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。     

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。     

p-STAT3通过上皮间质转化影响结肠癌的转移性研究

  目的   研究p-STAT3的活化在结肠癌中的表达与Snail、MMP2的相关性,及其在离体细胞实验中激活受抑后对结肠癌细胞迁移能力的影响并探讨其机制。  方法  免疫组织化学染色检测p-STAT3与Snail、MMP2的表达及其相关性,分析三者与TNM分期、远处转移、淋巴结转移及分化程度之间的关系;MTT实验筛选AG490对增殖无影响的浓度和时间,并用该浓度和时间进行后续实验;采用Western blot法检测AG490抑制p-STAT3活化后STAT3、Snail、MMP2蛋白表达情况;划痕实验观察p-STAT3活化受抑后,结肠癌细胞迁移情况。  结果  免疫组织化学染色结果表明,p-STAT3的表达与Snail、MMP2均存在相关性,且均与淋巴结转移相关（P < 0.05）。在两个结肠癌细胞系中,通过MTT实验,选出10 μM作为AG490的最佳作用浓度,并采用该浓度进行后续实验。AG490抑制p-STAT3活化后Snail、MMP2表达明显下降,而STAT3总蛋白表达无明显变化。且当AG490抑制p-STAT3活化后,细胞的迁移能力明显下降。  结论  p-STAT3可以通过上皮间质转化（epithelial-mesenchymal transition,EMT）促进结肠癌的转移。      

STAT3信号通路抑制剂AG490协同TCF21促进卵巢癌细胞凋亡的实验研究

目的:探讨信号转导与转录因子3(STAT3)信号通路抑制剂AG490对过表达转录因子21(TCF21)的卵巢癌细胞凋亡的影响。方法:卵巢癌细胞ES-2转染TCF21过表达载体,Western blot检测转染后细胞中TCF21表达情况。ES-2细胞分为对照组(未转染且正常培养)、过表达组(转染TCF21过表达载体)、抑制剂组(未转染的细胞经过STAT3信号通路抑制剂处理)、联合组(STAT3信号通路抑制剂处理过表达TCF21的细胞)。噻唑蓝(MTT)检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测STAT3、磷酸化STAT3(p-STAT3)、剪切后的Caspase-3(Cleaved Caspase-3)蛋白表达。结果:转染TCF21过表达载体的ES-2细胞中TCF21表达水平升高明显高于对照组(P<0.05)。过表达组和抑制剂组细胞增殖率和p-STAT3/STAT3水平明显低于对照组且明显高于联合组(P<0.05)。过表达组和抑制剂组细胞凋亡率和Cleaved Caspase-3水平明显高于对照组且明显低于联合组(P<0.05)。结论:STAT3信号通路抑制剂能够协同TCF21促进卵巢癌细胞凋亡并抑制卵巢癌细胞增殖。     

普鲁卡因联合肌成束蛋白1对膀胱癌细胞的影响及机制研究

目的探讨普鲁卡因联合肌成束蛋白1(FSCN1)对膀胱癌细胞增殖、凋亡和细胞周期的影响及作用机制。方法将阴性对照干扰小RNA(siRNA)和FSCN1 siRNA分别转染至人膀胱癌T24细胞,分别作为NC组和si-FSCN1 组,将仅加入LipofectamineTM 2000 的T24 细胞作为对照组。分别向NC 组和si-FSCN1 组T24 细胞中加入4 mmol/L 的普鲁卡因处理48 h,分别作为普鲁卡因组和普鲁卡因+si-FSCN1 组。噻唑蓝(MTT)法检测细胞增殖能力,流式细胞仪检测细胞周期分布情况和细胞凋亡率,蛋白质印迹法(Western blot)检测FSCN1、信号转导及转录激活因子3(STAT3)、磷酸化STAT3(p-STAT3)、细胞周期蛋白D1(cyclin D1)、Bcl-2 相关X蛋白(BAX)蛋白的相对表达量。结果普鲁卡因组、si-FSCN1组和普鲁卡因+si-FSCN1组细胞G0/G1期细胞比例、细胞凋亡率和BAX蛋白的相对表达量均高于对照组细胞,G2/M 期及S 期细胞比例、A值、p-STAT3 和cyclin D1 蛋白的相对表达量均低于对照组细胞,差异均有统计学意义(P﹤0.05);且普鲁卡因+si-FSCN1 组细胞G0/G1期细胞比例、细胞凋亡率和BAX蛋白的相对表达量均高于普鲁卡因组和si-FSCN1 组细胞,G2/M 期及S 期细胞比例、A值、p-STAT3 和cyclin D1蛋白的相对表达量均低于普鲁卡因组和si-FSCN1组细胞,差异均有统计学意义(P﹤0.05)。结论普鲁卡因或FSCN1 均可抑制膀胱癌细胞增殖,诱导细胞凋亡,将肿瘤细胞阻滞于G0/G1期,且可能通过STAT3 信号通路发挥协同作用。     

红景天苷通过JAK2/STAT3 通路影响宫颈鳞癌C33A细胞的增殖、侵袭和凋亡

目的：观察红景天苷对宫颈鳞癌C33A细胞增殖、侵袭及凋亡的影响并初步探讨其可能的机制。方法：将C33A细胞分为4 组：对照组、低剂量组（红景天苷50 μg/ml）、高剂量组（红景天苷150 μg/ml）、抑制剂AG490 组（JAK2/STAT3 信号通路抑制剂AG490 50 μmol/L）,采用MTT 法、EdU 标记实验、Transwell 实验、Rh123 染色和流式细胞术分别检测红景天苷和AG490 对C33A 细胞增殖、侵袭和凋亡的影响,Western blotting 检测红景天苷和AG490 对C33A 细胞中JAK2/STAT3 通路相关蛋白（p-JAK2、p-STAT3）和凋亡相关蛋白（Bax、Bcl-2、caspase-3）表达的影响。结果：与对照组相比,低剂量组C33A细胞的增殖和DNA的合成明显受到抑制（均P<0.05）、侵袭能力明显降低（均P<0.05）、Rh123 荧光强度明显减弱（均P<0.05）、线粒体膜结构受到破坏、细胞凋亡率明显增加（均P<0.05）;p-JAK2、p-STAT3、Bcl-2 水平显著下降（均P<0.05）、Bax、caspase-3 的表达水平显著升高（P<0.05）;与低剂量组相比,高剂量组、抑制剂组对C33A细胞增殖、侵袭、凋亡及相关蛋白表达的影响更为显著（P<0.05）;而高剂量组、抑制剂组间无显著差异。结论：红景天苷可以抑制C33A细胞的增殖和侵袭、促进细胞凋亡,其作用机制可能与抑制JAK2/STAT3 信号通路有关。     

阻断STAT3信号通路对恶性淋巴瘤细胞凋亡的影响

目的探讨阻断信号转导与转录激活因子3(STAT3)信号通路对恶性淋巴瘤细胞凋亡的影响。方法用0、200、400、800、1600 nmol/L的STAT3信号通路特异性阻断剂JSI-124作用于恶性淋巴瘤细胞株Raji,噻唑蓝(MTT)法检测细胞增殖水平,计算半数抑制浓度。用半数抑制浓度的JSI-124作用于Raji细胞,流式细胞术检测细胞凋亡和细胞周期,Western blot法检测细胞周期蛋白D1(cyclin D1)、STAT3、磷酸化的STAT3(p-STAT3)、活化的半胱氨酸天冬氨酸蛋白水解酶3(cleaved caspase 3)的蛋白相对表达水平。结果随着JSI-124作用浓度的升高,细胞存活率逐渐下降。计算半数抑制浓度为(615.62±52.98)nmol/L,故后续选用600 nm/L的JSI-124作用于恶性淋巴瘤细胞。600 nm/L作用组细胞凋亡率明显高于0 nm/L作用组(P﹤0.01)。600 nm/L作用组G0/G1期细胞所占比例明显低于0 nm/L作用组,G2/M期细胞所占比例明显高于0 nm/L作用组,差异均有统计学意义(P﹤0.01);两组S期细胞所占比例比较,差异无统计学意义(P﹥0.05)。600 nm/L作用组p-STAT3、cyclin D1蛋白相对表达水平明显低于0 nm/L作用组,cleaved caspase 3蛋白相对表达水平明显高于0 nm/L作用组,差异均有统计学意义(P﹤0.01)。结论阻断STAT3信号通路可促进恶性淋巴瘤细胞凋亡,抑制细胞增殖,将细胞周期阻滞在G2/M期。     

Genistein对人卵巢癌细胞系3AO抑制增殖和促进凋亡的作用及机制探讨

目的 研究genistein对人卵巢癌细胞系3AO抑制增殖和促进凋亡的作用，探讨其抗癌作用的机制。方法 应用MTT法检测genistein对3AO的生长抑制作用，电镜观察凋亡细胞及凋亡小体，FCM分析细胞周期及凋亡率，免疫细胞化学法检测细胞增殖凋亡调控相关蛋白的表达。结果 genistein可明显抑制3AO的增殖，且这种抑制作用呈时间及浓度依赖性。FCM分析发现genistein阻断3AO细胞生长于细胞周期的G2／M期，且24～72h可见明显亚G1峰。电镜观察到用药后凋亡细胞典型的形态学特征。免疫细胞化学结果显示，20pmol／L的genistein作用48h后，PCNA、bcl-2及Cyclin B1蛋白表达降低，而p21^WAP1/CIP1和bax及Cyclin B1蛋白表达增加。结论 genistein对卵巢癌细胞系3AO的生长有明显的呈时间及浓度依赖性的抑制作用，其阻断细胞的生长主要在细胞周期的G2／M期，且这种生长抑制作用与p21^WAF1／CIP1蛋白水平表达增加及PCNA cy-lin B1蛋白表达降低有关，且可通过下调bcl-2蛋白表达，上调bax蛋白表达诱导卵巢癌细胞凋亡。     

STAT3反义寡核苷酸对乳腺癌MCF-7细胞增殖和凋亡的影响

目的探讨STAT3反义寡核苷酸的抗肿瘤机制,为乳腺癌的基因治疗提供新的实验依据。方法应用阳离子脂质体介导STAT3反义寡核苷酸转染乳腺癌MCF-7细胞,在倒置显微镜下观察细胞生长情况及形态变化;采用MTT比色法检测细胞增殖状态;流式细胞仪检测细胞凋亡率及细胞周期;Western blot检测STAT3总蛋白和p-STAT3蛋白的表达。结果STAT3反义核苷酸转染乳腺癌细胞后,能抑制MCF-7细胞的增殖,促进MCF-7细胞的凋亡,STAT3蛋白的表达和磷酸化水平下降,出现G1期阻滞。结论STAT3反义核苷酸能抑制MCF-7细胞的增殖,促进MCF-7细胞的凋亡,这将为乳腺癌的治疗提供新的方向。     

STAT3 activation by IL-6 from mesenchymal stem cells promotes the proliferation and metastasis of osteosarcoma

We previously demonstrated that human mesenchymal stem cells (MSCs) promote the growth of osteosarcoma in the bone microenvironment. The aim of the present study was to further determine the effect of IL-6/STAT3 signaling on the progression of osteosarcoma. First, conditioned medium from MSCs was used to stimulate the growth of osteosarcoma cells (Saos-2) in vitro. We found that STAT3 was activated and that the activation could be blocked by an IL-6-neutralizing antibody. The inhibition of STAT3 in Saos-2 cells by siRNA or AG490 decreased cell proliferation, migration and invasion, down-regulated the mRNA expression of Cyclin D, Bcl-xL and Survivin and enhanced the apoptotic response. Furthermore, a nude mouse osteosarcoma model was established by injecting luciferase-labeled Saos-2 cells into the tibia, and the effect of STAT3 on tumor growth was determined by treating the mice with AG490. In vivo bioluminescence images showed that tumor growth was dramatically reduced in the AG490 group. In addition, STAT3 inhibition decreased the lung metastasis rate and prolonged the survival of these mice. After treatment with AG490, the protein levels of IL-6, p-STAT3 and PCNA were decreased, and the level of apoptosis in the tumor was increased. Altogether, these data indicate that MSCs in the bone microenvironment might promote the progression of osteosarcoma and protect tumor cells from drug-induced apoptosis through IL-6/STAT3 signaling.     

STAT3 is essential for Hodgkin lymphoma cell proliferation and is a target of tyrphostin AG17 which confers sensitization for apoptosis.

Classical Hodgkin lymphoma (cHL) is a distinct malignancy of the immune system. Despite the progress made in the understanding of the biology of cHL, the transforming events remain to be elucidated. Recently, we demonstrated that the Janus kinase inhibitor AG490 blocked cellular proliferation and STAT3 phosphorylation in cHL. To explore the potential of constitutively activated STAT3 as a drug target and its role in cHL pathogenesis, different cHL cell lines were analyzed. Treatment of cHL cells by the protein tyrosine kinase inhibitor AG17 was associated with inhibition of cellular proliferation and cell cycle arrest. AG17 treatment was accompanied by decreased levels of STAT3 phosphorylation, whereas NF-kappaB and p38/SAPK2 signaling were not inhibited. Incubation with AG17 or AG490 sensitized cHL cells to CD95/Fas/Apo-1 or staurosporine-mediated apoptosis. Coincubation of tyrphostins with staurosporine was accompanied by rapid complete inhibition of STAT3 phosphorylation. RNA interference directed against STAT3 in L428 and L1236 cHL cells demonstrated that STAT3 is essential for cell proliferation of these cHL cells. In conclusion, these findings support the concept that STAT3 signaling is important in the pathogenesis of cHL and tyrphostins are agents for developing new therapeutic strategies.     

Silibinin Inhibits Proliferation,Induces Apoptosis and Causes Cell Cycle Arrest in Human Gastric Cancer MGC803 Cells Via STAT3 Pathway Inhibition

Background: To investigate the effect of silibinin on proliferation and apoptosis in human gastric cancer cell line MGC803 and its possible mechanisms. Materials and Methods: Human gastric cancer cell line MGC803 cells were treated with various concentration of silibinin. Cellular viability was assessed by CCK-8 assayandapoptosis and cell cycle distribution by flow cytometry. Protein expression and mRNA of STAT3, and cell cycle and apoptosis regulated genes were detected by Western blotting and real-time polymerase chain reaction, respectively. Results: Silibinin inhibits growth of MGC803 cells in a dose- and time-dependent manner. Silibinin effectively induces apoptosis of MGC803 cells and arrests MGC803 cells in the G2/M phase of the cell cycle, while decreasing the protein expression of p-STAT3, and of STAT3 downstream target genes including Mcl-1, Bcl-xL, survivin at both protein and mRNA levels. In addition, silibinin caused an increase in caspase 3 and caspase 9 protein as well as mRNA levels. Silibinin caused G2/M phage arrest accompanied by a decrease inCDK1 and Cyclin B1 at protein and mRNA levels.. Conclusions: These results suggest that silibinin inhibits the proliferation of MGC803 cells, and it induces apoptosis and causes cell cycle arrest by down-regulating CDK1, cyclinB1, survivin, Bcl-xl, Mcl-1 and activating caspase 3 and caspase 9, potentially via the STAT3 pathway.     

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。