二烯丙基二硫对人结肠癌SW480细胞周期的阻滞作用

背景与目的:前期研究表明,二烯丙基二硫(diallyl disulfide.DADS)能有效在体内外抑制人结肠癌细胞增殖,并将细胞阻滞于G2/M期.本实验旨在进一步探讨DADS对人结肠癌SW480细胞周期阻滞作用的可能机制.方法:采用MTT、细胞计数法检测DADS对SW480细胞增殖的抑制作用;流式细胞术检测DADS对SW480细胞周期的影响;免疫细胞化学法检测DADS作用后PCNA、P53表达情况,蛋白印迹法分析P21WAF1、细胞周期蛋白B1(Cyclin B1)表达的改变.结果:不同浓度DADS作用SW480细胞24、48、72 h后.对细胞增殖的抑制作用及细胞群体倍增时间呈浓度、时间依赖性增加.流式细胞仪分析结果显示,DADS作用SW480细胞后,G0/G1期细胞含量降低.S期细胞比例变化不大,而G2/M期细胞比例则明显增加,且随着处理浓度的增加和处理时间的延长,其G2/M期细胞含量逐渐增加,与对照组比较差异有统计学意义(P<0.05).DADS作用后.免疫细胞化学检测显示,PCNA、P53蛋白表达明显下降.蛋白印迹分析显示,DADS作用SW480细胞后,Cyclin BI蛋白表达明显下降,P21WAF1蛋白的表达明显升高,均呈时间效应关系.结论:DADS可能通过下调PCNA、P53、Cyclin B1蛋白表达.上调P21WAF1蛋白表达引起SW480细胞G2/M期阻滞.     

二烯丙基二硫阻滞人胃癌细胞周期的作用及机制

 目的观察二烯丙基二硫(DADS)对人胃癌MGC803细胞周期的影响及其机制。方法应用流式细胞术分析人胃癌MGC803细胞在DADS处理前后细胞周期分布的变化。采用免疫组化链霉素抗生物素蛋白过氧化酶方法(SP)检测DADS处理前后p21WAF1、Rb、p53、p21ras、Cmyc基因的表达。结果20、25、30、35μ·ml-1DADS的不同浓度处理的MGC803细胞G1期细胞所占比例无影响,S期细胞数减少,而G2期细胞则明显增加,与未处理组比较有显著性差异(P<0.05)。DADS作用后的MGC803细胞,p21WAF1表达明显增加,而Rb表达呈弱阳性,p53、p21ras、Cmyc表达阴性。DMSO处理的MGC803细胞亦呈现同样改变。结论DADS可以阻滞MGC803细胞于G2/M期。其作用机制可能与上调p21WAF1、Rb基因表达,下调p53、ras、Cmyc基因表达有关。     

二烯丙基二硫对人鼻咽癌CNE2细胞周期的阻滞作用

目的 探讨二烯丙基二硫(DADS)对人鼻咽癌CNE2细胞周期的阻滞作用及分子机制.方法 体外培养CNE2细胞,采用MTT比色实验、细胞计数法、细胞形态学观察、流式细胞仪和Western blotting方法分析DADS对CNE2细胞的增殖抑制作用、细胞周期分布的影响及细胞周期相关蛋白p21WAF1的表达.结果 MTT法显示,不同浓度DADS(90、140、240、400 μmol/L)处理CNE2细胞48小时后,生长抑制率分别为3.3%、12.9%、28.3%、56.9%;细胞计数法表明,常规培养CNE2细胞群体倍增时间为24.5小时,DADS的浓度由90 μmol/L增加到400 μmol/L时,其细胞群体倍增时间由27.6小时延长到93.1小时;HE染色结果显示,不同浓度DADS处理CNE2细胞48小时后细胞异型性明显减少,核浆比例明显减少;流式细胞仪分析显示DADS呈浓度依赖性将CNE2细胞阻滞在G0/G1期;Western blotting分析表明,在细胞周期阻滞的同时有p21WAF1蛋白表达上调.结论 DADS对CNE2细胞的抑制增殖作用与G0/G1期阻滞有关,其分子机制可能与调节p21WAF1表达有关.     

二烯丙基二硫对裸鼠体内人结肠癌细胞增殖的影响

背景与目的:二烯丙基二硫(diallyl disulfide,DADS)在体外可抑制多种肿瘤细胞增殖,但在体内抗肿瘤作用的研究报道较少.本实验旨在研究DADS对裸鼠体内人结肠癌SW480细胞增殖的影响.方法:建立人结肠癌裸鼠移植瘤模型,实验动物分为DADS组及对照组.观察DADS作用后移植瘤体积和重量的变化,通过光镜和电镜观察移植瘤细胞形态学变化,通过免疫组织化学和形态定量图像分析系统检测瘤组织内增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)蛋白表达变化,用流式细胞术检测移植瘤细胞周期分布.结果:DADS在体内能明显抑制移植瘤的生长,相对肿瘤增殖率为49.85%;组织学分析显示DADS组瘤细胞异型性降低,核质比下降,胞浆内细胞器丰富,部分胞核呈退行性变,可见凋亡小体;免疫组化示两组移植瘤组织PCNA蛋白均有阳性表达,DADS组(149.02±4.26)与对照组(178.86±7.69)比较,裸鼠移植瘤组织内PCNA蛋白表达明显减弱(P＜0.05);流式细胞术分析显示,对照组移植瘤细胞中G2/M期细胞为18.8%,与对照组比较,DADS组G2/M期细胞(38.6%)显著增多.结论:DADS可体内抑制人结肠癌SW480细胞增殖,使癌细胞阻滞于G2/M期.     

熊果酸诱导结肠癌HT-29细胞株凋亡的实验研究

目的探讨熊果酸诱导结肠癌HT-29细胞株凋亡的作用机制.方法用不同浓度的熊果酸处理HT-29细胞,采用四甲基偶氮唑蓝(MTT)比色检测熊果酸对HT-29细胞的增殖抑制效应,采用形态学、TUNEL法、流式细胞术检测细胞凋亡的发生,应用免疫组织化学SP法检测凋亡相关基因caspase-9, bax表达的变化.结果熊果酸在体外对HT-29细胞有中度增殖抑制效应,在熊果酸作用下,HT-29细胞出现显著的细胞凋亡征象,TUNEL显示细胞固缩,核染色质聚集或断裂,形成凋亡小体.流式细胞术检测在G1期之前出现sub-G1峰,凋亡率最高为11.63%,熊果酸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和bax的表达依次增强.结论熊果酸在体外对HT-29细胞有诱导凋亡的作用.其作用机制可能是通过促进caspase-9的活化和bax的表达上调来实现.     

二烯丙基二硫对K562细胞增殖和细胞周期的影响

目的 探讨二烯丙基二硫( DADS)对人白血病K562细胞增殖和细胞周期的影响.方法 采用台盼蓝计数法、形态学观察、MTT分析法、流式细胞术、免疫组化法探讨DADS对K562细胞的作用及机制.结果 DADS能抑制K562细胞增殖;DADS处理后的K562细胞主要停滞在G2/M期;DADS处理后的K562细胞P21表达增加.结论 DADS有抑制K562细胞增殖作用,并使K562细胞生长阻滞在G2/M期.其作用机制可能与细胞周期蛋白依赖性激酶抑制剂P21参与其中有关.     

盐酸普鲁卡因对人结肠癌HT-29细胞的作用及其机制

目的探讨盐酸普鲁卡因对人结肠癌HT-29细胞生长的抑制作用及其机制。方法 应用不同浓度（0~10mmol/L）盐酸普鲁卡因处理HT-29细胞,通过倒置显微镜观察细胞形态学改变、四甲基偶氮唑盐比色法（MTT）检测细胞存活率、流式细胞术（FCM）观察细胞周期变化。结果倒置显微镜观察盐酸普鲁卡因处理后的HT-29细胞呈现缩小、皱缩、空泡、脱壁等增殖变慢的形态学特征;MTT结果分析表明盐酸普鲁卡因能显著抑制HT-29细胞增殖,其抑制率呈药物浓度及时间依赖性;FCM结果显示盐酸普鲁卡因可以使HT-29细胞的G0/G1期延长,S期缩短。结论盐酸普鲁卡因能够使HT-29细胞的生长阻滞在G0/G1期,从而显著抑制HT-29细胞的增殖,并且具有剂量、时间依赖效应。     

p38通路在二烯丙基二硫诱导人胃癌MGC803细胞G2/M期阻滞中的作用

背景与目的:研究表明p38通路参与了细胞G2/M期阻滞过程的调控,但对其调控机制研究很少.本研究旨在探讨p38通路在二烯丙基二硫(diallyldisulfide,DADS)诱导人胃癌MGC803细胞G2/M期阻滞中的作用及其机制.方法:采用MTT法检测MGC803细胞的生长活性;流式细胞仪检测药物对细胞周期分布的影响;Western blot法检测药物对P38蛋白、磷酸化P38蛋白、Cdc25C蛋白的表达.结果:MTT法检测结果显示,P38特异性抑制剂SB203580可拮抗DADS对MGC803细胞的抑制增殖作用.流式细胞仪分析表明,30 mg/L的DADS处理MGC803细胞24 h后,G2/M期的比率为39.4%,高于对照组的9.3%(P＜0.05);但用SB203580抑制P38的活性后,DADS诱导的G2/M期比率从39.4%降到21.2%(P＜0.05).Western blot结果表明,DADS处理MGC803细胞后,p38通路快速激活,磷酸化P38的表达与对照组相比,在20 min内升高3.52倍,而P38总蛋白量没有发生明显改变;DADS作用24 h后,Cdc25C磷酸酶表达降低了62%,而用SB203580抑制剂后,DADS对Cdc25C的抑制作用可以部分逆转(P＜0.05).结论:DADS可能通过激活p38通路使部分MGC803细胞停滞在G2/M期,p38通路调节Cdc25C磷酸酶的表达在DADS诱导MGC803细胞G2/M期阻滞中起着重要作用.     

大蒜内烯丙基硫在恶性肿瘤中的作用

大量流行病学研究显示,大蒜可以降低多种恶性肿瘤的发生率和死亡率,包括结肠癌、前列腺癌和胃癌等.大蒜的抗癌主要有效成分为烯丙基硫,其抗癌机制可能涉及调节信号转导通路、调控细胞周期、诱导细胞凋亡和分化,抑制肿瘤致癌基因的表达等.     

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。     

二烯丙基二硫化物诱导胃癌BGC823 细胞周期G2/M期阻滞的机制*

目的:以细胞周期作为抗癌药物新靶点的研究,可能是很有前途的。笔者的前期工作发现,二烯丙基二硫化物（diallyl disulfide,DADS）可抑制人胃癌BGC 823 细胞增殖,其增殖抑制与细胞周期G2/M期阻滞有关;DADS可能是通过抑制细胞分裂周期蛋白25C（Cell division cycle protein 25C,Cdc25C）、cyclinB 1 表达使部分BGC 823 细胞停滞在G2/M期,但G2/M期阻滞的机制还未完全阐明。本研究进一步探讨DADS诱导人胃癌BGC 823 细胞周期G2/M期阻滞的可能机制。方法:RT-PCR 检测Chk1 和Chk2 在mRNA 水平的改变;Western blot检测DADS处理BGC 823 细胞前后细胞周期相关蛋白ATM-RAD3 相关基因（ATM-RAD3-related gene,ATR ）、细胞周期检查点蛋白激酶1（checkpoint kinase1,Chk1）、细胞周期检查点蛋白激酶2（checkpoint kinase2,Chk2）表达和ATR 、Chk1、Chk2 的磷酸化程度;免疫共沉淀检测Chk1、Chk2 与Cdc25C 结合情况。结果:RT-PCR 检测显示,Chk1 和Chk2 的mRNA 水平在处理组与未处理组之间无显著性差异（P>0.05）。 Western blot检测显示,总Chk1 和Chk2 蛋白表达在细胞处理前后均无明显改变,但15mg/LDADS刺激BGC 823 细胞2h 后,处理组细胞Chk1 磷酸化程度明显增加,并呈时间依赖性（P<0.05）,而Chk2 磷酸化程度在处理组与未处理组之间无显著性差异（P>0.05）。 15mg/L DADS 作用15～120min,ATR 磷酸化程度明显增加,呈时间依赖性（P<0.05）,而ATR 表达无改变。免疫共沉淀分析表明,DADS 能促进BGC 823 细胞Chk1 与Cdc25C 结合,而对Chk2 与Cdc25C 结合无影响。结论:DAD诱导人胃癌BGC 823 细胞G2/M期阻滞与Chk1 的活化有关,DADS可能是通过激活ATR 、Chk1,调节Cdc25C 的表达引起人胃癌BGC 823 细胞G2/M期阻滞。      

Eupatorin and Salvigenin Potentiate Doxorubicin-Induced Apoptosis and Cell Cycle Arrest in HT-29 and SW948 Human Colon Cancer Cells

Background: Cancer persists as one of the world’s most pressing maladies. Notable points about chemotherapy are drug side effects which are almost universally encountered. Emerging knowledge focusing on mechanisms of toxicity due to chemotherapy has led to characterization of novel methods, including the exploitation of natural compounds, in combination therapies. Flavonoids are natural polyphenolic compounds that play protective roles against tumor cell development. The focus of this study was apoptotic effects of two flavonoids, eupatorin and salvigenin, in combination with doxorubicin on a cellular model of colon cancer. Method: Upon establishing a non-effective dose of doxorubicin, and effective doses of eupatorin (100μM) and salvigenin (150μM) via MTT, morphological features of apoptosis were distinguished using DAPI staining and cell cycle blockage in the sub-G1 phase. Apoptosis was determined by annexin/ PI and western blotting. ROS levels and MMP were measured to show any role of mitochondria in apoptosis. Results: Co-administration of flavonoids with doxorubicin induced apoptosis via the mitochondrial pathway as mitochondrial membrane potential and ROS production were changed. Annexin/PI analysis demonstrated that apoptosis frequency was increased with the combination treatments in colon cancer cells. Finally, the combination of these flavonoids with doxorubicin increased the Bax/Bcl-2 ratio, caspase-3 expression and PARP cleavage. Conclusion: Combination of flavonoids with doxorubicin induces apoptosis and enhances effect on cancer cells which might allow amelioration of side effects by dose lowering.     

鞘磷脂水解产物对结肠癌细胞中碱性鞘磷脂酶的影响

背景与目的： 碱性鞘磷脂酶(alk-SMase)是鞘磷脂(SM)新陈代谢和发挥抑制结肠癌作用的关键酶。通过体外实验探讨SM的水解产物的神经酰胺(Cer)和鞘氨醇(Sph)对人结肠癌细胞HT-29中alk-SMase表达的影响。 材料与方法： 分别用12.5、25、50 μmol/L的C2-Cer和Sph处理HT-29细胞12、24、48 h后,分别用Western blot法和RT-PCR检测细胞中alk-SMase的蛋白水平和mRNA表达水平的变化。实验并设DMSO溶剂对照组。 结果： C2-Cer和Sph作用HT-29细胞后,其alk-SMase蛋白及其mRNA的表达下调。 结论： Cer和Sph对HT-29细胞中alk-SMase的表达具有负反馈调节作用。     

Anti Cancer Effects of Cnidium officinale Makino Extract Mediated through Apoptosis and Cell Cycle Arrest in the HT-29 Human Colorectal Cancer Cell Line

The anti cancer properties and underlying cell death mechanism induced by the extract of the roots of Cnidium officinale Makino(COM) were investigated. The ethanolic extract of COM inhibited the proliferation of human colon cancer cells (HT-29) in both dose-dependent and time-dependent manners. Analysis of the cell cycle after treatment of HT-29 cells with various concentrations of COM showed that COM extract inhibited the cellular proliferation of HT-29 cells via G1 phase arrest of the cell cycle. The apoptotic effect of COM on HT-29 cells was confirmed by Annexin V- propidium iodide apoptosis test. RT-PCR and Western blot analysis both revealed that COM extract dose-dependently increased the expressions of p53, p21, Bax and Caspase-3. Anti-apoptotic factor Bcl-2 expression was down regulated as well as cyclin D1 and CDK4. These data suggest that COM has anti cancer properties by inducing apoptosis and cell cycle arrest in HT-29 cells and could have possible therapeutic potential against human colon adenocarcinoma.     

二烯丙基二硫对人胃癌SGC7901细胞Aurora-A、Aurora-B表达的影响

目的探讨二烯丙基二硫(DADS)对人胃癌SGC7901细胞中Aurora-A、Aurora-B表达的影响。方法体外培养SGC7901细胞,采用RT-PCR、Western blot和免疫细胞化学分析DADS 对SGC7901细胞中Aurora-A、Aurora-B表达的影响。结果RT-PCR显示用15 mg/L DADS处理SGC7901细胞24、48 h后,Aurora-A、Aurora-B mRNA表达明显下调(P<0.05);Western blot显示用15 mg/L DADS处理SGC7901细胞24、48 h后,Aurora-A、Aurora-B蛋白表达水平呈时间依赖性下降(P<0.05);免疫细胞化学显示用15 mg/L DADS处理SGC7901细胞24、48 h后,Aurora-A和Aurora-B蛋白表达明显降低。结论DADS可从基因和蛋白水平抑制SGC7901细胞Aurora-A、Aurora-B的表达。     

Effect of Root Extracts of Medicinal Herb Glycyrrhiza glabra on HSP90 Gene Expression and Apoptosis in the HT-29 Colon Cancer Cell Line

Colorectal cancer is one of the most common lethal cancer types worldwide. In recent years, widespread and large-scale studies have been done on medicinal plants for anti-cancer effects, including Glycyrrhiza glabra. The aim of this study was to evaluate the effects of an ethanol extract Glycyrrhiza glabra on the expression of HSP90, growth and apoptosis in the HT-29 colon cancer cell line. HT-29 cells were treated with different concentrations of extract (50,100,150, and 200 μg/ml). For evaluation of cell proliferation and apoptosis, we used MTT assay and flow cytometry technique, respectively. RT-PCR was also carried out to evaluate the expression levels of HSP90 genes. Results showed that Glycyrrhiza glabra inhibited proliferation of the HT-29 cell line at a concentration of 200 μg/ml and this was confirmed by the highest rate of cell death as measured by trypan blue and MTT assays. RT-PCR results showed down-regulation of HSP90 gene expression which implied an ability of Glycyrrhiza glabra to induce apoptosis in HT-29 cells and confirmed its anticancer property. Further studies are required to evaluate effects of the extract on other genes and also it is necessary to make an extensive in vivo biological evaluation and subsequently proceed with clinical evaluations.     

Effect of 5-Fluorouracil on Cell Cycle Regulatory Proteins in Human Colon Cancer Cell Line

We investigated the effects of 5-fluorouracil (5-FU) on cell cycle-regulating proteins in RPMI 4788 cells. 5-FU inhibited cell growth dose-dependently and this growth inhibition was accompanied with cell cycle accumulation in early S phase and increased expression of cyclin A. When cells were released from short-term treatment (3 or 24 h) with 5-FU, the cell cycle started to progress again and cyclin A protein levels decreased. Cyclin A-associated kinase activity assay showed that cyclin A-cyclin-dependent kinase (Cdk) 2 kinase activity was altered by 5-FU treatment concomitantly with the changes in cell cycle state seen in flow cytometric analysis. Furthermore, the elevation of cyclin A protein level by 5-FU treatment was observed in three other human cancer cell lines, DLD-1, H226Br and T.Tn. These results suggest that cyclin A protein levels in cancer cells are increased by 5-FU, and the cyclin A function and degradation mechanism remain normal.     

辣椒碱对结肠癌细胞自噬抑制作用的研究

[目的]研究辣椒碱对人结肠癌细胞SW480自噬的抑制作用。[方法]通过MTT法和克隆形成实验检测不同浓度辣椒碱处理的细胞增殖率,免疫荧光检测细胞自噬体的形成．Western Blot检测自噬标志物和信号通路蛋白的表达。通过蛋白组实验方法获取辣椒碱药物的相互作用蛋白。质谱技术进行蛋白鉴定及STRING数据库进行生物信息学分析。[结果]MTT结果表明100μmol／L浓度的辣椒碱能促进细胞增殖,该浓度的辣椒碱抑制了细胞自噬体的形成,抑制了LC3的活化,促进了p62的表达,提高了Akt和mTOR的磷酸化。质谱分析和STRING数据库查询发现了7个辣椒碱的相互作用蛋白,其中ATG2B与自噬存在重要联系。[结论]低浓度辣椒碱通过激活P13K-Akt信号通路及与ATG2B相互作用来抑制细胞自噬。     

Effect of Sesamin on Apoptosis and Cell Cycle Arrest in Human Breast Cancer MCF-7 Cells

Dietary prevention has been known to reduce breast cancer risk. Sesamin is one of the major components insesame seeds and has been widely studied and proven to have anti-proliferation and anti-angiogenic effects oncancer cells. In this study, the influence of sesamin was tested in the human breast cancer MCF-7 cell line forcell viability (MTT assay) and cell cycling (flow cytometry). Results showed that sesamin dose-dependently (1,10 and 50 μM) reduced the cell viability and increased LDH release and apoptosis (TUNEL assay). In addition,there was a significant increase of sub-G1 phase arrest in the cell cycle after sesamin treatment. Furthermore,sesamin increased the expression of apoptotic markers of Bax, caspase-3, and cell cycle control proteins, p53 andcheckpoint kinase 2. Taken together, these results suggested that sesamin might be used as a dietary supplementf or prevention of breast cancer by modulating apoptotic signal pathways and inhibiting tumor cell growth.