选择性环氧合酶2抑制剂塞来昔布对人子宫内膜癌细胞的凋亡诱导

目的 研究环氧合酶2(COX-2)选择性抑制剂塞来昔布诱导人子宫内膜癌细胞凋亡作用.方法 体外培养的人子宫内膜癌细胞HEC-1-B经塞来昔布处理后,采用Giemsa染色、流式细胞术和DNA Ladder观察HEC-1-B细胞凋亡变化;通过半定量RT-PCR方法检测HEC-1-B细胞COX-2及凋亡相关基因Fas、survivin的表达.结果 20 μmol/L塞来昔布处理HEC-1-B细胞后,Giemsa染色即可见细胞核固缩、碎裂,出现凋亡小体等细胞凋亡形态变化;流式细胞学检测示50 μmol/L塞来昔布可使HEC-1-B细胞凋亡率达46.9%,PI荧光直方图出现凋亡细胞峰,DNA电泳可见典型的细胞凋亡梯形条带.塞来昔布处理HEC-1-B细胞后,COX-2 mRNA表达下降,细胞凋亡相关基因Fas表达增加,细胞凋亡抑制基因surviyin表达下降,50 μmol/L塞来昔布可使survivin表达难以检出.结论 塞来昔布对HEC-1-B细胞有明显的凋亡诱导作用,通过抑制COX-2表达进而使Fas表达上调,survivin表达下调可能是其诱导细胞凋亡机制之一.     

紫苏醇对肺癌A549 细胞增殖和侵袭的抑制作用机制的研究

目的:探讨紫苏醇(Perillyl alcohol,PA)对肺癌A549 细胞增殖和侵袭的抑制作用机制,并阐明其对肿瘤血管生成信号的影响。方法:不同浓度PA 和厄洛替尼加入到A549 细胞中,利用溴化3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑(MTT)法测定药物组对A549 细胞的抑制作用,Transwell 法检测PA 对A549 细胞侵袭的抑制作用,采用间接荧光标记法测定细胞内活性氧(ROS)水平的变化;分光光度法检测PA 对细胞凋亡蛋白Caspase-3 活性的影响,Western blot 法检测A549 中VEGF、HIF-1 及COX-2 的表达,凝胶迁移或电泳迁移率实验测定A549 细胞中NF-κB 活性。结果:与空白对照组比较,随着浓度的增加(10、50、100 g/ ml),PA 和厄洛替尼对A549 细胞生长的抑制率在不断地增加,差异均有统计学意义(P<0.05),A549 细胞侵袭能力呈现不断下降的趋势,差异均有统计学意义(P<0.05),A549 细胞内活性氧水平随厄洛替尼浓度的增加变化不大,而ROS 水平随着PA 的浓度的增加而增加,在100 g/ ml 浓度的PA 下引起的ROS 百分率达到了(80.43±6.92)%,差异均有统计学意义(P<0.05)。细胞活力检测结果显示,随着PA 和厄洛替尼浓度的增加和作用时间的延长,A549 细胞中凋亡蛋白Caspase-3 活性明显增加(P<0.05),随着紫苏醇浓度的增加,COX-2、VEGF 和HIF-1 的表达呈不断降低的趋势,EMSA 检测结果显示,随着PA 浓度的增加,NF-κB 的条带面积不断减少。结论:PA 可能参与并促进了ROS 的生成和Caspase-3 活性增加,最终诱导A549 细胞的凋亡,PA 可能通过降低NF-κB 的表达,进而诱导COX-2、VEGF 等的表达减少,使血管生成滞后,能够有效地使细胞的穿透能力下降和凋亡发生。     

塞来昔布联合替吉奥对胃癌裸鼠移植瘤生长的抑制作用

目的 研究选择性环氧化酶-2抑制剂塞来昔布联合替吉奥对胃癌裸鼠皮下移植瘤生长的影响及可能机制.方法 建立胃癌裸鼠皮下移植瘤模型,成瘤后将裸鼠随机分为阴性对照组、塞来昔布组、替吉奥组、塞来昔布联合替吉奥组,连续给药21 d后,取材,测量肿瘤体积、计算抑瘤率,TUNEL检测肿瘤组织的凋亡率,免疫组化检测各组PCNA、Bcl-2、caspase-3的表达情况.结果 塞来昔布组、替吉奥组、联合给药组的抑瘤率分别为30.8％、50.1％、78.8％.各干预组较对照组凋亡率明显增加(P＜0.01),联合给药组较单药组凋亡率明显增加(P＜0.01).各干预组PCNA、Bcl-2的表达较对照组明显降低(P＜0.01),联合给药组较单药组明显降低(P＜0.05).各干预组caspase-3的表达较对照组明显升高(P＜0.05),联合给药组表达较单药组明显升高(P＜0.01).结论 塞来昔布、替吉奥均有明显的抗肿瘤作用,二者联合表现为协同作用,可能是抑制肿瘤细胞的增殖、促进凋亡所致.     

塞来昔布对鼻咽癌CNE-2Z细胞株CD44v6表达水平的影响

目的 研究塞来昔布(COX-2选择性抑制剂)对鼻咽癌CNE-2Z细胞侵袭能力的影响及可能机制.方法 噻唑蓝(MTT)法检测塞来昔布对鼻咽癌CNE-2Z细胞增殖活性的影响.不同浓度塞来昔布作用鼻咽癌CNE-2Z细胞24 h后,通过重组人工基膜实验检测细胞侵袭力的改变;RT-PCR检测鼻咽癌细胞CD44v6 mRNA的表达情况;用Western blot和免疫细胞化学检测鼻咽癌细胞CD44v6蛋白表达变化.结果 MTT法结果显示,塞来昔布对鼻咽癌CNE-2Z细胞增殖有明显抑制作用,呈剂量依赖性关系(P<0.01).塞来昔布作用细胞24 h后,肿瘤细胞的侵袭力明显下降(P<0.05).RT-PCR结果显示,塞来昔布可以明显抑制CD44v6 mRNA的表达水平(P<0.01).Western blot和免疫组化结果显示,随着药物浓度的增加,CD44v6蛋白表达水平逐渐减少(P<0.01).结论 塞来昔布可能通过下调CD44v6表达而降低鼻咽癌CNE-2Z细胞侵袭能力.     

环氧合酶-2抑制剂对非小细胞肺癌A549细胞VEGF表达的影响

目的探讨环氧合酶-2抑制剂对非小细胞肺癌A549细胞血管内皮生长因子(VEGF)表达的影响。方法加不同浓度塞来昔布与A549肺癌细胞共培养,MTT法检测细胞增殖,50μmol/L塞来昔布作用于A549细胞48 h,RT-PCR方法检测各组细胞的VEGF基因表达水平,Western blot方法检测各组细胞的VEGF蛋白表达。结果塞来昔布在体外对细胞株A549的生长抑制作用呈剂量、时间依赖性;各组细胞培养48 h后,相比于对照组,RT-PCR方法检测发现塞来昔布组细胞VEGF m RNA表达显著降低(P0.01),Western blot方法检测发现塞来昔布组细胞VEGF蛋白表达显著降低(P0.01)。结论塞来昔布可能通过调节VEGF的表达抑制A549细胞增殖。     

塞来昔布联合阿霉素促胃癌细胞凋亡作用的探讨

目的：探讨非甾体类抗炎药（NSAIDs）中选择性的环氧化酶-2（COX-2）抑制剂塞来昔布与常规化疗药物阿霉素（ADM）小剂量联合用药对MGC-803细胞的促凋亡或抑制增殖作用。方法：传代培养MGc-803细胞，用MTT法检测细胞的增殖程度；用荧光显微镜术，流式细胞术和DNA梯度检测细胞凋亡情况。结果：（1）荧光显微镜下，塞来昔布联合ADM用药组的MGc-803细胞出现典型的细胞凋亡形态学改变；（2）塞来昔布和ADM联合使用可以诱导细胞凋亡发生，DNA梯形条带较单独使用时更加明显：（3）流式细胞术显示单独使用塞来昔布的细胞凋亡率为18．1，阿霉素28．7％，小剂量塞来昔布和阿霉素联合使用为64．9％。结论：小剂量塞来昔布与ADM联合使用可以促进胃癌细胞凋亡。     

环氧合酶-2抑制剂对非小细胞肺癌A549细胞Bcl-2和Bax表达的影响

目的探讨环氧合酶-2抑制剂对非小细胞肺癌A549细胞Bcl-2和Bax表达的影响。方法加不同浓-1度塞来昔布与A549肺癌细胞共培养,MTT法检测细胞增殖,50μmol·L塞来昔布作用于A549细胞48 h,实时定量PCR方法检测各组细胞的Bcl-2和Bax基因表达水平,Western blot方法检测各组细胞的Bcl-2和Bax蛋白表达。结果塞来昔布在体外对细胞株A549的生长抑制作用呈剂量、时间依赖性;各组细胞培养48h后,相比于对照组,塞来昔布组细胞Bcl-2 m RNA和蛋白表达显著下调,Bax m RNA和蛋白表达显著上调,Bcl-2/Bax比值显著降低(<0.01)。结论塞来昔布可能通过调节Bcl-2和Bax的表达抑制A549细胞增殖。     

Survivin在塞来昔布诱导非小细胞肺癌细胞株凋亡中的作用

目的观察survivin在塞来昔布诱导非小细胞肺癌(NSCLC)细胞株凋亡中的作用。方法用MTT法检测细胞株的增殖,用流式细胞学、透射电镜检测细胞株的凋亡,用RT-PCR、W estern b lot检测survivin的mRNA及蛋白表达。结果①塞来昔布对NSCLC有明显的剂量依赖性和时间依赖性抑制作用,塞来昔布对鳞癌细胞株NC I-H520的抑制率明显高于对腺癌细胞株A549的抑制率;②塞来昔布诱导NSCLC凋亡,凋亡随剂量的增加而增加;③塞来昔布作用后survivin的mRNA及蛋白表达减少。结论塞来昔布可能是通过降低survivin的mRNA及蛋白表达水平,诱导NSCLC细胞凋亡。     

环氧合酶-2抑制剂对非小细胞肺癌A549细胞Fas表达的影响

目的探讨环氧合酶-2抑制剂对非小细胞肺癌A549细胞Fas表达的影响。方法加不同浓度塞来昔布与A549肺癌细胞共培养,MTT法检测细胞增殖,50μmol·L~(-1)塞来昔布作用于A549细胞48 h,RT-PCR方法检测各组细胞的Fas基因表达水平,Western blot方法检测各组细胞的Fas蛋白表达。结果塞来昔布在体外对细胞株A549的生长抑制作用呈剂量、时间依赖性;各组细胞培养48h后,相比于对照组,塞来昔布组Fas mR NA和蛋白在A549细胞的表达水平显著升高(P0.01)。结论 T塞来昔布抑制A549细胞增殖可能与上调Fas的表达相关。     

RNA干扰技术沉默STAT3对肺腺癌A549细胞生长抑制的作用

目的:利用RNAi技术研究STAT3对肺腺癌细胞A549的生长抑制作用,同时研究STAT3对CyclinDl,Bcl-2,BAX,Caspase-9的影响及其意义.方法:利用荧光定量PCR法检测后STAT3mRNA的表达,选取1条最能有效抑制STAT3mRNA的siRNA进行后续实验;利用AM-BLUE法检测STAT3siRNA的抑制率;利用流式细胞术(FCM)检测STAT3沉默前后肺腺癌A549细胞周期分布状况和凋亡情况;利用Western blot检测RNA干扰前后STAT3与CyclinDl ,Bcl-2,BAX,Caspase-9的蛋白表达.结果:STA3被沉默后,肺腺癌细胞A549生长被抑制(P<0.05);细胞处于Go/G1期(P<0.05),而S,G2/M期的细胞相对减少(P<0.05);凋亡细胞明显增多(P<0.05) ; STAT3基因被沉默后,CyclinDl、Bcl-2蛋白的表达明显降低(P<0.05),BAX,Caspase-9蛋白的表达增高(P<0.05).Person相关性分析显示,RNAi后人肺腺癌细胞A549中STAT3与CyclinDl,Bel-2蛋白表达呈正相关(P<0.05),STAT3与BAX蛋白表达呈负相关(P<0.05),STAT3与Caspase-9蛋白表达无相关性(P>0.05).结论:RNA干扰技术抑制A549细胞STAB基因后,可阻断A549的细胞周期,使细胞阻滞在Go/G1期,无法进人到S,M期.同时可诱导肺腺癌A549细胞的凋亡.其机制可能为STAT3siRNA抑制了STAB基因的表达,从而使CyclinDl、Bcl-2蛋白表达降低,BAX蛋白表达增高.     

Relationship between epidermal growth factor receptor (EGFR) mutation and serum cyclooxygenase-2 Level,and the synergistic effect of celecoxib and gefitinib on EGFR expression in non-small cell lung cancer cells

Epidermal growth factor receptor (EGFR) mutations occur mostly in patients with lung adenocarcinoma; such patients are also more likely to express cyclooxygenase-2 (COX-2), indicating a possible relationship between EGFR mutation and COX-2. The COX-2 and EGFR pathways mutually enhance their procarcinogenic effects in different tumor types. Therefore, simultaneous EGFR and COX-2 inhibition may be a promising therapeutic approach for patients with lung adenocarcinoma. We obtained tissue and serum samples from patients with non-small cell lung cancer (NSCLC) to detect the relationship between EGFR mutation and serum COX-2 level. Subsequently, gefitinib was combined with celecoxib to investigate the efficacy of inhibition in vitro in two NSCLC cell lines: HCC827 (del E746-A750) and A549 (wild-type EGFR). The cells were treated with gefitinib or celecoxib alone or with gefitinib plus celecoxib. Cell proliferation and apoptosis were assessed and correlated with expression of COX-2 and phosphorylated (p)-EGFR. The EGFR mutation rate of the high-COX-2 patients was significantly higher than that in the low-COX-2 patients. Multivariate analysis showed that high COX-2 levels were independently associated with EGFR mutation. Celecoxib and gefitinib inhibited cell growth in both cell lines. At sufficiently high concentrations, celecoxib plus gefitinib significantly mutually enhanced their anti-proliferative and apoptotic effects in both cell lines. At low concentrations, the combination had no additional effects on A549 cells. There was increased down regulation of COX-2 and p-EGFR when both cell lines were treated with high-concentration celecoxib plus gefitinib compared to either agent alone. This study demonstrates that high serum COX-2 levels may indicate EGFR mutations and that the efficacy of combined celecoxib and gefitinib is significantly greater in NSCLC cells with EGFR mutations; at high concentrations, the combination is efficacious in wild-type NSCLC cells.     

Effect of celecoxib combined with chemotherapy drug on malignant biological behaviors of gastric cancer

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has been reported to have antitumor effects. In some tumor models, the combination of celecoxib with chemotherapy agents has shown synergistic antitumor effect; however, the effect of celecoxib combination with tegafur/gimeracil/oteracil potassium on the malignant biological behaviors of gastric cancer in nude mice is unclear. In this study, female nude mice were subcutaneously transplanted with SGC-7901 gastric cancer cells. When the tumor model formed, the mice were divided into control group, celecoxib group, tegafur/gimeracil/oteracil potassium group, and the combination of both drug regimens group. Mice were treated for 3 weeks. Following treatment, the proliferating index was calculated, apoptosis related proteins, COX-2, vascular endothelial growth factor-C (VEGF-C) and lymphatic vessel density were quantified in tumor tissues by immunohistochemistry. Apoptosis was evaluated by TUNEL staining. The results revealed that celecoxib and tegafur/gimeracil/oteracil potassium alone significantly inhibited tumor growth. The combination of these two drugs showed a synergistic antitumor effect. Both celecoxib and tegafur/gimeracil/oteracil potassium alone inhibited proliferation and promoted apoptosis. The combination of these two drugs further enhanced this anticancer effect. Both celecoxib and the combination treatment inhibited lymphangiogenesis and the expression of COX-2 and VEGF-C. However, tegafur/gimeracil/oteracil potassium treatment had no obvious effect on lymphangiogenesis. These results suggested that the combination of celecoxib and tegafur/gimeracil/oteracil potassium produced a synergistic antitumor effect, possibly by inhibiting the proliferation of tumor cells and promoting apoptosis. Celecoxib and celecoxib in combination with tegafur/gimeracil/oteracil potassium possibly by reducing the expression of COX-2, in turn down-regulating the expression of VEGF-C, resulted in the inhibition of lymphangiogenesis.     

环氧合酶抑制剂塞来昔布促进酪氨酸激酶抑制剂STI571抗白血病效应的研究

目的：探讨环氧合酶-2(COX-2)抑制剂塞来昔布(celecoxib)促进酪氨酸激酶抑制剂STI571的抗白血病效应及其机制。方法：以不同浓度的celecoxib与STI571组合孵育K562细胞，光镜、荧光显微镜观察细胞形态，MTT比色法观察两者对细胞的生长抑制情况，流式细胞术作DNA倍体分析及细胞凋亡线粒体流式检测，半定量RT-PCR法分析相关基因的表达。结果：(1) Celecoxib可促进STI571抑制白血病细胞增殖的能力，0.25 μmol/L STI571与40.0、60.0 μmol/L的celecoxib联用抑制率分别为76.1%±1.6％、91.5%±0.4％，明显高于单药组60.0%±2.0％(0.25 μmol/L STI571)、34.7%±0.5％(40.0 μmol/L celecoxib)、49.8%±1.8％(60.0 μmol/L celecoxib) (P<0.05)。(2)单用celecoxib时未检测到细胞凋亡发生，但与STI571联用时可显著增强STI571诱导凋亡的能力，表现为明显凋亡形态学改变，亚G1期细胞及线粒体凋亡百分率显著增高，其中0.25 μmol/L STI571与40 μmol/L celecoxib联用时分别为20.6%±3.6％、31.3%±4.3％，高于STI571单药组(分别为3.0%±0.6％、3.2%±0.4％)(P<0.05)。(3)单用celecoxib、STI571对COX-2表达没有影响，而联用则COX-2显著下调(P<0.05)，但联用及单用对VEGF表达均没有影响(P>0.05)。结论：单用celecoxib仅抑制K562细胞增殖，而联用STI571可明显降低K562细胞对STI571的效应阈值，促进STI571诱导的增殖抑制和凋亡。     

赫赛汀对吉非替尼诱导肺癌A549细胞凋亡的影响

目的： 研究吉非替尼与赫赛汀联合应用对人肺腺癌A549 细胞凋亡的影响。方法: 应用MTT法,流式细胞仪Annexin V－PI 双标法、DAPI荧光染色等多项方法,体外研究吉非替尼与赫赛汀联合对A549 细胞的促凋亡作用。结果: 吉非替尼与赫赛汀单独应用及联合应用均可以明显抑制人肺腺癌A549细胞的生长，促进细胞凋亡,并呈浓度及时间依赖性,2者联合作用人肺腺癌A549 细胞24 h、48 h、72 h 的凋亡率显著高于单用吉非替尼或赫赛汀组(P<0.05),2者呈现出相加的抗瘤效果。结论: 吉非替尼与赫赛汀联合应用在体外对人肺腺癌A549 细胞有明显的促凋亡作用。     

塞来昔布诱导不表达环氧合酶-2的胃癌细胞凋亡研究

目的： 探讨选择性COX-2抑制剂celecoxib诱导COX-2不表达胃癌细胞MGC-803凋亡。 方法： 应用MTT法研究celecoxib对细胞存活率的影响；Hoechst33258染色，DNA 琼脂糖凝胶电泳和流式细胞术分别检测celecoxib处理后细胞形态学改变，DNA断裂情况和DNA含量的变化。 结果： COX-2高表达于AGS胃癌细胞株，而MGC-803中未检测到COX-2表达; 选择性COX-2抑制剂 celecoxib呈浓度和时间依赖性诱导MGC-803细胞凋亡。 结论： Celecoxib 可诱导不表达COX-2的胃癌细胞MGC-803凋亡。     

盐霉素增强吉非替尼诱导人肺腺癌细胞株A549凋亡的作用

 目的：探讨盐霉素联合吉非替尼诱导人肺腺癌细胞株A549凋亡的协同作用。方法：采用MTT的方法检测盐霉素对A549细胞生长的抑制作用;流式细胞术检测盐霉素对A549细胞凋亡和线粒体膜电位的影响;比色法检测caspase-3、-8和-9活性;Western bloting 分析细胞色素C、Bcl-2、p-EGFR、p-Akt和p-ERK蛋白水平。结果：盐霉素与吉非替尼单用均出现不同程度的细胞增殖抑制作用和诱导细胞凋亡作用;而盐霉素与吉非替尼联合作用,能更显著地抑制细胞增殖,且凋亡细胞显著增加（P<0.05）。盐霉素单独作用A549细胞,线粒体膜电位显著下降,细胞内活性氧和Ca2+在短期内显著升高,胞浆细胞色素C含量以及caspase-3、-8和-9活性均显著增加,与对照组比较差异均有统计学意义;吉非替尼单用则主要表现为对p-EGFR、p-Akt和p-ERK蛋白表达的抑制作用,而对胞浆细胞色素C含量以及caspase-3、-8和-9活性影响较少。Western blotting检测发现,联合用药组的Bcl- 2、p-EGFR、p-Akt和p-ERK蛋白表达量明显减少,但是对EGFR、Akt和ERK总蛋白水平无显著影响。结论：盐霉素与吉非替尼联用具有较好的协同作用,可能通过Bcl-2途径及线粒体凋亡途径诱导人肺腺癌A549细胞凋亡,提高A549细胞对吉非替尼的敏感性。     

NF-κB圈套寡核苷酸促进肺癌细胞A549凋亡的作用

目的:观察NF-κB圈套寡核苷酸对NF-κB活性的影响,研究其在肺癌细胞A549凋亡中的作用机制。方法: 用转染技术将FITC标记的NF-κB圈套寡核苷酸转入人肺癌细胞A549中,通过荧光倒置显微镜和共聚焦显微镜进行核内定位的观察,凝胶迁移率实验(EMSA)检测转染前后NF-κB活性的变化。生长曲线观察低活性NF-κB对细胞增殖的影响。流式细胞仪、TUNEL检测细胞凋亡率。Western blot检测NF-κB低活性引起的凋亡相关蛋白Bcl-2和Fas的改变。结果: NF-κB圈套寡核苷酸转染后定位于细胞核内,EMSA显示转染后NF-κB活性明显下降。A549细胞对NF-κB圈套寡核苷酸敏感,细胞生长速度减缓。转染NF-κB圈套寡核苷酸的A549细胞凋亡率增加。NF-κB 的低活性可以引起凋亡抑制蛋白Bcl-2表达水平下调和凋亡蛋白Fas表达增加。结论: NF-κB圈套寡核苷酸具有促进肺癌细胞A549凋亡的作用,其机制可能与NF-κB圈套寡核苷酸通过下调NF-κB的活性使凋亡蛋白Fas表达增加和凋亡抑制蛋白Bcl-2表达降低有关。     

Selective inhibition of cyclooxygenase-2 exacerbates methamphetamine-induced dopamine depletion in the striatum in rats

Neuroinflammatory processes associated with induction of cyclooxygenase (COX) have been implicated in the deleterious events resulting in neurodegeneration. The present study was designed to investigate the impact of acute methamphetamine (MA) administration on COX expression and prostaglandin E2 (PGE₂) production, and to evaluate the effect of selective COX-2 inhibition using celecoxib in MA-induced degeneration of dopaminergic terminal and cell apoptosis in the striatum. Male Sprague–Dawley rats were treated with either a neurotoxic regimen of methamphetamine hydrochloride (5 mg/kg, i.p., every 2 h for four injections) with or without celecoxib (7.5 mg/kg) or vehicle. COX-1 expression was not affected by MA, while both COX-2 protein expression and number of COX-2 positive cells in striatum were significantly reduced 24 h after MA treatment. However, after 72 h, a significant upregulation of COX-2 protein was detected. PGE₂ production was correlated with altered COX-2 levels. NFkappa-B (NFκB), a key regulator of COX-2 expression, was activated 72 h after MA administration, and was accompanied by increased Ikappa B (IκB) phosphorylation. Animals receiving MA exhibited an increase in apoptotic cells and notable reductions in dopamine (DA) content (63.9%) in immunoreactivity of tyrosine hydroxylase (TH) and neuron specific microtubule-associated protein 2 (MAP2) in striatum. Administration of celecoxib exacerbated MA-induced DA depletion, and did not affect MA-induced MAP2 damage, apoptosis or proliferation of glial cells. Our findings suggest that COX-2 containing cells are targets of the damage during earlier stages of MA-related neurotoxicity, and that the selective inhibition of COX-2 enzyme is harmful rather than protective. The COX-2 induction appears during the recovery period, and NFκB activation may be an important mechanism.