贝母素乙通过诱导G0/G1期阻滞抑制结肠癌细胞的增殖

目的：探讨贝母素乙对结肠癌HCT116细胞增殖的抑制作用及其分子机制。方法：利用不同浓度的贝母素乙处理人结肠癌细胞HCT116和正常结肠上皮细胞CCD841 CON,通过CCK-8法和结晶紫染色法检测贝母素乙对HCT116和CCD841CON细胞增殖活力的影响,流式细胞术和WB法检测贝母素乙对HCT116细胞周期及其细胞周期相关蛋白表达的影响。构建HCT116移植瘤裸鼠模型和AOM/DSS结肠癌小鼠模型,观察贝母素乙对小鼠模型肿瘤生长和OS的影响,免疫组化法和WB法检测对移植瘤或肿瘤组织中细胞周期相关蛋白CDK4、CDK6和cyclin D1表达的影响。结果：贝母素乙可显著抑制结肠癌HCT116细胞的增殖能力（P<0.01）,诱导HCT116细胞周期G0/G1期阻滞（P<0.01）,降低CDK4、CDK6和cyclin D1的蛋白表达水平（均P<0.01）。荷瘤小鼠实验结果显示,贝母素乙（0.75 mg/kg）显著抑制HCT116细胞移植瘤的生长并延长荷瘤裸鼠的OS(P<0.05或P<0.01),降低AOM/DSS模型小鼠的体质量、延长OS、减少癌变肠组织...     

外源性SHIP基因对K562细胞周期及其相关蛋白表达的影响

目的:探讨SHIP基因对人白血病细胞系K562细胞周期的调控作用.方法:用携带野生型SHIP基因及绿色荧光蛋白 (green fluorescent protein,GFP)基因的慢病毒及空载体慢病毒转染人白血病K562细胞.FCM检测转染效率、细胞凋亡率及细胞周期变化,实时荧光定量PCR(real-time fluorescent quantitative PCR,RFQ-PCR)检测SHIP mRNA水平的变化,Western 印迹法检测SHIP、cyclin D1、p21WAF1/CIPI、p27KIP1蛋白表达水平的变化.结果:与转染空载体组和未转染组相比,转染野生型SHIP基因的K562细胞增殖减慢,凋亡率明显上升(P＜0.05);细胞周期显示,G0/G1期延长,G1期前出现亚二倍体的凋亡峰,S期和G2/M期比例降低;cyclin D1表达降低,p21WAF1/CIPI和p27KIP1表达增加.结论:转染野生型SHIP基因可使细胞周期阻滞在G0/G1期,抑制白血病细胞增殖,促进白血病细胞凋亡.     

辛二酰苯胺异羟肟酸对结肠癌HCT116和SW480细胞增殖、周期和凋亡的影响

目的:研究辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid,SAHA)对结肠癌细胞系HCT116和SW480增殖、周期和凋亡的影响,并对其分子作用机制进行初步探讨.方法:将不同浓度SAHA分别处理结肠癌HCT116和SW480细胞后,MTT法检测SAHA对HCT116和SW480细胞增殖的影响,流式细胞仪检测HCT116和SW480细胞周期和细胞凋亡率,罗丹明(rhodamine) 123和二氯二氢荧光素二乙酸酯(DCFH-DA)法检测HCT116和SW480细胞线粒体跨膜电位(Aψm)和活性氧(ROS)水平,Real-time PCR和Western blotting法检测乙酰化组蛋白3(Ac-H3)、p21、CyclinD1、Bax和Bcl-2的mRNA和蛋白的表达水平.结果:SAHA作用于HCT116和SW480细胞48 h后,细胞增殖被抑制、细胞周期G1期比率升高、凋亡率升高(均P＜0.05),线粒体跨膜电位显著下降、细胞内ROS产生增多(均P＜0.05).与对照组比较,SAHA处理组p21和Bax mRNA增多、Cyclin D1和Bcl-2 mRNA表达量减少(均P＜0.05),相关蛋白Ac-H3、p21和Bax增多,CyclinD1和Bcl-2减少(均P＜0.05).结论:SAHA抑制结肠癌HCT116和SW480细胞增殖、阻滞细胞周期并诱导细胞凋亡,其机可能与调节p21、CyclinD1和Bcl-2家族基因的表达、促进组蛋白乙酰化有关.     

白桦脂酸对Jurkat白血病细胞增殖和凋亡的影响

目的 观察白桦脂酸对人T淋巴细胞白血病Jurkat细胞株细胞增殖、凋亡和细胞周期的影响,并探讨其分子机制.方法 以不同浓度的白桦脂酸处理Jurkat细胞,采用二苯基溴化四氮唑蓝(MTY)法检测细胞的增殖活性,Hoechst 33258染色和Annexin-V/PI双标法检测细胞凋亡,流式细胞仪检测细胞周期分布,逆转录聚合酶链反应(RT-PCR)法检测Jurkat细胞中cyclin D3和bcl-xlmRNA含量的变化,Western blot法检测cyclin D3和bcl-xl蛋白的表达.结果 白桦脂酸对Jurkat细胞有明显的增殖抑制作用,并呈时间和浓度依赖性.白桦脂酸可诱导Jurkat细胞凋亡,Hoechst 33258染色可见典型的凋亡小体;Annxin-V/PI双标法显示,20、60和100 μmol/L白桦脂酸作用于Jurkat细胞24 h时,细胞的早期凋亡率分别为8.7%±0.3%、28.0%±1.3%和33.6%±2.0%.白桦脂酸可使Jurkat细胞的细胞周期阻滞于G0/G1期.白桦脂酸可使Jurkat细胞中凋亡相关基因cyclin D3和bcl-xl mRNA及蛋白的含量均明显减少,并呈浓度依赖性.结论 白桦脂酸可抑制Jurkat细胞增殖并诱导其凋亡,可使细胞阻滞于G0/G1期;其可能通过下调cyclin D3和bcl-xl mRNA及蛋白的表达而发挥抗肿瘤作用.     

忍冬藤提取物抗结肠癌作用研究

【摘要】目的研究忍冬藤提取物的体外抗结肠癌作用。方法体外培养结肠癌HCT116和SW480细胞并用忍冬藤提取物干预,采用CCK8法检测忍冬藤提取物对HCT116和SW480细胞增殖的影响;采用Hochest法和Annexin V/PI法检测忍冬藤提取物及p53抑制剂PFT α对HCT116和SW480细胞凋亡的影响;JC 1法检测忍冬藤提取物对HCT116细胞线粒体膜电位的影响;Western Blot检测忍冬藤提取物对HCT116细胞相关蛋白表达水平的影响。结果相较于空白组,忍冬藤提取物可明显抑制结肠癌HCT116和SW480细胞增殖,并促进细胞凋亡（P<001）,促进HCT116细胞线粒体膜电位坍塌,明显上调HCT116细胞Bax/Bcl 2的比值（P<001）以及Cleaved PARP、Cyt C、Cleaved Caspase 3和p53蛋白表达（P<005,P<001）;相较于忍冬藤提取物组,忍冬藤提取物+PFT α明显抑制HCT116细胞凋亡（P<001）。结论忍冬藤提取物可通过诱导p53依赖的线粒体凋亡发挥抗结肠癌作用。     

KCa3．1表达改变对子宫内膜癌细胞增殖、细胞周期及凋亡的影响

目的:探讨钙激活性中电导钾离子通道(intermediate-conductance Ca2+- activated K+ channels,KCa3.1)表达水平和活性改变对子宫内膜癌细胞增殖、细胞周期及凋亡的影响.方法:将携有KCa3.1 RNA干扰(RNA interference,RNAi)片段的质粒转染子宫内膜癌HEC-1-A和Ishikawa细胞,应用实时荧光定量PCR(real time fluorogentic quantitative PCR,RFQ-PCR)及Western 印迹法检测KCa3.1的表达变化;应用MTT法、BrdU掺入法、FCM及Western 印迹法检测KCa3.1特异性阻断剂TRAM-34和KCa3.1 RNAi表达质粒干扰后,子宫内膜癌细胞株增殖、细胞周期、凋亡及cyclin D1、cyclin E及survivin蛋白表达的改变.结果:KCa3.1 RNAi表达质粒可以明显抑制KCa3.1 mRNA及蛋白的表达,与对照组相比差异有统计学意义(P＜0.01);TRAM-34及KCa3.1 RNAi可以明显抑制HEC-1-A和Ishikawa细胞的增殖,2株细胞的G0/G1期细胞百分比上升,S期细胞百分比下降,与对照组相比差异有统计学意义(P<0.05),但细胞凋亡率与对照组相比无明显差异(P＞0.05);cyclin D1、cyclin E及survivin的蛋白表达水平降低,与对照组相比差异有统计学意义(P<0.05).结论:抑制KCa3.1的表达及活性可以抑制子宫内膜癌细胞增殖,阻碍细胞周期进展,但对细胞凋亡无明显影响.     

4EGI-1通过下调Bcl-2诱导K-RAS基因突变型结直肠癌细胞凋亡

目的：研究真核细胞翻译起始因子4E(eukaryotic translation initiation factor4E,eIF4E)/eIF4G相互作用抑制剂4EGI-1对K-RAS基因突变型及野生型结直肠癌(colorectal cancer,CRC)细胞HCT116和DLD1增殖和凋亡的影响,及可能的作用机制。方法 ：采用MTT法和FCM法检测不同浓度的4EGI-1对K-RAS基因突变型及野生型HCT116和DLD1细胞(HCT116^KRASG13D、HCT116^KRASWT、DLD1^KRASG13D和DLD1^KRASWT)增殖及凋亡率的影响;蛋白质印迹法检测4EGI-1对HCT116^KRASG13D和HCT116^KRASWT细胞中凋亡相关蛋白caspase3和cleaved-caspase3及其底物多聚腺苷二磷酸核糖聚合酶(poly adenosine diphosphate-ribosepolymerase,PARP)和cleaved-PARP,...     

吲哚美辛诱导肝癌HepG2细胞凋亡及其对cyclin E蛋白表达的影响

目的　观察吲哚美辛诱导肝癌 Hep G2细胞凋亡 ,及其对 cyclin E蛋白的影响。方法　采用 MTT比色法观察吲哚美辛对肝癌 Hep G2细胞增殖的影响 ;采用流式细胞仪检测吲哚美辛对 Hep G2细胞周期分布的影响 ,同时结合透射电子显微镜观察吲哚美辛诱导肝癌 Hep G2细胞凋亡的作用 ;免疫细胞化学方法观察吲哚美辛对Hep G2细胞周期调控蛋白 cyclin E的影响。结果　吲哚美辛可抑制肝癌 Hep G2细胞的增殖 ,诱导其凋亡 ,改变细胞周期分布 ,使 G0 /G1 期细胞比例增高 ,S期比例降低 ,同时还可使 cyclin E蛋白表达下降。上述作用具有剂量和时间依赖性 (P<0 .0 5 )。结论　吲哚美辛可诱导肝癌 Hep G2细胞凋亡 ,改变细胞周期分布 ,影响细胞周期调控蛋白表达 ,从而抑制细胞增殖。     

体外Fas介导肿瘤细胞凋亡过程中cyclins/CDKs的变化规律及调节机制

目的:探讨Fas介导的细胞周期特异性细胞凋亡发生过程中,细胞周期蛋白(cyclins)和细胞周期依赖性蛋白激酶(cyclin-dependent kinases,CDKs)的变化规律,及其可能的调节机制.方法:以急性淋巴细胞白血病Molt-4细胞株为靶细胞,建立Fas介导的细胞周期特异性凋亡模型;采用cyclin/DNA双参数流式细胞术和Western印迹法检测cyclins的表达规律;应用Western印迹法检测CDK1的Thr-161、Tyr-15位点和CDK2的Thr-160位点磷酸化水平的变化.结果:重组人Fas配体(recombinant human Fas ligand,rhFasL)诱导Molt-4细胞的凋亡定位在G1期.在发生细胞凋亡效应前,cyclin D3水平明显升高,而cyclin E水平和CDK2的Thr-160位点磷酸化水平均明显下降;发生凋亡效应后,cyclin D3水平明显下降,而cyclin E水平升高,CDK2 的Thr-160位点磷酸化水平则明显下降,CDK1的Thr-161、Tyr-15位点磷酸化水平稍有下降.Cyclin A和Cyclin B1水平在诱导细胞凋亡过程中无明显变化.Roscovitine在特定浓度(5 μmol/L)下可下调CDK2 Thr-160位点和CDK1 Thr-161位点的磷酸化水平,与rhFasL共同作用后细胞凋亡的发生率明显升高.结论:Fas介导的细胞凋亡具有细胞周期特异性,并始动于晚G1期,是G1期cyclin E/CDK2活性下降以及晚G1期检测点监督的结果;cyclin D3/CDK复合体可能是决定细胞凋亡能否发生的关键.     

鱼藤素对淋巴瘤Daudi细胞株细胞增殖细胞凋亡的影响及其机制

目的观察鱼藤素对人Burkitt淋巴瘤Daudi细胞株细胞增殖、细胞凋亡和细胞周期的影响,并探讨其分子机制。方法四甲基偶氮唑蓝(MTY)法检测细胞增殖活性,Hoechst 33258染色和Annexin-V／PI双标法检测细胞凋亡,流式细胞仪检测细胞周期分布,Western blot检测细胞内cyclin D1和pRb的蛋白表达。结果鱼藤素对Daudi细胞具有明显的增殖抑制作用,而对正常人外周血单个核细胞(PBMC)抑制作用不明显。鱼藤素可以诱导Daudi细胞凋亡,Hoechst 33258染色可见典型凋亡小体。Annexin V／PI双标法显示,鱼藤素诱导细胞发生早期凋亡,并呈剂量依赖性,20、40、80 nmol／L鱼藤素作用24 h时,凋亡率分别为15．46％±0．62％、18．48％±2．98％和31．42％±1．43％。鱼藤素作用Daudi细胞后,主要使细胞周期聚积于G0／G1期,G0／G1期细胞比例随鱼藤素剂量增大而逐渐增高,40 nmol／L鱼藤素作用24 h达56．56％;相反,S期细胞比例随鱼藤素剂量增大而逐渐降低,对G2／M期细胞作用不明显。鱼藤素使cyclin D1及pRb蛋白表达降低,呈剂量依赖关系。结论鱼藤素抑制Daudi细胞增殖,使细胞阻滞于G0／G1期,并诱导细胞凋亡。其抗肿瘤机制可能与下调cyclin D1和pRb蛋白表达有关。     

Influence of p53 and p21Waf1 expression on G2/M phase arrest of colorectal carcinoma HCT116 cells to proteasome inhibitors

Ubiquitin-mediated protein degradation in vertebrates has been implicated in cell cycle control. In this report we explored the effects of proteasome inhibitors (MG132, lactacystin and ALLN) on cell cycle distribution. Colorectal carcinoma HCT116 cells were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in a dose-dependent manner. MG132 arrested HCT116 cells at G2/M phase, which was associated with drug-induced blockade of p53 degradation and/or induction of p53-related gene expression along with the accumulation of cyclin B, cyclin A and p21. MG132 treated HCT116 (wild-type) had a similar cell cycle distribution as the MG132 treated HCT116 (p53-/-) and HCT116 (p21-/-) cells, suggesting that p53 and p21 may not be essential for MG132-induced G2/M phase arrest. The release experiments from nocodazole-induced mitotic phase cells indicated that MG132 inhibits the proliferation of HCT116 cells via arrest in the G2 phase. In addition, when HCT116 cells were exposed to combination of sodium butyrate and MG132 enhanced cell growth inhibition and induction of apoptosis were observed.     

PRL-3对结直肠癌细胞迁移、侵袭能力的影响

目的:构建肝再生磷酸酶-3（phosphatase of regenerating liver-3,PRL-3）基因过表达的重组慢病毒载体,研究其对结直肠癌细胞系HCT116迁移、侵袭能力的影响。方法:PCR扩增PRL-3基因并克隆至pcDH载体,构建成pcDH-PRL-3过表达载体,再将其与慢病毒包装载体共转染293T细胞,包装成Lenti-PRL-3重组慢病毒载体,并感染结直肠癌细胞系HCT116。利用qPCR、Western分别从mRNA、蛋白水平检测PRL-3表达情况;通过划痕实验、Transwell实验研究过表达PRL-3对结直肠癌细胞系HCT116侵袭能力的影响。结果:成功构建Lenti-PRL-3重组慢病毒载体,并在结直肠癌细胞系HCT116过表达。划痕实验、Transwell实验结果表明过表达PRL-3能够促进HCT116细胞的迁移、侵袭。结论:构建Lenti-PRL-3重组慢病毒载体,在结直肠癌细胞系HCT116过表达PRL-3后能增加细胞的迁移、侵袭能力。     

miR-637抑制结肠癌HCT116细胞生长和迁移的机制研究

目的:分析miR-637对结肠癌HCT116细胞生长和迁移的影响。方法:分别构建miR-637过表达或者低表达的结肠癌HCT116细胞系。通过MTT方法检测miR-637对细胞增殖的作用,PI（propidium iodide）染色检测miR-637对细胞周期的影响,Annexin V-FITC/PI染色检测miR-637对细胞凋亡的作用。Western blot检测miR-637对细胞中CDK4和Bcl-2的影响。Transwell实验检测miR-637对细胞迁移的作用,进一步通过Western blot检测miR-637对细胞中MMP2的影响。结果:miR-637抑制HCT116细胞的增殖,抑制G1/S期进程,促进细胞凋亡,Western blot结果显示,miR-637抑制CDK4和Bcl-2在HCT116细胞中的表达。Transwell实验指出,miR-637抑制细胞的迁移,Western blot结果指出miR-637抑制MMP2在HCT116细胞中的表达。结论:miR-637可以通过抑制CDK4、Bcl-2和MMP2的表达抑制HCT116细胞的生长和迁移。     

Reduced expression of PNUTS leads to activation of Rb-phosphatase and caspase-mediated apoptosis

There is abundant evidence that Retinoblastoma (Rb) activity is important in the control of cell proliferation and apoptosis. Reversible phosphorylation of the Rb protein that is carried out by cyclin dependent kinases and Protein phosphatase 1 (PP1) regulates its functions. A PP1 interacting protein, PNUTS (Phosphatase Nuclear Targeting Subunit) is proposed to be a regulator of Rb phosphorylation. In this study, PNUTS knockdown in MCF7, SKA and HCT116 cancer cells causes a reduction in viability due to increased apoptosis. However, normal cells (MCF10A breast and CCD-18Co colon) do not exhibit reduced viability when PNUTS expression is diminished. PNUTS knockdown has no effect in Rb-null Saos-2 cells. However, when Rb is stably expressed in Saos-2 cells, PNUTS knockdown reduces cell number. Knockdown of PNUTS in p53-/- HCT116 cells indicates that p53 is dispensable for the induction of apoptosis. Loss of PNUTS expression results in increased Rb-phosphatase activity and Rb dephosphorylation. E2F1 dissociates from Rb in cells depleted of PNUTS and the resulting apoptosis is dependent on caspase-8. These results indicate that Rb phosphorylation state can be manipulated by targeting Rb phosphatase activity and suggest that PNUTS may be a potential target for therapeutic pro-apoptotic strategies.     

Expression of pRB, cyclin/cyclin-dependent kinases and E2F1/DP-1 in human tumor lines in cell culture and in xenograft tissues and response to cell cycle agents

Purpose: Cell cycle regulatory components are interesting targets for cancer therapy. Expression of pRb, cyclin D1, cdk4, cyclin E, cdk2, E2F1 and DP-1 was determined in MCF-7 and MDA-MB-468 breast carcinoma cells, H460 and Calu-6 non-small cell lung carcinoma cells, H82 and SW2 small cell lung carcinoma cells, HCT116 and HT29 colon carcinoma cells and LNCaP and DU-145 prostate carcinoma cells. Methods: For Western blotting, the ratio with actin expression was used to normalize the data; all lines were run on the same gels. Results: In cell culture, pRb was not detected in MB-468 and H82 was low in SW2 and DU-145 and highest in HCT116; in tumors, pRb was not detected in MB-468, H82, SW2, and DU-145 and was highest in LNCaP and Calu-6. Cyclin D1 was not detected in SW2 cells in culture, was low in MB-468 and H82, and was highest in LNCaP and H460; in tumors, cyclin D1 was low in MB-468, H460, SW2 and DU145, and was highest in LNCaP. In cell culture, cdk4 was lowest in Calu-6, HCT116, HT29 and DU-145 and highest in H82 and SW2; in tumors, cdk4 was low in MCF-7, MB-468, H460, Calu-6 and HCT116 and was very high in the SW2. Expression of cyclin E was very low in MCF-7 and HT29 and high in H460 in culture and was very low in MCF-7, H460, Calu-6, H82, HT29 and DU-145 in tumors and high in HCT116 and LNCaP. In cell culture, E2F1 was lowest in MB-468, Calu-6, HT29 and DU-145 cells and highest in LNCaP cells; in tumors, E2F1 was lowest in MCF-7, MB-468 and Calu-6 and highest in LNCaP. In cell culture, DP-1 was lowest in MB-468, HCT116 and HT29 and highest in SW2. The MCF-7 and MB-468 lines were most resistant to flavopiridol and olmoucine and the H460 and Calu-6 lines were most resistant to genistein. The SW2 tumor was most responsive to flavopiridol and olomoucine. Conclusions: There is a high degree of variability in the expression of cell cycle components in human tumor cell lines, resulting in complexity in predicting response to cell cycle directed agents. Received: 27 October 1999 / Accepted: 16 March 2000     

miR-340通过下调 CCND1 表达增加结直肠癌细胞对5-Fu的耐药

目的:探讨细胞周期蛋白D1(cyclin D1)的编码基因CCND1 miR-340介导的逆转结直肠癌细胞对5-氟尿嘧啶(5-Fu)耐药的机制.方法:采用瞬时转染技术将结直肠癌细胞HCT116、SW480株分别转染si-CCND1和miR340-mimic.应用MTT法检测转染后的结直肠癌细胞对5-Fu敏感性的变化,应用双荧光素酶试验验证CCND1对miR340参与的影响结直肠癌细胞对5-Fu敏感性的影响.结果:瞬时转染siCCND1和过表达miR-340后,结直肠癌HCT116和SW480细胞的IC50值均显著低于对照组(10,10 vs 20 μmol/L和20,20 vs 40 μmol/L,均P＜0.05).共转染CCND1 3'UTR野生质粒和miR-340 inhibitor的结直肠癌HCT116和SW48细胞荧光素酶的活性显著高于共转染空载体和mimic细胞(P＜0.01).结论:CCND1作为不良因子通过抑制miR340的表达进而发挥增加结直肠癌细胞对5-Fu耐药的作用.     

The cell cycle checkpoint kinase Chk2 is a negative regulator of mitotic catastrophe

Fusion between nonsynchronized cells leads to the formation of heterokarya which transiently activate Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 and enter the prophase of the cell cycle, where they arrest due to a loss of Cdk1/cyclin B1 activity, activate p53, disorganize centrosomes, and undergo apoptosis. Here, we show that the down regulation of Cdk1/cyclin B is secondary to the activation of the DNA structure checkpoint kinase Chk2. Thus, syncytia generated by the fusion of asynchronous HeLa cells contain elevated levels of active Chk2 but not Chk1. Chk2 bearing the activating phosphorylation on threonine-68 accumulates in BRCA1 nuclear bodies when the cells arrest at the G2/M boundary. Inhibition of Chk2 by transfection of a dominant-negative Chk2 mutant or a chemical inhibitor, debromohymenialdesine, stabilizes centrosomes, maintains high cyclin B1 levels, and allows for a prolonged activation of Cdk1. Under these conditions, multinuclear HeLa syncytia do not arrest at the G2/M boundary and rather enter mitotis and subsequently die during the metaphase of the cell cycle. This mitotic catastrophe is associated with the activation of the pro-apoptotic caspase-3. Inhibition of caspases allows the cells to go beyond the metaphase arrest, indicating that apoptosis is responsible for cell death by mitotic catastrophe. In another, completely different model of mitotic catastrophe, namely 14.3.3 sigma-deficient HCT116 colon carcinoma cells treated with doxorubicin, Chk2 activation was also found to be deficient as compared to 14.3.3 sigma-sufficient controls. Inhibition of Chk2 again facilitated the induction of mitotic catastrophe in HCT116 wild-type cells. In conclusion, a conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe, provided that the checkpoint kinase Chk2 is inhibited. Inhibition of Chk2 thus can sensitize proliferating cells to chemotherapy-induced apoptosis.