Identification of specific genes and pathways involved in NSAIDs-induced apoptosis of human colon cancer cells

INTRODUCTIONColorectal cancer(CRC)is the second most lethal cancer in the USA.Numerous studies suggest that nonsteroidal anti-inflammatory drugs(NSAIDs)have antineoplastic effects.Epidemiological studies have shown a reduction in the risk of death from colorectal cancer associated with the use of NSAIDs[1].Perhaps the most compelling evidence for the chemopreventive role of NSAIDs is provided by the clinical studies in patients with familial adenomatous polyposis(FAP),an inherite…     

舒林酸对结肠腺癌HT-29细胞增殖调控的实验研究

目的 观察舒林酸对结肠腺癌ＨＴ－２９细胞增殖的影响,探讨其作用机制。方法 采用ＭＴＴ比色法观察舒林酸对ＨＴ－２９细胞增殖的影响;采用流式细胞仪检测舒林酸对ＨＴ－２９细胞周期的影响,同时结合ＤＮＡ电泳和透射电镜观察舒林酸对ＨＴ－２９细胞有无促凋亡作用。结果 舒林酸可抑制ＨＴ－２９细胞增殖,使Ｇ０／Ｇ１期细胞比例增高,Ｓ期比例降低,７２ｈ后,ＨＴ－２９细胞凋亡分别上升至１２．５＾、１５．４＾和２４．２     

砷对人胶质瘤细胞周期调控相关基因表达的影响

目的比较三氧化二砷（As2O3）对两种不同p53表型胶质瘤细胞U87MG、T98G细胞周期调控相关基因表达的影响。方法应用功能分类细胞周期基因芯片技术，筛选出As2O3作用于两种胶质瘤细胞U87MG、T98G后与细胞周期调控表达有差异的基因，并对3条有共性表达的基因进行Western印迹分析；四甲基偶氮唑蓝（MTT）法检测细胞生长抑制率；流式细胞仪分析细胞周期改变。结果MTT分析表明：As2O2对2个细胞系的生长有抑制作用，且抑制率具有浓度和时间的依赖性，砷作用72h后，U87MG和T98G细胞系的生长抑制率为50％（IC50）时，其浓度分别为1．78、3．55μmol／L。As2O3可以将U87MG和T98G细胞周期阻滞于G0／G1和G2／M期。U87MG细胞在加入As2O3后，差异表达2倍以上的基因共11条，表达上调的有5条，下调的有6条。T98G细胞在加入As2O3后，差异表达的基因共13条，表达上调的有9条，下调的有4条。Western分析显示，As2O3能上调2个胶质瘤细胞系的P53蛋白水平，同时降低细胞周期蛋白B1、D1表达。结论As2O3通过将细胞周期阻滞于G0／G1和G2／M期来抑制U87MG和T98G细胞的增殖；p53、CyclinB、CylinD等基因表达与As2O3诱导胶质瘤细胞周期阻滞的发生有关。     

Growth inhibitory effect of 4-phenyl butyric acid on human gastric cancer cells is associated with cell cycle arrest

AIM: To investigate the growth effects of 4-phenyl butyric acid (PBA) on human gastric carcinoma cells and their mechanisms.METHODS: Moderately-differentiated human gastric carcinoma SGC-7901 and lowly-differentiated MGC-803 cells were treated with 5, 10, 20, 40, and 60 μmol/L PBA for 1-4 d. Cell proliferation was detected using the MTT colorimetric assay. Cell cycle distributions were examined using flow cytometry.RESULTS: The proliferation of gastric carcinoma cells was inhibited by PBA in a dose- and time-dependent fashion. Flow cytometry showed that SGC-7901 cells treated with low concentrations of PBA were arrested at the G₀/G₁ phase, whereas cells treated with high concentrations of PBA were arrested at the G₂/M phase. Although MGC-803 cells treated with low concentrations of PBA were also arrested at the G₀/G₁ phase, cells treated with high concentrations of PBA were arrested at the S phase.CONCLUSION: The growth inhibitory effect of PBA on gastric cancer cells is associated with alteration of the cell cycle. For moderately-differentiated gastric cancer cells, the cell cycle was arrested at the G₀/G₁ and G₂/M phases. For lowly-differentiated gastric cancer cells, the cell cycle was arrested at the G₀/G₁ and S phases.     

Effects of allitridi on cell cycle arrest of human gastric cancer cells

AIM: To determine the effect of allitridi on cell cycle of human gastric cancer (HGC) cell lines MGC803 and SGC7901 and its possible mechanism. METHODS: Trypan blue dye exclusion was used to evaluate the proliferation, inhibition of cells and damages of these cells were detected with electron microscope. Flow cytometry and cell mitotic index were used to analyze the change of cell cycle, immunohistochemistry, and RTPCR was used to examine expression of the p21~(WAF1) gene. RESULTS: MGC803 cell growth was inhibited by allitridi with 24 h IC_(50) being 6.4 μg/mL. SGC7901 cell growth was also inhibited by allitridi with 24 h IC_(50) being 7.3 μg/mL After being treated with allitridi at the concentration of 12 μg/mL for 24 h, cells were found to have direct cytotoxic effects, including broken cellular membrane, swollen and vesiculated mitochondria and rough endoplasmic reticula, and mass lipid droplet. When cells were treated with allitridi at the concentration of 3, 6, and 9 μg/mL for 24 h, the percentage of G_0/G_1 phase cells was decreased and that of G_2/M phase cells was significantly increased (P=0.002) compared with those in the group. When cells were treated with allitridi at the concentration of 6 μg/mL, cell mitotic index was much higher (P=0.003) than that of control group, indicating that allitridi could cause gastric cancer cell arrest in M phase. Besides, the expression levels of p21~(WAF1)gene of MGC803 cells and p21~(WAF1) gene of SGC7901 cells were remarkably upregulated after treatment. CONCLUSION: Allitridi can cause gastric cancer cell arrest in M phase, and this may be one of the mechanisms for inhibiting cell proliferation. Effect of allitridi on cells in M phase may be associated with the upregulation of p21~(WAF1) genes. This study provides experimental data for clinical use of allitridi in the treatment of gastric carcinoma.     

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721)to human umbilical vein endothelial cells (ECV-304),expression of adhesive molecule integrinβ1 in SMMC-7721cells and its contribution to this adhesive course.METHODS: Adhesive force of SMMC-7721 cells to endothelialcells was measured using micropipette aspiration technique.Synchronous G1 and S phase SMMC-7721 cells wereachieved by thymine-2-deoxyriboside and colchicinessequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronousrates of SMMC-7721 cells and expression of integrinβ1 inSMMC-7721 cells were detected by flow cytometer.RESULTS: The percentage of cell cycle phases of generalSMMC-7721 cells was 11.01% in G2/M phases, 53.51% inG0/G1 phase, and 35.48% in S phase. The synchronous ratesof G1 and S phase SMMC-7721 cells amounted to 74.09%and 98.29%, respectively. The adhesive force of SMMC-7721cells to endothelial cells changed with the variations ofadhesive time and presented behavior characteristics ofadhesion and de-adhesion. S phase SMMC-7721 cells had higheradhesive forces than G1 phase cells [(307.65±92.10)× 10-10Nvs (195.42±60.72)×10-10N, P＜0.01]. The expressivefluorescent intensity of integrinβ1 in G1 phase SMMC-7721cells was depressed more significantly than the values ofS phase and general SMMC-7721cells. The contribution ofadhesive integrinβ1 was about 53% in this adhesive course.CONCLUSION: SMMC-7721 cells can be synchronizedpreferably in G1 and S phases with thymine-2-deoxyribosideand colchicines. The adhesive molecule integrinβ1 expressesa high level in SMMC-7721 cells and shows differences invarious cell cycles, suggesting integrin β1 plays an importantrole in adhesion to endothelial cells. The change of adhesiveforces in different cell cycle SMMC-7721 cells indicatesthat S phase cells play predominant roles possibly whilethey interact with endothelial cells.     

Identification of fibroblast growth factor-8b target genes associated with early and late cell cycle events in breast cancer cells

Fibroblast growth factor-8 (FGF-8) is implicated in the development and progression of breast cancer and its levels are frequently elevated in breast tumors. The mechanisms driving FGF-8-mediated tumorigenesis are not well understood. Herein we aimed to identify target genes associated with FGF-8b-mediated breast cancer cell proliferation by carrying out a cDNA microarray analysis of genes expressed in estrogen receptor negative S115 breast cancer cells treated with FGF-8b for various time periods in comparison with those expressed in non-treated cells. Gene and protein expression was validated for selected genes by qPCR and western blotting respectively. Furthermore, using TRANSBIG data, the expression of human orthologs of FGF-8-regulated genes was correlated to the Nottingham prognostic index and estrogen receptor status. The analysis revealed a number of significantly up- and down-regulated genes in response to FGF-8b at all treatment times. The most differentially expressed genes were genes related to cell cycle regulation, mitosis, cancer, and cell death. Several key regulators of early cell cycle progression such as Btg2 and cyclin D1, as well as regulators of mitosis, including cyclin B, Plk1, survivin, and aurora kinase A, were identified as novel targets for FGF-8b, some of which were additionally shown to correlate with prognosis and ER status in human breast cancer. The results suggest that in stimulation of proliferation FGF-8b not only promotes cell cycle progression through the G1 restriction point but also regulates key proteins involved in chromosomal segregation during mitosis and cytokinesis of breast cancer cells.     

二甲双胍抑制人胰腺癌细胞株Patu8988的增殖

二甲双胍孵育胰腺癌细胞株Patu8988 72 h后应用CCK-8(Cell counting Kit-8)检测细胞增殖,流式细胞仪分析细胞周期,RT-PCR检测相关基因表达.干预后细胞增殖呈浓度依赖性抑制(r=0.994.,P＜0.05),细胞周期G0/G1期所占比例显著增加,G2/M期显著减少(均P＜0.05),相关基因MMP-3、CyclinD1、p53表达下调,Bax表达上调.结果表明二甲双胍抑制Patu8988细胞增殖,主要机制可能与阻滞细胞周期、影响相关基因表达有关.     

曲格列酮抑制宫颈癌SiHa细胞增殖中的作用

目的观察曲格列酮对宫颈癌SiHa细胞增殖的影响,并探讨S期激酶相关蛋白2（s-phase kinase associated protein 2,skp2）、p27在此过程中的作用。方法曲格列酮（0、100、200、400μg/ml）处理人宫颈癌SiHa细胞,采用四甲基偶氮唑蓝（MTT）法测定细胞增殖活性,膜联蛋白-V（Annexin V）-FITC法检测细胞凋亡;流式细胞术检测细胞周期;倒置显微镜观察SiHa细胞形态学变化;构建skp2表达质粒并转染SiHa细胞,Western blot检测蛋白的表达。结果曲格列酮明显抑制宫颈癌SiHa细胞增殖,且呈明显时间及浓度依赖性（P〈0.05）;曲格列酮增加SiHa细胞周期G1/S期阻滞,细胞凋亡率未见显著性变化（P〉0.05）;曲格列酮上调SiHa细胞p27表达,下调skp2表达;过表达skp2可明显抑制曲格列酮对p27表达的升高作用,同时抵消曲格列酮对细胞周期抑制的作用。结论曲格列酮通过调节细胞周期而非凋亡途径,抑制宫颈癌SiHa细胞增殖,其机制可能与调节skp2、p27蛋白表达有关。     

应用基因芯片技术筛选胰腺癌相关基因

目的应用基因芯片技术筛选胰腺癌相关基因。方法将14000种人类基因PCR产物按微矩阵排列点样于化学涂层的载玻片上，制成基因芯片。按一步法抽提4例胰腺癌和癌旁正常胰腺组织的总RNA，将等量的RNA分别逆转录合成荧光分子掺人的cDNA一链作探针，混合后杂交上述基因芯片。经严格洗片后用ScanArray 4000扫描仪扫描芯片荧光信号图像，每点上两种荧光信号的强度分别代表Cy5-dCTP和Cy3-dCTP的量，获得的荧光信号图像用计算机分析。结果按差异显著性标准，从14000个基因中筛选出在胰腺癌组织中共同差异表达基因189条，其中已知基因101条，新基因88条。在筛选出的已知基因中，有50条表达上调，51条表达下调。结论基因芯片技术的肿瘤基因表达谱分析能够高通量筛选胰腺癌相关基因。并高效对基因功能进行研究。胰腺癌基因表达谱的分析有助于认识肿瘤发病机制。     

曲古菌素A对人胃癌细胞增殖和细胞周期的作用

目的 观察曲古菌素A(TSA)对人胃癌细胞株SGC-7901细胞增殖及细胞周期的影响,探讨其可能的机制.方法 TSA干预人胃癌SGC-7901细胞24 h后.采用四甲基偶氮唑盐法检测其对细胞增殖的影响,流式细胞技术检测细胞周期,实时PCR检测细胞周期素D1和p21 mRNA的表达情况.结果 经TSA干预24 h后,人胃癌SGC-7901细胞增殖受抑制,TSA 0.1、0.5和2.0μmol/L组抑制率分别为3.52%±6.11%、13.29%±4.13%和14.24%±2.80%;同时TSA 0.5μmol/L组(71.26%±0.51%)和TSA 2.0μmol/L组(71.03%±0.12%)的G0/Gl期细胞比例明显高于对照组(51.12%±1.17%);TSA 0.5μmol/L组(13.55%±0.44%)和TSA 2.0 μmol/L组(10.63%±0.63%)的S期细胞比例明显低于对照组(34.60%±0.60%).出现G0/G1细胞周期阻滞.TSA干预后细胞周期相关基因细胞周期素D1 mRNA表达下调和p21 mRNA表达上调.结论 TSA通过调控细胞周期相关基因细胞周期素D1和p21的表达,抑制人胃癌SGC-7901细胞的增殖,引起G0/G1期细胞周期阻滞,最终影响肿瘤细胞的生长.     

基因芯片技术在食管鳞癌转移相关基因表达谱筛选中的应用

目的 研究人食管鳞癌转移相关基因表达谱,探讨食管鳞癌的转移机制。方法 选取392个与肿瘤转移相关的基因克隆,制备成肿瘤转移基因芯片。提取食管鳞癌组织以及正常食管组织RNA,反转录后标记为cDNA探针,与cDNA芯片杂交,经扫描及Quantarray 3．0软件分析后比较两种组织中的差异表达基因。结果 共筛查出差异表达基因58条,其中表达上调基因36条、下调基因22条,包括癌基因、抑癌基因、黏附分子、基质金属蛋白酶、信号转导因子、细胞代谢和免疫相关基因等。结论 基因芯片筛查食管鳞癌转移相关基因表达谱可为明确食管鳞癌转移机制提供重要参考。     

肝癌细胞系放射诱导的细胞凋亡和细胞周期的变化

目的：探讨肝癌细胞系放射诱导的细胞周期和细胞凋亡的变化特点．方法：研究细胞系为肝癌细胞系HepG2和SMMC-7721．对照细胞系为正常肝细胞系HL-7702、肺小细胞癌HCI-H460和肺腺癌A549．常规培养48h后接受4Gy射线照射,收获受照前(0h)和受照后6,12,24,36和48h的细胞,采用流式细胞术(FCM)检测各细胞系细胞周期和细胞凋亡．结果：4GyX线照射后,HepG2在照射后12h出现细胞凋亡高峰,射线诱导的细胞凋亡比率为45．16%(t=8．864,P＜0．0025),而SMMC-7721在24h达高峰,诱导的细胞凋亡比率为24．94％,HepG2较SMMC-7721射线诱导的细胞凋亡高峰出现早、比率高：HepG2和SMMC-7721与HCI-H460和A549变化较一致,凋亡变化的走势和峰值均与S期的相反,两株肝癌细胞可能均发生了射线诱导的有丝分裂前S期细胞凋亡．HepG2在照射后12h有明显的G_2/M期阻滞,可能有射线诱导的G_2/M期细胞损伤,发生了延迟的间期死亡．结论：两株肝癌细胞可能均发生了射线诱导的有丝分裂前S期细胞凋亡,HepG2可能伴有射线诱导的G_2/M期细胞损伤,发生了延迟的间期死亡．     

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than G1 phase cells [(307.65±92.10)×10^-10N vs (195.42±60.72)×10^-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in G1 and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.     

Distinct effects of different concentrations of sodium selenite on apoptosis, cell cycle, and gene expression profile in acute promyeloytic leukemia-derived NB4 cells

Selenium at a low concentration has a chemopreventive role against cancer, while at a high concentration, it exerts a direct antitumor effect. However, the mechanisms remain elusive. In this article, we discovered that Na₂SeO₃ at 20 μmol/l concentration could significantly inhibit the proliferation of NB4 cells, affect the cell cycle distribution of cell population, and induce cellular changes characteristic of apoptotic cells, while this same compound at 2 μmol/l concentration had no such effects. The mechanisms underlying these overt differences caused by treatment of different concentrations of selenium were further investigated. cDNA microarray analysis showed that after treatment by 20 μmol/l Na₂SeO₃, 34 genes were changed in expression, while treatment by 2 μmol/l Na₂SeO₃ resulted in the changes of 29 genes. Nine genes were regulated in both groups, among which three showed opposite changes caused by 2 and 20 μmol/l Na₂SeO₃. The majority of regulated genes did not coincide between the two experiment groups. In conclusion, 2 and 20 μmol/l Na₂SeO₃ could have different effects on NB4 cells, and some genes might be involved in the underlying mechanisms. Our findings could provide basis for further uncovering the molecular mechanisms of the chemopreventive and antitumor effects of selenium and, in turn, for probing the rationality of treating leukemia with selenium.     

基因芯片技术筛选卵巢癌紫杉醇耐药差异表达基因的研究

目的应用基因芯片技术研究人卵巢癌紫杉醇耐药细胞株OC3/Tax300与其敏感细胞系OC3之间的基因表达谱差异,筛选耐药相关基因.方法分别提取OC3/Tax300与OC3细胞的总RNA并纯化mRNA;将等量的mRNA逆转录,以Cy5和Cy3标记的cDNA做探针,在BiostarH140S基因表达谱芯片上进行杂交.扫描芯片荧光信号图像,用基因图像分析软件对扫描图像进行数字化处理和分析.结果共筛选出显著表达差异基因234种,其中217种基因表达水平下调,17种基因表达上调;下调基因主要为EB病毒编码核蛋白（EBNA-3）、信号蛋白（COP9）,上调的基因主要是酪氨酸激酶（JAK2）、热休克蛋白（HSPs）、还原型辅酶烟胺腺嘌呤二核苷酸（NADH）等.结论本研究筛选出的基因可能为进一步探讨卵巢癌肿瘤细胞耐药机制提供新的途径.