黄腐酚抑制人前列腺癌PC3细胞增殖机制初探

目的：初步探讨黄腐酚抑制人前列腺癌 PC3细胞增殖机制。方法：检测细胞周期和凋亡分析黄腐酚对 PC3细胞增殖的影响;Western blot 分析黄腐酚对 Notch 信号通路关键蛋白的作用。结果：黄腐酚可以抑制PC3细胞增殖,同时降低 Notch 信号通路活性。在黄腐酚对 Notch 信号通路的作用机制研究中发现,黄腐酚能够降低 GSK －3β活性,引起 Notch 信号通路活性降低。结论：黄腐酚可能是通过抑制 GSK －3β活性从而降低Notch 信号通路活性,最终抑制 PC3细胞的增殖。     

细胞外信号调节激酶1/2参与三氧化二砷诱导的胶质瘤细胞凋亡

目的:探讨细胞外信号调节激酶1/2(ERK1/2)信号通路在三氧化二砷诱导的胶质瘤细胞U251凋亡中的作用,为应用三氧化二砷治疗胶质瘤奠定基础。方法:50μmol/L三氧化二砷作用U251细胞,不同时间检测胶质瘤细胞增殖活性,半定量PCR检测ERK1/2mRNA表达,Western blot检测ERK1/2蛋白表达;Ho-echst33258染色及流式细胞术(FCM)检测细胞凋亡;转染ERK1/2上游激酶MEK1,应用ERK1/2激酶抑制剂U0126观察ERK1/2通路在肿瘤凋亡及增殖中的作用;比色分析法检测Caspase-3活性的变化。结果:三氧化二砷诱导胶质瘤细胞发生明显凋亡,抑制肿瘤细胞增殖;增加ERK1/2蛋白的表达,呈时间依赖性;阻断ERK1/2信号通路后胶质瘤细胞凋亡受到抑制,Caspase-3活性下降。结论:ERK1/2信号通路在三氧化二砷诱导的胶质瘤细胞凋亡中起重要作用。     

miR-214通过PTEN调控AKT信号通路抑制鼻咽癌细胞凋亡CSCD

目的探讨miR-214通过PTEN调控下游AKT信号通路激活对鼻咽癌细胞凋亡的影响。方法miR-214抑制剂转染鼻咽癌5-8F和6-10B细胞。MTT法评估细胞增殖情况。Annexin-V/PI染色法检测两种鼻咽癌细胞凋亡情况。Western blot和real-time PCR检测miR-214抑制剂对PTEN基因转录和蛋白表达以及对AKT信号通路和凋亡相关蛋白表达水平的影响。检测shRNA沉默PTEN基因转录和蛋白表达后,miR-214抑制剂对鼻咽癌细胞凋亡影响。结果miR-214抑制剂可抑制鼻咽癌细胞生长,诱导5-8F和6-10B细胞凋亡。miR-214通过靶向3'-UTR区域调控PTEN基因转录和蛋白表达。抑制miR-214可促进PTEN的表达,抑制AKT信号通路激活,进而调控下游细胞周期和凋亡相关蛋白表达。沉默PTEN基因转录和蛋白表达可逆转miR-214抑制剂对鼻咽癌细胞AKT信号通路活性和凋亡的影响。结论miR-214可通过直接靶向PTEN基因转录促进AKT信号通路激活抑制鼻咽癌细胞凋亡。     

MEK/ERK抑制剂影响胃癌细胞对5-FU敏感性及其机制的探讨

目的:探讨MEK/ERK特异性抑制剂PD98059在5-氟尿嘧啶(5-FU)抗胃癌细胞增殖中的作用。方法:应用5-FU与MEK/ERK抑制剂(PD98059)处理胃癌细胞,MTT法检测细胞增殖状态,流式细胞术检测细胞凋亡,蛋白质印迹法检测ERK和p-ERK的表达。结果:随着5-FU浓度的增加,5-FU对3种胃癌细胞系的增殖抑制率逐渐增大。5-FU作用MGC803细胞72 h的抑制率显著高于48和24 h,t值分别为16.477和25.349,P值分别为0.002和0.001。与对照相比,5-FU作用MGC803细胞48和72 h可引起显著的凋亡,t值分别为-20.576和-40.389,P值分别为0.015和0.001。5-FU处理胃癌MGC803细胞48 h,与对照相比pERK表达一过性下降然后升高。20μmol/L的PD98059联合5-FU作用48 h,可明显抑制pERK的表达,抑制率为22%;同时可明显增加细胞凋亡,增加率为20%。结论:MEK/ERK信号传导通路在5-FU作用胃癌细胞的过程中被激活,联合应用MEK/ERK信号通路抑制剂可部分逆转ERK的活化,增加胃癌细胞凋亡,从而增加胃癌细胞对5-FU的敏感性。     

MEK抑制剂增加乳腺癌细胞MCF-7对表柔比星的敏感性

背景与目的: 化疗是目前乳腺癌治疗的重要手段之一,通过抑制肿瘤细胞增殖,促进肿瘤细胞凋亡发挥抗肿瘤功效.有丝分裂原激活蛋白激酶(mitogen activated protein kinase,MAPK)信号传导通路作鼢用于核内转录因子,与细胞增殖和凋亡关系密切.因此MAPK信号传导通路在化疗中所起的作用日益受到人们的重视,期望抑制此通路能改善化疗药物的疗效,从而低毒高效的治疗肿瘤.本研究通过检测表柔比星与MEK抑制剂PD98059对人乳腺癌细胞细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)活性的影响(MEK、ERK分别是MAPK{~号传导通路中的环节),探讨MEK抑制剂PD98059在表柔比星抗人乳腺癌细胞中的作用.方法: 应用表柔比星与MEK抑制剂(PD98059)处理MCF-7及MCF-7/ADR细胞,应用Western印迹法检测MEK2和p-ERK表达情况,应用MTT法检测细胞增殖状态.结果: 表柔比星处理细胞后,MCF-7的ERK蛋白活性上升,加用MEK抑制剂PD98059,细胞对表柔比星的敏感性增加.结论: MAPK信号传导通路在表柔比星杀伤敏感细胞的过程中被激活,联合应用MAPK信号传导通路抑制剂可以增加表柔比星敏感性.     

LncRNA FENDRR通过调控ERK/MAPK影响肺鳞癌H226细胞增殖、迁移和凋亡

目的 探讨lncRNA FENDRR对肺鳞癌细胞H226的增殖、迁移、侵袭和凋亡的影响及分子机制。方法 qRT-PCR检测肺正常上皮细胞BEAS、肺腺癌细胞A549、H1299和肺鳞癌细胞H226中FENDRR的表达水平。将H226细胞分为FENDRR-siRNA组和FENDRR-siNC阴性对照组；CCK-8法检测细胞增殖活力，Transwell小室检测细胞迁移和侵袭能力；流式细胞术检测细胞凋亡；Western blot检测细胞MEK、p-MEK、ERK、p-ERK蛋白表达。结果 与肺正常上皮细胞相比，肺鳞癌细胞H226中FENDRR水平明显下降。下调肺鳞癌细胞H226中FENDRR的表达后，p-MEK和p-ERK蛋白表达水平显著增多，H226细胞增殖活力、迁移及侵袭能力显著增加，细胞凋亡率明显降低。加入ERK抑制剂U0126能逆转FENDRR表达下调对H226细胞的作用。结论 低表达FENDRR后，可以通过ERK/MAPK信号通路，促进H226细胞的增殖、迁移和侵袭，并抑制其凋亡。     

ERK通路在调节子宫内膜癌增殖凋亡侵袭中的作用

背景与目的 探讨胰岛素/细胞外信号调节激酶(ERK)信号通路在调节子宫内膜癌细胞增殖、凋亡和侵袭中的作用.方法 将无血清饥饿的子宫内膜癌Ishikawa3-H-12细胞分为空白对照组、1×10<'6>mol/L胰岛素单独刺激组以及不同浓度MEK抑制剂PD98059预处理后再用胰岛素刺激组.Western blot检测各组ERK磷酸化(P-EPK)水平,MTF试验观察细胞增殖、流式细胞仪观察细胞凋亡、transwell小室观察细胞侵袭情况.结果 胰岛素可引起子宫内膜癌细胞ERK活化并以浓度依赖模式被PD98059完全阻断.胰岛素显著促进子宫内膜癌细胞增殖.PD98059可以阻断胰岛素诱导的细胞增殖,在24 h、48 h、72 h三个时间点均有统计学差异(F=204.160;33.850;90.764,均P<0.001).胰岛素显著抑制子宫内膜癌细胞凋亡,并以浓度依赖模式为PD98059所阻断(F=165.99,P<0.001).胰岛素明显增加子宫内膜癌细胞侵袭,PD98059可以部分阻断胰岛素诱导的细胞侵袭(F=23.841,P<0.001).结论 胰岛素可以促进子宫内膜癌细胞增殖、抑制细胞凋亡,此过程是ERK途径依赖的;胰岛素可以增加子宫内膜癌细胞侵袭能力.此过程是部分ERK途径依赖的.     

姜黄素调控Notch信号通路对肺癌干细胞增殖及凋亡的影响

目的:研究姜黄素对肺癌干细胞增殖及凋亡的影响。方法:利用免疫磁珠标记肺癌表面标记物乙醛脱氢酶1(ALDH1)，从肺癌A549细胞系中分离肺癌干细胞；MTT实验检测姜黄素对肺癌干细胞增殖的影响；流式细胞仪检测姜黄素对肺癌干细胞凋亡的影响；Western blot检测Notch1和Hes1蛋白的表达情况；抑制剂γ-分泌酶抑制Notch信号通路研究对肺癌干细胞增殖及凋亡的影响。结果:利用免疫磁珠分选法分选出肺癌细胞系A549中ALDH1+肺癌干细胞。MTT检测结果显示，姜黄素能够呈浓度依赖性的抑制肺癌干细胞的增殖；经姜黄素处理后的肺癌干细胞，与对照组相比，凋亡率明显升高，Notch1和Hes1蛋白的表达明显下降。经γ-分泌酶抑制剂处理肺癌干细胞后，实验结果显示，γ-分泌酶抑制剂通过下调Notch1和Hes1蛋白的表达调控Notch信号通路抑制肺癌干细胞的增殖，诱导其凋亡。结论:姜黄素抑制肺癌干细胞的增殖，诱导其凋亡与Notch信号通路有关。     

选择性COX-2抑制剂对非小细胞肺癌细胞株毒性及分子机制的研究

目的：探讨选择性环氧化酶-2（cyclooxy-genase-2,COX-2）抑制剂Celecoxib对非小细胞肺癌（non-small cell lung cancer,NSCLC）细胞株增殖与凋亡的作用以及相关分子机制。方法：对3种NSCLC细胞株A549（腺癌）、GLC82（腺癌）和SW1573（肺泡细胞癌）使用MTT法测定细胞生长抑制率;Hoechest33258荧光染色观察细胞凋亡;流式细胞仪分析细胞凋亡及细胞周期分布;West-ern blot法检测COX-2、磷酸化AKT（p-AKT）、总丝苏氨酸激酶（serine/threonine kinase,AKT）、磷酸化ERK（p-ERK）及总细胞外信号调节激酶（ex-tracellular signal-regulated kinase,ERK）蛋白的表达。结果：Celecoxib抑制A549、GLC82和SW1573细胞增殖的IC50值分别为14.7、10.6和18.6μmol/L;Celecoxib对3种NSCLC细胞株都有较明显的凋亡诱导作用,Celecoxib作用12h凋亡率增加,24～48h更明显,Celecoxib10～40μmol/L作用可见凋亡率明显增加;Celecoxib作用于GLC82细胞后检测到胞内p-AKT及p-ERK蛋白表达下调。结论：COX-2抑制剂Celecoxib在体外能抑制细胞信号传导AKT及ERK通路的活化,诱导NSCLC细胞发生凋亡,抑制细胞增殖。     

唑来膦酸与紫杉醇体外协同抗肺腺癌细胞增殖及诱导凋亡的分子机制研究

  目的   探讨唑来膦酸（zoledronic acid,ZOL）及联合紫杉醇（paclitaxel,PTX）体外对人肺腺癌A-549细胞增殖影响及潜在机制。   方法   实验组包括不同浓度ZOL组、2 nM PTX组与ZOL联合应用PTX加药顺序不同3个组。MTT方法观测不同浓度ZOL与联合PTX对A-549细胞增殖的影响;流式细胞仪检测各组细胞凋亡;RT-PCR检测各组ERK和AKT mRNA的变化;Western blot检测各组ERK、AKT蛋白表达及磷酸化水平和Bcl-2的表达。   结果   :ZOL能抑制A-549细胞生长,该作用随ZOL浓度增加而增强;ZOL与PTX具有协同抗肿瘤作用,作用大小与两药加药顺序有关,产生最大抑制作用的顺序为先PTX后ZOL;RT-PCR结果显示不同组ERK和AKT mRNA表达水平分别为空白对照组最高,单药ZOL组次之,再次为PTX组,先PTX后ZOL组最低;Western blot结果显示各实验组中ERK、AKT蛋白表达水平无差异,但ERK和AKT磷酸化水平有差异,分别为空白对照组最高,单药ZOL组次之,再次为PTX组,先PTX后ZOL组最低,Bcl-2的表达水平亦是如此。   结论   ZOL联合PTX可能通过下调RAF/MEK/ ERK信号通路中ERK基因mRNA的表达,降低ERK磷酸化水平,抑制ERK激酶活性,进而抑制A-549细胞生长和增殖;同时可能通过下调PI3K/AKT信号通路中AKT基因mRNA的表达,降低AKT磷酸化水平,进而减弱AKT活性,进一步降低抗凋亡蛋白Bcl-2水平,从而起到促凋亡作用。      

Suppression of the notch signaling pathway by γ-secretase inhibitor GSI inhibits human nasopharyngeal carcinoma cell proliferation

Notch signaling has been suggested to be required for many human cancers. However, the role of Notch signaling in human nasopharyngeal carcinoma cells (NPC) remains unknown. Here, we report that Notch-1, Notch-2, Notch-3 and Notch-4 are all detected in NPC cells. Notch inhibitor, GSI, suppresses the levels of Notch-1, Notch-2 and Notch-4, but not Notch-3. In addition, GSI inhibits NPC cell proliferation by inducing the cell cycle arrest and apoptosis. Furthermore, GSI inhibits the AKT and MEK signaling, without affecting P38 and JNK1/2. Thus, NPC cells may up-regulate Notch signaling to maintain cell proliferation and targeting the Notch signaling pathway may offer a potential alternative strategy for the treatment of NPC.     

Notch and BCR signaling synergistically promote the proliferation of Raji B-lymphoma cells

The evolutionarily conserved Notch signaling pathway plays a pivotal role in cell proliferation, apoptosis, and cell fate decision from invertebrates to vertebrates, and is oncogenic in some human hematopoietic malignancies. To study the role of Notch signaling in B-lymphoma, we expressed a soluble fragment of human Delta-like1 (hDll1) in E. coli, which was shown to activate the Notch signaling. Incubation of Burkitt's lymphoma Raji cells with the soluble hDll1 led to gamma-secretase-dependent up-regulation of a Notch downstream gene, Hes1. This treatment synergized with B-cell receptor (BCR)-mediated signaling to promote proliferation of Raji cells in vitro, which was cancelled by GSI. We further showed that Notch signaling significantly repressed, while gamma-secretase inhibitor (GSI) enhanced, "natural" apoptosis of Raji cells. Because c-myc is a downstream gene of both Notch signaling and BCR signaling, and GSI blocked c-myc expression in the presence of hDll1 and anti-IgM, Notch signaling might interact with BCR signaling at the level of c-myc expression to regulate proliferation and apoptosis of B-lymphoma cells.     

Gamma secretase inhibitor blocks Notch activation and induces apoptosis in Kaposi's sarcoma tumor cells

Kaposi's sarcoma (KS) is a common neoplasm in HIV-1-infected individuals causing significant morbidity and mortality. Despite recent advances, the pathogenesis of this potentially life-threatening neoplasm remains unclear, and there is currently no cure for KS. Notch proteins are known to play a fundamental role in cell fate decisions including proliferation, differentiation, and apoptosis. It is, therefore, not surprising that Notch proteins have been implicated in tumorigenesis and appear to function as either oncogenes or tumor suppressor proteins depending on cellular context. In this report, we demonstrate elevated levels of activated Notch-1, -2, and -4 in KS tumor cells in vivo and in vitro compared to endothelial cells, the precursor of the KS cell. Notch activation was confirmed through luciferase reporter assays and localization of Hes (Hairy/Enhancer of Split)-1 and Hey (Hairy/Enhancer of Split related with YRPW)1 (primary targets of the Notch pathway) in KS cell nuclei. Studies using gamma-secretase inhibitors (GSI and LY-411,575), which block Notch activation, resulted in apoptosis in primary and immortalized KS cells. Similar studies injecting GSI into established KS cell tumors on mice demonstrated growth inhibition or tumor regression that was characterized by apoptosis in treated, but not control tumors. The results indicate that KS cells overexpress activated Notch and interruption of Notch signaling inhibits KS cell growth. Thus, targeting Notch signaling may be of therapeutic value in KS patients.     

Notch-1信号通路在甲状腺癌中作用的研究进展

Notch信号通路参与调控机体细胞的增殖、分化、凋亡等，人类Notch信号通路包括了四种受体及五种配体，其中Notch-1受体在甲状腺癌中发挥重要作用。然而，Notch-1在甲状腺乳头状癌中的作用仍存在争议，大多数学者认为Notch-1信号通路能够促进乳头状癌细胞增殖、侵袭转移，且其表达水平与甲状腺乳头状癌侵袭性相关。而对于甲状腺滤泡状癌、髓样癌及未分化癌细胞，活化Notch-1信号通路可抑制他们的增殖。众多研究表明Notch-1信号通路有望成为甲状腺癌重要治疗靶点。本文就Notch-1在甲状腺癌发生、发展中的作用进行综述。     

Role of stromal cell-mediated Notch signaling in CLL resistance to chemotherapy

Stromal cells are essential components of the bone marrow (BM) microenvironment that regulate and support the survival of different tumors, including chronic lymphocytic leukemia (CLL). In this study, we investigated the role of Notch signaling in the promotion of survival and chemoresistance of human CLL cells in coculture with human BM-mesenchymal stromal cells (hBM-MSCs) of both autologous and allogeneic origin. The presence of BM-MSCs rescued CLL cells from apoptosis both spontaneously and following induction with various drugs, including Fludarabine, Cyclophosphamide, Bendamustine, Prednisone and Hydrocortisone. The treatment with a combination of anti-Notch-1, Notch-2 and Notch-4 antibodies or γ-secretase inhibitor XII (GSI XII) reverted this protective effect by day 3, even in presence of the above-mentioned drugs. Overall, our findings show that stromal cell-mediated Notch-1, Notch-2 and Notch-4 signaling has a role in CLL survival and resistance to chemotherapy. Therefore, its blocking could be an additional tool to overcome drug resistance and improve the therapeutic strategies for CLL.     

Knockdown of Notch1 inhibits nasopharyngeal carcinoma cell growth and metastasis via downregulation of CCL2, CXCL16, and uPA

Notch pathway is a highly conserved cell signaling system that plays very important roles in controlling multiple cell differentiation processes during embryonic and adult life. Multiple lines of evidence support the oncogenic role of Notch signaling in several human solid cancers; however, the pleiotropic effects and molecular mechanisms of Notch signaling inhibition on nasopharyngeal carcinoma (NPC) remain unclear. In this study, we evaluated Notch1 expression in NPC cell lines (CNE1, CNE2, SUNE1, HONE1, and HK1) by real‐time quantitative PCR and Western blot analysis, and we found that CNE1 and CNE2 cells expressed a higher level of Notch1 compared with HONE1, SUNE1, and HK1 cells. Then Notch1 expression was specifically knocked down in CNE1 and CNE2 cells by Notch1 short hairpin RNA (shRNA). In Notch1 knockdown cells, cell proliferation, migration, and invasion were significantly inhibited. The epithelial‐mesenchymal transition of tumor cells was reversed in Notch1‐shRNA‐transfected cells, accompanied by epithelioid‐like morphology changes, increased protein levels of E‐cadherin, and decreased expression of vimentin. In addition, knockdown of Notch1 markedly inhibited the expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and chemokines C‐C motif chemokine ligand 2 and C‐X‐C motif chemokine ligand 16, indicating that these factors are downstream targets of Notch1. Furthermore, deleting uPA expression had similar effects as Notch1. Finally, knockdown of Notch1 significantly diminished CNE1 cell growth in a murine model concomitant with inhibition of cell proliferation and induction of apoptosis. These results suggest that Notch1 may become a novel therapeutic target for the clinical treatment of NPC.     

Dual antitumor mechanisms of Notch signaling inhibitor in a T-cell acute lymphoblastic leukemia xenograft model

Constitutive activation of Notch signaling is required for the proliferation of a subgroup of human T-cell acute lymphoblastic leukemias (T-ALL). Previous in vitro studies have demonstrated the therapeutic potential of Notch signaling inhibitors for treating T-ALL. To further examine this possibility, we applied a γ-secretase inhibitor (GSI) to T-ALL xenograft models. Treatment of established subcutaneous tumors with GSI resulted in partial or complete regression of tumors arising from four T-ALL cell lines that were also sensitive to GSI in vitro . To elucidate the mechanism of action, we transduced DND-41 cells with the active form of Notch1 (aN1), which conferred resistance to in vitro GSI treatment. Nevertheless, in vivo treatment with GSI induced a partial but significant regression of subcutaneous tumors that developed from aN1-transduced DND-41 cells, whereas it induced complete regression of tumors that developed from mock-transduced DND-41 cells. These findings indicate that the remarkable efficacy of GSI might be attributable to dual mechanisms, directly via apoptosis of DND-41 cells through the inhibition of cell-autonomous Notch signaling, and indirectly via disturbance of tumor angiogenesis through the inhibition of non-cell-autonomous Notch signaling. ( Cancer Sci 2009; 100: 2444–2450)