藤梨根提取物对人胃癌MKN－45细胞增殖及凋亡的影响

目的：探讨太白山藤梨根提取物（Radix Actinidiae extractive,RAE）在体外对胃癌MKN-45细胞增殖与凋亡的影响。方法：用0．01—100μg／ml浓度范围内的RAE和胃癌MKN-45细胞共培养24、48、72、96小时,采用MTF法检测RAE对MKN-45细胞增殖的影响;用流式细胞仪检测其对MKN-45细胞凋亡及细胞周期分布的影响;DNA琼脂糖凝胶电泳检测细胞凋亡。结果：RAE在体外能够显著抑制胃癌MKN-45细胞的增殖,且呈现浓度和时间依赖性;RAE对胃癌细胞的作用表现为周期特异性,使细胞阻滞于Go／G1期,进一步诱导细胞凋亡。对照组凋亡率为0．53％,1μg／ml、10μg／ml、100μg／ml浓度的RAE干预胃癌细胞48小时后均出现凋亡峰,细胞凋亡率分别为3．27％、8．97％、12．6％。DNA琼脂糖凝胶电泳检测细胞凋亡,不同浓度RAE处理的细胞样品中均可看到梯形凋亡条带,并呈现浓度依赖性。结论：RAE在体外对人胃癌MKN-45细胞有显著的抑制细胞增殖及诱导细胞凋亡作用,其作用机制可能是使胃癌细胞周期阻滞于G0／G1期。     

盐酸小檗碱对人胃癌细胞增殖和凋亡的影响

目的:研究盐酸小檗碱对人胃癌细胞及永生化胃上皮细胞增殖和凋亡的影响,初步探讨其中可能的分子机制。方法:利用不同浓度的小檗碱处理胃癌细胞系MKN-45和SGC-7901细胞,以及永生化胃上皮细胞系GES细胞,采用MTT法测定细胞活力,流式细胞术检测细胞周期和细胞凋亡(PI标记),Western blot检测细胞周期和凋亡相关蛋白的表达。结果:MTT结果显示与空白对照组相比,小檗碱处理后,细胞增殖速度明显受到抑制(P<0.05),并呈浓度依赖性,其中50μg/ml处理组最大抑制率为81.3%,这种抑制作用对胃癌细胞更为明显。流式细胞仪检测表明,小檗碱在较低浓度(10μg/ml)时即可诱导胃癌细胞出现G0/G1阻滞,S期细胞百分比减少和细胞凋亡(P<0.05),这种作用呈时间和浓度依赖性。Western blot结果提示小檗碱处理后,胃癌细胞Bcl-2蛋白表达水平降低,Bax及p53蛋白表达水平明显上调。结论:小檗碱可抑制胃癌细胞增殖,诱导胃癌细胞凋亡,其机制与阻滞细胞周期进展以及调控凋亡相关蛋白Bcl-2、Bax和p53密切相关。     

盐酸小檗碱对人胃癌细胞增殖和凋亡的影响

目的：研究盐酸小檗碱对人胃癌细胞及永生化胃上皮细胞增殖和凋亡的影响,初步探讨其中可能的分子机制。方法：利用不同浓度的小檗碱处理胃癌细胞系MKN-45和SGC-7901细胞,以及永生化胃上皮细胞系GES细胞,采用MTT法测定细胞活力,流式细胞术检测细胞周期和细胞凋亡（PI标记）,Western blot检测细胞周期和凋亡相关蛋白的表达。结果：MTT结果显示与空白对照组相比,小檗碱处理后,细胞增殖速度明显受到抑制（P〈0.05）,并呈浓度依赖性,其中50μg/ml处理组最大抑制率为81.3%,这种抑制作用对胃癌细胞更为明显。流式细胞仪检测表明,小檗碱在较低浓度（10μg/ml）时即可诱导胃癌细胞出现G0/G1阻滞,S期细胞百分比减少和细胞凋亡（P〈0.05）,这种作用呈时间和浓度依赖性。Western blot结果提示小檗碱处理后,胃癌细胞Bcl-2蛋白表达水平降低,Bax及p53蛋白表达水平明显上调。结论：小檗碱可抑制胃癌细胞增殖,诱导胃癌细胞凋亡,其机制与阻滞细胞周期进展以及调控凋亡相关蛋白Bcl-2、Bax和p53密切相关。     

丹参酮ⅡA对MKN-45细胞生长的影响

目的:研究丹参酮ⅡA(Tanshi-noneⅡA,TanⅡA)对人胃癌细胞株MKN-45的生长抑制作用。方法:选用人胃癌细胞株MKN-45,运用MTT法、流式细胞术(FCM)和逆转录聚合酶链式反应(RT-PCR)半定量法检测细胞增殖、细胞周期、细胞凋亡和各组p53mRNA表达。结果:TanⅡA可明显抑制MKN-45细胞的增殖,其抑制作用具有时间-效应和剂量-效应关系。当2·0μg/mL TanⅡA处理MKN-45细胞96h,增殖抑制率达(74·84±0·41)%。FCM检测肿瘤细胞DNA变化,观察到TanⅡA使MKN-45细胞周期阻滞于G2/M期,出现浓度依赖性的亚“G1”峰,同时野生型p53表达增加。结论:TanⅡA能有效地抑制人胃癌细胞株MKN-45的增殖,其可能的机制与凋亡有关。肿瘤防治杂志,2005,12(19):1465-1468     

金丝桃苷诱导人胃癌细胞MKN-45凋亡及其机制北大核心CSCD

目的探讨金丝桃苷(hyperoside,Hyp)对人胃癌MKN-45细胞增殖及凋亡的影响及其机制。方法体外培养MKN-45细胞,对照组不加药物,实验组分别加入不同浓度Hyp(12.5、25、50、100、200μg/ml)作用24、48 h,MTT法检测Hyp对细胞增殖的影响并筛选适宜药物浓度;流式细胞术检测细胞周期变化及凋亡率;Western blot法分析NF-κB P65、caspase-3、Bax、Bcl-2蛋白的表达情况。结果 Hyp可明显抑制MKN-45细胞的增殖,阻滞细胞于G0/G1期并促进其凋亡,Western blot结果显示,随着药物浓度的增高,Hyp可使NF-κB P65、Bcl-2蛋白表达降低,casepase-3、Bax蛋白表达增高。结论金丝桃苷可抑制MKN-45细胞增殖并诱导凋亡,其机制可能与该药物阻断细胞周期,抑制NF-κB通路,下调NF-κB P65、Bcl-2蛋白,上调casepase-3、Bax蛋白有关。     

塞来昔布抑制人胃癌细胞株增殖及其机制的实验研究

目的:研究特异性环氧化酶-2(COX-2)抑制剂塞来昔布对人胃癌细胞株MKN-45是否有抑制增殖、诱导凋亡作用并初步探讨其作用机制.方法:采用四氮唑盐比色法(MTT法)观察细胞增殖活力改变;流式细胞仪检测细胞凋亡率及细胞周期变化; 透射电镜观察细胞超微结构的改变; DNA提取行琼脂糖凝胶电泳观察DNA的"梯状"条带;免疫组化观察bcl-2基因和bax基因表达情况.结果:MTT法显示随着塞来昔布浓度和作用时间增加,细胞抑制率上升.人胃癌细胞株MKN-45在25、50、75、100μmol/L塞来昔布浓度下,24小时细胞抑制率分别为(16.39±1.430%、(23.55±0.11)%、(62.78±1.42)%、(87.53±0.27)%,48小时细胞抑制率分别为 (30.40±0.68)%、(46.18±1.80)%、(89.77±0.21)%、(97.94±0.18)%,(P＜0.05);流式细胞仪测定塞来昔布组出现凋亡峰,50、100μmol/ml浓度的塞来昔布干预24小时后,人胃癌细胞株MKN-45凋亡率分别为(25.5±2.66)%、(57.23±2.19)%;透射电镜观察显示塞来昔布组细胞呈凋亡表现,核固缩、碎裂,染色质浓缩,边集;塞来昔布(50μmol/L、100μmol/L)组培养48小时后DNA琼脂糖凝胶电泳可见典型的梯状条带,而对照组细胞DNA 未见梯状条带;免疫细胞化学法显示经塞来昔布干预后,凋亡蛋白Bax表达增加,而Bcl-2 表达减少,并随时间和药物浓度变化而明显.结论:塞来昔布呈剂量、时间依赖性地抑制MKN-45细胞增殖,促进MKN-45细胞凋亡.其机制之一可能是通过减少Bcl-2的表达、增加Bax的表达,从而启动细胞凋亡途径.     

莪术醇对人宫颈癌CASKI细胞增殖抑制及促凋亡作用的研究

目的:探讨莪术醇对人宫颈癌CASKI细胞体外增殖和凋亡的影响.方法:12.5μg/ml、25μg/ml、50μg/ml和100μg/ml不同浓度莪术醇作用于人宫颈癌CASKI细胞后,MTT法检测CASKI细胞的增殖抑制率;流式细胞仪分析细胞周期分布及细胞凋亡率变化;电镜观察肿瘤细胞凋亡的形态学特征.结果:12.5μg/ml、25μg/ml、50μg/ml和100μg/ml的莪术醇对宫颈癌CASKI细胞均有不同程度的增殖抑制作用,在一定范围内,具有浓度-时间依赖性.流式细胞仪分析结果显示:50μg/ml、100μg/ml莪术醇作用于宫颈癌CASKI细胞24h,可阻滞细胞周期于G2/M期,并诱导部分细胞凋亡.电镜下可见凋亡细胞及凋亡小体,且100μg/ml组细胞凋亡改变较50μg/ml组更明显.结论:莪术醇能明显抑制CASKI细胞的体外增殖,且可阻滞CASKI细胞周期于G2/M期并诱导细胞凋亡.     

泮托拉唑对胃癌细胞MKN-45增殖与凋亡的影响

目的 探讨泮托拉唑对胃癌细胞MKN-45增殖与凋亡的影响及相关机制.方法 采用0、10、20、40 μg/ml的泮托拉唑处理MKN-45细胞48 h;MTT法检测细胞活力;Hoechst染色检测细胞形态;流式细胞术检测细胞周期;Western blot检测细胞中B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞周期蛋白D1(Cyclin D1)及Cyclin E表达.结果 与0 μg/ml比较,10、20、40 μg/ml的泮托拉唑能明显降低MKN-45细胞活力(P﹤0.01),使细胞核发白,皱染,使细胞周期阻滞在G1期(P﹤0.01),下调Cyclin D1及Cyclin E的表达(P﹤0.01);与0 μg/ml比较,20、40 μg/ml的泮托拉唑能明显下调MKN-45细胞中Bcl-2的表达(P﹤0.01),上调Bax的表达(P﹤0.01).结论 泮托拉唑通过调控细胞周期蛋白及细胞凋亡相关蛋白表达抑制胃癌细胞MKN-45的增殖,并诱导细胞凋亡.     

莪术醇对人宫颈癌CASKI细胞增殖抑制及促凋亡作用的研究

目的：探讨莪术醇对人宫颈癌CASKI细胞体外增殖和凋亡的影响。方法：12．5μg/ml、25μg／ml、50μg／ml和100μg／ml不同浓度莪术醇作用于人宫颈癌CASKI细胞后,MTT法检测CASKI细胞的增殖抑制率;流式细胞仪分析细胞周期分布及细胞凋亡率变化;电镜观察肿瘤细胞凋亡的形态学特征。结果：12．5μg／ml、25μg/ml、50μg／ml和100μg／ml的莪术醇对宫颈癌CASKI细胞均有不同程度的增殖抑制作用,在一定范围内,具有浓度一时间依赖性。流式细胞仪分析结果显示：50μg／ml、100μg／ml莪术醇作用于宫颈癌CASKI细胞24h,可阻滞细胞周期于G2／M期,并诱导部分细胞凋亡。电镜下可见凋亡细胞及凋亡小体,且100μg／ml组细胞凋亡改变较50μg／ml组更明显。结论：莪术醇能明显抑制CASKI细胞的体外增殖,且可阻滞CASKI细胞周期于G2／M期并诱导细胞凋亡。     

三氧化二砷对胃癌BGC-823细胞增殖及细胞周期的影响

[目的]探讨三氧化二砷（AS2O3）对胃癌BGC-823细胞增殖的抑制作用及对细胞周期的影响。[方法]应用MTT方法检测三氧化二砷对胃癌BGC-823细胞增殖的抑制作用，流式细胞仪进行细胞周期解析及凋亡检测．[结果]三氧化二砷对胃癌BGC823细胞具有杀伤作用，且呈时间-浓度依赖性抑制肿瘤细胞的增殖．72h抑制细胞50％增殖的药物浓度约为3．3μmol／L，与对照组比差异显著（P=0．0048）；5μmol／LAs2O3作用24h，细胞形态学可见典型的凋亡细胞及M期阻滞；流式细胞仪进行细胞周期解析结果提示，G2/M期细胞由正常对照的25％增加到46．3％，出现了明显的G2/M期阻滞，亚二倍体凋亡细胞由4％增加到18％。[结论]三氧化二砷抑制胃癌细胞的增殖．诱导细胞周期阻滞及凋亡。     

Expression of Fas Antigen and Its Mediation of Apoptosis in Human Gastric Cancer Cell Lines

Fas, a member of the tumor necrosis factor receptor/nerve growth factor receptor family, induces apoptosis by crosslinking with Fas ligand or anti-Fas antibody in a variety of cultured cells. We examined the expression of Fas antigen and its mediation of apoptosis in six human gastric carcinoma cell lines. Flow cytometric analysis and western blotting revealed relatively high expression of Fas antigen in MKN-74 (wild-type p53 gene) and MKN-45 (wild-type), followed by MKN-1 (mutated), MKN-7 (mutated) and KATO-III (deleted). MKN-28 (mutated) showed minimal expression of the antigen. The expression was apparently enhanced by interferon-γ, except for MKN-1 and MKN-28. Anti-Fas antibody (100 ng/ml) induced nuclear fragmentation characteristic of apoptosis. Apoptosis occurred in a delayed fashion and the apoptotic index at 72 h was approximately 60% in MKN-74, 35% in MKN-45, and 20% in MKN-1 and KATO-III. A DNA ladder was noted in MKN-74 at 72 h. Expression levels of P53 and P21^Wafl did not change for up to 48 h in MKN-74. The biological effects did not correlate with endogenous Bcl-2 expression. These results indicated that a) Fas antigen is variably expressed in human cultured gastric carcinoma cells, b) the protein transduces an apoptotic signal which leads to delayed cell death, and c) susceptibility to the antibody correlates well with the expression level of Fas antigen.     

A model chemosensitivity test examining apoptosis in small specimens of gastric cancer

Background. Because chemosensitivity tests usually require a large amount of tissue, they are not used routinely in patients with unresectable gastric cancer. The aim of this study was to investigate whether apoptosis can be used as a sensitivity assay for chemosensitivity in small gastric cancer specimens. Methods. Apoptosis, detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick labeling (TUNEL), was investigated in small specimens of the MKN-1, MKN-45, and TMK-1 human gastric cancer cell lines as a marker of chemosensitivity following exposure to antineoplastic agents. Results. Doxorubicin (DXR), SN-38 (active metabolite of irinotecan), and paclitaxel (Taxol) induced DNA fragmentation in MKN-45 and TMK-1 cells, but not in MKN-1. In contrast, neither 5-fluorouracil (5-FU) nor cisplatin (CDDP) induced DNA fragmentation in any of the three cell lines. Small pieces cut from tumors implanted in nude mice were exposed to the antineoplastic agents in culture medium for 24 h, and the percentage of TUNEL-positive cancer cells (TUNEL positivity) was examined. TUNEL positivity in all three cancers increased after exposure to DXR, SN-38, and Taxol, but not after exposure to CDDP or 5-FU. MKN-45 showed the highest TUNEL positivity with SN-38 and Taxol, and TMK-1 TUNEL positivity was highest with DXR. MKN-45 and TMK-1 were the most sensitive to these three antineoplastic agents in vitro, while MKN-1, with the lowest TUNEL positivity, was the least sensitive to these three antineoplastic agents. TUNEL positivity after exposure to Taxol correlated with the antitumor effects of this compound in an animal model. Conclusion. These results suggest that, in small gastric cancer specimens where apoptosis is implicated, TUNEL positivity may be applicable to a chemosensitivity test. Received: January 28, 2000 / Accepted: April 14, 2000     

siRNA沉默PIM1基因表达对人胃癌MKN-45细胞增殖及凋亡的影响

目的:探讨PIM1 基因沉默对人胃癌 MKN-45 细胞增殖和凋亡的影响。方法:设计2条针对PIM1 mRNA靶序列的干扰RNA正义链和反义链及阴性对照序列,构建重组pSIREN-retroQ质粒载体,并转染胃癌 MKN-45 细胞。应用实时荧光定量PCR和蛋白质印迹法分别检测 PIM1在 mRNA水平及蛋白水平的表达。CCK-8 法检测胃癌 MKN-45 细胞的增殖,流式细胞分析胃癌细胞的凋亡,蛋白质印迹法检测凋亡相关蛋白Caspase 3和Caspase 9的表达。结果:重组克隆通过 PCR 扩增及测序,证明干扰序列插入正确。与正常对照组和阴性对照组相比,两个RNA干扰组胃癌 MKN-45 细胞PIM1在 mRNA及蛋白水平的表达量都显著降低（P<0.05）,细胞增殖能力下降（P<0.05）。此外,干扰组细胞凋亡明显增多（P<0.01）,Caspase 3和Caspase 9蛋白表达上调（P<0.01）。 结论: PIM1 siRNA能有效下调靶基因表达,产生特异性基因沉默效应;PIM1基因沉默显著抑制胃癌MKN-45 细胞的增殖并促进其凋亡。     

Docetaxel enhances the cytotoxicity of cisplatin to gastric cancer cells by modification of intracellular platinum metabolism

We have examined the combined anticancer effects of docetaxel (DOC) and cisplatin (CDDP) in vitro using the gastric cancer cell lines MKN-45, MKN-74, and TMK-1. Treatment of the cell lines with 30 microg/ml of DOC for 24 h followed by incubation with 3 or 10 microg/ml of CDDP for 24 h showed a clear synergistic effect. Sequence dependency of the agents was observed in these cell lines: DOC followed by CDDP (DC) showed a stronger antitumor effect than CDDP followed by DOC (CD) in all cell lines. To clarify the mechanism of action of the DC combination, total intracellular platinum (Pt) levels were evaluated after treatment with CDDP alone or combined with DC. For the MKN-45 and -74 cell lines, cells treated with DOC (10 microg/ml for 12 h) and then CDDP showed significantly increased intracellular Pt accumulation compared to cells treated with CDDP alone. We also investigated alterations in intracellular glutathione (GSH) concentration in response to DOC and CDDP. MKN-45 and -74 cells pretreated with DOC (10 microg/ml for 12 h) showed significantly increased intracellular GSH levels compared to cells administered CDDP only. To explain these findings, messenger RNA (mRNA) levels for multidrug resistance-associated protein-1 (MRP-1), the ATP-dependent pump for Pt-GSH complexes, were quantified in CDDP-treated MKN-45 cells with and without DOC pretreatment. While CDDP administration increased MRP-1 mRNA expression in MKN-45 cells, MRP-1 was not up-regulated after CDDP administration in DOC pretreated MKN-45 cells. Our results suggested that the enhanced CDDP toxicity due to DOC pretreatment may be related to the accumulation of intracellular Pt-GSH complexes, because DOC appears to suppress the MRP-1 up-regulation induced by CDDP exposure in gastric cancer cells.     

miR-875-5p通过靶向USF2抑制胃癌细胞的增殖、迁移和侵袭

目的:探讨miR-875-5p对胃癌细胞增殖、迁移和侵袭的影响及其机制。方法:采用qPCR法检测胃癌细胞BGC-823、HGC-27、MGC-803、SGC-7901、AGS、MKN-45和胃黏膜上皮细胞GES-1中miR-875-5p的表达水平。利用脂质体转染技术,分别将miR-875-5p模拟物/抑制剂(mimic/inhibitor)及其阴性对照质粒(miR-NC/Anti-miR-NC)转染至AGS细胞/MKN-45细胞,构建过表达/抑制miR-875-5p的细胞模型,空白对照组(Control组)不转染。通过CCK-8、克隆形成、Transwell等实验分别检测miR-875-5p表达变化对细胞增殖、克隆形成、迁移和侵袭的影响。采用双荧光素酶报告基因实验验证miR-875-5p与上游刺激因子2(USF2)的靶向关系,WB实验验证miR-875-5p对USF2的调控作用并检测USF2蛋白的表达。构建MKN-45细胞裸鼠移植瘤模型,验证miR-875-5p过表达对MKN-45细胞成瘤能力的影响。结果:miR-875-5p在6种胃癌细胞中表达水平显著低于胃黏膜上皮细胞GES-1(均P<0.01)。与Control组和miR-NC组相比,miR-875-5p mimic组AGS细胞的增殖、克隆形成率、迁移和侵袭细胞数,以及USF2蛋白的表达均显著降低(P<0.05或P<0.01);miR-875-5p inhibitor组MKN-45细胞的增殖、克隆形成率、迁移和侵袭细胞数,以及USF2蛋白的表达均显著提高(P<0.05或P<0.01)。双荧光素酶报告基因实验证明,miR-875-5p能够直接靶向USF2基因。体内成瘤实验结果表明,过表达miR-875-5p显著抑制MKN-45细胞移植瘤的生长(均P<0.01)。结论:miR-875-5p通过靶向USF2抑制胃癌细胞的增殖、迁移和侵袭。     

In-vitro effect of a combination of 5-fluorouracil (5-FU) and cisplatin (CDDP) on human gastric cancer cell lines: timing of cisplatin treatment

Abstract. Background: To date, we have few effective chemotherapeutic agents against advanced and recurrent gastric cancer. 5-Fluorouracil (5-FU) and cisplatin (CDDP) are the most widely used drugs, and their combination has demonstrated favorable outcomes. However, the method (especially the timing) of CDDP administration is not well established. Methods: We examined the in-vitro effect of a combination of 5-FU and CDDP on four human gastric cancer cell lines: MKN-1, MKN-28, MKN-45, and MKN-74. The cell lines were exposed to 50% of the inhibitory concentrations of 5-FU and CDDP for 72 h and 8 h, respectively. CDDP was applied before, simultaneously with, and after the start of treatment with 5-FU. Results: When CDDP was applied after 5-FU, the cytotoxic activity against MKN-28, MKN-45, and MKN-74 was significantly potentiated. Against MKN-1, the earlier the initiation of CDDP treatment, the stronger was the cytotoxic effect. Conclusion: Our results suggest that the cytotoxicity of a combination of 5-FU and CDDP against human gastric cancer cells is both cell-line- and schedule-dependent and is especially affected by the timing of the CDDP treatment. Received: August 22, 2001 / Accepted: December 7, 2001     

Antitumor activity of cis-diamminedichloroplatinum (II) depends on its time × concentration product against human gastric cancer cell lines in vitro

A pharmacodynamic study of cisplatin (DDP) was conducted using the gastric cancer cell lines MKN-45 and MKN-74 in vitro. Ten thousand tumor cells were incubated with 0.4–500 μg/ml DDP for 1–25 h, followed by recovery culture for a further 48 h. At the end of incubation, cell viability was detected by the MTT end-point, and the inhibition rate was compared in relation to the incubation time, DDP concentration, and the time × concentration product (area under the curve in vitro: AUC vitro). In both of the cell lines, the IC50 and IC90 values decreased as the exposure time increased, going a linear curve with a slope of almost — 1, and showing a typical log-log AUC vitro-dependent curve. These results indicate that the antitumor activity of DDP is dependent on its AUC vitro, suggesting the clinical usefulness of this drug when administered daily in small divided doses. © 1995 Wiley-Liss, Inc.