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医药导报
HERALD OF MEDICINE, 2018, 37(1): 20-26
doi: 10.3870/j.issn.1004-0781.2018.01.005
木犀草素调控非小细胞肺癌上皮间质转化的分子机制*
Molecular Mechanism of Luteolin-regulated Epithelial Mesenchymal Transition in Non-small Cell Lung Cancer
阮君山1,2,, 周欢1, 蒋宗胜3, 王少明1,2
1.福建医科大学省立临床医学院,福州 350001
2.福建省立医院中医药分子生物学实验室,福州 350001
3.福建医科大学药学院,福州 350001
RUAN Junshan1,2,, ZHOU Huan1, JIANG Zongsheng3, WANG Shaoming1,2
1.Clinical College of Fujian Medical University,Fuzhou 350001,China
2.Molecular Biology Laboratory of Traditional Chinese Medicine,Fujian Provincial Hospital,Fuzhou 350001,China
3.School of Pharmacy,Fujian Medical University,Fuzhou 350001,China

作者简介 阮君山(1976-),男,福建政和人,副主任药师,博士,研究方向:药物干预肿瘤转移的分子机制和医院药学。电话:0591-88216304,E-mail:ruanjunshan@sohu.com。
基金资助: 国家自然科学基金面上项目(81273647)
中图分类号: R282.71;R734.2     文献标识码: A     文章编号: 1004-0781(2018)01-0020-07
摘要:

目的 探讨木犀草素调控非小细胞肺癌上皮-间质转化(EMT)的分子机制。方法 首先借助分子对接技术研究木犀草素可能的作用靶点,再通过流式细胞检测技术验证对接的结果。结果 分子对接结果表明木犀草素与整合素家族具有很好的对接作用,其中与整合素αIIbβ3在对接过程中能量最低,为-15.11;流式细胞检测结果显示,木犀草素可以下调低氧所诱导的整合素表达。结论 木犀草素可以抑制整合素的表达,据此推测木犀草素可以通过整合素来调控EMT,为中医药防治肿瘤转移提供新的思路。
关键词: 木犀草素 ; 上皮-间质转化 ; 分子对接 ; 整合素

Abstract:

Objective To investigate the molecular mechanism of luteolin-regulated epithelial-mesenchymal transition(EMT) in non-small cell lung cancer. Methods Firstly,the potential target of luteolin was studied by molecular docking.The effect of luteolin on EMT markers was analyzed,and then molecular biology experiments were used to verify the results. Results The result of molecular docking showed that luteolin had a good docking effect on the integrin family,of which the lowest binding energy was -15.11 in docking with Integrin αIIbβ3.The result of flow cytometry showed that luteolin could down-regulate the expression of integrin induced by hypoxia. Conclusion Luteolin can regulate integrin expression ,which suggested that luteolin can regulate EMT through integrin.This conclusion may provide new methods in prevention of tumor metastasis for traditional Chinese medicine.
Key words: Luteolin ; Epithelial-mesenchymal transition ; Molecular docking ; Integrin

木犀草素(luteolin) 属于黄酮类植物化学物,其化学名为:3',4',5,7-四羟黄酮(C15H10O6,CAS491-70-3),其结构见图1。木犀草素主要存在夏枯草、白花蛇舌草、石见穿、石上柏、鱼腥草、败酱草、半枝莲、金银花、菊花、荆芥等中药。研究表明其具有抗菌、抗炎、保护心血管、解痉、祛痰、抑制酶活性、免疫调节、抗氧化、抗辐射、改善帕金森病、利尿、利胆、抗纤维化等药理作用[1-5]。近年来,木犀草素在抗肿瘤方面的作用引起关注。研究发现木犀草素能干预细胞生长周期,抑制肿瘤细胞增殖[5-6];可抑制DNA拓扑异构酶,增敏肿瘤坏死因子(TNF-α)和Trail诱导的多种肿瘤细胞凋亡[7];能增强Gefitinib对EGF 信号通路的调控[8];抑制血管生成、抑制前列腺癌PC3细胞的侵袭转移[9-10]。


图1 木犀草素的化学结构

Fig.1 Chemical stucture of luteolin

上皮间质转化(epithelial-mesenchymal transition,EMT)的发生是多种细胞因子生长因子共同作用的结果。大量的信号通过细胞膜表面受体将信号转导入细胞内,因此而造成肿瘤细胞EMT相关蛋白的表达发生改变。肿瘤细胞一旦由上皮形态的细胞转化为间质化形态,肿瘤的增殖能力和侵袭转移能力将显著增强[11-12]。研究表明,木犀草素能有效地干预肿瘤的侵袭转移,抑制肿瘤的恶性进程,与它在EMT中的干预作用有关,但其中的分子机制尚不明确。

分子对接(molecular docking)技术是近年来发展迅速的计算机辅助药物设计的一项新兴技术。其通过配体与受体相互作用的“锁-钥匙”原理,模拟小分子配体与受体生物大分子之间的潜在作用[13]。通过计算模拟,可以有效预测药物与其靶点的结合模式和亲和力,从而进行药物的虚拟筛选[14]。为了探寻木犀草素对EMT的作用靶点,笔者采用分子对接技术,对EMT过程中的重要分子蛋白及靶点进行了模拟筛选,并对筛选后的结果进行实验验证。

1 材料与方法
1.1 实验细胞

H1975、A549人非小细胞肺癌细胞株,购自中国科学院上海细胞库。

1.2 试剂

RPMI1640(加拿大维森特公司,批号:35000006);胎牛血清(加拿大维森特公司,批号:086-110);木犀草素,含量98%,购自西安和霖生物工程有限公司;兔抗人Integrin αIIbβ3抗体和山羊抗兔二抗购自美国Cell Signaling Technology公司。

1.3 木犀草素三维结构分子的准备

木犀草素的结构来源于NCBI网站(PubChem Compound,CID:5280805),在PubMed网站上下载后缀名为sdf的木犀草素文件,然后使用UCSF Chimera分子图形软件制备分子(Prepping Molecules),将木犀草素的平面结构转化为三维结构,对其结构进行加极性氢(essential hydrogen)、加电子、合并非极性氢原子、计算Gasteiger-Htiekel电荷等常规处理,最后使用MM2算法对木犀草素的三维结构进行能量最小化优化(迭代次数1 000次,最小的均方根RMS梯度为0.10 nm)。将能量最优化的即最稳定的木犀草素结构转化生成PDB格式文件,以备使用。使用AutoDockTools(ADT)软件定义木犀草素分子的柔性部分(可旋转键),以PDBQT格式存储,己备Autogrid使用。

1.4 对接参数的设置

使用Autodock软件,应用拉马克(LGA)遗传算法,将局部能量搜索与遗传算法相结合,以半经验势函数作为能量打分函数,搜索小分子构象和位置。对接选用的受体格点盒子大小为2.25 nm×2.25 nm×2.25 nm (60点×60点×60点),格点间距为0.375 nm,格点盒子中心位于各个蛋白活性位点的中心。为了提高底物分子在对接过程中的柔性,使每次计算更为充分,从而得到更精确的结果,采用了较大的运算参数,将该算法中的种群数ga_pop_size (此命令是设置种群中个体的数目,每个个体都是由基因型和相关的表型组成的复合体)从默认值100增大为150,能量评估的最大值ga_n μM_evals (此命令设置在遗传算法计算过程中能量评定的最大次数)由默认值2 500 000增大为10 000 000,运算循环数从默认值10增大为100,其余参数使用默认值。对接计算在3.75 nm×3.75 nm×3.75 nm的矩形框中进行。

1.5 对接受体的获取

参照文献[15-16],从专业数据库获取蛋白质结构。蛋白质数据库RCSB的PDB数据库(http://www.rcsb.org/pdb/home/home.do)是一个专业收集包括蛋白质和核酸在内的生物大分子三级结构的数据库,于1971年由美国的布鲁克海文(Brookhaven)国家实验室创建。这些是构成包括细菌、酵母、植物、苍蝇、其他动物以及人类在内的生命体的基本分子。因参与肿瘤EMT过程的几大蛋白的三维晶体结构信息均可在蛋白质数据库RCSB的PDB数据库中(http://www.rcsb.org/pdb/home/home.do)上获得。从BIDD(Bioinformatics and Drug Design group)治疗性靶标数据库(Therapeutic Target Database)中收集对接受体(调控EMT的关键蛋白),包括现有常见药物靶标,结合调控EMT的几大因子,在PDB数据库搜索晶体结构,选出共20个靶标,其PDB代码见表1。对接前将配体从蛋白——配体复合物中去除后,得到各个目的蛋白的空间结构及活性口袋空间位点。通过用户图形界面ADT1.5.4版本软件中Select功能下Select from string将各个膜蛋白三维结构文件去掉水分子;同理用Select功能下Select from string窗口中Residue键入ARG8,判定对这些膜蛋白中是否含有柔性残基白;用Edit功能下Hydrogens进行加氢、合并非极性的氢,计算Gasteiger-Huekel电荷。最后以PDB格式存储,进行格式转化成PDBQT,以备Autogrid使用。

表1 蛋白PDB号、活性口袋等相关参数
Tab.1 Parameters of protein PDB number and their active pockets
目的蛋白质 PDB X Y Z 长度 参考文献(第一作者、杂志名、年份)
TGF-betaR 2X7O 16.956 -4.82 2.512 342 ROTH GJ.J Med Chem,2010
EGFR 3IKA 98.27 14.08 23.68 331 ZHOU W.Nature,2009
VEGFR 2XIR -9.236 68.598 44.359 316 IYER SJ .Biol Chem,2010
HGFR 2UZX 46.876 -34.983 130.84 289 NIEMANN HH.Cell,2007
C-Met 3R7O 90.38 100.79 -45.784 307 RICKERT KW.J Biol Chem,2011
PDGFR 1GQ4 -89.2.56 92.143 5.671 90 KARTHIKEYAN S.J Biol Chem,2002
E-cadherin 2QVF 49.341 -19.34 94.43 213 HAUSSINGER D.EMBO J,2008
N-cadherin 1NCI 24.57 56.784 100.35 110 SHAPIRO L.Nature,1995
beta-catenin 3SLA 56.498 -155.37 24.46 168 EVRARD-TODESCHI N.J Chem Inf Model,2008
IGFR 2ZM3 159.40 35.76 98.358 308 EPA VC.Protein Eng Des Sel,2006
Integrin αVβ3 1JV2 239.03 1.676 -56.140 1649 XIONG JP.Science,2001
Integrin α6β1 1AOX 98.013 -69.396 -64.13 1078 EMSLEY J.J Biol Chem,1997
Integrin αIIbβ3 3FCS -43.654 154.93 -15.67 1649 ZHU J.Mol Cell,2008
Integrin αVβ6 3F7Q 90.256 -4.254 -67.356 896 DE PEREDA JM.EMBO J,2009
Integrin α1β1 1CK4 35.574 6.094 -57.835 198 NOLTE M.FEBS Lett,1999
Twist 1AO5 12.673 -45.670 111.56 237 TIMM DE.Protein Sci,1997
Snail 1Y62 -25.268 -178.63 9.352 160 UTSINTONG M.J Biomol Screen,2009
Slug 4MBA 79.36 -67.350 56.394 147 BOLOGNESI M.J Mol Biol,1989
Claudin-1 2QUO 100.43 -3.673 89.64 126 VAN ITALLIE CM.J Biol Chem,2008
Valentine 1PVL 67.386 -90.252 6.327 301 PEDELACQ JD.Structure,1999
Wnt 4FOA -17.954 11.393 -13.741 316 KAKUGAWA S.Nature,2015

表1 蛋白PDB号、活性口袋等相关参数

Tab.1 Parameters of protein PDB number and their active pockets

1.6 细胞培养

取对数生长期的A549、H1975细胞,磷酸盐缓冲液(PBS)洗涤2次,加入0.25%胰蛋白酶(含0.02%乙二胺四乙酸)2 mL,37 ℃消化5 min。倒置显微镜下观察细胞分散情况,防止消化过度。加入小牛血清0.1 mL终止消化,吹散细胞并转移至10 mL离心管中,1 000 r·min-1离心5 min,用新鲜培养液2 mL混悬,取少量进行细胞计数,并调整细胞浓度至1×106·L-1,接种于培养皿中,待细胞生长至70%~80%融合,除空白组外,实验组中加入木犀草素(分子量为286.23,为浅黄色粉末,稍溶于水,易溶于乙醇、苯、乙醚等有机溶剂)终浓度分别为0,5,10,15,25 μmol·L-1,低氧培养条件为5% 氧气、5%二氧化碳、90%氮气,培养24 h。将木犀草素溶于二甲亚砜(DMSO)中,配制成10 mmol·L-1木犀草素母液,经孔径0.2 μm滤器滤过,按每管500 μL 分装,-20 ℃存储。

1.7 流式细胞检测

用10 mmol·L-1乙二胺四乙酸收集不同浓度(5,10,15,25 μmol·L-1)木犀草素处理的人A549、H1975细胞和未经处理的正常对照组细胞,用羊血清在4 ℃下封闭10 min。所有细胞经PBS漂洗1次后,分别使用抗Integrin αIIbβ3单克隆抗体4 ℃处理30 min,然后用PBS漂洗2次。细胞用标记了藻红蛋白的二抗4 ℃处理20 min,再用PBS漂洗2次。用PBS重悬液0.5 mL上流式细胞仪进行分析,测定处理组和正常对照组细胞表面Integrin αIIbβ3的表达情况。

2 结果
2.1 木犀草素的作用靶点

分子对接结果显示木犀草素的作用靶点为Integrin。在PudMed下查询出目的蛋白的标准Mesh词汇,在Protein Data Bank数据库中搜索目的蛋白的晶体结构,其PDB代码、参数信息见表1。

虚拟对接过程中,Autodock软件将木犀草素的10个不同空间构象分别于目的受体/蛋白在活性口袋进行计算机拟合,之后软件给予木犀草素与蛋白结合评分,其对接示意图见图2,计算机虚拟对接情况见表2。


图2 木犀草素与目的蛋白结合示意图

Fig.2 Docking diagram of luteolin and target protein

表2 木犀草素与目的蛋白虚拟结合能量及结合参数
Tab.2 Virtually intermolecular energy and binding parameter between luteolin and target protein
目的蛋白质 PDB 对接能量 kI/(nmol·L-1) 分子间能量 内部能量 扭转能量 未对接扩展能量
Integrin αIIbβ3 3FCS -15.11 80.48 -7.79 -2.30 2.98 -2.30
Integrin αVβ6 3F7Q -14.08 106.65 -5.49 -1.50 2.09 -1.50
Integrin αVβ3 1JV2 -13.08 187.67 -3.76 -1.49 2.12 -1.59
Integrin α6β1 1AOX -12.98 207.56 -2.99 -2.19 3.03 -2.69
Integrin α1β1 1CK4 -11.34 209.89 -2.58 -0.28 1.79 -0.28
Valentine 1PVL -8.32 199.37 -2.95 -1.32 3.01 -1.39
beta-catenin 3SLA -7.24 99.87 -1.56 -0.24 -1.45 0.44
IGFR 2ZM3 -5.78 104.83 -3.69 -3.91 -2.48 1.56
TGF-betaR 2X7O -5.63 87.46 -11.12 -0.48 1.49 -0.48
HGFR 2UZX -3.55 100.79 -9.55 0.00 0.00 0.00
C-Met 3R7O -1.56 100.83 -2.67 -0.89 3.7 -2.93
Claudin-1 2QUO -1.46 200.43 -0.33 -3.07 1.29 -3.07
N-cadherin 1NCI -0.98 139.56 -3.7 -1.39 0.00 -2.78
Slug 4MBA -0.59 134.78 -4.59 -5.10 0.29 -2.50
Twist 1AO5 -0.34 194.68 -4.51 -1.87 0.13 -6.18
VEGFR 2XIR 1.14 200.02 -10.33 -0.37 1.19 -0.37
Wnt 4FOA 2.37 183.35 -8.48 -0.92 1.23 -3.62
E-cadherin 2QVF 4.67 110.56 -3.12 -2.59 1.03 -1.93
EGFR 3IKA 8.68 435.15 -8.68 0.00 0.00 0.00
Snail 1Y62 9.56 198.56 -4.76 -1.39 1.22 -5.19
PDGFR 1GQ4 10.79 89.23 -1.45 -1.57 0.00 -1.86

表2 木犀草素与目的蛋白虚拟结合能量及结合参数

Tab.2 Virtually intermolecular energy and binding parameter between luteolin and target protein

对接结果显示,木犀草素与PDGFR等调控EMT蛋白的对接能量为正值,提示木犀草素与这些蛋白的作用相比较与能量为负值的蛋白可能不是主要因素。对接能量为负值的结果中发现与Integrin等膜蛋白比较,木犀草素对E-cadherin和N-cadherin的对接能量较低,这也提示木犀草素对EMT的作用可能不是直接与E-cadherin或N-cadherin结合而发挥的。

进一步的分析发现,与Integrin的对接能量值比较,木犀草素对EMT调控的几个转录因子如Twist、Snail等对接的能量值亦较低,提示木犀草素对EMT调控可能不是通过binding这几个转录因子产生的。表2显示,木犀草素与Integrin αIIbβ3在对接过程中能量最低,因此选取木犀草素10个构象中结合自由能最低的构象用来做进一步的分析。Integrin αIIbβ3与木犀草素的相互作用见图3。


图3 Integrin αIIbβ3和木犀草素的相互作用

Fig.3 Interaction between Integrin αIIbβ3 and luteolin

图3中mu代表木犀草素(蓝色棍棒模型),红色部分为木犀草素与Integrin αIIbβ3结合部分,绿色部分表示木犀草素与Integrin αIIbβ3的结合键(氢键)。Binding Site为ASP359、THR364、HIS358、PRO327、ILE365、ARG358,两氢键Distance分别为2.206,2.081,氢键能量分别为-0.12, -1.71。从分子模拟的结构图预测了木犀草素与Integrin αIIbβ3可能存在的结合位点,因此Integrin αIIbβ3可能是其调控EMT作用的靶点。

2.2 流式细胞检测木犀草素对Integrin表达的影响

见图4。图4B与图4A比较显示,低氧诱导可以升高A549细胞表面的Integrin αIIbβ3表达。图C~F分别为在低氧的条件下,采用木犀草素终浓度为5,10,15,25 μmol·L-1孵育后A549细胞表面Integrin αIIbβ3的表达情况。流式细胞结果图显示,木犀草素对于低氧诱导的A549细胞表面Integrin αIIbβ3表达的增加具有抑制作用,并且这种对于Integrin αIIbβ3表达的下调呈现出剂量依赖性。


图4 流式细胞术检测木犀草素对A549细胞Integrin αIIbβ3表达的调控作用 A.正常对照组;B.低氧组;C.5 μmol·L-1木犀草素组;D.10 μmol·L-1木犀草素组;E.15 μmol·L-1木犀草素组;F.25 μmol·L-1木犀草素组

Fig.4 Effect of luteolin on the expression of Integrin αIIbβ3 in A549 cells detected by flow cytometry A.normal control group;B.hypoxia group;C.5 μmol·L-1 luteolin group;D.10 μmol·L-1 luteolin group;E.15 μmol·L-1 luteolin group;F.25 μmol·L-1 luteolin group

图5B与图5A相比的流式细胞结果显示低氧诱导可以升高H1975细胞表面的Integrin αIIbβ3表达。图5的C~F分别为在低氧的条件下,采用木犀草素终浓度为5,10,15,25 μmol·L-1孵育后H1975细胞表面Integrin αIIbβ3的表达情况。流式细胞结果图显示,木犀草素对于低氧诱导的H1975细胞表面Integrin αIIbβ3表达的增加具有抑制作用,并且这种对于Integrin αIIbβ3表达的下调呈现出剂量依赖性。


图5 流式细胞术检测木犀草素对H1975细胞Integrin αIIbβ3表达的调控作用 A.正常对照组;B.低氧组;C.5 μmol·L-1木犀草素组;D.10 μmol·L-1木犀草素组;E.15 μmol·L-1木犀草素组;F.25 μmol·L-1木犀草素组

Fig.5 Effect of luteolin on the expression of Integrin αIIbβ3 in H1975 cells detected by flow cytometry A.normal control group;B.hypoxia group;C.5 μmol·L-1 luteolin group;D.10 μmol·L-1 luteolin group;E.15 μmol·L-1 luteolin group;F.25 μmol·L-1 luteolin group

3 讨论

低氧微环境在EMT发生过程中发挥着重要作用[17-18]。研究表明低氧可以诱导细胞分泌多种细胞生长因子如TGF-β、FGF、VEGF来刺激肿瘤的发生发展[19-21],调节和改变所处微环境,微环境被激活,可使肿瘤细胞进一步发生EMT。细胞标志蛋白如E-cadherin表达下调和N-cadherin、Vimentin表达上调是EMT发生的标志,这一变化与生长因子、信号通路、转录因子以及微环境改变如低氧等多种因素有关,是肿瘤微环境中多种细胞因子和信号通路相互作用的结果。Integrin是肿瘤细胞表面重要的黏附分子,同时也介导了胞外信号向胞内转导的过程,这些细胞因子与Integrin结合后触发细胞内信号级联反应,促使细胞发生EMT。

本实验用分子对接技术预测了木犀草素和EMT相关的一些蛋白的对接情况,结果显示木犀草素可以很好地与Integrin发生对接。流式细胞检测的结果提示,木犀草素可以和细胞表面的Integrin特异性结合,从而减少了Integrin的表达,实验结果证实了分子对接的假设。

肿瘤的侵袭和转移过程可以由多种信号通路和相关的侵袭转移蛋白所调控,并且木犀草素作为小分子中药单体,可能存在着多个靶点。仅利用分子对接的结果,尚无法说明Integrin是木犀草素干预的唯一靶点。是否在肿瘤细胞内还存在其他的药物靶点尚不明确。但不可否认的是分子对接技术作为计算机辅助模拟药物及靶点设计具有极大的利用价值。通过分子对接找到药物的可能作用靶点进而通过实验进行验证,为实验提供了正确性和可行性,可以为中药分子的机制研究提供新的思路。

The authors have declared that no competing interests exist.
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关键词（key words）
木犀草素
上皮-间质转化
分子对接
整合素
Luteolin
Epithelial-mesenchymal tr...
Molecular docking
Integrin
作者
阮君山
周欢
蒋宗胜
王少明
RUAN Junshan
ZHOU Huan
JIANG Zongsheng
WANG Shaoming