42 KD(D)246810121416C o n tr o lG e mc i t a b in eE m o di nE m o di n+G e m c i t a b i n eF o l d c h a n g e (A c t i v e c a s p a s e 3)****（E ）Fig.2 Induction of apoptosis in SW1990 cells treated with gemcitabine (20μM) for 48h, emodin(40μM ) for 48h,ortheir combination(emodin (40μM) coincubat ed with gemcitabine(20μM) for 48h ) was determined by flowcytometry on annexinV-FITC,and cells were also viewed under a fluorescent microscopy. Emo:Emodin;Gem:Gemcitabine. (A)The percentages of apoptotic cells. (B)Representative dot-plots illustrating apoptotic status in SW1990 cells. “*”: p <0.05,compared with control.“**”: p <0.05,compared with single agent or control.(C) Cell lysates were assayed for caspase-3 activity as described in Section 2. Data are presented as mean ± S.E. of three independent experiments.*P < 0.05 compared with control; **P < 0.05 compared with control, emodin or gemcitabine treatment. (D) Active caspase 3 protein levels were investigated. Western immunoblotting for beta-actin protein was performed as loading control representative.(E) Quantified data of western blot werepresented. “*”: p <0.05,compared with control.“**”: p <0.05,compared with single agent or control.Emodin augments antitumor effect of gemcitabine in xenograft model of pancreatic cancerThe schematic overview of the study protocol was present in Fig. 3A.SW1990 cell xenografts were established in athymic mice within 3 weeks and the nude mice were randomly divided into four experimental groups(12 mice in each group) ,which were respectivelysubjectedtotreatmentwithcontrol(0.9%sodiumchloride),emodin(40mg/Kg),gemcitabine(125mg/Kg),or emodin(40mg/Kg)plusgemcitabine(80mg/Kg) every three days.At the beginning of the therapy,the tumor volume of all mice was(0.38±0.05)cm 3,no significant difference among groups(P>0.05). Emodin, gemcitabine, or emodin plus gemcitabine treatments dramatically suppressed tumor growth, and combination drug therapy was more effective in tumor inhibition than monotherapy with either agent (Fig. 3B). One week after the final injection, mice were sacrificed and solid tumors were presented (Fig. 3C). Consistent with in vitro results,both tumor weight and volume were suppressed by monotherapy with either agent or combination therapy with emodin plus gemcitabine compared with control, while combination treatment exhibited more efficient than single treatment (P < 0.05) (Table 1). These results demonstrated that emodin and gemcitabine combination treatment showed efficient tumor growth suppressionin pancreatic tumor inoculated mice.(A)00.511.522.53d0d6d12d18d24d30d37daysT u m o r v o l u m e /c m 3ControlEmodin(40mg/kg)Gemcitabine(125mg/kg)Emodin(40mg/kg)+Gemcitabine(80mg/kg)(B)Control Emodin Gemcitabine Emodin+Gemcitabine（C ）Fig.3 Effects of different treatments on tumor size (A) Schematic overview of the study protocol. (B)The tumorvolume of mice in different groups was measured on indicated days. (C)One week after the final injection (Day 37), mice were sacrificed and tumors were removed. Example solid tumors are shown.Table 1 Effect of combination treatment on tumor growth inhibition. One week after the final injection (Day37), mice were sacrificed, and tumor weight was measured. Tumor volume and inhibition rate were calculated as described in Materials and Methods.Group Drug dose (mg*kg-1)Tumor weight(g) Tumor volume(cm 3) Inhibition rate(%) sodium chloride0 2.38±0.15 2.45±0.16 0 emodin 40 1.68±0.13* 1.66±0.11* 37.5 gemcitabine 125 1.32±0.05* 1.20±0.12* 59.1 emodin+gemcitabine40(e)+80(g)0.56±0.07#0.57±0.08#90.8*P < 0.05 compared with sodium chloride treatment; #P < 0.05 compared with sodium chloride, emodin or gemcitabine treatment.Tunel staining shows emodin potentiates tumor cell apoptosis by gemcitabine We next examined the expression of the cell proliferation marker Ki-67 . The results in Fig. 3A and B showed that emodin in combination with gemcitabine significantly down-regulated the expression of Ki-67 in tumor tissues compared with other groups (P < 0.05) .As apoptosis is one major pathway which leads tumor cell to death, we investigated apoptotic cell death induced by drug treatment.In vivo,we found inhibition of cell growth was also correlated with apoptotic cell death.As shown by Fig. 4C, increased TUNEL-positive cells were observed in drug therapy groups, and combination treatment remarkably promoted the apoptotic cell death. Quantified datarevealed that combination therapy with emodin plus gemcitabine dramatically elevated integrated optical density (IOD) as compared with other groups (P < 0.01) (Fig. 4D).Aboveresults were consistent with our in vitro result.A. Control;B. Emodin;C. Gemcitabine;D. Emodin+Gemcitabine.(A)102030405060708090100Co n t r o lE mo di nGe m c i t a b i n eE mo di n +G e m c i t a b i n e P r o l i f e r a t i o n i n d e x (% k i 67+ c e l l s )****(B)Control Emodin Gemcitabine Emodin+Gemcitabine(C)5000100001500020000250003000035000C o nt r o lE m o di nG e mc i t a b in eG e mc i t a b in e +E mo di nI O D****(D)Fig. 4Determination of proliferation suppression and apoptotic cell death promoted by combinationtreatment. (A)Immunohistochemical analysis of proliferation marker Ki-67 indicates the inhibition of pancreatic cancer cell proliferation in emodin alone or in combination with gemcitabine-treated groups of animals. Arrows show Ki-67 positive cells.(×400) (B)Quantification of Ki-67+ cells. Columns,mean of triplicate;bars,SE. (C) Apoptosis of the tumor cell was demonstrated by TUNEL assay. Increased TUNEL-positive cells were found in combination treatment group. (×400) (D) TUNEL staining was further quantified and presented as average IOD level. *P < 0.01 compared with sodium chloride treatment; **P < 0.01 compared with sodium chloride, emodin or gemcitabine treatment.Immunohistochemistry shows Combination treatment reduces Bcl-2/Bax ratio, promotes caspase-3 activation and CytC releaseDecreased Bcl-2/Bax ratio, activation of caspase-3 and CytC release are recognized as the key events that mediate the apoptotic pathway. To clarify the mechanism of emodin plus gemcitabine-promoted apoptotic cell death in tumor tissues, the expression of anti-apoptotic protein Bcl-2, pro-apoptotic protein Bax, active caspase-3, and CytC were assessed by immunohistochemistry.As revealed by Fig. 5A-C, reduced Bcl-2/Bax ratio, but enhanced caspase-3 activation and CytC release from mitochondrial were observed after drug injection (P < 0.01 compared with control). Importantly, combination therapy further promoted the reduction of Bcl-2/Bax ratio (P < 0.01 compared with monotherapy with either agent) (Fig. 5B). The ratio of Bcl-2/Bax is essential for regulating CytC release from the mitochondria as well as caspase-3 cleavage. Immunohistochemical analysis confirmed the elevated cytosolic CytC level and activated caspase-3 protein expression in the combination treatment group (P < 0.01 compared with monotherapy or with either agent) (Fig. 5C).Control Emodin GemcitabineEmodin+GemcitabineBaxBcl-2CytCActive caspase3(A)123456Co n t r o lE m o d i nG e mc i t ab i n e E m o d i n+G e mc i t a b i n eM e a n o p t i c a l d e n s i t y （B c l -2/B a x ）****(B)0.020.040.060.080.10.120.140.160.18Cytochrome c Active caspase 3M e a n o p t i c a l d e n s i t yControl EmodinGemcitabineEmodin+Gemcitabine********(C)Fig. 5 Combination treatment reduced Bcl-2/Bax ratio, promoted caspase-3 activation and CytC release. Oneweek after the final injection (Day37), the protein expression of Bcl-2, Bax, active caspase-3, and CytC were assessed by immunohistochemistry(A) (×400). Quantified data were presented in (B), (C). *P < 0.01 compared with sodium chloride treatment; **P < 0.01 compared with sodium chloride, emodin or gemcitabine treatment.Further detection of Bcl-2/Bax ratio, caspase-3 activation and CytC releaseSimilar results to above in immunohistochemistry were obtained from western blot analysis (Fig. 6 A-E).Based on our results of western blot showing obviously reduced Bcl-2/Bax ratio and strongcaspase 3 activation by combination of emodin and gemcitabine(Fig. 6A-C),we assessed CytC release into cytosol by immunoblotting mitochondrial extract and mitochondria-free cytosolic extract(Fig. 6D). A significant decrease in the mitochondrial CytC level in the combination treatment was observed compared with other groups,while appearance of CytC in the cytosol significantly upregulated comared with other groups(Fig. 6E). These data demonstrated combination therapy with both emodin and gemcitabine promoted tumor cell apoptosis via downregulation of Bcl-2/Bax ratio, activation of caspase-3 and CytC release from mitochondria to cytosol.(A)1234567Bax Bcl-2Active caspase 3F o l d c h a n g eControl Emodin GemcitabineEmodin+Gemcitabin************(B)β-Actin （42KD ）β-Actin （42KD ） Bax (21KD)Bcl-2 (26KD) Active Caspase 3(17KD)。
脂联素通过上调PPARα
是影响胃癌患者预后不良的独立因素。
胃癌早期症状不典型，进展较快，寻找评估及监测患者病情进展的生物标志物尤为重要［23,24］，而本研究结果提示COX6B2可能具备作为肿瘤标志物的能力，并为胃癌的临床干预提供了新的靶点。
以上研究结果证明COX6B2在胃癌组织中的高表达参与胃癌的病理过程并影响患者远期预后。
新近研究发现细胞色素c 氧化酶与其他核编码蛋白构成氧化还原反应活性中心，其功能缺陷会导致线粒体的功能障碍，促进神经退行性病变的发生［25,26］，还可以通过调节PPAR 信号通路促进脂肪代谢，抑制肥胖［27］。
另外，COX6B2被证实与一些肿瘤的恶性进展相关，可以通过靶向ATP/purinergic 受体调节线粒体功能来促进胰腺癌的转移，是分化型甲状腺癌远处转移的新型生物标志物［13,15］，但其对胃癌的作用尚且不知。
我们对公共数据库富集发现COX6B2可以调控胃癌细胞周期。
接下来采用慢病毒转染方式干扰MGC-803和SGC-7901中COX6B2表达，CCK-8和细胞周期检测显示过表达COX6B2可促进细胞增殖且G1/S 期细胞比例显著增加，提示COX6B2可能通过调控细胞周期影响胃癌恶性增殖过程。
这一结果开辟了COX6B2调控肿瘤进展的途径，为日后COX6B2功能研究了提供新视角。
为进一步阐释COX6B2作用于胃癌细胞的机制，我们通过KEGG 富集分析COX6B2作用于胃癌细胞可能于p53信号通路相关。
p53是一种肿瘤抑制蛋白，可调节多种基因的表达，这些基因参与细胞凋亡、生长停滞、抑制细胞周期进程、分化和加速DNA 修复或衰老，以应对基因毒性或细胞应激［28,29］。
作为转录因子，p53可激活上百种基因表达，这些靶基因直接参与细胞周期的调控、DNA 损伤的修复，也与细胞衰老、分化及细胞凋亡有关［30,31］。
而p21是P53的靶点，它依赖于P53的活性。
故我们检测了胃癌细胞中p53及p21的蛋白水平，研究结果显示COX6B2可以显著抑制p53信号通路的活化，提示COX6B2可能是通过抑制p53信号通路发挥作用。
信迪利单抗联合盐酸安罗替尼用于晚期非小细胞肺癌疗效研究
本研究还将血清Ｌｐ－ＰＬＡ２、ＲＤＷ用于ＡＩＳ的诊断分析，ＲＯＣ曲线分析显示，二者联合应用诊断ＡＩＳ的ＡＵＣ达到０．９６１，明显高于单项指标应用，表明血清Ｌｐ－ＰＬＡ２联合ＲＤＷ对ＡＩＳ具有较高的诊断效能。
４ 结论综上所述，Ｌｐ－ＰＬＡ２、ＲＤＷ均与ＡＩＳ的发生发展、病情严重程度关系密切，二者联合用于ＡＩＳ诊断中具有较高的诊断效能。
临床在诊疗疑似ＡＩＳ患者时可密切关注患者的Ｌｐ－ＰＬＡ２、ＲＤＷ水平，发挥其早期诊断、病情严重程度监测的积极作用，使患者获得最佳预后。
参考文献［１］ 葛成东，张尊胜．脑白质高信号与急性缺血性脑卒中患者功能预后的相关性研究［Ｊ］．临床和实验医学杂志，２０１９，１８（２）：１９８－２０１．［２］ 中国老年医学学会急诊医学分会，中华医学会急诊医学分会卒中学组，中国卒中学会急救医学分会．急性缺血性脑卒中急诊急救中国专家共识（２０１８版）［Ｊ］．中华急诊医学杂志，２０１８，２７（７）：７２１－７２８．［３］ Ｒｏｄｒｉｇｕｅｚ－ＣｏｌｏｎＳＭ，ＭｏＪ，ＤｕａｎＹ，ｅｔａｌ．Ｍｅｔａｂｏｌｉｃｓｙｎｄｒｏｍｅｃｌｕｓｔｅｒｓａｎｄｔｈｅｒｉｓｋｏｆｉｎｃｉｄｅｎｔｓｔｒｏｋｅ：ｔｈｅａｔｈｅｒｏｓｃｌｅｒｏｓｉｓｒｉｓｋｉｎｃｏｍｍｕｎｉ ｔｉｅｓ（ＡＲＩＣ）ｓｔｕｄｙ［Ｊ］．Ｓｔｒｏｋｅ，２００９，４０（１）：２００－２０５．［４］ ＲａｍａｗａｔＢ，ＳａｌｕｊａＡ，ＢｈａｔａｃｈａｒｊｅｅＪ，ｅｔａｌ．ＡｃｕｔｅＩｓｃｈｅｍｉｃＳｔｒｏｋｅｉｓＡｓｓｏｃｉａｔｅｄｗｉｔｈＩｎｃｒｅａｓｅｄＳｅｒｕｍＬｅｖｅｌｓｏｆＰｒｏ－ｉｎｆｌａｍｍａｔｏｒｙＣｙｔｏｋｉｎｅｓ［Ｊ］．ＩｎｄｉａｎＪＣｌｉｎＢｉｏｃｈｅｍ，２０２０，３６（６）：８－１０．［５］ 姜旭，李云慧，万楠．急性缺血性脑卒中的发生与脂蛋白相关磷脂酶Ａ２的相关性分析［Ｊ］．中国实验诊断学，２０２０，２４（１０）：１６２７－１６３０．［６］ ＨｕＧＸ，ＺｈａｎｇＪ，ＴｉａｎＹＧ，ｅｔａｌ．ＤｉａｇｎｏｓｔｉｃｖａｌｕｅｏｆｊｏｉｎｔｄｅｔｅｃｔｉｏｎｏｆｈｏｍｏｃｙｓｔｅｉｎｅａｎｄＲＤＷＣＶｏｎａｃｕｔｅｍｙｏｃａｒｄｉａｌｉｎｆａｒｃｔｉｏｎ．［Ｊ］．ＥｕｒＲｅｖＭｅｄＰｈａｒｍＳｃｉ，２０１６，２０（１９）：４１２４－４１２８．［７］ 张贵涛，朱天瑞，张凤，等．红细胞分布宽度在急性脑梗死动脉硬化评估中的应用［Ｊ］．中国老年学杂志，２０１７，３７（１）：５９．［８］ 中华医学会神经病学分会，中华医学会神经病学分会脑血管病学组．中国急性缺血性脑卒中诊治指南２０１８［Ｊ］．中华神经科杂志，２０１８，５１（９）：６６６－６８２．［９］ 王洁，于蕾，郭淮莲．颈动脉硬化斑块内新生血管的超声造影评价及其与脑梗死患者病情严重程度及预后的关系［Ｊ］．中风与神经疾病杂志，２０１９，２６（８）：６７６－６８０．［１０］ＳｒｉｖａｓｔａｖａＡ，ＭｃｍａｈｏｎＫＤ，ＳｔｅｐａｎａｕｓｋａｓＲ，ｅｔａｌ．Ｄｅｎｏｖｏｓｙｎｔｈｅｓｉｓａｎｄｆｕｎｃｔｉｏｎａｌａｎａｌｙｓｉｓｏｆｔｈｅｐｈｏｓｐｈａｔａｓｅ－ｅｎｃｏｄｉｎｇｇｅｎｅａｃＩ－ＢｏｆｕｎｃｕｌｔｕｒｅｄＡｃｔｉｎｏｂａｃｔｅｒｉａｆｒｏｍＬａｋｅＳｔｅｃｈｌｉｎ（ＮＥＧｅｒｍａｎｙ）［Ｊ］．ＩｎｔＭｉｃｒｏｂｉｏｌ，２０１６，１８（４）：３９－４７．［１１］陈志华，张梅，李镒冲，等．我国成年人血压水平与心脑血管疾病行为危险因素及其聚集关系分析［Ｊ］．中华流行病学杂志，２０１８，３９（５）：６４０－６４５．［１２］ＭａｇｎｕｓＢ，ＨａｎｓｓｏｎＧＫ．Ａｎｔｉ－ｉｎｆｌａｍｍａｔｏｒｙｔｈｅｒａｐｉｅｓｆｏｒａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ［Ｊ］．ＮａｔｕｒｅＲｅｖｉｅｗｓＣａｒｄｉｏｌｏｇｙ，２０１５，１２（４）：１９９－２１１．［１３］ＵｌｒｉｃｈＣ，ＴｒｏｊａｎｏｗｉｃｚＢ，ＦｉｅｄｌｅｒＲ，ｅｔａｌ．ＤｉｆｆｅｒｅｎｔｉａｌＥｘｐｒｅｓｓｉｏｎｏｆＬｉｐｏｐｒｏｔｅｉｎ－ＡｓｓｏｃｉａｔｅｄＰｈｏｓｐｈｏｌｉｐａｓｅＡ２ｉｎＭｏｎｏｃｙｔｅＳｕｂｓｅｔｓ：ＩｍｐａｃｔｏｆＵｒｅｍｉａａｎｄＡｔｈｅｒｏｓｃｌｅｒｏｓｉｓ［Ｊ］．Ｎｅｐｈｒｏｎ，２０１７，１３５（３）：２３１－２４１．［１４］Ｂｏｎｎｅｆｏｎｔ－ＲｏｕｓｓｅｌｏｔＤ．Ｌｐ－ＰＬＡ２，ａｂｉｏｍａｒｋｅｒｏｆｖａｓｃｕｌａｒｉｎｆｌａｍｍａｔｉｏｎａｎｄｖｕｌｎｅｒａｂｉｌｉｔｙｏｆａｔｈｅｒｏｓｃｌｅｒｏｓｉｓｐｌａｑｕｅｓ［Ｊ］．ＡｎｎＰｈａｒｍＦｒ，２０１６，７４（３）：１９０－１９７．［１５］张晓红，刘向 ．脂蛋白相关磷脂酶Ａ２与心血管疾病的关系［Ｊ］．标记免疫分析与临床，２０１９，１２（５）：８９２－８９６．［１６］ＫｉｔａｍｕｒａＪ，ＵｅｎｏＨ，ＮａｇａｉＭ，ｅｔａｌ．ＢｌｏｏｄＰｒｅｓｓｕｒｅＶａｒｉａｂｉｌｉｔｙｉｎＡｃｕｔｅＩｓｃｈｅｍｉｃＳｔｒｏｋｅ：ＩｎｆｌｕｅｎｃｅｏｆＩｎｆａｒｃｔＬｏｃａｔｉｏｎｉｎｔｈｅＩｎｓｕｌａｒＣｏｒｔｅｘ［Ｊ］．ＥｕｒＮｅｕｒｏｌ，２０１８，７９（１－２）：９０－９９．［１７］刘希奇，李孝庆，姚彦，等．急性脑梗死患者血清ＣＸＣＬ１６ＧＤＦ１５Ｌｐ－ＰＬＡ２水平变化及临床意义［Ｊ］．河北医学，２０１９，２５（３）：５８０－５８４．［１８］Ｓｚｙｇｕｌａ－ＪｕｒｋｉｅｗｉｃｚＢ，ＳｚｃｚｕｒｅｋＷ，ＳｋｒｚｙｐｅｋＭ，ｅｔａｌ．ＲｅｄＢｌｏｏｄＣｅｌｌＤｉｓｔｒｉｂｕｔｉｏｎＷｉｄｔｈｉｎＥｎｄ－ＳｔａｇｅＨｅａｒｔＦａｉｌｕｒｅＰａｔｉｅｎｔｓＩｓＩｎｄｅ ｐｅｎｄｅｎｔｌｙＡｓｓｏｃｉａｔｅｄＷｉｔｈＡｌｌ－ＣａｕｓｅＭｏｒｔａｌｉｔｙＡｆｔｅｒＯｒｔｈｏｔｏｐｉｃＨｅａｒｔＴｒａｎｓｐｌａｎｔａｔｉｏｎ［Ｊ］．ＴｒａｎｓｐｌａｎｔＰｒｏｃｅｅｄ，２０１８，５０（７）：２０９５－２０９９．［１９］ＥｍｉｒｏｇｌｕＮ，ＣｅｎｇｉｚＦＰ，ＡＧＢａｈａｌ ，ｅｔａｌ．ＲｅｄＢｌｏｏｄＣｅｌｌＤｉｓｔｒｉｂｕｔｉｏｎＷｉｄｔｈａｎｄＮｅｕｔｒｏｐｈｉｌ－ｔｏ－ＬｙｍｐｈｏｃｙｔｅＲａｔｉｏｉｎＰａｔｉｅｎｔｓｗｉｔｈＣｕｔａｎｅ ｏｕｓＶａｓｃｕｌｉｔｉｓ［Ｊ］．ＡｎｎＣｌｉｎＬａｂＳｃｉ，２０１７，４７（２）：１６２－１６５．［２０］Ｇａｒｒｏｔｅ－ＦｉｌｈｏＳ，Ｂｅｒｎａｒｄｉｎｏ－ＮｅｔｏＭ，Ｐｅｎｈａ－ＳｉｌｖａＮ．ＩｎｆｌｕｅｎｃｅｏｆＥｒｙｔｈｒｏｃｙｔｅＭｅｍｂｒａｎｅＳｔａｂｉｌｉｔｙｉｎＡｔｈｅｒｏｓｃｌｅｒｏｓｉｓ［Ｊ］．ＣｕｒｒＡｔｈｅｒＲｅｐ，２０１７，１９（４）：１７．［２１］张茜，王萌，赖惠英，等．红细胞分布宽度对老年急性缺血性脑卒中患者的早期诊断价值［Ｊ］．中华老年医学杂志，２０２０，３９（５）：４９７－５００．（收稿日期：２０２１－１２－０６） ＤＯＩ：１０．３９６９／ｊ．ｉｓｓｎ．１６７１－４６９５．２０２２．０４．００６ 文章编号：１６７１－４６９５（２０２２）０４－０３５６－０５信迪利单抗联合盐酸安罗替尼用于晚期非小细胞肺癌疗效研究操思源 宋文灿 陶正平 （池州市人民医院肿瘤科 安徽 池州 ２４７１００）基金项目：安徽省自然科学基金项目（编号：１４０８０８５ＭＧ１４６） 【摘要】 目的 探讨信迪利单抗联合盐酸安罗替尼用于晚期非小细胞肺癌疗效研究。
p8基因中文及翻译
P8基因是最近发现的具有有丝分裂mitogenic活动的基因。
在急性胰腺炎、胰腺发育及再生中均可诱导表达。
然而，胰腺癌中却未见有关P8基因表达的报道。
本项研究旨在通过免疫组化研究包括38例胰腺癌在内的72例人胰腺组织中的p8基因表达。
38例胰腺癌中有27例（71%）p8基因过度表达（在1，000个细胞中阳性细胞>25%），而慢性胰腺炎中仅有17%。
粘液性囊腺瘤mucinous cystadenoma或正常胰腺组织中未见过度表达。
与其他病例相比胰腺癌中p8基因过度表达明显增高（p<0.05）.RT-PCR证实75%的胰腺癌中有P8 mRNA的过度表达（肿瘤/非肿瘤比例> 2）。
p8基因过度表达同样见于人胰腺癌细胞系(MIA PaCa-2 和PANC-1)。
研究结果表明p8基因参与了胰腺癌的发生，反应出其有丝分裂活动。
The p8 gene is a recently identified gene with mitogenic activity. Its expression is induced in acute pancreatitis, pancreatic development, and regeneration. However, the expression of p8 in pancreatic cancer is not reported. This study was designed to investigate p8 expression in 72 human pancreatic tissues, including 38 pancreatic cancers (PCs), by immunohistochemistry. The p8 was overexpressed (positive cells >25% in 1,000 cells) in 71% (27 of 38) of PCs, but in only 17% (3 of 18) of chronic pancreatitis cases. There was no overexpression in mucinous cystadenoma or in normal pancreas. The p8 overexpression rate in PCs was significantly higher than that in other conditions (p<0.05). Reverse transcription-PCR analysis confirmed p8 mRNA overexpression (tumor/nontumor ratio >2) in 75% (3of 4) 0f PCs. The p8 was over-expressed also in human pancreatic cancer lines (MIA PaCa-2 and PANC-1). These results suggest that p8 is involved in the development of pancreatic cancer, reflecting its mitogenic activity.。
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MOLECULARCARCINOGENESIS53:181–191(2014)InvolvementofAnnexinA8inthePropertiesofPancreaticCancer
HarumiHata,MasayukiTatemichi,*andToshioNakadateDepartmentofHygieneandPreventiveMedicine,ShowaUniversity,SchoolofMedicine,Tokyo,Japan
AlthoughAnnexinA8(ANXA8),amemberofasuperfamilyofcalciumandphospholipidbindingproteins,isphysio-logicallyexpressedinatissue-specificmanner,recentmicroarraystudiesreportedthatANXA8wasalsoectopicallyexpressedinpancreaticcancers.WeinvestigatedthemolecularmechanismofexpressionofANXA8incancercellsanditsfunctionalroleinpancreaticcancercells.ANXA8wasdiverselyexpressedinhumancancercelllines.Expressionwasenhancedbytreatmentwith5-aza-dCandbutyrate,andcorrelatedwithmethylationstatusatCpGinthepro-moter-exon1region.InhibitionofANXA8usingsiRNAinBxPC-3cellswhichexpressANXA8atahighlevelelevatedcaspase-3and-7activities.Ininvitroinvasionassay,inhibitionofANXA8usingsiRNAinBxPC-3reducedthenumbersofmigratingcells,anddown-regulatedHIF-1amRNAtranscription.OverexpressionofANXA8increasedthenumberofviablecellsandBrdUincorporationinPANC-1cells,whichexpressANXA8atalowlevel.ExpressionofANXA8wasinducedunderconditionsofnutrientdeprivation,andoverexpressionofANXA8showedresistanceagainstserumstarvationinPANC-1cells.Inapromoterassay,co-transfectionwiththeexpressionvectorofANXA8andthevectorofareportergenecontainingthepromoterofHIF-1aenhancedHIF-1apromoteractivity.Incontrast,thiseffectofANXA8wasinhibitedbyadministrationofBAPTA-AM,anintracellularCa2þchelator.Theseresultssuggestthatectop-
icANXA8expressionincancercellsmightinvolveanepigeneticmechanism.ANXA8mightplayanimportantroleincalciumfluctuation-mediatedHIF-1atranscriptionalactivationandcellviability.ß2012WileyPeriodicals,Inc.
Keywords:AnnexinA8;pancreaticcancer;epigenetics;HIF-1alpha
INTRODUCTIONThepoor5-yrsurvivalrateofpancreaticcancer,whichat3–6%istheworstprognosisofallthema-jorcancers[1],isduetothreefactors,thelackofeffectivemarkersforearlydiagnosis,aggressiveinva-sionandrapidrecurrencefromtheearlyclinicalstage,andresistancetochemotherapy.Pancreaticcancersdemonstrateuniqueproperties:thetumoritselfisextremelyhypovascularandhypoxic[2],andmostestablishedpancreaticcancercellsacquireremarkabletolerancetohypoxiaandnutrientdepri-vation[3].Acquisitionofthesepropertiesintheear-lystageofcarcinogenesisleadstoaggressiveinvasionandrapidmetastasis,inturnresultinginpoorprognosis.Clarificationofthemolecularbasisofthesecharacteristicsmightleadtothedevelop-mentofnewdiagnosticapproaches,andthediscov-eryofnewtherapeutictargets.Todate,however,thesefactorshavenotbeeninvestigatedatthemo-lecularlevel.Annexins(ANXs)areafamilyofCa2þ-dependent
phospholipid-bindingproteins.AllmembersoftheANXfamilyshareaconservedcoredomainthatisformedbyfouroreightrepeatsof70–80aminoacidscontainingCa2þbindingsites[4].ANXsarein-
volvedinnumerouscellprocesses,includingvesicletrafficking,calciumsignaling,cellgrowth,division,andapoptosis.Theyshowcelltype-specificexpres-sion,andtheN-terminaldomainisuniqueandconsideredtoberesponsibleforthefunctional
diversityofeachmember[5].Accumulatingevi-denceindicatesthatANXsplayavarietyofrolesintumordevelopment,progression,andresistancetochemotherapeuticagents[6–9],suggestingthatindi-vidualANXsmightrepresenttherapeutictargets.ANXA8(previouslyVAC-b),whichwasfirstdis-coveredinahumanplacentalcDNAlibrary,hasbeenshowntobindtophospholipidsinthepres-enceofCa2þandtopossessanticoagulantandphos-
pholipaseA2inhibitoryactivity,likeotherANXs[10].IncontrasttootherANXs,however,ANXA8ispoorlycharacterizedandexpressedatratherlowlev-elsinatissue-specificmanner[11].Amongfindingstodate,ANXA8isexpressedduringmousemamma-ryglandinvolutionandinpubertalductalepitheli-um[12],andisexpressedinosteoclastsandregulatescytoskeletalreorganizationinosteoclastsgeneratedonamineralizedmatrix[13].Recently,ANXA8hasbeenreportedtobeinvolvedinthereg-ulationofEGFreceptorlocalizationandactivity,similarlytoANXA1,ANXA2,andANXA6[14,15].ANXA8interactswithphosphatidylinositidesand
*Correspondenceto:DepartmentofHygieneandPreventiveMedicine,ShowaUniversity,SchoolofMedicine,1-5-8Hatanodai,Shinagawa-ku,Tokyo142-8555,Japan.
Received13February2012;Revised9August2012;Accepted23August2012
DOI10.1002/mc.21961Publishedonline21September2012inWileyOnlineLibrary(wileyonlinelibrary.com).
ß2012WILEYPERIODICALS,INC.