罗非昔布抑制胰腺癌细胞ERK、AP-1信号转导通路

目的研究环氧合酶-2抑制剂-罗非昔布对胰腺癌细胞株BXPC-3增殖的信号转导作用的影响.方法用免疫组化法检测裸小鼠胰腺癌移植瘤中c-Fos的表达,免疫印迹法检测BXPC-3中c-Fos、ERK蛋白的改变,采用EMSA技术检测罗非昔布对胰腺癌细胞AP-1活化的影响.结果罗非昔布可抑制裸小鼠胰腺癌移植瘤c-Fos的表达.随着罗非昔布浓度的增加,胰腺癌细胞中c-Fos、ERK蛋白逐渐下降.EMSA分析显示,20%胎牛血清能刺激胰腺癌细胞BXPC-3中AP-1的活化增加,罗非昔布能抑制这种刺激作用.结论罗非昔布可通过抑制胰腺癌细胞ERK、AP-1信号转导通路,抑制胰腺癌细胞增殖.     

塞来昔布对Lewis肺癌组织COX-2、VEGF、MVD、MMP-2表达的影响

目的探讨选择性环氧化酶(COX)-2抑制剂塞来昔布对Lewis肺癌移植瘤的抑制作用及其对肿瘤组织V.EGF、MVD、MMP-2表达的影响.方法将C57BL/6小鼠随机分为药物组和对照组,药物组于接种Lewis肺癌细胞后第二天开始给予含1/1 000塞来昔布的食物,连续24 d,计算抑瘤率.采用免疫组化的方法研究COX-2、VEGF、MVD、MMP-2的表达.并采用逆转录聚合酶链反应(RT-PCR)技术研究VEGF、MMP-2 mRNA的表达.结果含1/1 000塞来昔布食物饲养对Lewis肺癌移植瘤有明显抑制作用,抑瘤率为60.1%.免疫组化结果显示药物组肿瘤组织的COX-2积分平均值低于对照组,但无统计学差异(P＞0.05).药物组与对照组的VEGF积分为2.00±0.82和2.90±0.88(P＜0.05),MVD平均值为16.70±7.77和23.15±4.58(P＜0.05).但药物组与对照组比较,MMP-2的表达无显著性差异.RT-PCR结果表明药物组VEGF mRNA表达较对照组明显下调,而MMP-2 mRNA表达无明显改变.结论塞来昔布对Lewis肺癌移植瘤有明显抑制作用.其作用途径可能是通过抑制COX-2的活性,进而抑制VEGF的表达,减少新生血管形成,抑制肿瘤的生长.抑制肿瘤血管生长可能是塞来昔布肿瘤生长的又一机制.     

抑制NF-κB 对人肺癌裸鼠移植瘤生长及血管生成的影响

 目的 探讨核因子-κB(NF-κB)抑制剂吡咯烷二硫氨基甲酸(PDTC)对肺癌裸鼠移植瘤生长及血管生成的影响。方法 人肺癌A549细胞接种于裸鼠皮下，建立肺癌裸鼠移植瘤模型，分为对照组和PDTC组，比较移植瘤重量，计算抑瘤率；RT—PCR检测移植瘤环氧化酶-2(COX-2)基因表达，免疫组织化学法检测血管内皮生长因子(VEGF)和FⅧ蛋白表达，并计数微血管密度(MVD)。结果 PDTC组移植瘤重量显著低于对照组(P<0．01)，抑瘤率为41．03％；COX-2、VEGF表达及MVD计数均显著低于对照组(P<0．01，P<0．01，P<0．05)。结论 PDTC可抑制肺癌裸鼠移植瘤的生长，该作用可能与其抑制COX-2、VEGF表达，从而抑制血管生成有关。     

罗非昔布对裸鼠人胃癌组织中新生血管及血管内皮生长因子表达的影响

目的探讨罗非昔布对裸鼠人胃癌组织中新生血管及血管内皮生长因子（vascular endothelial growthfactor,VEGF）表达的影响。方法建立BALB/C nu/nu裸鼠人胃腺癌SGC-7901细胞株原位种植转移模型,在种植术后1周以罗非昔布0.2 mL,5 mg.kg-1.d-1给裸鼠灌胃,连续8周后,对荷瘤鼠行彩色多普勒探查,观察胃肿瘤新生血管的形态和数量,通过免疫组织化学Envision法及RT-PCR方法检测胃癌组织中的VEGF蛋白及VEGFmRNA的表达。结果各治疗组肿瘤血管体积[罗非昔布组（9.96±3.58）,5-FU组（9.74±2.83）及联合组（8.25±3.14）]与生理盐水对照组（13.10±4.00）比较,差异有统计学意义（P〈0.05）。VEGF蛋白表达及VEGFmRNA检测结果显示,各治疗组与生理盐水对照组比较,差异有统计学意义（P〈0.05）。结论环氧合酶-2抑制剂罗非昔布可以通过降低胃癌肿瘤组织中VEGF蛋白及VEGFmRNA的表达,减少肿瘤血管数量,抑制胃癌细胞的生长和转移。     

塞来昔布对实验性结肠癌原位移植瘤生长及血管形成的影响

目的:探讨塞来昔布对实验性结肠癌原位移植瘤生长及血管形成的影响。方法:使用对数生长期的人结肠癌细胞(HT-29)于裸鼠皮下接种成瘤后原位种植。术后随机分为对照组(C组)及塞来昔布高、中、低剂量组(H、M、L组)共四组进行研究。结果:24只裸鼠实验期间无1只死亡,成瘤率为100%,比较各组原位移植瘤体积和瘤质量差异有统计学意义,P值均<0·05。L、M和H组的抑瘤率分别为25·30%、38·80%和76·92%,与对照组比较差异有统计学意义,P=0·000,且存在明显的剂量依赖。干预组瘤组织中MVD、VEGFmRNA、MMP-2mRNA和匀浆上清中PGE2含量与对照组相比差异有统计学意义,P值分别为0·050、0·050、0·050和0·010,随着剂量增加,MVD、VEGFmRNA、MMP-2mRNA和匀浆上清中PGE2含量逐渐降低。瘤组织中PGE2含量与瘤质量,以及MVD与VEGFmRNA、MMP-2mRNA均存在显著的正相关性,r=0·8814,P=0·000;r=0·8573,P=0·000;r=0·6427,P=0·001。结论:塞来昔布通过抑制人结肠癌裸鼠原位移植瘤环氧化酶-2活性,抑制PGE2合成、VEGFmRNA和MMP-2mRNA表达,抑制肿瘤的血管形成,从而对结肠癌的生长有抑制作用。     

尼美舒利对人喉鳞癌Hep-2细胞祼鼠移植瘤CD44和MMP-7表达的影响

目的探讨尼美舒利对喉鳞状细胞癌裸鼠皮下移植模型的抑瘤作用及机制。方法建立人喉鳞癌Hep-2细胞株裸鼠移植瘤模型,应用尼美舒利处理裸鼠,观察肿瘤生长情况并绘制生长曲线,免疫组织化学技术检测移植瘤组织中COX-2、CD44和MMP-7蛋白表达,RT-PCR技术检测移植瘤组织中COX-2、CD44 和MMP-7mRNA表达情况。结果与对照组相比,治疗组肿瘤体积增长较对照组缓慢,体积抑瘤率为63.36%,重量抑瘤率达51.81%,肿瘤体积和重量均明显低于对照组（P均＜0.05）。实验组和对照组治疗前后裸鼠体重增长值差异无统计学意义（P＞0.05）。实验组COX-2、CD44和MMP-7蛋白及mRNA表达明显低于对照组,差异有统计学意义（P均＜0.05）。结论尼美舒利可有效抑制人喉鳞癌细胞系Hep-2裸鼠移植瘤的生长,其机制可能与抑制COX-2、CD44及MMP-7表达有关。     

姜黄素对人乳腺癌MDA-MB-231细胞裸鼠移植瘤放射增敏的作用

背景与目的:姜黄素(curcumin)能有效增强放射线对肿瘤的作用,但其作用机制尚不清楚。该研究旨在探讨姜黄素是否对人乳腺癌MDA-MB-231细胞裸鼠移植瘤有放射增敏效应,并探讨其协同抑制效应的机制。方法:人乳腺癌裸鼠移植瘤模型建立成功后,将裸鼠随机分为4组:对照组、药物组、放射组和联合组(姜黄素+放射),每组6只;检测裸鼠移植瘤的体积及体质量,计算抑瘤率,并绘制移植瘤的生长曲线;免疫组织化学法检测血管内皮生长因子(vascular endothelial growth factor,VEGF)表达;蛋白质印迹法(Western blot)检测基质金属蛋白酶-9(matrix metalloproteinases-9,MMP-9)及缺氧诱导因子-1α(hypoxiainducible factor-1α,HIF-1α)的蛋白表达。结果:联合组的抑瘤率[(71.62±6.11)%]显著高于放射组[(41.76±5.84)%](P<0.05)。免疫组化结果显示,单纯放射不能使VEGF表达下调(P>0.05),姜黄素联合放射能明显抑制VEGF的表达(P<0.01)。Western blot结果显示,姜黄素与放射均可抑制MMP-9的表达,两者联用较单纯放射效果更加显著(P<0.05);单纯放射不能抑制HIF-1α的表达(P>0.05),姜黄素联合放射HIF-1α蛋白表达显著降低(P<0.01)。结论:姜黄素可以增强乳腺癌裸鼠移植瘤的放射敏感性,其机制可能是通过下调VEGF、MMP-9和HIF-1α的蛋白表达而发挥作用。     

人巨噬细胞金属弹性蛋白酶对人胃癌细胞COX-2和VEGF体内表达的影响

目的：探讨体内人巨噬细胞金属弹性蛋白酶（HME）对人胃癌细胞环氧合酶-2和血管内皮生长因子（VEGF）表达的影响。方法：选取BALB/c nu/nu裸鼠24只构建人胃癌细胞SCG-7901裸鼠皮下移植模型,随机分为对照组和HME干预组,其中HME干预剂量分别为0.2mg/kg、0.4mg/kg、0.8mg/kg,对照组给予等体积0.9%氯化钠。干预6周后处死动物,测量鼠重、瘤重、肿瘤大小,计算抑瘤率;采用Western blot和免疫组织化学方法检测移植瘤组织中COX-2和VEGF的表达;CD34标记血管内皮细胞计数肿瘤微血管密度（MVD）。结果：与对照组相比各剂量组HME均能明显抑制皮下移植瘤生长（ P <0.05）;且在HME各剂量组中0.8mg/kg组抑瘤作用最明显。HME能明显抑制移植瘤组织中COX-2和VEGF的表达,并明显降低肿瘤MVD值（ P <0.05）。结论：HME通过抑制肿瘤微血管形成来抑制肿瘤的生长;HME抑瘤作用具有剂量依赖性。     

血管内皮抑制素对Calu-6裸鼠移植瘤的生物学效应

目的 探讨血管内皮抑制素对Calu-6裸鼠移植瘤生长及新生血管形成的影响.方法 在荷瘤裸鼠皮下注射不同剂量的血管内皮抑制素,观察注射后肿瘤体积的变化;应用免疫组化SABC法检测肿瘤组织中血管内皮生长因子(VEGF)、生存素(survivin)和环氧化酶-2(COX-2)蛋白的表达以及微血管密度(MVD)的变化;流式细胞术检测循环血管内皮细胞(CECs)的含量;应用逆转录聚合酶链反应(RT-PCR)和实时定量PCR检测外周血中CD146和CD105 mRNA的表达.结果经血管内皮抑制素治疗后,荷瘤鼠肿瘤体积明显减小;肿瘤组织中VEGF、survivin和COX-2蛋白的表达以及MVD均下降,且各治疗组与阳性对照组间的差异均有统计学意义(均P＜0.05);外周血中CECs、CD146和CD105 mRNA的含量均明显下降;活化CECs的含量与肿瘤组织中survivin和VEGF的表达以及MVD的变化均呈正相关.结论 血管内皮抑制素可通过下调移植瘤中VEGF、survivin和COX.2蛋白的表达以及减少MVD抑制肿瘤生长;活化CECs将可能作为理想的预测抗血管形成治疗预后的标记物应用于临床.     

环氧合酶-2抑制剂塞来昔布对人肝癌HepG2裸小鼠移植瘤生长和肿瘤血管生成的抑制作用

背景与目的:有研究证实,环氧合酶-2(cyclooxygenase-2,COX-2)与肿瘤,特别是与消化系统肿瘤形成的关系较为密切,而其抑制剂则具有抗肿瘤作用。本研究探讨特异性抑制剂塞来昔布对人肝癌HepG2裸小鼠移植瘤生长和肿瘤血管生成的抑制作用。方法:将肝癌细胞HepG2种植至裸鼠背侧皮下,4天后开始用塞来昔布治疗,58天后处死。观测裸鼠移植瘤的体积和质量。并采用免疫组化和逆转录-聚合酶链反应法(RT-PCR)检测裸鼠移植瘤组织中COX-2、血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)、碱性成纤维生长因子(basicfibroblastgrowthfactor,bFGF)和血管生成素-2(angiopoientin-2,Ang-2)的表达,用免疫组化法检测微血管密度(microvesseldensity,MVD)。结果:治疗组切除的移植瘤平均体积和质量分别是(709.11±108.53)mm3和(2.63±0.34)g,对照组分别为(1417.55±69.50)mm3和(5.32±0.98)g,两组比较差异有显著性(P<0.01)。治疗组肿瘤增殖率为55.21%。②对照组COX-2、VEGF、bFGF、Ang-2的表达和MVD值分别是4.50±0.25、5.43±0.58、4.03±0.47、5.53±0.54和128.24±9.82,而在塞来昔布组分别是2.42±0.29、2.80±0.30、2.23±0.41、2.88±0.25和29.23±1.52。两组相比较,COX-2、VEGF、bFGF、Ang-2的表达和MVD值差异有显著性(P<0.01)。COX-2与VEGF、bFGF、Ang-2和MVD有明显的相关性(r分别为0.862、0.882、0.857,P分别<0.01)。结论:塞来昔布有效抑制了人肝癌细胞HepG2裸小鼠移植瘤的生长和血管生成,其作用途径是抑制了COX-2的表达。     

Manumycin抗人肝癌细胞HepG2裸鼠移植瘤的作用及其对肿瘤血管生成的影响

背景与目的:法尼基转移酶抑制剂manumycin体内外抗胰腺癌、结肠癌和退行性甲状腺癌的研究已见报道。我们先前的体外研究发现,manumycin能抑制人肝癌HepG2细胞的增殖通路和生存通路。本研究拟探讨manumycin在裸小鼠体内的抗肝癌作用及其对肿瘤的血管生成的影响。方法:建立人肝癌HepG2细胞裸鼠移植瘤模型,接种后第8天按肿瘤大小随机分为5组:生理盐水对照组(20ml/kg)、阳性药对照组(CTX,25mg/kg)、0.1%Me2SO阴性对照组(20ml/kg)、manumycin低剂量组(2.5mg/kg)和高剂量组(5mg/kg)。测量各组肿瘤体积观察manumycin的体内抗瘤作用。采用免疫组化方法观察移植瘤的微血管密度(MVD),应用Westernblot法检测VEGF和b鄄FGF的蛋白水平变化,RT鄄PCR法检测VEGFmRNA的水平。结果:manumycin低剂量组和高剂量组的平均肿瘤体积比(V/V0)分别为0.68±0.09和0.59±0.04(n=5),与0.1%Me2SO阴性对照组(V/V0为1.38±0.21)相比,manumycin处理组肿瘤体积明显缩小(P<0.01)。与对照组相比较,manumycin处理组肿瘤组织MVD明显降低(P<0.01)。manumycin能明显抑制HepG2细胞及HepG2细胞裸鼠移植瘤VEGF蛋白的表达,而对b鄄FGF蛋白的表达影响不大。manumycin也明显抑制了HepG2细胞裸小鼠移植瘤VEGFmRNA的表达。结论:manumycin能明显抑制人肝癌HepG2细胞裸小鼠移植瘤的生长。下调VEGF表达,抑制肿瘤的血管生成可能是manumycin抗肿瘤作用的重要机理之一。     

Enhanced inhibitive effects of combination of rofecoxib and octreotide on the growth of human gastric cancer

Our previous studies indicated that cyclooxygenase-2 inhibitor or octreotide could suppress the proliferation of gastric adenocarcinoma in vitro or in vivo. The present study was aimed to find whether rofecoxib combined with octreotide could enhance the inhibitive effects on the growth of gastric cancer. The effect of rofecoxib or octreotide on proliferation of gastric cancer cell line was determined by 3H-thymidine ribotide incorporation. The TdT-mediated dUTP nick end-labeling assay was used to detect the apopotosis. To determine their synergic antineoplastic effects, the interaction between rofecoxib and octreotide on SGC-7901 cell was evaluated by the median effect plot. After orthotopical implantion of xenografts of human gastric cancer in stomach, nude mice were given rofecoxib plus octreotide for 8 weeks. Cyclooxygenase-2 in gastric cancer tissues was measured by immunohistochemistry. Combination of rofecoxib and octreotide presented synergistic effect (combination index < 1) in the majority of responses. The inhibitory rate for xenografts in nude mice was 89.7% in rofecoxib group. Combination of rofecoxib and octreotide enhanced inhibitory rate to 98.8%. The combination greatly increased the apoptotic index (78.20% +/- 6.45%) of the xenografts as compared with that of using rofecoxib alone (46.60% +/- 3.42%); the difference was very significant (p < 0.001). Rofecoxib could inhibit the activity of cyclooxygenase-2 in the tissue of gastric adenocarcinomas of nude mice. Our results indicate that combination of rofecoxib and octreotide significantly enhances the antiproliferative effect in gastric adenocarcinoma, which might have potential therapeutic value.     

Dihydroartemisinin inhibits angiogenesis in pancreatic cancer by targeting the NF-��B pathway

Purpose

Dihydroartemisinin (DHA) has recently shown antitumor activity in human pancreatic cancer cells. However, its effect on antiangiogenic activity in pancreatic cancer is unknown, and the mechanism is unclear. This study was aimed to investigate whether DHA would inhibit angiogenesis in human pancreatic cancer.

Methods

Cell viability and proliferation, tube formation of human umbilical vein endothelial cells (HUVECs), nuclear factor (NF)-??B DNA-binding activity, expressions of vascular endothelial growth factor (VEGF), interleukin (IL)-8, cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9 were examined in vitro. The effect of DHA on antiangiogenic activity in pancreatic cancer was also assessed using BxPC-3 xenografts subcutaneously established in BALB/c nude mice.

Results

DHA inhibited cell proliferation and tube formation of HUVECs in a time- and dose-dependent manner and also reduced cell viability in pancreatic cancer cells. DHA significantly inhibited NF-??B DNA-binding activity, so as to tremendously decrease the expression of NF-??B-targeted proangiogenic gene products: VEGF, IL-8, COX-2, and MMP-9 in vitro. In vivo studies, DHA remarkably reduced tumor volume, decreased microvessel density, and down-regulated the expression of NF-??B-related proangiogenic gene products.

Conclusions

Inhibition of NF-??B activation is one of the mechanisms that DHA inhibits angiogenesis in human pancreatic cancer. We also suggest that DHA could be developed as a novel agent against pancreatic cancer.     

Celecoxib inhibits angiogenesis by inducing endothelial cell apoptosis in human pancreatic tumor xenografts

Previous studies suggest that antagonists of cyclooxygenases 1 and 2 (COX-1, -2) inhibit angiogenesis in tumor xenografts, but the molecular mechanisms involved remain unclear. Here we characterized the effects of non-selective (indomethacin) and selective (NS398, celecoxib) cyclooxygenase inhibitors on parameters of angiogenesis in human pancreatic adenocarcinoma cells. COX-1 expression was constitutive in 9/9 pancreatic cancer cell lines, whereas COX-2 and cytosolic phospholipase A2 (cPLA2) expression were observed in 4/9 cell lines (BxPC3, Capan2, Cfpac1, and L3.6 pl). Production of the COX product, prostaglandin E2, correlated with expression of cPLA2 and COX-2 and was blocked by non-steroidal anti-inflammatory drugs (NSAIDs, indomethacin or NS398). In contrast to the findings of others, neither indomethacin nor NS398 affected tumor cell secretion of angiogenic factors (VEGF, bFGF, IL-8) at concentrations that produced maximal inhibition of PGE2 production, and higher concentrations increased angiogenic factor production. We also studied the effects of celecoxib in orthotopic L3.6 pl xenografts. Immunofluorescence analyses revealed high-level expression of COX-2 in endothelial cells in L3.6 pl xenografts that increased following therapy with celecoxib, whereas the tumor cells expressed uniformly low levels of COX-2. Celecoxib did not decrease tumor-associated VEGF levels in orthotopic human L3.6 pl xenografts, but the drug did decrease tumor microvessel density (MVD) and increase apoptosis in tumor-associated endothelial cells in a dose-dependent fashion. Together, our results demonstrate that the anti-angiogeneic effects of NSAIDs in human pancreatic cancer cells are exerted via direct effects on endothelial cells.     

Ellagic acid inhibits human pancreatic cancer growth in Balb c nude mice

Ellagic acid (EA) is a polyphenol found in several plants and fruits. The objectives of this study were to examine the molecular mechanisms by which EA inhibits pancreatic cancer growth in Balb C nude mice. PANC-1 cells were injected subcutaneously into Balb c nude mice, and tumor-bearing mice were treated with EA. The expression of Akt, Shh and Notch and their target gene products were measured by the immunohistochemistry and Western blot analysis. Treatment of PANC-1 xenografted mice with EA resulted in significant inhibition in tumor growth which was associated with suppression of cell proliferation and caspase-3 activation, and induction of PARP cleavage. EA inhibited the expression of Bcl-2, cyclin D1, CDK2, and CDK6, and induced the expression of Bax in tumor tissues compared to untreated control group. EA inhibited the markers of angiogenesis (COX-2, HIF1α, VEGF, VEGFR, IL-6 and IL-8), and metastasis (MMP-2 and MMP-9) in tumor tissues. Furthermore, treatment of mice with EA caused a significant inhibition in phospho-Akt, Gli1, Gli2, Notch1, Notch3, and Hey1. EA also reversed epithelial to mesenchymal transition by up-regulating E-cadherin and inhibiting the expression of Snail, MMP-2 and MMP-9. These data suggest that EA can inhibit pancreatic cancer growth, angiogenesis and metastasis by suppressing Akt, Shh and Notch pathways. In view of the fact that EA could effectively inhibit human pancreatic cancer growth by suppressing Akt, Shh and Notch pathways, our findings suggest that the use of EA would be beneficial for the management of pancreatic cancer.