青蒿不同溶剂萃取粗提物对结肠癌HT-29、LoVo细胞NF-κB活性的影响

目的:通过比较不同溶剂萃取青蒿的粗提物对结肠癌HT-29和LoVo细胞的核因子κB(NF-κB)活性的影响,寻找青蒿中对结肠癌细胞NF-κB活性有作用的萃取相.方法:分别用石油醚、氯仿、乙酸乙酯、正丁醇对青蒿水煎液进行萃取,并将萃取得到的粗提物分别处理结肠癌HT-29细胞和LoVo细胞,提取核蛋白后采用凝胶阻滞分析(EMSA)方法对NF-κB活性进行测定.结果:青蒿水煎液氯仿萃取相对HT-29细胞和LoVo细胞的NF-κB活性表达均有明显的抑制作用(P＜0.05),其它各萃取相对两种细胞NF-κB活性的影响与对照组比较无明显差异.结论:在不同溶剂萃取青蒿水煎液中,氯仿萃取相粗提物对结肠癌HT-29细胞和LoVo细胞的NF-κB活性具有抑制作用.     

Id1对结肠癌HT-29细胞VEGF表达及增殖的影响

目的探讨分化抑制因子１(inhibitor of differentiation-1,Id1)对结肠癌HT-29细胞VEGF表达的影响及Id1在结肠癌细胞增殖中的作用。方法HT-29转染Id1表达质粒及Id-1干扰序列后,采用RT-PCR方法和Western blot法检测VEGF mRNA和蛋白的表达,MTT法检测各组细胞增殖情况。结果 Id1过表达能显著上调VEGF mRNA和蛋白表达(P<0.01),促进细胞的生长(P<0.01);Id1抑制后能显著下调VEGFmRNA和蛋白表达(P<0.01),减缓细胞的生长(P<0.01)。结论在结肠癌HT-29细胞中Id1为VEGF的上游基因,Id1可通过正性调控VEGF影响结肠癌细胞增殖。     

阿司匹林对人结肠癌HT-29细胞肠分化标志物表达的影响

背景与目的：非甾体抗炎药阿司匹林临床防治结肠癌确切有效,近年来有研究认为其防治结肠癌最终通过对结肠癌干细胞调控而实现。当结肠癌干细胞分化成熟时,结肠癌将不再进展、甚至消退。本研究旨在探讨非甾体抗炎药阿司匹林对人结肠癌HT-29细胞株分化的影响。方法：采用活细胞计数试剂盒CCK-8检测不同浓度阿司匹林抑制HT-29细胞增殖的作用,得出IC₅₀;采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测阿司匹林IC₅₀干预24、48和72 h,肠分化标志物黏蛋白2(mucin 2,MUC2)、三叶因子3(trefoil factor 3,TFF3)、蔗糖酶-异麦芽糖酶(sucroseisomaltase)和溶菌酶(lysozyme)的mRNA表达情况;采用免疫细胞化学法检测MUC2蛋白在细胞中的表达情况;采用蛋白［质］印迹法(Western blot)检测sucrase-isomaltase及lysozyme的蛋白表达情况。结果：阿司匹林能明显抑制HT-29细胞的增殖,且呈时间和剂量依赖性;RTFQ-PCR和Western blot结果显示,阿司匹林干预HT-29细胞48、72 h,与对照组比较,肠杯状细胞标志物MUC2、TFF3的mRNA表达均上调(P<0.05),肠吸收细胞标志物sucrase-isomaltase和潘氏细胞标志物lysozyme的mRNA及蛋白表达均下调(P<0.05);免疫细胞化学实验结果显示,阿司匹林干预48 h,MUC2蛋白表达较对照组上调,差异有统计学意义(P<0.05)。结论：非甾体抗炎药阿司匹林能影响人结肠癌HT-29细胞肠分化标志物的表达,且可能导致其向杯状细胞表型分化。     

熊果酸诱导结肠癌HT-29细胞凋亡的实验研究

目的探讨熊果酸（UA）诱导结肠癌HT-29细胞凋亡的作用及机制。方法用不同浓度的UA处理HT-29细胞,采用四甲基偶氮唑蓝（MTT）比色法,检测UA对HT-29细胞的增殖抑制效应;采用形态学、TUNEL法和流式细胞术,检测细胞凋亡的发生;应用免疫组织化学S-P法,检测凋亡相关基因caspase-9和bcl-2的表达,并用病理图像分析软件进行半定量分析。结果UA在体外对HT-29细胞有中度增殖抑制效应,在UA作用下,HT-29细胞出现显著的细胞凋亡征象。TUNEL法显示细胞固缩,核染色质聚集或断裂,形成凋亡小体。流式细胞术检测结果显示,在G1期之前出现Sub—G1峰,凋亡率最高为11．63％,UA的作用具有浓度和时间依赖性。在HT-29细胞凋亡过程中,凋亡相关基因caspase-9的表达增强,bcl-2的表达减弱。结论凋亡是UA杀伤肿瘤细胞的机制之一;UA诱导结肠癌HT-29细胞凋亡主要与促进caspase-9的活化、下调bcl-2的表达有关。     

戊地昔布对人结肠癌HT-29细胞凋亡的影响

目的 观察戊地昔布（Valdecoxib）对COX-2高表达的人结肠癌HT-29细胞凋亡的影响。方法 将体外培养HT-29细胞分为正常组（C）、药物处理组（V）及溶剂对照组（S）。采用流式细胞术检测细胞凋亡,Western blot检测COX-2、caspase-3、cleaved caspase-3、Bcl-2、p38 MAPK、p-p38MAPK。结果 与正常组相比,药物处理组细胞凋亡明显增加(P<0.05),cleaved caspase-3和p-p38MAPK表达增高,Bax/Bcl-2比率明显升高(P<0.05)。Valdecoxib干预能够显著促进HT-29细胞凋亡,上调cleaved caspase-3和p-p38 MAPK的表达,增加Bax/Bcl-2比率。 结论 COX-2选择性抑制剂Valdecoxib能够促进HT-29细胞凋亡部分可能是通过激活p38MAPK信号通路而实现的。     

Bcl-2过表达对多柔比星诱导的膀胱癌细胞凋亡和NF-KB活化影响的观察

目的：探讨凋亡相关基因Bcl2过表达对多柔比星（ADM）诱导的膀胱癌细胞凋亡和NF—KB活化的影响。方法：采用脂质体转染法将Bcl-2基因转入膀胱癌细胞,G418筛选获得抗性亚克隆细胞株,RT—PCR检测Bcl-2基因的表达。用6．25、12．5和25μg／mL的ADM分别作用于细胞24h,流式细胞术检测细胞凋亡,蛋白质印迹法检测NF-kB活化情况。结果：建立分别稳定过表达Bcl-2基因和新霉素抗性基因（neo）的膀胱癌亚克隆株BIU87／Bcl-2和RIU87／neo,RT-PCR结果显示,H1U87／Bcl-2细胞的Bcl-2mRNA水平明显高于BIU87／neo和BIU87细胞,F=63．107,P〈0．0l。经6．25、12．5和25μg／mLADM作用24h后,BIU87／Bci-2细胞凋亡率较BIU87／neo细胞明显降低,t=11．216,JP〈0．01。与BIU87／neo细胞相比,ADM作用24h后BIU87／Bcl-2细胞胞质IKB明显减少,t=-O．255,P=0．018;而胞核NF—KBp65明显增多,t=2．088,P=O．049。结论：Bel-2基因能够抑制ADM诱导的膀胱癌BIU87细胞凋亡,其抗凋亡作用涉及NF—KB活化。     

丁酸钠对结肠癌 HT-29细胞的增殖抑制作用及 iNOS表达的影响

背景与目的：研究证明丁酸钠在体外对多种肿瘤细胞有抑制作用。本研究观察丁酸钠对结肠癌细胞株HT-29的生长抑制情况,以及对诱导型一氧化氮合成酶(iNOS)表达水平及其对一氧化氮(NO)分泌的影响。方法：采用不同浓度的丁酸钠对HT-29细胞进行干预．分别运用MTT法检测HT-29细胞株的增殖抑制情况,免疫细胞化学染色技术(SP法)对HT-29细胞胞浆内iNOS蛋白进行染色,图像分析系统检测胞浆内iNOS吸光度值(A值),Griess法检测HT-29细胞NO的分泌。结果：丁酸钠对HT-29细胞株增殖作用呈浓度、时间依赖性。在不同作用时间下,丁酸钠的IC50值不同(12h为15．4mmol／L．24h为5．7mmol／L,36h为2．5mmol／L,48h为0．9mmol／L)。丁酸钠也降低了HT-29细胞胞浆内iNOS的表达以及NO的分泌,亦呈浓度、时间依赖性。结论：丁酸钠能够抑制iNOS的表达水平,减少NO的分泌。这可能是它抑制结肠癌HT-29细胞增殖的作用机制之一。     

siRNA沉默livin的表达对人结肠癌HT-29细胞增殖、凋亡及侵袭的影响

目的:探讨小干扰RNA(small interference RNA,siRNA)沉默人结肠癌HT-29细胞livin表达对HT-29细胞增殖、凋亡和侵袭的影响。方法:合成靶向livin的双链siRNA(livin-siRNA),转染HT-29细胞,RT-PCR及Western blotting检测HT-29细胞中livin mRNA及蛋白的表达,MTT实验检测HT-29细胞的增殖,流式细胞术检测HT-29细胞周期分布及凋亡,细胞侵袭实验检测HT-29细胞侵袭性的变化,caspase-3活性检测试剂盒检测caspase-3活性的变化。结果:Livin-siRNA转染后48 h,与空白组、阴性对照组及脂质体组相比,livin-siRNA转染组HT-29细胞中livin mRNA水平明显下降(0.073±0.007 vs 0.395±0.082、0.423±0.025、0.418±0.032,P<0.05),其蛋白表达也明显下调(0.106±0.003 vs 0.456±0.065、0.473±0.078、0.491±0.045,P<0.05)。转染96 h后,livin-siRNA组HT-29细胞增殖能力明显低于对照组及脂质体组(0.564±0.102 vs0.833±0.127、0.860±0.153,P<0.05),且细胞凋亡率升高[(16.5±2.8)%vs(2.4±0.5)%、(3.7±1.0)%,P<0.05]。侵袭实验显示,livin-siRNA转染后,穿过Matrigel膜的HT-29细胞数量明显少于对照组及脂质体组[(31.3±4.5)vs(101.3±8.6)、(97.4±7.8)个,P<0.05)]。livin-siRNA组HT-29细胞的caspase-3活性低于对照组(0.160±0.023 vs 0.347±0.058,P<0.05)。结论:siRNA沉默livin的表达可抑制HT-29细胞的增殖,诱导细胞凋亡,抑制细胞的侵袭。     

染料木黄酮对人结肠癌细胞系HT-29细胞增殖的影响

目的:研究染料木黄酮对人结肠癌细胞系HT-29细胞增殖的影响.方法:采用MTT比色法测定染料木黄酮对人结肠癌细胞系HT-29细胞增殖的影响,应用流式细胞术检测染料木黄酮对HT-29细胞周期及凋亡的影响.结果:染料木黄酮能够显著抑制HT-29细胞的增殖,且具有浓度依赖性,其半数抑制浓度(IC50)为23μmol/L;染料木黄酮能够引起HT-29细胞发生G0/G1期阻滞,并能够诱导HT-29细胞发生凋亡;western结果显示染料木黄酮作用后Bax表达增强,而Bcl-2表达减弱.结论:染料木黄酮能够诱导人结肠癌HT-29细胞发生周期阻滞和凋亡,从而导致HT-29细胞增殖能力下降.     

NGX6对结肠癌细胞系HT-29基因表达谱的影响

背景与目的:结肠癌是最常见的恶性肿瘤之一,其发病机制仍未完全明了。本研究在前期工作的基础上探讨候选抑瘤基因NGX6对结肠癌细胞系HT-29基因表达谱的影响。方法:通过脂质体介导的基因转染构建稳定表达NGX6的结肠癌细胞系pcDNA3.1( )/NGX6/HT-29,以NGX6转染组为实验组,空白质粒转染组为对照组,利用高通量的表达谱芯片BiostarH-80sx1筛选差异表达基因,RT-PCR验证部分基因芯片的实验结果。结果:NGX6基因转染结肠癌细胞系HT-29后引起该细胞系基因表达谱广泛地改变,其中表达差异3倍以上的基因共377个,这些差异表达基因的功能涉及多个方面,包括细胞信号转导﹑细胞周期调控﹑细胞凋亡﹑细胞骨架和运动﹑基因转录和翻译、DNA损伤修复﹑蛋白质合成和代谢等。其中包括一些非常重要的信号转导通路上的分子改变,如Wnt/β-catenin信号通路上的DKK1、ILK、MMP1、COL11A1,ILK信号通路上的ILK,Eph-Ephrins信号通路上的EpHB2,RhoA信号通路上的ROCK2,RanGTPase信号通路上的RANBP1,以及RB/RBBP1信号通路上的RBBP1等。结论:NGX6转染引起了一些非常重要的信号转导通路上的分子改变,可能通过参与调节这些信号转导通路而发挥其抗肿瘤增殖和转移的作用。     

Changes of NF-KB activity in colon carcinoma cells treated with different crude extracts of abrotani herba

Objective: To study changes of NF-KB activity in colon carcinoma cell lines treated with different crude extracts of abrotani herba obtained through solvent extraction methods.Methods: Crude extracts of abrotani herba were extracted with ligarine, chloroform, acetoacetate and n-butanol in separating funnel.Exposure concentration of crude extracts were obtained through detecting viability of HT-29 cells by MTT.Then HT-29 cells and Lovo cells were treated with different crude extracts respectively.Changes of NF-KB activity in HT-29 cells and Lovo cells using different crude extracts were observed by EMSA.Results: Successfully extracted different crude extracts of abrotani herba and called them ligarine extract, chloroform extract,acetoacetate extract, n-butanol extract and remaining extract for short.NF-KB activity was significantly inhibited in HT-29 cells treated with chloroform extract, there were no significant differences in other groups compared with the control.The same change of NF-KB activity was observed in Lovo calls using different crude extracts of abrotani herba.Conclusion: NF-KB activity can be inhibited in colon carcinoma HT-29 calls and Lovo cells treated with chloroform extract obtained from abrotani herba through the method of solvent extraction.     

Changes of NF-κB activity in colon carcinoma cells treated with different crude extracts of abrotani herba

Objective  To study changes of NF-κB activity in colon carcinoma cell lines treated with different crude extracts of abrotani herba obtained through solvent extraction methods. Methods  Crude extracts of abrotani herba were extracted with ligarine, chloroform, acetoacetate and n-butanol in separating funnel. Exposure concentration of crude extracts were obtained through detecting viability of HT-29 cells by MTT. Then HT-29 cells and Lovo cells were treated with different crude extracts respectively. Changes of NF-κB activity in HT-29 cells and Lovo cells using different crude extracts were observed by EMSA. Results  Successfully extracted different crude extracts of abrotani herba and called them ligarine extract, chloroform extract, acetoacetate extract, n-butanol extract and remaining extract for short. NF-κB activity was significantly inhibited in HT-29 cells treated with chloroform extract, there were no significant differences in other groups compared with the control. The same change of NF-κB activity was observed in Lovo cells using different crude extracts of abrotani herba. Conclusion  NF-κB activity can be inhibited in colon carcinoma HT-29 cells and Lovo cells treated with chloroform extract obtained from abrotani herba through the method of solvent extraction. Supported by a grant from the Project of Tackling Key Problems in Science and Technology Foundation of Chongqing (No. 9398).     

Requirement of both mucins and proteoglycans in cell-cell dissociation and invasiveness of colon carcinoma HT-29 cells

Human colon carcinomas are characterized by an aberrant expression of mucins, which in some case leads to an abundant presence of mucus such as in mucinous and signet ring cell carcinomas. Cellular cloning of the human colon carcinoma cell line HT-29 (HT-29 STD), which is mainly composed of undifferentiated cells, yielded a highly mucin-secreting variant (HT-29 5M21). The latter cloned cells cultured on plastic display a polarized organization with an apical secretion of MUC5AC mucin (Lesuffleur et al., Int J Cancer 1998;76:383-92.). Our aim was to study these 2 cell-types as for the invasive and adhesive properties with regard to the function of E-cadherin. HT-29 STD cells were noninvasive in collagen type I, whereas HT-29 5M21 cells were invasive, and the latter behavior was connected to a loss of function of E-cadherin. Likewise, HT-29 5M21 cells were characterized by a cell-cell adhesion independent of E-cadherin, in contrast to the E-cadherin dependent cell-cell adhesion of HT-29 STD cells. Immunofluorescence of HT-29 5M21 cells cultured on collagen type I showed the disappearance of the polarized organization, with a redistribution of apical mucins to the entire cell surface. Treatment of HT-29 5M21 cells by 1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (GalNAcalpha-O-bn) or by beta-D-xyloside revealed that both mucins and proteoglycans were involved in the loss of E-cadherin function. The use of specific antibodies allowed to show that MUC5AC, MUC1 and heparan sulfate proteoglycans cooperated in the formation of a biological inhibitory complex towards the function of E-cadherin in this invasive HT-29 clone.     

RNAi沉默整合素连接激酶基因表达对结肠癌细胞HT-29增殖的影响

目的:利用RNA干扰(RNA interference,RNAi)技术,阻断结肠癌细胞系HT-29中整合素连接激酶(integrin-linked kinase,ILK)基因的表达,研究ILK基因沉默后对HT-29细胞系增殖产生的影响。方法:构建针对ILK基因的真核表达质粒pSUPER-neo-EGFP-ILK(pSNE-ILK),在脂质体介导下稳定转染人结肠癌细胞系HT-29细胞(HT-29/pSNE-ILK组),同时以无关质粒pSUPER-neo-EGFP-C(pSNE-C)转染HT-29细胞(HT-29/pSNE-C组)和未转染细胞(HT-29组)作为对照,分别应用RT-PCR技术及蛋白印迹法检测ILK mRNA及蛋白表达水平,四甲基偶氮唑盐(MTT)比色法检测细胞增殖状况。结果:稳定转染后,HT-29/pSNE-ILK组ILK mRNA及蛋白表达水平分别为0.16±0.11和0.24±0.07,HT-29/pSNE-C组分别为0.53±0.10和0.56±0.08,HT-29组分别为0.64±0.11和0.77±0.02,前组ILK mRNA及蛋白表达水平分别与后两组比较,差异均有统计学意义(P<0.05);稳定转染pSNE-ILK质粒后HT-29细胞中ILK mRNA及蛋白表达抑制率分别为69.8%和57.1%。MTT比色法检测显示,HT-29/pSNE-ILK组细胞增殖率明显下降(P<0.05)。结论:RNA干扰技术能有效抑制靶基因ILK的表达,进而可抑制结肠癌细胞系HT-29的增殖。     

结肠癌HT-29细胞中PIDD蛋白的表达及其对细胞耐药性的影响

目的:以结肠癌HT-29细胞株为载体,研究PIDD(p53-induced protein with a death domain)蛋白在结肠癌耐药中的作用机制.方法:构建并转染PIDD基因的小干扰RNA(small interfering RNA,siRNA)片段,用Western印迹法检测其干扰效果.通过MTT比色法检测细胞对化疗药物的敏感性,并用凝胶阻滞分析(electrophoretic mobility shift assay,EMSA)法检测核因子NF-κB活性的变化.结果:RNA干扰后给予细胞药物刺激,细胞核中PIDD表达下降,核因子NF-κB活性明显降低,细胞耐药性下降.结论:转染siRNA后,HT-29细胞对5-氟尿嘧啶(5-fluorouracil, 5-FU)的敏感性增加,此作用可能与细胞核PIDD复合体减少、核因子NF-κB活性减低及细胞凋亡增多有关.     

Induction of apoptosis by puerarin in colon cancer HT-29 cells

Puerarin was isolated from Pueraria radix and has beneficial effects on cardiovascular, neurological, and hyperglycemic disorders. The current study showed that puerarin also possessed anti-cancer properties. Methyl thiazolyl tetrazolium assay (MTT) assay revealed a dose-dependent reduction of HT-29 cellular growth in response to puerarin treatment. Apoptosis was observed following treatments ;with >or=25 microM puerarin, as reflected by the appearance of the subdiploid fraction and NDA fragmentations. We then investigated effects of puerarin on expression of apoptosis-associated genes and the results revealed an increase of bax and decreases of c-myc and bcl-2. Finally, puerarin treatment significantly increased the activation of caspase-3, a key executioner of apoptosis. These findings indicate that puerarin may act as a chemopreventive and/or chemotherapeutic agent in colon cancer cells by reducing cell viability and inducing apoptosis.     

Adhesion of HT-29 colon carcinoma cells to E-selectin results in increased tyrosine phosphorylation and decreased activity of c-src

Adhesion of metastatic cancer cells at secondary sites is known to be regulated by several families of adhesion proteins, including selectins and integrins. Colon carcinoma cells have been shown to tether to and roll on both stimulated endothelial cells and purified E-selectin. We have demonstrated that HT-29 human colon carcinoma cells adhere specifically to an E-selectin-lgG chimera. Upon adhesion to E-selectin, the amount of tyrosine phosphorylation of several proteins in HT-29 cell lysates increases compared with cells in bovine serum albumin-coated wells on phosphotyrosine Western blots; this increase is statistically significant. This effect is specific for adhesion to E-selectin, since addition of an E-selectin blocking monoclonal antibody (MAb), E3, to the wells causes a statistically significant decrease in tyrosine phosphorylation relative to E-selectin alone on phosphotyrosine Western blots. One protein that is affected this way has been identified as c-src. Kinase assays show a dose-dependent and statistically significant decrease in c-src activity upon adhesion to E-selectin, which correlates with an increase in phosphorylation of Tyr 527, the negative regulatory tyrosine. CnBr digestion of ³²P-labeled c-src shows an increase in phosphorylation of tyrosine 527 after adhesion to E-selectin. Our results may identify a signaling pathway involving the E-selectin ligand on HT-29 cells and c-src. Int. J. Cancer 72:645-653, 1997. © 1997 Wiley-Liss, Inc.