NS-398降低结肠癌细胞系HT-29体外侵袭力

研究环氧合酶-2选择性抑制剂NS-398对结肠癌细胞系HT-29体外侵袭力的作用。通过流式细胞术检测COX-2在HT-29细胞的表达,MTT法检测HT-29细胞与matrigel的黏附性及细胞活性,通过改良的Boyden小室法观察HT-29细胞侵袭重组基底膜的能力以及趋化运动能力。结果显示HT-29细胞COX-2表达阳性,存在NS-398作用的靶,0．1、1．0、10μmol／LNS-398可显著抑制HT-29细胞与matrigel的黏附性以及侵袭重组基底膜的能力和趋化运动能力,且上述作用与NS-398的毒性作用无关,提示NS-398具有抑制结肠癌细胞体外侵袭力的作用。     

选择性环氧合酶-2抑制剂对结肠癌细胞的生长抑制作用及机制探讨

目的：研究NS-398（一种选择性环氧合酶-2抑制剂）对结肠癌HT-29细胞的生长抑制作用并探讨其机制。 方法： 通过MTT法检测细胞的增殖情况，通过流式细胞术检测细胞凋亡率和细胞周期，通过RT-PCR检测bcl-2 mRNA和bax mRNA表达，通过共聚焦激光扫描显微镜观察细胞骨架成分F-actin的变化。 结果： NS-398对结肠癌HT-29细胞生长的抑制具有时间和剂量依赖性。流式细胞术结果显示NS-398可剂量依赖地诱导HT-29细胞凋亡，使其停滞于G0/G1期。经不同浓度的NS-398处理72 h后，bcl-2 mRNA 在HT-29细胞的表达降低，bcl-2／bax比值降低。细胞骨架成分F-actin主要分布在HT-29细胞核周围，呈环状结构，NS-398作用后细胞核周围的环状结构消失。 结论： NS-398可显著抑制结肠癌细胞的体外生长并诱导其凋亡，这与下调bcl-2／bax比值以及细胞骨架的破坏有关。     

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.     

CD15 mRNA在原发性肝细胞癌中的表达及其预后意义

目的：研究CD15mRNA表达与原发性肝细胞癌（HCC）侵袭转移和预后关系及与CD44v6和nm23H1的mRNA表达相关性。方法：应用原位杂交和免疫组织化学方法，检测分析HCC组织中CD15mRNA及其蛋白、CD44v6及nm23H的mRNA表达。结果：99例HCC中，CD15mRNA及其 蛋白、CD44和nm23H1的mRNA表达阳性率分别为38．4％、36．7％、41．4％和76．8％。CD15mRNA表达与其 蛋白表达一致，与CD44v6 mRNA表达呈正相关，与nm23H1 mRNA表达呈负相关。CD15mRNA及其蛋白、CD44和nm23H1的mRNA表达均与HCC侵袭转移倾向和患者预后相关。结论：检测CD15表达有可能成为HCC侵袭转移和预后判断的一项新的病理生物学指标。     

siRNA抑制nm23-H1基因表达对鼻咽癌6-10B细胞生物学行为的影响

目的：采用RNA干扰技术下调nm23-H1基因在鼻咽癌6-10B细胞中的表达,探讨nm23-H1表达下调对6-10B细胞生物学行为的影响。方法：采用脂质体法将nm23-H1基因siRNA(nm23-H1 siRNA)瞬间转染6-10B细胞,Western 印迹检测转染细胞中nm23-H1蛋白的表达水平,然后利用MTT法、流式细胞术和Transwell小室实验分别检测转染6-10B细胞的增殖、细胞周期和体外迁移侵袭等生物学行为的变化;测序检测6-10B细胞nm23-H1基因有无S120G 点突变。结果： nm23-H1 siRNA有效地下调nm23-H1基因的表达, nm23-H1 siRNA转染6-10B细胞的增殖能力增强,S期细胞增多,体外迁移和侵袭能力增强（P<0.05）。6-10B细胞nm23-H1 基因无S120G 点突变。结论：nm23-H1基因具有抑制人鼻咽癌细胞6-10B增殖和体外迁移侵袭的作用。     

NS-398对结肠癌细胞系HT-29超微结构的影响

目的选择性环氧合酶-2抑制剂NS-398可抑制结肠癌细胞系HT-29的体外侵袭力,本研究进一步观察其对HT-29细胞超微结构的影响。方法通过扫描电镜观察细胞表面微绒毛,通过激光共聚焦显微镜观察细胞骨架成分F-actin。结果未处理的HT-29细胞表面微绒毛和丝状伪足较多,其末端有分叉现象,NS-398处理后,细胞表面的微绒毛减少、皱缩呈小球样改变,以10μmol／L处理组明显。荧光标记的F-actin较集中地分布于HT-29细胞核周围,呈现“环状”结构,10μmol／LNS-398处理后细胞核周围的“环状”结构消失,荧光强度减弱。结论NS-398可显著改变HT-29细胞的超微结构,这可能是其抑制HT-29侵袭力的机制之一。     

COX-2表达下调抑制人结肠癌细胞系HT-29增殖并促进凋亡

目的探讨环氧化酶-2(COX-2)表达对大肠癌细胞HT-29细胞的细胞活力、增殖和凋亡等生物学行为的影响。方法利用脂质体将siRNA COX-2及空载体转入HT-29大肠癌细胞中,并设立对照组。48 h后,real-time PCR法检测COX-2、Bcl-2、Bax和基质金属蛋白酶2(MMP2)mRNA表达;Western blot检测COX-2及p-AKT表达;CCK-8法检测细胞活力;Annexin V/PI流式双染检测细胞凋亡;流式细胞计量术检测细胞周期;划痕实验及Transwell实验分别检测细胞迁移和侵袭能力。结果与对照组及空载体组比较,COX-2抑制组COX-2蛋白及mRNA表达量显著降低(P0.01);细胞活力下降(P0.01);细胞凋亡率提高,细胞周期阻滞于G期,细胞迁移及侵袭能力降低(P0.01);Bcl-2及MMP2 mRNA表达下调(P0.01);Bax mRNA表达上调(P0.01);p-AKT蛋白表达下调(P0.01)。结论 COX-2表达量下调后能显著的抑制细胞增殖、迁移与侵袭,并诱导细胞凋亡,可能与下调p-AKT有关。     

NS-398对肝癌细胞HepG2增殖和凋亡的影响

目的:探讨选择性环氧合酶II(COX-2)抑制剂NS-398对人肝癌细胞HepG2增殖和凋亡的影响。方法: 应用MTT法研究不同浓度NS-398对HepG2细胞增殖的影响,DNA梯状电泳(DNA ladder)检测凋亡的发生,流式细胞仪检测细胞周期的改变及凋亡百分率的变化,竞争性RT-PCR检测环氧合酶II COX-2 mRNA及抑凋亡基因bcl-2 mRNA表达的改变。结果: NS-398呈剂量依赖性抑制HepG2细胞增殖,并诱导其凋亡,细胞周期分析表明随着浓度增大S期细胞明显减少,有G₀/G₁期细胞累积现象,并伴有Bcl-2 mRNA表达的下调,而对COX-2 mRNA表达改变无明显影响,且COX-2表达改变与NS-398引起的HepG2细胞的增殖和凋亡均无相关性(相关系数分别为:r=0.056,P>0.05和r=0.119,P>0.05)。结论: NS-398能明显抑制HepG2细胞增殖并诱导其凋亡,与细胞G₀/G₁期阻滞以及bcl-2基因表达下调有关,而非依赖于抑制COX-2基因的表达。     

乳腺癌预后相关免疫组化指标分析

目的 研究BRCA1、MMP-2、PTEN、CD44v6、nm23-H1、Cath-D在乳腺癌组织中的表达及生物学意义,筛选与预后有关的最佳因子组合。方法 采用免疫组化S-P法标记上述指标。结果 （1）乳腺癌组织中PTEN、BRCA1、nm23-H1蛋白表达明显降低,表达水平与复发、淋巴结转移呈负相关,与生存期呈正相关。（2）CD44v6、MMP-2、肿瘤间质内Cath-D的蛋白表达增高并与淋巴结转移、复发呈正相关。（3）BRCA1蛋白与nm23-Hl、PTEN蛋白表达呈正相关,与MMP-2表达呈负相关。MMP-2蛋白表达与CD44v6表达呈正相关。结论 乳腺癌组织中MMP-2表达增强、nm23-H1和BRCA1表达抑制,可能作为预测肿瘤转移及判断预后的可靠指标。     

环氧合酶抑制剂NS-398增强放射诱导的肝癌细胞凋亡

目的：探讨环氧合酶-2（COX-2）选择性抑制剂NS-398对放射诱导的人肝癌细胞HepG2凋亡的影响及可能作用机制。 方法:应用MTT 法检测NS-398对细胞的抑制率;透射电子显微镜观察细胞凋亡的形态学变化,流式细胞术(FCM)定量检测细胞凋亡;实时荧光定量PCR检测凋亡相关基因bcl-2、bax及caspase-3 mRNA表达;Western blotting检测Bc1-2、Bax蛋白表达;比色法分析caspase-3酶活性变化。 结果:NS-398对HepG2细胞的生长抑制作用呈时间与剂量依赖性;电镜下处理组细胞呈现典型的凋亡形态学变化,NS-398显著增加放射诱导的细胞凋亡,上调bax mRNA、Bax蛋白及caspase-3mRNA 表达,并增强caspase-3酶活性,而Bcl-2 表达无明显变化（P>0.05）。 结论:NS-398能增加放射诱导的HepG2细胞凋亡,其机制可能与上调Bax、 caspase-3表达,上升Bax／Bcl-2比例,激活线粒体凋亡通路,活化caspase-3,最终诱导细胞凋亡相关。     

Inhibition of cyclooxygenase-2 suppresses invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 and CD44

Expression of cyclooxygenase-2 (COX-2) in tumors is known to be associated with enhanced angiogenesis, suppression of host immunity, and tumor invasion. In the present study, human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 were used to evaluate the effects of NS-398, a selective inhibitor of COX-2, and COX-2 antisense oligonucleotide (COX-2 AS) on the invasion activity of OSCC cells. Matrigel invasion assay revealed that the invasiveness of NA and HSC-4 was suppressed by treatment with either NS-398 or COX-2 AS. These reagents down-regulated the secretion of matrix metalloproteinase-2 (MMP-2) to culture supernatant as well as the expression of MMP-2 mRNA and protein. Membrane-type 1 matrix metalloproteinase (MT1-MMP), an activator of proMMP-2, was also down-regulated by treatment with these reagents. Furthermore, expression of CD44 on the surface of these cells was reduced by treatment with either NS-398 or COX-2 AS. In addition, MMP-2 antisense oligonucleotides reduced the expression of CD44 on the surface of both OSCC cell lines. These findings suggest that NS-398 and COX-2 AS suppress the invasiveness of OSCC cells via down-regulation of MMP-2 and CD44. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC patients.     

Inhibition of cyclooxygenase-2 suppresses invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 and CD44

Expression of cyclooxygenase-2 (COX-2) in tumors is known to be associated with enhanced angiogenesis, suppression of host immunity, and tumor invasion. In the present study, human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 were used to evaluate the effects of NS-398, a selective inhibitor of COX-2, and COX-2 antisense oligonucleotide (COX-2 AS) on the invasion activity of OSCC cells. Matrigel invasion assay revealed that the invasiveness of NA and HSC-4 was suppressed by treatment with either NS-398 or COX-2 AS. These reagents down-regulated the secretion of matrix metalloproteinase-2 (MMP-2) to culture supernatant as well as the expression of MMP-2 mRNA and protein. Membrane-type 1 matrix metalloproteinase (MT1-MMP), an activator of proMMP-2, was also down-regulated by treatment with these reagents. Furthermore, expression of CD44 on the surface of these cells was reduced by treatment with either NS-398 or COX-2 AS. In addition, MMP-2 antisense oligonucleotides reduced the expression of CD44 on the surface of both OSCC cell lines. These findings suggest that NS-398 and COX-2 AS suppress the invasiveness of OSCC cells via down-regulation of MMP-2 and CD44. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC patients.This revised version was published online in August 2005 with a corrected cover date.     

CD40 ligation triggers COX-2 expression in endothelial cells: evidence that CD40-mediated IL-6 synthesis is COX-2-dependent

OBJECTIVE: To investigate whether CD40-CD154 interactions on HUVEC can trigger COX-2 synthesis as well as PGE2 and PGI2 secretion in vitro and explore whether the CD40-triggered prostanoids provide costimulatory signals for IL-6 secretion in this cell type. MATERIALS AND METHODS: COX-2 protein expression was examined in HUVEC using Western blot analysis. ELISAs were employed to assess PGE2, PGI2 and IL-6 synthesis. RESULTS: We found that COX-2 expression is upregulated when HUVEC are cultured with CD154+ D1.1 cells but not CD154- B2.7 cells. This effect was specifically inhibited by anti-CD154 mAb, and was amplified by the presence of IFNgamma. Analysis of cell supernatants showed a concomitant rise in PGE2 and PGI2 secretion triggered by CD154+ D1.1 cells, or rsCD154. Use of selective (NS-398) and non-selective (ibuprofen) COX-2 inhibitors effectively inhibited prostanoid synthesis triggered by CD40 ligation. Reduction in prostanoid levels by NS-398 was accompanied by a reduction in IL-6 secretion levels triggered by CD40 ligation. Furthermore, exogenously added PGE2 triggered a dose-dependent IL-6 secretion, which was unaffected by NS-398. CONCLUSIONS: These studies demonstrate that CD40 ligation upregulates HUVEC COX-2 expression and function. Moreover, the data strongly suggest that CD154-induced IL-6 secretion in HUVEC is dependent on COX-2 activity.     

甲状腺滤泡癌和乳头状癌细胞黏附分子与转移抑制基因nm23-H1蛋白表达的关系

目的 探讨黏附分子CD44、上皮性钙黏附蛋白（E-cad )和转移抑制基因nm23-H1与甲状腺滤泡源性癌分化、浸润转移的关系。方法 采用免疫组织化学SP法和EnVision法检测42例滤泡癌和54例乳头状癌中CD44、E-cad和nm23-h1的表达。结果 CD44主要表达于癌细胞及浸润淋巴细胞膜,低分化滤泡癌和有转移乳头状癌CD44表达分别高于高分化滤泡癌和无转移乳头状癌,E-cad阳性物质和nm23-H1阳性率高于滤泡癌,转移癌的阳性率和表达强度低于原发灶。甲状腺滤泡原性癌CD44检测阳性率高于E-cad和nm23-H1。在滤泡癌中E-cad与nm23-H1的表达之间呈正相关关系,而在乳头状癌中呈正相关趋势。CD44与E-cad的表达、CD44与nm23-H1的表达之间在滤泡癌和乳头状癌均呈负相关趋势。结论 综合检测CD44、E-cad和nm23对甲状腺滤泡源性癌的诊断、预后评估具有一定参考价值。     

Interleukin-1 stimulates human uterine prostaglandin production through induction of cyclooxygenase-2 expression

PROBLEM: Uterine infection occurs in as much as 20% of preterm labor and results in increased decidual cytokines. The objective of this study was to examine the effect of interleukin-1 (IL-1) and the cyclooxygenase-2 (COX-2) inhibitor, NS-398, on myometrial prostaglandin (PG) production and COX-2 expression. METHOD OF STUDY: Human uterine myocytes were stimulated with IL-1 (0-50 ng/mL) over 24 hr. PGE2, PGF2alpha, and 6-keto F1alpha were measured by enzyme-linked immunosorbent assay. Both COX-1 and COX-2 proteins and mRNA were measured by western and northern blot, respectively. RESULTS: IL-1 increased PG production beginning at 6 hr, COX-2 protein increased beginning at 4 hr and continued to increase at 24 hr. COX-2 mRNA increased at 2 hr and peaked at 4 hr. NS-398 blocked PG production but had no effect on COX-2 protein or mRNA. CONCLUSIONS: IL-1 increases PG production by myometrium by increased COX-2 expression. NS-398 completely blocks IL-1-induced PG production. With intrauterine infection, IL-1 may induce labor through the autocrine production of uterotonic PGs.