AG490抑制人胰腺癌细胞侵袭转移的体外研究

目的探讨Janus激酶抑制剂AG490对高转移潜能人胰腺癌细胞系SW1990体外侵袭转移能力的影响及其机制。方法用AG490处州SW1990细胞,细胞侵袭测定试剂盒检测细胞侵袭能力,Western blot检测信号传导与转录激活因子3(STAT3)、磷酸化STAT3(p-STAT3)、基质金属蛋白酶-2(MMP-2)和血管内皮生长因子(VEGF)蛋白的表达,RT-PCR检测MMP-2 mRNA和VEGF mRNA的表达。结果20μmol／L AG490可显著抑制SW1990细胞侵袭能力,侵袭抑制率为(77．67±7．79)％。Western blot显示,p-STAT3、MMP-2和VEGF的蛋白表达在SW1990细胞中明显减低。RT- PCR显示,MMP-2 mRNA和VEGF mRNA在SW1990细胞中亦明显减低。结论AG490通过阻断STAT3活化,下调MMP-2和VEGF表达,抑制胰腺癌细胞体外侵袭转移能力。阻断STAT3信号转导通路可能为预防和治疗胰腺癌的侵袭转移提供一种新的策略。     

ERK信号通路参与RANKL诱导的乳腺癌细胞迁移

目的：探讨细胞外调节蛋白激酶（ERK）信号通路在RANKL诱导的乳腺癌细胞迁移中的作用.方法：流式细胞术检测MDA-MB-231细胞表面RANK蛋白的表达;western-blot检测RANKL刺激后磷酸化ERK（P-ERK）及ERK的表达;Transwell法测定RANKL刺激后细胞迁移能力的改变.结果：MDA-MB-231细胞表达RANK蛋白,RANKL诱导MDA-MB-231细胞迁移能力增强.RANKL刺激后MDA-MB-231细胞P-ERK表达升高,PD98059抑制RANKL诱导的细胞迁移.结论：ERK信号通路参与RANKL诱导的乳腺癌细胞迁移.     

JAK/STAT通路及其阻断剂AG490在淋巴瘤中的研究进展

JAK/STAT是多种细胞因子和生长因子信号转导的重要途径,参与细胞增殖、分化以及免疫调节等多个过程,在肿瘤中的持续性激活可促进肿瘤的发生发展。目前研究发现在淋巴瘤中有STAT3异常表达和活化,其活化与肿瘤生长、侵袭及转移有关。AG490是JAK2抑制剂,可有效地抑制其下游的STAT的活化,阻断JAK/STAT信号转导通路。既往研究证实AG490能抑制淋巴瘤细胞的增殖,促进其凋亡,可提高某些化疗药物的敏感性。本文就JAK/STAT通路及其阻断剂AG490在淋巴瘤中的研究进展作一综述。      

ERK信号通路参与RANKL诱导的乳腺癌细胞迁移

目的:探讨细胞外调节蛋白激酶(ERK)信号通路在RANKL诱导的乳腺癌细胞迁移中的作用.方法:流式细胞术检测MDA-MB-231细胞表面RANK蛋白的表达;western-blot检测RANKL刺激后磷酸化ERK(P-ERK)及ERK的表达;Transwell法测定RANKL刺激后细胞迁移能力的改变.结果:MDA-MB-231细胞表达RANK蛋白,RANKL诱导MDA-MB-231细胞迁移能力增强.RANKL刺激后MDA-MB-231细胞P-ERK表达升高,PD98059抑制RANKL诱导的细胞迁移.结论:ERK信号通路参与RANKL诱导的乳腺癌细胞迁移.     

下调SRGN基因表达对乳腺癌细胞凋亡及JAK/STAT信号通路的影响

目的 探讨小分子糖蛋白丝甘蛋白聚糖(SRGN)在乳腺癌细胞中的表达及下调其表达对细胞凋亡和JAK/STAT信号通路的影响.方法 以正常乳腺上皮细胞MCF-10A为对照,采用Western blot检测乳腺癌细胞MDA-MB-231、SMMC-7721、MCF7、HCC1569中SRGN蛋白的表达水平.通过脂质体LipofectamineTM2000将SRGN siRNA转染MDA-MB-231细胞(siRNA-SRGN组),以siRNA-Con为阴性对照(siRNA-Con组),并设置空白对照组,收集转染48 h的细胞,Western blot检测各组细胞中SRGN、活化的含半胱氨酸的天冬氨酸蛋白水解酶3 (cleaved caspase 3)以及JAK/STAT信号通路中磷酸化的蛋白酪氨酸激酶2(p-JAK2)和磷酸化的信号转导与转录因子3(p-STAT3)的表达水平;流式细胞仪检测各组细胞的凋亡率.结果 乳腺癌细胞中SRGN蛋白的表达水平高于正常乳腺上皮细胞(P﹤0.05);siRNA-SRGN组的SRGN蛋白表达水平低于空白对照组(P﹤0.05);与空白对照组比较,siRNA-SRGN组的细胞凋亡率升高,cleaved caspase 3蛋白表达水平升高,p-JAK2和p-STAT3蛋白表达水平均下降,差异均有统计学意义(P﹤0.05).结论 SRGN在乳腺癌细胞中的表达水平升高,通过RNA干扰抑制其表达后可诱导癌细胞凋亡,并下调JAK/STAT信号通路蛋白的表达水平.     

JAK抑制剂AG490诱导乳腺癌细胞凋亡及对Survivin表达的影响

背景与目的:已有研究证实Survivin在多种乳腺癌中可呈高表达且与肿瘤的恶性程度和患者预后密切相关,但是Survivin在乳腺癌细胞中高表达的相关机制尚有待深入了解。本实验拟研究JAK-STAT信号通路抑制剂AG490在人乳腺癌细胞MDA-MB-231中对Survivin表达以及对肿瘤细胞增殖、凋亡的影响。方法:以MDA-MB-231细胞为研究对象,应用AG490后,用Westernblot检测细胞中P-STAT3、Survivin的蛋白表达变化、RT-PCR检测SurvivinmRNA的变化;用MTT法检测细胞增殖活性的改变;用流式细胞术检测细胞凋亡的情况。结果:AG490使MDA-MB-231细胞中P-STAT3和Survivin的蛋白表达减少,同时细胞的增殖活性降低,凋亡增多。P-STAT3/β-actin用药前为0.74,AG49040μmol/L作用24h后为0.21;Survivin/β-actin用药前为1.09,40μmol/LAG490作用24h后为0.28。40μmol/LAG490作用24h后细胞出现“亚G1”峰,凋亡率为5.62%;AG49040μmol/L,作用48h后细胞凋亡率为28.81%。结论:AG490可诱导MDA-MB-231细胞凋亡、减少Survivin的表达。     

莪术醇对乳腺癌细胞增殖凋亡及JAK2/STAT3信号通路的影响

[目的]探讨莪术醇对乳腺癌细胞增殖凋亡及JAK2/STAT3信号通路的影响。[方法]以不同浓度(0、12.5、25、50、100μg/ml)莪术醇作用于乳腺癌MCF-7细胞,MTT法检测MCF-7细胞增殖抑制率,流式细胞术观察细胞周期凋亡分布,Hoechst33258染色法观察细胞凋亡形态学变化,逆转录聚合酶链反应法检测细胞酪氨酸激酶2(JAK2)、信号转导子和转录激活子3(STAT3)信使核糖核酸相对表达量,蛋白印迹法检测细胞磷酸化JAK2(P-JAK2)、磷酸化STAT3(P-STAT3)蛋白相对表达量。[结果]莪术醇对MCF-7细胞增殖具有明显抑制作用,且抑制率随药物作用时间及作用浓度的增加而上升。随着莪术醇作用浓度的增加,细胞凋亡率也有所上升[(4.27%±0.82%)、(4.54%±1.21%)、(19.32%±1.87%)、(29.58%±2.92%)、(33.74%±3.91%)、(42.94%±2.81%)]。细胞周期结果显示,莪术醇可将细胞周期阻滞在G0/G1期,从而抑制细胞增殖。Western blot结果显示,莪术醇可抑制JAK2、STAT3磷酸化的表达,而RT-PCR结果显示,莪术醇作用细胞后,JAK2、STAT3 mRNA表达受到明显抑制。[结论]莪术醇作用于乳腺癌MCF-7细胞G0/G1期,从而抑制细胞增殖,诱导细胞凋亡,其机制可能是通过JAK2/STAT3信号通路来实现。     

阻断STAT3信号转导通路对人胰腺癌细胞生长的影响

目的：探讨Janus激酶（Janus kinase，JAK）特异性抑制剂AG490阻断STAT3信号转导通路对高表达STAT3的人胰腺癌细胞系SW1990生长增殖的怍用及其机制。方法：使用AG490处理人胰腺癌细胞系SW1990，MTT法检测细胞增殖状态，流式细胞仪检测细胞凋亡，Western blot检测STAT3信号转导通路成员的表达。结果：AG490作用于人胰腺癌细胞系SW1990后，细胞增殖水平明显下降（P〈0．05）；细胞凋亡百分比显著升高（P〈0．05）；Western blot显示SW1990细胞中磷酸化STAT3（p-STAT3）、bcl-xL和cyclin D1蛋白表达明显降低（P〈0．05）。结论：STAT3信号转导通路在胰腺癌进展过程中起着重要作用；阻断STAT3信号转导通路可抑制人胰腺癌细胞增殖，促进其凋亡；以STAT3信号转导通路为靶点的治疗策略可能为胰腺癌治疗提供新的思路。     

JAK/STAT通路阻断剂AG490治疗肿瘤的研究进展

AG490是一种PTK抑制物,可阻断JAK-STAT3信号通路。而JAK-STAT3信号通路与肿瘤的增殖、凋亡密切相关。体内外研究表明将AG490与化疗药物协同作用于肿瘤,可增加肿瘤细胞凋亡百分比,提高化疗药物的敏感性。因此,JAK激酶抑制剂AG490作为以肿瘤细胞信号转导分子为靶点的小分子抑制剂可能成为新一代的抗肿瘤药物。     

Effects of inhibiting JAK on invasion and metastasis of the human breast cancer cells through ERK signaling transduction pathway

Objective: To explore the effects of Janus activated kinase (JAK) inhibitor AG490 on the phosphorylation of extracellular signal regulated protein kinase (ERK) in human breast cancer cells MDA-MB-231 and the roles of JAK in the invasion and metastasis of the human breast cancer cells through ERK signaling transduction pathways.Methods: MDA-MB-231 cells were treated with 20 (mol/L, 40 (mol/L, 80 (mol/L Janus kinase inhibitor AG490 for 24, 48 and 72 h. Proliferation and adhesion of MDA-MB-231 cells to matrigel were measured with MTT assay. When treated with 40 (mol/L AG490 for 24 h, the expressions of P-ERK and MMP-9 of cells were detected by Western-blot and invasion and metastasis of MDA-MB-231 cells were evaluated with transwell chamber.Results: After being treated with 20 (mol/L, 40 (mol/L, 80 (mol/L AG490 for 24, 48 and 72 h, the proliferation of MDA-MB-231 cells was inhibited in a dose-and time-dependent manner. MDA-MB-231 cells treated with 40 (mol/L AG490 for 30, 60, 90 and 120 min resulted in the increasing adhesion of cells to Matrigel in a time-dependent manner. However, capacity of adhesion in the group treated with AG490 was significantly decreased in comparison with the control group (P<0.01). The expression level of P-ERK and MMP-9 were decreased when treated with AG490. After treatment with 40 (mol/L AG490, in invasion assay, the number of cells in AG490 treated group to migrate to filter coated with Matrigel was reduced compared with control group (P<0.05). Meanwhile, in migration assay, the number of cells in AG490 treated group to migrate to filter was also decreased compared with control group (P<0.05).Conclusion: Our study indicates that JAK kinase could affect the activity of ERK signal transduction pathway through the phosphorylation of ERK. The inhibitory effects of JAK kinase on MMP-9 expression and invasion of breast cancer cells were associated with the down-regulation of the ERK signaling pathway.     

CKIs对人乳腺癌细胞侵袭转移的影响

目的利用CKI肽类似物抑制CDK激酶活性,以明确CKI对人乳腺癌细胞的作用,探讨其对乳腺癌细胞体外侵袭转移的影响。方法利用作用于CDK4／CDK6的CKI肽类似物p20和p21,分别干预人乳腺癌MDA-MB-231细胞株24h。然后应用细胞与纤维连结蛋白黏附实验测定肿瘤细胞黏附率,通过单层细胞划痕实验观察细胞转染前后迁移能力的变化,Transwell侵袭实验检测细胞体外侵袭力。结果人乳腺癌MDA-MB-231细胞株经CKI肽类似物p20和p21分别干预后,细胞黏附、迁移及侵袭能力明显减弱。结论 CDK4/CDK6激酶对乳腺癌侵袭转移过程具有重要的作用。CKI肽类似物可以抑制乳腺癌MDA-MB-231细胞的黏附、侵袭和迁移。因此CKI肽类似物可能阻抑肿瘤细胞的转移,显示此类细胞穿膜肽在恶性肿瘤治疗上的潜在作用。     

FRZB is Regulated by the Transcription Factor EGR1 and Inhibits the Growth and Invasion of Triple-Negative Breast Cancer Cells by Regulating the JAK/STAT3 Pathway

BackgroundTo explore the expression of frizzled related protein (FRZB) in triple-negative breast cancer (TNBC) and role of FRZB in TNBC cell growth and invasion.MethodsBreast cancer clinical data were downloaded from the Cancer Genome Atlas. FRZB and early growth response 1 (EGR1) mRNA levels in TNBC were measured by quantitative real-time polymerase chain reaction. FRZB protein level was measured by immunohistochemistry and western blot. Proliferation, migration, and invasion of TNBC cells were detected by colony formation, wound healing, and transwell assay, respectively. The protein levels of EGR1, E-cadherin, N-cadherin, Snail, p-JAK1/JAK1, p-JAK2/JAK2, and p-STAT3/STAT3 were measured by western blot. JASPAR was used to predict the binding site of FRZB and EGR1. The binding ability of FRZB and EGR1 was verified by dual-luciferase reporter gene assay and chromatin immunoprecipitation assay.ResultsFRZB was low expressed in TNBC tissues and cells. Silencing FRZB promoted cell proliferation, migration, invasion, and EMT and activated JAK/STAT pathway in MDA-MB-468 and MDA-MB-231 cells, but overexpression of FRZB acted opposite effects in MDA-MB-468 and MDA-MB-231 cells. EGR1 was low expressed in TNBC samples and positively correlated with FRZB. Moreover, EGR1 could recover the promotion of silencing FRZB on cell proliferation, migration, invasion, and JAK/STAT pathway in MDA-MB-468 cells, but silencing EGR1 led to the opposite results in MDA-MB-231 cells.ConclusionFRZB was low expressed in TNBC and was regulated by EGR1, and FRZB inhibited TNBC cell growth and invasion by regulating the JAK/STAT3 pathway.