表皮生长因子信号通路对前列腺癌细胞DU145黏附和迁移能力的影响

目的 探讨表皮生长因子（EGF）信号通路对雄激素非依赖型前列腺癌细胞株DU145黏附和迁移能力的影响。方法 应用细胞黏附能力测定和划痕实验测定EGF对DU145黏附和迁移能力的作用，应用流式细胞术测定EGF对DU145细胞表面整联蛋白α5和β1表达的影响，应用逆转录-聚合酶链反应（RT-PCR）和Western blot测定EGF对DUl45细胞整联蛋白α5和β1亚基总蛋白和mRNA表达的影响。结果 EGF可促进DUl45对纤连蛋白（Fn）的黏附和迁移作用，同时，Fn的受体a5β1的表达发生了变化，EGF上调细胞表面β1亚基的表达，而丝裂源激活蛋白激酶（MAPK）信号通路抑制剂PD98059则具有相反的作用，这种调节作用主要发生在mRNA水平上。结论 EGF可能通过MAPK信号通路，上调β1的表达，从而促进DU145细胞的侵袭能力。     

有丝分裂激活蛋白激酶在人前列腺癌细胞中的激活

目的 探讨前列腺癌（PCa)细胞中有丝分裂激活蛋白激酶（MAPK）的激活。方法 采用免疫沉淀和Western blot方法,检测人PCa细胞株LNCaP及DU－145中磷酸化MAPK的表达及雄激素或表皮生长因子（EGF）对其表达的影响。结果 在DU－145中,磷酸化MAPK表达本底水平比LNCaP高10倍。雄激素和EGF处理后8hLNCaP中MAPK激活水平升高,24h达最高,分别比本底高约10倍和5倍,在DU－145中仅EGF可促使MAPK激活水平升高,8h开始升高,24h达最高,比本底高约10倍。结论 在雄激素非依赖性人PCa细胞增生中,MAPK激活比在雄激素依赖性人PCa细胞中作用大,MAPK激活也参与了雄激素和EGF的增生刺激作用。     

EGF信号通路对雄激素非依赖性前列腺癌细胞株DU145细胞周期的影响

目的探讨EGF信号通路影响雄激素非依赖型前列腺癌细胞系DU145细胞周期分子学机理.方法MTT法检测EGF对DU145细胞增殖能力的影响,流式细胞仪检测细胞周期,Western印迹检测细胞周期调控因子的表达情况.结果EGF组细胞增殖能力显著升高,EGF组细胞在S期细胞比例为65.36%,明显高于对照组的44.32%,两者比较差异有统计学意义(P＜0.01);在蛋白质水平,EGF降低了p27表达,而CDK2、磷酸化Rb的表达不同程度增高,p16、p21表达无明显差异.结论EGF刺激导致其下游的信号通路活化,降低p27表达,并导致下游CDK2、Rb及Rb磷酸化水平的变化,最终促进了DU145细胞的增殖能力.     

肝癌细胞VEGF转录调控的信号传导通路

目的研究磷脂酰肌醇3-激酶(PI3K)和p42／p44丝裂源激活的蛋白激酶(MAPK)在肝癌细胞血管内皮生长因子(VEGF)转录调控中的作用。方法应用缺氧诱导剂氯化钴或重组人表皮生长因子(EGF)刺激HepG2细胞中VEGF的转录，在此过程中应用PI3K特异性阻断剂LY294002或p42／p44 MAPK特异性阻断剂PD98059干预，采用半定量逆转录PCR检测VEGF mRNA表达的变化。结果氯化钴或EGF可以诱导HepG2细胞中VEGF的表达。PI3K阻断剂LY294002可以在一定范围内浓度依赖性地抑制VEGF mRNA的表达，而p42／p44 MAPK阻断剂PD98059对VEGF mRNA的表达无抑制作用。结论肝癌细胞VEGF的转录调控受PI3K通路调控，而不受p42／p44 MAPK调控。     

SPAG9调控前列腺癌迁移侵袭能力的研究

目的探讨SPAG9对前列腺癌细胞迁移、侵袭能力的影响及其分子机制。方法应用RNA干扰技术下调SPAG9在前列腺癌细胞中的表达,采用Western blot方法评估SPAG9基因干扰效果。采用Transwell实验比较SPAG9干扰前后前列腺癌细胞DU145、PC3的迁移与侵袭能力的改变。用CCK8细胞增殖实验分析SPAG9基因沉默对前列腺癌细胞DU145、PC3增殖能力的影响。用Western blot实验研究SPAG9基因沉默对前列腺癌细胞中MMP-2及TIMP-2蛋白表达的影响。结果特异性SPAG9基因的干扰RNA(siRNA)能够有效沉默DU145、PC3前列腺癌细胞株中SPAG9蛋白的表达。SPAG9敲低后的前列腺癌细胞株迁移与侵袭能力显著降低,差异有统计学意义(P0.05)。siSPAG9组DU145和PC3细胞中MMP-2的蛋白表达水平比siCtrl组显著降低,两组比较差异有统计学意义(P0.05),而TIMP-2的表达量显著升高,差异有统计学意义(P0.05)。SPAG9基因沉默后并不影响DU145及PC3细胞的增殖,组间差异无统计学意义(P0.05)。结论SPAG9基因通过激活MMP-2信号通路增强前列腺癌细胞的迁移与侵袭能力。     

不同前列腺癌细胞株Talin-1表达情况和侵袭能力关系研究

目的探讨踝蛋白1(Talin-1)在不同前列腺癌细胞株中的表达情况和侵袭能力的关系。方法体外培养不同人前列腺癌细胞株(DU-145、PC-3、LNCaP)及人正常前列腺上皮细胞株(RWPE-1)。传代培养后利用MTT法检测各细胞株增殖能力。细胞划痕实验,Transwell小室侵袭和迁移实验检测各细胞株侵袭及迁移能力。RT-PCR技术检测各细胞株中Talin-1的mRNA表达水平。Western-blot技术检测各细胞株中Talin-1的蛋白表达水平。结果与RWPE-1细胞株相比,LNCaP细胞株、PC-3细胞株、DU145细胞株的增殖、侵袭和迁移能力明显增高(P0.05),其中LNCaP、PC-3、DU-145细胞株的增殖、迁移以及侵袭能力依次递增(P0.05)。前列腺癌LNCaP细胞株、PC-3细胞株、DU145细胞株中的Talin-1 mRNA及蛋白表达水平显著高于RWPE-1细胞株(P0.05);其中LNCaP、PC-3、DU-145细胞株中的Talin-1 mRNA以及蛋白表达水平依次递增(P0.05)。结论Talin-1与前列腺癌的增殖、侵袭和迁移能力相关;低表达Talin-1的前列腺癌的增殖、侵袭和迁移能力低,恶性度低;高表达Talin-1的前列腺癌的增殖、侵袭和迁移能力强,恶性度高。Talin-1可作为前列腺癌恶性度的预测指标之一。     

乳腺癌中雌激素诱导的细胞增殖与有丝分裂素激活蛋白激酶活性的关系

目的探讨雌二醇与有丝分裂素激活蛋白激酶(MAPK)信号通路的关系以及MAPK在乳腺癌细胞系中的表达情况。方法分别用上皮生长因子(EGF)和不同浓度的雌二醇诱导人类乳腺癌细胞系MCF-7细胞,诱导不同时间后用Western blot方法检测磷酸化ERK1／2的表达;用抗雌激素物质ICI182780和MAPK抑制剂PD98059作用于雌二醇诱导的MCF-7细胞,检测磷酸化ERK1／2的表达;用流式细胞仪检测雌二醇对MCF-7细胞周期的影响。结果MAPK信号通路的诱导剂EGF可以明显诱导磷酸化MAPK的表达;雌二醇也可以诱导磷酸化MAPK的表达。PD98059可以完全抑制雌二醇诱导的磷酸化ERK1／2水平,而ICI182780只能减少雌二醇诱导的磷酸化ERK1／2的表达。随着作用时间的延长,雌二醇可以使MCF-7细胞G2／M期的细胞数增加。结论雌二醇、雌激素受体与MAPK信号途径关系较为密切,MAPK是乳腺癌中重要的调节信号。     

低密度脂蛋白和氧化低密度脂蛋白诱导人肾小管上皮细胞转分化的通路研究

目的探讨低密度脂蛋白（LDL）和氧化（OX）LDL是否可诱导人肾小管上皮细胞转分化及其可能机制。方法体外培养的第2代肾小管上皮细胞随机分为（1）阴性对照组；（2）LDL（50mg／ml）组；（3）oxLDL（50mg／ml）组；（4）LDL（50mg／m1）＋PD98059（5μmol／L）组；（5）oxLDL（50mg／ml）＋PD98059（5μmol／L）组。应用形态学、免疫荧光、Western印迹观察LDL和oxLDL刺激小管细胞角蛋白（cytokerafin）、E-钙粘糖蛋白（cadhefin）、α-SMA和波形蛋白（vimenfin）表达的改变、Ⅰ型胶原产生的改变，及其与ERK1／2MAPK通路活化的关系。结果OXLDL较LDL有更强的致小管上皮细胞转分化的作用，与对照组相比。细胞中上皮细胞标记cytokeratin和E-cadherin表达减少，间充质细胞标记α-SMA和vimentin明显增加，Ⅰ型胶原产生增多（P〈0．05）。LDL和oxLDL刺激组细胞中ERK1／2MAPK和GSK-3β磷酸化活化较对照组明显增强（P〈0．05）；β-catenin发生细胞核内转移。MAPK抑制剂PD98059可抑制GSK-3β的磷酸活化，并几乎完全阻断oxLDL诱导的肾小管上皮细胞转分化，但对LDL诱导的小管上皮细胞转分化只有部分阻断作用。结论本研究首次在体外观察到（1）oxLDL可诱导肾小管细胞上皮细胞转分化和Ⅰ型胶原的产生，作用明显强于LDL；（2）ERK1／2MAPK、GSK-3β和β-catenin组成一条信号通路调节LDL和oxLDL诱导的小管上皮细胞转分化。     

TGFα和EGF对前列腺癌细胞系EGFR表达的调控作用

目的：探讨转化生长因子（TGF）α和表皮生长因子（EGF）对前列腺癌细胞系中表皮生长子受体（EGFR）表达的调控作用。方法：采用RT－PCR和Western印迹法分别对TGFα和EGF刺激前列腺癌细胞系LNCaP、PC3、ARCaP后EGFR mRNA表达及其蛋白水平进行定量分析。结果：EGF引起前列腺癌细胞系LNCaP、PC3、ARCaP的EGFR mRNA升高2－5倍,TGFα引起各细胞系EGFR mRNA升高2－10倍。TGFα使各细胞系EGFR不同程度升高;EGF处理的LNCaP EGFR蛋白水平略升高,PC3、ARCaP EGFR蛋白水平降低。结论：TGF／EGF-EGFR通路在前列腺癌发生发展中起重要作用;非依赖型前列腺癌中TGFα－EGFR自分泌环的作用可能强于EGF－EGFR自分泌环。     

TGF-β/Smads通路关键蛋白在5种前列腺癌细胞株中表达的鉴定及意义

目的:对多种不同人前列腺癌细胞株TGF-β/Smads信号通路的"开放"或"关闭"状态进行鉴定,初步探讨此通路在前列腺癌侵袭、转移中的作用及可能的机制。方法:用Western印迹法检测LNCaP、PC-3、DU145及AR-CaP亚细胞系IF11、IA8细胞中TGF-β/Smads通路的关键蛋白TGF-βⅡ型受体(TβRⅡ)、Smad2/3、磷酸化Smad2(p-Smad2)、Smad4的差异表达。结果:TβRⅡ在PC-3、DU145、IF11、IA8中表达较高,在LNCaP中表达极低;Smad2/3在所有细胞中表达均较高,但活性成分p-Smad2仅在PC-3、DU145中表达;Smad4在LNCaP、PC-3、DU145中表达较高,IF11、IA8中表达缺失。结论:不同转移潜能的前列腺癌细胞株TGF-β/Smads通路的"开闭"状态存在差异,仅PC-3、DU145细胞处于开放状态;前列腺癌细胞可能通过不同的方式改变TGF-β/Smads通路状态参与晚期肿瘤的侵袭、转移过程。     

TGF-beta1 calcium signaling increases alpha5 integrin expression in osteoblasts.

TGF-beta1 is a potent osteoactive factor and exhibits a wide variety of effects on osteoblasts, most of which are mediated through receptor associated Smad proteins. We have recently reported a novel TGF-beta1 intracellular Ca2+ signaling pathway in osteoblasts, and found that this signaling is required for the TGF-beta1 mediated enhancement of osteoblast adhesion to substrate. Given that interaction between the extracellular matrix protein fibronectin and alpha5beta1 integrin on the cell surface is principally responsible for osteoblast substrate adhesion, we examined here whether the TGF-beta1 stimulated Ca2+ signal is involved in this pathway. Our results show that, in primary human osteoblasts, the TGF-beta1 induced intracellular Ca2+ signal is responsible, in part, for the stimulation of expression of alpha5 integrin, but not of beta1 integrin or fibronectin. Increased levels of alpha5 integrin protein and mRNA were seen as early as 12 h after TGF-beta1 treatment, but were inhibited by co-treatment of cells with nifedipine, a selective L-type Ca2+ channel blocker. TGF-beta1 treatment increased both fibronectin and beta1 integrin protein production within 48 h, in a manner unaffected by co-treatment with nifedipine. Immunofluorescence observations revealed that TGF-beta1 treatment resulted in increased alpha5 integrin staining, and more prominent alpha5 integrin clustering, with increased co-localization with the actin cytoskeleton, effects that were blocked by co-treatment with nifedipine. The TGF-beta1 induced intracellular Ca2+ signal in human osteoblasts is thus an important mechanistic step in the regulation of alpha5 integrin expression, later contributing to enhanced cell adhesion.     

Antisense oligonucleotides to the integrin receptor subunit alpha(5) decrease fibronectin fragment mediated cartilage chondrolysis

OBJECTIVE: To investigate involvement of the integrin alpha(5) subunit of the classical fibronectin receptor in cartilage chondrolytic activities of fibronectin fragments (Fn-f). DESIGN: Bovine chondrocytes and cartilage explants were cultured in the presence of antisense oligonucleotide (ASO), or sense (SO) or scrambled sequence oligonucleotide (SCO) corresponding to the bovine alpha(5) subunit. The effects of the oligonucleotides on mRNA and protein expression of the alpha(5) subunit were analysed by rtPCR and Western blotting, respectively. To test effects on Fn-f activities, three different Fn-f were first added to serum or serum-free cultures, followed by addition of oligonucleotides and the effects on Fn-f mediated proteoglycan (PG) degradation, cartilage PG depletion and PG and general protein synthesis suppression were tested. RESULTS: The ASO decreased alpha(5) mRNA and protein expression to 69% and 55%, respectively, in monolayer cultures and decreased protein expression 67% in cartilage explants, while SO and SCO were ineffective. The ASO partially reversed the ability of the Fn-fs to suppress PG and general protein synthesis in cartilage explant and high density chondrocyte cultures. Concentrations of ASO from 1 nM to 5 microM effectively suppressed Fn-f activities in particular assays and the effects were reversible, while SO and SCO were not significantly effective. ASO also suppressed, in a dose-dependent and reversible fashion, the ability of the Fn-fs to enhance degradation and release of PG from cartilage explants. The ASO were also effective in suppressing the ability of an antibody to the alpha(5) subunit to enhance PG degradation, but were ineffective in blocking endotoxin or IL-1beta enhanced degradation. CONCLUSIONS: These data implicate the alpha(5) integrin subunit in Fn-f mediated activities, consistent with a role for the alpha(5)beta(1) integrin in this pathway.     

The integrin alpha5beta1 regulates alphavbeta3-mediated extracellular signal-regulated kinase activation

BACKGROUND: Integrin-mediated cell migration is essential for wound repair. Previous studies have shown that the interaction between integrins and the extracellular matrix (ECM) can initiate intracellular signaling pathways to regulate cell movement. Both the focal adhesion kinase (FAK) and the extracellular signal-regulated kinase/activated mitogen-activated protein kinase (ERK/MAPK) signaling pathways are required for efficient cell migration. Our previous work has shown that co-expression of the integrin alpha5beta1 inhibits alphavbeta3-mediated cell migration. We hypothesized that alpha5beta1 may regulate cell migration by modulating these alphavbeta3-mediated intracellular signaling events. METHODS: CHO B3 (alphavbeta3+) and B3C5 (alphavbeta3+/alpha5beta1+) cells were monitored by flow cytometry to determine integrin expression. Cells were allowed to migrate on fibrinogen (FBG)-coated transwells, with or without PD98059, an inhibitor of the ERK activator, mitogen-activated protein kinase kinase (MEK). Fixation, staining, and cell counting were used to quantify cell migration. Cells adherent to FBG were lysed and analyzed for FAK and ERK/MAPK activation by immunoblotting followed by image analysis densitometry. All experiments were repeated in triplicate. RESULTS: Treatment with PD98059 significantly decreased alphavbeta3-mediated cell migration on FBG (P = 0.0001) to a level comparable to untreated B3C5 cells. Following adhesion to FBG, B3 cells demonstrated a marked increase in ERK/MAPK activation compared to B3C5 cells. However, no significant difference was detected in FAK activation. CONCLUSION: Signaling through the ERK/MAPK pathway is required for efficient alphavbeta3-mediated migration on FBG. Inhibition of alphavbeta3-mediated migration by the integrin alpha5beta1 correlates with altered intensity and duration of ERK/MAPK activation, but not FAK activation, in response to adhesion. This suggests a mechanism for the regulatory effect of alpha5beta1 on alphavbeta3-mediated cell migration.     

EXOGENOUS PHOSPHOLIPASE C STIMULATES EPITHELIAL CELL MIGRATION AND INTEGRIN EXPRESSION IN VITRO

Phospholipase C secreted by bacterial pathogens has been identified as a virulence factor in several human diseases and has been implicated in impeding wound healing. The role of phospholipase C in the intracellular signal control of epithelial growth was studied in normal human skin keratinocytes cultured in conditions simulating aspects of wound healing. Bacillus cereus phospholipase C decreased cell-cell contact and increased cell migration resulting in disruption of the advancing epithelial sheet. Phospholipase C-induced migration was blocked by inhibitor of the phosphoinositol signal transduction pathway neomycin sulfate and protein kinase C inhibitor RO-31-8220. Induced migration was associated with elevated levels of matrix metalloproteinase-9 which, when blocked by tissue inhibitor of metalloproteinase-1, was accompanied by a loss of migration. Adhesion studies showed that phospholipase C treatment enhanced cell binding to fibronectin, vitronectin and collagen IV. Immunostained phospholipase C-stimulated cells cultured on fibronectin showed enhanced expression and relocation of the integrin subunits alpha(v), alpha5 and beta1. Confocal microscopy showed that phospholipase C-induced levels of integrin subunit beta1 were predominantly deposited on the basal surface of the cell apparently in focal contacts and associated with actin stress fibers. These results indicate that exogenous phospholipase C signaling from a bacterial source may play an important role in perturbing normal reepithelialization via altered expression of integrins and matrix metalloproteinase-9.     

Effect of cyclic strain and plating matrix on cell proliferation and integrin expression by ligament fibroblasts.

The role of cell surface integrins in cell migration, proliferation, and attachment to matrix molecules is well known. Integrin-matrix interactions have been implicated in mechanotransduction and load transmission from the outside to the inside of the cell. In this study, the effect of cyclic strain on the cell proliferation, attachment, and expression of integrin subunits beta1, beta3, and alpha5 was determined in anterior cruciate ligament (ACL) and medial collateral ligament (MCL) fibroblasts grown on polystyrene, Type I collagen, laminin, elastin, and fibronectin. ACL fibroblast proliferation was not affected by growth substrate whereas MCL cells reached confluence more rapidly on fibronectin compared with collagen or polystyrene. Exposure to 5% cyclic strain resulted in a significant decrease in ACL and MCL fibroblast proliferation on fibronectin and Type I collagen. MCL cells showed a greater strain-dependent inhibition of cells grown on a fibronectin substrate than those grown on collagen. This matrix-dependent effect of strain on cell proliferation was not seen with ACL cells. Attachment of ACL and MCL fibroblasts was stronger to fibronectin compared with Type I collagen, laminin, and polystyrene. In the absence of applied load, the expression of beta1, beta3, and alpha5 subunits was not substrate dependent and the expression of beta1 and alpha5 integrin subunits was higher in MCL cells than ACL cells on all substrates. In contrast, the expression of beta3 integrin subunit was higher in ACL cells than MCL cells. In response to 5% strain, beta1, and alpha5 expression increased in all fibroblasts with MCL cells having a higher magnitude of expression. beta3 expression showed a 90% increase in response to load when grown on laminin for both MCL and ACL fibroblasts and demonstrated no change in expression on Type I collagen or fibronectin. The duration of applied strain from 2 versus 22 h had no effect on cell proliferation or integrin expression.     

Laminin-1 and alpha6beta1 integrin regulate acinar morphogenesis of normal and malignant human prostate epithelial cells

BACKGROUND: Cell-matrix interactions via integrin receptors are critical for acinar morphogenesis. The non-tumorigenic, human prostate epithelial cell line RWPE-1 was used in a three-dimensional (3D) cell culture model to identify the matrix protein and its integrin receptor required for acinar morphogenesis. METHODS: 3D cultures, immunostaining, confocal microscopy, and Western blot analysis were used to examine acinar formation on matrix proteins and to determine integrin receptor expression. RESULTS: RWPE-1 cells differentiate into acini of polarized cells with a distinct lumen in 3D Matrigel culture. In contrast, the malignant WPE1-NB26 prostate epithelial cells form solid cell masses. In 3D gels of laminin-1, type IV collagen, or fibronectin, RWPE-1 cells form acini only in laminin-1. Anti-laminin-1 antibody reduces acinar formation in a dose-dependent manner. Polarized RWPE-1 cells showed basal expression of alpha6 and beta1 integrin subunits. Blocking antibodies to alpha6 or beta1 reduced acinar formation to 9 and 6 percent of control, respectively. The beta1 integrin colocalized with focal adhesion kinase (FAK). Inhibition of extracellular signal-regulated kinase kinase activity significantly reduced acinar formation to 38 percent of control, suggesting that beta1 integrin-mediated signal transduction may be regulated through a FAK pathway. CONCLUSIONS: While basal expression of alpha6beta1 integrin in RWPE-1 cells correlates with their ability to polarize and form acini, a decrease or loss of alpha6, and diffused beta1 expression in WPE1-NB26 cells correlates with loss of acinar-forming ability. Results show that laminin-1 and a functional alpha6beta1 integrin receptor are required for acinar morphogenesis. This novel 3D cell culture model is useful for elucidating regulation of acinar morphogenesis and its loss during prostate carcinogenesis.     

Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix

To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone marrow microenvironment and bone surfaces. Furthermore, these data suggest a possible role for the beta1 integrin subfamily during the development of stromal precursor cells into functional osteoblast-like cells.     

澳洲茄边碱对前列腺癌细胞增殖的抑制作用及机制研究

目的：研究澳洲茄边碱(solamargine,SM)对人前列腺癌激素非依赖细胞增殖的影响及可能的作用机制.方法用不同浓度 SM(0、1、2、4、6、8、10μmol/L)处理 DU145和 PC3细胞, MTT法检测 SM 对细胞生长的影响,Western blot 检测 SM 对相关信号通路蛋白 p38 MAPK、ERK1/2 MAPK、MUC1表达的影响.结果6μmol/L SM作用24 h后DU145和PC3细胞活力分别为(52.53±9.05)％、(56.28±2.36)％,并具有时间和剂量依赖;10μmol/L SM作用24 h后细胞活力分别为(27.36±2.72)％、(32.07±2.53)％.流式细胞术分析显示不同浓度 SM(0、4、6、8μmol/L)处理 PC3细胞能引起 PC3细胞阻滞在 G1期,G1期细胞比例分别为(52.61±0.50)％、(52.96±1.49)％、(66.16±2.84)％和(69.03±2.38)％.且能激活 MAPK信号通路减少下游蛋白 MUC1的表达.结论 SM能明显抑制DU145和PC3细胞生长,该作用可能与 MAPK信号通路的激活以及下游 MUC1蛋白表达下调有关.     

成纤维细胞生长因子受体1在胰腺癌细胞中的表达和调控

目的 研究成纤维细胞生长因子受体1(FGFR1)蛋白和mRNA在胰腺癌细胞系中的表达和调控机制.方法 采用Western免疫印迹实验、Northern印迹分析和RT-PCR检测FGFR1在胰腺癌细胞中的表达.使用外源性生长因子刺激细胞并使用激酶抑制剂阻断细胞内信号转导通路,观察FGFR1蛋白和mRNA在胰腺癌细胞中表达的变化情况.结果 FGFR1蛋白和mRNA在胰腺癌细胞系中均有不同程度的表达.生长因子刺激可上调FGFR1蛋白和mRNA的表达水平.其中IGF-1、EGF和FGF2显著增加Mia PaCa-2细胞FGFR1的表达,EGF和FGF2显著增加PANC-1细胞FGFR1的表达(P＜0.05).FGF2对FGFR1表达的调节具有时间依赖性.ERK1/2抑制剂UO126和p38 MAPK抑制剂SB203580降低了PANC-1细胞中FGFR1的蛋白和mRNA的表达水平. 结论生长因子可上调FGFR1在胰腺癌细胞中的表达水平,MAPK信号转导通路中的ERK1/2和p38 MAPK亚通路参与FGFR1表达的调节.     

Subsequent activation of mitogen-activated protein kinase after adhesion of transitional cell cancer cells to fibronectin

INTRODUCTION: In the process of tumor invasion and metastasis, interactions between tumor cells and extracellular matrix play a crucial role. Recently, it was shown that fibronectin binding to fibronectin receptor promotes mitogen-activated protein kinase (MAPK) activation after tyrosine phosphorylation of focal adhesion kinase (FAK). We investigated these signal transduction events in transitional cell cancer (TCC) cells. MATERIALS AND METHODS: (1) The adhesion of T24 cells, a fibronectin-receptor-positive TCC cell line, to fibronectin was investigated; (2) the MAPK activation after fibronectin stimulation in bladder cancer cell lines was examined by Western blotting using an antiactive MAPK antibody, and (3) FAK, Sos, and Grb-2 were also examined by Western blot analysis. RESULTS AND CONCLUSIONS: T24 cells adhered to fibronectin-coated dishes more quickly than to the noncoated dishes. Fibronectin stimulation induced activation of MAPK in T24, SCaBER, and HT1376 cells. However, activated MAPK was not detected in RT4 cells which do not express alpha(5)beta(1) integrin (major fibronectin receptor) after fibronectin stimulation. T24, SCaBER, and HT1376 expressed FAK and Sos. RT4 showed little FAK and Sos expression. Grb-2 was expressed in all cell lines. Adhesion of fibronectin-receptor-positive TCC cells to fibronectin activates the MAPK cascade, possibly resulting in activation of tumor cells.