Fungiform papilla pattern: EGF regulates inter-papilla lingual epithelium and decreases papilla number by means of PI3K/Akt, MEK/ERK, and p38 MAPK signaling

Fungiform papillae are epithelial taste organs that form on the tongue, requiring differentiation of papillae and inter-papilla epithelium. We tested roles of epidermal growth factor (EGF) and the receptor EGFR in papilla development. Developmentally, EGF was localized within and between papillae whereas EGFR was progressively restricted to inter-papilla epithelium. In tongue cultures, EGF decreased papillae and increased cell proliferation in inter-papilla epithelium in a concentration-dependent manner, whereas EGFR inhibitor increased and fused papillae. EGF preincubation could over-ride disruption of Shh signaling that ordinarily would effect a doubling of fungiform papillae. With EGF-induced activation of EGFR, we demonstrated phosphorylation in PI3K/Akt, MEK/ERK, and p38 MAPK pathways; with pathway inhibitors (LY294002, U0126, SB203580) the EGF-mediated decrease in papillae was reversed, and synergistic actions were shown. Thus, EGF/EGFR signaling by means of PI3K/Akt, MEK/ERK, and p38 MAPK contributes to epithelial cell proliferation between papillae; this biases against papilla differentiation and reduces numbers of papillae.     

Establishment and characterization of three novel human gastric cancer cell lines with differentiated intestinal phenotype derived from liver metastasis

Gastric cancers with liver metastasis are fatal diseases with rapid progression and poor patient outcome. To date, however, the molecular basis of their growth and metastasis remains essentially unknown, largely because of the presence of few available gastric cancer cell lines established from liver metastasis. In the present study, we developed two novel cultured cell lines (designated GLM-1 and GLM-2) and one transplantable line in nude mice (designated GLM-3) derived from liver metastasis of gastric cancer patients. These GLM cell lines share unique biological features such as differentiation, growth and metastasis. They form moderately differentiated tumors with CD10 positive and MUC2 negative intestinal absorptive phenotype when injected into nude mice. Their growth is stimulated by EGF and TGF-α in vitro like other gastric cancer cell lines. However, GLM cells differ from conventional gastric cancer cell lines in their high apoptotic rate, even in the absence of apoptosis inducing stimuli as revealed by Caspase3/7 assay and the TUNEL method. This apoptosis is further enhanced by phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), but not by MEK1/2 inhibitor (U0126), indicating the strong dependency of their survival on PI3K/Akt pathway rather than MAPK pathway, the major downstream signaling pathways of EGFR. GLM-1 cells can metastasize to the liver after intrasplenic injection, and GLM-3 cells have spontaneous lung metastatic potential after subcutaneous transplantation, respectively. These results indicate that the GLM series are the first cell lines reflecting the intestinal-type differentiated adenocarcinoma, a major subtype of gastric cancer with liver metastasis. Therefore, they would be excellent models for understanding the mechanism of metastatic growth and the development of a new molecular targeting therapy for gastric cancer with liver metastasis.     

米诺环素通过调控PI3K/Akt通路抑制硝普钠诱导的PC12细胞凋亡

 目的： 探讨PI3K/Akt信号通路在米诺环素（minocycline,MC）抑制硝普钠(sodium nitoprusside,SNP)诱导的PC12细胞凋亡中的作用。方法：将体外培养的PC12细胞分为4组：空白对照组、SNP组、MC+SNP组和PI3K抑制剂LY294002+ MC+SNP组。用四甲基偶氮唑盐（MTT）法检测细胞活力,流式细胞术检测细胞凋亡;Western blotting检测不同时点（0.5、1、2、3 h）各处理组PI3K/Akt通路蛋白p-Akt和Akt的表达。结果：SNP处理PC12细胞24 h能抑制细胞生长,加入10 μmol/L MC预处理30 min可明显提高细胞活力,降低细胞凋亡率（P<0.05）,抑制SNP诱导的PC12细胞凋亡。MC组的p-Akt表达高于其它组,而加入LY294002后可阻断MC的上述效应。结论：MC可通过调控PI3K/Akt通路抑制SNP诱导的PC12细胞凋亡。     

Fibroblast growth factor 8 induced downregulation of thrombospondin 1 is mediated by the MEK/ERK and PI3K pathways in breast cancer cells

Expression of fibroblast growth factor 8 (FGF-8) is increased in several forms of hormonal cancer. It was previously shown to regulate expression of thrombospondin 1 (TSP-1), an inhibitor of angiogenesis, in S115 breast cancer cells. Here, we studied the FGF-8-activated signalling pathways mediating TSP-1 repression in S115 cells and in non-tumorigenic MCF10A cells. Inhibition of FGF receptors or of MEK1/2 and PI3K with specific inhibitors (PD173074, U0126 or LY294002, respectively) restored TSP-1 mRNA expression in the presence of FGF-8 in S115 cells. Furthermore, U0126 and LY294002 increased TSP-1 mRNA expression in S115 cells over-expressing FGF-8. In MCF10A cells, FGF-8 treatment also decreased TSP-1 expression and the effect was dependent on active MEK1/2. In conclusion, FGF-8 suppresses TSP-1 expression through two independent pathways, MEK1/2 and PI3K. Repression of TSP-1 may be an important mechanism involved in induction of an angiogenic phenotype and growth of FGF-8-expressing breast cancer.     

欧前胡素通过下调c-met的表达提高肺癌CD133~+细胞亚群对吉非替尼的敏感性

目的:研究PC9 CD133~+细胞亚群对吉非替尼的耐药性并探讨欧前胡素提高吉非替尼抗肺癌活性的机制。方法:MTT法检测PC9细胞在吉非替尼和欧前胡素处理下的细胞活力。Western blot实验检测吉非替尼和欧前胡素对PC9细胞c-met表达水平、caspases活化水平及表皮生长因子受体(EGFR)、PI3K、AKT磷酸化水平的影响。流式细胞术检测欧前胡素和吉非替尼对PC9细胞系的CD133~+细胞亚群种群比例的影响及PC9细胞在二者处理下的凋亡率。结果:PC9 CD133~+细胞亚群对吉非替尼的敏感性显著低于PC9 CD133~-细胞亚群。吉非替尼能显著抑制PC9 CD133~-细胞亚群EGFR/PI3K/AKT的活化,但对PC9 CD133~+细胞亚群该通路的影响不大。吉非替尼单独处理能提高PC9细胞系中CD133~+细胞亚群的比例,然而联用欧前胡素后PC9 CD133~+细胞亚群的种群比例显著下降。Western blot实验表明欧前胡素能显著降低PC9 CD133~+细胞亚群的c-met蛋白表达水平表明c-met是欧前胡素的治疗靶点。MTT、Western blot、流式细胞术实验结果表明在PC9 CD133~+细胞亚群中,欧前胡素通过抑制c-met的表达提高吉非替尼对PI3K/AKT的抑制作用,从而诱导PC9 CD133~+细胞亚群发生caspases活化和凋亡。结论:欧前胡素通过下调c-met的表达提高肺癌CD133~+细胞亚群对吉非替尼的敏感性,两者存在协同抗肿瘤效应。     

Differential effect of FGF and PDGF on cell proliferation and migration in osteoblastic cells

It has been known that growth factors such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) can promote proliferation and migration in a variety of cell types including osteoblastic cells. However, the mechanism underlying their action has not been clearly defined. The present study was undertaken to examine the effect of FGF and PDGF on cell proliferation and migration and to determine the role of extracellular signal-regulated kinase (ERK) and Akt in action of FGF and PDGF in osteoblastic cells. FGF enhanced proliferation in a dose- and time-dependent manner, whereas it did not affect cell migration. FGF induced a transient activation of ERK, but not Akt, which was inhibited by an inhibitor of MEK, the upstream kinase of ERK, but not by inhibitors of PI3K/Akt (LY294002), epidermal growth factor receptor (EGFR, AG1478), and Src (PP2). FGF-induced proliferation was inhibited by inhibitors of MEK/ERK and Src pathways. Exposure of cells to FGF stimulated transition of cell cycle from the G1 phase to S phase and increased phosphorylation of Rb. FGF-induced phosphorylation of Rb was attenuated by inhibitors of MEK/ERK and Src pathways. Cell migration studies indicated that PDGF stimulated migration, but it had no effect on cell proliferation. PDGF induced activation of ERK and Akt. The ERK activatin was inhibited by the Src inhibitor and the Akt activation was inhibited by inhibitors of EGFR and Src. PDGF-induced migration was inhibited by inhibitors of MEK/ERK, PI3K/Akt, EGFR and Src pathways. Taken together, these findings suggest that the MEK/ERK and Src pathways play an important role in the FGF-induced proliferation and signaling pathways involving MEK/ERK, EGFR, Src and PI3K/Akt mediate the PDGF-induced migration. These data are of importance in understanding the roles of these growth factors in osteoblastic cell proliferation and migration.     

低表达S100钙离子结合蛋白A6促进食管腺癌SK-GT-4细胞的增殖和迁移

目的 探讨S100钙离子结合蛋白A6（S100A6）对食管腺癌SK-GT-4细胞增殖和迁移的影响。 方法 慢病毒转染,构建稳定细胞系shNC和shS100A6,Real-time PCR检测S100A6 mRNA表达情况;倒置显微镜和MTT检测细胞的增殖能力,Transwell检测细胞的迁移能力及U0126（MER1/2的抑制剂）、LY294002（PI3K抑制剂）对细胞增殖、迁移的影响;Western blotting检测细胞中S100A6、p-ERK、p-Akt及其下游参与增殖、迁移的相关蛋白的表达情况,检测U0126、LY294002对p-ERK、p-Akt及其下游参与增殖、迁移的相关蛋白表达的影响。 结果 构建了敲低S100A6的稳定细胞系,敲低S100A6促进了细胞的增殖和迁移,p-ERK、p-Akt水平升高,细胞周期抑制蛋白p21表达量下降、细胞周期蛋白D1(cyclinD1）表达升高,间质细胞标志蛋白——波形蛋白（vimentin）、β-连环蛋白（β-catenin）表达升高;U0126处理shS100A6细胞后,对细胞的增殖无影响,抑制细胞的迁移,p-ERK、β-catenin表达下降;LY294002处理shS100A6细胞后,抑制细胞的增殖和迁移,p-Akt表达下降,p21表达量升高,cyclinD1表达量下降,β-catenin表达下降。 结论 低表达S100A6促进细胞的增殖和迁移,其可能机制是通过p-Akt调控细胞周期的进程促进细胞增殖,通过激活p-Akt/p-ERK调控β-catenin促进细胞的迁移。     

乳腺癌细胞过表达HER2/neu时经PI3K和Ras/Raf/MEK/ERK信号通路下调野生型p53蛋白含量

目的 研究HER2/neu基因过表达对乳腺癌细胞野生型p53蛋白含量的影响并探讨其分子机制。方法 脂质体介导的HER2/neu基因转染MCF7细胞,G418筛选阳性克隆。Western印迹法鉴定HER2/neu蛋白表达,并同时检测p53及信号转导分子Akt,p-Akt,p-Raf,p-MEK,p-ERK蛋白含量,以及P13K信号通路抑制剂LY294002和MEK抑制剂U0126处理对p53蛋白含量的影响。逆转录-聚合酶链反应(RT-PCR)检测p53 mRNA水平。结果 成功建立稳定过表达HER2/neu的MCF7细胞(MCF7-neu3),其HER2/neu蛋白表达量是对照组MCF7细胞的13倍。MCF7-neu3中p-Akt,p-Raf,p-MEK,P-ERK蛋白含量分别较MCF7细胞升高了2.5倍、2倍、1.6倍和1.6倍(P<0.01),p53蛋白含量仅为对照组细胞的40％(P<0.01),但p53mRNA表达无明显差异。在经PI3K信号通路抑制剂LY294002及MEK抑制剂U0126处理24 h后,MCF7-neu3细胞p53蛋白含量分别上升了1.7和1.5倍(P<0.01),在LY294002和U0126处理48 h后,p53蛋白含量分别升高了4.7倍和5.3倍(P<0.01)。LY294002和U0126处理对MDA-MB-453细胞突变型p53蛋白含量不产生影响。结论 乳腺癌细胞中HER2/neu基因的过表达能够通过激活P13K和Ras/Raf/MEK/ERK通路导致野生型p53蛋白含量减少,可能是HER2/neu过表达乳腺癌患者预后不良及对治疗产生抗性的分子机制。     

PI3K/Akt通路在低氧诱导脂肪干细胞增殖和向内皮细胞分化中的作用

文题释义：PI3K/Akt信号通路：Akt是PI3K/Akt信号转导通路的核心,其分子质量约为60 kD,是原癌基因c-akt表达编码的一种丝氨酸/苏氨酸蛋白激酶。外源性抑制剂LY294002或wortmannin均可负向调节PI3K/Akt信号通路,导致第二信使PIP3逆行转化为PIP2,阻断其调节作用。 低氧：氧体积分数是机体微环境的重要组成部分,直接影响着细胞的成活和生物学行为。在体外培养过程中氧体积分数为1%-5%时即可为脂肪干细胞的增殖和功能提供足够的刺激,同时维持细胞形态和多向分化能力不变,因此可以认为低氧环境可以更好地为干细胞特性的维持提供信号传导。 背景：大量研究证明低氧可以促进干细胞的增殖和分化,但目前尚不清楚其通过何种途径发挥作用。 目的：观察低氧对脂肪干细胞增殖和向内皮细胞分化的影响,并探讨PI3K/Akt通路在此过程中的作用。 方法：取培养至第3代的Wistar大鼠脂肪干细胞,分为常氧组、低氧组、低氧+LY294002组,3组均在体积分数为20%O₂培养箱中培养24 h,然后低氧组转移至体积分数为2%O₂培养箱中进行培养,常氧组继续在体积分数为20%O₂培养箱中培养,低氧+LY294002组在低氧培养环境下的细胞培养基中加入终浓度为25 mmol/L的PI3K/Akt通路抑制剂LY294002。培养24 h后,Western blot检测p-Akt的表达明确PI3K/Akt通路的激活情况;CCK-8法检测各组脂肪干细胞的增殖情况;各组脂肪干细胞加入血管内皮细胞诱导培养基进行诱导分化10 d,分别通过qRT-PCR及抗CD31免疫荧光染色检测各组脂肪干细胞分化为内皮细胞的情况。 结果与结论：①第3代脂肪干细胞呈现出典型的梭形结构,流式细胞仪检测结果显示CD34和CD45表达阴性,CD105表达阳性,并可向成骨及成脂细胞方向分化;②低氧培养条件下p-Akt的表达明显升高,加入LY294002后p-Akt的表达受到明显抑制;③低氧组细胞增殖率明显高于常氧组和低氧+LY294002组(P < 0.05),低氧+ LY294002组脂肪干细胞的增殖率低于低氧组(P < 0.05);④低氧组内皮细胞基因及特异性蛋白CD31的表达明显高于常氧组和低氧+LY294002组(P < 0.05),低氧+LY294002组内皮细胞基因及特异性蛋白CD31的表达低于低氧组(P < 0.05),但仍高于常氧组(P < 0.05);⑤结果证实PI3K/Akt通路在低氧诱导的脂肪干细胞增殖及向内皮细胞分化过程中发挥重要作用。 ORCID: 0000-0001-8454-263X(殷令妮) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

PI3K/Akt通路在滋养细胞增殖中的调控机制研究

旨在探讨PI3K/Akt信号转导通路在滋养细胞增殖中的作用及具体调控机制。体外培养滋养细胞系EVT。应用MTT法检测不同浓度表皮生长因子（EGF）刺激后EVT的增殖情况;应用流式细胞技术检测不同处理组EVT凋亡情况。使用PI3K抑制剂LY294002处理细胞后,检测以上各项结果的变化。结果发现：1.随EGF浓度增高,EVT增殖呈现增强趋势,EGF在10ng/ml及其以上时效应明显;2.EGF能够显著降低EVT的凋亡发生;3.使用PI3K抑制剂LY294002明显逆转EGF的促进EVT增殖的效应。提示表皮生长因子可以活化滋养细胞的PI3K/Akt信号通路,进而促进细胞增殖,并且抑制其凋亡,PI3K抑制剂可以明显抑制EGF的促EVT增殖作用。     

Down-regulation of the PI3-kinase/Akt pathway by ERK MAP kinase in growth factor signaling

The ERK MAP kinase and PI3-kinase/Akt pathways are major intracellular signaling modules, which are known to regulate diverse cellular processes including cell proliferation, survival and malignant transformation. However, it has not been fully understood how these two pathways interact with each other. Here, we demonstrate that inhibition of the ERK pathway by the MEK inhibitor U0126 or PD98059 significantly potentiates EGF- and FGF-induced Akt phosphorylation at both Thr308 and Ser473. We also show that hyperactivation of the ERK pathway greatly attenuates EGF- and FGF-induced Akt phosphorylation. Furthermore, the enhanced Akt phosphorylation induced by U0126 is inhibited by the PI3-kinase inhibitor LY294002, and is accompanied by the up-regulation of Ras activity. These results suggest that the ERK pathway inhibition enhances Akt phosphorylation through the Ras/PI3-kinase pathway. Thus, our results demonstrate that the ERK pathway negatively modulates the PI3-kinase/Akt pathway in response to growth factor stimulation.     

S100A6通过PI3K/Akt信号通路促进人骨肉瘤细胞143B增殖和迁移

 目的：研究PI3K/Akt信号通路在S100A6介导的人骨肉瘤细胞143B的增殖和迁移中的作用。方法：首先制备重组的人S100A6蛋白（recombinant human S100A6, rhS100A6）;rhS100A6与PI3K抑制剂（LY294002和wortmannin）单独或同时处理143B细胞,其中rhS100A6的终浓度为30 mg/L,LY294002和wortmannin的终浓度分别为10 μmol/L和05 μmol/L;采用Western blotting分析143B细胞中PI3K/Akt信号通路相关分子总Akt（total Akt, t-Akt）及磷酸化Akt（phosphorylation of Akt, p-Akt）蛋白的表达变化,MTT检测细胞增殖,Transwell检测细胞迁移。 结果：（1）成功制备rhS100A6蛋白,rhS100A6显著增强143B细胞的增殖和迁移能力（P<005）;（2）rhS100A6上调143B细胞中Akt的磷酸化;（3）与rhS100A6组相比,rhS100A6与LY294002或wortmannin联合处理组143B细胞的p-Akt减少（P<005）,细胞的增殖和迁移能力降低,在不同时点细胞的增殖率下降103%～697%,细胞迁移率下降379%～416%,差异均有统计学意义（P<005）。 结论：S100A6促进人骨肉瘤细胞143B增殖和迁移的作用至少部分是通过激活PI3K/Akt信号通路实现的。     

U0126增强索拉非尼对K562细胞增殖抑制、凋亡及诱导分化作用

目的: 观察索拉非尼、索拉非尼联合MEK激酶抑制剂U0126对人慢性粒细胞白血病K562细胞株增殖、凋亡及分化的影响,并初步探讨其机制。方法: CCK-8法观察索拉非尼、U0126单用及不同浓度索拉非尼联合10 μmol/L U0126作用K562细胞48 h后细胞增殖活力变化;PI单染法及Hoechst 33342染色法观察索拉非尼、U0126单用及索拉非尼联合U0126对K562细胞株的凋亡诱导作用;细胞周期分析及联苯胺染色观察索拉非尼、U0126单用及低浓度索拉非尼联合U0126是否诱导K562细胞株向红系分化;采用免疫印迹法检测c-Myc蛋白表达。结果: MEK抑制剂U0126增强了索拉非尼对K562细胞增殖抑制、促凋亡及诱导分化作用。两药联用显著下调K562细胞c-Myc蛋白水平。结论: MEK抑制剂U0126增强索拉非尼对K562细胞增殖抑制、凋亡及诱导分化作用,这可能与其协同下调c-Myc蛋白有关。     

南蛇藤醇抑制宫颈癌细胞生物学行为的机制研究

目的　探讨南蛇藤醇抑制宫颈癌细胞增殖和侵袭的作用机制。 方法　将正常培养HeLa细胞分为对照组、南蛇藤醇低、中、高剂量组（终浓度分别为4 μmol/L、8 μmol/L和12 μmol/L）、阳性对照组（顺铂,终浓度为10 μmol/L）、LY294002组（LY294002,终浓度为20 μmol/L）和（南蛇藤醇+LY294002）组（南蛇藤醇和LY294002,终浓度分别为12 μmol/L和20 μmol/L）。MTT法检测各组细胞存活情况;流式细胞仪检测细胞凋亡情况;Transwell检测细胞的侵袭;蛋白质印迹法检测PI3K/AKT/NF-κB信号通路的表达;采用皮下注射HeLa细胞建立裸鼠模型,观察南蛇藤醇（120 mg/kg）对裸鼠移植瘤体积和重量的影响。 结果　与对照组相比,南蛇藤醇组、阳性对照组和LY294002组HeLa细胞的相对增殖率、侵袭细胞数均明显下降（P<0.001）,细胞凋亡率明显升高（P<0.001）,p-PI3K、p-AKT、NF-κB p65蛋白表达明显下调（P<0.001）,且随着南蛇藤醇浓度升高,其作用增强;与LY294002组相比,（南蛇藤醇+LY294002）组HeLa细胞的相对增殖率和侵袭细胞数明显下降（q=10.182,q=10.217,P均<0.001）,细胞凋亡率明显升高（q=23.636,P<0.001）;与对照组相比,南蛇藤醇组裸鼠体重、移植瘤的体积和重量明显下降（q=4.100,P<0.020;q=13.501,P<0.001;q=5.078,P=0.005）。 结论　南蛇藤醇可能通过抑制HeLa细胞的PI3K/Akt/NF-κB信号通路,抑制其恶性生物学行为。     

The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-speci?c inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.     

胰岛素样生长因子2通过磷酯酰肌醇-3激酶/蛋白激酶B信号通路调控人卵巢颗粒细胞增殖的机制

目的 检测胰岛素样生长因子2（IGF2）对人卵巢颗粒细胞（KGN细胞）增殖的调控作用及作用机制。 方法 将体外培养的KGN细胞,分不同浓度IGF2处理组（对照组和25 μg/L、50 μg/L、100 μg/L IGF2组)和磷酯酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)信号通路干预组（以LY294002干预处理将细胞分对照组、100μg/L IGF2组、LY294002组和IGF2+LY294002组）。采用MTS和5-乙炔基-2’-脱氧尿苷(EdU)法检测IGF2对KGN细胞增殖的影响,ELISA法检测细胞培养液上清中雌激素、孕激素的含量,Western blotting法检测各组胰岛素样生长因子受体1（IGF1R）、蛋白激酶B（Akt）、磷酸化的蛋白激酶B（p-Akt）及CYP19A1蛋白表达。 结果 随着IGF2的浓度梯度增高,KGN细胞的增殖率及雌激素、孕激素的分泌量逐渐升高,以 100 μg/L IGF2处理组的细胞增殖率和激素水平最高 (P<0.01),而抑制PI3K/Akt信号通路,细胞增殖率和激素的分泌量均明显降低(P<0.01);Western blotting结果显示,IGF1R、p-Akt及CYP19A1 蛋白表达水平随着IGF2的浓度梯度逐渐升高(P<0.05),而干预PI3K/Akt信号通路可影响上述蛋白的表达,与对照组相比,IGF2组和IGF2+ LY294002组IGF1R及p-Akt蛋白表达均明显升高（P<0.01）,CYP19A1在IGF2组明显升高（P<0.01）,LY294002组p-Akt及 CYP19A1蛋白明显降低（P<0.05）,IGF1R的表达差异无统计学意义。与IGF2组相比,IGF2+LY294002组p-Akt及CYP19A1蛋白表达降低（P<0.01）,IGF1R的表达差异无统计学意义,LY294002组p-Akt 及CYP19A1蛋白表达量均显著降低（P<0.01）。 结论 IGF2 可能由IGF1R介导通过PI3K/Akt 信号通路促进人卵巢颗粒细胞的增殖及分泌功能。     

索拉非尼联合柔红霉素对白血病K562细胞的抑制作用

目的:探讨小分子Raf激酶抑制剂索拉非尼(sorafenib)联合柔红霉素(DNR)对白血病细胞K562及U937的抑制作用及可能的分子机制。方法:MTT法测定索拉非尼和柔红霉素单独作用于K562和U937细胞的抑制率及DNR IC10联合不同浓度索拉非尼作用于K562及U937的联合抑制率;流式细胞AnnexinⅤ/PI法测定单药及联合用药后K562细胞的凋亡率以及Hoechst33258染色法观察单药及联合作用后细胞凋亡形态的改变;West-ern blotting法测定索拉非尼、DNR及U0126对K562及U937p-ERK1/2的影响;根据金氏方程证明2种药物联合抑制率及凋亡是否有协同作用。结果:MTT法测定索拉非尼联合DNR对K562及U937均有协同抑制作用(q1.15,P0.01);流式细胞AnnexinⅤ/PI和Hoechst33258染色法,均证明索拉非尼联合柔红霉素能联合诱导K562细胞凋亡(q1.15,P0.05),两者有明显的一致性;K562细胞的基础pERK1/2蛋白水平明显高于U937细胞(P0.01),索拉非尼和U0126都能够显著抑制K562细胞p-ERK1/2水平;U0126联合DNR存在协同抑制K562细胞的作用。结论:索拉非尼联合DNR作用于白血病细胞K562、U937存在协同抑制及凋亡诱导作用;DNR对U937的抑制作用明显高于K562;索拉非尼对K562的敏感性高于U937细胞;索拉非尼可能通过下调p-ERK1/2水平增加柔红霉素抗白血病细胞的效应。     

Fibroblast growth factor-2 and -4 promote the proliferation of bone marrow mesenchymal stem cells by the activation of the PI3K-Akt and ERK1/2 signaling pathways

Bone marrow mesenchymal stem cells (BMMSCs) have the capacity for self-renewal, and differentiation into a variety of cell types. They thus represent an attractive source of material for cell therapy. However, little is known about the mechanisms underlying the proliferation of BMMSCs. The purpose of this study was to identify the factors and signaling pathways involved in the proliferation of stem cell antigen-1(+) (Sca-1(+)) BMMSCs. Among the cytokines and growth factors examined in this study, fibroblast growth factor-2 (FGF-2) and FGF-4 significantly stimulated the proliferation of Sca-1(+) BMMSCs, as determined by bromodeoxyuridine incorporation. PI3K-Akt, ERK1/2, and JAK/STAT3 pathways were investigated after stimulation with FGF-2 or FGF-4 via Western blot analysis. No changes were observed in the total ERK1/2 and Akt; however, the pERK1/2 and pAkt levels were upregulated early within 15 min in the FGF-2- or FGF-4-treated Sca-1(+) BMMSCs. Moreover, the pERK1/2 and pAkt upregulation induced by FGF-2 and -4 were completely abolished by treatment with the MEK1/2 inhibitor, U0126 and the PI3K inhibitor, LY294002. However, no change in pJAK2 or total JAK2 levels was observed in the Sca-1(+) BMMSCs induced by FGF-2 or FGF-4. As a consequence of PI3K-Akt and ERK1/2, the upregulation of c-Jun in the Sca-1(+) BMMSCs, after stimulation with FGF-2 or FGF-4, was observed after 12 and 24 h. Moreover, the activation of c-Jun in FGF-2- and FGF-4-treated Sca-1(+) BMMSCs was significantly reduced by U0126. Taken together, these data suggest that FGF-2 and -4 promote the proliferation of Sca-1(+) BMMSCs by activation of the ERK1/2 and PI3K-Akt signaling pathways.     

Relationship between epidermal growth factor receptor (EGFR) mutation and serum cyclooxygenase-2 Level,and the synergistic effect of celecoxib and gefitinib on EGFR expression in non-small cell lung cancer cells

Epidermal growth factor receptor (EGFR) mutations occur mostly in patients with lung adenocarcinoma; such patients are also more likely to express cyclooxygenase-2 (COX-2), indicating a possible relationship between EGFR mutation and COX-2. The COX-2 and EGFR pathways mutually enhance their procarcinogenic effects in different tumor types. Therefore, simultaneous EGFR and COX-2 inhibition may be a promising therapeutic approach for patients with lung adenocarcinoma. We obtained tissue and serum samples from patients with non-small cell lung cancer (NSCLC) to detect the relationship between EGFR mutation and serum COX-2 level. Subsequently, gefitinib was combined with celecoxib to investigate the efficacy of inhibition in vitro in two NSCLC cell lines: HCC827 (del E746-A750) and A549 (wild-type EGFR). The cells were treated with gefitinib or celecoxib alone or with gefitinib plus celecoxib. Cell proliferation and apoptosis were assessed and correlated with expression of COX-2 and phosphorylated (p)-EGFR. The EGFR mutation rate of the high-COX-2 patients was significantly higher than that in the low-COX-2 patients. Multivariate analysis showed that high COX-2 levels were independently associated with EGFR mutation. Celecoxib and gefitinib inhibited cell growth in both cell lines. At sufficiently high concentrations, celecoxib plus gefitinib significantly mutually enhanced their anti-proliferative and apoptotic effects in both cell lines. At low concentrations, the combination had no additional effects on A549 cells. There was increased down regulation of COX-2 and p-EGFR when both cell lines were treated with high-concentration celecoxib plus gefitinib compared to either agent alone. This study demonstrates that high serum COX-2 levels may indicate EGFR mutations and that the efficacy of combined celecoxib and gefitinib is significantly greater in NSCLC cells with EGFR mutations; at high concentrations, the combination is efficacious in wild-type NSCLC cells.     

Angiotensin II regulation of TGF-beta in murine mesangial cells involves both PI3 kinase and MAP kinase

INTRODUCTION: Chronic activation of the angiotensin II (AngII) type 1 receptor (AT-1) is a central event in the development of chronic kidney disease (CKD), in part through enhanced expression of TGF-beta, and AT-1 receptor blockade inhibits the progression to CKD in a variety of disease states. The AT-1 receptor is a heptahelical Gaq/11-coupled receptor that initiates phospholipase C activity and release of intracellular calcium; recent data suggest that the AT-1 receptor can also activate the epidermal growth factor receptor (EGFR), although the roles of specific EGF-mediated signaling cascades in AT-1 effects on mesangial cell biology are uncertain. We hypothesized that 2 EGFR-activated pathways, PI3 kinase and MAP kinase, are stimulated by the AT-1 receptor and, in part, regulate the effects of AngII on TGF-beta1 levels in mesangial cells. METHODS: We examined the effects of AT-1 receptor activation on EGFR, PI3 kinase, and MAP kinase activation in murine mesangial cells. Upon achieving 60-80% confluence, the medium was changed to low-serum for 48 hr and cells were exposed to either the AT-1 receptor blocker, losartan, the EGFR blocker, AG1478, or control medium, and then stimulated with AngII. Similar experiments were performed using LY294002 and U0126, specific inhibitors of PI3 kinase and MEK, respectively. Total cellular protein lysates and RNA were isolated. Activation of the receptors and pathways was evaluated by immunoblotting and levels of TGF-beta mRNA were measured using real-time quantitative RT-PCR. RESULTS: AngII induced autophosphorylation of EGFR (pY1068) and activated Akt and ERK, downstream targets of PI3 kinase and MAP kinase, respectively. AngII-mediated EGFR autophosphorylation was inhibited by losartan and AG1478. AG1478 also inhibited both basal and AngII-mediated activation of Akt and ERK. Finally, AngII-mediated increase in TGF-beta mRNA was inhibited by losartan, AG1478, LY249002, and U0126. CONCLUSIONS: Stimulation of the AT-1 receptor in murine mesangial cells results in activation of the EGF receptor with subsequent signaling through PI3 kinase and MAP kinase, thereby regulating TGF-beta mRNA levels. These data suggest that AT-1 receptor signaling pathways through EGFR may serve as a therapeutic target to inhibit the development of CKD.