高等真菌来源化学小分子探针Grifolin信号转导机制研究
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摘要
高等真菌属于“创造系数”很高的生物资源。近年来,发现从高等真菌子实体、培养菌丝体和培养基中分离提取的一些低分子量次生代谢产物具有抗肿瘤先导化合物的特性。真菌次生代谢产物结构新颖,经过了“进化预选”(evolutionary pre-selection),具有高度的物种依赖性和特异性,使这些活性物质成为生物医学和天然创新药物研究的良好材料,在重大新药创新研究中具有不可替代性,其独特的化合物骨架和作用模式,已经成为发现新型天然抗肿瘤药物的理想化合物。
化学小分子探针是指能够高选择性地探测蛋白质的功能、结构以及与活性小分子作用模式的小分子化合物,分子量通常小于700Da。化学小分子探针可介入信号转导过程的研究,并能够干扰、改变或模拟信号转导过程,是阐述已知生物信号转导机理和揭示未知的生物信号转导过程的有效手段。
在前期研究中,我们在国际上率先从高等真菌地花菌子实体提取物中筛选得到新型抗肿瘤活性小分子探针grifolin。Grifolin为一种法尼基酚类化合物,分子式为C22H32O2,分子量为328,具有显著的广谱抗肿瘤活性,可诱导肿瘤细胞周期阻滞和凋亡发生,但其基于的分子机制并不十分清晰。为深入探讨grifolin发挥其生物学功能的信号转导分子机制,本课题从以下三个方面进行研究：
一、明确小分子探针Grifolin活化转录因子p53上调DAPK1表达
根据高通量凋亡相关基因表达谱筛查提供的线索：grifolin可显著上调死亡相关基因dapkl的表达,而DAPK1作为压力活化的肿瘤抑制蛋白,在促凋亡信号转导通路中扮演了重要角色。大量的临床样本检测及生物学实验数据显示,DAPK1表达缺失可能是肿瘤形成发展的重要起因。这启发我们在肿瘤细胞中对grifolin调节DAPK1蛋白表达分子机制进行深入探讨。
我们以人鼻咽鳞癌细胞CNE1为基本模型,通过RT-PCR、Western Blot等技术手段,分别从mRNA水平和蛋白质水平,证实grifolin可以剂量依赖性方式上调DAPK1的表达；进一步在多种肿瘤细胞系中证实这一结论。在此基础上,采用siRNA、EMSA、ChIP等策略,揭示grifolin上调DAPK1表达的分子机制。
通过Western blot检测发现,grifolin可显著上调转录因子p53Ser20和Ser392位磷酸化水平,而对其Ser15和Thr81位磷酸化状态无明显改变。p53作为重要的转录因子,其磷酸化修饰是p53重要的功能形式。由于Ser20和Ser392位磷酸化与p53转录活化密切相关,因此我们推断grifolin可能通过磷酸化活化p53,上调其转录活性。
生物信息学分析及文献显示,dapk1启动子区包含p53结合元件。我们采用siRNA策略,在CNE1细胞中转染p53 siRNA 72小时,可有效干扰p53蛋白表达,与此同时,DAPK1蛋白表达水平也显著下调；反之,在p53-/-H1299细胞中转染p53表达质粒pEGFP-C3-p53,在诱导p53表达的同时,DAPK1也明显上调,由此,证实转录因子p53与DAPK1蛋白表达呈正相关。
采用EMSA和ChIP策略,分别从体外和体内层面探讨grifolin是否通过促进p53与dapkl启动子区结合上调DAPK1的表达。EMSA结果显示,30μM grifolin处理CNE1 24小时后,其细胞核蛋白与dapkl基因DNA结合能力明显高于未处理CNE1细胞。Supershift-EMSA实验证实与dapkl基因DNA结合的转录因子为p53蛋白。ChIP实验表明,转录因子p53能特异性结合于dapkl基因启动子区。30μMgrifolin处理CNE1细胞24小时后,转录因子p53蛋白与dapkl基因DNA结合能力明显高于未处理组。
以上研究证实,小分子探针grifolin通过靶向p53-DAPK1通路,上调DAPK1表达,为我们深入探讨grifolin诱导肿瘤细胞凋亡的分子机制,提供了充分的实验依据。
二、证实Grifolin促进ERK1/2与DAPK1目互作用,抑制ERK1/2核积聚,诱导肿瘤细胞凋亡
在证实化学小分子探针grifolin可上调dapkl基因表达的基础上,进一步探讨其靶向DAPK1,发挥促凋亡生物学效应的分子机制。目前已知的与DAPK1死亡域相互作用的蛋白主要有两种,其中包括ERK激酶。由此提示,grifolin可能通过促进ERK1/2与DAPK1相互作用,抑制ERK1/2核内积聚,诱导肿瘤细胞凋亡。
运用LCFM、胞浆胞核蛋白抽提、Western blot等技术手段,分别从定性和定量层面确定,CNE1细胞经grifolin处理,对ERK1/2总蛋白水平无显著影响,但可以剂量依赖性方式促进ERK1/2蛋白胞浆羁留,抑制ERK1/2蛋白的核内分布。
通过LCFM、Co-IP和反式IP等策略,证实grifolin可有效促进ERK1/2和DAPK1蛋白间相互作用。
为探讨DAPK1是否介导grifolin诱导的肿瘤细胞凋亡效应,在CNE1细胞中转染DAPK1 siRNA, Western blot、FACS以及Caspase活性检测发现：DAPK1表达的抑制,有效逆转了grifolin诱导的Caspase-3,8,9的活化和凋亡效应。
由此提示,grifolin作为一种具有重要的诱导肿瘤细胞凋亡生物学功能的化学小分子探针,可能通过促进ERK1/2和DAPK1蛋白间作用,抑制ERK1/2蛋白核移位,发挥诱导肿瘤细胞凋亡的生物学效应。
三、明确Grifolin调节ERK1/2激酶活性,靶向ERK1/2-DAPK1-p21通路诱导肿瘤细胞G0/G1期阻滞
我们的前期研究发现grifolin不但对肿瘤细胞系和非肿瘤细胞系具有一定的选择性,而且能相对特异性地靶向丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPK)信号转导通路,以剂量依赖性方式抑制MAPK信号通路中ERK1/2激酶磷酸化,下调ERK1/2激酶的体内活性。上述这些重要发现为进一步证实grifolin是对肿瘤异常激活的MAPK信号分子有显著抑制作用的化学探针奠定了重要基础。
为了明确grifolin对ERK1/2通路的调节作用,我们利用伯基特淋巴瘤细胞Raji、人乳腺癌细胞MCF7、人宫颈鳞状上皮癌细胞Hela和人鼻咽鳞癌细胞CNE1等多种肿瘤细胞系为基本模型,通过亲和层析、激酶活性检测、Western Blot等技术手段,在证实grifolin在多种肿瘤细胞中抑制ERK1/2激酶磷酸化水平和体内激酶活性基础上,进一步明确grifolin调节ERK1/2激酶活性的作用机制。
研究显示,在Raji、MCF7和CNE1细胞中,grifolin可以显著抑制ERK1/2激酶磷酸化水平和激酶活性,但对ERK1/2蛋白表达无明显影响；在Hela细胞中,grifolin明显下调ERK1/2激酶磷酸化水平和激酶活性,同时下调ERK1/2蛋白表达。体外激酶实验证实grifolin可有效下调ERK2激酶活性。Grifolin-sepharose 4B pull down实验提示grifolin通过与ERK1/2蛋白结合干扰其激酶活性。ERK2激酶荧光淬灭实验进一步表明grifolin与ERK2蛋白可物理结合。当突变ERK2蛋白ATP结合空腔的Ile31、Val39、Leu156位氨基酸后,小分子探针grifolin无法与该突变体结合,即证实grifolin结合至ERK2蛋白的ATP结合空腔,以ATP竞争方式抑制ERK2激酶活性。
从细胞周期调控来阐明细胞增殖的分子基础,有助于确定化学小分子探针对相关蛋白特异性的功能效应。我们前期研究表明,grifolin能以剂量依赖性导致CNE1 G0/G1期细胞增多,阻滞细胞周期于G0/G1期。体内实验证实p21Cip1是DAPK家族ZIPk激酶的直接底物；而DAPK家族具有高度保守的催化域。研究表明,ERK及MAPK信号通路可调节DAPK1激酶活性。综上提示,grifolin可能通过靶向ERK1/2-DAPK1-p21信号通路,活化p21,诱导肿瘤细胞G0/G1期阻滞。
通过Western blot证明grifolin可有效诱导DAPK1激酶Ser308位的去磷酸化,活化DAPK1激酶。利用Western blot和IP等技术,证实grifolin可以剂量依赖性方式上调p21蛋白磷酸化水平,活化p21蛋白。进一步运用siRNA等策略,明确DAPK1介导grifolin诱导肿瘤细胞G0/G1期阻滞的生物学功能。
以上研究进一步证实grifolin以ATP竞争方式有效抑制ERK1/2激酶活性,通过ERK1/2-DAPK1-p21通路诱导肿瘤细胞G0/G1期阻滞。
综上,通过本课题的研究,证实grifolin是一种具有重要的诱导细胞凋亡及细胞周期阻滞生物学功能的化学小分子探针,即通过活化转录因子p53,上调DAPK1表达,并促进ERK1/2与DAPK1蛋白间相互作用,抑制ERK1/2的核积聚,诱导肿瘤细胞凋亡；通过靶向ERK1/2-DAPK1-p21通路,活化p21,诱导肿瘤细胞G0/G1期阻滞。
本研究揭示了grifolin诱导肿瘤细胞凋亡和周期阻滞的信号转导网络新组分,进一步阐明了真菌中小分子化合物与信号转导通路中多靶标蛋白间的相互作用,有利于认识化学小分子探针生物活性的本质；同时,为grifolin作为激酶抑制剂的候选小分子化合物提供了新的理论依据。
Higher fungi belong to biological source of higher "creative index". In recent years, some low molecular weight secondary metabolite products isolated from fruiting body, mycelin and medium of higher fungi exhibit properties of anti-tumor lead compounds. The secondary metabolites of fungi possess novel structures, higher specificity and species-dependence after evolutionary pre-selection. It makes these bioactive substances good material and irreplaceable for biomedicine and natural innovative drug development. The unique chemical-framework and action mode of bioactive secondary metabolites enable them to gradually become ideal chemicals in R&D of natural antitumor drugs.
Chemical low molecular probe is designated as chemicals that can highly selectively detect the structure, function of protein and the action mode. Its molecular weight is usually less than 700 Dalton. Chemical low-molecular probe may get involved in signal transduction, interfere, change or mimick the signal transduction progress and act as effective tool to elaborate the known biological signal transduction mechanism and explore the unknown transduction procedure.
In previous study, for the first time in the world we screened out a novel bioactive low molecular probe, grifolin, from extractions of the mushroom Albatrellus confluens. Grifolin is a Farnesyl phenolic compound, the molecular formula of which is C22H32O2, and the molecular weight is 328. It has been shown to inhibit the growth of some cancer cell lines, induce cell cycle arrest and significant apoptosis. However, the molecular signaling mechanism underlying the anticancer effect of this compound is not completely understood yet. To investigate the signal transduction mechanism of biological function of grifolin, the research is conducted from below three parts.
High-throughout scanning of apoptosis-related gene expression array provided a clue that the death-associated protein kinase 1 (DAPK1) was significantly up-regulated after grifolin treatment in CNE1 cells. DAPK1, as a stress-activated tumor suppressor protein, plays an important role in pro-apoptotic signal transduction pathways. Growing evidence of clinical sample detections and biological experiments suggest that loss of expression of DAPK1 might be an important initiator in tumorigenesis and development, which promotes us to conduct deeper research on molecular mechanism of DAPK1 protein expression regulated by grifolin in tumor cells.
We took human nasopharyngeal carcinoma cell CNE1 as a model, and observed that protein as well as mRNA level of DAPK1 was up-regulated by grifolin in a dose-dependent manner. It was further proved in other tumor cell lines. Based on it, we revealed the molecular mechanism of DAPK1 expression induced by grifolin via means of siRNA, over-expression, EMSA and ChIP assays.
By Western blot detection, we found that grifolin increased Ser392 and Ser20 phosphorylation level of transcription factor p53 protein, which could promote its transcription activity. While Ser15 and Thr81 phosphorylation levels of p53 had no detectable change after grifolin treatment. It suggests that grifolin may activate the transcription activity of p53 upon phosphorylation.
Bioinformatics analysis manifests the promoter region of dapkl includes p53 binding site. It effectively depleted the expression of p53 that introducing p53 siRNA to CNE1 for 72 hours. Meanwhile, DAPK1 protein expression was down-regulated significantly. Vice versa, introducing p53 expression vector pEGFP-C3-p53 to H1299, a p53-/-cell line, p53 protein was re-expressed and DAPK1 was up-regulated as well. Therefore, the expression of DAPK1 protein was identified to be positive proportional to p53 protein expression.
To evaluate whether grifolin promotes p53 binding to dapkl promoter region to up-regulate DAPK1 expression, we used EMS A and ChIP assays to detect in vitro and in vivo, respectively. We observed that nuclear protein treated with grifolin binding to dapkl DNA manifested higher than untreated CNE1 cells. Supershift-EMSA assay further demonstrated the transcript factor binding to dapkl DNA was p53 protein. The specificity of recruitment of p53 to dapkl promoter region was confirmed by ChIP assay. We found that compared with the untreated group,the recruitment of p53 to dapkl gene promoter increased after being treated with 30μM grifolin for 24 h.
The results above indicate that grifolin up-regulate the expression of DAPK1 via targeting p53-DAPK1 pathway and provide sound basis for revealing the molecular mechanism of apoptosis in tumor cells induced by grifolin.
On the basis of demonstrating that dapkl gene expression was upregulated by low molecular probe grifolin, we further investigated how it represents pro-apopotic effect by targeting DAPK1. Currently there are two known proteins interacting with death domain of DAPK1, including ERK kinase. It suggests that grifolin may promote the interaction of DAPK1 and ERK 1/2 to activate DAPK1 and inhibit the nuclear translocation of ERK 1/2, thus inducing tumor apoptosis.
By means of LCFM, separation of cytoplasmic and nuclear protein and Western blot, we observed the total protein level of ERK 1/2 did not change in CNE1 when treated with grifolin. Grifolin induced the cytoplasmic arrest of ERK 1/2, meantime, it restricted the nuclear translocation of ERK 1/2 in a dose-dependent manner. LCFM, Co-IP and reverse IP assays were used to determine that grifolin enhanced the interaction of DAPK1 and ERK 1/2 proteins. From the above experiments, we deduce that grifolin induces apoptosis by promoting the interaction of DAPK1 and ERK1/2 to block the nuclear accumulation of ERK 1/2.
To evaluate whether grifolin is involved in apoptotic effect induced by grifolin, DAPK1 siRNA was introduced into CNE1 cell. The results showed the suppression of DAPK1 expression effectively reversed the activation of Caspase-3,8,9 and apoptotic effect induced by grifolin.
The data above indicate that grifolin, a low molecular probe, exhibits biological activity of inducing apoptosis in tumor cells via promoting the interaction of ERK1/2 and DAPK1 proteins to inhibit the nuclear location of ERK1/2.
In our previous studies, we found grifolin had some selectivity for tumorous and non-tumorous cells and also relatively specifically targeted MAPK signal transduction pathway, moreover, down-regulated the phosphorylation level of ERK1/2 kinase in a dose-dependent manner. The above important findings suggest that grifolin has significant inhibitive effect on signal moleculars of MAPK pathway, which is frequently activated aberrantly in tumors.
In order to estimate whether grifolin regulate the ERK1/2 pathway, we detected the phosphorylation level of ERK1/2 and its kinase activity in human Burkitts Lymphoma cell line Raji, human breast cancer cell line MCF7, human cervical cancer cell line HeLa and nasopharyngeal carcinoma cell CNE1, respectively.
In Raji, MCF7 and CNE1 cells, grifolin significantly inhibited the phosphorylation level of ERK1/2 as well as its kinase activity, while had not much influence on ERK1/2 protein expression. In Hela, the phosphorylation level of ERK1/2 and its kinase activity as well as the total ERK1/2 protein were down-regulated by grifolin. In vitro kinase assay confirmed that grifolin can effectively down-regulate ERK2 kinase activity. Using grifolin-sepharose 4B pull down and fluorescence quench of ERK2 kinase assays, we demonstrated that grifolin interfered the ERK1/2 kinase activity via binding to it. When Ile 31, Val 39 and Leu 156 amino acids in ATP binding cavity of ERK2 protein are mutated simultaneously, grifolin can not bind to the ERK2 mutant, thus demonstrating that grifolin binds to the ATP cavity of ERK1/2 and inhibit the ERK2 kinase activity in ATP-competitive manner.
In previous study, we observed that grifolin induced G0/G1 arrest in tumor cells. In view of cell cycle regulation, it is beneficial to clarify the molecular basis of cell proliferation, and define specific target proteins related to chemical low molecular probe. In vivo experiment demonstrated p21 protein is direct substrate of DAPK family member ZIP kinase. It is known that DAPK family members possess high-conservative catalytic domain. ERK and mitogen-activated protein kinase (MAPK) signaling cascades, have been shown to regulate DAPK1 activity. Taken together, it suggests that grifolin may induce G0/G1 arrest via targeting ERK1/2-DAPK1-p21 signaling pathway to activate p21.
In this study, firstly we identified that grifolin effectively induced the dephosphorylation at Ser 308 of DAPK1 kinase to activate it. Secondly, we used Western blot and IP analysis to verify grifolin upregulated the phosphorylation level of p21 in a dose-dependent manner and activated it. Finally, the role of DAPK1 involved in G0/G1 arrest induced by grifolin was confirmed by introduing siRNA targeting DAPK1.
We demonstrated from above results that grifolin effectively inhibit the kinase activity of ERK1/2 in ATP-competitive manner and further induce G0/G1 arrest in tumor cells via ERK1/2-DAPK1 - p21 pathway.
In summary, we demonstrate that grifolin is an important chemical probe possessing the ability to induce apoptosis and cell cycle arrest in tumor cells. On one hand, it activates p53 to up-regulate the expression of DAPK1 and enhance the interaction of ERK1/2 and DAPK1 proteins to inhibit the nuclear accumulation of ERK1/2 and induce apoptosis in tumor cells. On the other hand, it activate p21 to induce G0/G1 arrest in tumor cells via targeting ERK1/2-DAPK1-p21.
The purpose of this research is to reveal the new components of signal transduction networks related to cell apoptosis and cell cycle arrest, and further clarify the interaction between low molecular chemicals from higher fungi and multi-protein of signaling pathway. It will explore the knowledge of biological active essence of low chemical probe. Meanwhile, it brings out new evidence for grifolin as kinase inhibitor candidate.
化学小分子探针是指能够高选择性地探测蛋白质的功能、结构以及与活性小分子作用模式的小分子化合物,分子量通常小于700Da。化学小分子探针可介入信号转导过程的研究,并能够干扰、改变或模拟信号转导过程,是阐述已知生物信号转导机理和揭示未知的生物信号转导过程的有效手段。
在前期研究中,我们在国际上率先从高等真菌地花菌子实体提取物中筛选得到新型抗肿瘤活性小分子探针grifolin。Grifolin为一种法尼基酚类化合物,分子式为C22H32O2,分子量为328,具有显著的广谱抗肿瘤活性,可诱导肿瘤细胞周期阻滞和凋亡发生,但其基于的分子机制并不十分清晰。为深入探讨grifolin发挥其生物学功能的信号转导分子机制,本课题从以下三个方面进行研究：
一、明确小分子探针Grifolin活化转录因子p53上调DAPK1表达
根据高通量凋亡相关基因表达谱筛查提供的线索：grifolin可显著上调死亡相关基因dapkl的表达,而DAPK1作为压力活化的肿瘤抑制蛋白,在促凋亡信号转导通路中扮演了重要角色。大量的临床样本检测及生物学实验数据显示,DAPK1表达缺失可能是肿瘤形成发展的重要起因。这启发我们在肿瘤细胞中对grifolin调节DAPK1蛋白表达分子机制进行深入探讨。
我们以人鼻咽鳞癌细胞CNE1为基本模型,通过RT-PCR、Western Blot等技术手段,分别从mRNA水平和蛋白质水平,证实grifolin可以剂量依赖性方式上调DAPK1的表达；进一步在多种肿瘤细胞系中证实这一结论。在此基础上,采用siRNA、EMSA、ChIP等策略,揭示grifolin上调DAPK1表达的分子机制。
通过Western blot检测发现,grifolin可显著上调转录因子p53Ser20和Ser392位磷酸化水平,而对其Ser15和Thr81位磷酸化状态无明显改变。p53作为重要的转录因子,其磷酸化修饰是p53重要的功能形式。由于Ser20和Ser392位磷酸化与p53转录活化密切相关,因此我们推断grifolin可能通过磷酸化活化p53,上调其转录活性。
生物信息学分析及文献显示,dapk1启动子区包含p53结合元件。我们采用siRNA策略,在CNE1细胞中转染p53 siRNA 72小时,可有效干扰p53蛋白表达,与此同时,DAPK1蛋白表达水平也显著下调；反之,在p53-/-H1299细胞中转染p53表达质粒pEGFP-C3-p53,在诱导p53表达的同时,DAPK1也明显上调,由此,证实转录因子p53与DAPK1蛋白表达呈正相关。
采用EMSA和ChIP策略,分别从体外和体内层面探讨grifolin是否通过促进p53与dapkl启动子区结合上调DAPK1的表达。EMSA结果显示,30μM grifolin处理CNE1 24小时后,其细胞核蛋白与dapkl基因DNA结合能力明显高于未处理CNE1细胞。Supershift-EMSA实验证实与dapkl基因DNA结合的转录因子为p53蛋白。ChIP实验表明,转录因子p53能特异性结合于dapkl基因启动子区。30μMgrifolin处理CNE1细胞24小时后,转录因子p53蛋白与dapkl基因DNA结合能力明显高于未处理组。
以上研究证实,小分子探针grifolin通过靶向p53-DAPK1通路,上调DAPK1表达,为我们深入探讨grifolin诱导肿瘤细胞凋亡的分子机制,提供了充分的实验依据。
二、证实Grifolin促进ERK1/2与DAPK1目互作用,抑制ERK1/2核积聚,诱导肿瘤细胞凋亡
在证实化学小分子探针grifolin可上调dapkl基因表达的基础上,进一步探讨其靶向DAPK1,发挥促凋亡生物学效应的分子机制。目前已知的与DAPK1死亡域相互作用的蛋白主要有两种,其中包括ERK激酶。由此提示,grifolin可能通过促进ERK1/2与DAPK1相互作用,抑制ERK1/2核内积聚,诱导肿瘤细胞凋亡。
运用LCFM、胞浆胞核蛋白抽提、Western blot等技术手段,分别从定性和定量层面确定,CNE1细胞经grifolin处理,对ERK1/2总蛋白水平无显著影响,但可以剂量依赖性方式促进ERK1/2蛋白胞浆羁留,抑制ERK1/2蛋白的核内分布。
通过LCFM、Co-IP和反式IP等策略,证实grifolin可有效促进ERK1/2和DAPK1蛋白间相互作用。
为探讨DAPK1是否介导grifolin诱导的肿瘤细胞凋亡效应,在CNE1细胞中转染DAPK1 siRNA, Western blot、FACS以及Caspase活性检测发现：DAPK1表达的抑制,有效逆转了grifolin诱导的Caspase-3,8,9的活化和凋亡效应。
由此提示,grifolin作为一种具有重要的诱导肿瘤细胞凋亡生物学功能的化学小分子探针,可能通过促进ERK1/2和DAPK1蛋白间作用,抑制ERK1/2蛋白核移位,发挥诱导肿瘤细胞凋亡的生物学效应。
三、明确Grifolin调节ERK1/2激酶活性,靶向ERK1/2-DAPK1-p21通路诱导肿瘤细胞G0/G1期阻滞
我们的前期研究发现grifolin不但对肿瘤细胞系和非肿瘤细胞系具有一定的选择性,而且能相对特异性地靶向丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPK)信号转导通路,以剂量依赖性方式抑制MAPK信号通路中ERK1/2激酶磷酸化,下调ERK1/2激酶的体内活性。上述这些重要发现为进一步证实grifolin是对肿瘤异常激活的MAPK信号分子有显著抑制作用的化学探针奠定了重要基础。
为了明确grifolin对ERK1/2通路的调节作用,我们利用伯基特淋巴瘤细胞Raji、人乳腺癌细胞MCF7、人宫颈鳞状上皮癌细胞Hela和人鼻咽鳞癌细胞CNE1等多种肿瘤细胞系为基本模型,通过亲和层析、激酶活性检测、Western Blot等技术手段,在证实grifolin在多种肿瘤细胞中抑制ERK1/2激酶磷酸化水平和体内激酶活性基础上,进一步明确grifolin调节ERK1/2激酶活性的作用机制。
研究显示,在Raji、MCF7和CNE1细胞中,grifolin可以显著抑制ERK1/2激酶磷酸化水平和激酶活性,但对ERK1/2蛋白表达无明显影响；在Hela细胞中,grifolin明显下调ERK1/2激酶磷酸化水平和激酶活性,同时下调ERK1/2蛋白表达。体外激酶实验证实grifolin可有效下调ERK2激酶活性。Grifolin-sepharose 4B pull down实验提示grifolin通过与ERK1/2蛋白结合干扰其激酶活性。ERK2激酶荧光淬灭实验进一步表明grifolin与ERK2蛋白可物理结合。当突变ERK2蛋白ATP结合空腔的Ile31、Val39、Leu156位氨基酸后,小分子探针grifolin无法与该突变体结合,即证实grifolin结合至ERK2蛋白的ATP结合空腔,以ATP竞争方式抑制ERK2激酶活性。
从细胞周期调控来阐明细胞增殖的分子基础,有助于确定化学小分子探针对相关蛋白特异性的功能效应。我们前期研究表明,grifolin能以剂量依赖性导致CNE1 G0/G1期细胞增多,阻滞细胞周期于G0/G1期。体内实验证实p21Cip1是DAPK家族ZIPk激酶的直接底物；而DAPK家族具有高度保守的催化域。研究表明,ERK及MAPK信号通路可调节DAPK1激酶活性。综上提示,grifolin可能通过靶向ERK1/2-DAPK1-p21信号通路,活化p21,诱导肿瘤细胞G0/G1期阻滞。
通过Western blot证明grifolin可有效诱导DAPK1激酶Ser308位的去磷酸化,活化DAPK1激酶。利用Western blot和IP等技术,证实grifolin可以剂量依赖性方式上调p21蛋白磷酸化水平,活化p21蛋白。进一步运用siRNA等策略,明确DAPK1介导grifolin诱导肿瘤细胞G0/G1期阻滞的生物学功能。
以上研究进一步证实grifolin以ATP竞争方式有效抑制ERK1/2激酶活性,通过ERK1/2-DAPK1-p21通路诱导肿瘤细胞G0/G1期阻滞。
综上,通过本课题的研究,证实grifolin是一种具有重要的诱导细胞凋亡及细胞周期阻滞生物学功能的化学小分子探针,即通过活化转录因子p53,上调DAPK1表达,并促进ERK1/2与DAPK1蛋白间相互作用,抑制ERK1/2的核积聚,诱导肿瘤细胞凋亡；通过靶向ERK1/2-DAPK1-p21通路,活化p21,诱导肿瘤细胞G0/G1期阻滞。
本研究揭示了grifolin诱导肿瘤细胞凋亡和周期阻滞的信号转导网络新组分,进一步阐明了真菌中小分子化合物与信号转导通路中多靶标蛋白间的相互作用,有利于认识化学小分子探针生物活性的本质；同时,为grifolin作为激酶抑制剂的候选小分子化合物提供了新的理论依据。
Higher fungi belong to biological source of higher "creative index". In recent years, some low molecular weight secondary metabolite products isolated from fruiting body, mycelin and medium of higher fungi exhibit properties of anti-tumor lead compounds. The secondary metabolites of fungi possess novel structures, higher specificity and species-dependence after evolutionary pre-selection. It makes these bioactive substances good material and irreplaceable for biomedicine and natural innovative drug development. The unique chemical-framework and action mode of bioactive secondary metabolites enable them to gradually become ideal chemicals in R&D of natural antitumor drugs.
Chemical low molecular probe is designated as chemicals that can highly selectively detect the structure, function of protein and the action mode. Its molecular weight is usually less than 700 Dalton. Chemical low-molecular probe may get involved in signal transduction, interfere, change or mimick the signal transduction progress and act as effective tool to elaborate the known biological signal transduction mechanism and explore the unknown transduction procedure.
In previous study, for the first time in the world we screened out a novel bioactive low molecular probe, grifolin, from extractions of the mushroom Albatrellus confluens. Grifolin is a Farnesyl phenolic compound, the molecular formula of which is C22H32O2, and the molecular weight is 328. It has been shown to inhibit the growth of some cancer cell lines, induce cell cycle arrest and significant apoptosis. However, the molecular signaling mechanism underlying the anticancer effect of this compound is not completely understood yet. To investigate the signal transduction mechanism of biological function of grifolin, the research is conducted from below three parts.
High-throughout scanning of apoptosis-related gene expression array provided a clue that the death-associated protein kinase 1 (DAPK1) was significantly up-regulated after grifolin treatment in CNE1 cells. DAPK1, as a stress-activated tumor suppressor protein, plays an important role in pro-apoptotic signal transduction pathways. Growing evidence of clinical sample detections and biological experiments suggest that loss of expression of DAPK1 might be an important initiator in tumorigenesis and development, which promotes us to conduct deeper research on molecular mechanism of DAPK1 protein expression regulated by grifolin in tumor cells.
We took human nasopharyngeal carcinoma cell CNE1 as a model, and observed that protein as well as mRNA level of DAPK1 was up-regulated by grifolin in a dose-dependent manner. It was further proved in other tumor cell lines. Based on it, we revealed the molecular mechanism of DAPK1 expression induced by grifolin via means of siRNA, over-expression, EMSA and ChIP assays.
By Western blot detection, we found that grifolin increased Ser392 and Ser20 phosphorylation level of transcription factor p53 protein, which could promote its transcription activity. While Ser15 and Thr81 phosphorylation levels of p53 had no detectable change after grifolin treatment. It suggests that grifolin may activate the transcription activity of p53 upon phosphorylation.
Bioinformatics analysis manifests the promoter region of dapkl includes p53 binding site. It effectively depleted the expression of p53 that introducing p53 siRNA to CNE1 for 72 hours. Meanwhile, DAPK1 protein expression was down-regulated significantly. Vice versa, introducing p53 expression vector pEGFP-C3-p53 to H1299, a p53-/-cell line, p53 protein was re-expressed and DAPK1 was up-regulated as well. Therefore, the expression of DAPK1 protein was identified to be positive proportional to p53 protein expression.
To evaluate whether grifolin promotes p53 binding to dapkl promoter region to up-regulate DAPK1 expression, we used EMS A and ChIP assays to detect in vitro and in vivo, respectively. We observed that nuclear protein treated with grifolin binding to dapkl DNA manifested higher than untreated CNE1 cells. Supershift-EMSA assay further demonstrated the transcript factor binding to dapkl DNA was p53 protein. The specificity of recruitment of p53 to dapkl promoter region was confirmed by ChIP assay. We found that compared with the untreated group,the recruitment of p53 to dapkl gene promoter increased after being treated with 30μM grifolin for 24 h.
The results above indicate that grifolin up-regulate the expression of DAPK1 via targeting p53-DAPK1 pathway and provide sound basis for revealing the molecular mechanism of apoptosis in tumor cells induced by grifolin.
On the basis of demonstrating that dapkl gene expression was upregulated by low molecular probe grifolin, we further investigated how it represents pro-apopotic effect by targeting DAPK1. Currently there are two known proteins interacting with death domain of DAPK1, including ERK kinase. It suggests that grifolin may promote the interaction of DAPK1 and ERK 1/2 to activate DAPK1 and inhibit the nuclear translocation of ERK 1/2, thus inducing tumor apoptosis.
By means of LCFM, separation of cytoplasmic and nuclear protein and Western blot, we observed the total protein level of ERK 1/2 did not change in CNE1 when treated with grifolin. Grifolin induced the cytoplasmic arrest of ERK 1/2, meantime, it restricted the nuclear translocation of ERK 1/2 in a dose-dependent manner. LCFM, Co-IP and reverse IP assays were used to determine that grifolin enhanced the interaction of DAPK1 and ERK 1/2 proteins. From the above experiments, we deduce that grifolin induces apoptosis by promoting the interaction of DAPK1 and ERK1/2 to block the nuclear accumulation of ERK 1/2.
To evaluate whether grifolin is involved in apoptotic effect induced by grifolin, DAPK1 siRNA was introduced into CNE1 cell. The results showed the suppression of DAPK1 expression effectively reversed the activation of Caspase-3,8,9 and apoptotic effect induced by grifolin.
The data above indicate that grifolin, a low molecular probe, exhibits biological activity of inducing apoptosis in tumor cells via promoting the interaction of ERK1/2 and DAPK1 proteins to inhibit the nuclear location of ERK1/2.
In our previous studies, we found grifolin had some selectivity for tumorous and non-tumorous cells and also relatively specifically targeted MAPK signal transduction pathway, moreover, down-regulated the phosphorylation level of ERK1/2 kinase in a dose-dependent manner. The above important findings suggest that grifolin has significant inhibitive effect on signal moleculars of MAPK pathway, which is frequently activated aberrantly in tumors.
In order to estimate whether grifolin regulate the ERK1/2 pathway, we detected the phosphorylation level of ERK1/2 and its kinase activity in human Burkitts Lymphoma cell line Raji, human breast cancer cell line MCF7, human cervical cancer cell line HeLa and nasopharyngeal carcinoma cell CNE1, respectively.
In Raji, MCF7 and CNE1 cells, grifolin significantly inhibited the phosphorylation level of ERK1/2 as well as its kinase activity, while had not much influence on ERK1/2 protein expression. In Hela, the phosphorylation level of ERK1/2 and its kinase activity as well as the total ERK1/2 protein were down-regulated by grifolin. In vitro kinase assay confirmed that grifolin can effectively down-regulate ERK2 kinase activity. Using grifolin-sepharose 4B pull down and fluorescence quench of ERK2 kinase assays, we demonstrated that grifolin interfered the ERK1/2 kinase activity via binding to it. When Ile 31, Val 39 and Leu 156 amino acids in ATP binding cavity of ERK2 protein are mutated simultaneously, grifolin can not bind to the ERK2 mutant, thus demonstrating that grifolin binds to the ATP cavity of ERK1/2 and inhibit the ERK2 kinase activity in ATP-competitive manner.
In previous study, we observed that grifolin induced G0/G1 arrest in tumor cells. In view of cell cycle regulation, it is beneficial to clarify the molecular basis of cell proliferation, and define specific target proteins related to chemical low molecular probe. In vivo experiment demonstrated p21 protein is direct substrate of DAPK family member ZIP kinase. It is known that DAPK family members possess high-conservative catalytic domain. ERK and mitogen-activated protein kinase (MAPK) signaling cascades, have been shown to regulate DAPK1 activity. Taken together, it suggests that grifolin may induce G0/G1 arrest via targeting ERK1/2-DAPK1-p21 signaling pathway to activate p21.
In this study, firstly we identified that grifolin effectively induced the dephosphorylation at Ser 308 of DAPK1 kinase to activate it. Secondly, we used Western blot and IP analysis to verify grifolin upregulated the phosphorylation level of p21 in a dose-dependent manner and activated it. Finally, the role of DAPK1 involved in G0/G1 arrest induced by grifolin was confirmed by introduing siRNA targeting DAPK1.
We demonstrated from above results that grifolin effectively inhibit the kinase activity of ERK1/2 in ATP-competitive manner and further induce G0/G1 arrest in tumor cells via ERK1/2-DAPK1 - p21 pathway.
In summary, we demonstrate that grifolin is an important chemical probe possessing the ability to induce apoptosis and cell cycle arrest in tumor cells. On one hand, it activates p53 to up-regulate the expression of DAPK1 and enhance the interaction of ERK1/2 and DAPK1 proteins to inhibit the nuclear accumulation of ERK1/2 and induce apoptosis in tumor cells. On the other hand, it activate p21 to induce G0/G1 arrest in tumor cells via targeting ERK1/2-DAPK1-p21.
The purpose of this research is to reveal the new components of signal transduction networks related to cell apoptosis and cell cycle arrest, and further clarify the interaction between low molecular chemicals from higher fungi and multi-protein of signaling pathway. It will explore the knowledge of biological active essence of low chemical probe. Meanwhile, it brings out new evidence for grifolin as kinase inhibitor candidate.
引文
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