朱　悅，周逸嬋，朱國(guó)琴，李劍萍，焦　政，李曉林，邵　耘，孫為豪

(1.南京醫(yī)科大學(xué)第一附屬醫(yī)院老年消化科，江蘇 南京　210029；2.鹽城市第一人民醫(yī)院腫瘤科，江蘇 鹽城　224005)

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熊果酸抑制胃癌細(xì)胞COX-2表達(dá)的信號(hào)轉(zhuǎn)導(dǎo)研究

朱悅1，周逸嬋1，朱國(guó)琴1，李劍萍2，焦政1，李曉林1，邵耘1，孫為豪1

(1.南京醫(yī)科大學(xué)第一附屬醫(yī)院老年消化科，江蘇 南京210029；2.鹽城市第一人民醫(yī)院腫瘤科，江蘇 鹽城224005)

目的進(jìn)一步明確熊果酸(ursolic acid,UA)抑制胃癌細(xì)胞環(huán)氧化酶-2(cycloxygenase-2,COX-2)表達(dá)的信號(hào)轉(zhuǎn)導(dǎo)通路。方法人胃腺癌細(xì)胞株SGC-7901和MKN-45常規(guī)培養(yǎng)于RPMI-1640培養(yǎng)液中，細(xì)胞長(zhǎng)至亞單層后分別加抗氧化劑N-乙酰-L半胱氨酸(NAC)、單磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase, AMPK)激活劑5-氨基咪唑-4-甲酰胺核苷酸(AICAR)、AMPK抑制劑compound C和信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄活化因子3(signal transducer and activator of transcription 3, STAT3)抑制劑WP1066預(yù)處理后再加UA連續(xù)培養(yǎng)24 h，Western blot檢測(cè)AMPK、STAT3磷酸化水平和COX-2蛋白表達(dá)。結(jié)果抗氧化劑NAC和AMPK抑制劑compound C有效地阻斷了UA抑制STAT3磷酸化和COX-2表達(dá)的作用，AMPK激活劑AICAR抑制STAT3磷酸化和COX-2表達(dá)，AICAR和UA聯(lián)合作用大于單用，STAT3抑制劑WP1066對(duì)UA誘導(dǎo)的AMPK磷酸化無明顯影響，WP1066單獨(dú)或聯(lián)合UA均可抑制STAT3磷酸化和COX-2表達(dá)且聯(lián)合作用大于單用。結(jié)論UA通過ROS/AMPK/STAT3信號(hào)轉(zhuǎn)導(dǎo)通路抑制胃癌細(xì)胞COX-2表達(dá)。

熊果酸；胃癌；活性氧；單磷酸腺苷激活的蛋白激酶；信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄活化因子3；環(huán)氧化酶-2

胃癌是影響我國(guó)人民生命健康最嚴(yán)重的惡性腫瘤之一，是癌癥死亡的主要原因。根治性手術(shù)是目前治療胃癌的最有效方法，但手術(shù)不能完全避免局部復(fù)發(fā)和遠(yuǎn)處轉(zhuǎn)移的可能，防止腫瘤復(fù)發(fā)和轉(zhuǎn)移的輔助治療越來越受到國(guó)內(nèi)外學(xué)者的重視。熊果酸(ursolic acid, UA)是廣泛存在于白花蛇舌草、女貞子、烏梅和夏枯草等天然植物中的一種五環(huán)三萜類化合物，具有抑制腫瘤細(xì)胞增殖、誘導(dǎo)細(xì)胞凋亡、抗血管生成、抗促癌、抗突變和調(diào)節(jié)氧化應(yīng)激等作用，近年來已成為腫瘤化學(xué)預(yù)防研究的熱點(diǎn)[1-3]。臨床前研究顯示，UA在轉(zhuǎn)基因腫瘤動(dòng)物模型和人癌裸鼠移植模型中的抑瘤作用明顯[3-4]，具有良好的臨床應(yīng)用前景。我們的前期研究發(fā)現(xiàn)，UA促進(jìn)胃癌細(xì)胞內(nèi)活性氧(reactive oxygen species, ROS)生成，誘導(dǎo)單磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase, AMPK)磷酸化，抑制信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄活化因子3(signal transducer and activator of transcription 3, STAT3)磷酸化和環(huán)氧化酶-2(cycloxygenase-2,COX-2)表達(dá)[5-6]。UA通過下調(diào)COX-2表達(dá)而抑制胃癌細(xì)胞增殖、誘導(dǎo)凋亡[6-7]。然而，UA抑制胃癌細(xì)胞COX-2表達(dá)的信號(hào)轉(zhuǎn)導(dǎo)通路尚不清楚。本研究選擇2株不同分化程度但COX-2高表達(dá)的SGC-7901和MKN-45胃癌細(xì)胞株作為研究對(duì)象，觀察ROS/AMPK/STAT3信號(hào)轉(zhuǎn)導(dǎo)通路在UA影響SGC-7901和MKN-45胃癌細(xì)胞株COX-2表達(dá)中的作用，進(jìn)一步探討UA抑制胃癌細(xì)胞COX-2表達(dá)的具體機(jī)制。

1　材料與方法

1.1藥物和試劑UA、AMPK激活劑5-氨基咪唑-4-甲酰胺核苷酸(AICAR)、二甲基亞砜(DMSO)、碘化丙啶(PI)、苯甲磺酰氟(phenylmethanesulfonyl fluoride，PMSF) 和抑肽酶為美國(guó)Sigma-Aldrich公司產(chǎn)品；AMPK抑制劑compound C和STAT3抑制劑WP1066為德國(guó)Merck公司產(chǎn)品；RPMI 1640培養(yǎng)液和胎牛血清為美國(guó)Gibco BRL公司產(chǎn)品；蛋白質(zhì)定量BCA試劑盒為美國(guó)Pierce公司產(chǎn)品；抗氧化劑N-乙酰-L半胱氨酸(NAC)為江蘇碧云天生物技術(shù)公司產(chǎn)品；兔抗人AMPK和磷酸化AMPK(p-AMPK)、乙酰輔酶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單克隆抗體為美國(guó)Cell Signaling Technology公司產(chǎn)品；辣根過氧化物酶標(biāo)記的山羊抗兔IgG抗體為美國(guó)Bioworld Technology公司產(chǎn)品。PVDF膜為美國(guó)Millipore公司產(chǎn)品；ECL發(fā)光試劑盒為英國(guó)Amersham公司產(chǎn)品；其它試劑為國(guó)產(chǎn)分析純級(jí)。

1.2細(xì)胞和培養(yǎng)人胃腺癌中分化細(xì)胞株SGC-7901購(gòu)自中國(guó)科學(xué)院上海生科院細(xì)胞資源中心，人胃腺癌低分化細(xì)胞株MKN-45購(gòu)自南京凱基生物科技發(fā)展有限公司。SGC-7901和MKN-45細(xì)胞常規(guī)傳代培養(yǎng)于含10%胎牛血清、100 kU·L-1青霉素和100 mg·L-1鏈霉素的RPMI 1640培養(yǎng)液中，37℃、5% CO2及飽和濕度的二氧化碳培養(yǎng)箱中培養(yǎng)生長(zhǎng)。隔天換液，3 d傳代1次。

1.3藥物配制UA、AICAR、compound C和WP1066先以DMSO溶解，NAC用超純水溶解，而后均以RPMI 1640培養(yǎng)液稀釋至所需濃度，DMSO在培養(yǎng)液中的濃度不超過0.1%，0.22 μm的微孔濾膜過濾除菌后4℃保存?zhèn)溆谩?/p>

1.4實(shí)驗(yàn)分組將傳代后處于對(duì)數(shù)生長(zhǎng)期的細(xì)胞分為對(duì)照(control)組、UA(30 μmol·L-1)組、抗氧化劑(NAC, 5 mmol·L-1)組、NAC+UA組、AMPK激活劑(AICAR, 0.5 mmol·L-1)組、AICAR+UA組、AMPK抑制劑(compound C, 2.5 μmol·L-1)組、compound C+UA組、STAT3抑制劑(WP1066, 5 μmol·L-1)組、WP1066+UA組。NAC+UA組NAC預(yù)處理30 min后UA再干預(yù)培養(yǎng)24 h；AICAR+UA組AICAR預(yù)處理2 h后UA再干預(yù)培養(yǎng)24 h；compound C+UA組compound C預(yù)處理1 h后UA再干預(yù)培養(yǎng)24 h；WP1066+UA組WP1066預(yù)處理1 h后UA再干預(yù)培養(yǎng)24 h。

1.5細(xì)胞蛋白提取和Western blot檢測(cè)各組干預(yù)培養(yǎng)后的細(xì)胞用預(yù)冷的PBS洗滌3次，以100 μL細(xì)胞裂解液(PBS內(nèi)含：Nonidet P-40 1%，脫氧膽酸鈉5 g·L-1，SDS 1 g·L-1，PMSF 0.1 g·L-1和抑肽酶10 mg·L-1)4℃處理60 min。細(xì)胞裂解物經(jīng)11 000×g4℃離心10 min后取上清，BCA試劑盒測(cè)定其蛋白濃度。常規(guī)進(jìn)行SDS-PAGE電泳后轉(zhuǎn)印至PVDF膜，室溫封閉2 h，分別加入一抗(APMK抗體、p-AMPK抗體、ACC抗體、p-ACC抗體、STAT3抗體、p-STAT3抗體、COX-2抗體和β-actin抗體)，4℃孵育過夜，辣根過氧化物酶標(biāo)記的山羊抗兔IgG抗體為第二抗體，4℃孵育2 h，ECL發(fā)光，使用ImageJ(National Institute of Health, Bethesda, MD)圖像分析軟件對(duì)蛋白電泳帶的灰度進(jìn)行半定量分析。

2　結(jié)果

2.1UA和抗氧化劑NAC對(duì)胃癌細(xì)胞AMPK、STAT3磷酸化和COX-2表達(dá)的影響Western blot檢查結(jié)果顯示，抗氧化劑NAC有效抑制UA誘導(dǎo)的AMPK磷酸化，ACC是AMPK的下游直接效應(yīng)靶蛋白，p-ACC被認(rèn)為是觀察AMPK被激活的最佳指標(biāo)[8](Fig 1)，UA抑制STAT3磷酸化和COX-2表達(dá)的作用被NAC所逆轉(zhuǎn)(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激活劑AICAR對(duì)胃癌細(xì)胞AMPK、STAT3磷酸化和COX-2表達(dá)的影響Western blot檢查結(jié)果顯示，AMPK激活劑AICAR單用或聯(lián)合UA均可誘導(dǎo)SGC-7901和MKN-45細(xì)胞AMPK磷酸化(Fig 3)，抑制STAT3磷酸化和COX-2表達(dá)，聯(lián)合作用大于單藥作用(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抑制劑compound C對(duì)胃癌細(xì)胞AMPK、STAT3磷酸化和COX-2表達(dá)的影響AMPK抑制劑compound C有效抑制UA誘導(dǎo)的AMPK磷酸化(Fig 5)，UA抑制STAT3磷酸化和COX-2表達(dá)的作用被compound C所逆轉(zhuǎn)(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抑制劑WP1066對(duì)胃癌細(xì)胞AMPK、STAT3磷酸化和COX-2表達(dá)的影響STAT3抑制劑WP1066對(duì)UA誘導(dǎo)的AMPK磷酸化無明顯影響(Fig 7)，WP1066單用或聯(lián)合UA均可抑制SGC-7901和MKN-45細(xì)胞STAT3磷酸化和COX-2表達(dá)，且聯(lián)合作用大于單藥作用(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　討論

ROS是氧在機(jī)體代謝過程中產(chǎn)生的中間產(chǎn)物及其衍生物，參與細(xì)胞增殖、分化、轉(zhuǎn)化和凋亡以及細(xì)胞內(nèi)重要信號(hào)途徑的轉(zhuǎn)導(dǎo)[9]，近年來研究表明，抗腫瘤藥物誘導(dǎo)細(xì)胞凋亡與其引起腫瘤細(xì)胞內(nèi)ROS水平增加密切相關(guān)[10-12]。AMPK是細(xì)胞能量代謝的主要調(diào)節(jié)器[13],激活A(yù)MPK可誘導(dǎo)胃癌和胰腺癌等腫瘤細(xì)胞發(fā)生凋亡[14-15]，UA和白藜蘆醇(resveratrol)的抗腫瘤作用與ROS依賴的AMPK活化有關(guān)[5,16-17]。

AMPK除參與細(xì)胞內(nèi)的能量代謝調(diào)節(jié)外還調(diào)控一些基因的轉(zhuǎn)錄和表達(dá)[18]，本研究結(jié)果顯示，UA誘導(dǎo)AMPK磷酸化，抑制STAT3磷酸化和COX-2表達(dá)；抗氧化劑NAC能逆轉(zhuǎn)UA對(duì)STAT3磷酸化和COX-2表達(dá)的抑制作用，提示ROS是UA抑制STAT3活性和COX-2表達(dá)的重要介質(zhì)[5]。AMPK激活劑AICAR是AMP類似物，明顯抑制STAT3磷酸化和COX-2表達(dá)，UA與AICAR聯(lián)合使用對(duì)STAT3磷酸化和COX-2表達(dá)的抑制作用和單用UA組相比差異有顯著性，提示UA聯(lián)合AICAR后AMPK得到進(jìn)一步的激活。而AMPK抑制劑compound C則逆轉(zhuǎn)UA對(duì)STAT3磷酸化和COX-2表達(dá)的抑制作用，進(jìn)一步證明UA通過AMPK信號(hào)通路抑制胃癌細(xì)胞STAT3活化和COX-2表達(dá)。

我們前期研究發(fā)現(xiàn)，JAK2/STAT3和PI3K/Akt信號(hào)途徑能誘導(dǎo)胃癌細(xì)胞COX-2的表達(dá)[19]，而UA能阻斷STAT3通路抑制多發(fā)性骨髓瘤細(xì)胞增殖[20]。STAT3在各種類型的人胃癌細(xì)胞株和胃癌組織中都有較高的活性，JAK/STAT信號(hào)轉(zhuǎn)導(dǎo)途徑可能在胃癌的發(fā)生、發(fā)展中起重要的作用[21]。因此，我們推測(cè)UA通過ROS/AMPK/STAT3通路下調(diào)COX-2表達(dá)。當(dāng)然，由于細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)機(jī)制十分復(fù)雜，UA抑制胃癌細(xì)胞COX-2表達(dá)尚不排除有其它通路的存在，也不排除不同通路之間存在關(guān)聯(lián)(如cross-talk等)，要全面了解其機(jī)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

國(guó)家自然科學(xué)基金面上項(xiàng)目(No 81372659)

朱悅(1990-)，女，碩士生，研究方向：胃癌防治，E-mail: 421892415@qq.com；孫為豪(1963-)，男，教授，博士生導(dǎo)師，研究方向：胃腸道腫瘤的分子生物學(xué)，通訊作者，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

網(wǎng)絡(luò)出版時(shí)間：2016-6-20 11:49網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/detail/34.1086.R.20160620.1149.018.html