低聚葡萄籽原花青素联合顺铂对A549细胞增殖及细胞周期的影响

目的:探讨低聚葡萄籽原花青素(oligomer grape seed proanthocyanidins,O-GSP)联合顺铂(cisdichlorodiamineplatinum(Ⅱ),DDP)对A549细胞增殖、细胞周期及细胞周期相关蛋白表达的影响。方法:体外培养A549细胞,四甲基偶氮唑蓝法检测DDP和O-GSP单独及联合用药对A549细胞存活率的影响;流式细胞仪检测O-GSP联合DDP对A549细胞周期的影响;Western Blot检测促细胞周期相关蛋白CDK 4、Cyclin D1和p-Rb的表达。结果:DDP(≥5 mg/L)和O-GSP(≥16 mg/L)单独用药均对A549细胞增殖有抑制作用,且两者同时作用对细胞增殖抑制具有联合作用。5 mg/L DDP单独用药可以于S期阻滞A549细胞,4 mg/L O-GSP单独用药可以于G_0/G_1期阻滞A549细胞,两者联合可显著降低细胞周期调控蛋白CDK 4、Cyclin D1和p-Rb的表达(P0.05)。结论:O-GSP可以联合DDP抑制A549细胞增殖,且这种联合作用与Cyclin D1-CDK 4-Rb通路介导的细胞周期调控有关。     

羧甲基壳聚糖与阿霉素联合对Hela 细胞的抑制作用研究

目的：探讨羧甲基壳聚糖与阿霉素联合应用对人宫颈癌Hela 细胞增殖的影响。方法：采用四甲基偶氮唑蓝(MTT)比色法测定单独和合用羧甲基壳聚糖与阿霉素对癌细胞的生长抑制作用,应用金氏公式进行联合用药分析。结果：羧甲基壳聚糖和阿霉素均可有效的抑制癌细胞生长,且同时给药对癌细胞可产生单纯相加至增强的协同杀伤效果。序贯给药法中先给阿霉素后给羧甲基壳聚糖结果为协同作用,先给羧甲基壳聚糖后给阿霉素为拮抗作用。结论：羧甲基壳聚糖与阿霉素同时联合用药可产生协同作用,序贯联合用药产生单向协同作用。     

二氢杨梅素对骨肉瘤细胞增殖的抑制作用

本研究旨在探讨二氢杨梅素对骨肉瘤细胞增殖的抑制作用及其机制。采用MTT法检测不同浓度的二氢杨梅素溶液对骨肉瘤细胞增殖的抑制作用；采用倒置显微镜、Hoechst 33258荧光染色法和流式细胞术检测不同浓度的二氢杨梅素溶液对骨肉瘤细胞形态、凋亡和周期的影响；并进一步采用Western Blot法分析细胞周期蛋白和凋亡蛋白表达的影响。研究结果表明二氢杨梅素对骨肉瘤细胞增殖具有显著的抑制作用，细胞形态发生了明显变化，呈显著的剂量依赖型，其半数抑制浓度（IC50）值为24.41±1.25 μM。细胞周期实验结果表明，经二氢杨梅素处理后，G0/G1期细胞数百分比从60.20%下降到21.50%，而G2/M细胞数百分比从11.60%增加到45.30%，细胞周期蛋白Cyclin B1表达下降引起细胞周期阻滞；此外，抗凋亡蛋白Bcl-2表达和促凋亡蛋白Bax表达的比例显著下降，从而诱导细胞发生凋亡。结果表明二氢杨梅素具有显著的抗骨肉瘤增殖活性，可开发一种潜在的治疗骨肉瘤的功能食品或药物。     

麦麸结合态多酚对肝癌HepG-2细胞增殖及凋亡效应研究

目的 本文主要研究麦麸结合态多酚对肝癌HepG-2细胞的抑制效应。方法 小麦麸皮粉碎后,采用丙酮-碱消化法,获得麦麸结合态多酚物质,命名为：WBBP,用福林酚比色法测定该多酚的含量及提取得率;通过MTT法检测WBBP处理对肝癌HepG-2细胞增殖的抑制效应,倒置显微镜观察WBBP处理对HepG-2肝癌细胞形态学的影响;采用流式细胞仪检测WBBP处理对HepG-2肝癌细胞周期、凋亡情况以及线粒体膜电位的影响;利用caspases试剂盒检测WBBP作用下肝癌HepG-2细胞中Caspase-3、Caspase-8、Caspase-9的酶活性变化。结果显示,不同浓度的WBBP通过诱导HepG-2肝癌细胞周期在S期阻滞,显著抑制了细胞增殖,且具有浓度依赖性;同时WBBP通过caspase依赖的线粒体凋亡路径诱导HepG-2肝癌细胞凋亡率以浓度依赖的方式增加。结论 麦麸结合态多酚WBBP能够通过显著抑制HepG-2肝癌细胞的增殖,并诱导细胞凋亡来发挥抗肝癌效应。     

熊猫豆提取物抑制肿瘤活性及诱导结肠癌细胞Caco-2凋亡机制研究

为了探究熊猫豆的抑制肿瘤活性及其诱导Caco-2凋亡的机制,采用80%丙酮浸提法制备熊猫豆粗提物,通过MTT法测定其对人结肠癌细胞Caco-2增殖的抑制效果,琼脂糖凝胶电泳、流式细胞术、酶联免疫分析等技术与方法检测细胞周期、DNA及凋亡相关蛋白相对表达量的变化。结果发现,与空白对照组相比,0.1~1.0 mg/m L熊猫豆丙酮提取物对Caco-2细胞增殖有明显的抑制作用,且呈现剂量效应关系,IC50为0.72 mg/m L。0.4、0.6、0.8 mg/m L熊猫豆丙酮提取物处理72 h后的Caco-2细胞,可以观察到典型的凋亡细胞的梯状DNA条带,细胞周期G1期延长,Cyt C、caspase-9、caspase-3蛋白的相对表达量均有显著上升。熊猫豆丙酮能够抑制结肠癌细胞Caco-2增殖,这种抑制作用是通过阻滞细胞周期和诱导线粒体通路介导的细胞凋亡实现的。     

灵芝多糖联合5-FU对人结肠癌HCT-116细胞增殖及凋亡的影响

目的：探讨灵芝多糖(GLP)联合5-氟尿嘧啶(5-FU)对人结肠癌HCT-116细胞增殖抑制及凋亡的影响。方法：采用MTT法测定单独和联用GLP与5-FU对人结肠癌HCT-116细胞的协同抑制作用,应用联合指数法分析两药物之间相互作用,Hoechst33258荧光染色观察细胞的形态学改变,碘化吡啶(PI)标记流式细胞术(FCM)检测GLP联合5-FU对HCT-116细胞周期和凋亡抑制率的变化。结果：MTT法测定结果显示GLP和5-FU单独用药均能显著抑制HCT-116细胞增殖。GLP(＞659μg/mL)联用5-FU(＞1.6μg/mL)联用呈协同效应(CI＜1),与5-FU单独用药组相比有极显著差异(P＜0.01),并呈时间依赖性。给药48h后,Hoechst33258荧光染色可见典型的凋亡形态学特征。FCM检测凋亡显示两药联合作用24h后,5mg/mL GLP联合50μg/mL 5-FU的凋亡率为36.14%,高于5-FU(50μg/mL)单独用药组(26.07%);1.25mg/mL GLP联合12.5μg/mL 5-FU、2.5mg/mL GLP联合25μg/mL 5-FU、5mg/mL GLP联合50μg/mL 5-FU作用48h后,凋亡率分别为20.95%、32.87%、30.01%,均高于5-FU(50μg/mL)单独用药组(14.51%)。联合用药组可使HCT-116细胞阻滞于S期。结论：较高质量浓度的GLP与5-FU联用具有协同抑制人结肠癌细胞株HCT-116细胞增殖,诱导细胞凋亡,阻滞细胞周期作用。     

高良姜素对肝癌细胞HepG-2的凋亡效应

以人肝癌细胞 HepG-2 为研究对象,探讨高良姜素对肝癌细胞增殖及凋亡的影响。研究不同浓度的高良姜素对HepG-2细胞毒性及形态学、凋亡率、细胞周期及线粒体膜电位、胞内钙离子稳态的影响。结果表明:5.4、10.8和21.6 μg/mL高良姜素使细胞增殖受到抑制,48 h光密度(optical density,OD)值分别为1.295、1.170、1.043,呈现典型的凋亡形态学变化,细胞周期阻滞于DNA合成前期(first gap,G1)。出现凋亡细胞且凋亡率均呈浓度和时间依赖性,48 h凋亡率分别为23.34%、33.15%、44.15%。细胞线粒体膜电位降低,空白对照细胞线粒体膜电位相对荧光强度为27.32,5.4、10.8和21.6 μg/mL高良姜素处理组分别为11.26、7.23、3.17;细胞内钙离子浓度增大,空白对照胞内钙离子浓度相对荧光强度为3.82,5.4、10.8和21.6 μg/mL高良姜素处理组分别为6.83、11.63、18.73;胞内钙离子稳态被打破。高良姜素可通过阻滞细胞周期进程,降低线粒体膜电位,打破细胞内钙离子稳态来诱导肝癌细胞HepG-2凋亡。     

鼓槌石斛毛兰素诱导人结肠癌Caco-2细胞凋亡

目的:研究毛兰素对人结肠癌Caco-2细胞增殖的抑制作用及其诱导细胞凋亡的分子机制。方法:采用SRB法检测毛兰素对Caco-2细胞增殖的抑制作用,用Hoechst33342染色法观察细胞的形态学改变,流式细胞术检测细胞的凋亡率和细胞周期;蛋白免疫印迹法(Western Blot)检测细胞凋亡相关蛋白Caspase-3的表达水平。结果:与对照相比,毛兰素能抑制结肠癌细胞Caco-2的增殖,且抑制率随着药物浓度与时间增加,呈剂量时间效应,48 h半数抑制浓度IC50为0.845μg/m L;毛兰素能诱导Caco-2细胞凋亡,并诱导细胞周期阻滞于G_2期;Caspase-3活性裂解片段表达增高。结论:毛兰素对肿瘤细胞的增殖具有一定抑制作用且通过线粒体途径诱导Caco-2细胞凋亡。     

草苁蓉提取物抑制肺癌细胞增殖分子机制的研究

草苁蓉提取物可通过促进肿瘤细胞凋亡抑制A549肺癌细胞增殖。继续探讨草苁蓉提取物(BRE)抑制肺癌细胞增殖的分子机制。采用流式细胞术检测A549细胞周期分布和细胞凋亡,免疫细胞化学法检测凋亡相关蛋白Bax、Bc1-2、P53和Fas蛋白的表达,ELISA法检测sFAS蛋白的表达。结果表明,BRE改变肺癌细胞周期分布,使多数细胞阻滞于G0/G1期。同时,BRE诱导肺癌细胞凋亡,明显增加Fas表达,增加Bax表达和降低Bc1-2表达,但不改变P53表达以及sFas蛋白水平。提示,BRE抗肺癌细胞增殖作用与其改变细胞周期分布、增高Fas表达和降低Bcl-2/Bax比值而诱导肺癌细胞凋亡相关。     

乙醇-水相中酪蛋白水解物修饰及对2个性质的影响

利用Alcalase水解酪蛋白,制备水解度为11.6％、IC5值为42.8 mg/L的酪蛋白水解物.在乙醇-水体系中采用Alcalase催化类蛋白反应修饰酪蛋白水解物,固定反应时间6h,优化得到酶添加量、乙醇体积分数、底物质量浓度、反应温度分别为:8.36 kU/g,56.8％,568 g/L,37.5℃.制备不同反应程度的修饰产物,评估其ACE抑制活性及Zn2+螯合能力变化,发现修饰产物的ACE抑制活性得到改善,抑制最高达到62.5％(IC50达到27.7 mg/L),但是与反应程度有关;Zn2+螯合能力则由4.22 mg/g降低至1.97～3.86 mg/g.修饰产物的Zn2+螯合能力与类蛋白反应程度无关,与ACE抑制活性也不存在相关性.     

Induction of the cell cycle arrest and apoptosis by flavonoids isolated from Korean Citrus aurantium L. in non-small-cell lung cancer cells

This study investigated the anti-proliferative and apoptotic effect of flavonoids isolated from Korean Citrus aurantium L. using A549 lung cancer cells. Flavonoids potently inhibited of A549 cells in a dose-dependent manner, whereas flavonoids had a weak inhibitory effect on proliferation of WI-38 cells. Flow cytometry and Western blot analysis showed that flavonoids induced cell cycle arrest at the G2/M checkpoint by controlling the proteins expression level of cyclin B1, cdc2, cdc25c and p21^WAF1/CIP1. Also, flavonoids induced apoptosis through the regulation of the expression of caspases, cleaved PARP and Bax/Bcl-xL ratio. The activity of caspase-3 on A549 cells increased in a dose-dependent manner. These results clearly indicated that the anti-cancer effect of flavonoids on A549 cells follows multiple cellular pathways through G2/M arrest and the induction of apoptosis.     

In vitro growth inhibition of mouse mammary epithelial tumor cells by methylseleninic acid: involvement of protein kinases

Methylseleninic acid (MSeA) is a synthetic organoselenium form known to be effective against mammary carcinogenesis in vivo. Using the synchronized mouse mammary epithelial tumor cell (TM6) model, we have previously shown that 5 microM MSeA significantly inhibits cell growth and induces a reversible growth arrest in the G1 phase. In the present study, we examined the effects of MSeA on Rb, cyclin dependent kinase 2 (cdk2), cdk4, cyclin E and cyclin D1. Growth arrest of cells was accompanied by a reduction in total cdk2 kinase and cyclin E-associated cdk2 kinase activities. The p27 levels associated with cdk2 were elevated during the cell cycle. In addition, growth inhibition correlated with a relative increase in the hypophosphorylated form of Rb in MSeA-treated cells and Egr1 was elevated in MSeA-treated cells. The Kinetworks Protein Kinase Screen (KPKS 1.0) was used to examine 75 protein kinases. MSeA treatment resulted in differential expression of several protein-serine/threonine kinases, protein-tyrosine kinases and protein-threonine/tyrosine kinases. Some of these kinases are being reported for the first time as being altered by MSeA. The outcome of these experiments will be of significance since these kinases are known to be involved in survival and/or apoptotic pathways of tumor cells.     

Shallot and licorice constituent isoliquiritigenin arrests cell cycle progression and induces apoptosis through the induction of ATM/p53 and initiation of the mitochondrial system in human cervical carcinoma HeLa cells

This study is the first to investigate the anticancer effect of isoliquiritigenin (ISL) in human cervical carcinoma HeLa cells. The results reveal that ISL inhibits HeLa cells by blocking cell cycle progression in the G2/M phase and inducing apoptosis. Blockade of cell cycle is associated with increased activation of ataxia telangiectasia‐mutated (ATM). Activation of ATM by ISL phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute‐2 (MDM2) interaction. In addition, ISL‐mediated G2/M phase arrest was also associated with decreases in the amounts of cyclin B, cyclin A, cdc2, and cdc25C, and increases in the phosphorylation of Chk2, cdc25C, and cdc2. The specific ATM inhibitor caffeine significantly decreased ISL‐mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. ISL induced apoptotic cell death is associated with changes in the expression of Bax and Bak, decreasing levels of Bcl‐2 and Bcl‐X_L, and subsequently triggering mitochondrial apoptotic pathway. In addition, pretreatment of cells with caspase‐9 inhibitor blocked ISL‐induced apoptosis, indicating that caspase‐9 activation is involved in ISL‐mediated HeLa cell apoptosis. These findings suggest that ISL may be a promising chemopreventive agent against human uterine cervical cancer.     

Gallic acid induces G2/M phase cell cycle arrest via regulating 14‐3‐3β release from Cdc25C and Chk2 activation in human bladder transitional carcinoma cells

Scope: Cell cycle regulation is a critical issue in cancer treatment. Previously, gallic acid (GA) has been reported to possess anticancer ability. Here, we have evaluated the molecular mechanism of GA on cell cycle modulation in a human bladder transitional carcinoma cell line (TSGH‐8301 cell). Methods and results: Using flow cytometer analysis, exposure of the cells to 40 μM GA resulted in a statistically significant increase in G2/M phase cells, which was accompanied by a decrease in G0/G1 phase cells. GA‐treated cells resulted in significant growth inhibition in a dose‐dependent manner accompanied by a decrease in cyclin‐dependent kinases (Cdk1), Cyclin B1, and Cdc25C, but significant increases in p‐cdc2 (Tyr‐15) and Cip1/p21 by western blotting. Additional mechanistic studies showed that GA induces phosphorylation of Cdc25C at Ser‐216. This mechanism leads to its translocation from the nucleus to the cytoplasm resulting in an increased binding with 14‐3‐3β. When treated with GA, phosphorylated Cdc25C can be activated by ataxia telangiectasia‐mutated checkpoint kinase 2 (Chk2). This might be a DNA damage response as indicated by Ser‐139 phosphorylation of histine H2A.X. Furthermore, treatment of the cells with a Chk2 inhibitor significantly attenuated GA‐induced G2/M phase arrest. Conclusion: These results indicate that GA can induce cell cycle arrest at G2/M phase via Chk2‐mediated phosphorylation of Cdc25C in a bladder transitional carcinoma cell line.     

九香虫防御性物质水溶液成分分析及其对 LO2 细胞活性的影响

研究了九香虫的防御性物质水溶液中挥发性成分及其相对百分含量,以及该水溶液对人肝LO2细胞活性的研究。采用温水浸泡法获得九香虫的防御性物质水溶液;GC-MS获得该水溶液的挥发性成分组成,并用面积归一法计算各成分的相对百分含量;利用MTT法、流式细胞术研究该水溶液对LO2细胞的影响。研究表明,九香虫防御性物质水溶液中挥发性成分包含19种化合物,其中相对百分含量最高的为反-2-己烯醛（88.54%）,其次为2-甲基-4-戊烯醛（5.16%）、十三烷（3.43%）等;该水溶液作用LO2细胞后,IC50为3.64 μL,呈剂量依赖性;细胞周期结果显示,G2/M期细胞比例明显降低,而G0/G1期细胞比例明显升高,且差异具有统计学意义。九香虫防御性物质水溶液成分主要是烯醛和烷烃类化合物;该物质能够抑制人肝LO2细胞的体外增殖,可能与其细胞周期阻滞有关。     

Characterization of an active GST-human Cdc2 fusion protein kinase expressed in the fission yeast Schizosaccharomyces pombe: A new approach to the study of cell cycle control proteins

Characterization of cdk (c yclin d ependent k inases) substrates and studies of their regulation require purified enzymatic complexes of cdc2-related catalytic and cyclin regulatory subunits. We produced human Cdc2 kinase in the fission yeast Schizosaccharomyces pombe as a fusion protein with glutathione S-transferase (GST). The GST-human Cdc2p fusion protein was active in vivo since it rescued a temperature-sensitive allele of cdc2. The fusion protein was purified using a one-step chromatography procedure with glutathione–Sepharose and exhibited a catalytic activity in vitro. Yeast cyclin B and suc1 were found in association with GST-Cdc2. A 17-fold stimulation of GST-Cdc2 kinase activity was obtained by incubation of recombinant human cyclin A with the S. pombe cellular extract prior to affinity purification. This indicates that cyclin concentration is limiting in this overexpression system. These findings describe a fast and easy production of active recombinant human Cdc2 kinase in yeast that can be used for biochemical studies.     

皮蛋模拟胃肠道消化物对HepG2细胞的影响

皮蛋是中国传统食品,其水解物具有抗氧化、抗炎和抗癌等功效,具有良好的医学前景,但目前皮蛋发挥抗癌功效的途径及作用机制尚不清楚.探究了皮蛋模拟胃肠道消化物(preserved eggs simulated gastrointestinal digests,PESD)对人肝癌HepG2细胞增殖和细胞周期的影响.将皮蛋经体外...     

亚硒酸钠与白藜芦醇联合应用对肺癌细胞的协同效应分析

目的:观察亚硒酸钠与白藜芦醇单独及联合应用对肺癌细胞的影响,并通过细胞周期探讨其作用机理。方法:用四甲基偶氮唑盐法检测亚硒酸钠和白藜芦醇单独及联合应用对细胞增殖和存活的影响;用流式细胞术测定细胞的凋亡率及细胞周期。结果:亚硒酸钠和白藜芦醇对肺癌细胞A549均有拮抗作用,且随浓度的增加,拮抗效果越显著,当亚硒酸钠浓度大于等于5μmol/L时,拮抗效果极显著(P0.01);当白藜芦醇浓度大于等于65μmol/L时,拮抗效果极显著(P0.01)。亚硒酸钠对A549细胞的半致死率(lethal dose of 50%,LD_(50))为4.759μmol/L,白藜芦醇对A549细胞的LD_(50)为45.24μmol/L。然而亚硒酸钠和白藜芦醇联合用药作用于A549细胞的拮抗效果优于单独用药,且低剂量的亚硒酸钠和白藜芦醇联合就能发挥出高剂量亚硒酸钠对A549细胞的拮抗作用效果,有效地降低了亚硒酸钠对细胞的毒副作用。另外,亚硒酸钠联合白藜芦醇作用可以阻滞A549细胞G0/G1期,最终导致细胞凋亡。