尼美舒利对结肠癌细胞株PGE2及VEGF表达的影响

目的:探讨选择性环氧化酶2(COX-2)抑制剂对COX-2高表达的结肠癌细胞株HT-29增殖和凋亡的影响,明确以COX-2为靶点治疗结肠癌的作用途径。方法:将选择性COX-2抑制剂尼美舒利(nimesulide)按不同浓度作用于结肠癌细胞系HT-29,用MTT(四甲基偶氮唑蓝比色法)法分别于0、24、48、72h检测细胞增殖状态;流式细胞仪观察尼美舒利对细胞凋亡的影响,进一步采用ELISA法检测药物作用后前列腺素E2(PGE2)的表达,免疫组化法测定内皮细胞生长因子(VEGF)表达阳性率。结果:尼美舒利对结肠癌细胞系HT-29呈时间、剂量依赖性方式抑制细胞增殖,促进其凋亡。PEG2及VEGF表达水平随作用时间延长而下降。结论:选择性COX-2抑制剂可能通过PGE2与VEGF途径影响结肠癌细胞HT-29的增殖与凋亡,是其治疗结肠癌的分子机制。     

阿司匹林对宫颈癌Hela细胞COX-2mRNA表达的抑制作用

目的研究COX-2非选择性抑制剂阿司匹林（Aspirin）对宫颈癌细胞株Hela的细胞增殖的抑制作用及COX-2mRNA表达量的影响。方法Hela细胞分4组培养在6孔板上设对照组及实验组，观察不同剂量阿司匹林作用后细胞的增殖状态及采用逆转录-聚合酶链反应（RT—PcR）检测阿司匹林作用前后Hela细胞COX-2mRNA量的变化。结果阿司匹林能抑制Hela细胞增殖及细胞中COX-2mRNA的转录，且抑制强度随阿司匹林的浓度的提高而增加。结论体外阿司匹林抑制癌细胞Hela生长，并从mRNA水平抑制COX-2蛋白的表达，对宫颈癌可能有潜在治疗意义。     

塞来昔布联合 PDTC 对人结肠癌细胞 HT-29 的抑制增殖和促凋亡作用及其机制

目的研究塞来昔布联合吡咯烷二硫氨基甲酸（PDTC）对人结肠癌细胞HT-29增殖、凋亡的影响及其对COX-2、NF-κB、Caspase-3基因表达的影响,探讨塞来昔布联合用药抗肿瘤的机制。方法用不同浓度的塞来昔布（30、60、120、240μmol／L）（单药组）以及不同浓度塞来昔布联合不同浓度的PDTC（50、100μmol／L）（联合组）分别处理人结肠癌细胞,采用CCK-8法检测细胞的增殖情况,流式细胞术分析细胞凋亡情况,实时荧光定量PCR检测各组细胞COX-2、NF-κB、Caspase-3表达的变化。结果塞来昔布单药组对结肠癌细胞具有抑制增殖、促进凋亡作用,其作用随着药物浓度、给药时间的增加而增强（P均〈0．05）;同时检测到COX-2、NF-κBKBmRNA的表达降低（P均〈0．05）,Caspase-3表达升高（P〈0．05）,且具有浓度依赖性;联合组较同一塞来昔布浓度的单药组上述作用更明显（P均〈0．05）。结论塞来昔布单药组和联合组均能抑制人结肠癌细胞的增殖,促进其凋亡;联合组作用强于单药组;其机制可能与COX-2、NF-κB的下调和Caspase-3表达上调有关。     

粉防己碱对人结肠癌细胞株HT-29增殖与凋亡的影响

目的：通过观察粉防己碱对人结肠癌细胞株HT-29增殖与凋亡的影响．初步探讨粉防己碱对结肠癌的体外抗肿瘤效应。方法：采用MTT比色法观察粉防己碱对HT-29细胞的增殖抑制效应，利用细胞DNA琼脂糖凝胶电泳及细胞凋亡荧光染色法检测粉防己碱诱导肿瘤细胞凋亡的作用．以免疫细胞化学法检测粉防己碱对Bct-2和BAX表达水平的影响。结果：MTT比色法显示粉防己碱对人结肠癌细胞株HT-29增殖有抑制作用。其抑制效应具有剂量依赖的特点，细胞凋亡荧光染色法、琼脂糖凝胶电泳表明粉防己碱可诱导HT-29细胞凋亡，免疫细胞化学法显示粉防己碱上调BAX基因表达．下调Bcl-2基因表达。结论：粉防己碱对人结肠癌细胞株HT-29增殖的抑制效应具有剂量依赖性，并可诱导细胞凋亡，其抗肿瘤效应可能与凋亡相关基因表达的调控有关     

茶多酚对人结肠癌细胞株Caco-2凋亡及RhoA蛋白活性的影响

目的探讨茶多酚对人结肠癌细胞株Caco-2凋亡及RhoA蛋白活性的影响。方法将传代后的人结肠癌细胞株Caco-2细胞分为实验组和对照组,实验组加入不同浓度的茶多酚（25、50、100和200μmol／L）,对照组加入等量RPMI-1640培养液,采用四甲基偶氮唑盐（MTr）法测定茶多酚对Caco-2增殖抑制的影响,流式细胞术（FCM）检测细胞凋亡指数,Pull．down法检测人结肠癌细胞株Caco-2细胞内的RhoA蛋白活性。结果不同浓度的茶多酚对人结肠癌细胞株Caco-2有明显的抑制作用,且呈剂量及时间依赖性;Pull-down法检测结果显示,茶多酚可以降低人结肠癌细胞株Caco-2细胞内的RhoA蛋白活性,且随浓度增加其蛋白活性降低。结论茶多酚可促进人结肠癌细胞株Caco-2的凋亡,其可能通过降低RhoA蛋白活件来实现。     

Celecoxib抗肝癌作用及临床意义

目的：观察特异性环氧合酶-2（cyclooxygenase-2，COX-2）抑制剂Celecoxib对肝癌细胞增殖和凋亡的影响，探讨应用于肝癌治疗的临床意义。方法：用MTT法观察Celecoxib对肝癌细胞增殖的影响；透射电镜及流式细胞仪观察Celecoxib诱导肝癌细胞凋亡的作用、对细胞周期的影响及P-gp表达的变化；用RT-PCR技术观察COX-2、Survivin mRNA药物处理后表达的变化。结果：Celecoxib对肝癌细胞抑制增殖、诱导凋亡呈时间和剂量依赖性；细胞周期分布改变，G0／G1期细胞比例增加；在Survivin高表达的肝癌细胞凋亡过程中，Survivin基因的表达显著下调，MDR／P-gp表达减弱。结论：Celecoxib可能通过诱导细胞凋亡、降低COX-2表达、影响细胞周期分布，发挥抗肿瘤作用；并且可能通过下调Survivin、COX-2表达而对MDR细胞同样有效，有重要的临床应用意义。     

5-杂氮-2’-脱氧胞苷对人HL-60细胞DAPK基因表达的影响

本研究旨在观察甲基化转移酶抑制剂5-杂氮-2’-脱氧胞苷（5-aza-2＇-deoxycytidine,5-aza-2dC）对人急性髓系白血病细胞株HL．60凋亡及死亡相关蛋白激酶（deathassociatedproteinkinase,DAPK）基因表达的影响,探讨5-aza-2dC治疗AML的作用机制。不同浓度的5-aza-2dC处理HL-60细胞后,采用瑞式染色法观察5-aza-2dC对HL-60细胞形态的影响,流式细胞术检测5-aza-2dC对HL-60细胞凋亡的影响,RT—PCR法检测5-aza-2dC对HL击0细胞DAPK基因表达的影响。结果表明,①5-aza-2dC呈浓度依赖性地促进HL-60细胞凋亡;②经5-aza-2dC处理后,HL-60细胞DAPK基因表达水平较处理前呈剂量依赖性地增加。结论：随着5-aza-2dC作用浓度的增加,HL-60细胞的凋亡率及DAPK基因表达水平均逐渐增加,提示DAPK基因可能是5-aza-2dC诱导HL-60细胞凋亡的调控基因之     

熊果酸诱导脑胶质瘤U251细胞株凋亡及其机制

目的：探讨熊果酸(Ursolic acid, UA)诱导脑胶质瘤U251细胞株凋亡的作用及机制。方法：培养脑胶质瘤细胞株(U251)，应用流式细胞仪观察UA对细胞周期的影响；琼脂糖凝胶电泳观察细胞DNA的变化；Western-blot测定COX-2及凋亡相关蛋白Bcl-2、Bax表达。放射免疫分析法测定COX-2催化产物前列腺素E2(prostaglandin E2, PGE2)。结果：流式细胞术及琼脂糖凝胶电泳均显示UA诱导U251细胞凋亡，细胞核DNA 呈梯状降解。同时COX-2蛋白表达及其催化生成产物PGE2浓度下降，凋亡相关蛋白Bcl-2表达减少，而Bax无明显变化。结论：熊果酸能明显诱导U251细胞凋亡，其机制可能与阻滞细胞周期、抑制COX-2表达、减少PGE2生成及下调凋亡抑制蛋白Bcl-2表达有关。     

CyclinL2在化疗药物杀伤胃癌细胞的增敏作用

目的：探讨cyclinL2基因在化疗药物顺铂（DI）P）、氟尿嘧啶（5-Fu）和多西紫杉醇（Doc）诱导下的胃癌细胞凋亡中的作用。方法选用胃腺癌BCG-823细胞，MTT法检测DDP、5-Fu和Doe不同药物浓度对胃癌细胞生长的抑制作用，以及CyclinL2转染、反义cyclinL2转染后对该细胞生长的抑制作用的影响，同时利用流式细胞术定量检测细胞凋亡。结果不同浓度的化疗药物对该胃癌细胞生长均有明显的抑制作用，并有浓度的依赖性。细胞凋亡率和cydinL2基因表达率成正相关。结论CydinL2基因在化疗药物诱导胃癌细胞凋亡中起重要作用。     

辛伐他汀对人结肠癌细胞增殖的影响

目的体外观察辛伐他汀对人结肠癌HT-29、colo-320细胞增殖、细胞周期及环氧合酶-2（cyclooxygenase-2，COX-2）蛋白表达的影响。方法采用噻唑蓝（MTT）法观察辛伐他汀对结肠癌HT-29、colo-320细胞增殖的影响，流式细胞仪（FCM）研究辛伐他汀对细胞周期的作用，免疫细胞化学法观察辛伐他汀对COX-2蛋白表达的影响。结果体外辛伐他汀可抑制结肠癌HT-29（F=15．400，P〈0．001）、colo-320（F=5．162，P=0．004）细胞的增殖，辛伐他汀处理组内G0／G1期细胞增多，辛伐他汀可抑制结肠癌HT-29（F=5．699，P=0．002）、colo-320（F=7．214，P=0．001）细胞COX-2蛋白的表达。结论体外辛伐他汀对colo-320细胞增殖有抑制作用，该作用可能与其使细胞生长阻滞于G0／G1期及抑制COX-2蛋白表达有关。     

环氧化酶2抑制剂对体外缺氧大鼠心肌细胞凋亡的影响

目的观察体外培养心肌急性缺氧条件下环氧化酶-2(COX2)抑制剂与细胞凋亡间的关系.方法分离、培养Wistar大鼠心肌细胞.以第4～6代心肌细胞24份样本(每份1×105个细胞)随机分为正常对照组、单纯缺氧组、缺氧+COX-2抑制剂(NS-398,20 μmol/L)组及缺氧+阿司匹林(100 μg/L)组.缺氧前30 min加入干预药物或等量二甲亚砜.以蛋白质免疫印迹法检测心肌细胞中COX-1/2表达;流式细胞仪检测心肌细胞凋亡率;放射免疫法(RIA)检测心肌细胞培养液中6-酮-前列腺素F1α(6-ketoPGF1α)、血栓素B2(TXB2)含量.结果各组心肌细胞中COX-1表达差异无显著性;COX2表达均较正常对照组增高,阿司匹林与NS-398均不抑制COX2表达.心肌细胞凋亡率由高到低依次为缺氧+NS-398组、缺氧+阿司匹林组、单纯缺氧组和正常对照组;缺氧+NS-398组与其他各组间比较差异均有显著性(P均<0.05).各组心肌细胞培养液内TXB2水平由低到高依次为正常对照组、缺氧+NS-398组、缺氧+阿司匹林组和单纯缺氧组;缺氧+NS-398组与单纯缺氧组比较差异有显著性(P<0.05);6-keto-PGF1α水平由低到高依次为正常对照组、缺氧+NS-398组、缺氧+阿司匹林组和单纯缺氧组;缺氧+NS-398组与单纯缺氧组比较差异有显著性(P<0.05);TXB2/6-keto-PGF1α比值差异无显著性.结论急性缺氧可直接诱导培养心肌细胞表达COX-2,而COX-1表达不增加,缺氧使培养液内COX-2作用产物TXB2、6keto-PGF1α水平升高,此效应无中性粒细胞、巨噬细胞等炎症细胞参与.COX-2抑制剂NS-398可降低缺氧心肌培养液内TXB2、6-keto-PGF1α升高程度,但不改变TXB2/6keto-PGF1α比值.急性缺氧可直接诱导心肌细胞凋亡,NS398可显著增加缺氧培养心肌凋亡率,此效应不需要其他组织细胞参与.     

The effects of acetylsalicylic acid on proliferation,apoptosis, and invasion of cyclooxygenase-2 negative colon cancer cells

BACKGROUND: Acetylsalicylic acid (ASA, aspirin), the most common nonsteroidal anti-inflammatory drug (NSAID), has been shown to have a protective effect against the incidence and mortality of colorectal cancer. However, the mechanism of its anticancer function remains unclear. The aim of this study was to determine the effects of acetylsalicylic acid on proliferation, apoptosis, and invasion in human cyclooxygenase-2 (COX-2) negative colorectal cancer cell lines. MATERIALS AND METHODS: After treatment with various concentrations of ASA, cell proliferation was measured in the human colon cancer cell line SW480. Apoptotic cells were identified by transmission electron microscopy, acridine orange staining, and flow cytometry. The invasive potential of SW480 cells was detected using an in vitro invasion assay. The production of carcinoembryonic antigen was measured by microparticle enzyme immunoassay. Expression of Bcl2, Bax, CD44v6, and nm23 were evaluated by immunocytochemistry. RESULTS: ASA significantly inhibited the proliferation of SW480 cells and stimulated apoptosis. Production of carcinoembryonic antigen and the invasive potential of SW480 cells were also inhibited by ASA. After treatment with ASA, down-regulation of Bcl2 and CD44v6 expression and up-regulation of nm23 expression were observed in SW480 cells. No obvious effect of ASA was found on Bax expression. CONCLUSION: Our findings reveal that ASA inhibits the proliferation and promotes apoptosis in the human colon cancer cell line SW480. Down-regulation of Bcl2 expression might represent a potential mechanism by which ASA induces apoptosis in this COX-2 negative colon cancer cell line. Our results also suggest that ASA decreases the invasive potential of these colon cancer cells. Decreased CEA content and CD44v6 expression and elevated nm23 expression may contribute to the effect of ASA on invasive potential of SW480 colon cancer cells.     

Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin

BACKGROUND: The mechanisms for the variability in antiplatelet effects of aspirin are unclear. Immature (reticulated) platelets may modulate the antiplatelet effects of aspirin through uninhibited cyclooxygenase (COX)-1 and COX-2. Objectives: To evaluate the role of reticulated platelets in the antiplatelet effects of aspirin. METHODS: Sixty healthy volunteers had platelet studies performed before and 24 h after a single 325-mg dose of aspirin. Platelet studies included light transmission aggregometry; P-selectin and integrin alpha(IIb)beta(3) expression, and serum thromboxane B(2) (TxB(2)) levels. Reticulated platelets and platelet COX-2 expression were measured using flow cytometry. RESULTS: Subjects were divided into tertiles based on the percentage of reticulated platelets in whole blood. Baseline platelet aggregation to 1 microg mL(-1) collagen, and postaspirin aggregations to 5 microm and 20 microm ADP and collagen, were greater in the upper than in the lower tertile of reticulated platelets. Stimulated P-selectin and integrin alpha(IIb)beta(3) expression were also higher in the upper tertile both before and after aspirin. Platelet COX-2 expression was detected in 12 +/- 7% (n = 10) of platelets in the upper tertile, and in 7 +/- 3% (n = 12) of platelets in the lower two tertiles (P = 0.03). Postaspirin serum TxB(2) levels were higher in the upper (5.5 +/- 4 ng mL(-1)) than in the lower tertile (3.2 +/- 2.5 ng mL(-1), P = 0.03), and decreased even further with ex vivo additional COX-1 and COX-2 inhibition. The incidence of aspirin resistance (>or= 70% platelet aggregation to 5 microm ADP) was significantly higher in the upper tertile (45%) than in the lower tertile (5%, P < 0.0001). CONCLUSIONS: Reticulated platelets are associated with diminished antiplatelet effects of aspirin and increased aspirin resistance, possibly because of increased reactivity, and uninhibited COX-1 and COX-2 activity.     

EFFECTS OF p53 GENE THERAPY COMBINED WITH CYCLOOXYGENASE-2 INHIBITOR ON CYCLOOXYGENASE-2 GENE EXPRESSION AND GROWTH INHIBITION OF HUMAN LUNG CANCER CELLS

1 INTRODUCTIONCOXis the key enzyme involved in the synthesis of prostanoids,a collective termfor the PGs andthromboxanes.Of the two major isoforms of the COXenzyme,COX-1 is ubiquitous and constitutively ex-pressedin virtually all normal tissues.In contras…     

Evaluation of antitumor effects of aspirin and LGK974 drugs on cellular signaling pathways,cell cycle and apoptosis in colorectal cancer cell lines compared to oxaliplatin drug

Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies. Despite recent advances in the treatment for CRC, resistance to chemotherapy drugs and recurrence of the tumor are among the main problems for treatment in this cancer. The MTT assay was performed to assess the cytotoxic effects of drugs on CRC cell lines (SW742 and SW480) and normal colon cells. Three-dimensional culture (spheroid) was also used to evaluate the effect of drugs on tumor cell masses. The rate of expression of genes was also evaluated using Real-Time PCR. The analysis of the results demonstrated that aspirin and LGK974 have cytotoxic effects on CRC cell lines, and in the IC50 dose, they disintegrate the cancerous cell masses. These drugs reduce the invasion and increase apoptosis in SW742 and SW480 cell lines. A decrease in the expression of WNT, AXIN, TCF and APC genes and an increase in the expression of β-catenin gene in the WNT signaling pathway were revealed. The genes involved in the MAPK signaling pathway such as ERK, JNK, KRAS and MEK showed a decrease in expression and a increase in expression of RAF gene. In the apoptotic pathway, increased expression of BAX and decreased expression of BCL-2 were reported. Also, decreased expression of P53, cyclin D1 and COX-2 was observed. This study demonstrates that aspirin and LGK974 could be effective in inhibiting the signaling pathways of WNT and MAPK, arresting cell cycle and inducing apoptosis in CRC cell lines.     

Cyclooxygenase-2 contributes to N-methyl-D-aspartate-mediated neuronal cell death in primary cortical cell culture

Cyclooxygenase isozymes (COX-1 and COX-2) are found to be constitutively expressed in brain, with neuronal expression of COX-2 being rapidly induced after numerous insults, including cerebral ischemia. Because overactivation of N-methyl-D-aspartate (NMDA) receptors has been implicated in the cell loss associated with ischemia, we characterized the expression of the COX isozymes in murine mixed cortical cell cultures and used isozyme-selective inhibitors to determine their relative contribution to NMDA receptor-stimulated prostaglandin (PG) production and excitotoxic neuronal cell death. Immunocytochemical analysis of mixed cortical cell cultures revealed that COX-2 expression was restricted to neurons, whereas COX-1 was expressed in both neurons and astrocytes. Brief exposure to NMDA (5 min; 100 microM) elicited a time-dependent accumulation of PGs in the culture medium that preceded neuronal cell death and correlated with the induction of COX-2 mRNA. COX-1 expression remained unchanged. Flurbiprofen, a nonselective COX-1/COX-2 inhibitor, blocked NMDA-stimulated PG production and attenuated neuronal death in a concentration-dependent manner. Similar results were obtained with the specific COX-2 inhibitor NS-398 (10-30 microM) but not with the selective COX-1 inhibitor valeryl salicylate (10-300 microM). Inhibition of total constitutive COX activity with aspirin (100 microM, 1.5 h) before NMDA exposure did not prevent subsequent NMDA-mediated neuronal cell death. However, neuronal injury in aspirin-pretreated cultures was attenuated by flurbiprofen administration after NMDA exposure. Finally, the protection afforded by COX-2 inhibition was specific for NMDA because neither flurbiprofen nor NS-398 protected neurons against kainate-mediated neurotoxicity. Together, these results support the conclusion that newly synthesized COX-2 protein contributes to NMDA-induced neuronal injury.     

大蒜素对LM-8细胞Bcl-2及Bax表达的影响

背景:有研究证实,大蒜素对LM-8细胞增殖及凋亡有影响.目的:观察大蒜素干预C3H小鼠骨肉瘤细胞株LM-8细胞Bcl-2、Bax凋亡蛋白的表达,以及LM-8细胞的形态学变化与Bcl-2及Bax变化的关系.设计:随机分组,非盲法,细胞水平体外实验.单位:实验于2006-09/2007-05在华中科技大学同济医学院附属协和医院中心实验室完成.材料:实验所用C3H小鼠LM-8骨肉瘤细胞株购自解放军第四军医大学实验动物中心.大蒜素为武汉汇海公司产品,是从大蒜球茎中分离出的硫化物.Cell Counting Kit-8试剂购于上海同仁化学研究所,Bcl-2和Bax抗体及SP试剂盒购于福州迈新生物技术开发公司.方法:以含体积分数为0.1新生牛血清的1640完全培养基,培养LM-8细胞,制作细胞爬片.SP免疫细胞组织化学技术检测细胞爬片中Bcl-2和Bax蛋白表达;倒置显微镜下观察5.0,10.0和15.0 mg/L质量浓度大蒜素作用前后LM-8细胞形态变化;将LM-8细胞调整至7.5×107L-1接种于96孔板,用1.0,5.0,10.0和15.0 mg/L质量浓度的大蒜素处理细胞,设立不加任何处理因素的对照组及不含细胞和药物的培养基空白组,孵育24,48,72 h后取出培养板,加入CCK-8测定吸光度值,计算LM-8细胞生长抑制率:以5.0,10.0和15.0 mg/L质量浓度大蒜素干预LM-8细胞24,48,72 h,消化离心收集细胞,以流式细胞仪分析细胞凋亡率的变化.主要观察指标:LM-8细胞中Bcl-2和Bax蛋白表达;LM-8细胞形态及增殖、凋亡情况.结果:①Bcl-2和Bax蛋白表达:大蒜素可以降低Bcl-2表达,增强Bax表达,经15.0mg/L大蒜素作用72h,Bcl-2和Bax蛋白表达阳性率及Bcl-2/Bax表达与对照组比较,差异有显著性意义(P＜0.01).②LM-8细胞的形态:经不同质量浓度大蒜素干预后均出现明显凋亡表现.③LM-8细胞的增殖:大蒜素能明显抑制LM-8细胞的增殖,当大蒜素浓度从1.0 mg/L增加到15.0 mg/L,LM-8细胞72 h时生长抑制率由(23.87±3.26)%增至(58.32±5.38)%,在72 h其药物半数抑制率浓度是11.09 mg/L.④LM-8细胞的凋亡:5.0,10.0和15.0 mg/L质量浓度的大蒜素作用72 h后可诱导LM-8细胞凋亡,且呈浓度依赖性(P＜0.05).结论:①大蒜素可抑制LM-8细胞增殖,该效应与大蒜素的浓度和时间有关.②大蒜素可诱导LM-8细胞凋亡,作用呈浓度依赖性.③大蒜素抑制LM-8细胞增殖和诱导LM-8细胞凋亡的机制可能与下调Bcl-2的表达和上调Bax的表达有关.