NS398对胰腺癌细胞周期及其蛋白依赖性激酶抑制物p21Wafl/cipl、p27Kipl/pic2的影响

目的:观察选择性环氧合酶-2(COX-2)抑制剂NS398对胰腺癌细胞周期及细胞周期相关蛋白p21Wafl/cipl、p27Kipl/pic2转录和表达的影响,探讨NS398的抗胰腺癌机制.方法:以NS398和前列腺素E2(PGE2)处理SW1990人胰腺癌细胞,分别采用MTT法检测细胞活力,酶联免疫分析法(ELISA)检测细胞内PGE2含量,流式细胞仪(FCM)检测细胞周期变化,并以半定量逆转录聚合酶链式反应(RT-PCR)和Western印迹检测COX-2及细胞周期相关蛋白p21Wafl/cipl、p27Kipl/pic2的mRNA和蛋白水平.结果:NS398抑制胰腺癌细胞生长,并呈剂量依赖性减少细胞内PGE2的生成;NS398诱导细胞周期相关蛋白p21Wafl/cipl和p27Kipl/pic2转录和表达的升高并诱导部分细胞阻滞在G0/G1期(较对照组升高11%);10nmol/L的外源性PGE2可增加胰腺癌细胞的活力,但并不能拮抗NS398对细胞活力的抑制作用或细胞周期分布的改变,以及COX-2、p21Wafl/cipl、p27Kipl/pic2的转录和表达.结论:NS398可能通过增强p21Wafl/cipl和p27Kipl/pic2的表达而诱导细胞周期的阻滞,从而抑制胰腺癌细胞的生长活力.但NS398并非通过抑制COX-2的唯一机制,还可能存在其它非COX-2途径.     

葡萄糖对内皮细胞环氧化酶2mRNA表达的影响及辛伐他汀、NS398的干预作用

目的 研究葡萄糖对人脐静脉内皮细胞(ECV304)环氧化酶2(COX-2)的表达及前列腺素E2(PGE2)含量的影响,探讨辛伐他汀、选择性COX-2抑制剂NS398对其的干预作用.方法 采用逆转录-聚合酶链反应法及放射免疫分析法检测不同浓度的葡萄糖、辛伐他汀、NS398单独或联合作用ECV304细胞24h后细胞COX-2mRNA表达及培养上清液中的PGE2含量的变化.结果 与对照组相比,葡萄糖(15、25、30mM)呈剂量依赖方式诱导细胞COX-2mRNA表达增加(P<0.05);25mM的葡萄糖增加细胞培养液中PGE2浓度明显高于对照组(P<0.01);1、10、100μM的辛伐他汀和10、20μM的NS398均可使高糖(25mM)诱导的COX-2mRNA表达下调及PGEμ浓度下降(P<0.05).结论 高糖可诱导ECV304细胞COX-2mRNA表达,并增加细胞培养液中PGE2含量;而辛伐他汀、NS398能抑制高糖的诱导作用.     

食管上皮癌变过程中细胞周期蛋白E细胞周期蛋白依赖性激酶2表达及DNA含量的研究

<正>本文探讨食管上皮癌变过程中DNA含量的变化,以及细胞周期蛋白E(cyclinE)和细胞周期蛋白依赖性激酶2(cy-clin-dependent kinase2,CDK2)表达的变化及其意义。     

罗格列酮对2型糖尿病模型小鼠肾组织中mPGES-1/PGE_2水平的影响

目的:研究罗格列酮(RSG)给药不同时间对2型糖尿病模型小鼠肾组织中膜结合型前列腺素E2合酶-1/前列腺素E2(m PGES-1/PGE2)水平的影响。方法:将小鼠随机均分为正常对照组、模型组、RSG 1周组、RSG 4周组、RSG 8周组和RSG 12周组,每组6只,后5组小鼠分别建立2型糖尿病模型。RSG各组小鼠腹腔注射RSG 4 mg/(kg·d),分别连续给药1、4、8、12周,末次给药后分别收集每只小鼠24 h尿液,检测其24 h尿量及尿液中PGE2、前列腺素D2(PGD2)、血栓素B2(TXB2)水平;再检测肾组织中环氧合酶(COX)-1、COX-2、m PGES-1、m PGES-2、15-羟基前列腺素脱氢酶(15-PGDH)、过氧化物酶体增殖物激活受体γ(PPAR-γ)蛋白水平及前列腺素E2受体1(EP1)及EP4m RNA水平。结果:与正常对照组比较,模型组小鼠24 h尿量明显增多,且尿液中PGE2、PGD2、TXB2水平明显升高(P<0.05),经RSG治疗后能以时间依赖性方式降低PGE2水平。糖尿病模型组小鼠较正常对照组小鼠肾组织中m PGES-1、COX-2、PPAR-γ蛋白水平明显升高(P<0.05),而m PGES-2、COX-1、15-PGDH和EP1、EP4m RNA水平未见明显改变。给予RSG治疗后能使模型小鼠的PPAR-γ、m PGES-1水平呈阶梯式降低,与给药时间呈正相关,但差异无统计学意义;EP4m RNA水平明显降低(P<0.05)。结论:RSG有望通过下调m PGES-1/PGE2/EP4表达水平来改善2型糖尿病肾脏病变。     

前列腺素E2及其相关蛋白在难愈性创面修复中的作用研究

前列腺素(prostaglandin,PG)E₂具有扩张血管、舒缓平滑肌的作用,已在消化道黏膜创面修复中发挥重要作用。环氧合酶是PGE₂的主要合成酶,而PG转运蛋白和15-羟基PG脱氢酶是其主要的代谢蛋白。该文通过综述PGE₂、环氧合酶、PG转运蛋白和15-羟基PG脱氢酶在创面修复中的作用,以探讨PGE₂为核心的创面修复特点,为未来选择较好的促进创面修复的药物靶点提供一些思路和手段。     

青蒿琥酯对人结肠癌SW620细胞增殖、细胞周期及细胞周期素依赖性激酶抑制蛋白的影响

目的观察青蒿琥酯对人结肠癌细胞系SW620细胞增殖、细胞周期及细胞周期素依赖性激酶抑制蛋白(p16、p21、p27)表达的变化,探讨ART可能的作用机制。方法:不同浓度青蒿琥酯作用SW620细胞后,MTT法检测细胞增殖情况,流式细胞仪检测细胞周期,Westernblot分别检测细胞周期素依赖性激酶抑制蛋白(p16、p21、p27)蛋白变化。结果:在一定浓度范围之内,青蒿琥酯能抑制SW620细胞增殖,细胞周期发生G1—S期阻滞,并上调p21、p27的蛋白、mRNA水平,但对p16的蛋白、mRNA水平没有影响。结论:青蒿琥酯可抑制人结肠癌细胞增殖,其机制与上调p21、p27蛋白有关。     

帕瑞昔布钠对宫颈癌细胞COX2/PGE2通路及细胞生物学行为的影响

目的 探讨帕瑞昔布钠对宫颈癌HeLa细胞环氧合酶2/前列腺素E2(COX2/PGE2)通路及增殖、凋亡、侵袭行为的影响.方法 体外培养人宫颈癌HeLa细胞,分为对照组(不加药物)、低浓度帕瑞昔布钠组(10μmol/L帕瑞昔布钠)、中浓度帕瑞昔布钠组(40μmol/L帕瑞昔布钠)和高浓度帕瑞昔布钠组(160μmol/L帕...     

原花青素对RAW264.7细胞膜相关前列腺素E2合成酶-1表达的影响

目的 探讨原花青素对RAW264.7细胞膜相关前列腺素E2合成酶-1(mPGES-1)表达的影响.方法 酶免疫测定法(EIA)检测原花青素对PGE2生成的影响,逆转录聚合酶链反应(RT-PCR)检测mPGES-1mRNA的表达,Western blotting检测mPGES-1蛋白的表达.结果 脂多糖(LPS)可以促进RAW264.7细胞PGE2的生成同时上调mPGES-1mRNA和蛋白的表达,而原花青素(4、20mg·L-1)下调LPS诱导的RAW264.7细胞mPGE-1mRNA和蛋白的表达,从而抑制LPS诱导的RAW264.7细胞PGE2的生成.结论 原花青素在mRNA和蛋白水平抑制LPS诱导的RAW264.7细胞mPGES-1表达从而减少PGE2的合成,这可能是原花青素抗炎的机制之一.     

胃肠道肿瘤细胞E2F-1转录因子活性对细胞周期的影响

目的:探讨癌细胞E2F-1因子的表达状态及其与细胞周期的关系.方法:采用免疫荧光组织化学染色法检测8株胃肠道肿瘤细胞系E2F-1转录因子的表达,并采用流式细胞术检测肿瘤细胞周期的变化.结果:依据E2F-1阳性表达的程度不同,将8株肿瘤细胞系分为E2F-1高表达组(包括HGC-27、BGC-823、SGC-7901、HT29)和E2F-1低表达组(包括MKN45、MKN28、SH288、SW-620).E2F-1高表达组G1/G0期、S期、细胞凋亡比例分别为(33.48±4.61)%、(63.91±5.02)%、(1.50±0.56)%,低表达组分别为(58.58±3.41)%、(35.38±4.03)%、(5.61±1.44)%,2组间比较差异有统计学意义(t分别为6.04、5.99和2.44,p<0.05或P<0.01).结论:肿瘤细胞E2F-1因子的表达状态对细胞周期有明显的影响.E2F-1阳性的肿瘤细胞则倾向于更高的增殖速率.     

Skp2-siRNA干预对Eca-109食管癌细胞系p21和p27蛋白及细胞周期的影响

目的探讨Skp2-siRNA干扰后食管癌Eca-109细胞株Skp2、p21和p27蛋白表达情况及其对细胞增殖调控的影响。方法经Skp2-siRNA干扰食管癌Eca-109细胞株24、48和72 h后,用免疫细胞化学法检测Skp2、p21和p27蛋白的表达情况,流式细胞仪检测细胞增殖周期的分布。结果食管癌Eca-109细胞株主要分布在G1期,Skp2蛋白表达下降（P＆lt;0.05或＆lt;0.01）,p21和p27蛋白的表达增高（P＆lt;0.05或＆lt;0.01）。结论运用Skp2-siRNA技术可以有效降低对p21和p27蛋白的降解,促进细胞停滞在G1期,降低细胞增殖活性。     

选择性环氧化酶2抑制剂NS-398对胃癌细胞AGS增殖的抑制作用

目的观察选择性环氧化酶2(COX-2)抑制剂NS-398对胃癌细胞AGS增殖的抑制作用,并探讨其相关机制。方法 NS-398 25,50和100μmol·L-1作用于AGS细胞0～48 h,用CCK-8法检测细胞存活率。NS-398 50μmol·L-1作用于AGS细胞48 h,用流式细胞仪检测细胞凋亡率,实时荧光定量PCR检测Notch信号通路相关基因的表达,Western印迹法检测Notch胞内结构域(NICD)及下游靶基因NF-κB和毛蛋白和断裂1增强子(Hes-1)蛋白表达。结果 NS-398 25,50和100μmol·L-1能抑制胃癌细胞AGS增殖,并呈时间(r时间=-1.00,P=0.003,50μmol·L-1)和浓度(r浓度=-0.999,P=0.027,48 h)依赖性。NS-39850μmol·L-1作用48 h,AGS细胞凋亡率为(20.1±3.5)%,比正常对照组(3.5±1.4)%明显增加(P<0.05);Notch信号通路受体Notch1和Notch2及Notch信号通路配体δ样1(DLL1)和锯齿状1(JAG1)mRNA表达无明显变化,Notch下游靶基因Hes-1和NF-κB mRNA表达较正常对照组明显减少(P<0.05);NICD,Hes-1和NF-κB蛋白表达明显减少(P<0.05)。结论选择性COX-2抑制剂NS-398可能通过抑制Notch信号途径抑制胃癌细胞AGS增殖。     

Effects of cyclooxygenase 2 inhibitors on biological traits of nasopharyngeal carcinoma cells

AIM: To investigate effects of cyclooxygenase 2 (COX-2) inhibitors on nasopharyngeal carcinoma (NPC) cells and on angiogenesis in vitro. METHODS: Human NPC cell lines (CNE1, CNE2 and SUNE) were treated with nimesulide or celecoxib. MTT assay and colony formation assay were performed to observe antiproliferation activity of COX-2 inhibitors to NPC cell lines. Cell cycle arrest and apoptosis of NPC cell strains were tested by flow cytometry assay, microscopic morphology observation, and DNA fragmentation assay. The effect of COX-2 inhibitor on angiogenesis was tested by chick chorioallantoic membrane (CAM) model. RESULTS: Nimesulide (Nim) and celecoxib (Cel) could antiproliferate NPC cell lines with IC50 182 μmo1/LNim-SUNE, 78 vmol/LNim-CNE1, 175μmoI/LNim-CNE2, 7.2 μmol/LCet-SUNE, 8.1 μmoI/LCet-CNE1, and 7.6 μmol/LCet-CNE2. The antiproliferation presented dose-dependent (Nim 5-400 μmol/L, Cel 0.5-80 μmol/L) and time-dependent manner (Nim IC50 562 μmol/L24 h, 316 μmol/L 48 h,50.1 μmol/L240h). Nim and Cel arrested SUNE and CNE1 cell cycle at phase G2/M (cell aggregation rate 28.9 %-45.1%y.Nim25-200μmol-12h-SUNM,18.9 %-26.2 %Nim25-200μmol-24h-SUNM, 28.8%-35.6 %Nim25-200μmol-48h-SUNM,30.4 %-16.4 %Nim25-200μmol-12h-SUNM,21.2 %-19.7 %Nim25-200μmol-24h-SUNM, 31.1%- 19.9%Nim25-200μmol-12h-SUNM, 20.5 %-34.1%Nim25-200μmol-12h-SUNM,25.2 %-26.9 %Nim25-200μmol-12h-SUNM, 11.5 %-7.1% Nim25-200μmol-12h-SUNM,Apoptosis shape and apoptosis strap displayed in NPC cells after treatment with Nim and Cel. Nim had a feature of anti-angiogenesis on CAM model. CONCLUSION:Nim and Cel could suppress proliferation of squamous epithelium NPC cell (SUNE, CNE1 and CNE2) through blocking cell cycle and inducing cell apoptosis. Nim could apparently suppress CAM angiogenesis induced bySUNE cell.     

Selective prostaglandin G/H synthase (PGHS)-2 inhibitors show greater inhibitory activities on human PGHS-2 than on murine PGHS-2 in intact cells

Excess eicosanoid formation during inflammation has been attributed to the expression of the gene coding for the inducible isoform of prostaglandin G/H synthase (PGHS-2). Human and murine PGHS-2 proteins differ in 73 out of the 604 amino acids. When comparing the inhibitory effects of a panel of PGHS-inhibitors in a whole cell human and murine PGHS-2 assay carried out under identical conditions, classical NSAIDs with the exception of aspirin and tenoxicam showed similar inhibitory effects on both human and murine PGHS-2 enzymes. However, the PGHS-2 selective inhibitors nimesulide, flosulide and NS398 showed a much greater inhibition of human PGHS-2. We suggest that these differences could be due to the genetic differences of human and murine PGHS-2. Formerly R&D Division of Hafslund Nycomed Pharma AG, Austria.     

Magnolol elicits activation of the extracellular signal-regulated kinase pathway by inducing p27KIP1-mediated G2/M-phase cell cycle arrest in human urinary bladder cancer 5637 cells

Magnolol has been reported to play a role in antitumor activity. However, the relevant pathway integrating cell cycle regulation and signaling pathways involved in growth inhibition in cancer cells remains to be identified. In the present study, magnolol treatment of these cells resulted in significant dose-dependent growth inhibition together with apoptosis, G1- and G2/M-phase cell cycle arrest at a 60 microM (IC50) dose in 5637 bladder cancer cells. In addition, magnolol treatment strongly induced p27KIP1 expression, and down-regulated expression of cyclin-dependent kinases (CDKs) and cyclins. Moreover, treatment with magnolol-induced phosphorylation of ERK, p38 MAP kinase, and JNK. Among the pathway inhibitors examined, only PD98059, an ERK-specific inhibitor, blocked magnolol-dependent p27KIP1 expression. Blockade of ERK function consistently reversed magnolol-mediated inhibition of cell proliferation and decreased G2/M cell cycle proteins, but not G1 cell cycle proteins. Furthermore, magnolol treatment increased both Ras and Raf activation. Transfection of cells with dominant negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed magnolol-induced ERK activity and p27KIP1 expression. Finally, the magnolol-induced reduction in cell proliferation and G2/M cell cycle proteins was also abolished in the presence of RasN17 and RafS621A mutant genes. These data demonstrate that the Ras/Raf/ERK pathway participates in p27KIP1 induction, leading to a decrease in the levels of cyclin B1/Cdc2 complexes and magnolol-dependent inhibition of cell growth. Overall, these novel findings concerning the molecular mechanisms of magnolol in 5637 bladder cancer cells provide a theoretical basis for therapeutic treatment of malignancies.     

Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

Context: Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong’s Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture.

Objectives: This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells.

Materials and methods: Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0?µmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot.

Results: Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC₅₀) values were 0.186, 0.158 and 0.123?µmol/ml at 24, 48 and 72?h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5?μmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with down-regulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of pro-apoptotic Bax and p53 proteins.

Conclusion: Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.     

Effects of genistein on cell proliferation and cell cycle arrest in nonneoplastic human mammary epithelial cells: involvement of Cdc2, p21(waf/cip1), p27(kip1), and Cdc25C expression

Genistein, a soy isoflavone, has been reported to inhibit the multiplication of numerous neoplastic cells, including those in the breast. However, there is limited information on the effect of genistein on nonneoplastic human breast cells. In the present studies, genistein inhibited proliferation of, and DNA synthesis in, the nonneoplastic human mammary epithelial cell line MCF-10F with an IC(50) of approximately 19-22 microM, and caused a reversible G2/M block in cell cycle progression. Genistein treatment (45 microM) increased the phosphorylation of Cdc2 by 3-fold, decreased the activity of Cdc2 by 70% after 8 hr, and by 24 hr reduced the expression of Cdc2 by 70%. In addition, genistein enhanced the expression of the cell cycle inhibitor p21(waf/cip1) by 10- to 15-fold, increased p21(waf/cip1) association with Cdc2 by 2-fold, and increased the expression of the tumor suppressor p53 by 2.8-fold. Genistein did not alter the expression of p27(kip1) significantly. Furthermore, genistein inhibited the expression of the cell cycle-associated phosphatase Cdc25C by 80%. From these results, we conclude that genistein inhibits the growth of nonneoplastic MCF-10F human breast cells by preventing the G2/M phase transition, induces the expression of the cell cycle inhibitor p21(waf/cip1) as well as its interaction with Cdc2, and inhibits the activity of Cdc2 in a phosphorylation-related manner. Down-regulation of the cell cycle-associated phosphatase Cdc25C combined with up-regulation of p21(waf/cip1) expression appear to be important mechanisms by which genistein decreases Cdc2 kinase activity and causes G2 cell cycle arrest.     

基于靶蛋白结构的CDK2小分子抑制剂研究进展

细胞周期蛋白依赖性激酶(CDKs)活性的精确调控对细胞的增殖和转录至关重要,对于一些增殖性疾病特别是恶性肿瘤,细胞的生长和增殖失控与CDKs活性异常密切相关。对CDK2蛋白结构的认识,特别是一些抑制剂与CDK2复合物的晶体学认识,极大的推动了小分子抑制剂的设计和开发。本文对一些骨架相异的有临床开发价值的小分子抑制剂与CDK2激酶的相互作用进行了解析,对激酶的各结合区特征做了归纳,进而分析了目前该领域的研究状况和趋势。     

钠钾泵抑制剂通过调节细胞周期相关蛋白的生成介导肝癌HepG2细胞周期S期阻滞与凋亡

目的研究钠泵抑制剂哇巴因(ouabain)和华蟾毒配基(cinobufogenin)对人肝癌HepG2细胞增殖的抑制作用及细胞周期的改变,初步分析其机制。方法以人肝癌细胞HepG2为靶细胞,MTT比色法检测哇巴因和华蟾毒配基对HepG2细胞增殖的影响;Hoechst 33342荧光染色检测细胞形态学变化;流式细胞术检测细胞周期;实时定量PCR和Western blot检测CyclinA1、CDK2、PCNA和p21CIP1表达的变化。结果哇巴因和华蟾毒配基可明显抑制HepG2细胞增殖,抑制作用呈时间-浓度依赖性。荧光染色显示药物处理24h后,细胞呈现典型的凋亡形态特征;细胞周期分析显示,实验组S期细胞比例升高,实时定量PCR和Western blot结果显示:哇巴因和华蟾毒配基可下调CyclinA1、CDK2和PC-NA的表达(P<0.05),上调p21CIP1的表达(P<0.05)。结论钠泵抑制剂可抑制肝癌HepG2细胞的增殖,引起细胞周期S期阻滞,诱导细胞凋亡,这与其调节细胞周期相关蛋白的生成关系密切。     

Quercetin induces cell cycle G1 arrest through elevating Cdk inhibitors p21 and p27 in human hepatoma cell line (HepG2)

Quercetin is a flavonoid ubiquitously found in nature. The therapeutic effect of quercetin on human hepatoma cell line (HepG2) was evaluated in this study. Various groups were incubated with different doses of quercetin for 12-, 24-, 48- and 72-h time duration and compared with control groups. Dose- and time-dependent inhibition in HepG2 proliferation was found with quercetin treatment. At 48 h of incubation, 61.78% of the cells were arrested at G(1) phase with 25 microM/l quercetin while 89.62% were arrested at G(1) phase with 50 microM/l quercetin. Furthermore, the results indicate that quercetin increased the content of Cdk inhibitor p21 protein, which was correlated with the elevation in p53 levels during 12 h of incubation. In addition, quercetin also increased the level of Cdk inhibitor p27 protein during 24 h of incubation. From our results it can be concluded that quercetin blocks cell cycle progression at G(1) phase and exerts its growth-inhibitory effect through the increase of Cdk inhibitors p21 and p27 and tumor suppressor p53 in HepG2.