氯化钴体外诱导人胃癌细胞COX-2表达的研究

研究证实,多种恶性肿瘤组织内存在低氧区,其产生与肿瘤生长迅速、肿瘤血管结构异常等有关。而低氧可诱导包括血管内皮生长因子(VEGF)在内的多种促血管生长因子表达,促进肿瘤生长。环氧合酶(COX)是催化花生四烯酸转化为前列腺素类代谢中的限速酶,环氧合酶-2(COX-2)呈诱导性表达,为其同工酶,在多种肿瘤组织中表达升高。多项细胞水平的研究表明,低氧可以刺激多种细胞表达COX-2,还可以诱导多种细胞COX-2、VEGF共表达;     

乙型肝炎病毒感染对原发性肝细胞癌血管内皮生长因子、环氧合酶-2蛋白表达的影响

目的:探讨乙型肝炎病毒(HBV)感染对肝细胞癌(HCC)中血管内皮生长因子(VEGF)、环氧合酶-2(COX-2)蛋白表达水平的影响和肿瘤血管形成的临床病理意义.方法:利用血清学指标检测40例HCC的HBV感染情况,采用快速免疫组化法检测HCC中VEGF、COX-2的蛋白表达,用抗CD34单克隆抗体显示血管内皮细胞,根据CD34阳性的血管内皮细胞计数测定肿瘤微血管密度(MVD).结果:HBV感染组中VEGF、COX-2蛋白以及MVD的阳性表达率均高于非HBV感染组,差异有显著性意义(P<0.05).VEGF和COX-2表达呈正相关(r=0.618,P<0.01).结论:HBV可能通过上调VEGF、COX-2等血管形成因子表达,共同促进了肿瘤血管的生成,从而促进HCC的生长、浸润和转移.     

前列腺素E2在环氧合酶-2促胰腺癌新生血管生成中的介导作用

目的 探讨环氧合酶-2(COX-2)促进胰腺癌新生血管生成过程中前列腺素E2(PGE2)的介导作用，进一步揭示COX-2促进胰腺癌生长的机制。方法 体外培养胰腺癌细胞株PC-3，分别应用酶联免疫吸附(ELISA)和放射免疫(RIA)等方法，检测选择性COX-2抑制剂Celebrex对胰腺癌PC3细胞血管内皮生长因子(VEGF)和PGR表达的调节作用，并观察外源性PGE2对Celebrex调节VEGF表达的干预。建立裸鼠PC3细胞移植瘤，Western印迹检测Celebrex对胰腺癌组织VEGF表达的影响，RIA测定Celebrex对胰腺癌组织PGB变化的调节。结果 随着Celebrex作用浓度的提高以及作用时间的延长，PC3细胞分泌的VEGF和PGE2受到抑制，呈时间和剂量依赖性。外源性PGE2显著上调Celebrex作用后PC-3细胞VEGF蛋白表达，呈剂量依赖性，体内实验表明，Celebrex可显著抑制移植瘤组织VEGF和PGE2的表达。结论 COX-2参与了PC-3细胞VEGF分泌的调节，进而促进胰腺癌新生血管形成，而PGE2则在该过程中起着重要的介导作用。     

胃癌组织环氧合酶-2表达与血管内皮生长因子的关系

目的 观察胃癌环氧合酶-2和血管内皮生长因子的表达及相互关系。方法 应用免疫组化法检测胃癌组织COX-2和VEGF的表达状况。结果 32例胃癌中发现COX-2表达阳性22例(68.7％),其中伴淋巴结转移者COX-2表达阳性率80.9％(17/21),高于不伴淋巴结转移者的45.4％(5/11)(P<0.05)。而不同胃癌分期与组织类型之间COX-2表达无显著性差异(P>0.05)。32例胃癌中VEGF表达阳性20例(62.5％),其中COX-2阳性胃癌VEGF表达阳性率77.3％(17/22),而COX-2阴性胃癌VEGF阳性率30％(3/10),两组比较具有显著性差异(P<0.05)。结论 胃癌组织存在COX-2过度表达,且与VEGF表达相关。     

大肠肿瘤COX-2、VEGF和MVD的表达及其意义

目的探讨环氧合酶-2（COX-2）与肿瘤的血管生成、转移、细胞增殖,病理类型、患者预后的关系,深入研究COX-2选择性抑制剂对大肠肿瘤防治的作用机制,方法采用免疫组化方法检测大肠肿瘤中COX-2、血管内皮生长;因子（WXGF）、CD34、增殖细胞核抗原（PCNA）的表达情况。结果COX-2和VEGF的表达在大肠癌均有显著增加,分别为78％和76％。大肠癌中COX-2的表达与VEGF的表达相关。COX-2阳性的大肠癌中微血管密度（MVD）是阻性肿瘤中的3．9倍,VEGF阳性的大肠癌中MVD是阻性肿瘤中的3.2位。结论COX-2和VEGF在肠道腺癌和结直肠癌形成早期就已开始表达,并在腺瘤的增生、癌变,肿瘤的形成、生长和血管形成中起重要作用。     

慢性缺氧改变肺内动脉平滑肌细胞环氧合酶基因的表达

目的研究慢性缺氧对肺动脉平滑肌细胞环氧合酶基因表达的影响.方法根据常氧(PaO2152mmHg)及慢性缺氧(PaO240±5nmHg)的不同培养条件,将平滑肌细胞分为常氧组和慢性缺氧组,采用半定量RT-PCR技术检测大鼠肺内动脉平滑肌细胞环氧合酶(COX)基因的表达及其对急性缺氧刺激的反应.结果COX-1mRNA的表达不受缺氧及传代的影响,而COX-2mRNA的表达随慢性缺氧时间延长而增加,在4、6代慢性缺氧培养组均高于同代常氧组水平(P＜0.05).急性缺氧后COX-2mRNA增加的幅度在慢性缺氧组均大于同代常氧组,以第四代最为显著.结论慢性缺氧可增强急性缺氧时肺内动脉平滑肌细胞COX-2基因的表达,在慢性缺氧所致肺血管对缺氧的反应性降低中可能起作用.     

5-LOX mRNA和COX-2 mRNA在结直肠癌中的表达及二者的相互关系

目的 研究5脂氧合酶(5-LOX)和环氧合酶2(COX-2)在结直肠癌中的表达及二者的相互关系.方法 用RT-PCR检测39例结直肠癌标本中5-LOX mRNA和COX-2 mRNA的表达.结果 5-LOX mRNA和COX-2 mRNA在结直肠癌组织中的表达率分别为84.6%(33/39)及76.9%(30/39),均与结直肠癌临床分期、肿瘤大小、肿瘤分化程度有关.但两者无相关性(P＞0.05).结论 5-LOX mRNA和COX-2 mRNA在结直肠癌组织中表达增高,提示了在研究结直肠癌治疗和预防时可采取对代谢途径抑制这一新方向.     

喉癌组织中COX-2、VEGF、MVD的表达及意义

采用免疫组化法检测50例喉癌组织中诱导型环氧合酶（COX-2）、血管内皮生长因子（VEGF）的表达，用CD34。标记新生血管内皮细胞，观察微血管密度（MVD）。喉癌组织中COX-2、VEGF的阳性表达率分别为62％和68％，MVD为（51．62±16．64）条／200倍视野。COX-2和VEGF的表达与喉癌病理学分级及临床分期有关。表明喉癌组织中COX-2呈高表达，COX-2与肿瘤新生血管形成有关，检测癌组织中COX-2、VEGF有助于喉癌的绢织学分级和预后判断。     

美洛昔康对结肠癌细胞VEGF和angiopoietin-2表达的影响

目的:研究COX-2选择性抑制剂美洛昔康(meloxicam)对结肠癌细胞HT-29生长及血管内皮生长因子(VEGF)和血管生成素-2(angiopoietin-2,Ang-2)表达的影响.方法:分别用100,200,400,800μmol/L美洛昔康对细胞进行干预后,采用CCK-8活细胞计数法检测细胞增殖,流式细胞术检测细胞周期,ELISA测定细胞培养上清液中VEGF,Ang-2的蛋白含量,实时荧光定量PCR检测细胞COX-2,VEGF,Ang-2mRNA含量.结果:美洛昔康作用不同时间后,对HT-29细胞具有细胞毒作用,细胞增殖活性随浓度增加、时间延长逐渐降低(P值:0.000→0.029;0.000→0.043),呈现量-效、时-效关系.并且美洛昔康呈浓度依赖性改变细胞周期分布,G0/G1期细胞比例增加(P值:0.000→0.015).上清液中VEGF和Ang-2蛋白含量明显降低,存在时间和浓度依赖性(P值:0.000→0.018;0.000→0.028).细胞COX-2,VEGF和Ang-2mRNA表达亦明显降低(P值:0.000→0.025),也存在浓度依赖性.结论:美洛昔康能够在蛋白、核酸水平上抑制结肠癌细胞分泌VEGF和Ang-2,从而抑制肿瘤血管生成.     

尼美舒利对人胃癌SGC-7901细胞PGE2合成及COX-2、VEGF表达的影响

目的观察尼美舒利对人胃癌SGC-7901细胞前列腺素E2（PGE2）合成及环氧合酶（COX）-2、血管内皮生长因子（VEGF）表达的影响，探讨其抑制胃癌血管生成的机制。方法对人胃癌SGC-7901细胞进行培养．免疫组织化学、放射免疫法检测不同浓度尼美舒利作用后细胞COX-2、PGE2、VEGF的表达。结果与阴性对照组相比。尼美舒利100、200μmol／L作用48h后SGC-7901细胞的COX-2、VEGF表达明显降低（P〈0．05），COX-2与VEGF的表达呈正相关（r=0．8080，P〈0．05）；随着尼美舒利浓度的升高，PGE2分泌逐渐降低。结论抑制COX-2的活性与表达、减少PGE。的合成及由此引起的VEGF表达降低，在抑制胃癌血管生长中可能具有一定作用。     

环氧合酶-2抑制剂对结肠癌细胞株的体外研究

目的 观察选择性环氧合酶-2（COX-2）抑制剂对COX-2高表达的结肠癌细胞株HT-29增殖和凋亡的影响，明确以COX2为靶点治疗结肠癌的作用途径以及与COX-2活性、表达水平的相关关系。方法 将选择性COX-2抑制剂NS-398作用于结肠癌细胞系HT29，运用MTT法检测细胞增殖状态。流式细胞仪观察NS-398对细胞凋亡的影响。进一步用逆转录聚合酶链式反应（RT-PCR）检测药物作用前后HT-29中COX-2mRNA表达。ELISA法测定前列腺素E2（PGE2）水平。Western blot检测药物作用前后细胞周期素D1、Bcl-2的表达。结果 结肠癌细胞系HT-29中COX-2 mRNA高表达，NS-398呈时间和剂量依赖性抑制HT-29细胞增殖，促进其凋亡。加入NS-398的HT-29细胞中COX-2mRNA表达水平无明显变化（P〉0．05），PGE2却显著下降（P〈0．01）。72h时空白组与NS-398（75μmol／L）处理组细胞周期素D1、Bcl-2表达水平比值分别为2．21和3．25（P〈0．01），两者表达水平随作用时间延长而下降。结论 选择性COX-2抑制剂NS-398不影响结肠癌细胞COX-2 mRNA表达水平，而与其活性相关（PGE2水平）．可能通过细胞周期素D1、Bcl-2影响结肠癌细胞系HT-29的增殖与凋亡，揭示了COX-2为靶点治疗结肠癌的分子机制。     

Induction of cyclooxygenase-1 and -2 modulates angiogenic responses to engagement of alphavbeta3

Prostaglandins and cyclooxygenase (COX) have been implicated in the angiogenesis that occurs around tumours, but how they are induced is unclear. Prostaglandin formation is regulated by the availability of arachidonic acid and/or COX activity that in turn are controlled by activation of G-protein-coupled receptors or kinase receptors. Adhesion receptors provide another potential level of control as they transduce a variety of "outside-in" signals implicated in inflammation. We examined whether engagement of the vitronectin receptor (alphavbeta3) modulated prostacyclin (PGI2) formation in human umbilical vein endothelial cells (EC). Engagement of EC alphavbeta3 by vitronectin (versus fibronectin or gelatin) or by monoclonal antibodies (mAbs) LM609 and LIBS6, enhanced PGI2 generation and also induced expression of both COX-1 and -2 isoforms. Alphavbeta3 engagement also led to vascular endothelial cell growth factor (VEGF) generation and EC proliferation that was attenuated by inhibition of both COX-1 and COX-2. COX-1 inhibition also prevented new vessel formation in an in vitro model of angiogenesis that is alphavbeta3 dependent. Inhibition of angiogenesis by the COX-1 inhibitor was partially reversed by removal of the inhibitor or by addition of the stable analogue of PGI2, iloprost. These findings strongly indicate that alphavbeta3-mediated angiogenesis is partly due to induction of both isoforms of COX.     

Human fallopian tubes express prostacyclin (PGI) synthase and cyclooxygenases and synthesize abundant PGI

Animal studies unequivocally support the indispensable role of prostaglandin (PG) and cyclooxygenase (COX) in ovulation and implantation. Available data also suggest that PG and COX may be important in the transport of embryos. The effects of PGE(2) and PGF(2alpha) on the contractility of human tubal muscle have been studied extensively; the expression of COX in human fallopian tubes was also reported. Despite all these, two fundamentally important questions remained to be answered: 1) which PGs are produced by human fallopian tubes; and 2) which COX isoform(s) is expressed by the fallopian tubes. We used reverse-phase HPLC to study the metabolism of [1-(14)C] arachidonic acid by the fallopian tubes. We found that 6 keto-PGF(1alpha), a stable metabolite of prostacyclin (PGI), and PGE(2) constituted 56% +/- 10% and 35% +/- 10% (mean +/- SEM, four samples), respectively, of total eicosanoids synthesized. Western blot analysis revealed the expression of both COX isoforms. Immunohistochemistry study showed that both COX-1 and -2 were localized to nonciliated epithelia and tubal smooth muscle. In addition, COX-2 was also expressed in ciliated epithelial cells. Western blot analysis revealed the expression of PGI synthase (PGIS) and PGI receptor by fallopian tubes. Immunohistochemistry confirmed the expression of PGIS by luminal epithelia, tubal smooth muscle, vascular endothelial cells, and vascular smooth muscle cells. Iloprost, a PGI analog, inhibited the activities of circular and longitudinal muscles of the fallopian tube. Thus, the fallopian tube expresses both COX isoforms and PGIS. Furthermore, it is a source and a target of PGI. PGI and COX may be important to gamete function, embryo transport, and embryo development.     

COX-2 expression is correlated with VEGF-C, lymphangiogenesis and lymph node metastasis in human cervical cancer

Lymphangiogenesis has been shown to promote lymph node metastasis in cancers, making it an important target in cancer therapy. Vascular endothelial growth factor (VEGF)-C is upregulated in various tumors/cancers and is one of the most potent growth factors for inducing lymphangiogenesis and promoting lymph node metastasis (LNM). Likewise, cyclooxygenase (COX)-2 plays major roles in carcinogenesis, tumor growth and metastasis via multiple mechanisms including inactivation of host antitumor immunity and promotion of tumor cell migration, tumor cell invasiveness and tumor-associated angiogenesis and lymphangiogenesis. We previously demonstrated an association between COX-2 and VEGF-C in an in vitro model of lung cancer. However, little is known about the regulation of VEGF-C by COX-2 in cervical cancer. In this study, we measured the COX-2 and VEGF-C expressions by immunohistochemistry in 23 LNM-positive and 20 LNM-negative cervical cancer specimens. We then examined the correlations among the expressions and the lymphatic microvessel density (LMVD) and ultrastructural changes to the lymphatic vessel walls by enzyme histochemical staining and electron microscopy. In addition, we used the HeLa cervical cancer cell line to explore the in vitro regulation of VEGF-C by COX-2 and its metabolite, PGE₂, using siRNA-mediated gene silencing and EP receptor blockade. The LNM-positive specimens exhibited significantly higher VEGF-C expression, COX-2 expression and LMVD than the LNM-negative specimens. Furthermore, there were strong correlations between the levels of COX-2 expression and the levels of VEGF-C expression and secretion and a significant positive association between the LMVD and LNM. siRNA-mediated knockdown of COX-2 expression inhibited VEGF-C mRNA expression while EP1 and EP4 receptor antagonists reduced the VEGF-C protein level and tyrosine phosphorylation of Src kinase. Moreover, inhibition of Src kinase with the tyrosine kinase inhibitor PP1 attenuated VEGF-C expression. Collectively, our data provide evidence for a clinical association between COX-2 and VEGF-C expressions in cervical cancer. EP1 and EP4 receptors may be involved in the COX-2-mediated regulation of VEGF-C protein and mRNA expressions. Src may be a downstream mediator of EP1 and EP4 receptors. COX-2 inhibition may diminish LNM by suppressing VEGF-C-mediated lymphangiogenesis.     

环氧合酶-2与P-糖蛋白在人肝癌细胞中的表达及其意义

目的 探讨环氧合酶(COX)-2在肝细胞癌发生、发展中的作用及其对P-糖蛋白(P-gp)表达的影响. 方法 收集2003年10月-2005年6月因肝癌切除术切取的肝癌组织标本,用RTPCR、免疫组织化学方法分别检测COX-2 mRNA和COX-2蛋白在肝癌组织和腹部手术取得的正常肝组织中的表达.同时检测多药耐药基因1 mRNA和P-gp在肝癌组织中的表达情况,并分析COX-2与P-gp在肝癌组织中表达的相关性.各样本率间比较采用x2检验、用精确概率法分析组间差异,样本间均数的比较用t检验,用Spearman' s等级相关公式分析COX-2和P-gp的相关性.结果 共收集肝癌组织52例,正常肝组织20例.正常肝组织中未见COX-2表达,COX-2表达在中、低分化肝癌组织的阳性率(94.1％)高于高分化肝癌组织(38.9％,x2= 6.80,P＜0.01);在HBsAg阳性的肝癌组织中的表达(91.7％)高于HBsAg阴性的肝癌组织(62.5％,x2= 4.70,P＜0.05);在合并肝硬化的肝癌组织中的表达(96.7％)高于无肝硬化的肝癌组织(63.6％,x2= 7.51,P＜0.01);P-gp在癌组织中的表达(86.6％)高于相应的癌旁组织(30.7％,x2= 64.42,P＜0.01).RT-PCR检测结果显示COX-2 mRNA在正常肝组织中无表达,而多药耐药基因l mRNA在肝癌组织和正常肝组织中均有表达.同时表达COX-2和多药耐药基因1为42例,两者呈正相关(r=0.563,P＜0.01).结论 本研究结果提示COX-2参与了以P-gp介导的肝癌的多药耐药机制.     

Effect of azoxymethane and curcumin on transcriptional levels of cyclooxygenase-1 and -2 during initiation of colon carcinogenesis

Background. Curcumin is well documented as an effective colonic chemopreventive agent in preclinical studies. Inhibition of arachidonic acid metabolism has been considered one of anticarcinogenic mechanisms of curcumin. We recently reported resistance of middle-aged F344 male rats to inhibition of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) by curcumin (Nutr Cancer, 48, 37–43). It was important to confirm this finding and to find potential mechanisms responsible, as loss of preventive activity of curcumin due to aging was a novel finding, with important implications for human intervention trials. Methods. To confirm our previous findings, and investigate arachidonic acid metabolism as a potential mechanism of age-related differences in response to curcumin, middle-aged F344 male rats were given AOM injections after being fed their experimental diets, 0.6% curcumin or control diet. Colonic ACF were evaluated and colonic levels of cyclooxygenase (COX)-1 and 2 mRNA and prostaglandin E2 (PGE2) were measured. Next, we investigated the short-term effect of AOM and curcumin on arachidonic acid metabolism in young rats. Six week-old rats were given injections of either AOM or untreated following their experimental diets. Colonic COX-1 and COX-2 mRNA as well as PGE2 levels were measured shortly after AOM treatment. Lastly, three different ages of F344 rats were treated with either AOM or saline, and colonic COX-1 and COX-2 mRNA levels were measured shortly after the injections to find if aging alters the effect of AOM on COX mRNA expression. Results. In middle-aged rats, dietary curcumin did not reduce the number of ACF and surprisingly increased colonic levels of COX-2 mRNA. Colonic COX-2 and PGE2 levels were also significantly increased in young rats fed curcumin after AOM injections. Interestingly, AOM did not affect COX-2 but decreased COX-1 expression in all ages. Conclusions. Our study suggests that during initiation, AOM inhibits colonic COX-1 expression without affecting COX-2 and dietary curcumin may increase COX-2 expression to compensate AOM-induced reduction of COX-1 expression.     

尼美舒利和5-氟尿嘧啶联用对胃癌细胞的协同抑制作用及机制

目的 体外研究选择性环氧合酶-2（COX-2）抑制剂尼美舒利和5-氯尿嘧啶（5-FU）对胃癌细胞的抑制作用及机制。方法 以胃癌细胞株MKN45、MKN28为研究对象．观察尼美舒利和5-FU单独或联合应用对细胞增殖、凋亡和细胞周期的影响。应用MTT法检测细胞增殖，流式细胞仪检测细胞凋亡（FITC-Annexin-V/PI双标记）和细胞剧期，RT-PCR观察朋药前后COX-2 mRNA在两株细胞中的表达，Western免疫印迹法观察经两种药物单独和联合作用48h后细胞内凋亡相关蛋白Bax和Bcl-2的表达。结果 在MKN45和MKN28细胞中均可观察到不同水平的COX-2 mRNA表达，尼美舒利和5FU联合应用可明显抑制COX-2 mRNA表达。尼美舒利可抑制两株细胞的增殖并诱导凋亡。尼美舒利和5-FU具有协同抑制细胞增殖及诱导凋亡的作用，该作用与两种药物作用顺序无关，但在联用时作用最强。两药协同抑制增殖的作用主要通过协同杀伤和诱导凋亡而实现。5-FU增强了凋亡诱导蛋白Bax的表达，而尼美舒利则减少凋亡抑制蛋白Bcl-2的表达。两药联用可明显抑制胃癌细胞株生长。结论 选择性环氧合酶-2抑制剂尼美舒利和5-FU通过抑制COX-2 mRNA的表达硬增强Bax／Bcl-2的表达比率诱导胃癌细胞凋亡．从而对胃癌细胞起到协同抑制增殖的作用。     

Cyclooxygenase 1 is required for pH control at the mouse gastric surface

BACKGROUND: Endogenous cyclooxygenase (COX) activity is required to maintain a relatively alkaline surface pH at the gastric luminal surface. AIMS: The purpose of this study was to determine which COX isoform, COX-1 or COX-2, is responsible for regulating the protective surface pH gradient and to test if COX inhibitors also had non-COX mediated effects in vivo. METHODS: Immunofluorescence and western blot analysis showed constitutive expression of both COX isoforms in the normal mouse stomach. We used in vivo confocal microscopy to measure pH near the mucosal surface of anaesthetised COX-1 (-/-), COX-2 (-/-), or wild-type mice of the same genetic background. RESULTS: When the gastric mucosal surface was exposed and superfused (0.2 ml/min) with a weakly buffered saline solution (pH 3) containing the pH indicator Cl-NERF, the pH directly at the gastric surface and thickness of the pH gradient were similar in wild-type and COX-2 (-/-) mice, but COX-1 (-/-) mice had a significantly thinner pH gradient. Addition of indomethacin had minimal effects on the residual surface pH gradient in COX-1 (-/-) mice, suggesting no role for COX-2 in surface pH regulation. Whole stomach perfusion studies demonstrated diminished net alkali secretion in COX-1 (-/-) mice, and application of SC-560 or rofecoxib to wild-type mice and mutant mice confirmed that only COX-1 inhibition reduced alkali secretion. CONCLUSION: COX-1 is the dominant isoform regulating the normal thickness of the protective surface pH gradient in mouse stomach.