熊果酸抑制胃癌细胞COX-2表达的信号转导研究

朱　悦，周逸婵，朱国琴，李剑萍，焦　政，李晓林，邵　耘，孙为豪

(1.南京医科大学第一附属医院老年消化科，江苏 南京　210029；2.盐城市第一人民医院肿瘤科，江苏 盐城　224005)



熊果酸抑制胃癌细胞COX-2表达的信号转导研究

朱悦1，周逸婵1，朱国琴1，李剑萍2，焦政1，李晓林1，邵耘1，孙为豪1

(1.南京医科大学第一附属医院老年消化科，江苏 南京210029；2.盐城市第一人民医院肿瘤科，江苏 盐城224005)

目的进一步明确熊果酸(ursolic acid,UA)抑制胃癌细胞环氧化酶-2(cycloxygenase-2,COX-2)表达的信号转导通路。方法人胃腺癌细胞株SGC-7901和MKN-45常规培养于RPMI-1640培养液中，细胞长至亚单层后分别加抗氧化剂N-乙酰-L半胱氨酸(NAC)、单磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase, AMPK)激活剂5-氨基咪唑-4-甲酰胺核苷酸(AICAR)、AMPK抑制剂compound C和信号转导与转录活化因子3(signal transducer and activator of transcription 3, STAT3)抑制剂WP1066预处理后再加UA连续培养24 h，Western blot检测AMPK、STAT3磷酸化水平和COX-2蛋白表达。结果抗氧化剂NAC和AMPK抑制剂compound C有效地阻断了UA抑制STAT3磷酸化和COX-2表达的作用，AMPK激活剂AICAR抑制STAT3磷酸化和COX-2表达，AICAR和UA联合作用大于单用，STAT3抑制剂WP1066对UA诱导的AMPK磷酸化无明显影响，WP1066单独或联合UA均可抑制STAT3磷酸化和COX-2表达且联合作用大于单用。结论UA通过ROS/AMPK/STAT3信号转导通路抑制胃癌细胞COX-2表达。

熊果酸；胃癌；活性氧；单磷酸腺苷激活的蛋白激酶；信号转导与转录活化因子3；环氧化酶-2

胃癌是影响我国人民生命健康最严重的恶性肿瘤之一，是癌症死亡的主要原因。根治性手术是目前治疗胃癌的最有效方法，但手术不能完全避免局部复发和远处转移的可能，防止肿瘤复发和转移的辅助治疗越来越受到国内外学者的重视。熊果酸(ursolic acid, UA)是广泛存在于白花蛇舌草、女贞子、乌梅和夏枯草等天然植物中的一种五环三萜类化合物，具有抑制肿瘤细胞增殖、诱导细胞凋亡、抗血管生成、抗促癌、抗突变和调节氧化应激等作用，近年来已成为肿瘤化学预防研究的热点[1-3]。临床前研究显示，UA在转基因肿瘤动物模型和人癌裸鼠移植模型中的抑瘤作用明显[3-4]，具有良好的临床应用前景。我们的前期研究发现，UA促进胃癌细胞内活性氧(reactive oxygen species, ROS)生成，诱导单磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase, AMPK)磷酸化，抑制信号转导与转录活化因子3(signal transducer and activator of transcription 3, STAT3)磷酸化和环氧化酶-2(cycloxygenase-2,COX-2)表达[5-6]。UA通过下调COX-2表达而抑制胃癌细胞增殖、诱导凋亡[6-7]。然而，UA抑制胃癌细胞COX-2表达的信号转导通路尚不清楚。本研究选择2株不同分化程度但COX-2高表达的SGC-7901和MKN-45胃癌细胞株作为研究对象，观察ROS/AMPK/STAT3信号转导通路在UA影响SGC-7901和MKN-45胃癌细胞株COX-2表达中的作用，进一步探讨UA抑制胃癌细胞COX-2表达的具体机制。

1　材料与方法

1.1药物和试剂UA、AMPK激活剂5-氨基咪唑-4-甲酰胺核苷酸(AICAR)、二甲基亚砜(DMSO)、碘化丙啶(PI)、苯甲磺酰氟(phenylmethanesulfonyl fluoride，PMSF) 和抑肽酶为美国Sigma-Aldrich公司产品；AMPK抑制剂compound C和STAT3抑制剂WP1066为德国Merck公司产品；RPMI 1640培养液和胎牛血清为美国Gibco BRL公司产品；蛋白质定量BCA试剂盒为美国Pierce公司产品；抗氧化剂N-乙酰-L半胱氨酸(NAC)为江苏碧云天生物技术公司产品；兔抗人AMPK和磷酸化AMPK(p-AMPK)、乙酰辅酶A羧化酶(ACC)和磷酸化ACC(p-ACC)、STAT3和 磷酸化STAT3(p-STAT3)、COX-2和β-actin单克隆抗体为美国Cell Signaling Technology公司产品；辣根过氧化物酶标记的山羊抗兔IgG抗体为美国Bioworld Technology公司产品。PVDF膜为美国Millipore公司产品；ECL发光试剂盒为英国Amersham公司产品；其它试剂为国产分析纯级。

1.2细胞和培养人胃腺癌中分化细胞株SGC-7901购自中国科学院上海生科院细胞资源中心，人胃腺癌低分化细胞株MKN-45购自南京凯基生物科技发展有限公司。SGC-7901和MKN-45细胞常规传代培养于含10%胎牛血清、100 kU·L-1青霉素和100 mg·L-1链霉素的RPMI 1640培养液中，37℃、5% CO2及饱和湿度的二氧化碳培养箱中培养生长。隔天换液，3 d传代1次。

1.3药物配制UA、AICAR、compound C和WP1066先以DMSO溶解，NAC用超纯水溶解，而后均以RPMI 1640培养液稀释至所需浓度，DMSO在培养液中的浓度不超过0.1%，0.22 μm的微孔滤膜过滤除菌后4℃保存备用。

1.4实验分组将传代后处于对数生长期的细胞分为对照(control)组、UA(30 μmol·L-1)组、抗氧化剂(NAC, 5 mmol·L-1)组、NAC+UA组、AMPK激活剂(AICAR, 0.5 mmol·L-1)组、AICAR+UA组、AMPK抑制剂(compound C, 2.5 μmol·L-1)组、compound C+UA组、STAT3抑制剂(WP1066, 5 μmol·L-1)组、WP1066+UA组。NAC+UA组NAC预处理30 min后UA再干预培养24 h；AICAR+UA组AICAR预处理2 h后UA再干预培养24 h；compound C+UA组compound C预处理1 h后UA再干预培养24 h；WP1066+UA组WP1066预处理1 h后UA再干预培养24 h。

1.5细胞蛋白提取和Western blot检测各组干预培养后的细胞用预冷的PBS洗涤3次，以100 μL细胞裂解液(PBS内含：Nonidet P-40 1%，脱氧胆酸钠5 g·L-1，SDS 1 g·L-1，PMSF 0.1 g·L-1和抑肽酶10 mg·L-1)4℃处理60 min。细胞裂解物经11 000×g4℃离心10 min后取上清，BCA试剂盒测定其蛋白浓度。常规进行SDS-PAGE电泳后转印至PVDF膜，室温封闭2 h，分别加入一抗(APMK抗体、p-AMPK抗体、ACC抗体、p-ACC抗体、STAT3抗体、p-STAT3抗体、COX-2抗体和β-actin抗体)，4℃孵育过夜，辣根过氧化物酶标记的山羊抗兔IgG抗体为第二抗体，4℃孵育2 h，ECL发光，使用ImageJ(National Institute of Health, Bethesda, MD)图像分析软件对蛋白电泳带的灰度进行半定量分析。

2　结果

2.1UA和抗氧化剂NAC对胃癌细胞AMPK、STAT3磷酸化和COX-2表达的影响Western blot检查结果显示，抗氧化剂NAC有效抑制UA诱导的AMPK磷酸化，ACC是AMPK的下游直接效应靶蛋白，p-ACC被认为是观察AMPK被激活的最佳指标[8](Fig 1)，UA抑制STAT3磷酸化和COX-2表达的作用被NAC所逆转(Fig 2)。

Fig 1　Effects of NAC on ursolic acid(UA)-induced AMPK and ACC phosphorylation in gastric cancer cells

Fig 2　Effects of NAC on UA-inhibited STAT3 phosphorylation and COX-2 expression in gastric cancer cells

2.2UA和AMPK激活剂AICAR对胃癌细胞AMPK、STAT3磷酸化和COX-2表达的影响Western blot检查结果显示，AMPK激活剂AICAR单用或联合UA均可诱导SGC-7901和MKN-45细胞AMPK磷酸化(Fig 3)，抑制STAT3磷酸化和COX-2表达，联合作用大于单药作用(Fig 4)。

Fig 3　Effects of AMPK activator AICAR(5-amino-4-imidazolecarboxamide riboside-1-b-D-ribofuranoside) on UA-induced AMPK and ACC phosphorylation in gastric cancer cells

Fig 4　Effects of AMPK activator AICAR on UA-inhibited STAT3 phosphorylation and COX-2 expression in gastric cancer cells

2.3UA和AMPK抑制剂compound C对胃癌细胞AMPK、STAT3磷酸化和COX-2表达的影响AMPK抑制剂compound C有效抑制UA诱导的AMPK磷酸化(Fig 5)，UA抑制STAT3磷酸化和COX-2表达的作用被compound C所逆转(Fig 6)。

Fig 5　Effects of AMPK inhibitor compound C on UA-induced AMPK and ACC phosphorylation in gastric cancer cells

Fig 6　Effects of AMPK inhibitor Compound C on UA-inhibited STAT3 phosphorylation and COX-2 expression in gastric cancer cells

2.4UA和STAT3抑制剂WP1066对胃癌细胞AMPK、STAT3磷酸化和COX-2表达的影响STAT3抑制剂WP1066对UA诱导的AMPK磷酸化无明显影响(Fig 7)，WP1066单用或联合UA均可抑制SGC-7901和MKN-45细胞STAT3磷酸化和COX-2表达，且联合作用大于单药作用(Fig 8)。

Fig 7　Effects of STAT3 inhibitor WP1066 on UA-induced AMPK and ACC phosphorylation in gastric cancer cells

Fig 8　Effects of STAT3 inhibitor WP1066 on UA-inhibited STAT3 phosphorylation and COX-2 expression in gastric cancer cells

3　讨论

ROS是氧在机体代谢过程中产生的中间产物及其衍生物，参与细胞增殖、分化、转化和凋亡以及细胞内重要信号途径的转导[9]，近年来研究表明，抗肿瘤药物诱导细胞凋亡与其引起肿瘤细胞内ROS水平增加密切相关[10-12]。AMPK是细胞能量代谢的主要调节器[13],激活AMPK可诱导胃癌和胰腺癌等肿瘤细胞发生凋亡[14-15]，UA和白藜芦醇(resveratrol)的抗肿瘤作用与ROS依赖的AMPK活化有关[5,16-17]。

AMPK除参与细胞内的能量代谢调节外还调控一些基因的转录和表达[18]，本研究结果显示，UA诱导AMPK磷酸化，抑制STAT3磷酸化和COX-2表达；抗氧化剂NAC能逆转UA对STAT3磷酸化和COX-2表达的抑制作用，提示ROS是UA抑制STAT3活性和COX-2表达的重要介质[5]。AMPK激活剂AICAR是AMP类似物，明显抑制STAT3磷酸化和COX-2表达，UA与AICAR联合使用对STAT3磷酸化和COX-2表达的抑制作用和单用UA组相比差异有显著性，提示UA联合AICAR后AMPK得到进一步的激活。而AMPK抑制剂compound C则逆转UA对STAT3磷酸化和COX-2表达的抑制作用，进一步证明UA通过AMPK信号通路抑制胃癌细胞STAT3活化和COX-2表达。

我们前期研究发现，JAK2/STAT3和PI3K/Akt信号途径能诱导胃癌细胞COX-2的表达[19]，而UA能阻断STAT3通路抑制多发性骨髓瘤细胞增殖[20]。STAT3在各种类型的人胃癌细胞株和胃癌组织中都有较高的活性，JAK/STAT信号转导途径可能在胃癌的发生、发展中起重要的作用[21]。因此，我们推测UA通过ROS/AMPK/STAT3通路下调COX-2表达。当然，由于细胞内信号转导机制十分复杂，UA抑制胃癌细胞COX-2表达尚不排除有其它通路的存在，也不排除不同通路之间存在关联(如cross-talk等)，要全面了解其机制需大量而深入的研究。

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Signal transduction pathway of ursolic acid inhibiting COX-2 expression in gastric cancer cells

ZHU Yue1, ZHOU Yi-chan1, ZHU Guo-qin1, LI Jian-ping2,JIAO Zheng1, LI Xiao-lin1, SHAO Yun1, SUN Wei-hao1

(1.DeptofGeriatricGastroenterology,theFirstAffiliatedHospitalofNanjingMedicalUniversity,Nanjing210029,China;2.DeptofOncology,theFirstPeople′sHospitalofYanchengCity,YanchengJiangsu224005,China)

AimOur previous study has found that ursolic acid(UA) increased intracellular reactive oxygen species(ROS) production and adenosine monophosphate-activated protein kinase(AMPK) phosphorylation, inhibited signal transducer and activator of transcription 3(STAT3) phosphorylation and cyclooxygenase-2(COX-2) expression in gastric cancer cells. However, the molecular mechanism by which UA inhibits COX-2 expression in gastric cancer cells has not been fully clarified. In this study we aimed to further clarify the signal transduction pathways involved in the UA-mediated inhibition of COX-2 expression in gastric cancer cells.MethodsHuman gastric cancer cell lines SGC-7901 and MKN-45 were routinely cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum. Sub-confluent cell cultures were pre-treated with antioxidant N-acetylcysteine(NAC), AMPK activator 5-amino-4-imida- zolecarboxamide-riboside(AICAR), AMPK inhibitor compound C, or STAT3 inhibitor WP1066 and then treated with or without UA for 24 h. The expression of AMPK and phosphorylated AMPK(p-AMPK), STAT3 and phosphorylated STAT3(p-STAT3), as well as COX-2 was detected by Western blot analysis.ResultsAntioxidant NAC and AMPK inhibitor compound C blocked UA-induced inhibition of STAT3 phosphorylation and down-regulation of COX-2 expression in gastric cancer cells. Both AMPK activator AICAR and UA inhibited STAT3 phosphorylation and COX-2 expression; the combination of two drugs resulted in further reduction. STAT3 inhibitor WP1066 did not affect UA-induced AMPK phosphorylation, whereas it inhibited STAT3 phosphorylation and COX-2 expression. The inhibitory effects on the STAT3 phosphorylation and COX-2 expression were significantly enhanced when SGC-7901 and MKN-45 cells were treated simultaneously with WP1066 plus UA.ConclusionUA inhibits COX-2 expression in gastric cancer cells, which may be mediated through ROS/AMPK/STAT3 signal transduction pathway.

ursolic acid; gastric cancer; reactive oxygen species; adenosine monophosphate-activated protein kinase; signal transducer and activator of transcription 3; cyclooxygenase-2

2016-01-07,

2016-03-17

国家自然科学基金面上项目(No 81372659)

朱悦(1990-)，女，硕士生，研究方向：胃癌防治，E-mail: 421892415@qq.com；孙为豪(1963-)，男，教授，博士生导师，研究方向：胃肠道肿瘤的分子生物学，通讯作者，E-mail：swh@njmu.edu.cn

10.3969/j.issn.1001-1978.2016.07.009

A

1001-1978(2016)07-0925-08

R284.1；R345.4；R345.57；R735.202.2；R977.3；R977.6

网络出版时间：2016-6-20 11:49网络出版地址：http://www.cnki.net/kcms/detail/34.1086.R.20160620.1149.018.html