CisoDGR 多肽对乳腺癌 MCF －7细胞增殖、凋亡的影响及其机制探讨

目的：探讨 CisoDGR 多肽对乳腺癌 MFC －7细胞增殖和凋亡的影响及可能机制。方法：采用 MTT 法检测 CisoDGR 多肽对乳腺癌 MCF －7细胞的抑制率;流式细胞术检测 CisoDGR 多肽对 MCF －7细胞凋亡的影响;Western blot 法检测 Caspase －3、Bcl －2蛋白的表达。结果：CisoDGR 多肽能明显抑制乳腺癌 MCF －7细胞的增殖,诱导细胞凋亡,并存在一定的时－效及量－效关系。其对凋亡的诱导作用可能与 Caspase －3蛋白表达增加及 Bcl －2蛋白表达下降有关。结论：CisoDGR 多肽具有抑制 MCF －7细胞增殖,诱导 MCF －7细胞凋亡的作用,其作用机制可能与 Caspase －3、Bcl －2蛋白的表达变化有关。     

应用DNA微阵列研究斑蝥酸钠诱导K562细胞凋亡前后的基因变化

目的: 研究斑蝥酸钠体外诱导人红白血病K562细胞凋亡的作用,应用DNA微阵列技术探讨K562细胞凋亡过程中基因表达的变化.方法: 用不同浓度的斑蝥酸钠处理K562细胞,运用MTT染色观察、DNA凝胶电泳检测细胞凋亡,并提取斑蝥酸钠作用K562细胞前后的总RNA,逆转录成eDNA,分别用cy3/Cy5标记对照组和处理组,与含162个cDNA片段的K562表达谱芯片杂交.结果: 斑蝥酸钠强烈抑制K562细胞生长,2.5μg/ml的斑蝥酸钠即可诱导细胞凋亡.K562细胞经斑蝥酸钠作用后,大部分基因片段的表达都有所降低,其中37个基因片段的表达降低较为显著(CyS/Cy3<0.5).这些表达下调的基因片段包括转导因子、tRNA合成酶、代谢酶、信号通路蛋白、激酶等.结论: 斑蝥酸钠能有效诱导K562细胞凋亡,我们制备的K562细胞表达谱芯片可以成功地用于基因表达的研究.     

基因促进TRAIL诱导乳腺癌MCF 7细胞的凋亡

目的:研究抑制促分裂原活化蛋白激酶/细胞外信号调节激酶激酶3(mitogenactivated protein/extracellular signal regulated kinasekinase 3,MEKK 3)基因表达促进TRAIL诱导乳腺癌MCF7细胞凋亡的作用，寻找乳腺癌临床治疗新策略。方法：应用MTT法检测人TRAIL对MCF7细胞生长的抑制作用。合成MEKK3siRNA，应用脂质体介导MEKK3siRNA转染入人乳腺癌细胞MCF7，以RTPCR和Western blotting法检测MCF7细胞MEKK 3 mRNA和蛋白的表达。应用MTT法和流式细胞术检测MEKK3siRNA与TRAIL联合处理后MCF7细胞的增殖和凋亡。结果：TRAIL具有抑制MCF7细胞增殖作用，但其抑制作用较弱。MEKK3siRNA转染后能有效而稳定地抑制MCF7细胞中 MEKK3 mRNA和蛋白的表达(P<0.01)。TRAIL与MEKK3siRNA联合处理MCF7细胞较TRAIL单独处理更明显地抑制细胞增殖活力(P<0.05)，更明显地增加细胞凋亡率(P<001)。结论：siRNA沉默 MEKK3基因能显著促进TRAIL对乳腺癌MCF7细胞凋亡的诱导作用，为探讨乳腺癌治疗新方案提供了实验依据。     

188Re诱导乳腺癌ER-75-30细胞凋亡及其与bcl-2、bax基因表达的研究

目的 研究放射性核素^188铼（^188Re）诱导乳腺癌ER－75－30细胞凋亡及其与bcl-2和bax基因表达的关系。方法 应用光镜、电`镜流式细胞仪和免疫组化图像分析技术，检测不同浓度^188Re作用于体外培养的乳腺癌ER－75－30细胞后，不同时间点诱导细胞凋亡情况及其与bcl-2和bax基因表达情况。结果^188Re能诱导乳腺癌ER－75－30细胞发生凋亡形态学变化，并且随着^188Re浓度增大和作用时间延长，凋亡率增加，bcl-2表达减弱，bax表达增强。结论 ^188Re能诱导乳腺癌ER－75－30细胞凋亡且具有浓度、时间和周期依赖性，bcl-2和bax基因均参与了^188Re诱导的细胞凋亡过程。     

CD/5-FC系统对小鼠胃癌细胞杀伤作用的研究

目的：探讨CD5／5－FC自杀基因系统对小鼠胃癌细胞的体外杀伤作用及其机制。方法：以逆转录病毒载体介导CD基因转导小鼠胃癌细胞株MFC,采用MTT法测定CD／5－FC系统的体外杀伤作用及其“旁观者”效应;利用Hoechst 33258荧光染色法、琼脂糖凝胶电泳法检测凋亡细胞,同时采用半定量RT－PCR技术检测凋亡相关基因的表达。结果：转导CD基因可使MFC对5－FC高度敏感,并且显示出强大的“旁观者”效应。经5－FC作用后,荧光染色可见到典型的凋亡细胞核形态学改变,琼脂糖凝胶电泳出现典型的DNA梯度,同时发现凋亡相关基因bcl-2基因表达下调,而bax与caspase-3基因表达上调。结论：CD／5－FC自杀基因系统对小鼠胃癌细胞MFC具有强大的杀伤作用,体外杀伤作用是通过诱导MFC的凋亡来完成。     

Notch信号通路相关基因与乳腺癌发生发展的研究新进展

Notch信号通路是一条进化上十分保守的信号转导途径,广泛存在于生物进化过程中,相邻细胞间通过Notch受体与配体的相互作用转导细胞信号,调节细胞的增殖、分化和凋亡,影响器官形成和形态的发生。近年大量研究表明Notch信号分子的异常表达在乳腺癌发生过程中起着重要的调控作用,但该基因表达异常的内在机制还不清楚,本文就Notch基因表达及其表达机制与乳腺癌的发生关系进行综述。     

Notch信号通路相关基因与乳腺癌发生发展的研究新进展

Notch信号通路是一条进化上十分保守的信号转导途径,广泛存在于生物进化过程中,相邻细胞间通过Notch受体与配体的相互作用转导细胞信号,调节细胞的增殖、分化和凋亡,影响器官形成和形态的发生。近年大量研究表明Notch信号分子的异常表达在乳腺癌发生过程中起着重要的调控作用,但该基因表达异常的内在机制还不清楚,本文就Notch基因表达及其表达机制与乳腺癌的发生关系进行综述。     

神经酰胺诱导人乳腺癌细胞凋亡的研究

目的 :观察神经酰胺对人乳腺癌细胞株MCF 7及耐多柔比星细胞株MCF 7/ADR的诱导凋亡作用及其对bcl 2基因表达的影响 ,探讨神经酰胺诱导凋亡的机制。方法 :采用MTT法检测细胞生存率 ,用流式细胞术检测凋亡率 ,运用流式细胞术、RT PCR方法检测bcl 2基因表达。结果 :神经酰胺对MCF 7、MCF 7/ADR细胞有明显的抑制生长作用。 2 5 μmol/LC6 神经酰胺作用48h后 ,MCF 7及MCF 7/ADR细胞凋亡率分别为( 2 0 70± 2 66) %、( 12 3 9± 1 76) % ,前者凋亡率高于后者 ,P <0 0 1。流式细胞术检测 ,MCF 7、MCF 7/ADR细胞bcl 2蛋白阳性率分别为 ( 2 7 5 9± 2 94) %、( 79 5 6±3 2 0 ) %。MCF 7/ADR细胞经神经酰胺作用后 ,bcl 2蛋白及mRNA水平均降低。结论 :神经酰胺对MCF 7、MCF 7/ADR细胞有抑制生长及诱导凋亡作用 ,神经酰胺诱导凋亡可能是通过下调bcl 2基因表达而实现的     

腺病毒介导IL-24基因诱导人脑胶质瘤U87MG细胞的凋亡

目的：研究腺病毒介导IL-24基因表达载体(Ad-IL-24)对人脑胶质瘤U87MG细胞的抑制作用,初步探讨其作用机制。方法：将本科室构建的Ad-IL-24感染U87MG细胞,RT-PCR法检测IL-24基因的表达,MTT法和流式细胞术检测U87MG细胞的生长和凋亡,激光共聚焦显微镜观察U87MG细胞凋亡的形态学变化,RT-PCR法检测Bax、Bcl-2基因mRNA的表达,Western blotting检测caspase-3的活化。结果：Ad-IL-24感染U87MG细胞后,IL-24在U87MG细胞中有明显表达,并抑制U87MG细胞生长、诱导其凋亡、出现典型的凋亡细胞核形态学改变。Ad-IL-24可上调U87MG细胞中Bax基因、下调Bcl-2基因的表达,并诱导caspase-3蛋白的活化。结论：Ad-IL-24可诱导U87MG细胞凋亡,其机制可能与上调Bax基因、下调Bcl-2基因表达,并活化caspase-3有关。     

Grb2-SH3抑制剂peptidimer-c对K562细胞凋亡相关基因表达的影响

 目的　分析peptidimer-c对K562细胞基因表达谱的影响,初步探讨peptidimer-c诱导K562细胞凋亡和抑制生长的可能机制。方法　应用锥虫蓝染色法计数经不同浓度peptidimer-c分别作用不同时间后的K562活细胞数;通过透射电子显微镜观察peptidimer-c作用前后K562细胞的超微结构;应用Human U133 Plus 3.0基因表达谱芯片检测peptidimer-c作用前后K562细胞的差异表达基因;并用反转录聚合酶链反应（RT-PCR）验证芯片结果。结果　peptidimer-c能诱导K562细胞凋亡和抑制生长,peptidimer-c作用于K562细胞后引起大量基因表达的变化,差异表达基因有529个,其中上调455个,下调74个,影响细胞凋亡的基因包括JUN、AXUD1、TNFRSF10B等表达明显上调;RT-PCR验证选取的15个基因的表达差异与芯片结果一致。结论　peptidimer-c可通过上调肿瘤坏死因子及其受体家族成员和JUN家族,启动K562细胞凋亡。     

p27kip1高表达乳腺癌细胞株MCF-7基因表达谱的差异分析

目的:利用基因芯片技术筛选p27kip1转染乳腺癌MCF-7细胞的差异表达基因,探讨p27kip1调控肿瘤细胞增殖和细胞周期的分子机制。方法:将含p27cDNA的质粒在脂质体介导下体外转染乳腺癌MCF-7细胞,利用Affy-metrix公司的人类基因组基因芯片U133A分别检测表达p27kip1蛋白和空载体的乳腺癌细胞株的基因表达谱,用生物信息学软件进行比较分析,对差异表达基因进行功能分类。结果:在含22277个基因cDNA的芯片上差异表达的基因有1379条,其中表达谱发生明显变化(2倍以上)的基因173条,占总检测基因的0·78%。157条基因表达明显上升(SLR>1),占总检测基因的0·705%;16条基因表达明显下降(SLR<-1),占总检测基因的0·072%。基因表达差异主要集中在物质代谢、细胞增殖、细胞周期调控、细胞分化与凋亡、免疫反应、信号传导、蛋白转运、调节转录等方面。结论:多基因共同参与了p27kip1抑制肿瘤细胞过度增殖以及促进细胞凋亡的作用。对于p27kip1相关基因群的研究有助于认识p27kip1蛋白在细胞周期调控乃至整个细胞癌变过程中作用的分子机制。     

基因芯片技术筛查并鉴定乳腺癌细胞中MAGE-A11的相关基因

目的：应用高通量基因芯片技术筛查乳腺癌细胞中黑色素瘤相关抗原（melanoma antigen,MAGE）-A11 的相关基因,并从数量和功能两方面加以验证。方法：采用基因芯片技术筛选乳腺癌MCF-7、MDA-MB-231 和BT-549 中MAGE-A11 下游靶基因的mRNA的差异表达,对有代表性的基因进行了聚类分析,并利用qRT-PCR进行验证。以CCK-8 法、细胞划痕实验和Transwell实验检测MAGE-A11 对乳腺癌细胞中增殖、迁移和侵袭功能的影响。结果：3 种乳腺癌细胞过表达MAGE-A11 导致1 608个下游基因差异表达,主要涉及蛋白泛素化、细胞增殖和凋亡、肿瘤侵袭和转移。基因芯片中典型高表达的ZNF-451、CENPTJ、CDK13、API5 和LMO7 在qRT-PCR 在验证结果中也显著高于对照组（P<0.01）,低表达的SHPRH、PML、MARK2、LIMA1 和ANGPTL4也显著低于对照组（P<0.01）。转染MAGE-A11 组的乳腺癌细胞MCF-7、MDA-MB-231 和BT-549 72 h 的增殖能力较对照组明显增强（均P<0.01）,培养48 h 后与对照组相比,转染MAGE-A11 的3 种细胞划痕出现明显愈合（P<0.05 或P<0.01）,穿膜数较对照组明显增多（均P<0.01）。结论：在MCF-7、MDA-MB-231 和BT-549 三种乳腺癌细胞中筛查到涉及蛋白泛素化、细胞增殖和凋亡、肿瘤侵袭和迁移等生物功能众多的表达差异基因,对其中10 种典型差异基因从数量和功能两方面进行验证,并得到初步确认。     

长链非编码RNA STMN1P2在乳腺癌中的作用及机制研究

背景与目的:长链非编码RNA(long non-coding RNA,lncRNA)在肿瘤进展中发挥重要调控作用,我们的前期研究通过lncRNA表达谱芯片筛选发现,微管不稳定蛋白1假基因2(stathmin 1 pseudogene 2,STMN1P2)在乳腺癌中高表达,但其生物学功能以及在乳腺癌中的作用尚未见报道.探...     

Expression patterns of fatty acid binding proteins in breast cancer cells

We studied the expression levels of fatty acid binding proteins in breast normal and cancer cell lines. Liver fatty acid binding protein (L-FABP) and intestine fatty acid binding proteins were shown to be up-regulated in breast cancer cell lines while adipose- and epidermal-fatty acid binding proteins were down regulated in breast cancer cells compared to normal breast cell lines. We have previously shown that blocking the expression of L-FABP resulted in remarkable effects on apoptosis and cell proliferation of prostate cancer cell lines (Hammamieh et al 2004). To study the mechanism of effect of the liver fatty acid binding protein in breast cancer cells, we designed an antisense oligodeoxynucleotide to block the production of liver-FABP in MCF-7 cells. The antisense was shown to inhibit the expression of L-FABP and to induce apoptosis in MCF-7 cells. This study examines the mechanism by which L-FABP antisense regulates proliferation and apoptosis in breast cancer cell lines. We used human cDNA microarrays to explore differentially expressed genes in MCF-7 breast cancer cells treated with L-FABP antisense oligonucleotide. Some of the genes that were differentially expressed were confirmed using quantitative RT-PCR. Genes related to cell growth, proliferation and angiogenesis showed significant variations. This suggests a possible use of these antisense ODNs as therapeutic agents for breast cancer in future.     

Inhibitory effect of conjugated dienoic derivatives of linoleic acid and beta-carotene on the in vitro growth of human cancer cells.

The effects of physiologic concentrations of conjugated linoleic acid (CLA) and beta-carotene were assessed on human (M21-HPB, malignant melanoma; HT-29, colorectal; MCF-7, breast) cancer cells. The incubation of cancer cells with CLA showed significant reductions in proliferation (18-100%) compared to control cultures. M21-HPB and MCF-7 cell mortality was dose- and time-dependent. beta-Carotene was inhibitory to breast cells only. MCF-7 cells supplemented with CLA incorporated significantly less [3H]leucine (45%), [3H]uridine (63%) and [3H]thymidine (46%) than control cultures. M21-HPB and HT-29 cells supplemented with CLA incorporated less [3H]leucine (25-30%). These in vitro results suggest that CLA and beta-carotene may be cytotoxic to human cancer cells in vivo.     

Transient over-expression of estrogen receptor-�� in breast cancer cells promotes cell survival and estrogen-independent growth

Estrogen receptor-?? (ER??) positive breast cancer frequently responds to inhibitors of ER?? activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ER?? in ER??-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ER?? over-expression in ER??-positive breast cancer cells, we over-expressed ER?? in the MCF-7 breast cancer cell line using adenovirus gene transduction. ER?? over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ER?? transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ER??-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ER?? remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ER?? could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.     

Molecular cloning and expression of proto-oncogene FRAT1 in human cancer

FRAT1 and FRAT2 genes, clustered in human chromosome 10q24, are human homologues to mouse proto-oncogene Frat1, which promotes carcinogenesis through activation of the WNT - beta-catenin - TCF signaling pathway. FRAT1 and FRAT2 mRNAs are up-regulated together in a gastric cancer cell line TMK1, and also in 2 out of 10 cases of primary gastric cancer. Here, we isolated FRAT1 cDNA (AB074890), which showed two amino-acid substitutions (Gln57X and His58Asp) compared with human FRAT1 cDNA previously reported by another group (U58975). The Gln57-His58 FRAT1 allele isolated in this study was also identified in human genome draft sequences. FRAT1 mRNA was almost ubiquitously expressed in human pancreatic cancer cell lines. Expression level of FRAT1 mRNA was relatively higher in esophageal cancer cell lines TE2, TE3, TE4, a cervical cancer cell line SKG-IIIa, and breast cancer cell lines MCF-7 and T-47D. Expression level of FRAT1 mRNA was not significantly changed after all-trans retinoic-acid treatment in NT2 cells with the potential of neuronal differentiation. Expression of FRAT1 mRNA in MCF-7 cells derived from breast cancer was down-regulated by beta-estradiol. This is the first report on isolation of FRAT1 cDNA derived from the more common FRAT1 allele, and also on regulation of FRAT1 mRNA in human cancer cells.     

Gene expression profiling revealed survivin as a target of 3,3'-diindolylmethane-induced cell growth inhibition and apoptosis in breast cancer cells

The phytochemical indole-3-carbinol (I3C), found in cruciferous vegetables, and its major acid-catalyzed reaction product 3,3'-diindolylmethane (DIM) showed anticancer activity mediated by its pleiotropic effects on cell cycle progression, apoptosis, carcinogen bioactivation, and DNA repair. To further elucidate the molecular mechanism(s) by which 3,3'-diindolylmethane exerts its effects on breast cancer cells, we have used microarray gene expression profiling analysis. We found a total of 1,238 genes altered in 3,3'-diindolylmethane-treated cells, among which 550 genes were down-regulated and 688 genes were up-regulated. Clustering analysis showed significant alterations in some genes that are critically involved in the regulation of cell growth, cell cycle, apoptosis, and signal transduction, including down-regulation of survivin. Previous studies have shown that antiapoptotic protein survivin is overexpressed in many human cancers, including breast cancer. However, very little or no information is available regarding the consequence of down-regulation of survivin for cancer therapy. We, therefore, hypothesized that down-regulation of survivin as observed by 3,3'-diindolylmethane could be an important approach for the treatment of breast cancer. We have tested our hypothesis using multiple molecular approaches and found that 3,3'-diindolylmethane inhibited cell growth and induced apoptosis in MDA-MB-231 breast cancer cells by down-regulating survivin, Bcl-2, and cdc25A expression and also caused up-regulation of p21(WAF1) expression, which could be responsible for cell cycle arrest. Down-regulation of survivin by small interfering RNA before 3,3'-diindolylmethane treatment resulted in enhanced cell growth inhibition and apoptosis, whereas overexpression of survivin by cDNA transfection abrogated 3,3'-diindolylmethane-induced cell growth inhibition and apoptosis. These results suggest that targeting survivin by 3,3'-diindolylmethane could be a new and novel approach for the prevention and/or treatment of breast cancer.     

cDNA microarray analysis of endothelial cells in response to green tea reveals a suppressive phenotype

Green tea may prevent cancer, partially by inhibiting tumor angiogenesis. Our previous studies showed that green tea extract was effective in inhibiting breast cancer and endothelial cell proliferation in vitro, and suppressed xenograft size and decreased the tumor vessel density in vivo. Here, we set out to further investigate the molecular mechanisms of this observed angiogenesis suppression. We utilized cDNA microarray technology to profile the global changes in endothelial cellular gene expression in response to green tea. HUVEC (human umbilical vein endothelial cells) were exposed in vitro to green tea for either 6 or 48 h. Only statistically significantly differentially expressed genes were analyzed. Gene profiling demonstrated a global down-regulation of multiple genes involved in endothelial cell growth, signal transduction and oxidation, accompanied by up-regulation of several apoptotic genes. We validated these observations by showing positive correlations with biological assays of cellular proliferation, cell cycle, and apoptosis. The anti-oxidant characteristics of green tea and its metabolites were confirmed in the ORAC (oxygen radical absorbance capacity) assay. cDNA microarray revealed that green tea has an overall suppressive effect on multiple pathways in endothelial cells. This study contributes to the comprehensive analysis of the molecular effects of green tea on endothelial cells, and provides insight into genes that may be important in chemoprevention.