选择性COX-2抑制剂塞来昔布对乳腺癌的放疗增敏作用及其机制研究

[目的]探讨选择性环氧化酶-2（COX-2）抑制剂塞来昔布对人乳腺癌细胞的放疗增敏作用及其可能的机制。[方法]取对数生长期MDA-MB-231细胞株,实验分为对照组、药物组、照射组及实验组（照射线＋塞来昔布）。流式细胞术（FCM）检测细胞周期变化和各组凋亡率;克隆形成实验检测放射增敏作用;Western blot法检测COX-2、VEGF、Bcl-2蛋白表达。[结果]不同浓度塞来昔布对MDA-MB-231细胞有明显抑制作用,且呈剂量依赖性（F=5.56,P=0.030）。FCM检测结果显示,随塞来昔布浓度增加,细胞周期明显变化（G0/G1期比例增高,S期比例降低）。实验组凋亡率较照射组明显高（31.20%±1.27%vs 17.99%±1.01%,t=2.43,P=0.025）。克隆形成实验显示,实验组与照射组相比较,反映放射敏感性指标的Dq、Do和SF2显著降低。塞来昔布能抑制COX-2、VEGF和Bcl-2蛋白表达（P〈0.05）。[结论]选择性COX-2抑制剂塞来昔布对人乳腺癌MDA-MB-231细胞有明显的放射增敏作用,其可能的机制是抑制COX-2表达、诱导细胞凋亡、调节细胞周期分布和抑制细胞血管生成因子生成。     

塞来昔布对不同COX-2表达肿瘤细胞株放射增敏作用及其机制研究

目的探讨选择性环氧化酶抑制剂塞来昔布对不同COX-2蛋白表达水平人类肿瘤细胞株的放射增敏作用及其发生机制。方法选用人乳腺癌细胞系MCF-7(低COX-2蛋白表达)和肺癌细胞系A549(高COX-2蛋白表达)为研究对象。RT-PCR检测COX-2表达,MTT法及成克隆分析法测定塞来昔布对两种细胞系的细胞毒作用及放射增敏作用及流式细胞仪(FCM)检测细胞周期分布、细胞凋亡指数。结果塞来昔布作用于MCF-7细胞30h后的IC_(50)值为(43.7±1.12)μmol/L,作用于A549细胞24 h后的IC_(50)值为(37.5±0.80)μmol/L。塞来昔布联合射线作用于A549细胞,与单纯照射组比较显示了放射增敏作用(P<0.05),而在MCF-7细胞中则没有观察到这些现象。细胞周期检测发现,两种细胞各组都出现了不同的周期分布。塞来昔布组G_0 G_1期细胞比例增加,照射组的细胞则大多处于G_2 M期。不同浓度的塞来昔布10～80μmol/L作用6h后,均未观察到明显的凋亡现象,两种细胞照射组与照射加药组测得的凋亡均无差异。结论塞来昔布对MCF-7、A549细胞都有增殖抑制作用,并呈时问和剂量依赖性,其作用不完全呈COX-2依赖。塞来昔布能增加A549细胞放射敏感性,其机制可能与抑制COX-2蛋白、改变细胞周期分布有关。     

塞来昔布对人三阴性乳腺癌细胞MDA-MB-231迁移、侵袭及黏附性的影响

背景与目的:三阴性乳腺癌(triple-negative breast cancer,TNBC)由于其高侵袭和高转移特性,是目前乳腺癌中预后最差的一种亚型.大量研究表明塞来昔布(celecoxib)具有很好的抗肿瘤活性,可以抑制肿瘤生长、减少肿瘤血管发生,防止肿瘤的早期转移.为了进一步明确塞来昔布与TNBC细胞侵袭活性之间的关系,本实验研究塞来昔布作用下人TNBC细胞MDA-MB-231黏附、迁移及体外侵袭能力的变化,并初步探讨其可能的机制.方法:以不同浓度的塞来昔布(0、40、80、120 μmol/L)作用MDA-MB-231细胞24 h后,采用细胞基质黏附实验检测塞来昔布对MDA-MB-231细胞黏附能力的影响;采用细胞划痕实验检测塞来昔布对MDA-MB-231细胞平面迁移能力的影响;采用Transwell侵袭小室实验检测塞来昔布对MDA-MB-231细胞体外侵袭能力的影响;RT-PCR检测塞来昔布作用前后基质金属蛋白酶(matrix metalloproteinases,MMPs)MMP-2和MMP-9 mRNA的表达情况.结果:经80、120 μmol/L塞来昔布作用24 h后,MDA-MB-231细胞与基质的黏附能力分别为(50.2±7.3)%和(31.5±2.2)%,与对照组(96.8±10.9)%相比显著下降,差异具有统计学意义(P<0.05).Transwell侵袭小室实验结果显示,经80、120 μmol/L塞来昔布作用后MDA-MB-231细胞趋化运动明显减少,穿过人工基底膜的细胞较对照组显著降低(P<0.05).划痕实验结果显示,经塞来昔布作用后MDA-MB-231细胞平面迁移到损伤区的细胞数明显减少(P<0.05).RT-PCR结果显示,经塞来昔布作用MDA-MB-231细胞后,MMP-2和MMP-9 mRNA的表达水平显著降低(P<0.05).结论:塞来昔布能显著地抑制MDA-MB-231细胞黏附、细胞体外的迁移和侵袭能力,该作用可能与抑制MMP-2和MMP-9 mRNA的表达有关.     

塞来昔布对宫颈癌Hela细胞放射增敏效应的研究

目的:探讨塞来昔布(Celecoxib)对人宫颈癌Hela细胞的放射增敏作用及其机制.方法:用四唑盐比色法(MTT)观察塞来昔布对Hela细胞的生长抑制情况;克隆形成实验检测放射增敏作用,流式细胞仪检测单用塞来昔布、放射以及二者联合使用时Hela细胞凋亡率和细胞周期变化.结果:MTT检测结果提示塞来昔布对宫颈癌Hela细胞的抑制作用呈剂量和放射强度依赖性;克隆形成实验提示塞来昔布对Hela细胞株有一定程度的放射增敏效应,塞来昔布+放射(D+R)组较单纯放射(R)组相比,放射敏感性指标SER、Do、Dq均呈现不同程度的下调;流式细胞分析显示:Hela细胞周期分布及凋亡率在塞来昔布(D)组、单纯放射(R)组及塞来昔布+放射(D+R)组中均有差异,表现为G0/G1期增加,S期减少,凋亡率增加.结论:塞来昔布对人宫颈癌Hela细胞有明显的放射增敏作用 ,为临床放疗及与塞来昔布联合运用提供了实验依据.     

塞来昔布通过阻断NF-кB信号通路诱导人乳腺癌细胞MDA-MB-231细胞周期阻滞的相关研究

背景与目的:研究表明塞来昔布能抑制人乳腺癌细胞增殖、诱导其凋亡,但其对肿瘤细胞周期的作用机制尚不明确。本实验研究塞来昔布是否通过阻断NF-кB信号通路对人乳腺癌细胞MDA-MB-231增殖及周期产生影响。方法:MTT法和流式细胞术观察塞来昔布对MDA-MB-231细胞增殖、周期分布的影响;应用RT-PCR和Western印迹法检测经塞来昔布干预该细胞24h后,细胞周期相关因子及NF-кB信号途径中p-IκBα的变化。结果:塞来昔布可显著抑制MDA-MB-231细胞生长,呈时间剂量依赖性。高浓度塞来昔布可改变细胞进程,将其阻滞于G0/G1期。塞来昔布作用24h后,细胞周期相关蛋白CyclinD1、CDK4呈剂量依赖性表达下降(P<0.05)。同时,细胞中p-IκBα表达随药物浓度增加而下降(P<0.05)。结论:塞来昔布能显著抑制MDA-MB-231细胞增殖,诱导细胞G0/G1期阻滞,其作用机制可能是通过抑制IκBα的磷酸化阻断NF-кB信号通路,进而下调其下游基因CyclinD1对细胞周期进行调控。     

塞来昔布对宫颈癌Hela细胞放射增敏效应的研究

目的：探讨塞来昔布（Celecoxib）对人宫颈癌Hela细胞的放射增敏作用及其机制。方法：用四唑盐比色法（M＇IT）观察塞来昔布对Hela细胞的生长抑制情况;克隆形成实验检测放射增敏作用,流式细胞仪检测单用塞来昔布、放射以及二者联合使用时Hela细胞凋亡率和细胞周期变化。结果：M＇IT检测结果提示塞来昔布对宫颈癌Hela细胞的抑制作用呈剂量和放射强度依赖性;克隆形成实验提示塞来昔布对Hela细胞株有一定程度的放射增敏效应,塞来昔布＋放射（D＋R）组较单纯放射（R）组相比,放射敏感性指标SER、Do、Dq均呈现不同程度的下调;流式细胞分析显示：Hela细胞周期分布及凋亡率在塞来昔布（D）组、单纯放射（R）组及塞来昔布＋放射（D＋R）组中均有差异,表现为G0／G1期增加,S期减少,凋亡率增加。结论：塞来昔布对人宫颈癌Hela细胞有明显的放射增敏作用,为临床放疗及与塞来昔布联合运用提供了实验依据。     

塞来昔布通过阻断NF—κB信号通路诱导人乳腺癌细胞MDA—MB-231细胞周期阻滞的相关研究

背景与目的:研究表明塞来昔布能抑制人乳腺癌细胞增殖、诱导其凋亡,但其对肿瘤细胞周期的作用机制尚不明确.本实验研究塞来昔布是否通过阻断NF-κ B信号通路对人乳腺痛细胞MDA-MB-231增殖及周期产生影响.方法:MTT法和流式细胞术观察塞来昔布对MDA-MB-231细胞增殖、周期分布的影响:应用RT-PCR和Western印迹法检测经塞来昔布干预该细胞24 h后,细胞周期相关因子及NF-κ B信号途径中p-Ⅰκ B α的变化.结果:塞来昔布可显著抑制MDA-MB-231细胞生长,呈时间剂量依赖性.高浓度塞来昔布可改变细胞进程,将其阻滞于G0/G1期.塞来昔布作用24 h后,细胞周期相关蛋白CyclinD1、CDK4呈剂量依赖性表达下降(P<0.05).同时,细胞中P-Ⅰκ B α表达随药物浓度增加而下降(P<0.05).结论:塞来昔布能显著抑SUMDA-MB-231细胞增殖,诱导细胞G0/G1期阻滞,其作用机制可能是通过抑制Ⅰκ B α的磷酸化阻断NF-κB信号通路,进而下调其下游基因CyclinD1对细胞周期进行调控.     

塞来昔布对胃癌SGC7901细胞放射敏感性的实验研究

目的:探讨环氧合酶2(COX-2)抑制剂塞来昔布对胃癌SGC7901细胞株的放射增敏作用及其机制。方法:MTT法检测塞来昔布对胃癌SGC7901细胞株的抑制作用,计算出塞来昔布的半数抑制浓度(IC50);克隆形成实验用于检测20%IC50这个浓度的塞来昔布对胃癌SGC细胞是否具有放射敏感性;流式细胞术(FCM)分析细胞周期的分布情况。结果:MTT实验显示塞来昔布对SGC7901细胞株的抑制率随浓度的升高而升高,48 h的IC50是34.38μmol/L;克隆形成实验显示,照射组+药物组与单纯照射组相比,反映放射敏感性指标的存活分数(SF2)、平均致死剂量(D0)及准阈剂量(Dq)均下降,放射增敏比(SER)升高。FCM检测细胞周期G2和M期细胞比例增加,S期细胞比例减少。结论:塞来昔布能增加胃癌SGC7901细胞的放射敏感性,其机制可能与其抑制肿瘤细胞亚致死性损伤修复能力和促进肿瘤细胞周期再分布有关。     

环氧化酶-2抑制剂对肺癌细胞的增殖抑制和放射增敏的实验研究

目的研究环氧化酶-2(COX-2)抑制剂塞来昔布对肺癌A549细胞增殖、凋亡的影响及其对A549细胞的放射增敏作用.方法噻唑蓝(MTT)比色法检测塞来昔布对肺癌A549细胞存活的影响;流式细胞分析法(FCM)检测对A549细胞周期时相影响及凋亡的作用;体外培养克隆形成率的方法检测塞来昔布对A549细胞的放射增敏作用.结果塞来昔布对肺癌A549细胞有明显的增殖抑制作用,这种抑制作用有时间和剂量依赖性,50μmol/L处理24和72 h的抑制率分别为(4.27±1.33)%、(10.63±2.23)%,100μmol/L处理分别为(33.47±8.02)%、(61.97±7.32)%,主要是抑制增殖,使细胞聚集在G0/G1期,100μmol/L处理72 h还有细胞死亡.塞来昔布无凋亡诱导作用,也不增加放射诱导的凋亡.体外培养克隆存活曲线分析显示,100μmol/L塞来昔布处理24和72 h降低了各个照射剂量点的存活分数(72 h比24 h作用强),但在照射高剂量区更明显.对照、100μmol/L处理24、72 h在2 Gy剂量点的存活分数(SF2)分别为(0.53±0.06)、(0.52±0.11)和(0.46±0.05),处理24和72 h的放射增敏比为1.27和1.7(D0值比).结论塞来昔布对肺癌A549细胞有明显的增殖抑制作用,呈时间和剂量依赖性,使细胞聚集在G0/G1期,但无诱导凋亡作用.塞来昔布对肺癌A549细胞有放射增敏作用,在高照射剂量区增敏作用更为明显.     

塞来昔布对胃癌BGC823细胞的放射增敏作用

目的：探讨环氧化酶2（COX-2）抑制剂塞来昔布对人胃癌BGC823细胞株的放射增敏作用及其机制。方法：MTT实验测定塞来昔布对胃癌BGC823细胞株的抑制率,计算半数抑制浓度（IC50）。克隆形成实验检测塞来昔布联合不同剂量X线照射后细胞的存活率,计算放射敏感性相关参数,评价增敏效果。流式细胞仪（FCM）分析细胞周期分布情况。结果：MTT实验显示塞来昔布对BGC823细胞作用24h、48h、72h的IC50分别是162.6μmol/L、95.4μmol/L、62.1μmol/L;克隆形成实验测得联合组的D0、Dq及SF2均明显低于单纯照射组（1.705VS 2.185,1.032VS 2.327,0.495 VS 0.745）,SER为1.3。流式细胞仪检测联合组G0/G1期细胞增多,S期细胞减少。结论：塞来昔布能增强胃癌BGC823细胞的放射敏感性,其机制可能与减少肿瘤细胞亚致死性损伤修复和细胞周期再分布有关。     

Radiosensitivity enhancement by combined treatment of celecoxib and gefitinib on human lung cancer cells.

PURPOSE: To characterize the radiation-enhancing effects and underlying mechanisms of combined treatment with celecoxib, a cyclooxygenase-2 selective inhibitor, and gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, in human lung cancer cells. EXPERIMENTAL DESIGN: Clonogenic cytotoxicity assays and clonogenic radiation survival assays after treatments with celecoxib and gefitinib with or without radiation were done on three human lung cancer cell lines. Synergisms after combined treatment with celecoxib, gefitinib, and radiation were investigated using isobologram and statistical analyses according to an independent action model. Alterations in apoptosis and cell cycle were measured to identify the mechanisms underlying the cell killing or radiation-enhancing effects of celecoxib and gefitinib combination treatment. Western blots for phosphorylated EGFR, EGFR, cyclooxygenase-2, and G(2) checkpoint molecules were conducted after treatment with celecoxib and/or gefitinib with or without radiation. RESULTS: Combination celecoxib, gefitinib, and radiation treatments were shown to be synergistic in causing clonogenic cell deaths in all cell lines tested, but the nature of synergism was cell type specific. The combined drug treatments induced apoptosis in an additive manner in A549 cells and in a synergistic manner in NCI-H460 and VMRC-LCD cells. Celecoxib or gefitinib attenuated radiation-induced G(2)-M arrest, and combined drug treatment additively attenuated radiation-induced G(2)-M arrest in all cell lines. Radiation-induced checkpoint kinase (Chk) 1 and Chk2 phosphorylation were inhibited by celecoxib and gefitinib treatment, respectively. CONCLUSIONS: Combined celecoxib and gefitinib treatments were shown to synergistically enhance the effect of radiation on lung cancer cells. The mechanisms underlying these synergistic effects seem to involve the synergistic enhancement of apoptosis and cooperative attenuation of radiation-induced G(2)-M arrest, possibly via Chk1 and Chk2 inhibition, by the combined drug treatments.     

塞来昔布对脑胶质瘤细胞放射增敏效应的研究

[目的]观察COX-2选择性抑制剂塞来昔布（Celecoxib）对三种脑胶质瘤细胞放射增敏作用。[方法]选择适当浓度的COX-2选择性抑制剂Celecoxib进行放射增敏实验，设置单纯放射（R）组和放射＋药物（R＋D）组，采用集落形成法分别检测Celecoxib对脑胶质瘤SHG-44细胞株，767细胞以及大鼠脑胶质瘤C6细胞株的放射增敏效应，并绘制细胞存活曲线。[结果]Celecoxib对3种胶质瘤细胞株均显示出放射增敏效应，R＋D组与R组相比较，反映放射敏感性指标的SF2、D0.01、D0、Dq均出现下降。其中tSF2=8．315，P=0．014；tD0.01=9．28，P=0．011；tD0=3．985，P=0．058；tDq=3．348，P=O．075。[结论]Celecoxib在体外实验中可以提高脑胶质瘤的放射敏感性。Celecoxib有可能成为治疗胶质瘤的放射增敏剂。     

塞来昔布联合奥曲肽对人胃癌多药耐药细胞生长的影响

背景与目的:环氧合酶-2(cyclooxygenase,COX-2)抑制剂塞来昔布及奥曲肽(octreotide)均对肿瘤细胞有抑制作用,本研究旨在探讨塞来昔布及其与奥曲肽联合对人胃癌多药耐药细胞株SGC7901/ADR生长的影响。方法:塞来昔布组:塞来昔布浓度为1×10-4～1×10-8mol/L;奥曲肽组:奥曲肽浓度为1×10-5～1×10-9mol/L;合用组:塞来昔布在上述浓度范围内加1×10-6mol/L的奥曲肽;对照组:无血清RPMI-1640培养液。比较各实验组对SGC7901、SGC7901/ADR细胞生长的影响。采用3H-胸腺嘧啶核苷(3H-TdR)掺入法检测细胞的增殖;免疫细胞化学检测增殖细胞核抗原(proliferatingcellnuclearantigen,PCNA)的表达;DNA末端原位标记染色法(TUNEL法)及流式细胞仪检测细胞凋亡。结果:塞来昔布较对照组明显降低SGC7901/ADR细胞的3H-TdR掺入,掺入值分别为(471.3±79.7)cpm及(917.5±130.8)cpm,塞来昔布与奥曲肽联合应用时,3H-TdR掺入值较单独用药(220.0±19.7)cpm下降53.3%。两药联合对SGC7901/ADR细胞DNA合成的抑制效应与塞郑文斌,等.塞来昔布联合奥曲肽来昔布的浓度呈显著正相关(r=0.996,P<0.001)。单用塞来昔布或联合用药都能明显降低SGC7901/ADR细胞PCNA的表达。单用塞来昔布时SGC7901/ADR细胞凋亡率为32.9%,当其与奥曲肽合用,凋亡率增加至5     

Inhibition of cyclooxygenase-2 activity by celecoxib does not lead to radiosensitization of human prostate cancer cells in vitro.

PURPOSE: To evaluate the potential radiosensitizing effect of the specific COX-2 inhibitor celecoxib (Celebrex) on prostate carcinoma cells in vitro. MATERIALS AND METHODS: The influence of celecoxib (concentration range 5 to 75 microM) on radiation-induced cellular and clonogenic survival was investigated in prostate carcinoma cell lines PC-3, DU145, LNCaP and normal prostate epithelial cells (PrEC). Western blot analysis and ELISA were used to determine the impact of radiation alone or radiation combined with celecoxib treatment on COX-2 expression and prostaglandin E2 synthesis. To evaluate induction of celecoxib-induced apoptosis cell cycle analysis has been performed. RESULTS: Celecoxib (5, 10 and 25 microM) in combination with single-dose irradiation of 2 Gy induced a significant radiosensitization in normal prostate epithelial cells which could not be observed for any of the prostate carcinoma cell lines investigated. Increased COX-2 protein expression in PC-3 cells was obvious only after IR with 15 Gy, while PGE2 production was elevated following irradiation (2-15 Gy) in a dose-dependent manner. Treatment with celecoxib alone or in combination with IR led to a dose-dependent increase in COX-2 protein expression. Nevertheless pre-treatment with celecoxib caused a marked reduction of radiation-induced enzyme activity as tested at the level of PGE2 production, both in PC-3 and DU145 cells. Following fractionated irradiation with single doses of 2 Gy, elevated COX-2 protein expression as well as enhanced PGE2 production was observed already after the second fraction in PC-3 cells. Pre-treatment with celecoxib reduced the amount of PGE(2) significantly, but not of COX-2 protein. CONCLUSIONS: Our data obtained for the human prostate cancer cell lines do not indicate that a marked inhibition of prostaglandin E2 synthesis by celecoxib leads to enhanced radiosensitization. Thus, in terms of radiosensitization the analysed prostate cancer cells can be classified as non-responders to celecoxib treatment.     

Celecoxib 在人癌裸鼠移植瘤抗癌与放射增敏的作用机制研究

 目的　探讨选择性环氧化酶-2 抑制剂celecoxib 抗癌及放射增敏作用的可能机制。方法　构建人宫颈癌裸鼠移植瘤模型,celecoxib 和/ 或移植瘤局部放射干预,检测移植瘤生长延缓、移植瘤组织中环氧化酶-2 与前列腺素E2 含量的变化,并分析此变化与移植瘤生长延缓的关系。结果　移植瘤最大径从8mm 生长至10mm 所需的时间,空白对照组为(4. 42 ±0. 78) d ,单纯放射组为(6. 25 ±0. 70) d ,celecoxib组为(7. 14 ±1. 06) d ,celecoxib + 放射联合组为(10. 62 ±2. 06) d ;Western Blot 显示4 组移植瘤组织中环氧化酶22 水平无明显差异;前列腺素E2 (pg/ 100mg) 含量分别为:空白对照组69. 07 ±5. 42 ,单纯放射组47. 4 ±15. 94 ,单纯celecoxib 组28. 62 ±4. 48 ,celecoxib + 放射联合组为43. 2 ±11. 73 ,与空白对照组比较差异均有显著性( P = 0. 009 、0. 005 、0. 026) ;但celecoxib + 放射联合组与单纯放射组的前列腺素E₂ 含量无显著差异( P = 0. 28) ;celecoxib 引起的移植瘤生长延缓与前列腺素E₂ 含量降低正相关( r = 0. 741) 。结论　Celecoxib及移植瘤局部放射均能延缓移植瘤生长,不影响环氧化酶-2量的表达,但均能引起前列腺素E₂ 含量的降低;celecoxib 引起的移植瘤生长延缓可能与环氧化酶-2 活性被抑制后前列腺素E₂ 含量的降低有关。     

Celecoxib induced tumor cell radiosensitization by inhibiting radiation induced nuclear EGFR transport and DNA-repair: a COX-2 independent mechanism

PURPOSE: The purpose of the study was to elucidate the molecular mechanisms mediating radiosensitization of human tumor cells by the selective cyclooxygenase (COX)-2 inhibitor celecoxib. METHODS AND MATERIALS: Experiments were performed using bronchial carcinoma cells A549, transformed fibroblasts HH4dd, the FaDu head-and-neck tumor cells, the colon carcinoma cells HCT116, and normal fibroblasts HSF7. Effects of celecoxib treatment were assessed by clonogenic cell survival, Western analysis, and quantification of residual DNA damage by gammaH(2)AX foci assay. RESULTS: Celecoxib treatment resulted in a pronounced radiosensitization of A549, HCT116, and HSF7 cells, whereas FaDu and HH4dd cells were not radiosensitized. The observed radiosensitization could neither be correlated with basal COX-2 expression pattern nor with basal production of prostaglandin E2, but was depended on the ability of celecoxib to inhibit basal and radiation-induced nuclear transport of epidermal growth factor receptor (EGFR). The nuclear EGFR transport was strongly inhibited in A549-, HSF7-, and COX-2-deficient HCT116 cells, which were radiosensitized, but not in FaDu and HH4dd cells, which resisted celecoxib-induced radiosensitization. Celecoxib inhibited radiation-induced DNA-PK activation in A549, HSF7, and HCT116 cells, but not in FaDu and HH4dd cells. Consequentially, celecoxib increased residual gammaH2AX foci after irradiation, demonstrating that inhibition of DNA repair has occurred in responsive A549, HCT116, and HSF7 cells only. CONCLUSIONS: Celecoxib enhanced radiosensitivity by inhibition of EGFR-mediated mechanisms of radioresistance, a signaling that was independent of COX-2 activity. This novel observation may have therapeutic implications such that COX-2 inhibitors may improve therapeutic efficacy of radiation even in patients whose tumor radioresistance is not dependent on COX-2.     

Inhibition of DNA repair as a mechanism of enhanced radioresponse of head and neck carcinoma cells by a selective cyclooxygenase-2 inhibitor, celecoxib

PURPOSE: Previously, we reported that inhibitors of cyclooxygenase-2 (COX-2) enzyme enhanced murine and human tumor cell response to radiation in vitro and in vivo. However, the molecular mechanisms mediating the effects of COX-2 inhibitors are not clear. The present study was designed to investigate the ability of celecoxib, a selective COX-2 inhibitor, to sensitize human head-and-neck cancer cell line, HN5, to radiation, and examine its effects on DNA repair, which may be a potential mechanism of radiosensitization. METHODS AND MATERIALS: Cells were assessed for the effect of celecoxib (5-50 microM), by 3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyltetrazolium bromide assay for growth inhibition and by clonogenic cell survival assay for the radiosensitizing effect. Kinase assay and Western analysis were conducted to assess the effect of celecoxib on DNA-dependent protein kinase catalytic subunit (PKcs) and Ku proteins. Electrophoretic mobility shift assays (EMSA) were performed to determine the DNA-binding activity of Ku/DNA-PKcs protein complex and nuclear factor kappa B (NFkappaB). RESULTS: Celecoxib (10 and 50 microM, for 2 days) inhibited the HN5 cell growth and significantly enhanced the cell radiosensitivity in a dose-dependent manner. It also reduced the shoulder region on the radiation-survival curve, suggesting that inhibition of DNA repair processes may have occurred. Western blot analysis demonstrated that celecoxib downregulated the expression of Ku70 protein and inhibited the kinase activity of DNA-PKcs, which are involved in the double-stranded DNA-break repair machinery. By EMSA, it was further shown that celecoxib reduced DNA-binding activity of Ku/DNA-PKcs protein complex. In addition, celecoxib inhibited the constitutively active NFkappaB and the radiation-induced NFkappaB in HN5 cells, suggesting that NFkappaB may play a role in mediating the effects of celecoxib. CONCLUSIONS: Celecoxib strongly enhanced the sensitivity of HN5 carcinoma cells to radiation, which, mechanistically, can be attributed to the inhibition of DNA repair processes in radiation-damaged cells.     

塞来昔布抑制人肺腺癌细胞的增殖和表皮生长因子受体的表达*

目的:探讨不同浓度的环氧化酶-2 抑制剂塞来昔布（Celecoxib ）在不同时间点对肺腺癌A549 细胞株增殖和表皮生长因子受体（EGFR）表达的影响。方法:人肺腺癌A549 细胞株培养于含10% 胎牛血清RPMI1640培养基中。实验细胞分组如下:A组,正常对照;B 组,Celecoxib（12.5 μ mol/L）;C 组Celecoxib（25μ mol/L）;D 组Celecoxib（50μ mol/L）,E 组Celecoxib（75μ mol/L）,均以RPMI1640培养液配置。使用不同浓度塞来昔布（12.5、25、50和75μ mol/L）处理肺癌A549 细胞24、48、72h 后,四甲基偶氮唑盐比色法（MTT）测定细胞增殖抑制率;AnnexinⅤ/PI 染色法与Hoechst33258 染色法检测细胞凋亡率;流式细胞仪检测药物作用周期;Real-time RT-PCR 法检测EGFR mRNA 的表达情况。结果:塞来昔布明显抑制了A549 细胞的生长,呈时间、剂量依赖性。塞来昔布组细胞凋亡率明显高于正常对照组（P<0.01）,且呈剂量依赖性,S 期细胞比例明显减少（P<0.01）,G0/G1 期细胞比例明显增加（P<0.01）,提示塞来昔布能将大多数A549 细胞阻滞于G0/G1 期。塞来昔布组细胞EGFR mRNA 表达明显减弱（P<0.05）,且呈浓度依赖性,各浓度间差异具有统计学意义（P<0.05）。 结论:塞来昔布显著抑制A549 细胞生长,可能机制是通过促进凋亡、增强G0/G1 期阻滞、下调细胞中EGFR mRNA 的表达,从而为应用塞来昔布治疗肺腺癌,以及塞来昔布和表皮生长因子受体抑制剂联用提供了一定的实验依据。      

Combination of radiation and celebrex (celecoxib) reduce mammary and lung tumor growth

The selective cyclooxygenase (COX)-2 inhibitor, celecoxib, alone and in combination with radiation was investigated in vitro and in vivo. Murine mammary tumor line (MCa-35) and human lung carcinoma line (A549) have high and low basal levels of COX-2 protein, respectively. Treatment of both tumor cells with celecoxib alone resulted in a dose- and time-dependent reduction of cell number (clonogenic cell death) and tumor cell growth rate in vitro; however, inhibition of tumor cell growth by celecoxib was not correlated with the reduction of COX-2 protein in tumor cells. Although both tumor cell types had similar DNA damage after celecoxib treatment, significant induction of tumor cell apoptosis was only observed in MCa-35. Celecoxib-mediated radiation sensitization also occurred in MCa-35 cells determined by clonogenic assay, in part due to a G2/M arrest at 8 to 24 hours after treatment. The tumor growth inhibitory effects of celecoxib were also studied in vivo. It was found that celecoxib inhibited both tumor growth after intragastric administration of celecoxib (5 daily doses of 50 mg/kg). Combined with a single 30-Gy dose of radiation, celecoxib resulted in additive effects on A549 tumors. Celecoxib-treated A549 tumors had marginal reduction of total and perfused blood vessels compared with untreated controls. Reduction of tumor angiogenic cytokine and growth factor mRNA was associated with decreased perfused vessels. Finally, reduction of vascular endothelial growth factor protein after celecoxib was also observed in both tumor lines by Western blot. Our results indicate that the selective inhibition of COX-2 combined with radiation has potential application in radiotherapy, and celecoxib-mediated antitumor effects may act through different mechanisms including direct inhibition of tumor cell proliferation, alteration of tumor cell cycle, and antiangiogenesis.