环氧化酶-2抑制剂塞来昔布对人舌鳞癌Tca8113细胞生长抑制及诱导凋亡的作用
|
摘要
舌癌是口腔癌中最常见的恶性肿瘤,约占口腔癌的40%以上,发病率男性约为0.5～0.6/10万,女性约为0.4～0.5/10万。近年来舌癌的发病率呈明显上升趋势,发病低龄化。舌癌的发病因素目前尚不十分清楚,可能与局部创伤、吸烟、嗜酒等因素有关。由于舌体组织是一个肌性活动器官,淋巴及血运丰富,且舌癌多呈浸润性生长,早期容易发生淋巴结转移,预后较差。因此,研究舌癌发生、发展的机理,探索有效的预防及治疗的方法,乃是舌癌防治研究的重要领域。
流行病学调查发现,长期服用阿司匹林(Aspirin)和其它非甾体类抗炎药物(Non-steroidal antiinflammatory drugs,NSAIDs)治疗其他疾病的人群,可明显降低结直肠癌的发病率,降低结肠息肉的大小和数量,并可降低结肠息肉恶变的危险性。进一步研究发现,NSAIDs不但能抑制肠道上皮肿瘤的发生,还具有预防结肠以外上皮癌发生的作用,如食道癌、胃癌和乳腺癌,同时还可以预防前列腺癌、膀胱癌以及头颈部鳞癌的发生。NSAIDs的这种作用主要与抑制环氧化酶-2(Cycloxygenase-2,COX-2)酶的活性有关,因而认为抑制COX-2酶的活性,是预防及治疗恶性肿瘤的热点之一。
本课题从病理形态学、细胞生物学、实验动物学的角度,通过免疫组织化学、分子生物学和医学统计学等方法,研究了选择性COX-2抑制剂塞来昔布(Celecoxib)对人舌鳞状细胞癌(Human tongue squamous cell carcinoma,HTSCC)Tca8113细胞的生长抑制及诱导凋亡的作用。首先采用免疫组化的方法研究了人正常口腔粘膜、不典型增生上皮、舌鳞状细胞癌及有、无癌转移的颈部淋巴结组织中COX-2、血管内皮生长因子-C(Vascular endothelial growth factor-C,VEGF-C)的表达及舌癌组织中的微血管密度(Microvessel Density,MVD),探讨COX-2和VEGF-C蛋白在HTSCC组织中的表达强度与临床诊断及预后分析中的相互关系及意义;运用分子生物学方法观察塞来昔布抑制Tca8113细胞生长及诱导凋亡的作用,研究塞来昔布增强抗癌药物顺铂(Cisplatin,CDDP)及博莱霉素(Bleomycin,BLM)对Tca8113细胞的杀伤作用;建立裸鼠Tca8113细胞皮下移植瘤动物模型,观察塞来昔布与CDDP联合应用后对裸鼠移植瘤的生长抑制作用,探讨选择性COX-2抑制剂在口腔癌预防及治疗方面的应用价值。
一、COX-2和VEGF-C在人舌鳞癌组织中的表达及与颈淋巴结转移的关系
目的:通过观察COX-2及VEGF-C在人正常口腔黏膜、不典型增生上皮、舌癌以及有、无癌转移的颈部淋巴结组织中的表达,研究两者与舌癌发生、血管生成及颈部淋巴结转移之间的关系。方法:搜集2000年至2006年间我院病理科保存的46例舌鳞状细胞癌石蜡标本,其中男性26例,女性20例,年龄28～81岁,平均(53.78±11.43)岁。标本经病理学证实,均为高～中分化鳞状细胞癌。46例患者中,伴颈淋巴结转移者13例,所有患者术前均未接受放疗或化疗。另取8例外伤清创保留的口腔粘膜作为正常对照。采用S-P免疫组化染色法,通过半定量方法,研究COX-2和VEGF-C在人正常口腔黏膜、不典型增生上皮、舌鳞癌及颈部淋巴结组织中的表达。根据免疫反应的强度分为(—)、(+)、(++)、(+++)4个等级。根据免疫染色情况,通过计数的方法,计算CD_(34)标记的微血管数量,研究肿瘤组织中微血管密度(MVD)。结果:COX-2在人正常口腔粘膜上皮不表达或弱表达,在增生和不典型增生的鳞状上皮中表达逐渐增强,在大部分舌鳞癌组织及转移淋巴结中高表达。VEGF-C在人正常口腔粘膜及不典型增生鳞状上皮中弱表达,在舌鳞癌组织中表达增强。COX-2及VEGF-C在有、无淋巴结转移的原发灶癌组织中的表达强度之间差异均有显著性意义,发生淋巴结转移组的舌癌原发灶组织中COX-2和VEGF-C的表达强于未发生淋巴结转移组。在46例人舌鳞癌组织中,COX-2和VEGF-C之间的表达强度呈正相关(r_s=0.874,P=0.000)。CD_(34)染色表明,人舌鳞癌组织中新生微血管主要分布在在癌组织周围与正常组织之间,舌癌组织中COX-2、VEGF-C表达的强弱同舌癌组织中MVD值均呈正相关(P=0.000)。结论:COX-2与VEGF-C的强阳性表达与舌鳞状细胞癌的生物学行为密切相关,COX-2和/或VEGF-C可以作为临床判断肿瘤的侵袭性、转移潜能及估计预后重要的参考指标,其在肿瘤发生中的作用机制有待于进一步探讨。
二、COX-2抑制剂塞来昔布对人舌鳞癌Tca8113细胞生长抑制及诱导凋亡的作用
目的:观察选择性COX-2抑制剂塞莱昔布对人舌鳞癌Tca8113细胞生长抑制及诱导凋亡的作用。方法:Tca8113细胞常规培养24 h后,分别加入含有不同浓度塞来昔布的培养液,并以塞来昔布敏感细胞人肝癌Hep-G2细胞株作为阳性对照,其药物终浓度分别为2.5、5、10、20、40、80μmol/L。培养24、48、72h后,采用MTT方法测定细胞的OD值,计算细胞生长抑制率;采用倒置显微镜、光学显微镜及透射电镜观察药物处理后的细胞形态学及超微结构变化;采用流式细胞仪检测分析细胞周期变化;应用荧光显微镜观察凋亡细胞的形态学特征;应用Annexin V-FITC/PI双标记法检测细胞早期凋亡的变化。结果:COX-2蛋白在Tca8113细胞呈强阳性表达,塞来昔布在抑制细胞生长的同时,抑制Tca8113细胞COX-2蛋白的表达。塞来昔布主要以剂量依赖方式抑制Tca8113细胞的生长、增殖,当药物浓度达到≥40μmol/L时,这种作用最为明显。塞来昔布抑制Tca8113细胞生长增殖的作用,与阻滞细胞周期进程有关,其主要阻滞Tca8113细胞从G_1期细胞向S期细胞进程,导致G_0/G_1期细胞的数量增加,从(53.53±0.93)%增加至(66.70±0.98)%(40μmol/L,24 h,P=0.000)和(58.77±1.35)%(80μmol/L,24h,P=0.001),而S期和G_2/M期细胞数量相应减少,S期细胞从(35.73±0.76)%减少至(25.97±1.23)%(40μmol/L,24 h,P=0.000)和(28.63±1.12)%(80μmol/L,24 h,P=0.000)。G_2/M期细胞的比例则随G_0/G_1期和S期细胞比例的变化而改变。塞来昔布这种对细胞周期的阻滞作用,主要发生在药物作用后的24h左右。倒置显微镜观察:塞来昔布处理后,Tca8113细胞生长明显受到抑制,细胞突起减少、皱缩、胞浆颗粒增加,部分细胞呈固缩状态,并可见少数细胞碎裂死亡。光学显微镜观察发现,随着塞莱昔布的浓度增加及培养时间的延长,细胞形态开始发生改变,出现细胞皱缩、胞体变小,核浆比例减小、核仁数目变少,胞浆内有空泡产生,并出现凋亡细胞。透射电镜下观察显示,随着塞来昔布浓度的增加及作用时间的延长,Tca8113细胞出现胞体变圆、细胞电子密度升高、细胞器变性、细胞核染色质浓缩等改变,但各部分膜结构完整,细胞表面绒毛消失脱落,为早期凋亡细胞,并可见到凋亡细胞前期与后期变化并存,药物进一步作用,则形成中、后期凋亡细胞,并形成大量凋亡小体。AO/EB荧光双染检测发现,塞来昔布处理后可见早期凋亡细胞,细胞被AO及EB双重染色,早期表现为细胞核被染色呈黄绿色荧光,浓聚成新月形或颗粒状,位于细胞的一侧;随着药物浓度增加和作用时间延长,细胞凋亡现象明显加重,表现为细胞核被EB深染呈桔红色,核固缩和偏位,细胞膜可见出芽、起泡以及太阳花形变化;严重时可见细胞坏死,坏死细胞体积增大,呈不均匀的橙红色荧光,轮廓不清、解体或接近解体。Annexin V-FITC/PI双标记法流式细胞术检测细胞早期凋亡结果显示,对照组24、48 h细胞早期凋亡率分别为0%及(0.93±0.11)%;塞来昔布40、80μmol/L处理24 h后的早期凋亡率分别为(3.86±0.23)%、(15.25±0.87)%;48h后早期凋亡率分别为(7.17±0.64)%和(20.06±1.06)%,与对照组相比,早期凋亡细胞增多有显著性意义(P均=0.000)。结论:COX-2抑制剂塞来昔布主要以剂量依赖的方式,通过抑制细胞周期进程、诱导凋亡的作用抑制Tca8113细胞生长、增殖,其作用机制有待进一步研究。
三、COX-12抑制剂塞来昔布增强抗癌药物对人舌鳞癌Tca8113细胞的杀伤作用
目的:研究COX-2抑制剂塞来昔布与抗癌药物联合应用后能否增强抗癌药物对人舌鳞癌Tca8113细胞的杀伤作用。方法:Tca8113细胞常规培养24h后,分别加入含有不同浓度的塞来昔布、CDDP及BLM培养液,其塞来昔布药物终浓度分别为2.5、5、10、20、40、80μmol/L,CDDP药物终浓度分别为0、3.125、6.25、12.5、50 mg/L,BLM药物终浓度分别为0、3.125、6.25、12.5、50mg/L,并以人肝癌Hep-G2细胞株作为塞来昔布敏感细胞系进行阳性对照。继续培养24、48、72 h后,采用噻唑蓝法(MTT)测定细胞OD值,计算细胞生长抑制率,计算上述药物对Tca8113细胞抑制作用的IC_(50),依据IC_(50)选择塞来昔布作用的最佳药物浓度(药物浓度＜IC_(50))。本研究采用塞来昔布(10μmol/L)分别与CDDP(3.12、6.25、12.5mg/L)和BLM(3.12、12.5、50 mg/L)联合作用Tca8113细胞。培养24、48、72 h后,MTT测定联合用药对Tca8113细胞的细胞毒性作用,经S-N-K法统计学检验后,再用金正钧法计算q值,判断塞来昔布与抗癌药物联合应用后对Tca8113细胞的杀伤作用,当q=1±0.15为相加作用,q＞1.15为协同作用,q＜0.85为拮抗作用。同时采用流式细胞仪检测联合用药后对Tca8113细胞周期进程的影响,光镜下观察联合用药后Tca8113细胞的形态学变化。结果:小剂量的塞来昔布(10μmol/L)与CDDP、BLM联合应用后,其增强CDDP及BLM对Tca8113细胞的杀伤作用有显著性意义,塞来昔布与CDDP、BLM联合应用后对Tca8113细胞杀伤方面的相互作用,随时间变化而作用方式不同,在药物作用后24h以相加作用为主,48h以协同作用为主,而72h后又以相加作用为主。塞来昔布与CDDP及BLM联合应用后,可显著增强CDDP及BLM阻滞Tca8113细胞周期进程的作用,其阻滞G_1期细胞进入S期的作用最为明显,导致G_0/G_1细胞的数量增加,S期及G_2/M期细胞减少。G_0/G_1期细胞数量从抗癌药单用时的(58.53±1.00)%增加至(61.83±0.50)%(CDDP,P=0.001),及从(56.5±0.96)%增加至(60.93±0.32)%(BLM,P=0.000),S期和G_2/M期细胞数量减少,S期细胞数量从(31.3±0.70)%减少至(29.63±0.64)%(CDDP,P=0.175)和(30.3±1.95)%减少至(30.57±0.78)%(BLM,P=0.849),G_2/M期细胞也随G_0/G_1期和S期细胞数量的变化而发生相应改变。结论:小剂量的塞来昔布即可增强CDDP及BLM对Tca8113细胞的杀伤作用,抑制细胞生长与增殖,增强对细胞周期进程的阻滞作用,其与CDDP和BLM联合应用后对Tca8113细胞的杀伤作用呈协同作用或相加作用。
四、动物体内观察COX-2抑制剂塞来昔布联合顺铂的抗癌作用
目的:进一步研究COX-2抑制剂塞来昔布单独应用或与抗癌药联合应用对Tca8113细胞裸鼠荷瘤的生长抑制作用。方法:将Tca8113细胞(2×10~6个细胞)接种于32只Balb/c裸鼠皮下,一周后,确认裸鼠皮下种植部位已成瘤后,将裸鼠随机分为4组:对照组、塞来昔布组、CDDP组及塞来昔布+CDDP联合用药组,每组8只。塞来昔布组采用灌胃给药,剂量为200mg/kg·d~(-1);CDDP组采用腹腔注射给药,剂量为5 mg/kg·w~(-1);塞来昔布+CDDP组采用塞来昔布(200mg/kg·d~(-1),灌胃)+CDDP(5 mg/kg·w~(-1),腹腔注射);对照组予腹腔注射生理盐水,口服灭菌蒸馏水。给药后每隔7天测量移植瘤的直径并计算体积。给药后35天处死裸鼠,测移植瘤质量,计算抑瘤率。光镜及电镜观察裸鼠移植瘤组织形态学及细胞超微结构变化;免疫组织化学检测移植瘤组织COX-2、VEGF-C蛋白的表达;荧光定量PCR检测各组移植瘤组织中COX-2、VEGF-C mRNA的表达,并通过析因方差分析检验塞来昔布和CDDP两药对裸鼠移植瘤生长、移植瘤组织中COX-2、VEGF-C mRNA表达的交互作用。结果:Tca8113细胞接种裸鼠皮下后的7天,注射点确认有肿瘤生长。从给药后14天开始,与对照组相比,各药物处理组移植瘤生长较慢,其中以塞来昔布+CDDP组移植瘤生长明显慢于对照组及单用药物组。实验终止时对照组移植瘤体积及瘤体质量明显大于其它各组,而塞来昔布+CDDP组移植瘤的体积及瘤体质量最小。对照组和塞来昔布组在不同时间之间瘤体体积变化有差异有显著性,说明随着时间的延长,移植瘤体积逐渐增大。但CDDP和塞来昔布+CDDP联合应用后不同时间之间差异无显著性意义,提示CDDP及塞来昔布+CDDP具有显著的抑制Tca8113细胞裸鼠移植瘤生长的作用。各实验组从第14天开始,与对照组相比,其抑制肿瘤生长作用有显著性意义。而塞来昔布+CDDP组联合应用的抑制移植瘤生长的作用,与CDDP单用相比,差异有显著性意义(P=0.034),说明塞来昔布有增强CDDP抗肿瘤的作用。移植瘤质量结果表明,塞来昔布、CDDP及塞来昔布+CDDP组的抑瘤率分别为15.63%、37.5%及82.81%。通过析因方差分析方法对各实验组与对照组抑瘤情况分析,塞来昔布和CDDP抑制裸鼠Tca8113细胞移植瘤生长的主效应均有统计学意义(P=0.000),二者交互作用显著(P=0.001),说明两者在抑制裸鼠Tca8113移植瘤生长方面具有协同增强作用。移植瘤组织学光镜观察见:塞来昔布组肿瘤细胞凋亡现象多见,部分区域可见坏死灶;CDDP组表现为细胞变性和坏死,可以见到细胞肿胀、液化、质膜破裂,部分细胞出现核溶解、消失等表现,但凋亡细胞很少;塞来昔布+CDDP组则可见到肿瘤细胞大片凝固性坏死,并可见较多的凋亡细胞。透射电镜观察:塞来昔布组及塞来昔布+CDDP组均可见到凋亡细胞,但以塞来昔布+CDDP组凋亡细胞多见,表现为细胞皱缩、细胞器变性、电子密度升高、体积变小,但膜结构完整;核染色质浓缩,呈均质状、碎裂及核仁消失。免疫组织化学染色观察发现,对照组COX-2及VEGF-C蛋白均呈强阳性表达,塞来昔布及塞来昔布+CDDP组COX-2及VEGF-C蛋白的表达明显减弱。经析因方差分析对各实验组与对照组COX-2和VEGF-C mRNA表达循环域值(Ct)的倍比关系(2~(-ΔΔCt)值)分析发现,塞来昔布抑制COX-2 mRNA表达方面的作用较弱,其与CDDP对COX-2基因表达的抑制方面无交互增强作用,提示选择性COX-2抑制剂塞来昔布对COX-2的抑制作用,可能主要表现在蛋白水平,抑制其酶的活性。但对VEGF-C基因表达方面,塞来昔布与CDDP主效应均有统计学意义,两者有交互增强作用。这也提示COX-2抑制剂单独应用或与CDDP联合应用后抑制肿瘤血管形成的作用,可能与抑制VEGF-C基因表达有关。结论:COX-2抑制剂塞来昔布单独应用,虽然与对照组相比其抑制Tca8113细胞裸鼠移植瘤生长的作用有显著性意义,但其抑制肿瘤生长的作用仍然较弱;其与CDDP联合应用后,抑制裸鼠移植瘤的生长有显著性意义,两者在抑制移植瘤生长方面有交互增强的作用。塞来昔布对COX-2 mRNA表达的抑制作用较弱,但对VEGF-C mRNA表达的抑制作用有显著性意义,与CDDP联合应用后,对VEGF-C mRNA表达的抑制呈交互增强作用。塞来昔布与CDDP联合应用后抑制移植瘤生长的同时,促进移植瘤组织的变性、坏死及诱导肿瘤细胞凋亡。塞来昔布抑制移植瘤生长的作用可能与抑制COX-2蛋白表达有关,其具体作用机制尚待进一步研究。
总之,本研究发现,COX-2与VEGF-C在舌癌组织中呈强阳性表达,并与舌鳞状细胞癌的生物学行为密切相关,COX-2和/或VEGF-C可以作为临床判断肿瘤的侵袭性、转移潜能及估计预后重要的参考指标。选择性COX-2抑制剂塞来昔布主要以剂量依赖方式通过抑制细胞周期进程、诱导凋亡从而抑制人舌鳞癌Tca8113细胞生长。同时,塞来昔布能增强抗癌药CDDP和BLM对Tca8113细胞的杀伤作用,塞来昔布与CDDP、BLM联合应用后对Tca8113细胞的杀伤作用呈协同或相加作用。大剂量的塞来昔布(200 mg/kg·d~(-1))单独应用,能够抑制裸鼠Tca8113细胞移植瘤的生长,与CDDP联合应用后,能增强CDDP对裸鼠移植瘤的生长抑制作用。塞来昔布在抑制裸鼠移植瘤生长的同时,抑制肿瘤组织COX-2和VEGF蛋白的表达。塞来昔布与CDDP联合应用能增强抑制Tca8113细胞裸鼠移植瘤VEGF-C mRNA表达,两者之间有交互增强作用,而塞来昔布对COX-2 mRNA的表达则无明显抑制作用,其与CDDP联合应用后对COX-2 mRNA的表达无交互增强作用,提示COX-2抑制剂塞来昔布的抗肿瘤作用可能主要是抑制COX-2酶的活性。塞来昔布抑制裸鼠移植瘤生长的同时,促进移植瘤组织的变性、坏死及诱导肿瘤细胞凋亡。以上这些研究结果,为今后更深入研究COX-2与口腔癌的发生,以及选择性COX-2抑制剂在口腔癌的预防及治疗方面的作用,奠定了良好的工作基础。
Carcinoma of the tongue,one of the most common oral malignant tumors, accounts for over 40%of the oral carcinomas with an incidence of about 0.5～0.6/100 000 in males,and of 0.4～0.5/100 000 in females.Recently,the occurrence of tongue carcinoma tends to be significantly increasing even in younger population.The pathogenic factors still remain unknown.Local injury,smoking, alcoholic drinking and precancerous lesion may be involved in the pathogenesis.The tongue is an active muscle organ with abundant blood supply and lymph drainage, which facilitates its infiltrative growth and lymphatic metastasis at its early stage and thus makes it more difficult in its treatment.Therefore,it becomes more and more important to elucidate the pathogenic mechanism,and to explore the efficient preventive measures of the carcinoma of tongue.
Epidemiological investigation revealed that patients with a long-term administration of aspirin or other non-steroidal anti-inflammatory drugs(NSAIDs) had a significantly lower incidence of colorectal cancer with less colonic polyp size and number,which reduced the risks of colonic polyp canceration.Further investigations confirmed that NSAIDs could not only inhibit gut epithelial tumors, but also prevent the pathogenesis of epithelioma other than colorectum.Such effects of NSAIDs might be mainly associated with their inhibition of the activity of cycloxygenase-2(COX-2).Therefore,selective COX-2 inhibitor is becoming a hot spot in the prevention and treatment of malignant tumors.
In the present study,we investigated the inhibitory effects of Celecoxib,a selective COX-2 inhibitor,on Tca8113 cell line of human tongue squamous cell carcinoma(HTSCC) by immunohistochemistry,molecular biological and statistical methods in view of pathology,cell biology and experimental zoology. Immunohistochemistry was used in our study to investigate the expression of COX-2 and vascular endothelial growth factor-C(VEGF-C) in normal human oral mucosa, atypical hyperplastic epithelium,HTSCC and metastatic lymph nodes,which may be of important clinical value in the diagnosis and prognosis of the tongue carcinoma. Molecular biological techniques were employed to detect the effects of Celecoxib on growth inhibition and apoptosis of Tca8113 cell line.The role of COX-2 inhibitor Celecoxib in enhancing the cytotoxicity of anticancer agents in Tca8113 cell line was also detected.We established an animal tumor model through subcutaneously implantation of Tca8113 cells so as to observe the effect of Celecoxib along with CDDP on transplanted tumor growth in nude mice and the histomorphological changes of tumor-bearing tissues and organs,which may provide us primarily clinical value for COX-2 in the prevention and treatment of oral cancers.
Ⅰ.Expression of COX-2 and VEGF-C in human tongue squamous cell carcinoma and its correlation with lymph node metastasis
Objective:To observe the coexpression of COX-2/VEGF-C in normal oral mucosa,atypical hyperplastic epithelium and HTSCC,and to evaluate the correlation of this coexpression with canceration of tongue,and with lymph node metastasis, respectively.Methods:46 cases(26 males and 20 females,ages ranging from 28 to 81 with an average age of 53.78±11.43) of tongue squamous cell carcinoma specimen were collected from the Department of Pathology of Nanfang Hospital from 2000 to 2006.Pathological analysis confirmed that all the cases were advanced to moderately differentiated squamous cell carcinoma;13 of the 46 cases had cervical lymph node metastasis.None of the patients received radiotherapy or chemotherapy before surgery.8 cases of oral mucosa from traumatic patients were used as control. Methods:S-P immunohistochemistry staining was performed to determine COX-2 and VEGF-C expression in normal oral mucosa,cancer tissue and lymph nodes by semi-quantitative method.Based on the intensity of immunoreaction,intensity levels were classified into(-),(+),(++),(+++).CD_(34) marked microvessel numbers were calculated by immunostaining to study microvessel density(MVD) in the cancer tissues.Results:COX-2 and VEGF-C showed—or+in normal oral mucosa,while it showed obvious strong expression in hyperplasia and atypical hyperplastic epithelium, and overexpression in most cancer tissues and metastasis lymph nodes.Expression level of COX-2 and VEGF-C demonstrated a significant difference in primary focus cancer tissues with or without lymph nodes metastasis.The result showed a positive correlation between the expression level of COX-2 and VEGF-C in carcinoma tissues of tongue.Their correlation coefficient(r_s) was 0.874(P=0.000).CD34 staining showed that newly formed microvessels were mainly distributed in tissues surrounding the carcinoma;buffy granules were found in the cytoplasm of vascular endothelial cells,while microvessels were hardly found in cancer tissues.The expression level of COX-2 and VEGF-C showed a positive correlation with MVD. Conclusions:The coexpression of COX-2 and VEGF-C had close relationship to biological behaviors in human tongue squamous cell carcinoma.It may be an important marker in evaluating the prognosis,metastasis and invasion in HTSCC. However,the mechanism of both proteins in carcinogenesis in HTSCC needs further investigation.
Ⅱ.The growth inhibition and apoptotic induction of COX-2 inhibitor Celecoxib on Tca8113 cell line of HTSCC
Objective:To observe whether Celecoxib could inhibit the growth and induce apoptosis of Tca8113 cells.Methods:24 h after incubation of Tca8113 cells,different concentrations of Celecoxib were added into the cells,with the final concentrations being 2.5,5,10,20,40,80μmol/L,respectively.Humen hepatic carcinoma cell line Hep-G2 as a Celecoxib-sensitive cells used as a positive control.MTT was used to determine the OD values at 570 nm for calculating cell growth inhibition rate after incubation for 24,48 and 72 h,respectively.Inverted microscope,optical microscope and transmission electron microscope were used to observe cell morphological changes before and after the treatment.Cell cycle phase distribution was detected by flow cytometer(FCM);morphology of apoptosis cells was observed by fluorescence microscopy;Annexin V-FITC/PI double labeling method was employed to detect early stage cell apoptosis.Results:COX-2 protein was strongly expressed in Tca8113 cells and was suppressed by Celecoxib.The growth and proliferation of Tca8113 cells treated with Celecoxib was inhibited in a dose-dependent manner,which reached its valley value at the concentration of 40μmol/L.Celecoxib's growth inhibitory effect was associated with the blockage of cell cycle.Celecoxib blocked Tca8113 cell transmission from G_1 stage to S stage in a dose-dependent manner, resulting in the accumulation of G_0/G_1 stage cells,from(53.53±0.93)%to (66.70±0.98)%(40μmol╱L,24 h,P=0.000) and to(58.77±1.35)%(80μmol/L,24 h, P=0.001),while cells at S stage reduced from(35.73±0.76)%to(25.97±1.23)%(40μmol/L,24 h,P=0.000) and to(28.63±1.12)%(80μmol/L,24 h,P=0.000).Cells at G_2/M stages also decreased significantly in accordance with the changes of the percentage in cells at both G_0/G_1 and S stages.This blockage of cell cycles mainly occurred at about 24 h.Inverted microscopic analysis showed that Tca8113 cells treated by Celecoxib was significantly inhibited,showing with less cytoplasmic processes,cell shrinkage,increase of cytoplasmic granules,nuclear pycnosis,even dead cell fragments.In HE staining sections showes cell shrinkage,reduction of karyoplasmic ratio and nucleolus number,occurrence of cytoplasmic vacuoles,and finally apoptotic cells.In electron microscopy,after exposure to the Celecoxib, Tca8113 cells became rounded in shape with higher electron dense,degenerated organelles,and condensed nuclear chromatin,but intact cell boundary with the increase of Celecoxib levels and the incubating durations.The Tca8113 cells lost their superficial villi and became early-staged apoptotic cells.Meanwhile,we also found the co-existence of the early-and later-staged apoptotic cells.Further exposure to the agent results in mid- even later- staged apoptotic cells with many apoptotic bodies.AO/EB fluorescence double staining showed that early-staged apoptotic cells were found AO-and EB-positive in experiment groups with flavo-green fluorescence on one side of the nuclei,forming a crescent-shaped or a granular structure.With the increase of concentrations and exposure periods,apoptosis became more prominent with the nuclei being stained reddish yellow by EB,karyopyknotic and eccentric. Membranous budding and vacuolization occurred,forming a sunflower-shaped structure.Cell necrosis was found under severe conditions.FCM analysis of V-FITC/PI-double labeled Annexin revealed that Celecoxib(40,80μmol/L) treatment resulted in significant early apoptosis with cell apoptosis rates of (3.86±0.23)%,(15.25±0.87)%and(7.17±0.64)%,(20.06±1.06)%at 24,48 h of incubation,respectively,as compared with control ones of 0%and(1.057±0.10)%at 24,48 h(P=0.000).The results indicate that Celecoxib inhibit cell proliferation in a dose-dependent manner.Conclusion:Celecoxib inhibits growth and proliferation of Tca8113 cells mainly in a dose-dependent manner by inhibiting cell cycle process and by inducing cell apoptosis.
Ⅲ.COX-2 inhibitor Celecoxib enhances the lethal effects of anticancer agents in Tca8113 cell line of HTSCC
Objective:To explore the role of Celecoxib in enhancing the lethal effects of anticancer agents in Tca8113 cell line.Methods:24 h after cultivation of Tca8113 cell line,different concentrations of Celecoxib,CDDP and BLM were added into the cells, with the final concentrations being 2.5,5,10,20,40,80μmol/L(Celecoxib),0, 3.125,6.25,12.5,50 mg/L(CDDP) and 0,3.125,6.25,12.5,50 mg/L(BLM), respectively.MTT was used to calculate cell growth inhibition rate after incubation for 24,48,72 h,respectively.According to IC_(50) of the above agents,optimal concentration for interaction of Celecoxib was selected(drug concentration＜IC_(50)). We combined Celecoxib(10μmol/L) with CDDP(3.12,6.25,12.5 mg/L) and BLM (3.12,12.5,50 mg/L) to treat Tca8113 cells,respectively.Lethal effects of different drug applications in Tca8113 cells after incubation for 24,48 and 72 h were measured by MTT.Flow cytometry(FCM) was used to evaluate the effects of combined drug applications on cell cycles.Experimental data were statistically processed by S-N-K test first.Jin Zheng Jun's Method was used to calculate q values,the criterion for evaluating the interaction of Celecoxib and anticancer drugs,q=1±0.15 indicates additive effect;q＞1.15 means synergistic effect;and q＜0.85 refers to antagonism. Results:Celecoxib(10μmol/L) combined either with CDDP or with BLM showed synergism or additive lethal effect on Tca8113 cell line.The way of interaction varied with the time elapsed.Additive effect was dominant after 24 h's and 72 h's incubation, while synergism was dominant between 48～72 h.Celecoxib with CDDP or BLM could notably enhance the effect of CDDP and BLM by blocking cell cycle after 24 h. The effect was most significant when it blocked G_1-staged cells into S stage,thus increasing G_0/G_1 cells distribution and reducing S-staged and G_2/M-staged cells.The percentage of G_0/G_1-staged cells increased from(58.53±1.00)%(CDDP only) to (61.83±0.50)%(CDDP+Celecoxib,P=0.001) and from(56.5±0.96)%(BLM only) to (60.93±0.32)%(BLM+Celecoxib,P=0.000),accompanied by the reduced cells at S and G_2/M stages.Cell at S stage reduced from(31.3±0.70)%to(29.63±0.64)% (CDDP,P=0.175) and(30.3±1.95)%to(30.57±0.78)%(BLM,P=0.849).Cells at G_2/M stage changed with cells at G_0/G_1 and S stages.Conclusions:Combination of Celecoxib with anticancer drug CDDP or BLM could obviously increase the lethal effect of CDDP and BLM on Tca8113 cells by inhibiting their growth and survival. Low doses of Celecoxib could significantly enhance the lethal effect of anticancer drugs CDDP and BLM on Tca8113 cells by inhibiting cell growth and proliferation through cell cycle blockage.The ways of these interactions on inhibiting Tca8113 cell growth were synergistic or/and additive.
Ⅳ.In vivo study of the antitumous effects of Celecoxib combined with cisplatin on Tca8113 cell xenografts
Objective To further investigate whether Celecoxib combined with anticancer drugs can inhibit the growth of Tca8113 cell line-transplanted tumor in tumor-bearing nude mice.Methods:Inoculate Tca8113 cells(2×10~6 cells) into 32 Balb/c nude mice subcutaneously.Nude mice were divided randomly into 4 groups(8 mice each): control,Celecoxib treated,CDDP treated and Celecoxib+CDDP treated.Intragastric administration of Celecoxib,200 mg/kg·d~(-1),intraperitoneal injection of CDDP 5 mg/kg·w~(-1),or both was applied to mice in different groups.In control group, intraperitoneal injection of normal saline and oral administration of sterile purified water were performed.Body weights and tumor sizes were measured for calculating the volume of the nude mice every seven days.Nude mice were sacrificed at 35 d after drug administration.Tumor masses were dissected and weighed for calculating tumor inhibition rates.Histological and electron microscopic analysis were used to observe nude mice tumor tissues.Immunohistochemistry staining was employed for COX-2 and VEGF-C protein expression in tumor tissues.Fluorescent quantitive real-time RT-PCR was used to detect COX-2 and VEGF-C mRNA expression in transplanted tumor tissues of each group,t 7 days after inoculation of Tca8113 cells into nude mice,tumor growth was confirmed in the injection sites in all groups.Since 14 days after administration of drugs,transplanted tumors in drug-treated groups grew slowly as compared with other groups,of which is most significant in Celecoxib+CDDP group.The above-mentioned fact indicates that tumor growth be inhibited by either Celecoxib or CDDP,or both,and that Celecoxib might act in accordance with CDDP.When the experiment terminated,the average length and short diameter of the tumor in control group were notably larger than those of other treatment groups.Tumors in control group grew most quickly with an average weight of(1.28±0.15)g,while tumors in Celecoxib+CDDP group were lightest with an average weight of(0.22±0.09)g.Tumor inhibition rates were 15.63%,37.5%and 82.81%in groups of Celecoxib-treated,CDDP-treated and Celecoxib+CDDP-treated, respectively.In light microscopy,tumor cell ischemic necrosis and prominent apoptosis in some regions were found in Celecoxib-treated group.Typical cytotoxic phenomenon occurred in CDDP-treated group,showing cell degeneration and necrosis,cellular swelling,liquefaction,plasmarrhexis,karyolysis even nuclear deprivation.Apoptosis was rare.In Celecoxib+CDDP-treated group,coagulation necrosis of tumor cells and more apoptotic cells were seen.Under TEM,Apoptotic cells were found in Celecoxib-treated group,especially in Celecoxib+CDDP-treated group.Cell shrinkage,organdie degeneration,higher electron density,lesser cell volume with intact plasmalemma,condensed but homogenous nuclear chromatin, nuclear fragmentation and absence of nucleoli.Immunohistochemical results showed that the expression of COX-2 and VEGF-C was strongly positive in control group, but significantly weaker in both Celecoxib-treated and Celecoxib+CDDP-treated group.The factorial analysis of variance demonstrated that both Celecoxib and CDDP showed their main effect and interaction on growth inhibition of xenografts.The multiproportion of cycle threshold(Ct) of COX-2 mRNA and VEGF-C mRNA in each treat group,compared with control,was also analysised with factorial analysis of variance.The results indicated that both Celecoxib and CDDP showed their significanct main effect and interaction on suppression of VEGF-C mRNA expression, but no significance of main effect or interaction on suppression of COX-2 mRNA expression.Conclusion:COX-2 inhibitor Celecoxib could not only inhibit nude mice transplanted tumor growth,but also enhance the effect of cytotoxic agent CDDP on growth inhibition of xenografts of Tca8113 cell line.
In conclusion,in the present study,we found that COX-2 and VEGF-C were strongly positiveexpression in tongue carcinoma tissues,and were closely correlated with the biological behaviors of squamous cell carcinoma of tongue.Selective COX-2 inhibitor Celecoxib could inhibit growth and proliferation of Tca8113 cells of HTSCC by blocking cell cycle proceeding and by inducing cell apoptosis.Meanwhile, Celecoxib could enhance the cytotoxicity of CDDP and BLM on Tca8113 cells. Celecoxib together with CDDP and BLM showed synergism or additive effects of cytotoxcity on Tca8113 cells;Celecoxib could inhibit Tca8113 cell transplanted tumor growth,and after combined application with CDDP,it manifested more significant inhibition of nude mice tumor growth Our results provide better basis for our further study of COX-2 and oral cancer,and for the prevention of COX-2 inhibitor in oral cancer.
流行病学调查发现,长期服用阿司匹林(Aspirin)和其它非甾体类抗炎药物(Non-steroidal antiinflammatory drugs,NSAIDs)治疗其他疾病的人群,可明显降低结直肠癌的发病率,降低结肠息肉的大小和数量,并可降低结肠息肉恶变的危险性。进一步研究发现,NSAIDs不但能抑制肠道上皮肿瘤的发生,还具有预防结肠以外上皮癌发生的作用,如食道癌、胃癌和乳腺癌,同时还可以预防前列腺癌、膀胱癌以及头颈部鳞癌的发生。NSAIDs的这种作用主要与抑制环氧化酶-2(Cycloxygenase-2,COX-2)酶的活性有关,因而认为抑制COX-2酶的活性,是预防及治疗恶性肿瘤的热点之一。
本课题从病理形态学、细胞生物学、实验动物学的角度,通过免疫组织化学、分子生物学和医学统计学等方法,研究了选择性COX-2抑制剂塞来昔布(Celecoxib)对人舌鳞状细胞癌(Human tongue squamous cell carcinoma,HTSCC)Tca8113细胞的生长抑制及诱导凋亡的作用。首先采用免疫组化的方法研究了人正常口腔粘膜、不典型增生上皮、舌鳞状细胞癌及有、无癌转移的颈部淋巴结组织中COX-2、血管内皮生长因子-C(Vascular endothelial growth factor-C,VEGF-C)的表达及舌癌组织中的微血管密度(Microvessel Density,MVD),探讨COX-2和VEGF-C蛋白在HTSCC组织中的表达强度与临床诊断及预后分析中的相互关系及意义;运用分子生物学方法观察塞来昔布抑制Tca8113细胞生长及诱导凋亡的作用,研究塞来昔布增强抗癌药物顺铂(Cisplatin,CDDP)及博莱霉素(Bleomycin,BLM)对Tca8113细胞的杀伤作用;建立裸鼠Tca8113细胞皮下移植瘤动物模型,观察塞来昔布与CDDP联合应用后对裸鼠移植瘤的生长抑制作用,探讨选择性COX-2抑制剂在口腔癌预防及治疗方面的应用价值。
一、COX-2和VEGF-C在人舌鳞癌组织中的表达及与颈淋巴结转移的关系
目的:通过观察COX-2及VEGF-C在人正常口腔黏膜、不典型增生上皮、舌癌以及有、无癌转移的颈部淋巴结组织中的表达,研究两者与舌癌发生、血管生成及颈部淋巴结转移之间的关系。方法:搜集2000年至2006年间我院病理科保存的46例舌鳞状细胞癌石蜡标本,其中男性26例,女性20例,年龄28～81岁,平均(53.78±11.43)岁。标本经病理学证实,均为高～中分化鳞状细胞癌。46例患者中,伴颈淋巴结转移者13例,所有患者术前均未接受放疗或化疗。另取8例外伤清创保留的口腔粘膜作为正常对照。采用S-P免疫组化染色法,通过半定量方法,研究COX-2和VEGF-C在人正常口腔黏膜、不典型增生上皮、舌鳞癌及颈部淋巴结组织中的表达。根据免疫反应的强度分为(—)、(+)、(++)、(+++)4个等级。根据免疫染色情况,通过计数的方法,计算CD_(34)标记的微血管数量,研究肿瘤组织中微血管密度(MVD)。结果:COX-2在人正常口腔粘膜上皮不表达或弱表达,在增生和不典型增生的鳞状上皮中表达逐渐增强,在大部分舌鳞癌组织及转移淋巴结中高表达。VEGF-C在人正常口腔粘膜及不典型增生鳞状上皮中弱表达,在舌鳞癌组织中表达增强。COX-2及VEGF-C在有、无淋巴结转移的原发灶癌组织中的表达强度之间差异均有显著性意义,发生淋巴结转移组的舌癌原发灶组织中COX-2和VEGF-C的表达强于未发生淋巴结转移组。在46例人舌鳞癌组织中,COX-2和VEGF-C之间的表达强度呈正相关(r_s=0.874,P=0.000)。CD_(34)染色表明,人舌鳞癌组织中新生微血管主要分布在在癌组织周围与正常组织之间,舌癌组织中COX-2、VEGF-C表达的强弱同舌癌组织中MVD值均呈正相关(P=0.000)。结论:COX-2与VEGF-C的强阳性表达与舌鳞状细胞癌的生物学行为密切相关,COX-2和/或VEGF-C可以作为临床判断肿瘤的侵袭性、转移潜能及估计预后重要的参考指标,其在肿瘤发生中的作用机制有待于进一步探讨。
二、COX-2抑制剂塞来昔布对人舌鳞癌Tca8113细胞生长抑制及诱导凋亡的作用
目的:观察选择性COX-2抑制剂塞莱昔布对人舌鳞癌Tca8113细胞生长抑制及诱导凋亡的作用。方法:Tca8113细胞常规培养24 h后,分别加入含有不同浓度塞来昔布的培养液,并以塞来昔布敏感细胞人肝癌Hep-G2细胞株作为阳性对照,其药物终浓度分别为2.5、5、10、20、40、80μmol/L。培养24、48、72h后,采用MTT方法测定细胞的OD值,计算细胞生长抑制率;采用倒置显微镜、光学显微镜及透射电镜观察药物处理后的细胞形态学及超微结构变化;采用流式细胞仪检测分析细胞周期变化;应用荧光显微镜观察凋亡细胞的形态学特征;应用Annexin V-FITC/PI双标记法检测细胞早期凋亡的变化。结果:COX-2蛋白在Tca8113细胞呈强阳性表达,塞来昔布在抑制细胞生长的同时,抑制Tca8113细胞COX-2蛋白的表达。塞来昔布主要以剂量依赖方式抑制Tca8113细胞的生长、增殖,当药物浓度达到≥40μmol/L时,这种作用最为明显。塞来昔布抑制Tca8113细胞生长增殖的作用,与阻滞细胞周期进程有关,其主要阻滞Tca8113细胞从G_1期细胞向S期细胞进程,导致G_0/G_1期细胞的数量增加,从(53.53±0.93)%增加至(66.70±0.98)%(40μmol/L,24 h,P=0.000)和(58.77±1.35)%(80μmol/L,24h,P=0.001),而S期和G_2/M期细胞数量相应减少,S期细胞从(35.73±0.76)%减少至(25.97±1.23)%(40μmol/L,24 h,P=0.000)和(28.63±1.12)%(80μmol/L,24 h,P=0.000)。G_2/M期细胞的比例则随G_0/G_1期和S期细胞比例的变化而改变。塞来昔布这种对细胞周期的阻滞作用,主要发生在药物作用后的24h左右。倒置显微镜观察:塞来昔布处理后,Tca8113细胞生长明显受到抑制,细胞突起减少、皱缩、胞浆颗粒增加,部分细胞呈固缩状态,并可见少数细胞碎裂死亡。光学显微镜观察发现,随着塞莱昔布的浓度增加及培养时间的延长,细胞形态开始发生改变,出现细胞皱缩、胞体变小,核浆比例减小、核仁数目变少,胞浆内有空泡产生,并出现凋亡细胞。透射电镜下观察显示,随着塞来昔布浓度的增加及作用时间的延长,Tca8113细胞出现胞体变圆、细胞电子密度升高、细胞器变性、细胞核染色质浓缩等改变,但各部分膜结构完整,细胞表面绒毛消失脱落,为早期凋亡细胞,并可见到凋亡细胞前期与后期变化并存,药物进一步作用,则形成中、后期凋亡细胞,并形成大量凋亡小体。AO/EB荧光双染检测发现,塞来昔布处理后可见早期凋亡细胞,细胞被AO及EB双重染色,早期表现为细胞核被染色呈黄绿色荧光,浓聚成新月形或颗粒状,位于细胞的一侧;随着药物浓度增加和作用时间延长,细胞凋亡现象明显加重,表现为细胞核被EB深染呈桔红色,核固缩和偏位,细胞膜可见出芽、起泡以及太阳花形变化;严重时可见细胞坏死,坏死细胞体积增大,呈不均匀的橙红色荧光,轮廓不清、解体或接近解体。Annexin V-FITC/PI双标记法流式细胞术检测细胞早期凋亡结果显示,对照组24、48 h细胞早期凋亡率分别为0%及(0.93±0.11)%;塞来昔布40、80μmol/L处理24 h后的早期凋亡率分别为(3.86±0.23)%、(15.25±0.87)%;48h后早期凋亡率分别为(7.17±0.64)%和(20.06±1.06)%,与对照组相比,早期凋亡细胞增多有显著性意义(P均=0.000)。结论:COX-2抑制剂塞来昔布主要以剂量依赖的方式,通过抑制细胞周期进程、诱导凋亡的作用抑制Tca8113细胞生长、增殖,其作用机制有待进一步研究。
三、COX-12抑制剂塞来昔布增强抗癌药物对人舌鳞癌Tca8113细胞的杀伤作用
目的:研究COX-2抑制剂塞来昔布与抗癌药物联合应用后能否增强抗癌药物对人舌鳞癌Tca8113细胞的杀伤作用。方法:Tca8113细胞常规培养24h后,分别加入含有不同浓度的塞来昔布、CDDP及BLM培养液,其塞来昔布药物终浓度分别为2.5、5、10、20、40、80μmol/L,CDDP药物终浓度分别为0、3.125、6.25、12.5、50 mg/L,BLM药物终浓度分别为0、3.125、6.25、12.5、50mg/L,并以人肝癌Hep-G2细胞株作为塞来昔布敏感细胞系进行阳性对照。继续培养24、48、72 h后,采用噻唑蓝法(MTT)测定细胞OD值,计算细胞生长抑制率,计算上述药物对Tca8113细胞抑制作用的IC_(50),依据IC_(50)选择塞来昔布作用的最佳药物浓度(药物浓度＜IC_(50))。本研究采用塞来昔布(10μmol/L)分别与CDDP(3.12、6.25、12.5mg/L)和BLM(3.12、12.5、50 mg/L)联合作用Tca8113细胞。培养24、48、72 h后,MTT测定联合用药对Tca8113细胞的细胞毒性作用,经S-N-K法统计学检验后,再用金正钧法计算q值,判断塞来昔布与抗癌药物联合应用后对Tca8113细胞的杀伤作用,当q=1±0.15为相加作用,q＞1.15为协同作用,q＜0.85为拮抗作用。同时采用流式细胞仪检测联合用药后对Tca8113细胞周期进程的影响,光镜下观察联合用药后Tca8113细胞的形态学变化。结果:小剂量的塞来昔布(10μmol/L)与CDDP、BLM联合应用后,其增强CDDP及BLM对Tca8113细胞的杀伤作用有显著性意义,塞来昔布与CDDP、BLM联合应用后对Tca8113细胞杀伤方面的相互作用,随时间变化而作用方式不同,在药物作用后24h以相加作用为主,48h以协同作用为主,而72h后又以相加作用为主。塞来昔布与CDDP及BLM联合应用后,可显著增强CDDP及BLM阻滞Tca8113细胞周期进程的作用,其阻滞G_1期细胞进入S期的作用最为明显,导致G_0/G_1细胞的数量增加,S期及G_2/M期细胞减少。G_0/G_1期细胞数量从抗癌药单用时的(58.53±1.00)%增加至(61.83±0.50)%(CDDP,P=0.001),及从(56.5±0.96)%增加至(60.93±0.32)%(BLM,P=0.000),S期和G_2/M期细胞数量减少,S期细胞数量从(31.3±0.70)%减少至(29.63±0.64)%(CDDP,P=0.175)和(30.3±1.95)%减少至(30.57±0.78)%(BLM,P=0.849),G_2/M期细胞也随G_0/G_1期和S期细胞数量的变化而发生相应改变。结论:小剂量的塞来昔布即可增强CDDP及BLM对Tca8113细胞的杀伤作用,抑制细胞生长与增殖,增强对细胞周期进程的阻滞作用,其与CDDP和BLM联合应用后对Tca8113细胞的杀伤作用呈协同作用或相加作用。
四、动物体内观察COX-2抑制剂塞来昔布联合顺铂的抗癌作用
目的:进一步研究COX-2抑制剂塞来昔布单独应用或与抗癌药联合应用对Tca8113细胞裸鼠荷瘤的生长抑制作用。方法:将Tca8113细胞(2×10~6个细胞)接种于32只Balb/c裸鼠皮下,一周后,确认裸鼠皮下种植部位已成瘤后,将裸鼠随机分为4组:对照组、塞来昔布组、CDDP组及塞来昔布+CDDP联合用药组,每组8只。塞来昔布组采用灌胃给药,剂量为200mg/kg·d~(-1);CDDP组采用腹腔注射给药,剂量为5 mg/kg·w~(-1);塞来昔布+CDDP组采用塞来昔布(200mg/kg·d~(-1),灌胃)+CDDP(5 mg/kg·w~(-1),腹腔注射);对照组予腹腔注射生理盐水,口服灭菌蒸馏水。给药后每隔7天测量移植瘤的直径并计算体积。给药后35天处死裸鼠,测移植瘤质量,计算抑瘤率。光镜及电镜观察裸鼠移植瘤组织形态学及细胞超微结构变化;免疫组织化学检测移植瘤组织COX-2、VEGF-C蛋白的表达;荧光定量PCR检测各组移植瘤组织中COX-2、VEGF-C mRNA的表达,并通过析因方差分析检验塞来昔布和CDDP两药对裸鼠移植瘤生长、移植瘤组织中COX-2、VEGF-C mRNA表达的交互作用。结果:Tca8113细胞接种裸鼠皮下后的7天,注射点确认有肿瘤生长。从给药后14天开始,与对照组相比,各药物处理组移植瘤生长较慢,其中以塞来昔布+CDDP组移植瘤生长明显慢于对照组及单用药物组。实验终止时对照组移植瘤体积及瘤体质量明显大于其它各组,而塞来昔布+CDDP组移植瘤的体积及瘤体质量最小。对照组和塞来昔布组在不同时间之间瘤体体积变化有差异有显著性,说明随着时间的延长,移植瘤体积逐渐增大。但CDDP和塞来昔布+CDDP联合应用后不同时间之间差异无显著性意义,提示CDDP及塞来昔布+CDDP具有显著的抑制Tca8113细胞裸鼠移植瘤生长的作用。各实验组从第14天开始,与对照组相比,其抑制肿瘤生长作用有显著性意义。而塞来昔布+CDDP组联合应用的抑制移植瘤生长的作用,与CDDP单用相比,差异有显著性意义(P=0.034),说明塞来昔布有增强CDDP抗肿瘤的作用。移植瘤质量结果表明,塞来昔布、CDDP及塞来昔布+CDDP组的抑瘤率分别为15.63%、37.5%及82.81%。通过析因方差分析方法对各实验组与对照组抑瘤情况分析,塞来昔布和CDDP抑制裸鼠Tca8113细胞移植瘤生长的主效应均有统计学意义(P=0.000),二者交互作用显著(P=0.001),说明两者在抑制裸鼠Tca8113移植瘤生长方面具有协同增强作用。移植瘤组织学光镜观察见:塞来昔布组肿瘤细胞凋亡现象多见,部分区域可见坏死灶;CDDP组表现为细胞变性和坏死,可以见到细胞肿胀、液化、质膜破裂,部分细胞出现核溶解、消失等表现,但凋亡细胞很少;塞来昔布+CDDP组则可见到肿瘤细胞大片凝固性坏死,并可见较多的凋亡细胞。透射电镜观察:塞来昔布组及塞来昔布+CDDP组均可见到凋亡细胞,但以塞来昔布+CDDP组凋亡细胞多见,表现为细胞皱缩、细胞器变性、电子密度升高、体积变小,但膜结构完整;核染色质浓缩,呈均质状、碎裂及核仁消失。免疫组织化学染色观察发现,对照组COX-2及VEGF-C蛋白均呈强阳性表达,塞来昔布及塞来昔布+CDDP组COX-2及VEGF-C蛋白的表达明显减弱。经析因方差分析对各实验组与对照组COX-2和VEGF-C mRNA表达循环域值(Ct)的倍比关系(2~(-ΔΔCt)值)分析发现,塞来昔布抑制COX-2 mRNA表达方面的作用较弱,其与CDDP对COX-2基因表达的抑制方面无交互增强作用,提示选择性COX-2抑制剂塞来昔布对COX-2的抑制作用,可能主要表现在蛋白水平,抑制其酶的活性。但对VEGF-C基因表达方面,塞来昔布与CDDP主效应均有统计学意义,两者有交互增强作用。这也提示COX-2抑制剂单独应用或与CDDP联合应用后抑制肿瘤血管形成的作用,可能与抑制VEGF-C基因表达有关。结论:COX-2抑制剂塞来昔布单独应用,虽然与对照组相比其抑制Tca8113细胞裸鼠移植瘤生长的作用有显著性意义,但其抑制肿瘤生长的作用仍然较弱;其与CDDP联合应用后,抑制裸鼠移植瘤的生长有显著性意义,两者在抑制移植瘤生长方面有交互增强的作用。塞来昔布对COX-2 mRNA表达的抑制作用较弱,但对VEGF-C mRNA表达的抑制作用有显著性意义,与CDDP联合应用后,对VEGF-C mRNA表达的抑制呈交互增强作用。塞来昔布与CDDP联合应用后抑制移植瘤生长的同时,促进移植瘤组织的变性、坏死及诱导肿瘤细胞凋亡。塞来昔布抑制移植瘤生长的作用可能与抑制COX-2蛋白表达有关,其具体作用机制尚待进一步研究。
总之,本研究发现,COX-2与VEGF-C在舌癌组织中呈强阳性表达,并与舌鳞状细胞癌的生物学行为密切相关,COX-2和/或VEGF-C可以作为临床判断肿瘤的侵袭性、转移潜能及估计预后重要的参考指标。选择性COX-2抑制剂塞来昔布主要以剂量依赖方式通过抑制细胞周期进程、诱导凋亡从而抑制人舌鳞癌Tca8113细胞生长。同时,塞来昔布能增强抗癌药CDDP和BLM对Tca8113细胞的杀伤作用,塞来昔布与CDDP、BLM联合应用后对Tca8113细胞的杀伤作用呈协同或相加作用。大剂量的塞来昔布(200 mg/kg·d~(-1))单独应用,能够抑制裸鼠Tca8113细胞移植瘤的生长,与CDDP联合应用后,能增强CDDP对裸鼠移植瘤的生长抑制作用。塞来昔布在抑制裸鼠移植瘤生长的同时,抑制肿瘤组织COX-2和VEGF蛋白的表达。塞来昔布与CDDP联合应用能增强抑制Tca8113细胞裸鼠移植瘤VEGF-C mRNA表达,两者之间有交互增强作用,而塞来昔布对COX-2 mRNA的表达则无明显抑制作用,其与CDDP联合应用后对COX-2 mRNA的表达无交互增强作用,提示COX-2抑制剂塞来昔布的抗肿瘤作用可能主要是抑制COX-2酶的活性。塞来昔布抑制裸鼠移植瘤生长的同时,促进移植瘤组织的变性、坏死及诱导肿瘤细胞凋亡。以上这些研究结果,为今后更深入研究COX-2与口腔癌的发生,以及选择性COX-2抑制剂在口腔癌的预防及治疗方面的作用,奠定了良好的工作基础。
Carcinoma of the tongue,one of the most common oral malignant tumors, accounts for over 40%of the oral carcinomas with an incidence of about 0.5～0.6/100 000 in males,and of 0.4～0.5/100 000 in females.Recently,the occurrence of tongue carcinoma tends to be significantly increasing even in younger population.The pathogenic factors still remain unknown.Local injury,smoking, alcoholic drinking and precancerous lesion may be involved in the pathogenesis.The tongue is an active muscle organ with abundant blood supply and lymph drainage, which facilitates its infiltrative growth and lymphatic metastasis at its early stage and thus makes it more difficult in its treatment.Therefore,it becomes more and more important to elucidate the pathogenic mechanism,and to explore the efficient preventive measures of the carcinoma of tongue.
Epidemiological investigation revealed that patients with a long-term administration of aspirin or other non-steroidal anti-inflammatory drugs(NSAIDs) had a significantly lower incidence of colorectal cancer with less colonic polyp size and number,which reduced the risks of colonic polyp canceration.Further investigations confirmed that NSAIDs could not only inhibit gut epithelial tumors, but also prevent the pathogenesis of epithelioma other than colorectum.Such effects of NSAIDs might be mainly associated with their inhibition of the activity of cycloxygenase-2(COX-2).Therefore,selective COX-2 inhibitor is becoming a hot spot in the prevention and treatment of malignant tumors.
In the present study,we investigated the inhibitory effects of Celecoxib,a selective COX-2 inhibitor,on Tca8113 cell line of human tongue squamous cell carcinoma(HTSCC) by immunohistochemistry,molecular biological and statistical methods in view of pathology,cell biology and experimental zoology. Immunohistochemistry was used in our study to investigate the expression of COX-2 and vascular endothelial growth factor-C(VEGF-C) in normal human oral mucosa, atypical hyperplastic epithelium,HTSCC and metastatic lymph nodes,which may be of important clinical value in the diagnosis and prognosis of the tongue carcinoma. Molecular biological techniques were employed to detect the effects of Celecoxib on growth inhibition and apoptosis of Tca8113 cell line.The role of COX-2 inhibitor Celecoxib in enhancing the cytotoxicity of anticancer agents in Tca8113 cell line was also detected.We established an animal tumor model through subcutaneously implantation of Tca8113 cells so as to observe the effect of Celecoxib along with CDDP on transplanted tumor growth in nude mice and the histomorphological changes of tumor-bearing tissues and organs,which may provide us primarily clinical value for COX-2 in the prevention and treatment of oral cancers.
Ⅰ.Expression of COX-2 and VEGF-C in human tongue squamous cell carcinoma and its correlation with lymph node metastasis
Objective:To observe the coexpression of COX-2/VEGF-C in normal oral mucosa,atypical hyperplastic epithelium and HTSCC,and to evaluate the correlation of this coexpression with canceration of tongue,and with lymph node metastasis, respectively.Methods:46 cases(26 males and 20 females,ages ranging from 28 to 81 with an average age of 53.78±11.43) of tongue squamous cell carcinoma specimen were collected from the Department of Pathology of Nanfang Hospital from 2000 to 2006.Pathological analysis confirmed that all the cases were advanced to moderately differentiated squamous cell carcinoma;13 of the 46 cases had cervical lymph node metastasis.None of the patients received radiotherapy or chemotherapy before surgery.8 cases of oral mucosa from traumatic patients were used as control. Methods:S-P immunohistochemistry staining was performed to determine COX-2 and VEGF-C expression in normal oral mucosa,cancer tissue and lymph nodes by semi-quantitative method.Based on the intensity of immunoreaction,intensity levels were classified into(-),(+),(++),(+++).CD_(34) marked microvessel numbers were calculated by immunostaining to study microvessel density(MVD) in the cancer tissues.Results:COX-2 and VEGF-C showed—or+in normal oral mucosa,while it showed obvious strong expression in hyperplasia and atypical hyperplastic epithelium, and overexpression in most cancer tissues and metastasis lymph nodes.Expression level of COX-2 and VEGF-C demonstrated a significant difference in primary focus cancer tissues with or without lymph nodes metastasis.The result showed a positive correlation between the expression level of COX-2 and VEGF-C in carcinoma tissues of tongue.Their correlation coefficient(r_s) was 0.874(P=0.000).CD34 staining showed that newly formed microvessels were mainly distributed in tissues surrounding the carcinoma;buffy granules were found in the cytoplasm of vascular endothelial cells,while microvessels were hardly found in cancer tissues.The expression level of COX-2 and VEGF-C showed a positive correlation with MVD. Conclusions:The coexpression of COX-2 and VEGF-C had close relationship to biological behaviors in human tongue squamous cell carcinoma.It may be an important marker in evaluating the prognosis,metastasis and invasion in HTSCC. However,the mechanism of both proteins in carcinogenesis in HTSCC needs further investigation.
Ⅱ.The growth inhibition and apoptotic induction of COX-2 inhibitor Celecoxib on Tca8113 cell line of HTSCC
Objective:To observe whether Celecoxib could inhibit the growth and induce apoptosis of Tca8113 cells.Methods:24 h after incubation of Tca8113 cells,different concentrations of Celecoxib were added into the cells,with the final concentrations being 2.5,5,10,20,40,80μmol/L,respectively.Humen hepatic carcinoma cell line Hep-G2 as a Celecoxib-sensitive cells used as a positive control.MTT was used to determine the OD values at 570 nm for calculating cell growth inhibition rate after incubation for 24,48 and 72 h,respectively.Inverted microscope,optical microscope and transmission electron microscope were used to observe cell morphological changes before and after the treatment.Cell cycle phase distribution was detected by flow cytometer(FCM);morphology of apoptosis cells was observed by fluorescence microscopy;Annexin V-FITC/PI double labeling method was employed to detect early stage cell apoptosis.Results:COX-2 protein was strongly expressed in Tca8113 cells and was suppressed by Celecoxib.The growth and proliferation of Tca8113 cells treated with Celecoxib was inhibited in a dose-dependent manner,which reached its valley value at the concentration of 40μmol/L.Celecoxib's growth inhibitory effect was associated with the blockage of cell cycle.Celecoxib blocked Tca8113 cell transmission from G_1 stage to S stage in a dose-dependent manner, resulting in the accumulation of G_0/G_1 stage cells,from(53.53±0.93)%to (66.70±0.98)%(40μmol╱L,24 h,P=0.000) and to(58.77±1.35)%(80μmol/L,24 h, P=0.001),while cells at S stage reduced from(35.73±0.76)%to(25.97±1.23)%(40μmol/L,24 h,P=0.000) and to(28.63±1.12)%(80μmol/L,24 h,P=0.000).Cells at G_2/M stages also decreased significantly in accordance with the changes of the percentage in cells at both G_0/G_1 and S stages.This blockage of cell cycles mainly occurred at about 24 h.Inverted microscopic analysis showed that Tca8113 cells treated by Celecoxib was significantly inhibited,showing with less cytoplasmic processes,cell shrinkage,increase of cytoplasmic granules,nuclear pycnosis,even dead cell fragments.In HE staining sections showes cell shrinkage,reduction of karyoplasmic ratio and nucleolus number,occurrence of cytoplasmic vacuoles,and finally apoptotic cells.In electron microscopy,after exposure to the Celecoxib, Tca8113 cells became rounded in shape with higher electron dense,degenerated organelles,and condensed nuclear chromatin,but intact cell boundary with the increase of Celecoxib levels and the incubating durations.The Tca8113 cells lost their superficial villi and became early-staged apoptotic cells.Meanwhile,we also found the co-existence of the early-and later-staged apoptotic cells.Further exposure to the agent results in mid- even later- staged apoptotic cells with many apoptotic bodies.AO/EB fluorescence double staining showed that early-staged apoptotic cells were found AO-and EB-positive in experiment groups with flavo-green fluorescence on one side of the nuclei,forming a crescent-shaped or a granular structure.With the increase of concentrations and exposure periods,apoptosis became more prominent with the nuclei being stained reddish yellow by EB,karyopyknotic and eccentric. Membranous budding and vacuolization occurred,forming a sunflower-shaped structure.Cell necrosis was found under severe conditions.FCM analysis of V-FITC/PI-double labeled Annexin revealed that Celecoxib(40,80μmol/L) treatment resulted in significant early apoptosis with cell apoptosis rates of (3.86±0.23)%,(15.25±0.87)%and(7.17±0.64)%,(20.06±1.06)%at 24,48 h of incubation,respectively,as compared with control ones of 0%and(1.057±0.10)%at 24,48 h(P=0.000).The results indicate that Celecoxib inhibit cell proliferation in a dose-dependent manner.Conclusion:Celecoxib inhibits growth and proliferation of Tca8113 cells mainly in a dose-dependent manner by inhibiting cell cycle process and by inducing cell apoptosis.
Ⅲ.COX-2 inhibitor Celecoxib enhances the lethal effects of anticancer agents in Tca8113 cell line of HTSCC
Objective:To explore the role of Celecoxib in enhancing the lethal effects of anticancer agents in Tca8113 cell line.Methods:24 h after cultivation of Tca8113 cell line,different concentrations of Celecoxib,CDDP and BLM were added into the cells, with the final concentrations being 2.5,5,10,20,40,80μmol/L(Celecoxib),0, 3.125,6.25,12.5,50 mg/L(CDDP) and 0,3.125,6.25,12.5,50 mg/L(BLM), respectively.MTT was used to calculate cell growth inhibition rate after incubation for 24,48,72 h,respectively.According to IC_(50) of the above agents,optimal concentration for interaction of Celecoxib was selected(drug concentration＜IC_(50)). We combined Celecoxib(10μmol/L) with CDDP(3.12,6.25,12.5 mg/L) and BLM (3.12,12.5,50 mg/L) to treat Tca8113 cells,respectively.Lethal effects of different drug applications in Tca8113 cells after incubation for 24,48 and 72 h were measured by MTT.Flow cytometry(FCM) was used to evaluate the effects of combined drug applications on cell cycles.Experimental data were statistically processed by S-N-K test first.Jin Zheng Jun's Method was used to calculate q values,the criterion for evaluating the interaction of Celecoxib and anticancer drugs,q=1±0.15 indicates additive effect;q＞1.15 means synergistic effect;and q＜0.85 refers to antagonism. Results:Celecoxib(10μmol/L) combined either with CDDP or with BLM showed synergism or additive lethal effect on Tca8113 cell line.The way of interaction varied with the time elapsed.Additive effect was dominant after 24 h's and 72 h's incubation, while synergism was dominant between 48～72 h.Celecoxib with CDDP or BLM could notably enhance the effect of CDDP and BLM by blocking cell cycle after 24 h. The effect was most significant when it blocked G_1-staged cells into S stage,thus increasing G_0/G_1 cells distribution and reducing S-staged and G_2/M-staged cells.The percentage of G_0/G_1-staged cells increased from(58.53±1.00)%(CDDP only) to (61.83±0.50)%(CDDP+Celecoxib,P=0.001) and from(56.5±0.96)%(BLM only) to (60.93±0.32)%(BLM+Celecoxib,P=0.000),accompanied by the reduced cells at S and G_2/M stages.Cell at S stage reduced from(31.3±0.70)%to(29.63±0.64)% (CDDP,P=0.175) and(30.3±1.95)%to(30.57±0.78)%(BLM,P=0.849).Cells at G_2/M stage changed with cells at G_0/G_1 and S stages.Conclusions:Combination of Celecoxib with anticancer drug CDDP or BLM could obviously increase the lethal effect of CDDP and BLM on Tca8113 cells by inhibiting their growth and survival. Low doses of Celecoxib could significantly enhance the lethal effect of anticancer drugs CDDP and BLM on Tca8113 cells by inhibiting cell growth and proliferation through cell cycle blockage.The ways of these interactions on inhibiting Tca8113 cell growth were synergistic or/and additive.
Ⅳ.In vivo study of the antitumous effects of Celecoxib combined with cisplatin on Tca8113 cell xenografts
Objective To further investigate whether Celecoxib combined with anticancer drugs can inhibit the growth of Tca8113 cell line-transplanted tumor in tumor-bearing nude mice.Methods:Inoculate Tca8113 cells(2×10~6 cells) into 32 Balb/c nude mice subcutaneously.Nude mice were divided randomly into 4 groups(8 mice each): control,Celecoxib treated,CDDP treated and Celecoxib+CDDP treated.Intragastric administration of Celecoxib,200 mg/kg·d~(-1),intraperitoneal injection of CDDP 5 mg/kg·w~(-1),or both was applied to mice in different groups.In control group, intraperitoneal injection of normal saline and oral administration of sterile purified water were performed.Body weights and tumor sizes were measured for calculating the volume of the nude mice every seven days.Nude mice were sacrificed at 35 d after drug administration.Tumor masses were dissected and weighed for calculating tumor inhibition rates.Histological and electron microscopic analysis were used to observe nude mice tumor tissues.Immunohistochemistry staining was employed for COX-2 and VEGF-C protein expression in tumor tissues.Fluorescent quantitive real-time RT-PCR was used to detect COX-2 and VEGF-C mRNA expression in transplanted tumor tissues of each group,t 7 days after inoculation of Tca8113 cells into nude mice,tumor growth was confirmed in the injection sites in all groups.Since 14 days after administration of drugs,transplanted tumors in drug-treated groups grew slowly as compared with other groups,of which is most significant in Celecoxib+CDDP group.The above-mentioned fact indicates that tumor growth be inhibited by either Celecoxib or CDDP,or both,and that Celecoxib might act in accordance with CDDP.When the experiment terminated,the average length and short diameter of the tumor in control group were notably larger than those of other treatment groups.Tumors in control group grew most quickly with an average weight of(1.28±0.15)g,while tumors in Celecoxib+CDDP group were lightest with an average weight of(0.22±0.09)g.Tumor inhibition rates were 15.63%,37.5%and 82.81%in groups of Celecoxib-treated,CDDP-treated and Celecoxib+CDDP-treated, respectively.In light microscopy,tumor cell ischemic necrosis and prominent apoptosis in some regions were found in Celecoxib-treated group.Typical cytotoxic phenomenon occurred in CDDP-treated group,showing cell degeneration and necrosis,cellular swelling,liquefaction,plasmarrhexis,karyolysis even nuclear deprivation.Apoptosis was rare.In Celecoxib+CDDP-treated group,coagulation necrosis of tumor cells and more apoptotic cells were seen.Under TEM,Apoptotic cells were found in Celecoxib-treated group,especially in Celecoxib+CDDP-treated group.Cell shrinkage,organdie degeneration,higher electron density,lesser cell volume with intact plasmalemma,condensed but homogenous nuclear chromatin, nuclear fragmentation and absence of nucleoli.Immunohistochemical results showed that the expression of COX-2 and VEGF-C was strongly positive in control group, but significantly weaker in both Celecoxib-treated and Celecoxib+CDDP-treated group.The factorial analysis of variance demonstrated that both Celecoxib and CDDP showed their main effect and interaction on growth inhibition of xenografts.The multiproportion of cycle threshold(Ct) of COX-2 mRNA and VEGF-C mRNA in each treat group,compared with control,was also analysised with factorial analysis of variance.The results indicated that both Celecoxib and CDDP showed their significanct main effect and interaction on suppression of VEGF-C mRNA expression, but no significance of main effect or interaction on suppression of COX-2 mRNA expression.Conclusion:COX-2 inhibitor Celecoxib could not only inhibit nude mice transplanted tumor growth,but also enhance the effect of cytotoxic agent CDDP on growth inhibition of xenografts of Tca8113 cell line.
In conclusion,in the present study,we found that COX-2 and VEGF-C were strongly positiveexpression in tongue carcinoma tissues,and were closely correlated with the biological behaviors of squamous cell carcinoma of tongue.Selective COX-2 inhibitor Celecoxib could inhibit growth and proliferation of Tca8113 cells of HTSCC by blocking cell cycle proceeding and by inducing cell apoptosis.Meanwhile, Celecoxib could enhance the cytotoxicity of CDDP and BLM on Tca8113 cells. Celecoxib together with CDDP and BLM showed synergism or additive effects of cytotoxcity on Tca8113 cells;Celecoxib could inhibit Tca8113 cell transplanted tumor growth,and after combined application with CDDP,it manifested more significant inhibition of nude mice tumor growth Our results provide better basis for our further study of COX-2 and oral cancer,and for the prevention of COX-2 inhibitor in oral cancer.
引文
[1] Botting RM. Cyclooxygenase: Past, present and future. A tribute to John R. Vane (1927-2004) [J]. J Therm Biol, 2006, 31 (1-2): 208-219.
[2] Hemler M, Lands WE. Purification of the cyclooxygenase that forms prostaglandins: demonstration of two forms of iron in the holoenzyme[J]. J Biol Chem, 1976,251(18):5575-9.
[3] DeWitt DL and Smith WL. Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence[J]. Proc Natl Acad Sci USA, 1988, 85(14):5056.
[4] Merlie JP, Fagan D, Mudd J. et al. Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase)[J]. J Biol Chem, 1988,263(8):3550-3.
[5] Yokoyama C, Takai T, Tanabe T. Primary structure of sheep prostaglandin endoperoxide synthase deduced from cDNA sequence[J]. FEBS Lett, 1988,231(2): 347-51.
[6] Picot D, Loll PJ, Garavito RM. The X-ray crystal structure of the membrane protein prostaglandin H_2 synthase-1[J]. Nature (Lond.), 1994,367 (6460):243-9.
[7] Malkowski MG, Ginell SL, Smith WL, et al. The productive conformation of arachidonic acid bound to prostaglandin synthase[J]. Science, 2000, 289(5486):1933-7.
[8] Chandrasekharan NV, Dai H, Roos KL. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs cloning,structure and expression[J]. Proc Natl Acad Sci USA, 2002,99(21):13926-31.
[9] Rosen GD, Birkenmeier TM, Raz A, et al. Identification of a cyclooxygenase-related gene and its potential role in prostaglandin formation[J]. Biochem Biophys Res Commun, 1989,164(3):1358-65.
[10] Raz A, Wyche A, Needleman P. Temporal and pharmacological division of fibroblast cyclooxygenase expression into transcriptional and translational phases[J]. Proc Natl Acad Sci U S A, 1989, 86(5): 1657-61.
[11] Fu JY, Masferrer JL, Seibert K et al. The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes[J]. J Biol Chem, 1990,265(28):16737-40.
[12] Simmons DL, Levy DB, Yannoni Y, et al. Identification of a phorbol ester-repressible v-src-inducible gene[J]. Proc Natl Acad Sci USA, 1989, 86(4):1178-82.
[13] Xie WL, Chipman JG, Robertson DL, et al. Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing[J]. Proc Natl Acad Sci USA, 1991, 88(7):2692-6.
[14] Kujubu DA, Fletcher BS, Varnum B.C, et al. TIS10, a phorbol ester tumor promoter- inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/ cyclooxygenase homologue[J]. J Biol Chem, 1991,266(20):12866-72.
[15] O'Banion MK, Sadowski HB, Winn V, et al. A serum- and glucocorticoid-regulated 4-kilobase mRNA encodes a cyclooxygenase-related protein[J]. J Biol Chem, 1991,266(34): 23261-7.
[16] Sirois J, Richards JS. Purification and characterisation of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles[J]. J Biol Chem,1992,267(9):6382-8.
[17] de Groot DJ, de Vries EG, Groen HJ, et al. Non-steroidal anti-inflammatory drugs to potentiate chemotherapy effects: from lab to clinic[J].Crit Rev Oncol Hematol, 2007,61(1):52-69.
[18] Dempke W, Rie C, Grothey A, et al. Cyclooxygenase-2: a novel target for cancer chemotherapy?[J]. J Cancer Res Clin Oncol, 2001,127(7):411-7.
[19] Chen CC, Sun YT, Chen JJ, et al. Tumor necrosis factor-alphainduced cyclooxygenase-2 expression via sequential activation of ceramide-dependent mitogen-activated protein kinases, and IkappaB kinase 1/2 in human alveolar epithelial cells[J]. Mol Pharmacol, 2001, 59(3):493-500.
[20] Howe LR, Subbaramaiah K, Brown AM, et al. Cyclooxygenase-2: a target for the prevention and treatment of breast cancer [J]. Endocr Relat Cancer, 2001, 8(2):97-114.
[21] Kulkarni S, Rader JS, Zhang F, et al. Cyclooxygenase-2 is overexpressed in human cervical cancer [J]. Clin Cancer Res, 2001, 7(2):429-34.
[22] Shiota G, Okubo M, Noumi T, et al. Cyclooxygenase-2 expression in hepatocellular carcinoma [J]. Hepatogastroenterology, 1999,46(25):407-12.
[23] Yoshimura R, Sano H, Masuda C, et al. Expression of cyclooxygenase-2 in prostate carcinoma [J]. Cancer, 2000, 89(3):589-96.
[24] Shirahama T, Arima J, Akiba S, et al. Relation between cyclooxygenase-2 expression and tumor invasiveness and patient survival in transitional cell carcinoma of the urinary bladder [J]. Cancer, 2001, 92(1):188-93.
[25] Renkonen J, Wolff H, Paavonen Y. Expression of cyclo-oxygenase-2 in human tongue carcinoma and its precursor lesions[J]. Virchows Arch, 2002, 440(6):594-7.
[26] Sung JJ, Leung WK, Go MY, et al. Cyclooxygenase-2 expression in Helicobacter pylori-associated premalignant and malignant gastric lesions [J].Am J Pathol, 2000,157(3):729-35.
[27] Hosomi Y, Yokose T, Hirose Y, et al. Increased cyclooxygenase 2 (COX-2) expression occurs frequently in precursor lesions of human adenocarcinoma of the lung [J]. Lung Cancer, 2000, 30(2):73-81.
[28] Achiwa H, Yatabe Y, Hida T, et al. Prognostic significance of elevated cyclooxygenase 2 expression in primary, resected lung adenocarcinomas[J].Clin Cancer Res, 1999,5 (5):1001-5.
[29] Thun MJ, Namboodiri MM, Heath Jr CW, et al. Aspirin use and reduced risk of fatal colon cancer[J]. N Engl J Med, 1991,325(23):1593-6.
[30] Chan AT, Giovannucci EL, Meyerhardt JA, et al. Aspirin dose and duration of use and risk of colorectal cancer in men[J]. Gastroenterology, 2008,134(1):21-8.
[31] Genkinger JM, De Vivo I, Stampfer MJ, et al. Nonsteroidal antiinflammatory drug use and risk of bladder cancer in the health professionals follow-up study[J]. Int J Cancer, 2007,120(10):2221-5.
[32] Jacobs EJ, Rodriguez C, Mondul AM, et al. A large cohort study of aspirin and other nonsteroidal anti-inflammatory drugs and prostate cancer incidence[J]. J Natl Cancer Inst, .2005, 97(13):975-80.
[33] Fortuny J, Johnson CC, Bohlke K, et al. Use of anti-inflammatory drugs and lower esophageal sphincter-relaxing drugs and risk of esophageal and gastric cancers[J]. Clin Gastroenterol Hepatol, 2007, 5(10):1154-9.
[34] Shiotani H, Denda A, Yamamoto K, et al. Increased expression of cyclooxygenase-2 protein in 4-nitroquinoline-1-oxide-induced rat tongue carcinomas and chemopreventive efficacy of a specific inhibitor, nimesulide[J].Cancer Res, 2001, 61:1451 -6.
[35] Mitchell JA,Akarasereenont P,Thiemermann C,et al. Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase[J]. Proc Natl Acad Sci USA, 1993, 90(24):11693-7.
[36] Bombardier C, Laine L, Reicin A, et al.Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis.VIGOR Study Group [J].N Engl J Med, 2000,343(21):1520-8.
[37] Nussmeier NA, Whelton AA, Brown MT, et al. Complications of the COX-2 inhibitors parecoxib and valdecoxib after cardiac surgery[J]. N Engl J Med,2005,352(11):1081-91.
[38] Lanas A, Garcia-Rodriguez LA, Arroyo MT, et al. Risk of upper gastrointestinal ulcer bleeding associated with selective cyclo-oxygenase-2 inhibitors,traditional non-aspirin non-steroidal anti-inflammatory drugs, aspirin and combinations[J]. Gut, 2006,55(12):1731-8.
[39] Meric JB, Rottey S, Olaussen K, et al. Cyclooxygenase-2 as a target for anticancer drug development[J]. Crit Rev Oncol Hematol, 2006, 59(1):51-64.
[40] Kurumbail RG,Stevens AM,Gierse JK, et al. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents[J]. Nature, 1996,384:(6610):644-8.
[41] Kang KB, Wang TT, Woon CT, et al. Enhancement of glioblastoma radioresponse by a selective COX-2 inhibitor Celecoxib:Inhibition of tumor angiogenesis with extensive tumor necrosis[J].Int J Radiation Oncology Biol Phys,2007,67(3):888-896.
[42]Ohneseit PA,Krebiehl G,Dittmann K,et al.Inhibition of cyclooxygenase-2activity by Celecoxib does not lead to radiosensitization of human prostate cancer cells in vitro[J].Radiother Oncol,2007,82(2):229-38.
[43]Goldenberg MM.Celecoxib,a selective cyclooxygenase-2 inhibitor for the treatment of rheumatoid arthritis and osteoarthritis[J].Clin Ther,1999,21(9):1497-513.
[44]Penning TD,Talley JJ,Bertenshaw SR,et al.Synthesis and biological evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors:identification of 4-[5-(4-methylphenyl)-3-(trifluoromethy1)-1H-pyrazol-1-y1]benze nesulfonamide(SC-58635,celecoxib)[J].J Med Chem,1997,40(9):1347-65.
[45]Brooks PM,Day RO.COX-2 inhibitors[J].Med J Aust,2000,173(8):433-6.
[46]Hawkey CJ.COX-2 inhibitors[J].Lancet,1999,353(9149):307-14.
[47]Steinbach G,Lynch PM,Phillips RK,et al.The effect of celecoxib,a cyclooxygenase-2 inhibitor,in familial adenomatous polyposis[J].N Eng1 J Med,2000,342(26):1946-52.
[48]Kismet K,Akay MT,Abbasoglu O,et al.Celecoxib:a potent cyclooxygenase-2inhibitor in cancer prevention[J].Cancer Detect Prev,2004,28(2):127-42.
[49]Wang Z,Fuentes CF,Shapshay SM.Antiangiogenic and chemopreventive activities of Celecoxib in oral carcinoma cell[J].Laryngoscope,2002,112(5):839-43.
[50]Terakado N,Shintani S,Yano J,et al.Overexpression of cyclooxygenase-2 is associated with radioresistance in oral squamous cell carcinoma[J].Oral Oncol,2004,40(4):383-9.
[51]Hashitani S,Urade M,Nishimura N,et al.Apoptosis induction and enhancement of cytotoxicity of anticancer drugs by Celecoxib,a selective cyclooxygenase-2 inhibitor,in human head and neck carcinoma cell line[J].Int J Oncol, 2003,23(3):665-72.
[52] Kwak YE, Jeon NK, Kim J, et al. The cyclooxygenase-2 selective inhibitor Celecoxib suppresses proliferation and invasiveness in the human oral squamous carcinoma[J]. Ann N Y Acad Sci, 2007,1095:99-112.
[53] Kyzas PA, Stefanou D, Agnantis NJ. COX-2 expression correlates with VEGF-C and lymph node metastases in patients with head and neck squamous cell carcinoma[J]. Mod Pathol, 2005,18(1):153-60.
[54] Soumaoro LT, Uetake H, Takagi Y, et al. Coexpression of VEGF-C and Cox-2 in Human Colorectal Cancer and its Association With Lymph Node Metastasis[J]. Dis Colon Rectum, 2006,49(3): 392-8.
[55] Franchi A, Massi D, Santucci M, et al. Inducible nitric oxide synthase activity correlates with lymphangiogenesis and vascular endothelial growth factor-C expression in head and neck squamous cell carcinoma[J]. J Pathol, 2006,208(3):439-45.
[56] O-charoenrat P, Rhys-Evans P, Eccles S A. Expression of Vascular Endothelial Growth Factor Family Members in Head and Neck Squamous Cell Carcinoma Correlates with Lymph Node Metastasis[J]. Cancer, 2001,92(3):556-68.
[57] Weidner N, Semphe JP, Welch WR, et al. Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma [J]. N Eng J Med, 1991,324(1):1-8.
[58] Yoshida K, Hirose Y, Tanaka T, et al. Inhibitory effects of troglitazone, a peroxisome proliferator- activated receptor γ ligand, in rat tongue carcinogenesis initiated with 4-nitroquinoline l-oxide[J]. Cancer Sci, 2003,94(4): 365-71.
[59] Hayes A, Scase T, Miller J, et al. COX-1 and COX-2 Expression in Feline Oral Squamous Cell Carcinoma[J]. J Comp Path, 2006,135(2-3):93-9.
[60] Shibata M, Kodani I, Osaki M, et al. Cyclo-oxygenase-1 and -2 expression in human oral mucosa, dysplasias and squamous cell carcinomas and their pathological significance[J]. Oral Oncol, 2005, 41(3): 304-12.
[61] Joukov V, Pajusola K, Kaipainen A, et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases[J]. EMBO J, 1996,15(7): 290-8.
[62] Liu CH, Chang SH, Narko K, et al. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice[J]. J Biol Chem, 2001,276(21):18563-9.
[63] Masferrer JL, Leahy KM, Koki AT, et al. Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors[J]. Cancer Res, 2000,60(5):1306-11.
[64] Kakiuchi Y, Tsuji S, Tsujii M. Cyclooxygenase-2 activity altered the cell-surface carbohydrate antigens in colon cancer cells and enhanced liver metastasis[J]. Cancer Res, 2002, 62(5):1567-72.
[65] Sedivy R, Beck-Mannagetta J, Haverkampf C, et al. Expression of vascular endothelial growth factor-C correlates with the lymphatic microvessel density and the nodal status in oral squamous cell cancer[J]. J Oral Pathol Med, 2003,32(8):455-60.
[66] Gallo O, Franchi A, Magnelli L, et al. Cyclooxygenase-2 Pathway Correlates with VEGF Expression in Head and Neck Cancer. Implications for Tumor Angiogenesis and Metastasis[J]. Neoplasia, 2001,3(1): 53-61.
[67] Schliephake H. Prognostic relevance of molecular markers of oral cancer-A review[J]. Int J Oral Maxillofac Surg, 2003,32(3):233-45.
[68] Konno H, Abe J, Kaneko T, et al .Urokinase receptor and vascular endothelial growth factor are synergistically associated with the liver metastasis of colorectal cancer [J]. Jpn J Cancer Res, 2001, 92(5):516-23.
[69] Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors[J], Nat Rev Cancer, 2002,2(10):727-39.
[70] Daniel TO, Liu H, Morrow JD, et al. Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis[J].Cancer Res, 1999, 59(18):4574-7.
[71] Tsujii M, Kawano S, Tsuji S, et al. Cyclooxygenase regulates angiogenesis induced by colon cancer cells[J]. Cell, 1998,93(5):705-16.
[72] Ferrara N, Houck KA, Jakeman LB, et al. The vascular endothelial growth factor family of polypeptides[J]. J Cell Biochem, 1991,47(3):211-8.
[73] Kato H, Yoshikawa M, Miyazaki T, et al. Expression of vascular endothelial growth factor (VEGF) and its receptors (Fit-1 and Flk-1) in esophageal squamous cell carcinoma[J]. Anticancer Res, 2002,22(6C):3977-84.
[74] Smith BD, Smith GL, Carter D, et al. Prognostic significance of vascular endothelial growth factor protein levels in oral and oropharyngeal squamous cell carcinoma[J]. J Clin Oncol, 2000,18(10):2046-52.
[75] Tae K, El-Naggar A K, Yoo Enise, et al. Expression of Vascular Endothelial Growth Factor and Microvessel Density in Head and Neck Tumorigenesis[J].Clinical Cancer Research, 2000,6(7):2821-8.
[76] Teknos TN, Cox C, Barrios MA, et al. Tumor angiogenesis as a predictive marker for organ preservation in patients with advanced laryngeal carcinoma[J].aryngoscope, 2002,112(5):844-51.
[77] Johnstone S, Logan RM. The role of vascular endothelial growth factor (VEGF) in oral dysplasia and oral squamous cell carcinoma[J]. Oral Oncol, 2006, 42(4):337-42
[78] Su JL, Shih JY, Yen ML, et al. Cyclooxygenase-2 induces EP1- and HER-2/Neu-dependent vascular endothelial growth factor-C up-regulation: a novel mechanism of lymphangiogenesis in lung adenocarcinoma[J]. Cancer Res,2004,64(2):554-64.
[79] Beasley NJ, Prevo R, Banerji S, et al. Intratumoral lymphangiogenesis and lymph node metastases in head and neck cancer[J]. Cancer Res, 2002, 62(5):1315-20.
[80] Shintani S, Li C, Ishikawa T, et al. Expression of vascular endothelial growth factor A, B, C, and D in oral squamous cell carcinoma[J]. Oral Oncol, 2004,40(1):13-20.
[81] Kardosh A, Blumenthal M, Wang WJ, et al. Differential effects of selective COX-2 inhibitors on cell cycle regulation and proliferation of glioblastoma cell lines[J]. Cancer Biol Ther, 2004, 3(1):55-62.
[82] Narayanan BA, Condon MS, Bosland MC, et al. Suppression of N-methyl-N- nitrosourea/testosterone-induced rat prostate cancer growth by celecoxib:effects on cyclooxygenase-2,cell cycle regulation,and apoptosis mechanism(s)[J].Clin Cancer Res,2003,9(9):3503-13.
[83]Gr(o|¨)sch S,Tegeder I,Niederberger E,et al.COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2inhibitor celecoxib[J].FASEB J,2001,15(4):2742-4.
[84]Maier TJ,Schilling K,Schmidt R,et al.Cyclooxygenase-2(COX-2)-dependent and -independent anticarcinogenic effects of celecoxib in human colon carcinoma cells[J].Biochem Pharmacol,2004,67(8):1469-78.
[85]Han C,Leng J,Demetris AJ,et al.Cyclooxygenase-2 promotes human cholangiocarcinoma growth:evidence for cyclooxygenase-2-independent mechanism in celecoxib-mediated induction of p21waf1/cip1 and p27kip1 and cell cycle arrest[J].Cancer Res,2004,64(4):1369-76.
[86]Bock JM,Menon SG,Sinclair LL,et al.Celecoxib toxicity is cell cycle phase specific[J].Cancer Res,2007,67(8):3801-8.
[87]Basu GD,Pathangey LB,Tinder TL,et al.Cyclooxygenase-2 inhibitor induces apoptosis in breast cancer cells in an in vivo model of spontaneous metastatic breast cancer[J].Mol Cancer Res,2004,2(11):632-42.
[88]Arico S,Pattingre S,Bauvy C,et al.Celecoxib induces apoptosis by inhibiting 3-phosphoinositide-dependent protein kinase-1 activity in the human colon cancer HT-29 cell line[J].J Biol Chem,2002,277(31):27613-21.
[89]Zhu J,Huang JW,Tseng PH,et al.From the cyclooxygenase-2 inhibitor celecoxib to a novel class of 3-phosphoinositide-dependent protein kinase-1inhibitors[J].Cancer Res,2004,64(12):4309-18.
[90]Kulp SK,Yang YT,Hung CC,et al.3-phosphoinositide-dependent protein kinase-1/Akt signaling represents a major cyclooxygenase-2-independent target for celecoxib in prostate cancer cells[J].Cancer Res,2004,64(4):1444-51.
[91]Zhou BP,Liao Y,Xia W,et al.Cytoplasmic localization of p21Cipl/WAF1 by Akt-induced phosphorylation in HER-2/neu- overexpressing cells[J].Nat Cell Biol,2001,3(3):245-52.
[92] Gao N, Flynn DC, Zhang Z, et al. Gl cell cycle progression and the expression of G1 cyclins are regulated by PI3K/AKT/mTOR/p70S6K1 signaling in human ovarian cancer cells[J]. Am J Physiol Cell Physiol, 2004,287(2):C281-91.
[93] Liang J, Slingerland JM. Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression[J]. Cell Cycle, 2003,2(4):339-45.
[94] Liang J, Zubovitz J, Petrocelli T, et al. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest[J]. Nat Med, 2002,8(10):1153-60.
[95] Viglietto G, Motti ML, Bruni P, et al. Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer[J]. Nat Med, 2002, 8(10):1136-44.
[96] Pene F, Claessens YE, Muller O, et al. Role of the phosphatidylinositol 3-kinase/ Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma[J]. Oncogene, 2002,21(43):6587-97.
[97] Zhang Z, Lai GH, Sirica AE. Celecoxib-induced apoptosis in rat cholangiocarcinoma cells mediated by Akt inactivation and Bax translocation[J].Hepatology, 2004,39(4): 1028-37.
[98] Kundu N, Smyth MJ, Samsel L, et al. Cyclooxygenase inhibitors block cell growth, increase ceramide and inhibit cell cycle[J]. Breast Cancer Res Treat,2002, 76(1):57-64.
[99] Ruvolo PP. Intracellular signal transduction pathways activated by ceramide and its metabolites[J]. Pharmacol Res, 2003,47(5):383-92.
[100] Ostrowski J, Wocial T, Skurzak H, et al. Do altering in ornithine decarboxylase activity and gene expression contribute to antiproliferative properties of COX inhibitors[J]? Br J Cancer, 2003, 88(7):1143-51.
[101]Gerner EW, Meyskens FL Jr. Polyamines and cancer: old molecules, new understanding[J]. Nat Rev Cancer, 2004, 4(10):781-92.
[102]Wolter F, Ulrich S, Stein J. Molecular mechanisms of the chemopreventive effects of resveratrol and its analogs in colorectal cancer: key role of polyamines[J]? J Nutr, 2004,134(12):3219-22.
[103]Hsu AL,Ching TT,Wang DS,et al.The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bc1-2[J].J Biol Chem,2000,275(15):11397-403.
[104]Lim ML,Lum MG,Hansen TM,et al.On the release of cytochrome c from mitochondria during cell death signaling[J].J Biomed Sci,2002,9(6 Pt 1):488-506.
[105]Green DR,Evan GI.A matter of life and death[J].Cancer Cell,2002,1(1):19-30.
[106]Jendrossek V,Handrick R,Belka C.Celecoxib activates a novel mitochondrial apoptosis signaling pathway[J].FASEB J,2003,17(11):1547-9.
[107]Lin MT,Lee RC,Yang PC,et al.Cyclooxygenase-2 inducing Mcl-1-dependent survival mechanism in human lung adenocarcinoma CL1.0 cells.Involvement of phosphatidylinositol 3-kinase/Akt pathway[J].J Biol Chem,2001,276(52):48997-9002.
[108]Nam DH,Park K,Park C,et al.Intracranial inhibition of glioma cell growth by cyclooxygenase-2 inhibitor celecoxib[J].Oncol Rep,2004,11(2):263-8.
[109]Wun T,McKnight H,Tuscano JM.Increased cyclooxygenase-2(COX-2):a potential role in the pathogenesis of lymphoma[J].Leuk Res,2004,28(2):179-90.
[110]Dandekar DS,Lopez M,Carey RI,et al.Cyclooxygenase-2 inhibitor celecoxib augments chemotherapeutic drug-induced apoptosis by enhancing activation of caspase-3 and -9 in prostate cancer cells[J].Int J Cancer,2005,115(3):484-92.
[111]Catalano A,Graciotti L,Rinaldi L,et al.Preclinical evaluation of the nonsteroidal anti-inflammatory agent celecoxib on malignant mesothelioma chemoprevention[J].Int J Cancer,2004,109(3):322-8.
[112]Kern MA,Schubert D,Sahi D,et al.Proapoptotic and antiproliferative potential of selective cyclooxygenase-2 inhibitors in human liver tumor cells[J].Hepatology,2002,36(4 Pt 1):885-94.
[113]Kim SH,Song SH,Kim SG,et al.Celecoxib induces apoptosis in cervical cancer cells independent of cyclooxygenase using NF-kappaB as a possible target[J]. J Cancer Res Clin Oncol, 2004,130(9):551-60.
[114] Liu X, Yue P, Zhou Z, et al. Death receptor regulation and celecoxib-induced apoptosis in human lung cancer cells. J Natl Cancer Inst, 2004,96(23): 1769-80.
[115] Lin HP, Kulp SK, Tseng PH, et al. Growth inhibitory effects of celecoxib in human umbilical vein endothelial cells are mediated through G1 arrest via multiple signaling mechanisms[J]. Mol Cancer Ther. 2004,3(12):1671-80.
[116]Hanada M, Feng J, Hemmings BA. Structure, regulation and function of PKB/AKT-a major therapeutic target[J]. Biochim Biophys Acta, 2004,1697(1-2): 3-16.
[117] Lin CF, Chen CL, Chang WT, et al. Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramideand etoposide- induced apoptosis[J]. J Biol Chem, 2004, 279(39):40755-61.
[118]Stoica BA, Movsesyan VA, Knoblach SM, et al. Ceramide induces neuronal apoptosis through mitogen-activated protein kinases and causes release of multiple mitochondrial proteins[J]. Mol Cell Neurosci, 2005,29(3):355-71.
[119] Gomez Del Pulgar T, Velasco G, Sanchez C, et al. De novo-synthesized ceramide is involved in cannabinoid-induced apoptosis[J]. Biochem J, 2002,363(Pt 1):183-8.
[120]Siskind LJ. Mitochondrial ceramide and the induction of apoptosis[J]. J Bioenerg Biomembr, 2005, 37(30): 143-53.
[121] Johnson AJ, Hsu AL, Lin HP, et al. The cyclo-oxygenase-2 inhibitor celecoxib perturbs intracellular calcium by inhibiting endoplasmic reticulum Ca2+-ATPases: a plausible link with its anti-tumour effect and cardiovascular risks[J]. Biochem J, 2002,366(Pt 3):831-7.
[122] Wang S, El-Deiry WS. Cytochrome c: a crosslink between the mitochondria and the endoplasmic reticulum in calcium-dependent apoptosis[J]. Cancer Biol Ther, 2004,3(1):44-6.
[123]Matsuyama M, Yoshimura R, Mitsuhashi M, et al. 5-Lipoxygenase inhibitors attenuate growth of human renal cell carcinoma and induce apoptosis through arachidonic acid pathway[J]. Oncol Rep, 2005,14(1):73-9.
[124]Shureiqi I,Xu X,Chen D,et al.Nonsteroidal anti-inflammatory drags induce apoptosis in esophageal cancer cells by restoring 15-lipoxygenase-1expression[J].Cancer Res,2001,61(12):4879-84.
[125]Shureiqi I,Jiang W,Zuo X,et al.The 15-lipoxygenase-1 product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-delta to induce apoptosis in colorectal cancer cells[J].Proc Nat1 Acad Sci USA,2003,100(17):9968-73.
[126]Wu J,Xia HH,Tu SP,et al.15-Lipoxygenase-1 mediates cyclooxygenase-2inhibitor-induced apoptosis in gastric cancer[J].Carcinogenesis,2003,24(2):243-7.
[127]Brabender J,Park J,Metzger R,et al.Prognostic significance of cyclooxygenase 2 mRNA expression in non-small cell lung cancer[J].Ann Surg,2002,235(6):440-3.
[128]Shariat SF,Kim JH,Ayala GE,et al.Cyclooxygenase-2 is highly expressed in carcinoma in situ and T1 transitional cell carcinoma of the bladder[J],J Urol,2003,169(3):938-42.
[129]Choe MS,Chen Z,Klass CM,et al.Enhancement of docetaxel-induced cytotoxicity by blocking epidermal growth factor receptor and cyclooxygenase-2 pathways in squamous cell carcinoma of the head and neck[J].Clin Cancer Res,2007,13(10):3015-23.
[130]郭建友,霍海如,姜廷良。衡量联合用药作用研究方法评价[J]。中药药理与临床,2005,21(3):60-64.
[131]Awara WM,E1-Sisi AE,E1-Sayad ME,et al.The potential role of cyclooxygenase-2 inhibitors in the treatment of experimentally-induced mammary tumour:does Celecoxib enhance the anti-tumour activity of doxorubicin?[J].Pharmacol Res,2004,50(5):487-98.
[132]Mizutani Y,Nakanishi H,Li YN,et al.Enhanced sensitivity of bladder cancer cells to cisplatin mediated cytotoxicity and apoptosis in vitro and in vivo by the selective cyclooxygenase-2 inhibitor JTE-522[J].J Urol,2004,172(4Pt 1):1474-9.
[133] Raju U, Ariga H, Dittmann K, et al. Inhibition of DNA repair as a mechanism of enhanced radioresponse of head and neck carcinoma cells by a selective cyclooxygenase-2 inhibitor, Celecoxib[J]. Int J Radiat Oncol Biol Phys, 2005,63(2): 520-8.
[134] Dawson SJ, Michael M, Biagi J, et al. A phase I/II trial of Celecoxib with chemotherapy and radiotherapy in the treatment of patients with locally advanced oesophageal cancer[J]. Invest New Drugs, 2007,25(2):123-9.
[135] Lim YJ, Rhee JC, Bae YM, et al. Celecoxib attenuates 5-fluorouracil-induced apoptosis in HCT-15 and HT-29 human colon cancer cells[J].World J Gastroenterol, 2007,13(13):1947-52.
[136] Lin J, Hsiao PW, Chiu TH, et al. Combination of cyclooxygenase-2 inhibitors and oxaliplatin increases the growth inhibition and death in human colon cancer cells[J]. Biochemical Pharmacology, 2005, 70(5): 658-7.
[137]Naruse T, Nishida Y, Ishiguro N. Synergistic effects of meloxicam and conventional cytotoxic drugs in human MG-63 osteosarcoma cells[J]. Biomed Pharmacother, 2007,61(6):338-46.
[138] van Wijngaarden J, van Beek E, van Rossum G, et al. Celecoxib enhances doxorubicin- induced cytotoxicity in MDA-MB231 cells by NF-kappaB-mediated increase of intracellular doxorubicin accumulation[J]. Eur J Cancer,2007,43(2): 433-42.
[139] Wall NR,O'Connor DS, Plescia J, et al. Suppression of surviving phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis[J].Cancer Res, 2003,63 (1):230-5.
[140]Altieri DC. The molecular basis and potential role of survivin in cancer diagnosis and therapy[J]. Trends Mol Med, 2001, 7 (12):542-7.
[141] Rodel C, Haas J, Groth A, et al. Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: survivin as a radioresistance factor[J]. Int J Radiat Oncol Biol Phys, 2003, 55(5):1341-7.
[142]Krysan K, Merchant FH, Zhu L, et al. COX-2-dependent stabilization of survivin in non-small cell lung cancer[J]. FASEB J, 2004,18(1):206-8.
[143]Krysan K, Dalwadi H, Sharma S, et al. Cyclooxygenase 2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer[J].Cancer Res, 2004,64(18):6359-62.
[144] Violette S, Poulain L, Dussaulx E, et al. Resistance of colon cancer cells to long-term 5-fluorouracil exposure is correlated to the relative level of Bcl-2 and Bcl-X(L) in addition to Bax and p53 status[J]. Int J Cancer, 2002, 98(4):498-504.
[145] Zhang L, Yu J, Park BH, et al. Role of BAX in the apoptotic response to anticancer agents[J]. Science, 2000,290(5493):989-92.
[146] Zhou XM, Wong BC, Fan XM, et al. Non-steroidal anti-inflammatory drugs induce apoptosis in gastric cancer cells through up-regulation of bax and bak[J].Carcinogenesis, 2001,22(9):1393-7.
[147] Andrews P, Zhao X, Allen J, et al. A comparison of the effectiveness of selected non-steroidal anti-inflammatory drugs and their derivatives against cancer cells in vitro[J]. Cancer Chemother Pharmacol, 2008, 61 (2):203-14.
[148] Kang HK,Lee E,Pyo H, et al. Cyclooxygenase-independent down-regulation of multidrug resistance-associated protein-1 expression by celecoxib in human lung cancer cells[J]. Mol Cancer Ther, 2005,4(9):1358-63.
[149] Puhlmann U,Ziemann C,Ruedell G, et al. Impact of the cyclooxygenase system on doxorubicin-induced functional multidrug resistance 1 overexpression and doxorubicin sensitivity in acute myeloid leukemic HL-60 cells[J]. J Pharmacol Exp Ther, 2005, 312(1):346-54.
[150] Chen WS, Liu JH, Liu JM, et al. Sequence-dependent effect of a cyclooxygenase-2 inhibitor on topoisomerase I inhibitor and 5-fluorouracil-induced cytotoxicity of colon cancer cells[J]. Anticancer Drugs, 2004, 15(3):287-94.
[151] Chang DZ, Abbruzzese JL. Capecitabine plus oxaliplatin vs infusional 5-fluorouracil plus oxaliplatin in the treatment of colorectal cancer. Pro: The CapeOx regimen is preferred over FOLFOX [J]. Clin Adv Hematol Oncol,2005,3(5):400-4.
[152]Hochster H, Chachoua A, Speyer J, et al. Oxaliplatin with weekly bolus fluorouracil and low-dose leucovorin as first-line therapy for patients with colorectal cancer[J]. J Clin Oncol, 2003,21(14):2703-7.
[153]Teicher BA, Korbut TT, Menon K, et al. Cyclooxygenase and lipoxygenase inhibitors as modulators of cancer therapies[J]. Cancer Chemother Pharmacol,1994, 33(6): 515-22.
[154]Hattori K, Matsushita R, Kimura K, et al. Synergistic effect of indomethacin with adriamycin and cisplatin on tumor growth[J]. Biol Pharm Bull, 2001,24(10): 1214-7.
[155] Miller RA, Woodburn KW, Fan Q, et al. Motexafin gadolinium: a redox active drug that enhances the efficacy of bleomycin and doxorubicin[J]. Clin Cancer Res, 2001,7(10):3215-21.
[156]Knapp DW, Glickman NW, Widmer WR, et al. Cisplatin versus cisplatin combined with piroxicam in a canine model of human invasive urinary bladder cancer[J]. Cancer Chemother Pharmacol, 2000,46(3):221-6.
[157] Lin E, Morris JS, Ayers GD. Effect of celecoxib on capecitabine-induced hand-foot syndrome and antitumor activity[J]. Oncology (Williston Park), 2002,16(12 Suppl No14):31-7.
[158]Kobayashi S, Okada S, Hasumi T, et al. The marked anticancer effect of combined VCR, MTX, and indomethacin against drug-resistant recurrent small cell lung carcinoma after conventional chemotherapy: report of a case[J]. Surg Today, 1999,29(7):666-9.
[159]Patel VA, Dunn MJ, Sorokin A. Regulation of MDR-1 (P-glycoprotein) by cyclooxygenase-2[J]. J Biol Chem, 2002,277(41):38915-20.
[160]Ratnasinghe D, Daschner PJ, Anver MR, et al. Cyclooxygenase-2,P-glycoprotein-170 and drug resistance, is chemoprevention against multidrug resistance possible[J]? Anticancer Res, 2001,21(3C):2141-7.
[161] Cheng HF, Wang CJ, Moeckel GW, et al. Cyclooxygenase-2 inhibitor blocks expression of mediators of renal injury in a model of diabetes and hypertension[J]. Kidney Int, 2002, 62(3):929-39.
[162] Agarwal B, Swaroop P, Protiva P, et al. Cox-2 is needed but not sufficient for apoptosis induced by Cox-2 selective inhibitors in colon cancer cells[J].Apoptosis, 2003, 8(6):649-54.
[163] Chao JI, Kuo PC, Hsu TS. Down-regulation of survivin in nitric oxide-induced cell growth inhibition and apoptosis of the human lung carcinoma cells[J]. J Biol Chem, 2004, 279(19):20267-76.
[164] Blanc-Brude OP, Mesri M, Wall NR, et al. Therapeutic targeting of the survivin pathway in cancer: initiation of mitochondrial apoptosis and suppression of tumor-associated angiogenesis[J]. Clin Cancer Res, 2003, 9(7):2683-92.
[165] Wall NR, O'Connor DS, Plescia J, et al. Suppression of survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis[J].Cancer Res, 2003, 63(1):230-5.
[166] Chiou SK, Jones MK, Tarnawski AS. Survivin - an anti-apoptosis protein: its biological roles and implications for cancer and beyond[J]. Med Sci Monit,2003,9(4):PI25-9.
[2] Hemler M, Lands WE. Purification of the cyclooxygenase that forms prostaglandins: demonstration of two forms of iron in the holoenzyme[J]. J Biol Chem, 1976,251(18):5575-9.
[3] DeWitt DL and Smith WL. Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence[J]. Proc Natl Acad Sci USA, 1988, 85(14):5056.
[4] Merlie JP, Fagan D, Mudd J. et al. Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase)[J]. J Biol Chem, 1988,263(8):3550-3.
[5] Yokoyama C, Takai T, Tanabe T. Primary structure of sheep prostaglandin endoperoxide synthase deduced from cDNA sequence[J]. FEBS Lett, 1988,231(2): 347-51.
[6] Picot D, Loll PJ, Garavito RM. The X-ray crystal structure of the membrane protein prostaglandin H_2 synthase-1[J]. Nature (Lond.), 1994,367 (6460):243-9.
[7] Malkowski MG, Ginell SL, Smith WL, et al. The productive conformation of arachidonic acid bound to prostaglandin synthase[J]. Science, 2000, 289(5486):1933-7.
[8] Chandrasekharan NV, Dai H, Roos KL. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs cloning,structure and expression[J]. Proc Natl Acad Sci USA, 2002,99(21):13926-31.
[9] Rosen GD, Birkenmeier TM, Raz A, et al. Identification of a cyclooxygenase-related gene and its potential role in prostaglandin formation[J]. Biochem Biophys Res Commun, 1989,164(3):1358-65.
[10] Raz A, Wyche A, Needleman P. Temporal and pharmacological division of fibroblast cyclooxygenase expression into transcriptional and translational phases[J]. Proc Natl Acad Sci U S A, 1989, 86(5): 1657-61.
[11] Fu JY, Masferrer JL, Seibert K et al. The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes[J]. J Biol Chem, 1990,265(28):16737-40.
[12] Simmons DL, Levy DB, Yannoni Y, et al. Identification of a phorbol ester-repressible v-src-inducible gene[J]. Proc Natl Acad Sci USA, 1989, 86(4):1178-82.
[13] Xie WL, Chipman JG, Robertson DL, et al. Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing[J]. Proc Natl Acad Sci USA, 1991, 88(7):2692-6.
[14] Kujubu DA, Fletcher BS, Varnum B.C, et al. TIS10, a phorbol ester tumor promoter- inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/ cyclooxygenase homologue[J]. J Biol Chem, 1991,266(20):12866-72.
[15] O'Banion MK, Sadowski HB, Winn V, et al. A serum- and glucocorticoid-regulated 4-kilobase mRNA encodes a cyclooxygenase-related protein[J]. J Biol Chem, 1991,266(34): 23261-7.
[16] Sirois J, Richards JS. Purification and characterisation of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles[J]. J Biol Chem,1992,267(9):6382-8.
[17] de Groot DJ, de Vries EG, Groen HJ, et al. Non-steroidal anti-inflammatory drugs to potentiate chemotherapy effects: from lab to clinic[J].Crit Rev Oncol Hematol, 2007,61(1):52-69.
[18] Dempke W, Rie C, Grothey A, et al. Cyclooxygenase-2: a novel target for cancer chemotherapy?[J]. J Cancer Res Clin Oncol, 2001,127(7):411-7.
[19] Chen CC, Sun YT, Chen JJ, et al. Tumor necrosis factor-alphainduced cyclooxygenase-2 expression via sequential activation of ceramide-dependent mitogen-activated protein kinases, and IkappaB kinase 1/2 in human alveolar epithelial cells[J]. Mol Pharmacol, 2001, 59(3):493-500.
[20] Howe LR, Subbaramaiah K, Brown AM, et al. Cyclooxygenase-2: a target for the prevention and treatment of breast cancer [J]. Endocr Relat Cancer, 2001, 8(2):97-114.
[21] Kulkarni S, Rader JS, Zhang F, et al. Cyclooxygenase-2 is overexpressed in human cervical cancer [J]. Clin Cancer Res, 2001, 7(2):429-34.
[22] Shiota G, Okubo M, Noumi T, et al. Cyclooxygenase-2 expression in hepatocellular carcinoma [J]. Hepatogastroenterology, 1999,46(25):407-12.
[23] Yoshimura R, Sano H, Masuda C, et al. Expression of cyclooxygenase-2 in prostate carcinoma [J]. Cancer, 2000, 89(3):589-96.
[24] Shirahama T, Arima J, Akiba S, et al. Relation between cyclooxygenase-2 expression and tumor invasiveness and patient survival in transitional cell carcinoma of the urinary bladder [J]. Cancer, 2001, 92(1):188-93.
[25] Renkonen J, Wolff H, Paavonen Y. Expression of cyclo-oxygenase-2 in human tongue carcinoma and its precursor lesions[J]. Virchows Arch, 2002, 440(6):594-7.
[26] Sung JJ, Leung WK, Go MY, et al. Cyclooxygenase-2 expression in Helicobacter pylori-associated premalignant and malignant gastric lesions [J].Am J Pathol, 2000,157(3):729-35.
[27] Hosomi Y, Yokose T, Hirose Y, et al. Increased cyclooxygenase 2 (COX-2) expression occurs frequently in precursor lesions of human adenocarcinoma of the lung [J]. Lung Cancer, 2000, 30(2):73-81.
[28] Achiwa H, Yatabe Y, Hida T, et al. Prognostic significance of elevated cyclooxygenase 2 expression in primary, resected lung adenocarcinomas[J].Clin Cancer Res, 1999,5 (5):1001-5.
[29] Thun MJ, Namboodiri MM, Heath Jr CW, et al. Aspirin use and reduced risk of fatal colon cancer[J]. N Engl J Med, 1991,325(23):1593-6.
[30] Chan AT, Giovannucci EL, Meyerhardt JA, et al. Aspirin dose and duration of use and risk of colorectal cancer in men[J]. Gastroenterology, 2008,134(1):21-8.
[31] Genkinger JM, De Vivo I, Stampfer MJ, et al. Nonsteroidal antiinflammatory drug use and risk of bladder cancer in the health professionals follow-up study[J]. Int J Cancer, 2007,120(10):2221-5.
[32] Jacobs EJ, Rodriguez C, Mondul AM, et al. A large cohort study of aspirin and other nonsteroidal anti-inflammatory drugs and prostate cancer incidence[J]. J Natl Cancer Inst, .2005, 97(13):975-80.
[33] Fortuny J, Johnson CC, Bohlke K, et al. Use of anti-inflammatory drugs and lower esophageal sphincter-relaxing drugs and risk of esophageal and gastric cancers[J]. Clin Gastroenterol Hepatol, 2007, 5(10):1154-9.
[34] Shiotani H, Denda A, Yamamoto K, et al. Increased expression of cyclooxygenase-2 protein in 4-nitroquinoline-1-oxide-induced rat tongue carcinomas and chemopreventive efficacy of a specific inhibitor, nimesulide[J].Cancer Res, 2001, 61:1451 -6.
[35] Mitchell JA,Akarasereenont P,Thiemermann C,et al. Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase[J]. Proc Natl Acad Sci USA, 1993, 90(24):11693-7.
[36] Bombardier C, Laine L, Reicin A, et al.Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis.VIGOR Study Group [J].N Engl J Med, 2000,343(21):1520-8.
[37] Nussmeier NA, Whelton AA, Brown MT, et al. Complications of the COX-2 inhibitors parecoxib and valdecoxib after cardiac surgery[J]. N Engl J Med,2005,352(11):1081-91.
[38] Lanas A, Garcia-Rodriguez LA, Arroyo MT, et al. Risk of upper gastrointestinal ulcer bleeding associated with selective cyclo-oxygenase-2 inhibitors,traditional non-aspirin non-steroidal anti-inflammatory drugs, aspirin and combinations[J]. Gut, 2006,55(12):1731-8.
[39] Meric JB, Rottey S, Olaussen K, et al. Cyclooxygenase-2 as a target for anticancer drug development[J]. Crit Rev Oncol Hematol, 2006, 59(1):51-64.
[40] Kurumbail RG,Stevens AM,Gierse JK, et al. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents[J]. Nature, 1996,384:(6610):644-8.
[41] Kang KB, Wang TT, Woon CT, et al. Enhancement of glioblastoma radioresponse by a selective COX-2 inhibitor Celecoxib:Inhibition of tumor angiogenesis with extensive tumor necrosis[J].Int J Radiation Oncology Biol Phys,2007,67(3):888-896.
[42]Ohneseit PA,Krebiehl G,Dittmann K,et al.Inhibition of cyclooxygenase-2activity by Celecoxib does not lead to radiosensitization of human prostate cancer cells in vitro[J].Radiother Oncol,2007,82(2):229-38.
[43]Goldenberg MM.Celecoxib,a selective cyclooxygenase-2 inhibitor for the treatment of rheumatoid arthritis and osteoarthritis[J].Clin Ther,1999,21(9):1497-513.
[44]Penning TD,Talley JJ,Bertenshaw SR,et al.Synthesis and biological evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors:identification of 4-[5-(4-methylphenyl)-3-(trifluoromethy1)-1H-pyrazol-1-y1]benze nesulfonamide(SC-58635,celecoxib)[J].J Med Chem,1997,40(9):1347-65.
[45]Brooks PM,Day RO.COX-2 inhibitors[J].Med J Aust,2000,173(8):433-6.
[46]Hawkey CJ.COX-2 inhibitors[J].Lancet,1999,353(9149):307-14.
[47]Steinbach G,Lynch PM,Phillips RK,et al.The effect of celecoxib,a cyclooxygenase-2 inhibitor,in familial adenomatous polyposis[J].N Eng1 J Med,2000,342(26):1946-52.
[48]Kismet K,Akay MT,Abbasoglu O,et al.Celecoxib:a potent cyclooxygenase-2inhibitor in cancer prevention[J].Cancer Detect Prev,2004,28(2):127-42.
[49]Wang Z,Fuentes CF,Shapshay SM.Antiangiogenic and chemopreventive activities of Celecoxib in oral carcinoma cell[J].Laryngoscope,2002,112(5):839-43.
[50]Terakado N,Shintani S,Yano J,et al.Overexpression of cyclooxygenase-2 is associated with radioresistance in oral squamous cell carcinoma[J].Oral Oncol,2004,40(4):383-9.
[51]Hashitani S,Urade M,Nishimura N,et al.Apoptosis induction and enhancement of cytotoxicity of anticancer drugs by Celecoxib,a selective cyclooxygenase-2 inhibitor,in human head and neck carcinoma cell line[J].Int J Oncol, 2003,23(3):665-72.
[52] Kwak YE, Jeon NK, Kim J, et al. The cyclooxygenase-2 selective inhibitor Celecoxib suppresses proliferation and invasiveness in the human oral squamous carcinoma[J]. Ann N Y Acad Sci, 2007,1095:99-112.
[53] Kyzas PA, Stefanou D, Agnantis NJ. COX-2 expression correlates with VEGF-C and lymph node metastases in patients with head and neck squamous cell carcinoma[J]. Mod Pathol, 2005,18(1):153-60.
[54] Soumaoro LT, Uetake H, Takagi Y, et al. Coexpression of VEGF-C and Cox-2 in Human Colorectal Cancer and its Association With Lymph Node Metastasis[J]. Dis Colon Rectum, 2006,49(3): 392-8.
[55] Franchi A, Massi D, Santucci M, et al. Inducible nitric oxide synthase activity correlates with lymphangiogenesis and vascular endothelial growth factor-C expression in head and neck squamous cell carcinoma[J]. J Pathol, 2006,208(3):439-45.
[56] O-charoenrat P, Rhys-Evans P, Eccles S A. Expression of Vascular Endothelial Growth Factor Family Members in Head and Neck Squamous Cell Carcinoma Correlates with Lymph Node Metastasis[J]. Cancer, 2001,92(3):556-68.
[57] Weidner N, Semphe JP, Welch WR, et al. Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma [J]. N Eng J Med, 1991,324(1):1-8.
[58] Yoshida K, Hirose Y, Tanaka T, et al. Inhibitory effects of troglitazone, a peroxisome proliferator- activated receptor γ ligand, in rat tongue carcinogenesis initiated with 4-nitroquinoline l-oxide[J]. Cancer Sci, 2003,94(4): 365-71.
[59] Hayes A, Scase T, Miller J, et al. COX-1 and COX-2 Expression in Feline Oral Squamous Cell Carcinoma[J]. J Comp Path, 2006,135(2-3):93-9.
[60] Shibata M, Kodani I, Osaki M, et al. Cyclo-oxygenase-1 and -2 expression in human oral mucosa, dysplasias and squamous cell carcinomas and their pathological significance[J]. Oral Oncol, 2005, 41(3): 304-12.
[61] Joukov V, Pajusola K, Kaipainen A, et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases[J]. EMBO J, 1996,15(7): 290-8.
[62] Liu CH, Chang SH, Narko K, et al. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice[J]. J Biol Chem, 2001,276(21):18563-9.
[63] Masferrer JL, Leahy KM, Koki AT, et al. Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors[J]. Cancer Res, 2000,60(5):1306-11.
[64] Kakiuchi Y, Tsuji S, Tsujii M. Cyclooxygenase-2 activity altered the cell-surface carbohydrate antigens in colon cancer cells and enhanced liver metastasis[J]. Cancer Res, 2002, 62(5):1567-72.
[65] Sedivy R, Beck-Mannagetta J, Haverkampf C, et al. Expression of vascular endothelial growth factor-C correlates with the lymphatic microvessel density and the nodal status in oral squamous cell cancer[J]. J Oral Pathol Med, 2003,32(8):455-60.
[66] Gallo O, Franchi A, Magnelli L, et al. Cyclooxygenase-2 Pathway Correlates with VEGF Expression in Head and Neck Cancer. Implications for Tumor Angiogenesis and Metastasis[J]. Neoplasia, 2001,3(1): 53-61.
[67] Schliephake H. Prognostic relevance of molecular markers of oral cancer-A review[J]. Int J Oral Maxillofac Surg, 2003,32(3):233-45.
[68] Konno H, Abe J, Kaneko T, et al .Urokinase receptor and vascular endothelial growth factor are synergistically associated with the liver metastasis of colorectal cancer [J]. Jpn J Cancer Res, 2001, 92(5):516-23.
[69] Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors[J], Nat Rev Cancer, 2002,2(10):727-39.
[70] Daniel TO, Liu H, Morrow JD, et al. Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis[J].Cancer Res, 1999, 59(18):4574-7.
[71] Tsujii M, Kawano S, Tsuji S, et al. Cyclooxygenase regulates angiogenesis induced by colon cancer cells[J]. Cell, 1998,93(5):705-16.
[72] Ferrara N, Houck KA, Jakeman LB, et al. The vascular endothelial growth factor family of polypeptides[J]. J Cell Biochem, 1991,47(3):211-8.
[73] Kato H, Yoshikawa M, Miyazaki T, et al. Expression of vascular endothelial growth factor (VEGF) and its receptors (Fit-1 and Flk-1) in esophageal squamous cell carcinoma[J]. Anticancer Res, 2002,22(6C):3977-84.
[74] Smith BD, Smith GL, Carter D, et al. Prognostic significance of vascular endothelial growth factor protein levels in oral and oropharyngeal squamous cell carcinoma[J]. J Clin Oncol, 2000,18(10):2046-52.
[75] Tae K, El-Naggar A K, Yoo Enise, et al. Expression of Vascular Endothelial Growth Factor and Microvessel Density in Head and Neck Tumorigenesis[J].Clinical Cancer Research, 2000,6(7):2821-8.
[76] Teknos TN, Cox C, Barrios MA, et al. Tumor angiogenesis as a predictive marker for organ preservation in patients with advanced laryngeal carcinoma[J].aryngoscope, 2002,112(5):844-51.
[77] Johnstone S, Logan RM. The role of vascular endothelial growth factor (VEGF) in oral dysplasia and oral squamous cell carcinoma[J]. Oral Oncol, 2006, 42(4):337-42
[78] Su JL, Shih JY, Yen ML, et al. Cyclooxygenase-2 induces EP1- and HER-2/Neu-dependent vascular endothelial growth factor-C up-regulation: a novel mechanism of lymphangiogenesis in lung adenocarcinoma[J]. Cancer Res,2004,64(2):554-64.
[79] Beasley NJ, Prevo R, Banerji S, et al. Intratumoral lymphangiogenesis and lymph node metastases in head and neck cancer[J]. Cancer Res, 2002, 62(5):1315-20.
[80] Shintani S, Li C, Ishikawa T, et al. Expression of vascular endothelial growth factor A, B, C, and D in oral squamous cell carcinoma[J]. Oral Oncol, 2004,40(1):13-20.
[81] Kardosh A, Blumenthal M, Wang WJ, et al. Differential effects of selective COX-2 inhibitors on cell cycle regulation and proliferation of glioblastoma cell lines[J]. Cancer Biol Ther, 2004, 3(1):55-62.
[82] Narayanan BA, Condon MS, Bosland MC, et al. Suppression of N-methyl-N- nitrosourea/testosterone-induced rat prostate cancer growth by celecoxib:effects on cyclooxygenase-2,cell cycle regulation,and apoptosis mechanism(s)[J].Clin Cancer Res,2003,9(9):3503-13.
[83]Gr(o|¨)sch S,Tegeder I,Niederberger E,et al.COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2inhibitor celecoxib[J].FASEB J,2001,15(4):2742-4.
[84]Maier TJ,Schilling K,Schmidt R,et al.Cyclooxygenase-2(COX-2)-dependent and -independent anticarcinogenic effects of celecoxib in human colon carcinoma cells[J].Biochem Pharmacol,2004,67(8):1469-78.
[85]Han C,Leng J,Demetris AJ,et al.Cyclooxygenase-2 promotes human cholangiocarcinoma growth:evidence for cyclooxygenase-2-independent mechanism in celecoxib-mediated induction of p21waf1/cip1 and p27kip1 and cell cycle arrest[J].Cancer Res,2004,64(4):1369-76.
[86]Bock JM,Menon SG,Sinclair LL,et al.Celecoxib toxicity is cell cycle phase specific[J].Cancer Res,2007,67(8):3801-8.
[87]Basu GD,Pathangey LB,Tinder TL,et al.Cyclooxygenase-2 inhibitor induces apoptosis in breast cancer cells in an in vivo model of spontaneous metastatic breast cancer[J].Mol Cancer Res,2004,2(11):632-42.
[88]Arico S,Pattingre S,Bauvy C,et al.Celecoxib induces apoptosis by inhibiting 3-phosphoinositide-dependent protein kinase-1 activity in the human colon cancer HT-29 cell line[J].J Biol Chem,2002,277(31):27613-21.
[89]Zhu J,Huang JW,Tseng PH,et al.From the cyclooxygenase-2 inhibitor celecoxib to a novel class of 3-phosphoinositide-dependent protein kinase-1inhibitors[J].Cancer Res,2004,64(12):4309-18.
[90]Kulp SK,Yang YT,Hung CC,et al.3-phosphoinositide-dependent protein kinase-1/Akt signaling represents a major cyclooxygenase-2-independent target for celecoxib in prostate cancer cells[J].Cancer Res,2004,64(4):1444-51.
[91]Zhou BP,Liao Y,Xia W,et al.Cytoplasmic localization of p21Cipl/WAF1 by Akt-induced phosphorylation in HER-2/neu- overexpressing cells[J].Nat Cell Biol,2001,3(3):245-52.
[92] Gao N, Flynn DC, Zhang Z, et al. Gl cell cycle progression and the expression of G1 cyclins are regulated by PI3K/AKT/mTOR/p70S6K1 signaling in human ovarian cancer cells[J]. Am J Physiol Cell Physiol, 2004,287(2):C281-91.
[93] Liang J, Slingerland JM. Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression[J]. Cell Cycle, 2003,2(4):339-45.
[94] Liang J, Zubovitz J, Petrocelli T, et al. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest[J]. Nat Med, 2002,8(10):1153-60.
[95] Viglietto G, Motti ML, Bruni P, et al. Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer[J]. Nat Med, 2002, 8(10):1136-44.
[96] Pene F, Claessens YE, Muller O, et al. Role of the phosphatidylinositol 3-kinase/ Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma[J]. Oncogene, 2002,21(43):6587-97.
[97] Zhang Z, Lai GH, Sirica AE. Celecoxib-induced apoptosis in rat cholangiocarcinoma cells mediated by Akt inactivation and Bax translocation[J].Hepatology, 2004,39(4): 1028-37.
[98] Kundu N, Smyth MJ, Samsel L, et al. Cyclooxygenase inhibitors block cell growth, increase ceramide and inhibit cell cycle[J]. Breast Cancer Res Treat,2002, 76(1):57-64.
[99] Ruvolo PP. Intracellular signal transduction pathways activated by ceramide and its metabolites[J]. Pharmacol Res, 2003,47(5):383-92.
[100] Ostrowski J, Wocial T, Skurzak H, et al. Do altering in ornithine decarboxylase activity and gene expression contribute to antiproliferative properties of COX inhibitors[J]? Br J Cancer, 2003, 88(7):1143-51.
[101]Gerner EW, Meyskens FL Jr. Polyamines and cancer: old molecules, new understanding[J]. Nat Rev Cancer, 2004, 4(10):781-92.
[102]Wolter F, Ulrich S, Stein J. Molecular mechanisms of the chemopreventive effects of resveratrol and its analogs in colorectal cancer: key role of polyamines[J]? J Nutr, 2004,134(12):3219-22.
[103]Hsu AL,Ching TT,Wang DS,et al.The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bc1-2[J].J Biol Chem,2000,275(15):11397-403.
[104]Lim ML,Lum MG,Hansen TM,et al.On the release of cytochrome c from mitochondria during cell death signaling[J].J Biomed Sci,2002,9(6 Pt 1):488-506.
[105]Green DR,Evan GI.A matter of life and death[J].Cancer Cell,2002,1(1):19-30.
[106]Jendrossek V,Handrick R,Belka C.Celecoxib activates a novel mitochondrial apoptosis signaling pathway[J].FASEB J,2003,17(11):1547-9.
[107]Lin MT,Lee RC,Yang PC,et al.Cyclooxygenase-2 inducing Mcl-1-dependent survival mechanism in human lung adenocarcinoma CL1.0 cells.Involvement of phosphatidylinositol 3-kinase/Akt pathway[J].J Biol Chem,2001,276(52):48997-9002.
[108]Nam DH,Park K,Park C,et al.Intracranial inhibition of glioma cell growth by cyclooxygenase-2 inhibitor celecoxib[J].Oncol Rep,2004,11(2):263-8.
[109]Wun T,McKnight H,Tuscano JM.Increased cyclooxygenase-2(COX-2):a potential role in the pathogenesis of lymphoma[J].Leuk Res,2004,28(2):179-90.
[110]Dandekar DS,Lopez M,Carey RI,et al.Cyclooxygenase-2 inhibitor celecoxib augments chemotherapeutic drug-induced apoptosis by enhancing activation of caspase-3 and -9 in prostate cancer cells[J].Int J Cancer,2005,115(3):484-92.
[111]Catalano A,Graciotti L,Rinaldi L,et al.Preclinical evaluation of the nonsteroidal anti-inflammatory agent celecoxib on malignant mesothelioma chemoprevention[J].Int J Cancer,2004,109(3):322-8.
[112]Kern MA,Schubert D,Sahi D,et al.Proapoptotic and antiproliferative potential of selective cyclooxygenase-2 inhibitors in human liver tumor cells[J].Hepatology,2002,36(4 Pt 1):885-94.
[113]Kim SH,Song SH,Kim SG,et al.Celecoxib induces apoptosis in cervical cancer cells independent of cyclooxygenase using NF-kappaB as a possible target[J]. J Cancer Res Clin Oncol, 2004,130(9):551-60.
[114] Liu X, Yue P, Zhou Z, et al. Death receptor regulation and celecoxib-induced apoptosis in human lung cancer cells. J Natl Cancer Inst, 2004,96(23): 1769-80.
[115] Lin HP, Kulp SK, Tseng PH, et al. Growth inhibitory effects of celecoxib in human umbilical vein endothelial cells are mediated through G1 arrest via multiple signaling mechanisms[J]. Mol Cancer Ther. 2004,3(12):1671-80.
[116]Hanada M, Feng J, Hemmings BA. Structure, regulation and function of PKB/AKT-a major therapeutic target[J]. Biochim Biophys Acta, 2004,1697(1-2): 3-16.
[117] Lin CF, Chen CL, Chang WT, et al. Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramideand etoposide- induced apoptosis[J]. J Biol Chem, 2004, 279(39):40755-61.
[118]Stoica BA, Movsesyan VA, Knoblach SM, et al. Ceramide induces neuronal apoptosis through mitogen-activated protein kinases and causes release of multiple mitochondrial proteins[J]. Mol Cell Neurosci, 2005,29(3):355-71.
[119] Gomez Del Pulgar T, Velasco G, Sanchez C, et al. De novo-synthesized ceramide is involved in cannabinoid-induced apoptosis[J]. Biochem J, 2002,363(Pt 1):183-8.
[120]Siskind LJ. Mitochondrial ceramide and the induction of apoptosis[J]. J Bioenerg Biomembr, 2005, 37(30): 143-53.
[121] Johnson AJ, Hsu AL, Lin HP, et al. The cyclo-oxygenase-2 inhibitor celecoxib perturbs intracellular calcium by inhibiting endoplasmic reticulum Ca2+-ATPases: a plausible link with its anti-tumour effect and cardiovascular risks[J]. Biochem J, 2002,366(Pt 3):831-7.
[122] Wang S, El-Deiry WS. Cytochrome c: a crosslink between the mitochondria and the endoplasmic reticulum in calcium-dependent apoptosis[J]. Cancer Biol Ther, 2004,3(1):44-6.
[123]Matsuyama M, Yoshimura R, Mitsuhashi M, et al. 5-Lipoxygenase inhibitors attenuate growth of human renal cell carcinoma and induce apoptosis through arachidonic acid pathway[J]. Oncol Rep, 2005,14(1):73-9.
[124]Shureiqi I,Xu X,Chen D,et al.Nonsteroidal anti-inflammatory drags induce apoptosis in esophageal cancer cells by restoring 15-lipoxygenase-1expression[J].Cancer Res,2001,61(12):4879-84.
[125]Shureiqi I,Jiang W,Zuo X,et al.The 15-lipoxygenase-1 product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-delta to induce apoptosis in colorectal cancer cells[J].Proc Nat1 Acad Sci USA,2003,100(17):9968-73.
[126]Wu J,Xia HH,Tu SP,et al.15-Lipoxygenase-1 mediates cyclooxygenase-2inhibitor-induced apoptosis in gastric cancer[J].Carcinogenesis,2003,24(2):243-7.
[127]Brabender J,Park J,Metzger R,et al.Prognostic significance of cyclooxygenase 2 mRNA expression in non-small cell lung cancer[J].Ann Surg,2002,235(6):440-3.
[128]Shariat SF,Kim JH,Ayala GE,et al.Cyclooxygenase-2 is highly expressed in carcinoma in situ and T1 transitional cell carcinoma of the bladder[J],J Urol,2003,169(3):938-42.
[129]Choe MS,Chen Z,Klass CM,et al.Enhancement of docetaxel-induced cytotoxicity by blocking epidermal growth factor receptor and cyclooxygenase-2 pathways in squamous cell carcinoma of the head and neck[J].Clin Cancer Res,2007,13(10):3015-23.
[130]郭建友,霍海如,姜廷良。衡量联合用药作用研究方法评价[J]。中药药理与临床,2005,21(3):60-64.
[131]Awara WM,E1-Sisi AE,E1-Sayad ME,et al.The potential role of cyclooxygenase-2 inhibitors in the treatment of experimentally-induced mammary tumour:does Celecoxib enhance the anti-tumour activity of doxorubicin?[J].Pharmacol Res,2004,50(5):487-98.
[132]Mizutani Y,Nakanishi H,Li YN,et al.Enhanced sensitivity of bladder cancer cells to cisplatin mediated cytotoxicity and apoptosis in vitro and in vivo by the selective cyclooxygenase-2 inhibitor JTE-522[J].J Urol,2004,172(4Pt 1):1474-9.
[133] Raju U, Ariga H, Dittmann K, et al. Inhibition of DNA repair as a mechanism of enhanced radioresponse of head and neck carcinoma cells by a selective cyclooxygenase-2 inhibitor, Celecoxib[J]. Int J Radiat Oncol Biol Phys, 2005,63(2): 520-8.
[134] Dawson SJ, Michael M, Biagi J, et al. A phase I/II trial of Celecoxib with chemotherapy and radiotherapy in the treatment of patients with locally advanced oesophageal cancer[J]. Invest New Drugs, 2007,25(2):123-9.
[135] Lim YJ, Rhee JC, Bae YM, et al. Celecoxib attenuates 5-fluorouracil-induced apoptosis in HCT-15 and HT-29 human colon cancer cells[J].World J Gastroenterol, 2007,13(13):1947-52.
[136] Lin J, Hsiao PW, Chiu TH, et al. Combination of cyclooxygenase-2 inhibitors and oxaliplatin increases the growth inhibition and death in human colon cancer cells[J]. Biochemical Pharmacology, 2005, 70(5): 658-7.
[137]Naruse T, Nishida Y, Ishiguro N. Synergistic effects of meloxicam and conventional cytotoxic drugs in human MG-63 osteosarcoma cells[J]. Biomed Pharmacother, 2007,61(6):338-46.
[138] van Wijngaarden J, van Beek E, van Rossum G, et al. Celecoxib enhances doxorubicin- induced cytotoxicity in MDA-MB231 cells by NF-kappaB-mediated increase of intracellular doxorubicin accumulation[J]. Eur J Cancer,2007,43(2): 433-42.
[139] Wall NR,O'Connor DS, Plescia J, et al. Suppression of surviving phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis[J].Cancer Res, 2003,63 (1):230-5.
[140]Altieri DC. The molecular basis and potential role of survivin in cancer diagnosis and therapy[J]. Trends Mol Med, 2001, 7 (12):542-7.
[141] Rodel C, Haas J, Groth A, et al. Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: survivin as a radioresistance factor[J]. Int J Radiat Oncol Biol Phys, 2003, 55(5):1341-7.
[142]Krysan K, Merchant FH, Zhu L, et al. COX-2-dependent stabilization of survivin in non-small cell lung cancer[J]. FASEB J, 2004,18(1):206-8.
[143]Krysan K, Dalwadi H, Sharma S, et al. Cyclooxygenase 2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer[J].Cancer Res, 2004,64(18):6359-62.
[144] Violette S, Poulain L, Dussaulx E, et al. Resistance of colon cancer cells to long-term 5-fluorouracil exposure is correlated to the relative level of Bcl-2 and Bcl-X(L) in addition to Bax and p53 status[J]. Int J Cancer, 2002, 98(4):498-504.
[145] Zhang L, Yu J, Park BH, et al. Role of BAX in the apoptotic response to anticancer agents[J]. Science, 2000,290(5493):989-92.
[146] Zhou XM, Wong BC, Fan XM, et al. Non-steroidal anti-inflammatory drugs induce apoptosis in gastric cancer cells through up-regulation of bax and bak[J].Carcinogenesis, 2001,22(9):1393-7.
[147] Andrews P, Zhao X, Allen J, et al. A comparison of the effectiveness of selected non-steroidal anti-inflammatory drugs and their derivatives against cancer cells in vitro[J]. Cancer Chemother Pharmacol, 2008, 61 (2):203-14.
[148] Kang HK,Lee E,Pyo H, et al. Cyclooxygenase-independent down-regulation of multidrug resistance-associated protein-1 expression by celecoxib in human lung cancer cells[J]. Mol Cancer Ther, 2005,4(9):1358-63.
[149] Puhlmann U,Ziemann C,Ruedell G, et al. Impact of the cyclooxygenase system on doxorubicin-induced functional multidrug resistance 1 overexpression and doxorubicin sensitivity in acute myeloid leukemic HL-60 cells[J]. J Pharmacol Exp Ther, 2005, 312(1):346-54.
[150] Chen WS, Liu JH, Liu JM, et al. Sequence-dependent effect of a cyclooxygenase-2 inhibitor on topoisomerase I inhibitor and 5-fluorouracil-induced cytotoxicity of colon cancer cells[J]. Anticancer Drugs, 2004, 15(3):287-94.
[151] Chang DZ, Abbruzzese JL. Capecitabine plus oxaliplatin vs infusional 5-fluorouracil plus oxaliplatin in the treatment of colorectal cancer. Pro: The CapeOx regimen is preferred over FOLFOX [J]. Clin Adv Hematol Oncol,2005,3(5):400-4.
[152]Hochster H, Chachoua A, Speyer J, et al. Oxaliplatin with weekly bolus fluorouracil and low-dose leucovorin as first-line therapy for patients with colorectal cancer[J]. J Clin Oncol, 2003,21(14):2703-7.
[153]Teicher BA, Korbut TT, Menon K, et al. Cyclooxygenase and lipoxygenase inhibitors as modulators of cancer therapies[J]. Cancer Chemother Pharmacol,1994, 33(6): 515-22.
[154]Hattori K, Matsushita R, Kimura K, et al. Synergistic effect of indomethacin with adriamycin and cisplatin on tumor growth[J]. Biol Pharm Bull, 2001,24(10): 1214-7.
[155] Miller RA, Woodburn KW, Fan Q, et al. Motexafin gadolinium: a redox active drug that enhances the efficacy of bleomycin and doxorubicin[J]. Clin Cancer Res, 2001,7(10):3215-21.
[156]Knapp DW, Glickman NW, Widmer WR, et al. Cisplatin versus cisplatin combined with piroxicam in a canine model of human invasive urinary bladder cancer[J]. Cancer Chemother Pharmacol, 2000,46(3):221-6.
[157] Lin E, Morris JS, Ayers GD. Effect of celecoxib on capecitabine-induced hand-foot syndrome and antitumor activity[J]. Oncology (Williston Park), 2002,16(12 Suppl No14):31-7.
[158]Kobayashi S, Okada S, Hasumi T, et al. The marked anticancer effect of combined VCR, MTX, and indomethacin against drug-resistant recurrent small cell lung carcinoma after conventional chemotherapy: report of a case[J]. Surg Today, 1999,29(7):666-9.
[159]Patel VA, Dunn MJ, Sorokin A. Regulation of MDR-1 (P-glycoprotein) by cyclooxygenase-2[J]. J Biol Chem, 2002,277(41):38915-20.
[160]Ratnasinghe D, Daschner PJ, Anver MR, et al. Cyclooxygenase-2,P-glycoprotein-170 and drug resistance, is chemoprevention against multidrug resistance possible[J]? Anticancer Res, 2001,21(3C):2141-7.
[161] Cheng HF, Wang CJ, Moeckel GW, et al. Cyclooxygenase-2 inhibitor blocks expression of mediators of renal injury in a model of diabetes and hypertension[J]. Kidney Int, 2002, 62(3):929-39.
[162] Agarwal B, Swaroop P, Protiva P, et al. Cox-2 is needed but not sufficient for apoptosis induced by Cox-2 selective inhibitors in colon cancer cells[J].Apoptosis, 2003, 8(6):649-54.
[163] Chao JI, Kuo PC, Hsu TS. Down-regulation of survivin in nitric oxide-induced cell growth inhibition and apoptosis of the human lung carcinoma cells[J]. J Biol Chem, 2004, 279(19):20267-76.
[164] Blanc-Brude OP, Mesri M, Wall NR, et al. Therapeutic targeting of the survivin pathway in cancer: initiation of mitochondrial apoptosis and suppression of tumor-associated angiogenesis[J]. Clin Cancer Res, 2003, 9(7):2683-92.
[165] Wall NR, O'Connor DS, Plescia J, et al. Suppression of survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis[J].Cancer Res, 2003, 63(1):230-5.
[166] Chiou SK, Jones MK, Tarnawski AS. Survivin - an anti-apoptosis protein: its biological roles and implications for cancer and beyond[J]. Med Sci Monit,2003,9(4):PI25-9.