shRNA干扰HIF-1α对人宫颈癌细胞黏附分子表达的影响

目的在细胞和移植瘤水平研究shRNA干扰低氧诱导因子-1α(hypoxia inducible factor-1,HIF-1α)对宫颈腺癌HeLa细胞中黏附分子CD44v6及上皮型钙黏素(E-cadherin)表达的影响.方法将人宫颈癌Hela细胞株(记为H0细胞)、转染pGenesil-1空白质粒的HeLa细胞株(记为H1细胞)及转染HIF-1α-shRNA质粒的HeLa细胞株(记为H2细胞)分别行常氧培养及低氧(1%O2)培养,应用Western blotting检测每组HeLa细胞中HIF-1α、CD44v6、E-cadherin及β连接素(β-catenin)在细胞水平的表达情况,应用免疫组织化学法检测HIF-1α、CD44v6、E-cadherin及β-catenin在上述3种HeLa细胞裸鼠移植瘤模型瘤组织水平的表达情况.结果Western blotting分析显示,H0和H1细胞低氧组检测到HIF-1α、β-catenin、CD44v6蛋白呈强阳性,而E-cadherin表达减弱;常氧状态下H0、H1和H2组及低氧下H2组HIF-1α无表达、E-cadherin表达呈阳性.免疫组化分析发现H0及H1组移植瘤组织中HIF-1α、E-cadherin、β-catenin及CIM4v6的表达分别为0.651±0.057、0.198±0.015、0.465±0.061及0.687±0.102,0.671±0.069、0.201±0.020、0.452±0.038及0.701±0.071,H2组分别为0.194±0.023、0.548±0.045、0.421±0.052及0.187±0.057,H2组与H0和H1组间HIF-1α、E-cadherin、及CD44v6表达的差异有统计学意义(P＜0.05);相关性分析提示HIF-1α与E-cadherin、CD44v6的表达有一定相关性.结论通过shRNA干扰抑制人宫颈癌HeLa细胞HIF-1α,可一定程度上下调低氧环境中宫颈癌HeLa细胞黏附分子CD44v6及上调E-cadherin的表达.     

RNA干扰HIF-1α增强宫颈癌BALB/c小鼠移植瘤放射敏感性

背景与目的：缺氧诱导因子-1α（HIF-1α）是一种重要的缺氧应答调控因子，在维持细胞能量代谢、肿瘤血管生成及转移、细胞增殖与凋亡等诸多方面起着重要作用。在子宫内膜癌、卵巢癌，食道癌等肿瘤中，国内外有大量研究报道，但均未有关于HIF-1α与宫颈癌移植瘤放疗敏感性关系的报道。本研究探讨RNA干扰乏氧诱导因子-1α增强宫颈癌BALB／c小鼠移植瘤放射敏感性的作用。方法：构建干扰HIF-1α质粒HIF-1α-shRNA，采用Western blot方法观察有氧与乏氧状态下RNA干扰HIF-1α蛋白的变化。将20只裸鼠分随机为两组．其中对照组接种含pGenesil-1空白质粒人宫颈癌HeLa细胞（命名为H0），实验组接种含HIF—1α-shRNA质粒的HeLa细胞（命名为H1），待裸小鼠右后大腿外侧皮下移植瘤长至直径8．0mm时，在两组中各取5只进行照射，计算两组裸鼠移植瘤长至14．0mm时的生长延缓时间。结果：①Western blot分析乏氧状态下对照组检测到HIF—1α蛋白质条带，常氧状态下实验组和对照组及乏氧状态下的实验组均未检测到HIF-1α蛋白质表达。②两组移植瘤从8．0mm生长到14．0mm时生长延缓时间分别为8d及14d（P〈0．05）。结论：RNA干扰H1F—1α可增强BALB／c小鼠宫颈癌移植瘤的放射敏感性。     

组蛋白去乙酰化酶抑制剂对乏氧宫颈癌HeLa细胞放射敏感度的影响

目的:观察组蛋白去乙酰化酶抑制剂曲古霉素A(trichostatin A,TSA)对乏氧状态下人宫颈癌HeLa细胞放射敏感度的影响。方法:应用不同浓度TSA作用经乏氧预处理的人宫颈癌HeLa细胞12、24、48和72 h,MTT法检测HeLa细胞的增殖率,计算半数抑制浓度(half inhibitory concentration,IC50)和IC10值;克隆形成实验检测TSA(IC10)作用24 h对乏氧HeLa细胞的放射增敏效应;免疫细胞化学法检测TSA(IC10)对HeLa细胞中乏氧诱导因子-1α(hypoxia-inducible factor-1 alpha,HIF-1α)和血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白表达的影响。结果:TSA对乏氧宫颈癌HeLa细胞的增殖率有明显的抑制作用,且随着TSA浓度的增加和作用时间的延长,其抑制作用逐渐增强。与乏氧照射组比较,TSA(IC10)作用乏氧HeLa细胞24 h,可增加细胞的放射敏感度(P<0.05)。乏氧HeLa细胞中HIF-1α和VEGF蛋白的表达明显高于常氧组细胞;与乏氧组比较,TSA(IC10)作用乏氧HeLa细胞24 h后,细胞中HIF-1α和VEGF蛋白表达下调。结论:TSA可能通过抑制乏氧HeLa细胞中HIF-1α和VEGF蛋白的表达而发挥放射增敏效应。     

HIF-1α沉默对鼻咽癌CNE-1细胞移植瘤生长和转移的影响

目的 低氧诱导因子-1α(hypoxia inducible factor-1alpha,HIF-1α)在鼻咽癌组织中存在高表达,与淋巴结转移和临床分期偏晚密切相关,是鼻咽癌患者独立的不良预后因子.前期研究显示,沉默HIF-1α可有效降低鼻咽癌CNE-1细胞的黏附和侵袭能力.本研究通过探讨沉默HIF-1α对鼻咽癌CNE-1细胞移植瘤生长和转移能力的影响,进一步为临床提供参考和实验依据.方法 采用RNA干扰(RNA interference,RNAi)技术沉默鼻咽癌CNE-1细胞中HIF-1α表达,RT-PCR检测其沉默效果;15只雄性裸鼠随机分为未转染组、阴性对照组和RNAi组,每组5只.将CNE-1细胞分别接种于裸鼠右大腿外侧皮下,建立鼻咽癌CNE-1细胞裸鼠移植瘤模型,观察各组成瘤时间并记录其生长情况,待分别形成肉眼可见的肿瘤后再培养20 d.收集移植瘤组织及裸鼠两侧腹股沟淋巴结.测量移植瘤大小并计算其体积.采用HE染色,计算各组淋巴结转移率及阳性率;蛋白质印迹法检测3组移植瘤中HIF-1α、E-钙粘蛋白(E-cadherin)及CX-CR4蛋白的表达.结果 RNAi可有效沉默鼻咽癌CNE-1细胞中HIF-1α表达;成功构建了裸鼠移植瘤模型;未转染组、阴性对照组和RNAi组裸鼠成瘤时间分别为(8.3±1.2)、(7.8±1.5)和(15.6±2.1)d,差异有统计学意义,F=21.181,P=0.002;最终成瘤体积分别为(1184.4±145.8)、(1254.0±130.5)和(322.7±118.2) mm3,F=82.081,P＜0.001.淋巴结转移率及阳性率均明显降低;与未转染组及阴性对照组比较,HIF-1α(P＜0.001)和CXCR4 (P=0.001)蛋白在RNAi组中表达明显降低,而E-cadherin则明显增强,P=0.003.结论 RNAi沉默HIF-1α可有效抑制鼻咽癌CNE-1细胞移植瘤的生长和转移,其机制可能与上调E-cadherin、下调CXCR4蛋白表达有关.     

赖氨酰氧化酶表达干扰对乳腺癌裸鼠移植瘤生长影响及机制研究

目的:探讨抑制乳腺癌细胞MD-MB-231中赖氨酰氧化酶(lysyl oxidase,LOX)的表达对乳腺癌裸鼠移植瘤生长的影响及可能机制。方法:设计合成特异性LOX基因慢病毒干扰载体(LOX-RNAi-LV)转染乳腺癌细胞MDA-MB-231,采用荧光定量PCR和蛋白质印迹法检测LOX mRNA和蛋白的表达;建立乳腺癌裸鼠原位移植模型,观察移植瘤的生长情况。采用免疫组化方法检测移植瘤组织中LOX蛋白及肿瘤增殖、转移相关蛋白Ki-67、MMP-2、MMP-9和HIF-1α的表达。结果:转染LOX-RNAi-LV的人乳腺癌细胞MDA-MB-231中LOX mRNA和蛋白的相对表达量分别为0.102±0.083和0.156±0.004,与空白对照组(0.945±0.158和0.916±0.007)相比,表达率均明显下调,抑制率分别为89.2%和83.0%。裸鼠原位接种癌细胞后6d均有肿瘤生成,40d后接种转染LOX-RNAi-LV的MDA-MB-231细胞的裸鼠移植瘤体积为(108.89±26.61)mm3,质量为(0.117±0.021)g;均明显低于空白对照组(400.15±79.81)mm3,(0.433±0.068)g,以及阴性对照组(380.15±65.81)mm3,(0.404±0.053)g,差异有统计学意义,P值均为0.000。移植瘤组织中LOX、Ki-67、MMP-2、MMP-9和HIF-1α蛋白相对表达水平分别为0.198±0.036、0.347±0.054、0.379±0.048、0.335±0.067和0.307±0.073,较空白对照组和阴性对照组明显降低,P值均<0.01。结论:干扰MDA-MB-231中LOX的表达,可抑制乳腺癌裸鼠移植瘤的生长侵袭,LOX可能通过调节Ki-67、MMP-2、MMP-9和HIF-1α蛋白的表达,在乳腺癌的侵袭转移中发挥作用。     

非小细胞肺癌中Snail、HIF-1α和E-cadherin表达的临床意义及相关性

目的探讨Snail、乏氧诱导因子-1α(HIF-1α)、E-钙黏素(E-cadherin)在非小细胞肺癌(NSCLC)中表达的临床意义及相关性。方法采用免疫组织化学法检测62例NSCLC患者肺癌组织和20例癌旁组织Snail、HIF-1α、E-cadherin蛋白表达,并分析与临床病理因素的关系。结果肺癌组织中Snail、HIF-1α及E-cadherin阳性表达率分别为64.5%、59.7%和54.8%,癌旁组织分别为15%、10%和100%。三种蛋白在肺癌组织及癌旁组织表达差异有统计学意义(P<0.05)。Snail和HIF-1α表达升高及E-cadherin降低与肺癌临床分期和淋巴结转移有关(P<0.05)。HIF-1α和Snail表达均与E-cadherin表达呈负相关性(P<0.05),但HIF-1α和Snail表达未显示相关性(P>0.05)。结论 Snail和HIF-1α表达升高、E-cadherin表达降低与肺癌发生发展和转移有关,Snail、HIF-1α和E-cadherin检测有助于评估肺癌恶性程度及预后。     

过表达缺氧诱导因子-1α的结直肠癌裸鼠可视化模型的建立

目的:建立一种荧光可视化显像且稳定过表达缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)的裸鼠结直肠癌移植瘤模型。方法:对不同的人结直癌细胞系(SW480、SW620、LOVO及HCT116细胞)用缺氧诱导剂Co Cl2处理,根据HIF-1α被诱导表达的情况选择合适的靶细胞;将HIF-1α的c DNA序列克隆至慢病毒表达质粒p LV-TRC-EGFP,构建p LVHIF1α-EGFP慢病毒表达质粒,并包装过表达HIF-1α的慢病毒颗粒Lenti-HIF1α-EGFP。将Lenti-HIF1α-EGFP感染SW480细胞,嘌呤霉素筛选稳定过表达HIF-1α的细胞株SW480HIF-1α,Western blotting检测SW480HIF-1α细胞内HIF-1α及其下游蛋白VEGF、M1型丙酮酸激酶(pyruvate kinase expression M1,PKM1)的表达情况,Transwell实验检测其迁移能力。裸鼠腹腔注射SW480HIF-1α建立结直肠癌移植瘤模型,倒置荧光显微镜观察裸鼠腹腔移植瘤结节。结果:常氧条件下4种结直肠癌细胞均不表达HIF-1α,经Co Cl2诱导后,除SW480细胞系以外的细胞均可被诱导表达HIF-1α,故选择SW480细胞作为研究靶细胞。成功构建稳定过表达HIF-1α的细胞株SW480HIF-1α,SW480HIF-1α细胞内HIF-1α、VEGF和PKM1的表达水平均高于野生型SW480细胞,其迁移能力较野生型SW480细胞显著增强[观察视野内迁移细胞数:(250±11)vs(50±5)个,P<0.01]。SW480HIF-1α组裸鼠肠壁形成肿瘤结节数量显著多于SW480组[(15±4)vs(4±1)个,P<0.05]。结论:成功建立了高表达HIF-1α的荧光可视化裸鼠移植瘤模型,为进一步的相关功能学研究以及药物筛选提供条件。     

反义寡核苷酸对鼻咽癌细胞乏氧条件下放射敏感性的影响

目的:探讨乏氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)在乏氧状态下对鼻咽癌CNE-1细胞放射敏感性的影响.方法:氯化钴处理CNE-1细胞模拟乏氧条件下的培养传代,通过脂质体将不同HIF-1α反义寡核苷酸酸导入CNE-1细胞中,根据转染复合物的不同将CNE-1细胞分为反义联合组、正义联合组、脂质体组和单纯放疗组(放疗对照组).X射线单次照射,照射条件为照射率4 Gy/min,共8Gy,细胞照射后继续乏氧条件下培养24 h.MTT法检测CNE-1细胞的存活率,Western blotting检测CNE-1细胞中HIF-1α和VEGF蛋白的表达.结果:在乏氧条件下,反义联合组的鼻咽癌CNE-1细胞存活率明显大于正义联合组和单纯放疗组.Western blotting结果显示,与正义联合组和单纯放疗组相比,反义联合组CNE-1细胞中HIF-1α蛋白表达显著下降(0.162 ±0.055vs 0.872 ±0.191,0.768±0.217,0.863±0.245,P＜0.05);同时反义联合组CNE-1细胞中VEGF蛋白的表达量较正义联合组和单纯放疗组明显减少(0.364±0.078 vs 1.165±0.346,1.068±0.379,1.087±0.266,P＜0.05).结论:HIF-1α反义寡核苷酸能有效抑制HIF-1α的表达,对乏氧状态下的鼻咽癌CNE-1细胞具有放射增敏作用.     

长程缺氧对结肠癌细胞增殖的影响

目的:体外观察长程缺氧环境对结肠癌细胞SW480和HCT116增殖能力的影响并探讨其可能的作用机制。方法:缺氧培养结肠癌SW480和HCT116细胞14～28d,采用MTT法和平板克隆法检测缺氧条件下2种结肠癌细胞增殖能力的改变;激光共聚焦显微镜下观察缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)和Wnt/β-catenin信号通路关键因子β-catenin的表达;蛋白质印迹法检测HIF-1α和β-catenin、糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)、Ser9被磷酸化的GSK-3β[phospho-glycogen synthase kinase-3β(Ser9),pGSK-3β(Ser9)]以及增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达。以常氧下培养的这2种细胞作为对照。结果:细胞生长曲线和平板克隆检测结果均显示,长程缺氧适应后SW480和HCT116细胞的增殖能力较常氧下明显增强(P<0.01);激光共聚焦显微镜下观察发现,HCT116细胞在缺氧条件下HIF-1α的表达部位发生改变;蛋白质印迹法检测发现,与常氧组比较长程缺氧组细胞中HIF-1α(P<0.05)、β-catenin(P<0.05)和pGSK-3β(Ser9)(P<0.01)蛋白的表达均上调,而总GSK-3β的表达无明显变化,PCNA在缺氧条件下表达上调(与常氧组比较,在SW480细胞中P<0.05,在HCT116细胞中P<0.01)。结论:缺氧适应可促进结肠癌细胞SW480和HCT116的增殖,这可能是HIF-1α与Wnt/β-catenin信号转导通路共同作用的结果。     

罗勒胶囊对大鼠移植性肝癌TACE术后肿瘤组织HIF-1α和MMP及血清AFP表达的影响

目的:观察罗勒胶囊对大鼠移植性肝癌TACE术后肿瘤组织中HIF-1α、MMP-2、MMP-9及血清AFP表达的影响.方法:建立大鼠移植性肝癌模型,造模成功后随机分为非TACE对照组、TACE+NS对照组、TACE+罗勒胶囊大剂量组、TACE+罗勒胶囊中剂量组和TACE+罗勒胶囊小剂量组5组,除对照组外,其余4组大鼠均进行TACE术,术后分别给予不同剂量罗勒胶囊灌胃治疗.10 d后处死大鼠,取出瘤块,免疫组化法检测瘤块中HIF-1α、MMP-2和MMP-9的表达情况,应用酶联免疫吸附试验(ELISA)法测定大鼠血清中AFP表达情况.结果:与TACE+生理盐水组比较罗勒胶囊大剂量组瘤质量明显降低(P<0.05),与非TACE对照组比较,TACE+NS组HIF-1α、MMP-2和MMP-9的表达均增高;罗勒胶囊治疗组HIF-1α、MMP-2和MMP-9表达较TACE+NS组表达显著降低,并呈现一定的剂量依赖性.TACE+NS组较非TACE组血清中AFP表达显著降低.罗勒胶囊大、中剂量治疗组较非TACE组血清中AFP显著降低,而与TACE+NS组比较,差异无统计学意义,P>0.05.肝癌组织中的HIF-1α与MMP-2的表达成正相关,P<0.01.结论:罗勒胶囊对大鼠移植性肝癌具有改善TACE术后乏氧的作用,并通过降低术后肿瘤乏氧微环境中HIF-1α和MMP-2及MMP-9的表达,而起到TACE术对肝移植瘤的抑制作用.     

低氧诱导因子-1αmRNA在胰腺癌组织中的表达与胰腺癌生物学特征的关系

背景与目的低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)是广泛存在于多种细胞的转录因子,对低氧时肿瘤细胞相关基因的表达发挥着重要的调控作用.本研究拟检测HIF-1α mRNA在胰腺癌中的表达,初步探讨其与胰腺癌血管形成、胰腺癌细胞增殖分化和转移等生物学特征的关系.方法采用逆转录多聚酶链反应(RT-PCR)法检测HIF-1α在胰腺癌组织和癌旁组织中的表达,采用免疫组化SP法检测survivin、PCNA和CD34在胰腺癌组织和癌旁组织中的表达.Spearman相关关系检验分析HIF-1α mRNA表达水平与survivin、PCNA、CD34之间的关系.结果HIF-1α mRNA在胰腺癌组织(0.77±0.04)与癌旁组织(0.56±0.01)中的表达具有极显著性差异(P<0.01);HIF-1α的表达水平在不同分化程度的肿瘤细胞中无显著性差异(P>0.05).JPS分期Ⅰ～Ⅱ期和Ⅲ～Ⅳ期之间HIF-1α mRNA表达水平(0.79±0.05 vs.0.76±0.04)具有显著性差异(P<0.05).HIF-1α表达与PCNA、survivin、CD34表达之间密切相关(r分别为1.00、0.91、0.68,P<0.01).结论HIF-1α的表达水平与胰腺癌血管形成以及胰腺癌细胞的增殖、凋亡和转移等有显著关系.     

Comparison of Beta-catenin with TGF-beta1, HIF-1alpha and Patients’ Disease-free Survival in Human Colorectal Cancer

Beta-catenin accumulation is suppressed by TGF-beta1 (transforming growth factor beta1) in intestinal epithelium suggesting negative feedback between these two factors. Besides that, beta-catenin interacts with HIF-1alpha (hypoxia-inducible factor-1alpha) at the promoter region of HIF-1 target genes. Our study was aimed at comparison of beta-catenin with HIF-1alpha, TGF-beta1, Ki67 and survival of sporadic colorectal cancer patients. Expressions of beta-catenin, TGF-beta1, HIF-1alpha, Ki67 were evaluated in triads of specimens of each primary tumor of 72 sporadic colorectal cancers with immunohistochemistry due to limited availability of tissue material. Disease-free survival was analyzed in case of all 100 beta-catenin stained tumors, in 85 cancers stained for HIF-1 and in 72 neoplasms with TGFbeta1 staining. Beta-catenin, TGF-beta1 and HIF-1alpha accumulated in 72 colorectal cancer cells. Beta-catenin correlated both with HIF-1alpha and TGF-beta1 in all colorectal cancers (p < 0.009, r = 0.307 and p = 0.003, r = 0.342, respectively) and in subgroups of different clinico-pathological profile. Beta-catenin failed to correlate with Ki67. In case of beta-catenin, TGF-beta1 and HIF-1alpha, disease-free survival curves failed to show any statistically significant differences between groups of marker negative tumors, cancers with low expression and neoplasms with higher protein expression. Positive correlations between beta-catenin and TGF-beta1 may indicate ineffective attempts of TGF-beta1 to reduce intracellular level of beta-catenin in colorectal cancer. Associations between beta-catenin and HIF-1alpha reflect previously detected interactions between HIF-1alpha with beta-catenin and are confirmative for presence of such reactions in human colorectal cancer.     

过表达miR-129-5p通过靶向MAPK1抑制宫颈癌HeLa细胞恶性生物行为

[摘要] 目的：探讨miR-129-5p 对宫颈癌HeLa细胞侵袭、迁移和EMT的作用及其机制。方法：选取宫颈癌HeLa细胞,利用生物信息学预测软件筛选miR-129-5p 的靶基因,双荧光素酶报告基因验证miR-129-5p 和MAPK1 的靶向关系。将miR-129-5p mimic、miR-129-5p inhibitor 和pcDNA-MAPK1 单独或联合转染到HeLa细胞,用qPCR检测HeLa细胞中miR-129-5p 和MAPK1 的表达水平,用Transwell、划痕愈合实验分别检测HeLa 细胞的侵袭、迁移能力,WB检测细胞中E-cadherin、N-cadherin、MAPK1、STAT3 和Bcl-xL的表达。构建裸鼠HeLa细胞皮下移植瘤模型,观察miR-129-5p 过表达对移植瘤生长的影响,WB检测移植瘤组织中EMT及MAPK1 通路相关蛋白的表达。结果：miR-129-5p 与MAPK1 在3’UTR区存在结合位点,过表达miR-129-5p 靶向抑制MAPK1（P<0.01）。与对照组相比,miR-129-5p mimic 组侵袭细胞数目减少（P<0.01）,划痕愈合率降低（均P<0.01）;细胞中Ecadherin表达上调而N-cadherin、MAPK1、STAT3 和Bcl-xL 表达下调（均P<0.01）;共转染MAPK1 可逆转上述现象。成功建立裸鼠HeLa 细胞移植瘤模型,与对照组相比,miR-128-3p mimic 组肿瘤质量减轻（P<0.01）;瘤组织中E-cadherin 表达水平上调而N-cadherin、MAPK1、STAT3 和Bcl-xL 的表达下调（均P<0.01）。结论：过表达miR-129-5p 通过靶向MAPK1 抑制宫颈癌HeLa细胞的侵袭、迁移和EMT。     

Hypoxia-inducible factor-1alpha obstructs a Wnt signaling pathway by inhibiting the hARD1-mediated activation of beta-catenin

Although a splice variant of mouse mARD1s was found to acetylate and destabilize hypoxia-inducible factor-1alpha (HIF-1alpha), human hARD1 has no such activities. Nonetheless, hARD1 has been reported to bind directly with HIF-1alpha. Here, we addressed the functional significance of the hARD1-HIF-1alpha interaction. Because hARD1 acetylates and activates beta-catenin, we examined whether HIF-1alpha regulates the hARD1-mediated activation of Wnt signaling. It was found that HIF-1alpha binds hARD1 through the oxygen-dependent degradation domain and, in so doing, dissociates hARD1 from beta-catenin, which prevents beta-catenin acetylation. In LiCl-stimulated HEK293 or cancer cell lines with active Wnt signaling, beta-catenin acetylation and activity were suppressed in hypoxia, and these suppressions were mediated by HIF-1alpha. Moreover, HIF-1alpha disruption of hARD1/beta-catenin repressed TCF4 activity, resulting in c-Myc suppression and p21(cip1) induction. In addition, we confirmed that the HIF-1alpha NH(2) terminal inactivates TCF4 by directly binding beta-catenin. In conclusion, HIF-1alpha was found to inactivate the Wnt signaling by binding to hARD1 or beta-catenin, which may contribute to the hypoxia-induced growth arrest of tumor cells.     

大肠腺癌组织中的Glut1和HIF-1α蛋白表达及其与癌细胞增殖的相关性

背景与目的:恶性肿瘤细胞表现出微环境缺氧和糖代谢增加,缺氧时葡萄糖转运蛋白1(glucosetransporter-1,Glut1)和缺氧诱导因子-1α(hypoxia-induciblefactor1alpha,HIF鄄1α)促进糖代谢增加。两者在大肠腺癌中表达的关联性分析及与增殖活性的关系尚未见报道,本研究旨在探讨两者在大肠腺癌组织中的表达及与细胞增殖活性的关系。方法:采用免疫组织化学方法检测Glut1、HIF-1α、增殖细胞核抗原(proliferatingcellnuclearantigen,PCNA)在60例大肠腺癌组织及20例正常大肠组织的表达水平,并比较它们之间相关性。结果:Glut1、HIF-1α在大肠腺癌组织中的表达阳性率分别为58.3%和73.3%,在正常大肠组织中无表达,与正常大肠组织表达比较差异有显著性(均P<0.01),PCNA在大肠腺癌组织中的表达(65.25±16.35)显著高于正常组织中的表达(15.20±3.47)(P<0.01)。Glut1、HIF-1α蛋白表达与PCNA呈正相关(r1=0.409,P<0.01和r2=0.323,P<0.05),与淋巴结转移、Dukes分期相关。结论:Glut1和HIF-1α蛋白的过表达共同参与了大肠腺癌的发生、发展,与大肠腺癌细胞增殖活性密切相关。     

Combination strategy targeting the hypoxia inducible factor-1 alpha with mammalian target of rapamycin and histone deacetylase inhibitors.

PURPOSE: The hypoxia-inducible factor-1 alpha (HIF-alpha) is a key regulator of tumor angiogenesis. Mammalian target of rapamycin (mTOR) and histone deacetylase (HDAC) inhibitors suppress tumor-induced angiogenesis by reducing tumor HIF-1 alpha protein expression. Thus, we hypothesized that combination treatment of rapamycin and the HDAC inhibitor LBH589 has greater antiangiogenic and antitumor activity compared with single agents. EXPERIMENTAL DESIGN: To evaluate the effect of LBH589 and rapamycin on HIF-1 alpha in human prostate PC3, renal C2 carcinoma cell lines, and endothelial cells (human umbilical vein endothelial cells), we did Western blot analysis. To determine the antitumor activity of LBH589 and rapamycin, cell proliferation assays and xenograft experiments were conducted. RESULTS: Western blotting showed that combination treatment of human umbilical vein endothelial cells, C2 and PC3, significantly reduced HIF-1 alpha protein expression compared with single agents. Treatment with rapamycin resulted in inhibition of the downstream signals of the mTOR pathway and increased phosphorylation of Akt in C2 cells, whereas the constitutively activated Akt in PC3 cells was not modulated. LBH589 decreased both constitutively expressed and rapamycin-induced phosphorylated Akt levels in PC3 and C2 cell lines. In clonogenic assays, the combination treatment had a greater inhibitory effect in PC3 cells (93 +/- 1.4%) compared with single agents (66 +/- 9% rapamycin and 43 +/- 4% LBH589). Combination of rapamycin and LBH589 significantly inhibited PC3 and C2 in vivo tumor growth and angiogenesis as measured by tumor weight and microvessel density. CONCLUSIONS: Combination treatment of mTOR and HDAC inhibitors represents a rational therapeutic strategy targeting HIF-1 alpha that warrants clinical testing.     

The effect of hypoxic microenvironment on matrix metalloproteinase expression in xenografts of human oral squamous cell carcinoma

Hypoxia is a common environmental stress factor and is associated with physiological and pathological conditions related to cancer invasion and metastasis. The process of cancer cell invasion involves degradation of the extracellular matrix. Here, we examine the effect of hypoxic microenvironment on matrix metalloproteinase expression in human oral squamous cell carcinoma under in vitro and in vivo conditions. At first, the expression levels of HIF-1alpha and matrix metalloproteinase (MMP) proteins in human oral squamous cell carcinoma cell lines, SAS and HSC-2 cultured under hypoxic or normoxic condition, were assessed by Western blotting. Enzyme activity and mRNA of MMP under hypoxic or normoxic condition were also investigated. Then the SAS and HSC-2 cells were transplanted subcutaneously into immunodeficient mice and the correlation between hypoxia and protein expression for MMPs, HIF-1alpha and Ki-67 were assessed. Hypoxic region was detected by in situ hypoxic probe, pimonidazole. MMP proteins and mRNA in both SAS and HSC-2 cells were increased under hypoxic condition. In xenograft, MMP-2 was expressed in tumor tissue, especially in hypoxic region. In contrast, MMP-9 expression was recognized in tumor tissue, especially neighboring stromal tissues containing blood vessels. Our study suggests that the hypoxic microenvironment in human oral squamous cell carcinoma plays important roles in expression for HIF-1alpha and MMPs, and proliferative activity of tumor cells.     

低氧对前列腺癌PC3细胞上皮间质转化的影响

 目的 探讨低氧对前列腺癌细胞上皮间质转化的影响。方法 分别在常氧（常氧组）和低氧（低氧组）条件下培养PC3细胞24 h,细胞增殖MTT实验检测低氧对前列腺癌PC3细胞增殖力的影响,Transwell侵袭实验评估低氧对前列腺癌PC3细胞侵袭力的影响,Western blot检测HIF-1α、E-cadherin、N-cadherin和Vimentin的表达水平。另外,在常氧条件下用siRNA抑制HIF-1α表达（干扰组）,用Western blot检测E-cadherin、N-cadherin和Vimentin的表达水平变化。结果 与常氧组比较,低氧组前列腺癌PC3细胞的增殖力和侵袭力增加,HIF-1α表达水平增加（P=0.0004）,HIF-1α向核内转位;N-cadherin（P<0.0001）和Vimentin（P<0.0001）的表达水平显著增加,E-cadherin的表达水平显著下降（P<0.0001）。同时,干扰组跟常氧组比较,抑制HIF-1α表达使N-cadherin（P=0.0002）和Vimentin（P=0.0002）的表达水平显著下降,而使E-cadherin的表达水平显著增加（P<0.0001）。结论 低氧可能通过调节HIF-1α表达促进前列腺癌PC3细胞的上皮间质转化。