MEKK3在TNF-α刺激后恶性肿瘤细胞产生IL-6作用中的初步研究
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摘要
恶性肿瘤是危害人类健康和生命的头号杀手,许多细胞因子与恶性肿瘤密切相关。大量研究发现,肿瘤细胞自身分泌或接受致炎因子(TNF-a等)刺激后产生的细胞因子IL-6,与恶性肿瘤的发生发展、侵袭转移及化疗抵抗作用等预后不良密切相关。在查阅大量文献和原有工作的基础上,本研究应用RNA干涉(siRNA)、基因克隆、基因转染、Western-blot、荧光实时定量PCR、RT-PCR和ELISA技术,探讨有丝分裂原激活的蛋白激酶MEKK3在TNF-a刺激后的恶性肿瘤细胞所产生的IL-6中的作用,旨在为阐明恶性肿瘤细胞中TNF-a介导的IL-6产生的机制和发现可能的治疗药物作用的靶点奠定基础。
肺癌是高度恶性的肿瘤,其死亡率占恶性肿瘤的第一位；乳腺癌是世界各地女性中最为常见的恶性肿瘤,也是导致女性死亡的主要原因之一。因此,本课题选择肺癌和乳腺癌细胞进行研究。
本研究第一部分首先应用ELISA方法检测肺癌A549细胞和乳腺癌MDA-MB-231细胞接受TNF-a刺激后IL-6的产生。结果表明：在接受TNF-a刺激后,A549细胞和MDA-MB-231细胞均能产生较高水平的IL-6。在此基础上,应用Western-blot和RT-PCR方法分别检测了A549细胞和MDA-MB-231细胞接受TNF-a刺激后MEKK3的磷酸化及MEKK3基因表达的改变,以判断其是否是TNF-a信号途径的成分。Western-blot结果表明：A549细胞接受TNF-a刺激后10min,MEKK3即发生磷酸化,30min达高峰,2h后恢复正常；MDA-MB-231细胞接受TNF-a刺激后30min发生磷酸化,1h达高峰,2h后亦恢复正常。RT-PCR结果显示：与未刺激的样本对比,两种细胞接受TNF-a刺激后,MEKK3 mRNA表达均明显增加。Western-blot结果和RT-PCR结果均表明：MEKK3可被TNF-a激活、MEKK3是TNF-a信号途径的信号分子,其接受TNF-a刺激后磷酸化的出现与改变及其mRNA表达的改变相似于文献报道的其他蛋白激酶因接受细胞因子刺激而活化后的磷酸化和基因表达的改变。
以第一部分的结果为依据,本研究的第二、三、四部分着眼于探讨MEKK3是否参与TNF-a刺激后恶性肿瘤细胞所产生的IL-6的调控。
本研究第二部分应用Ambion公司siRNA设计软件设计针对MEKK3基因4个不同部位siRNA靶点的模板DNA序列,在体外合成相应的DNA片段后,以BamHI及HindⅢ酶切位点分别克隆入pSilencer 4.1-CMV hygro载体,构建成四个针对MEKK3基因的siRNA表达载体—pSilencer4.1-MEKK3siRNA,再以脂质体(Lipofectamine2000)分别将其转染入A549细胞,应用Western-blot检测MEKK3 siRNA表达载体对MEKK3表达的抑制。结果发现,四个MEKK3 siRNA表达载体对MEKK3的表达均有抑制作用,其中pSilencer4.1-MEKK3 siRNA2的抑制作用最为显著,抑制率达84%,因此选择将其分别转染入A549和MDA-MB-231细胞,潮霉素筛选后获得的抗性细胞克隆株,经荧光实时定量PCR测定后,结果显示MEKK3 siRNA2/A549和MEKK3 siRNA2/MDA-MB-231稳定克隆株中MEKK3的表达均获得了80%以上的抑制,保证了后续的研究。
本研究第三部分首先应用RT-PCR,扩增MEKK3基因的全长cDNA序列,通过KpnI和XhoI酶切位点克隆入pcDNA3.1/hygro(+)载体中,构建pcDNA3.1/hygro(+)-MEKK3真核表达载体,并转染入A549和MDA-MB-231细胞中。Western-blot结果显示MEKK3蛋白在此两种细胞中均表达良好。应用潮霉素筛选获得的两种细胞的稳定细胞克隆株,经荧光实时定量PCR检测后发现,其MEKK3 mRNA的表达水平与空载体稳定细胞克隆株相比,均有显著增加(分别增加了195%和177%)。结果表明pcDNA3.1/hygro(+)-MEKK3真核表达载体构建成功,且成功地获得了MEKK3高表达稳定细胞克隆株,进一步保证了后续的研究。
本研究第四部分分别应用RT-PCR和ELISA检测TNF-a刺激未转染的A549和MDA-MB-231细胞(野生株)、MEKK3低表达的MEKK3 siRNA2/A549和MEKK3 siRNA2/MDA-MB-231稳定细胞克隆株、将MEKK3基因重新转染入MEKK3低表达的MEKK3 siRNA2/A549和MEKK3 siRNA2/MDA-MB-231并经RT-PCR验证恢复了MEKK3表达的细胞、A549和MDA-MB-231siRNA阴性对照稳定细胞克隆株、MEKK3高表达的稳定克隆株及pcDNA3.1空载体稳定细胞克隆株中IL-6的表达或产生。结果表明：MEKK3低表达的MEKK3siRNA2/A549和MEKK3 siRNA2/MDA-MB-231稳定细胞克隆株IL-6 mRNA的表达及IL-6的产生均明显低于野生株或阴性对照克隆株,差异非常显著(P均<0.01)；此两细胞株恢复MEKK3表达后,其IL-6mRNA的表达和IL-6的产生均获得了大幅度的提高,其结果高于野生株或阴性对照克隆株,差异非常显著(P均<0.01)；A549和MDA-MB-231 MEKK3高表达稳定细胞克隆株接受TNF-a刺激后,其IL-6的产生均明显高于两者的野生株或空载体对照株,差异非常显著(P<0.01)。
初步结果提示：MEKK3是TNF-a介导的肺癌和乳腺癌细胞中IL-6产生的信号转导途径的重要成分,MEKK3与TNF-a介导的肺癌和乳腺癌细胞中IL-6的产生密切相关,并在TNF-a介导的肺癌和乳腺癌细胞产生IL-6中起重要的调控作用,其下游信号途径有待进一步研究。
Cancer is well known to hazard human health and life, and many cytokines are closely related with it.A large number of studies have shown that the cancer can autocrine or generate a large number of IL-6 by the proinflammatory factor (TNF-a,LPS etc.)stimulating, which is the incidence of cancer developmen,invasion and metastasis and is closely related to poor prognosis.On account of the literature and previous work, this study investigated the role of the mitogen-activated protein kinase MEKK3 in the IL-6 production of the cancer cells by TNF-a-induced to clarity the mechanism of the production of IL-6 by TNF-a-induced and find a possible role of therapeutic drug targets by using RNA interference, gene cloning, gene transfection, Westernblot, real-time fluorescence quantitative PCR, RT-PCR and ELISA techniques.
Lung cancer is the most common and highly malignant tumors,and the first one which lead to death; breast cancer is not only the most common malignant tumor of women all over the world, but also one of the main cause of females death, therefore, the topic select lung cancer and breast cancer cells for study.
In the first part of this study, the production of IL-6 in tne wild-type A549 lung cancer and breast cancer MDA-MB-231 cells after TNF-astimulating was detected by ELISA.The results showed that:after the stimulation of TNF-a, A549 and MDA-MB-231 cells can produce higher levels of IL6.0n this basis, the changes of phosphorylation and gene expression changes of MEKK3 were detectd in the A549 and MDA-MB-231 cells receiving TNF-a stimulated by using Western blot and RT-PCR, in order to determine whether is the TNF-a signaling pathway components.Western-blot results showed that:in the A549 accepting TNF-a stimulated, the phosphorylation of MEKK3 happened after 10min、30min peaked and 2h returned to normal; in the MDA-MB-231 accepting TNF-a stimulated, the phosphorylation of MEKK3 happened after 30min、1h peaked and 2h recoveryed normal.RT-PCR results showed that:compared with non-stimulated samples, after two kinds of cells receiving TNF-a stimulated 6h and 8h, the expression of MEKK3 mRNA were significantly increased.Western blot and RT-PCR results showed that: MEKK3 can be actived by TNF-a; MEKK3 is a molecule of the TNF-a signaling pathway, the changes about the phosphory-lation and its mRNA expression after accepting TNF-a stimulated is entirely similar to the phosphorylation and gene expression changes of other protein kinase reported in the literature due to accept cytokines stimulating.
In the first part results of this study based, the second> third and fourth parts focused on exploring the role of the mitogen-activated protein kinase MEKK3 in the IL-6 production of the cancer cells by TNF-a-induced.
In the second part of this study, four different siRNA template DNA sequence targeting in MEKK3 gene were designed by the application of Ambion's siRNA design software, synthesized the corresponding DNA fragments in vitro, cloned into the pSilencer 4.1-CMV hygro vector with its two restriction enzyme BamHI and HindlⅢcloning point to construct four targeted MEKK3 gene siRNA expression vector—pSilencer4.1-MEKK3 siRNA, then were transfected into A549 cells with Lipofectamine2000, Western-blot was carried out to analyze the suppression of MEKK3 in siRNA expression vectors. The results showed that these siRNA expression vector silenced,especially the suppression ratio in pSilencer4.1-MEKK3 siRNA2 expression vector was 85%.So transfecting it into A549 and MDA-MB-231, pSilencer4.1-MEKK3 siRNA2-harboring cell lines were acquired by hygromycin B selecting transfected cells, Real-time fluorescence quantitative PCR found that the suppression ratio of their MEKK3mRNA was more than 80%, so ensuring the follow-up study.
In the third part of this study, The full length cDNA sequences of MEKK3 were obtained by RT-PCR., then cloned into pcDNA3.1/hygro (+) vector with KpnⅠand XhoⅠrestriction sites to construct pcDNA3.1/hygro (+)-MEKK3 eukaryotic expression vector, and transfected into A549 and MDA-MB-231 cells. Western-blot showed that the expression of MEKK3 protein were decreased in these two cells. pcDNA3.1/hygro(+)-MEKK3-harboring cell lines were acquired by hygromycin B selecting transfected cells. Real-time fluorescence quantitative PCR found that the expression level of their MEKK3 mRNA was increased comparing with empty vector stable clone.The results showed that pcDNA3.1/hygro (+)-MEKK3 eukaryotic expression vector was successfully constructed, and pcDNA3.1/hygro (+)-MEKK3-harboring cell lines were acquired, so ensuring the follow-up study.
In the fourth part of this study, RT-PCR and ELISA were carried out to detect the expression of IL6 mRNA and the production of IL6 in the non-transfected A549 and MDA-MB-231 cells (wild type), down-expressed MEKK3 siRNA2/A549 and MDA-MB-231 stable cell clones, the MEKK3 gene in transiently transfected into down-expressed MEKK3 siRNA2/A549 and MEKK3 siRNA2/MDA-MB-231 cells which the expression of MEKK3 was restored by RT-PCR analying, A549 and MDA-MB-231 siRNA negative-control stable cell clone,over-expressed MEKK3 stable cell clone and pcDNA3.1 empty vector stable cell clone after TNF-a stimulating.The results showed that:the expression of IL6 mRNA and the production of IL6 in the down-expressed MEKK3 siRNA2/A549 and MDA-MB-231 stable cell clones were significantly lower than the wild strain or the negative control clone, the difference was significant (P<0.01); the two cell lines was restored the expression of MEKK3,their expression of IL6 mRNA and production of IL6 have largly improved, and were similar to the wild strains (P> 0.05), the production of IL6 in the over-expressed MEKK3 A549 and MDA-MB-231 stable cell clon were significantly higher.
The preliminary results indicated that MEKK3 is closely related to the IL-6 production of the lung cancer and breast cancer cells by TNF-a-induced, and played an important role in regulating the IL-6 production of the lung cancer and breast cancer cells accepting TNF-a stimulated, the relevant mechanisms and downstream signaling pathways remains to be further studied.
肺癌是高度恶性的肿瘤,其死亡率占恶性肿瘤的第一位；乳腺癌是世界各地女性中最为常见的恶性肿瘤,也是导致女性死亡的主要原因之一。因此,本课题选择肺癌和乳腺癌细胞进行研究。
本研究第一部分首先应用ELISA方法检测肺癌A549细胞和乳腺癌MDA-MB-231细胞接受TNF-a刺激后IL-6的产生。结果表明：在接受TNF-a刺激后,A549细胞和MDA-MB-231细胞均能产生较高水平的IL-6。在此基础上,应用Western-blot和RT-PCR方法分别检测了A549细胞和MDA-MB-231细胞接受TNF-a刺激后MEKK3的磷酸化及MEKK3基因表达的改变,以判断其是否是TNF-a信号途径的成分。Western-blot结果表明：A549细胞接受TNF-a刺激后10min,MEKK3即发生磷酸化,30min达高峰,2h后恢复正常；MDA-MB-231细胞接受TNF-a刺激后30min发生磷酸化,1h达高峰,2h后亦恢复正常。RT-PCR结果显示：与未刺激的样本对比,两种细胞接受TNF-a刺激后,MEKK3 mRNA表达均明显增加。Western-blot结果和RT-PCR结果均表明：MEKK3可被TNF-a激活、MEKK3是TNF-a信号途径的信号分子,其接受TNF-a刺激后磷酸化的出现与改变及其mRNA表达的改变相似于文献报道的其他蛋白激酶因接受细胞因子刺激而活化后的磷酸化和基因表达的改变。
以第一部分的结果为依据,本研究的第二、三、四部分着眼于探讨MEKK3是否参与TNF-a刺激后恶性肿瘤细胞所产生的IL-6的调控。
本研究第二部分应用Ambion公司siRNA设计软件设计针对MEKK3基因4个不同部位siRNA靶点的模板DNA序列,在体外合成相应的DNA片段后,以BamHI及HindⅢ酶切位点分别克隆入pSilencer 4.1-CMV hygro载体,构建成四个针对MEKK3基因的siRNA表达载体—pSilencer4.1-MEKK3siRNA,再以脂质体(Lipofectamine2000)分别将其转染入A549细胞,应用Western-blot检测MEKK3 siRNA表达载体对MEKK3表达的抑制。结果发现,四个MEKK3 siRNA表达载体对MEKK3的表达均有抑制作用,其中pSilencer4.1-MEKK3 siRNA2的抑制作用最为显著,抑制率达84%,因此选择将其分别转染入A549和MDA-MB-231细胞,潮霉素筛选后获得的抗性细胞克隆株,经荧光实时定量PCR测定后,结果显示MEKK3 siRNA2/A549和MEKK3 siRNA2/MDA-MB-231稳定克隆株中MEKK3的表达均获得了80%以上的抑制,保证了后续的研究。
本研究第三部分首先应用RT-PCR,扩增MEKK3基因的全长cDNA序列,通过KpnI和XhoI酶切位点克隆入pcDNA3.1/hygro(+)载体中,构建pcDNA3.1/hygro(+)-MEKK3真核表达载体,并转染入A549和MDA-MB-231细胞中。Western-blot结果显示MEKK3蛋白在此两种细胞中均表达良好。应用潮霉素筛选获得的两种细胞的稳定细胞克隆株,经荧光实时定量PCR检测后发现,其MEKK3 mRNA的表达水平与空载体稳定细胞克隆株相比,均有显著增加(分别增加了195%和177%)。结果表明pcDNA3.1/hygro(+)-MEKK3真核表达载体构建成功,且成功地获得了MEKK3高表达稳定细胞克隆株,进一步保证了后续的研究。
本研究第四部分分别应用RT-PCR和ELISA检测TNF-a刺激未转染的A549和MDA-MB-231细胞(野生株)、MEKK3低表达的MEKK3 siRNA2/A549和MEKK3 siRNA2/MDA-MB-231稳定细胞克隆株、将MEKK3基因重新转染入MEKK3低表达的MEKK3 siRNA2/A549和MEKK3 siRNA2/MDA-MB-231并经RT-PCR验证恢复了MEKK3表达的细胞、A549和MDA-MB-231siRNA阴性对照稳定细胞克隆株、MEKK3高表达的稳定克隆株及pcDNA3.1空载体稳定细胞克隆株中IL-6的表达或产生。结果表明：MEKK3低表达的MEKK3siRNA2/A549和MEKK3 siRNA2/MDA-MB-231稳定细胞克隆株IL-6 mRNA的表达及IL-6的产生均明显低于野生株或阴性对照克隆株,差异非常显著(P均<0.01)；此两细胞株恢复MEKK3表达后,其IL-6mRNA的表达和IL-6的产生均获得了大幅度的提高,其结果高于野生株或阴性对照克隆株,差异非常显著(P均<0.01)；A549和MDA-MB-231 MEKK3高表达稳定细胞克隆株接受TNF-a刺激后,其IL-6的产生均明显高于两者的野生株或空载体对照株,差异非常显著(P<0.01)。
初步结果提示：MEKK3是TNF-a介导的肺癌和乳腺癌细胞中IL-6产生的信号转导途径的重要成分,MEKK3与TNF-a介导的肺癌和乳腺癌细胞中IL-6的产生密切相关,并在TNF-a介导的肺癌和乳腺癌细胞产生IL-6中起重要的调控作用,其下游信号途径有待进一步研究。
Cancer is well known to hazard human health and life, and many cytokines are closely related with it.A large number of studies have shown that the cancer can autocrine or generate a large number of IL-6 by the proinflammatory factor (TNF-a,LPS etc.)stimulating, which is the incidence of cancer developmen,invasion and metastasis and is closely related to poor prognosis.On account of the literature and previous work, this study investigated the role of the mitogen-activated protein kinase MEKK3 in the IL-6 production of the cancer cells by TNF-a-induced to clarity the mechanism of the production of IL-6 by TNF-a-induced and find a possible role of therapeutic drug targets by using RNA interference, gene cloning, gene transfection, Westernblot, real-time fluorescence quantitative PCR, RT-PCR and ELISA techniques.
Lung cancer is the most common and highly malignant tumors,and the first one which lead to death; breast cancer is not only the most common malignant tumor of women all over the world, but also one of the main cause of females death, therefore, the topic select lung cancer and breast cancer cells for study.
In the first part of this study, the production of IL-6 in tne wild-type A549 lung cancer and breast cancer MDA-MB-231 cells after TNF-astimulating was detected by ELISA.The results showed that:after the stimulation of TNF-a, A549 and MDA-MB-231 cells can produce higher levels of IL6.0n this basis, the changes of phosphorylation and gene expression changes of MEKK3 were detectd in the A549 and MDA-MB-231 cells receiving TNF-a stimulated by using Western blot and RT-PCR, in order to determine whether is the TNF-a signaling pathway components.Western-blot results showed that:in the A549 accepting TNF-a stimulated, the phosphorylation of MEKK3 happened after 10min、30min peaked and 2h returned to normal; in the MDA-MB-231 accepting TNF-a stimulated, the phosphorylation of MEKK3 happened after 30min、1h peaked and 2h recoveryed normal.RT-PCR results showed that:compared with non-stimulated samples, after two kinds of cells receiving TNF-a stimulated 6h and 8h, the expression of MEKK3 mRNA were significantly increased.Western blot and RT-PCR results showed that: MEKK3 can be actived by TNF-a; MEKK3 is a molecule of the TNF-a signaling pathway, the changes about the phosphory-lation and its mRNA expression after accepting TNF-a stimulated is entirely similar to the phosphorylation and gene expression changes of other protein kinase reported in the literature due to accept cytokines stimulating.
In the first part results of this study based, the second> third and fourth parts focused on exploring the role of the mitogen-activated protein kinase MEKK3 in the IL-6 production of the cancer cells by TNF-a-induced.
In the second part of this study, four different siRNA template DNA sequence targeting in MEKK3 gene were designed by the application of Ambion's siRNA design software, synthesized the corresponding DNA fragments in vitro, cloned into the pSilencer 4.1-CMV hygro vector with its two restriction enzyme BamHI and HindlⅢcloning point to construct four targeted MEKK3 gene siRNA expression vector—pSilencer4.1-MEKK3 siRNA, then were transfected into A549 cells with Lipofectamine2000, Western-blot was carried out to analyze the suppression of MEKK3 in siRNA expression vectors. The results showed that these siRNA expression vector silenced,especially the suppression ratio in pSilencer4.1-MEKK3 siRNA2 expression vector was 85%.So transfecting it into A549 and MDA-MB-231, pSilencer4.1-MEKK3 siRNA2-harboring cell lines were acquired by hygromycin B selecting transfected cells, Real-time fluorescence quantitative PCR found that the suppression ratio of their MEKK3mRNA was more than 80%, so ensuring the follow-up study.
In the third part of this study, The full length cDNA sequences of MEKK3 were obtained by RT-PCR., then cloned into pcDNA3.1/hygro (+) vector with KpnⅠand XhoⅠrestriction sites to construct pcDNA3.1/hygro (+)-MEKK3 eukaryotic expression vector, and transfected into A549 and MDA-MB-231 cells. Western-blot showed that the expression of MEKK3 protein were decreased in these two cells. pcDNA3.1/hygro(+)-MEKK3-harboring cell lines were acquired by hygromycin B selecting transfected cells. Real-time fluorescence quantitative PCR found that the expression level of their MEKK3 mRNA was increased comparing with empty vector stable clone.The results showed that pcDNA3.1/hygro (+)-MEKK3 eukaryotic expression vector was successfully constructed, and pcDNA3.1/hygro (+)-MEKK3-harboring cell lines were acquired, so ensuring the follow-up study.
In the fourth part of this study, RT-PCR and ELISA were carried out to detect the expression of IL6 mRNA and the production of IL6 in the non-transfected A549 and MDA-MB-231 cells (wild type), down-expressed MEKK3 siRNA2/A549 and MDA-MB-231 stable cell clones, the MEKK3 gene in transiently transfected into down-expressed MEKK3 siRNA2/A549 and MEKK3 siRNA2/MDA-MB-231 cells which the expression of MEKK3 was restored by RT-PCR analying, A549 and MDA-MB-231 siRNA negative-control stable cell clone,over-expressed MEKK3 stable cell clone and pcDNA3.1 empty vector stable cell clone after TNF-a stimulating.The results showed that:the expression of IL6 mRNA and the production of IL6 in the down-expressed MEKK3 siRNA2/A549 and MDA-MB-231 stable cell clones were significantly lower than the wild strain or the negative control clone, the difference was significant (P<0.01); the two cell lines was restored the expression of MEKK3,their expression of IL6 mRNA and production of IL6 have largly improved, and were similar to the wild strains (P> 0.05), the production of IL6 in the over-expressed MEKK3 A549 and MDA-MB-231 stable cell clon were significantly higher.
The preliminary results indicated that MEKK3 is closely related to the IL-6 production of the lung cancer and breast cancer cells by TNF-a-induced, and played an important role in regulating the IL-6 production of the lung cancer and breast cancer cells accepting TNF-a stimulated, the relevant mechanisms and downstream signaling pathways remains to be further studied.
引文
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[5]Domingo-Domenech J, Oliva C, Rovira A, et al. Interleukin-6, a nuclear factor-K B target, predicts resistance to docetaxel in hormone-independent prostate cancer and nuclear factor-K B inhibition by PS-1145 enhances docetaxel antitumor activity. Clin Cancer Res,2006,12(18):5578-5586
[6]Liao WC, Lin JT, Wu CY, et al. Serum interleukin-6 level but not genotype predicts survival after resection in stages Ⅱ and Ⅲ gastric carcinoma. Clin Cancer Res,2008,14(2):428-434
[7]Salem M, Elbaz O, Zahran M, et al. Malignancy:identification of predictors of disease status and progression in patients with myeloma (MM). Haematologica 2000,5, 41-45.
[8]Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in Inflamma-tionand cancer. Eur J Cancer 2005;41 (16):2502-2512.
[9]Yang L, Wang L, Lin HK, et al. Interleukin-6 differentially
regulates androgen receptor transactivation via PI3K-Akt, STAT3, and MAPK, three distinct signal pathways in prostate cancer cells. Biochem Biophys Res Commun 2003,305,462-469.
[10]Coussens LM, Werb Z. Inflammation and cancer. Nature.2002,420, 860-867.
[11]Grivennikov S, Karin E, Terzic J, et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell,2009,15(2):103-113
[12]Szlosarek PW, Grimshaw MJ, Kulbe H, et al. Expression and regulation of tumor necrosis factor a in normal and malignant ovarian epithelium. Mol Cancer Ther,2006,5(2):382-390
[13]Wang J, Tokoro T, Higa S, et al. Anti-inflammatory effect of pitavastatin on NF-kappaB activated by TNF-alpha in hepatocellular carcinoma cells. Biol Pharm Bull,2006, 29(4):634-639
[14]Asschert JG, Vellenga E, Ruiters MH, et al. Regulation of spontaneous and TNF/IFN-induced IL-6 expression in two human ovarian-carcinoma cell lines. Int J Cancer,1999,82(2):244-249
[15]Wang JY and Kitajima I. Pitavastatin inactivates NF-K B and decreaseds IL-6 production through Rho kinase pathway in MCF-7 cells. Oncology Reports,2007,17:1149-1154
[16]Inamura K and Ishikawa Y. Lung cancer progression and metastasis from the prognostic point of view. Clin Exp Metastasis.2010 Feb, Epub ahead of print
[17]Voogd AC, Duijm LE and Coebergh JW. More breast cancer death in 2008:stepping up prevention. Ned Tijdschr Geneeskd. 2010:154(6):A1550-1553.
[18]Yang J, Lin Y, Guo Z and et al. The essential role of MEKK3 in TNF-induced NF-κ B activation. Nature Immunology,2000,2:620-624
[19]Huang Q, Yang J, Walker C and et al. Differential regulation of
interleukin-1 receptor and Toll-like receptor signaling by MEKK3. Nature immunology, 2004, 5:98-103
[20]Kim K, Duramad 0, Qin X and et al. MEKK3 is essential for lipopolysaccharide-induced interleukin-6 and granulocyte-macrophage colony-stimulating factor production in macrophages. Immunology,2006,120:242-250
[21]Blank JL, Gerwins P, Elliott EM, Sather S, Johnson GL. Molecular cloning of mitogen-activated protein/ERK kinase kinases(MEKK) 2 and 3. Regulation of sequential phosphorylation pathways involving mitogen-activated protein kinase and c-Jun kinase. J BiolChem 1996; 271:5361-5368.
[22]Blonska M, You Y, Geleziunas R and et al. Restoration of NF-κ B Activation by Tumor Necrosis Factor Alpha Receptor Complex-Targeted MEKK3 in Receptor-Interacting Protein-Deficient Cells. Molecular and Cellular Biology,2004,24: 10757-10765
[23]Raungeaud J, Gupta S, Rogers JS, et al. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem,1995,270(13):7420-7426
[24]McCartney RR and Schmidt MC. Regulation of Snfl kinase. Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit. J Biol Chem.2001,276(39):36460-36466
[25]Helczynska K, Larsson AM, Mengelbier LH, et al. Hypoxia-inducible factor-2a correlates to distant recurrence and poor outcome in invasive breast cancer. Cancer Res 2008,68(22):9212-9220
[26]Nakajima T, Kinoshita S, Sasagawa T, etal. Phosphorylation at Threonine-235 by a ras dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6. Proc Natl Acad Sci USA.1993.90:2207
[27]Chik CL, Mackova M, Price D, et al. Adrenergic regulation and diurnal rhythm of p38 mitogen-activated protein kinase phosphorylation in the rat pineal gland. Endocrinology,2004, 145(11):5194-5201
[28]TanumaN, ShimaH, Nakamura K, et al. Protein tyrosine phosphatase epsilonC selectively inhibits interleukin-6 and interleukin-10-induced JAK-STAT signalling. Blood 2001,98:3030-3034.
[29]Zhao H, Ardelt B, Ardelt W, et al. The cytotoxic ribonuclease onconase targets RNA interference (siRNA). Cell Cycle.2008, 7(20):3258-3261.
[30]Cai CM, Sun BC,Liu XY. Short hairpin RNA targeting vascular endothelial growth factor effectively inhibits expression of vascular endothelial growth factor in human retinal pigment epithelium. Zhonghua Yan Ke Za Zhi.2006,42(4):334-337.
[31]Kim D, Garrett SH, Sens MA, etal. Metallothionein isoform 3 and proximal tubule vectorial active transport. Kidney Int. 2002,61 (2):464-472.
[32]Su B, Cheng J, Yang J, etal. MEKK2 is required for T-cell receptor signals in JNK activation and interleukin-2 gene expression. J Biol Chem.2001,276(18):14784-14790
[33]SuganumaM, Okabe S, Kurusu M, etal. Discrete roles of cytokines, TNF-alpha, IL-1, IL-6 in tumour promotion and cell transformation. Int J Oncol.2002,20:131-136.
[34]Starr R, Hilton DJ. Negative regulation of the JAK/STAT pathway. Bio-essays 1999,21:47-52.
[35]Szlosarek PW, Grimshaw MJ, Kulbe H, et al. Expression and regulation of tumor necrosis factor a in normal and malignant ovarian epithelium. Mol Cancer Ther,2006,5(2):382-390.
[36]Conze D, Weiss L, Regen PS, etal. Autocrine production of
interleukin-6 causes multidrug resistance in breast can cer cells. Cancer Res，2001,61f24): 8851-8858.
[37]Levy Y, Tsapis A, Brouet JC:Interleukin-6 antisense oligo nucleotides inhibit the growth of human myeloma cell lines. J Clin Invest.199.188:696
[38]Musselman DL, Miller AH, Porter MR, et al. Higher than normal plasma interleukin-6 concentrations in cancer patients with depression:preliminary findings. Am J Psychiatry 2001;158(8):1252-1257.
[39]Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in inflammation and cancer. Eur J Cancer 2005;41(16):2502-2512
[40]Balasubramanian SP, Azmy IA, Higham SE, et al. Interleukin-6 gene polymorphisms and breast cancer:a case control study and systematic literature review. BMC Cancer 2006:6:186-192
[1]Turner NA. Sinfield JK. Porter KE. TNF-α and IL-1 induce IL-6 expression in human cardiac fibroblasts:Role of p38 MAP kinase subtypes. Journal of Molecular and Cellular Cardiology.2008.44:714
[2]Hong DS. Angelo LS. Kurzrock R. Interleukin-6 and its receptor in cancer:implications for Translational Therapeutics. Cancer,.2007, 110(9):1911-1928.
[3]BoulangerMJ, Chow DC,8revnova EE, etal. Hexameric structure and assembly of the interleukin-6/IL-6 alpha-reeeptor/gp130 complex. [J].Sc/ence,2003,300(5628):2101-2104.
[4]Howlett M, Menheniott TR, JuddLM. etal. Cytokine signalling via gp130 in gastric cancer. Biochim Biophys Acta.2009,1793(11):1623-1633.
[5]0'Shea J J, Gadina M, Schreiber RD. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway [J]. Cell,2002,109:121-131.
[6]Coffer PJ, Koenderman L, Groot RP. The role of STATs in myeloid differentiation and leukemia [J]. Oncogene,2000,19 (21):2511-2522.
[7]Drucker I, Klajman A, Revel M, etal. Interleukin-6 and interleukin-6 receptor secretion by chronic lymphatic leukaemia and normal B lymphocytes:effect of PMA and PWM. Mediators Inflamm.1997;6(2): 147-153.
[8]Hideaki Ishikawa, Maged S, Mahmoud. etal:Proliferation of immature myeloma cells by interleukin-6 is associated with CD45 expression in human multiple myeloma.2000.39(1):51-55
[9]Conze D, Weiss L, Regen PS, et al. Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells. Cancer Research,2001,61:8851-8858.
[10]Domingo-Domenech J, Oliva C, Rovira A, et al. Interleukin 6, a nuclear factor-κB target, predicts resistance to docetaxel in hormone-independent prostate cancer and nuclear factor-KB
inhibition by PS-1145 enhances docetaxel antitumor activity. Clin Cancer Res,2006,12(18):5578-5586
[11]Liao WC, Lin JT, Wu CY, et al. Serum interleukin-6 level but not genotype predicts survival after resection in stages II and III gastric carcinoma. Clin Cancer Res,2008,14(2):428-434.
[12]Salem M, Elbaz 0, Zahran M, et al. Malignancy:identification of predictors of disease status and progression in patients with myeloma (MM). Haematologica 2000,5,41-45.
[13]Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in Inflamma-tion and cancer. Eur J Cancer 2005:41(16):2502-2512.
[14]Yang L, Wang L, Lin HK, et al. Interleukin-6 differentially regulates androgen receptor transactivation via PI3K-Akt, STAT3, and MAPK, three distinct signal pathways in prostate cancer cells. Biochem Biophys Res Commun 2003,305,462-469.
[15]Coussens LM, Werb Z. Inflammation and cancer. Nature.2002,420, 860-867.
[16]Grivennikov S, Karin E, Terzic J, et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell,2009,15(2):103-113
[17]Szlosarek PW, Grimshaw MJ, Kulbe H, et al. Expression and regulation of tumor necrosis factor a in normal and malignant ovarian epithelium. Mol Cancer Ther,2006,5(2):382-390
[18]Wang J, Tokoro T, Higa S, et al. Anti-inflammatory effect of pitavastatin on NF-kappaB activated by TNF-alpha in hepatocellular carcinoma cells. Biol Pharm Bull,2006,29(4):634-639
[19]Asschert JG, Vellenga E, Ruiters MH, et al. Regulation of spontaneous and TNF/IFN-induced IL-6 expression in two human ovarian-carcinoma cell lines. Int J Cancer,1999,82(2):244-249
[20]Wang JY and Kitajima I. Pitavastatin inactivates NF-κB and decreaseds IL-6 production through Rho kinase pathway in MCF-7 cells. Oncology Reports,2007,17:1149-1154.
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[2]Frassanito MA, Cusmai A, Iodice G, et al. Autocrine interleukin-6 production and highly malignant multiple myeloma:relation with resistance to drug-induced apoptosis. Blood 2001,97,483-489.
[3]Pucci S, Paola M, Fabiola S， et al. Interleukin-6 affects cell death escaping mechanisms acting on Bax-Ku70-Clusterin interactions in human colon cancer progression. Cell Cycle,2009,8(3):473-481
[4]Conze D, Weiss L, Regen PS, et al. Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells. Cancer Research,2001,61:8851-8858
[5]Domingo-Domenech J, Oliva C, Rovira A, et al. Interleukin-6, a nuclear factor-K B target, predicts resistance to docetaxel in hormone-independent prostate cancer and nuclear factor-K B inhibition by PS-1145 enhances docetaxel antitumor activity. Clin Cancer Res,2006,12(18):5578-5586
[6]Liao WC, Lin JT, Wu CY, et al. Serum interleukin-6 level but not genotype predicts survival after resection in stages Ⅱ and Ⅲ gastric carcinoma. Clin Cancer Res,2008,14(2):428-434
[7]Salem M, Elbaz O, Zahran M, et al. Malignancy:identification of predictors of disease status and progression in patients with myeloma (MM). Haematologica 2000,5, 41-45.
[8]Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in Inflamma-tionand cancer. Eur J Cancer 2005;41 (16):2502-2512.
[9]Yang L, Wang L, Lin HK, et al. Interleukin-6 differentially
regulates androgen receptor transactivation via PI3K-Akt, STAT3, and MAPK, three distinct signal pathways in prostate cancer cells. Biochem Biophys Res Commun 2003,305,462-469.
[10]Coussens LM, Werb Z. Inflammation and cancer. Nature.2002,420, 860-867.
[11]Grivennikov S, Karin E, Terzic J, et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell,2009,15(2):103-113
[12]Szlosarek PW, Grimshaw MJ, Kulbe H, et al. Expression and regulation of tumor necrosis factor a in normal and malignant ovarian epithelium. Mol Cancer Ther,2006,5(2):382-390
[13]Wang J, Tokoro T, Higa S, et al. Anti-inflammatory effect of pitavastatin on NF-kappaB activated by TNF-alpha in hepatocellular carcinoma cells. Biol Pharm Bull,2006, 29(4):634-639
[14]Asschert JG, Vellenga E, Ruiters MH, et al. Regulation of spontaneous and TNF/IFN-induced IL-6 expression in two human ovarian-carcinoma cell lines. Int J Cancer,1999,82(2):244-249
[15]Wang JY and Kitajima I. Pitavastatin inactivates NF-K B and decreaseds IL-6 production through Rho kinase pathway in MCF-7 cells. Oncology Reports,2007,17:1149-1154
[16]Inamura K and Ishikawa Y. Lung cancer progression and metastasis from the prognostic point of view. Clin Exp Metastasis.2010 Feb, Epub ahead of print
[17]Voogd AC, Duijm LE and Coebergh JW. More breast cancer death in 2008:stepping up prevention. Ned Tijdschr Geneeskd. 2010:154(6):A1550-1553.
[18]Yang J, Lin Y, Guo Z and et al. The essential role of MEKK3 in TNF-induced NF-κ B activation. Nature Immunology,2000,2:620-624
[19]Huang Q, Yang J, Walker C and et al. Differential regulation of
interleukin-1 receptor and Toll-like receptor signaling by MEKK3. Nature immunology, 2004, 5:98-103
[20]Kim K, Duramad 0, Qin X and et al. MEKK3 is essential for lipopolysaccharide-induced interleukin-6 and granulocyte-macrophage colony-stimulating factor production in macrophages. Immunology,2006,120:242-250
[21]Blank JL, Gerwins P, Elliott EM, Sather S, Johnson GL. Molecular cloning of mitogen-activated protein/ERK kinase kinases(MEKK) 2 and 3. Regulation of sequential phosphorylation pathways involving mitogen-activated protein kinase and c-Jun kinase. J BiolChem 1996; 271:5361-5368.
[22]Blonska M, You Y, Geleziunas R and et al. Restoration of NF-κ B Activation by Tumor Necrosis Factor Alpha Receptor Complex-Targeted MEKK3 in Receptor-Interacting Protein-Deficient Cells. Molecular and Cellular Biology,2004,24: 10757-10765
[23]Raungeaud J, Gupta S, Rogers JS, et al. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem,1995,270(13):7420-7426
[24]McCartney RR and Schmidt MC. Regulation of Snfl kinase. Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit. J Biol Chem.2001,276(39):36460-36466
[25]Helczynska K, Larsson AM, Mengelbier LH, et al. Hypoxia-inducible factor-2a correlates to distant recurrence and poor outcome in invasive breast cancer. Cancer Res 2008,68(22):9212-9220
[26]Nakajima T, Kinoshita S, Sasagawa T, etal. Phosphorylation at Threonine-235 by a ras dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6. Proc Natl Acad Sci USA.1993.90:2207
[27]Chik CL, Mackova M, Price D, et al. Adrenergic regulation and diurnal rhythm of p38 mitogen-activated protein kinase phosphorylation in the rat pineal gland. Endocrinology,2004, 145(11):5194-5201
[28]TanumaN, ShimaH, Nakamura K, et al. Protein tyrosine phosphatase epsilonC selectively inhibits interleukin-6 and interleukin-10-induced JAK-STAT signalling. Blood 2001,98:3030-3034.
[29]Zhao H, Ardelt B, Ardelt W, et al. The cytotoxic ribonuclease onconase targets RNA interference (siRNA). Cell Cycle.2008, 7(20):3258-3261.
[30]Cai CM, Sun BC,Liu XY. Short hairpin RNA targeting vascular endothelial growth factor effectively inhibits expression of vascular endothelial growth factor in human retinal pigment epithelium. Zhonghua Yan Ke Za Zhi.2006,42(4):334-337.
[31]Kim D, Garrett SH, Sens MA, etal. Metallothionein isoform 3 and proximal tubule vectorial active transport. Kidney Int. 2002,61 (2):464-472.
[32]Su B, Cheng J, Yang J, etal. MEKK2 is required for T-cell receptor signals in JNK activation and interleukin-2 gene expression. J Biol Chem.2001,276(18):14784-14790
[33]SuganumaM, Okabe S, Kurusu M, etal. Discrete roles of cytokines, TNF-alpha, IL-1, IL-6 in tumour promotion and cell transformation. Int J Oncol.2002,20:131-136.
[34]Starr R, Hilton DJ. Negative regulation of the JAK/STAT pathway. Bio-essays 1999,21:47-52.
[35]Szlosarek PW, Grimshaw MJ, Kulbe H, et al. Expression and regulation of tumor necrosis factor a in normal and malignant ovarian epithelium. Mol Cancer Ther,2006,5(2):382-390.
[36]Conze D, Weiss L, Regen PS, etal. Autocrine production of
interleukin-6 causes multidrug resistance in breast can cer cells. Cancer Res，2001,61f24): 8851-8858.
[37]Levy Y, Tsapis A, Brouet JC:Interleukin-6 antisense oligo nucleotides inhibit the growth of human myeloma cell lines. J Clin Invest.199.188:696
[38]Musselman DL, Miller AH, Porter MR, et al. Higher than normal plasma interleukin-6 concentrations in cancer patients with depression:preliminary findings. Am J Psychiatry 2001;158(8):1252-1257.
[39]Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in inflammation and cancer. Eur J Cancer 2005;41(16):2502-2512
[40]Balasubramanian SP, Azmy IA, Higham SE, et al. Interleukin-6 gene polymorphisms and breast cancer:a case control study and systematic literature review. BMC Cancer 2006:6:186-192
[1]Turner NA. Sinfield JK. Porter KE. TNF-α and IL-1 induce IL-6 expression in human cardiac fibroblasts:Role of p38 MAP kinase subtypes. Journal of Molecular and Cellular Cardiology.2008.44:714
[2]Hong DS. Angelo LS. Kurzrock R. Interleukin-6 and its receptor in cancer:implications for Translational Therapeutics. Cancer,.2007, 110(9):1911-1928.
[3]BoulangerMJ, Chow DC,8revnova EE, etal. Hexameric structure and assembly of the interleukin-6/IL-6 alpha-reeeptor/gp130 complex. [J].Sc/ence,2003,300(5628):2101-2104.
[4]Howlett M, Menheniott TR, JuddLM. etal. Cytokine signalling via gp130 in gastric cancer. Biochim Biophys Acta.2009,1793(11):1623-1633.
[5]0'Shea J J, Gadina M, Schreiber RD. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway [J]. Cell,2002,109:121-131.
[6]Coffer PJ, Koenderman L, Groot RP. The role of STATs in myeloid differentiation and leukemia [J]. Oncogene,2000,19 (21):2511-2522.
[7]Drucker I, Klajman A, Revel M, etal. Interleukin-6 and interleukin-6 receptor secretion by chronic lymphatic leukaemia and normal B lymphocytes:effect of PMA and PWM. Mediators Inflamm.1997;6(2): 147-153.
[8]Hideaki Ishikawa, Maged S, Mahmoud. etal:Proliferation of immature myeloma cells by interleukin-6 is associated with CD45 expression in human multiple myeloma.2000.39(1):51-55
[9]Conze D, Weiss L, Regen PS, et al. Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells. Cancer Research,2001,61:8851-8858.
[10]Domingo-Domenech J, Oliva C, Rovira A, et al. Interleukin 6, a nuclear factor-κB target, predicts resistance to docetaxel in hormone-independent prostate cancer and nuclear factor-KB
inhibition by PS-1145 enhances docetaxel antitumor activity. Clin Cancer Res,2006,12(18):5578-5586
[11]Liao WC, Lin JT, Wu CY, et al. Serum interleukin-6 level but not genotype predicts survival after resection in stages II and III gastric carcinoma. Clin Cancer Res,2008,14(2):428-434.
[12]Salem M, Elbaz 0, Zahran M, et al. Malignancy:identification of predictors of disease status and progression in patients with myeloma (MM). Haematologica 2000,5,41-45.
[13]Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in Inflamma-tion and cancer. Eur J Cancer 2005:41(16):2502-2512.
[14]Yang L, Wang L, Lin HK, et al. Interleukin-6 differentially regulates androgen receptor transactivation via PI3K-Akt, STAT3, and MAPK, three distinct signal pathways in prostate cancer cells. Biochem Biophys Res Commun 2003,305,462-469.
[15]Coussens LM, Werb Z. Inflammation and cancer. Nature.2002,420, 860-867.
[16]Grivennikov S, Karin E, Terzic J, et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell,2009,15(2):103-113
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