蛋白酪氨酸激酶抑制剂Genistein抑制肺癌细胞A549体外侵袭作用的研究

观察蛋白酪氨酸激酶抑制剂Genistein对人肺腺癌细胞株A549细胞侵袭能力的影响,探讨Genistein抑制肺癌细胞侵袭的可能机制。以不同浓度Genistein（20μmol/L和40μmol/L）作用于A549细胞3 d后,分别用基质胶侵袭模型、黏附基质分析、Transwell小室趋化运动模型、细胞骨架蛋白染色及RT-PCR法来研究药物处理后细胞侵袭、黏附、运动、聚合型骨架蛋白（F-actin）以及基质金属蛋白酶基因表达的改变。经Genistein处理后,A549细胞的F-actin聚合减少,侵袭能力明显下降,趋化运动能力降低,基质金属蛋白酶抑制剂（TIMP-1）基因相对表达量增加,但黏附率没有降低。Genistein可降低肺癌细胞的迁移、侵袭能力。F-actin聚合减少,TIMP-1的相对表达量增加,可能是Genistein抑制肺癌细胞侵袭的机制之一。     

2,3-吲哚醌对MCF-7和HT-29细胞株的增殖抑制和凋亡诱导

目的:研究2,3-吲哚醌(2,3-dioxoindoline,ISA)对人乳腺癌细胞株MCF-7和人大肠癌细胞株HT-29的增殖与凋亡影响.方法:应用MTT比色法检测ISA对MCF-7和HT-29的生长抑制情况;Hochest33258荧光染色法检测细胞凋亡情况,Westem Blotting 法检测凋亡相关基因bcl-2和bax在蛋白水平上的表达;RT-PCR两步法检测bcl-2和bax在mRNA水平上的变化.结果:①ISA在一定浓度范围内能明显抑制细胞生长;②荧光染色可观察到细胞核染色质浓集,核浓缩核碎裂,并出现典型的凋亡小体;③bcl-2 mRNA及蛋白的表达随药物浓度增大而逐渐减少,各实验处理组与阴性对照组比较存在统计学差异(P＜0.05或P＜0.01).bax mRNA及蛋白的表达随药物浓度增大而无明显变化,各组间比较不存在统计学差异(P＞0.05).结论:ISA能够抑制MCF-7和HT-29细胞增殖,并能进一步诱导其凋亡,其作用机制可能是与下调bcl-2表达有关.     

5-Aza-CdR对人胃癌细胞株生物学行为及RASSFlA mRNA表达的影响

观察不同浓度的5-氮-2′-脱氧胞苷(5-Aza-CdR)对人胃癌细胞株BGC-823、SGC-7901、MKN-28生长及RASSF1A mRNA表达的影响。方法:分别以0．4μmol/L、1．6μmol/L、6．4μmol/L、25．6μmol/L、102．4μmol/L浓度的5-Aza-CdR处理人胃癌细胞株BGC-823、SGC-7901、MKN-28,MTT比色法测定72h时间段的吸光度值、计算抑制率,流式细胞仪检测5-Aza-CdR对胃癌细胞株生长周期及凋亡的影响,RT-PCR检测5-Aza-CdR处理前、后抑癌基因RASSF1A mRNA的表达。结果:5-Aza-CdR抑制体外培养人胃癌细胞株BGC-823、SGC-7901、MKN-28生长,呈浓度依赖性;5-Aza-CdR能有效诱导BGC-823、SGC-7901、MKN-28细胞凋亡;RT-PCR检测人胃癌细胞株SGC-7901、MKN-28无RASSF1A mRNA表达,经5-Aza-CdR处理后基因重新表达, BGC-823处理前后RASSF1A mRNA均有表达。结论:新型抑癌基因RASSF1A与胃癌的发生相关,5-Aza-CdR能抑制胃癌细胞株的增殖,并促进凋亡,其机制可能与RASSF1A基因的重新表达有关。     

脂多糖对人支气管上皮细胞STAT1、STAT3、STAT4、STAT6表达的影响

目的观察脂多糖对人支气管上皮细胞16HBESTAT1、STAT3、STAT4、STAT6表达的影响。方法采用普通RT—PCR检测16HBE细胞STAT1、STAT3、STAT4、STAT6的mRNA表达;Western印迹检测16HBE细胞STAT1、STAT4、STAT6的蛋白表达。分别采用不同浓度的脂多糖在不同的时间点处理16HBE细胞,采用Real—timePCR的方法检测16HBE细胞STAT1、STAT3、STAT4、STAT6的mRNA表达。结果1μg／m1的LPS处理16HBE细胞1h组、0．25μg／m1的LPS处理16HBE细胞4h组、1μg／ml的LPS处理16HBE细胞4h组STAT1、STAT4的mRNA表达较正常对照组显著增高（P〈0．01）;0．25μg／ml的LPS处理16HBE细胞2h组、1μg／ml的LPS处理16HBE细胞2h组、10μg／ml的LPS处理16HBE细胞2h组STAT1、STAT4的mRNA表达较正常对照组有所增高（P〈0．05）;1μg/ml的LPS处理16HBE细胞1h组STAT6的mRNA表达较正常对照组显著增高（P〈0．01）。所有LPS处理16HBE细胞组STAT3的mRNA表达均较正常对照组减低。结论人支气管上皮细胞表达STAT1、STAT3、STAT4、STAT6的mRNA和STAT1、STAT4、STAT6的蛋白,一定剂量的脂多糖在某些时间点分别刺激了人支气管上皮细胞STAT1、sTAT4、STAT6的mRNA表达。     

TNF—α对哮喘模型大鼠气道平滑肌细胞增殖及ERK1／2活性的影响

目的探讨TNF—α对哮喘大鼠气道平滑肌细胞（ASMCs）增殖及对ASMCs上ERK1／2mRNA、p-ERK1／2表达水平的影响。方法通过对哮喘模型大鼠ASMCs培养,分别以0．2μg／L、1．0μg/L、20μg/L TNF-α干预ASMCs生长。采用流式细胞仪、MTT法检测ASMCs增殖情况,观察不同浓度TNF—α对ASMCs增殖的影响。RT-PCR检测ASMCs上ERK1／2mRNA表达,免疫细胞化学染色法检测磷酸化ERK1／2蛋白的表达及定位。结果哮喘组ASMCsS期比例、A值、ERK1／2mRNA、p-ERK1／2蛋白的表达量分别为（34．45±2．08）％、（0．550±0．010）、（0．995±0．118）、（130．77±4．16）,与对照组（11．17±0．96）％、（0．292±0．008）、（0．576±0．098）、（163．82±1．38）比较均显著增高（均P〈0．01）。各TNF—α干预组ASMCs的S期比例、A值、ERK1／2mRNA和p-ERK1／2蛋白表达量与哮喘组比较均显著降低（均P〈0．01）,0．2μg/L和1．0μg／LTN-α组p-ERK1／2蛋白表达量高于对照组（P〈0．01）,20μg／L TNF-α组p-ERK1／2蛋白表达量与对照组比较无差异（P〉0．05）。结论与正常鼠相比,慢性哮喘大鼠气道平滑肌细胞增殖明显,处于S期的细胞比例明显增高。经TNF—α干预后,慢性哮喘大鼠气道平滑肌细胞处于S期的细胞比例减少,增殖减弱,TNF-α可能抑制慢性哮喘大鼠气道平滑肌细胞增殖。TNF—α可下调慢性哮喘大鼠气道平滑肌细胞上ERK1／2mRNA及p-ERK1／2表达,TNF-α可能通过抑制ERK信号转导通道的活性对气道平滑肌细胞的增殖进行调控。     

表没食子儿茶素没食子酸酯对人结肠癌HT-29细胞G1期的阻滞作用

目的:研究表没食子儿茶素没食子酸酯(Epigallaocatechin-3-gallate,EGCG)时人结肠癌HT-29细胞增殖的影响.方法:实验分为EGCG不同浓度处理组和阴性对照组.采用MTT比色法检测EGCG(30μg/mL、40μg/mL、50μg/mL、60μg/mL、70μg/mL)对HT-29细胞的生长影响;应用流式细胞术分析EGCG对HT-29细胞周期分布的影响;免疫印迹观测EGCG对HT-29细胞p38MAPK、cyclinD1蛋白表达的影响.结果:MTT比色结果显示.不同浓度EGCG(30μg/ml、40μg/ml、50μg/ml、60μg/ml)对HT-29细胞具有明显的生长抑制作用,并呈剂量-效应依赖关系(P<0.05);流式细胞术分析显示,EGCG诱导人结肠癌细胞G1期阻滞,且随着处理时间的延长,其诱导周期阻滞的效应越明显(P<0.05);蛋白免疫印迹显示.总的p38MAPK不随处理时间和浓度的改变而改变,但是磷酸化的p38MAPK蛋白的表达随处理时间和处理浓度的增加而明显增加,而CyclinD1蛋白的表达随处理浓度的增加而明显减少.结论:EGCG诱导HT-29细胞G1期阻滞,抑制细胞增殖,可能与活化p38MAPK,下调CyclinD1蛋白表达有关.     

依托咪酯通过调控miR-3666/UBQLN1基因表达抑制乳腺癌细胞增殖和诱导凋亡的机制

本研究旨在探讨依托咪酯对乳腺癌细胞增殖、凋亡的影响及其机制,选取依托咪酯(0.2μg/mL,0.4μg/mL和0.8μg/mL)处理乳腺癌细胞SK-BR-3,筛选最适浓度0.8μg/mL。将依托咪酯+anti-miR-NC组(转染anti-miR-NC)、依托咪酯+anti-miR-3666组(转染anti-miR-3666)、miR-NC组(转染miR-NC)、miR-3666组(转染anti-miR-3666)、anti-miR-NC组(转染anti-miR-NC)和anti-miR-3666组(转染anti-miR-3666)用脂质体法转染至SK-BR-3细胞,部分组用0.8μg/mL依托咪酯处理。细胞计数(CCK-8)法、流式细胞术、免疫印迹(Western blotting)、实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测细胞的抑制率、凋亡率、miR-3666、泛素1(UBQLN1)、细胞周期素D1(CyclinD1)、细胞周期依赖性蛋白激酶抑制因子1A(p21)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关的X基因(Bax)的表达;双荧光素酶报告基因检测实验检测细胞的荧光活性。依托咪酯(0.2μg/mL,0.4μg/mL和0.8μg/mL)明显的增强对乳腺癌细胞的抑制率和凋亡率,并上调miR-3666,下调UBQLN1,均呈明显的浓度依赖性(p<0.05)。miR-3666能够明显的抑制野生型UBQLN1细胞的荧光活性。过表达miR-3666具有与依托咪酯相似的增强乳腺癌细胞抑制率和凋亡率,下调CyclinD1、Bcl-2,上调p21、Bax的作用。抑制miR-3666明显的减弱依托咪酯对乳腺癌细胞的抑制和凋亡的作用。依托咪酯可抑制乳腺癌细胞增殖,促进凋亡,其机制与上调miR-3666进而靶向抑制UBQLN1有关,将可为依托咪酯用于乳腺癌的治疗提供更充分的理论依据。     

在食管癌细胞增殖过程中蛋白酶激活受体-2(PAR-2)对细胞周期蛋白D1(CyclinD1)的调控机制*

目的:探讨在食管癌细胞增殖过程中蛋白酶激活受体-2(PAR-2)影响细胞周期蛋白D1(CyclinD1)的机制。方法:方法:使用食管癌EC109细胞株,实验分为:空白对照组、PAR-2激动组(加入激动剂SLIGKV)、PAR-2反激动组(加入反激动剂VKGILS)、PAR-2 shRNA组(PAR-2shRNA成功转染)和MAPK抑制组(加入阻滞剂PD98059);取对数生长期的细胞,以6×10⁴cells/ml的密度接种于培养瓶中,置于孵育箱中培养24 h后使用PBS清洗3次,更换为无血清的1640培养基培养24 h,之后各实验组按实验设计分别加入所需试剂,每组设置3个复孔,于孵育箱中继续培养24 h,采用RT-PCR法检测PAR-2、ERK1、CyclinD1的mRNA的表达水平;采用Western blot法检测PAR-2、ERK1、p-ERK1、CyclinD1的蛋白表达水平。结果:与空白对照组相比,PAR-2激动组PAR-2mRNA、ERK1 mRNA、和CyclinD1 mRN表达明显升高(P＜0.05),PAR-2、p-ERK1和CyclinD1蛋白表达升高(P＜0.05);PAR-2 shRNA组PAR-2mRNA、ERK1 mRNA和CyclinD1 mRNA表达降低(P＜0.05),PAR-2、p-ERK1和CyclinD1蛋白表达降低(P＜0.05);MAPK抑制组ERK1 mRNA和CyclinD1 mRNA表达明显降低(P＜0.05),p-ERK1和CyclinD1表达降低(P＜0.05)。结论: PAR-2可通过MAPK通路调节CyclinD1的表达从而促进食管癌细胞EC109的增殖。     

Antizyme1基因转染对K562细胞增殖与凋亡的影响

研究鸟氨酸脱羧酶抗酶蛋白对人红白血病K562细胞增殖、三氧化二砷（ As2O3）诱导凋亡时的影响。方法: 定点突变技术构建缺失frameshift位点的pEGFP-N1-AZ1-mutation重组表达载体。脂质体法转染K562细胞,通过G418筛选获得稳定表达antizyme1的K562pAZ1m细胞系。采用不同浓度的As2O3处理细胞,通过MTT法检测细胞增殖,流式细胞术分析细胞周期及凋亡变化。并通过RT-PCR方法检测antiyme1转染对cyclin D1和survivin基因表达的影响。结果：获得稳定表达antizyme1的K562-AZ1m细胞株后,其增殖能力明显减慢。CyclinD1基因表达降低,细胞主要停滞于G0/G1期。在 As2O3的诱导作用下,细胞凋亡增多,survivin基因表达降低。结论：AZ1基因能够抑制K562细胞增殖,通过对cyclinD1的负调控使细胞周期停滞于G0/G1期。并可能通过下调survivin表达来加强 As2O3对其的诱导凋亡作用     

PI3K/AKT信号传导通路对鼻咽癌细胞增殖和凋亡的影响

目的研究PI3K/AKT信号传导途径对人低分化鼻咽癌细胞系在CNE-2Z生长、增殖和凋亡的作用。方法应用wort mannin抑制PI3K的活性,绘制生长曲线、MTT观察wort mannin对CNE-2Z细胞生长、增殖的作用,以免疫细胞化学分别检测wort mannin处理培养后的CNE-2Z细胞中PI3K、AKT和bcl-2的表达,RT-PCR检测wort mannin处理后的细胞bcl-2 mRNA的表达情况,ELISA法检测wort mannin处理后的CNE-2Z细胞中的磷酸化的AKT的含量,流式细胞仪检测wort mannin处理培养后的CNE-2Z细胞的凋亡。结果 wort mannin对CNE-2Z细胞生长、增殖具有抑制作用,而且呈剂量依赖性。wort mannin作用后CNE-2Z细胞的PI3K和bcl-2的蛋白表达均减弱,而AKT的表达无明显变化。wort-mannin抑制CNE-2Z细胞bcl-2 mRNA的表达。wort mannin能显著抑制磷酸化的AKT的表达,并呈一定的剂量依赖性。Wort mannin还可以诱导CNE-2Z细胞的凋亡。结论 PI3K的抑制剂wort mannin可以有效抑制鼻咽癌CNE-2Z细胞的生长和增殖,诱导CNE-2Z细胞凋亡。     

Induction of Bcl-xL Expression by Human T-Cell Leukemia Virus Type 1 Tax through NF-κB in Apoptosis-Resistant T-Cell Transfectants with Tax

Human T-cell leukemia virus type 1 (HTLV-1) Tax is thought to play a pivotal role in immortalization of T cells. We have recently shown that the expression of Tax protected the mouse T-cell line CTLL-2 against apoptosis induced by interleukin-2 (IL-2) deprivation and converted its growth from being IL-2 dependent to being IL-2 independent. In this study, we demonstrate that constitutive expression of bcl-xl but not bcl-2, bcl-xs, bak, bad, or bax was associated with apoptosis resistance after IL-2 deprivation in CTLL-2 cells that expressed Tax. Transient-transfection assays showed that bcl-x promoter was transactivated by wild-type Tax. Similar effects were observed in mutant Tax retaining transactivating ability through NF-kappaB. Deletion or substitution of a putative NF-kappaB binding site identified in the bcl-x promoter significantly decreased Tax-induced transactivation. This NF-kappaB-like element was able to form a complex with NF-kappaB family proteins in vitro. Furthermore, Tax-induced transactivation of the bcl-x promoter was also diminished by the mutant IkappaBalpha, which specifically inhibits NF-kappaB activity. Our findings suggest that constitutive expression of Bcl-x(L) induced by Tax through the NF-kappaB pathway contributes to the inhibition of apoptosis in CTLL-2 cells after IL-2 deprivation.     

P2Y receptors activate neuroprotective mechanisms in astrocytic cells

Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT-PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor--genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.     

Nerve Growth Factor Determines Survival and Death of PC12 Cells by Regulation of the bcl-x, bax, and caspase-3 Genes

We investigated the effects of nerve growth factor (NGF) and NGF withdrawal on expression of members of the bcl-2 family of genes and caspase-3 in PC12 cells. NGF regulated several members of the bcl-2 family and caspase-3 in a manner consistent with its effect on apoptosis in PC12 cells. Levels of bcl-xl, bcl-xs, and caspase-3 mRNAs were increased by NGF treatment. The increases in caspase-3 and bcl-xs levels should have disposed the cells toward apoptosis but were opposed by the simultaneous increase in bcl-xl level. NGF withdrawal resulted in abrupt down-regulation of bcl-xl and up-regulation of bax, favoring apoptosis. Forced expression of bcl-xl after NGF withdrawal was sufficient to prevent cell death. Cell death was rapid when NGF was withdrawn after 5 days of treatment but relatively slow when NGF was withdrawn after only 1 or 2 days of treatment. This was consistent with the reduced accumulation of caspase-3 mRNA with shorter NGF treatments. These results indicate that Bcl-xl, Bcl-xs, Bax, and caspase-3 are important regulators of apoptosis in PC12 cells. Furthermore, regulation of their mRNA levels is implicated in the signal transduction of NGF.     

Genistein enhances the cisplatin-induced inhibition of cell growth and apoptosis in human malignant melanoma cells

Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy-refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 microM) did not induce apoptosis by itself but did enhance cisplatin-induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti-apoptotic proteins, bcl-2 and bcl-xL, and one pro-apoptotic protein, apoptotic protease activating factor-1 (Apaf-1), were examined. Genistein alone or cisplatin alone generally did not alter bcl-2 expression or bcl-xL expression, but slightly increased Apaf-1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl-2 and bcl-xL protein and increased Apaf-1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients.     

A Phosphatidylcholine Phospholipase C Inhibitor, D609, Blocks Interleukin-3 (IL-3)-Inducedbcl-2Expression But Notc-mycExpression in Human IL-3-Dependent Cells

Activation of the interleukin-3 (IL-3) receptor is required for the induction of cell proliferation and suppression of apoptosis in primitive hematopoietic progenitor cells. A rapid activation of tyrosine kinases and a phosphatidylcholine-specific phospholipase C has been observed in these cells in response to IL-3. The signal transduction cascades regulating cell proliferation and the suppression of apoptosis are poorly understood. Using human IL-3-dependent TF-1 cells, we have found that the tyrosine kinase inhibitor genistein blocks both the IL-3 suppression of apoptosis and the expression of the cell survival genebcl-2.In addition, we have found that D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C, also inhibits IL-3-induced expression of thebcl-2gene without affecting IL-3-induced tyrosine phosphorylation. D609 also drove these cells into apoptosis even in the presence of IL-3. Significantly, genistein inhibited the IL-3 induction of bothbcl-2andc-mycgene. The latter gene is related to the induction of cell proliferation. D609, however, blocked the induction only of the cell survival genebcl-2.Thus, phosphatidylcholine hydrolysis appears linked to the induction of genes related to cell survival. These data fit with the hypothesis that there is a bifurcation in the signaling pathways downstream of IL-3 receptor-induced tyrosine phosphorylation.     

Regulation of A1/Bfl-1 expression in peripheral splenic B cells

We analyzed here the expression of the prosurvival Bcl-2 homologue A1 in peripheral B cell compartment. We observed that A1 mRNA are highly expressed in peripheral B cells as compared with other anti-apoptotic genes of the Bcl-2 family such as bcl-xl and bcl-2 itself. The expression of A1 is up-regulated in immature B cells at the transition between transitional type 1 (T1) and type 2 (T2) cells, and remained highly expressed in mature (M) B cells. We, therefore, analyzed the effect of B cell antigen receptor (BCR) and BAFF receptor (BAFF-R) engagement on the regulation of A1 in total B220(+) cells but also FACS-sorted immature T1, T2 and M B cells. We demonstrated that only BCR engagement up-regulated the expression of A1 mRNA and protein. These results suggest that A1 may play a key role in antigen-dependent signals that are required for survival and/or proliferation of peripheral B cells.