PPARγ受体与消化道肿瘤相关性的研究进展

过氧化物酶体增殖物激活受体-γ(PPARγ)是由配体激活的一类核转录因子,属于II型核受体超家族成员之一。经研究发现,PPARγ在多种肿瘤组织中均有所表达,而且它在调控细胞分化、诱导细胞凋亡和抑制细胞增殖中发挥重要的转录调节作用。激活后的PPARγ可以调控多种核内靶基因的表达,抑制肿瘤细胞的形成、生长与增殖等,与消化道肿瘤的发生、发展及预后有着密切的关系。     

EGCG通过抑制wnt/β-catenin信号通路调节猪骨骼肌卫星细胞的增殖和分化

为了阐明Wnt/β-catenin信号通路在猪骨骼肌卫星细胞增殖分化中的作用,利用Wnt/β-catenin信号通路抑制剂(-)-表没食子儿茶素没食子酸酯(EGCG)处理猪骨骼肌卫星细胞,采用MTT、流式细胞术、免疫荧光和Western印迹等方法检测了细胞增殖和分化情况.结果显示,与对照组相比,EGCG以时间、浓度依赖方式抑制猪骨骼肌卫星细胞的增殖.流式细胞术检测细胞周期结果表明,与对照组相比,经EGCG处理后,猪骨骼肌卫星细胞的G1期细胞比例上升,而G2和S期细胞比例下降,这说明细胞被阻滞在G1期,细胞的增殖受到抑制.免疫荧光检测分化过程中MyHC的表达,与对照组相比,EGCG促进猪骨骼肌卫星细胞的分化,并降低增殖标志基因MyoD以及细胞周期蛋白D的表达量,而提高了分化标志基因MyoG和MyHC的表达量.在猪骨骼肌卫星细胞增殖分化过程中,EGCG降低β-联蛋白的表达量,且核内的β-联蛋白明显减少.结果表明,EGCG通过抑制Wnt/β-catenin信号通路抑制猪骨骼肌卫星细胞的增殖,促进其分化.     

雌激素快速激活MAPK途径促进前列腺间质细胞的增殖

为研究雌激素促进前列腺间质细胞增殖的机理,使用雌二醇（E2）或BSA雌二醇（BSA-E2）处理前列腺间质细胞系wpmy-1,用MTT法及细胞计数法检测细胞增殖情况;用实时RT-PCR以及Western印迹方法检测细胞增殖核抗原（PCNA）的表达水平;用抗磷酸化ERK1/2抗体检测细胞内MAPK途径的激活情况.结果显示,2种形式的雌二醇均能促进前列腺间质细胞系wpmy-1的增殖,并且显著上调增殖相关核抗原PCNA的表达水平;2种形式的雌激素均能快速激活wpmy-1细胞内的MAPK信号通路;MAPK途径的特异性抑制剂PD98059能够显著降低雌激素对细胞增殖以及PCNA表达水平的上调作用.结果表明,雌激素能够通过膜受体快速激活前列腺间质细胞中的MAPK信号通路,进而促进前列腺间质细胞的增殖.     

过表达ER恢复雌激素对子宫内膜癌KLE细胞中Notch信号通路的调控

目的：通过观察雌激素对子宫内膜癌KLE细胞中Notch信号通路的影响,探讨过表达雌激素核受体（estrogenreceptor,ER）是否可以恢复雌激素对Notch信号通路的调控作用,继而调节细胞增殖活性。方法：MTT检测雌激素及Notch信号通路对细胞增殖活性的影响;RT．PCR及Westem．blotting检测雌激素及Notch通路抑制剂DAPT对Notch表达的影响;质粒的抽提及转染使KLE细胞中的雌激素核受体ER过表达。结果：雌激素呈剂量依赖效应促进KLE细胞的增殖活性,其中以雌激素浓度为1．0×10-9M时最明显（相对于对照组为1．25±0．026,P〈0．05）;抑制Notch信号通路的表达可以明显下调KLE细胞的增殖活性（0．76±0．02,P〈0．05）;在KLE细胞中,雌激素对Notch的表达没有明显的调控作用,但是将其雌激素核受体过表达后,雌激素可明显上调Notch的表达,并显著促进细胞的增殖活性（1．24±0．02,P〈0．05）。结论：在ER阴性的子宫内膜癌细胞中过表达ER,可以恢复雌激素对Notch信号通路的调控,从而进一步的调控细胞增殖活性。     

粪肠球菌脂磷壁酸通过 激活NF-κB活化NLRP3炎性体

目的检测粪肠球菌脂磷壁酸(LTA)对NLRP3炎性体的活化机制。方法粪肠球菌LTA及NF-κB抑制剂作用于小鼠巨噬细胞RAW264.7上,运用Western blot及ELISA法检测NLRP3炎性体相关因子mRNA及蛋白的表达,检验LTA对NLRP3的活化是否借助NF-κB信号通路,免疫荧光染色检测NF-κB的核转位。结果LTA可直接活化RAW264.7细胞的NLRP3炎性体。LTA作用于细胞后NLRP3、Caspase-1和IL-1β蛋白的表达明显高于对照组(P0.05)。NF-κB抑制剂可有效抑制NF-κB P65的核转位,而一旦NF-κB信号通路被抑制,NLRP3炎性体蛋白的表达均明显降低。结论 LTA能直接激活小鼠巨噬细胞系RAW264.7的NLRP3炎性体的表达,NF-κB信号通路参与此过程。     

EWS蛋白质抑制核因子κB的转录活性

目的:研究EWS蛋白质是否参与核因子κB(NF-κB)信号通路,以及EWS蛋白质对NF-κB转录活性的影响。方法:在真核细胞中表达Flag-EWS,利用Western印迹检测其表达;通过双萤光素酶光报告系统,研究EWS蛋白质对NF-κB转录活性的影响及其发挥作用的分子水平。结果:Western印迹检测到相对分子质量为95×103的Flag-EWS能够在真核细胞中正确表达,过表达EWS蛋白质能够抑制TNFα、IL-1β及poly(I:C)激活的NF-κB转录活性;EWS蛋白质能够抑制由过表达HA-TRAF2或HA-p65激活的NF-κB转录活性,其抑制NF-κB转录活性发生在p65转录因子水平。结论:过表达EWS能够抑制多种刺激激活的NF-κB转录活性,这种抑制作用发生在p65转录因子水平。     

Shh信号参与调控BMP9诱导的间充质干细胞成骨分化

目的：观察sonic hedgehog（Shh）信号通路在骨形态发生蛋白9（BMP9）诱导的小鼠间充质干细胞（MSCs）C3H10T1/2和C2C12成骨分化中的作用,并初步探讨其作用机制。方法：Shh信号通路抑制剂Cyclopamine和激活剂Purmorphamine以及过表达Shh腺病毒分别作用于BMP9处理的C3H10T1/2和C2C12细胞,碱性磷酸酶（ALP）检测早期成骨指标ALP,茜素红S染色检测晚期成骨指标钙盐沉积,RT-PCR检测Shh信号相关基因以及成骨关键转录因子的表达,Western blot检测Shh的表达,荧光素酶报告基因检测Smad1/5/8的转录调控活性。结果：BMP9促进Shh信号相关基因的表达,激活Shh信号可增强BMP9诱导的C3H10T1/2和C2C12细胞早晚期成骨分化并促进了BMP9诱导的Smad荧光素酶活性,抑制Shh信号后作用相反。结论：激活Shh信号通路可促进BMP9诱导的小鼠MSCs成骨分化,抑制其活性后作用相反。     

PPAR-γ作用及其相关信号转导途径

过氧化物酶增殖物激活受体(peroxisomeproliferater-activatedreceptor,PPAR)是一类配体激活的核转录因子超家族成员,包括PPAR-α、PPAR-β/δ和PPAR-γ三种表型,其中以PPAR-γ的研究最为深入。PPAR-γ通过JAK-STAT、激活蛋白-1(AP-1)、NF-κB、活化T细胞核因子信号通路(NFAT)来抑制炎症反应;通过抑制泡沫细胞(foamcell)的分化、炎症反应以及细胞增殖来抑制动脉粥样硬化的发生发展;通过磷脂酰肌醇-3激酶(PI3K)、瘦素、脂链素等信号通路来参与糖稳态的调节;通过细胞周期的调控来影响肿瘤生长;参与脂肪细胞分化并与肥胖密切相关。明确这些相关信号通路以及相关细胞因子的作用,可对相关疾病机制及防治进一步提供有力依据和干预途径。     

白眉蝮蛇去整合素Adinbitor对Akt信号转导通路及对SSMC7721细胞增殖、迁移和凋亡的影响

研究白眉蝮蛇去整合素adinbitor对蛋白激酶B(Akt)通路信号分子的影响及对SSMC7721细胞增殖、迁移及凋亡的影响.采用MTT法检测adinbitor对SSMC7721细胞增殖的作用;Hoechst33258试剂盒检测adinbitor对SSMC7721凋亡的影响; Transwell 检测adinbitor对SSMC7721细胞迁移的作用;Western印迹检测adinbitor对信号分子Akt及磷酸化蛋白激酶B（p-Akt）、核因子κB（NF-κB）的抑制蛋白IκB-α及NF-κBp65的影响;分光光度法检测其对半胱天冬蛋白酶-3 （caspase -3）活性的影响.结果显示,adinbitor可显著抑制SSMC7721细胞的迁移和增殖(与对照组比较,P＜005),促进凋亡. 在浓度梯度adinbitor作用下,Akt 表达量基本不变,但其磷酸化受到抑制.细胞浆内IκB α表达增加,细胞核内NF-κBp65表达减少,caspase-3活化倍数平均在2.24～3.85之间,以上作用均呈现剂量依赖性.结果说明,adinbitor可通过抑制Akt相关信号转导分子的作用而抑制SSMC7721细胞增殖和迁移,并促进其凋亡.     

瘦素对血管平滑肌细胞核转录因子κB的影响

目的探讨瘦素(leptin)对血管平滑肌细胞(VSMCs)核转录因子(NF-κB)的影响。方法贴块法原代培养VSMCs;分别采用MTT法检测leptin对VSMCs增殖活性的影响,免疫荧光-共聚焦显微镜和蛋白印迹技术检测Lep-tin对VSMCs中NF?κB的细胞内定位和VSMCs核中NF?κB的蛋白含量。结果leptin对VSMCs具有促进细胞增殖的作用,这种促增殖活性具有剂量依赖性;Leptin能促进VSMCs中的NF?κB从胞质转移至细胞核,并且这种作用也具有剂量依赖性。结论Leptin能促进VSMCs增殖,能激活VSMCs中NF?κB,使其从胞质转位至胞核;同时,目前已有的研究已经证明NF?κB与平滑肌细胞的增殖和迁移关系密切,提示Leptin促进VSMCs的增殖作用中可能有NF?κB信号传导通路的参与。     

GYY4137, a novel hydrogen sulfide-releasing molecule,likely protects against high glucose-induced cytotoxicity by activation of the AMPK/mTOR signal pathway in H9c2 cells

Diabetic cardiomyopathy (DCM) has become a major cause of diabetes-related morbidity and mortality. Increasing evidences have proved that hydrogen sulfide (H₂S) fulfills a positive role in regulating diabetic myocardial injury. The present study was designed to determine whether GYY4137, a novel H₂S-releasing molecule, protected H9c2 cells against high glucose (HG)-induced cytotoxicity by activation of the AMPK/mTOR signal pathway. H9c2 cells were incubated in normal glucose (5.5 mM), 22, 33, and 44 mM glucose for 24 h to mimic the hyperglycemia in DCM in vitro. Then we added 50, 100, and 200 μM GYY4137, and measured the cell viability, lactate dehydrogenase (LDH) enzyme activity, and mitochondrial membrane potential (MMP). 0.5 mM 5-amino-4-imidazole-carboxamide riboside (AICAR, an AMPK activator) and 1 mM adenine 9-β-d-arabinofuranoside (Ara-A, an AMPK inhibitor) were used to identity whether the AMPK/mTOR signal pathway was involved in GYY4137-mediated cardioprotection. We demonstrated that HG decreased cell viability and increased LDH enzyme activity in a concentration-dependent manner. 33 mM HG treatment for 24 h was chosen as our model group for further study. Both 100 and 200 μM GYY4137 treatments significantly attenuated HG-induced cell viability decrement, LDH enzyme activity increase, and MMP collapse. AICAR had similar effects to GYY4137 treatment while Ara-A attenuated GYY4137-mediated cardioprotection. Importantly, both GYY4137 and AICAR increased AMPK phosphorylation and decreased mTOR phosphorylation compared with the HG model group while Ara-A attenuated GYY4137-mediated AMPK phosphorylation increase and mTOR phosphorylation decrement. In conclusion, we propose that GYY4137 likely protects against HG-induced cytotoxicity by activation of the AMPK/mTOR signal pathway in H9c2 cells.     

AICAR inhibits cancer cell growth and triggers cell-type distinct effects on OXPHOS biogenesis, oxidative stress and Akt activation

The AMP-activated protein kinase agonist AICAR mimics a low intracellular energy state and inhibits the proliferation of cancer cells by different mechanisms, which may depend on the bioenergetic signature of these cells. AICAR can also stimulate mitochondrial biogenesis in myoblasts, neurons and HeLa cells. Yet, whether the reactivation of oxidative phosphorylation biogenesis by AICAR contributes to the growth arrest of cancer cells remains undetermined. To investigate this possibility, we looked at the impact of 24- and 48-hour treatments with 750 μM AICAR on human cancer cell lines (HeLa, DU145, and HEPG2), non-cancer cells (EM64, FM14, and HLF), embryonic cells (MRC5) and Rho(0) cells. We determined the bioenergetic profile of these cells and assessed the effect of AICAR on oxidative phosphorylation biogeneis, cell viability and cell proliferation, ROS generation, mitochondrial membrane potential and apoptosis induction. We also followed possible changes in metabolic regulators such as Akt and Hif1-α stabilization which might participate to the anti-proliferative effect of AICAR. Our results demonstrated a strong and cancer-specific anti-growth effect of AICAR that may be explained by three different modes according to cell type: the first mode included stimulation of the mitochondrial apoptotic pathway however with compensatory activation of Akt and upregulation of oxidative phosphorylation. In the second mode of action of AICAR Akt phosphorylation was reduced. In the third mode of action, apoptosis was activated by different pathways. The sensitivity to AICAR was higher in cells with a low steady-state ATP content and a high proliferation rate.     

Aminoimidazole Carboxamide Ribonucleotide (AICAR) Inhibits the Growth of Retinoblastoma In Vivo by Decreasing Angiogenesis and Inducing Apoptosis

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an analog of AMP is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. Recently, we showed that AICAR-induced AMPK activation inhibits the growth of retinoblastoma cells in vitro by decreasing cyclins and by inducing apoptosis and S-phase arrest. In this study, we investigated the effects of AMPK activator AICAR on the growth of retinoblastoma in vivo. Intraperitoneal injection of AICAR resulted in 48% growth inhibition of Y79 retinoblastoma cell tumors in mice. Tumors isolated from mice treated with AICAR had decreased expression of Ki67 and increased apoptotic cells (TUNEL positive) compared with the control. In addition, AICAR treatment suppressed significantly tumor vessel density and macrophage infiltration. We also showed that AICAR administration resulted in AMPK activation and mTOR pathway inhibition. Paradoxically observed down-regulation of p21, which indicates that p21 may have a novel function of an oncogene in retinoblastoma tumor. Our results indicate that AICAR treatment inhibited the growth of retinoblastoma tumor in vivo via AMPK/mTORC1 pathway and by apoptogenic, anti-proliferative, anti-angiogenesis mechanism. AICAR is a promising novel non-chemotherapeutic drug that may be effective as an adjuvant in treating Retinoblastoma.     

P13K—Akt—mTORC1信号途径在细胞生长增殖中的调控作用

P13K-AKT—mTORCl信号途径在细胞生长增殖中起重要调控作用,P13K-Akt—mTORl信号途径能够调节细胞周期相关蛋白基因的表达来调控细胞的增殖;同时,P13K—Akt-mTORl信号途径也能够调控细胞的生长和大小;P13K-Akt-mTORCl信号途径的异常活化与肿瘤发生紧密相关。就P13K—AKT-mTORCl信号途径在细胞生长增殖中的作用作一综述。     

Activation of AMP-activated Protein Kinase Suppresses Oxidized Low-density Lipoprotein-induced Macrophage Proliferation

Macrophage-derived foam cells play important roles in the progression of atherosclerosis. We reported previously that ERK1/2-dependent granulocyte/macrophage colony-stimulating factor (GM-CSF) expression, leading to p38 MAPK/ Akt signaling, is important for oxidized low density lipoprotein (Ox-LDL)-induced macrophage proliferation. Here, we investigated whether activation of AMP-activated protein kinase (AMPK) could suppress macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages was assessed by [³H]thymidine incorporation and cell counting assays. The proliferation was significantly inhibited by the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and restored by dominant-negative AMPKα1, suggesting that AMPK activation suppressed macrophage proliferation. AICAR partially suppressed Ox-LDL-induced ERK1/2 phosphorylation and GM-CSF expression, suggesting that another mechanism is also involved in the AICAR-mediated suppression of macrophage proliferation. AICAR suppressed GM-CSF-induced macrophage proliferation without suppressing p38 MAPK/Akt signaling. GM-CSF suppressed p53 phosphorylation and expression and induced Rb phosphorylation. Overexpression of p53 or p27^kip suppressed GM-CSF-induced macrophage proliferation. AICAR induced cell cycle arrest, increased p53 phosphorylation and expression, and suppressed GM-CSF-induced Rb phosphorylation via AMPK activation. Moreover, AICAR induced p21^cip and p27^kip expression via AMPK activation, and small interfering RNA (siRNA) of p21^cip and p27^kip restored AICAR-mediated suppression of macrophage proliferation. In conclusion, AMPK activation suppressed Ox-LDL-induced macrophage proliferation by suppressing GM-CSF expression and inducing cell cycle arrest. These effects of AMPK activation may represent therapeutic targets for atherosclerosis.