二氫青蒿素通過(guò)抑制SIRT1的表達(dá)而增強(qiáng)5-氟尿嘧啶對(duì)胃癌的抗腫瘤活性

萬(wàn) 斌， 曹恒斌， 俞根華

(湖州市中心醫(yī)院 1藥學(xué)部， 2放療介入科， 浙江 湖州 313000)

二氫青蒿素通過(guò)抑制SIRT1的表達(dá)而增強(qiáng)5-氟尿嘧啶對(duì)胃癌的抗腫瘤活性

萬(wàn) 斌1△， 曹恒斌1， 俞根華2

(湖州市中心醫(yī)院1藥學(xué)部，2放療介入科， 浙江 湖州 313000)

目的探討二氫青蒿素對(duì)5-氟尿嘧啶治療胃癌的輔助作用并研究其機(jī)制。方法實(shí)驗(yàn)分為對(duì)照組、二氫青蒿素組、5-氟尿嘧啶組、5-氟尿嘧啶聯(lián)合二氫青蒿素組和5-氟尿嘧啶+二氫青蒿素+SIRT1質(zhì)粒組。MTT法檢測(cè)胃癌細(xì)胞系BGC-823在5-氟尿嘧啶聯(lián)合二氫青蒿素處理下的細(xì)胞活力。Western blot實(shí)驗(yàn)檢測(cè)5-氟尿嘧啶聯(lián)合二氫青蒿素對(duì)BGC-823細(xì)胞SIRT1和NADPH氧化酶表達(dá)水平，caspase-9和caspase-3活化水平及凋亡信號(hào)調(diào)節(jié)激酶1(ASK1)和c-Jun氨基末端激酶(JNK)蛋白磷酸化水平的影響。流式細(xì)胞術(shù)檢測(cè)BGC-823細(xì)胞在5-氟尿嘧啶和二氫青蒿素聯(lián)合處理下的活性氧簇(ROS)生成水平和細(xì)胞凋亡率。結(jié)果二氫青蒿素處理能顯著抑制BGC-823細(xì)胞SIRT1的表達(dá)并增加NADPH氧化酶的蛋白水平，明顯提高BGC-823細(xì)胞對(duì)5-氟尿嘧啶的敏感性，降低5-氟尿嘧啶的半數(shù)抑制濃度；轉(zhuǎn)染SIRT1表達(dá)質(zhì)粒后，二氫青蒿素聯(lián)合5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性受到顯著抑制(P<0.05)。二氫青蒿素能明顯促進(jìn)5-氟尿嘧啶對(duì)BGC-823細(xì)胞生成ROS的誘導(dǎo)效應(yīng)和ASK1及JNK的磷酸化(P<0.05)。用ROS清除劑N-乙酰半胱氨酸(NAC)或JNK特異性抑制劑SP600125處理后，二氫青蒿素聯(lián)合5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性和caspase-9及caspase-3的活化均受到明顯抑制(P<0.05)。另外，NAC能顯著抑制二氫青蒿素聯(lián)合5-氟尿嘧啶對(duì)JNK磷酸化的促進(jìn)作用，而SP600125卻不能影響B(tài)GC-823細(xì)胞ROS的產(chǎn)生，表明JNK是ROS的下游分子。結(jié)論二氫青蒿素聯(lián)合5-氟尿嘧啶通過(guò)SIRT1/NADPH氧化酶/ROS/JNK通路誘導(dǎo)胃癌細(xì)胞發(fā)生caspase依賴的凋亡。

二氫青蒿素； SIRT1蛋白； 5-氟尿嘧啶； BGC-823細(xì)胞； 活性氧簇； JNK信號(hào)通路

胃癌是臨床上最常見(jiàn)的消化道惡性腫瘤，近些年來(lái)的發(fā)病率呈上升趨勢(shì)。多數(shù)早期胃癌患者無(wú)明顯癥狀，因此很多患者在確診時(shí)已發(fā)生腫瘤的轉(zhuǎn)移和擴(kuò)散[1-2]。對(duì)于這些中晚期患者而言，化療在胃癌的治療中是不可替代的重要方法。然而隨著化療藥物的反復(fù)使用，腫瘤細(xì)胞對(duì)化療的敏感性往往會(huì)逐漸降低，而且大劑量的化療藥物對(duì)患者有很大的副作用[3-4]，因此采用一些輔助治療手段降低化療藥物的劑量并提高腫瘤細(xì)胞對(duì)化療的敏感性具有十分重要的意義。

5-氟尿嘧啶(5-fluorouracil，5-FU)是一種抗代謝化療藥物，在腫瘤細(xì)胞內(nèi)能轉(zhuǎn)化氟尿嘧啶脫氧核苷酸，從而抑制胸腺嘧啶核苷酸合成酶的活性干擾腫瘤細(xì)胞DNA的合成[5-6]。5-FU具有廣譜的抗腫瘤活性，是目前治療胃癌的一線化療藥物。然而，5-FU的副作用較大，且持續(xù)使用會(huì)使胃癌細(xì)胞對(duì)5-FU的敏感性逐漸降低[7-8]，因此，提高胃癌細(xì)胞對(duì)5-氟尿嘧啶的敏感性是提高化療效果的有效方法。

二氫青蒿素(dihydroartemisinin， DHA)是青蒿素衍生物，據(jù)報(bào)道有一定的抗腫瘤效應(yīng)[9-10]，然而其是否能增強(qiáng)5-氟尿嘧啶的抗腫瘤活性至今仍很少報(bào)道。本研究探討二氫青蒿素對(duì)5-氟尿嘧啶化療胃癌的輔助治療作用并研究其機(jī)制。

材 料 和 方 法

1 細(xì)胞培養(yǎng)

人胃癌細(xì)胞系BGC-823購(gòu)于美國(guó)模式培養(yǎng)物保存中心(American Type Culture Collection，ATCC)，在含10%胎牛血清的RPMI-1640培養(yǎng)基中，在37 °C恒溫培養(yǎng)箱中培養(yǎng)并通入5% CO2。

2 實(shí)驗(yàn)試劑

5-FU、DHA、MTT、N-乙酰半胱氨酸(N-acetylcysteine，NAC)、SP600125、二氫乙啶(dihydroethidium，DHE)和凋亡檢測(cè)試劑盒購(gòu)于Sigma-Aldrich；RPMI-1640培養(yǎng)基購(gòu)于Gibco；蛋白提取液、抗SIRT1、NADPH氧化酶p47亞基、p-ASK1、p-JNK、cleaved caspase-9、cleaved caspase-3和GAPDH抗體購(gòu)于Cell Signaling Technology；ECL試劑盒購(gòu)于Pierce；pcDNA3.1和Lipofectamine 2000購(gòu)于Invitrogen。

3 方法

3.15-氟尿嘧啶半數(shù)有效濃度(IC50)的測(cè)定 將BGC-823細(xì)胞按每孔5×103接種在96孔板上，孵育過(guò)夜，用0～4 μmol/L 5-氟尿嘧啶及50 μmol/L二氫青蒿素處理腫瘤細(xì)胞48 h，后加入20 μmol/L MTT (5 g/L)， 37 ℃恒溫培養(yǎng)箱中培養(yǎng)4 h，移除孔內(nèi)培養(yǎng)基，加入100 μL DMSO，570 nm波長(zhǎng)下測(cè)定吸光度(A)值。細(xì)胞活力結(jié)果用5-氟尿嘧啶處理組與對(duì)照組的A值比值表示。繪制細(xì)胞活力曲線，根據(jù)曲線計(jì)算5-氟尿嘧啶對(duì)BGC-823細(xì)胞的IC50。

3.2SIRT1重組質(zhì)粒構(gòu)建和轉(zhuǎn)染 人SIRT1基因的開(kāi)放閱讀框架序列經(jīng)PCR擴(kuò)增后以分子克隆的方法與pcDNA3.1連接后，構(gòu)建成SIRT1重組過(guò)表達(dá)質(zhì)粒。SIRT1過(guò)表達(dá)質(zhì)粒用脂質(zhì)體2000按試劑操作說(shuō)明書(shū)步驟進(jìn)行轉(zhuǎn)染，簡(jiǎn)要步驟如下：將2 ng/L空質(zhì)?；騍IRT1質(zhì)粒用脂質(zhì)體2000進(jìn)行包裹后加入到無(wú)血清培養(yǎng)基進(jìn)行混合。將貼壁的BGC-823細(xì)胞置于無(wú)血清培養(yǎng)基孵育6 h后，棄去無(wú)血清培養(yǎng)基，并加入新鮮的含10%胎牛血清的RPMI-1640培養(yǎng)基培養(yǎng)24 h。

3.3二氫青蒿素聯(lián)合5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性 空質(zhì)?；騍IRT1質(zhì)粒轉(zhuǎn)染的BGC-823細(xì)胞按每孔5×103接種在96孔板上，孵育過(guò)夜，之后加入2 μmol/L 5-氟尿嘧啶、50 μmol/L二氫青蒿素、2 mmol/L NAC和50 μmol/L SP600125處理細(xì)胞48 h，之后加入20 μL MTT (5 g/L) 37 ℃恒溫培養(yǎng)箱中培養(yǎng)4 h，移除孔內(nèi)培養(yǎng)基，加入100 μL DMSO，570 nm波長(zhǎng)下測(cè)定吸光度(A)值。細(xì)胞活力=A實(shí)驗(yàn)組/A對(duì)照組×100%。

3.4Western blot實(shí)驗(yàn) 空質(zhì)粒或SIRT1質(zhì)粒轉(zhuǎn)染的BGC-823細(xì)胞加入2 μmol/L 5-氟尿嘧啶、50 μmol/L二氫青蒿素、2 mmol/L NAC和50 μmol/L SP600125處理細(xì)胞48 h。之后用蛋白提取液提取BGC-823細(xì)胞中的總蛋白質(zhì)。將等量的總蛋白質(zhì)行12.5% SDS-PAGE。分離完畢后通過(guò)電轉(zhuǎn)方法將蛋白質(zhì)從分離膠上轉(zhuǎn)到PVDF膜上，用抗SIRT1、NADPH氧化酶p47亞基、p-ASK1、p-JNK、cleaved caspase-9、cleaved caspase-3和GAPDH抗體孵育過(guò)夜，之后再用帶辣根過(guò)氧化物酶的 II 抗孵育2 h，蛋白條帶用ECL試劑盒顯色發(fā)光。

3.5ROS水平的檢測(cè) DHE可自由透過(guò)活細(xì)胞膜進(jìn)入細(xì)胞內(nèi)，并被細(xì)胞內(nèi)的超氧陰離子型ROS氧化，形成氧化乙啶，氧化乙啶可摻入染色體DNA中，產(chǎn)生紅色熒光，因此紅色熒光強(qiáng)度越強(qiáng)，則ROS水平越高[11]。在空質(zhì)?；騍IRT1質(zhì)粒轉(zhuǎn)染的BGC-823細(xì)胞中加入2 μmol/L 5-氟尿嘧啶、50 μmol/L二氫青蒿素、2 mmol/L NAC和50 μmol/L SP600125處理細(xì)胞48 h。細(xì)胞處理完畢后用DHE試劑按說(shuō)明書(shū)步驟對(duì)BGC-823細(xì)胞進(jìn)行染色，之后用生理鹽水將細(xì)胞清洗3次，用流式細(xì)胞術(shù)檢測(cè)細(xì)胞的紅色熒光強(qiáng)度。

3.6流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡 在空質(zhì)?；騍IRT1質(zhì)粒轉(zhuǎn)染的BGC-823細(xì)胞中加入2 μmol/L 5-氟尿嘧啶、50 μmol/L二氫青蒿素、2 mmol/L NAC和50 μmol/L SP600125處理細(xì)胞48 h。之后按照凋亡試劑盒說(shuō)明書(shū)步驟將碘化丙啶(PI)和Annexin V加入細(xì)胞中孵育20 min，采用流式細(xì)胞術(shù)檢測(cè)腫瘤細(xì)胞的凋亡，Annexin V陽(yáng)性細(xì)胞即為凋亡細(xì)胞。

4 統(tǒng)計(jì)學(xué)處理

所有實(shí)驗(yàn)重復(fù)3次，實(shí)驗(yàn)數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，并用SPSS 15.0統(tǒng)計(jì)分析軟件進(jìn)行處理。兩組間均數(shù)的比較采用 Student’st檢驗(yàn)，多組間均數(shù)的比較采用單因素方差分析(one-way ANOVA)，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 二氫青蒿素通過(guò)下調(diào)SIRT1的表達(dá)增強(qiáng)5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性

MTT實(shí)驗(yàn)結(jié)果顯示，二氫青蒿素聯(lián)合處理能顯著提高5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性，降低5-氟尿嘧啶對(duì)BGC-823細(xì)胞的IC50(P<0.05)，見(jiàn)圖1A，表明二氫青蒿素輔助治療對(duì)胃癌的5-氟尿嘧啶化療有協(xié)同效應(yīng)。Western blot實(shí)驗(yàn)結(jié)果顯示二氫青蒿素顯著抑制BGC-823細(xì)胞中SIRT1蛋白的表達(dá)(P<0.05)，見(jiàn)圖1B，提示二氫青蒿素發(fā)揮協(xié)同效應(yīng)的機(jī)制可能和SIRT1的下調(diào)有關(guān)。MTT實(shí)驗(yàn)結(jié)果顯示轉(zhuǎn)染SIRT1表達(dá)質(zhì)粒后，二氫青蒿素聯(lián)合5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性明顯降低(P<0.05)，見(jiàn)圖1C，表明二氫青蒿素通過(guò)下調(diào)SIRT1的表達(dá)增強(qiáng)5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性。

2 二氫青蒿素通過(guò)下調(diào)SIRT1的表達(dá)促進(jìn)5-氟尿嘧啶對(duì)BGC-823細(xì)胞ROS的誘導(dǎo)產(chǎn)生

Western blot實(shí)驗(yàn)結(jié)果顯示二氫青蒿素顯著增加BGC-823細(xì)胞中NADPH氧化酶蛋白的表達(dá)水平(P<0.05)，然而轉(zhuǎn)染SIRT1質(zhì)粒后，NADPH氧化酶蛋白的表達(dá)水平明顯下降，見(jiàn)圖2A，提示SIRT1是NADPH氧化酶的負(fù)調(diào)節(jié)因子。流式細(xì)胞實(shí)驗(yàn)結(jié)果顯示，二氫青蒿素能明顯增強(qiáng)5-氟尿嘧啶對(duì)BGC-823細(xì)胞ROS的誘導(dǎo)產(chǎn)生，然而轉(zhuǎn)染SIRT1質(zhì)粒后，二氫青蒿素和5-氟尿嘧啶聯(lián)合治療的BGC-823細(xì)胞ROS的產(chǎn)生明顯減少，見(jiàn)圖2B，表明二氫青蒿素通過(guò)下調(diào)SIRT1的表達(dá)促進(jìn)5-氟尿嘧啶對(duì)BGC-823細(xì)胞ROS的誘導(dǎo)產(chǎn)生。流式細(xì)胞實(shí)驗(yàn)結(jié)果顯示ROS清除劑NAC[11]處理能明顯清除BGC-823細(xì)胞中的ROS，同時(shí)，MTT實(shí)驗(yàn)結(jié)果顯示BGC-823細(xì)胞用NAC處理后，二氫青蒿素聯(lián)合5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性顯著降低(P<0.05)，見(jiàn)圖2C，表明二氫青蒿素能通過(guò)SIRT1/ROS途徑提高5-氟尿嘧啶的抗胃癌活性。另外，Western blot實(shí)驗(yàn)結(jié)果顯示，二氫青蒿素聯(lián)合5-氟尿嘧啶顯著誘導(dǎo)BGC-823細(xì)胞caspase-9和caspase-3的活化，而SIRT1質(zhì)粒或NAC均能明顯抑制它們的活化(P<0.05)，見(jiàn)圖2D，提示二氫青蒿素能通過(guò)SIRT1/NADPH氧化酶/ROS途徑提高caspase對(duì)凋亡信號(hào)的敏感性。

3 二氫青蒿素聯(lián)合5-氟尿嘧啶通過(guò)ROS/JNK通路誘導(dǎo)BGC-823細(xì)胞發(fā)生caspase依賴的凋亡

Western blot結(jié)果顯示，二氫青蒿素聯(lián)合5-氟尿嘧啶能顯著誘導(dǎo)BGC-823細(xì)胞ASK1和其下游JNK蛋白的磷酸化，而JNK特異性抑制劑SP600125[12]和NAC均能抑制JNK的磷酸化(P<0.05)，見(jiàn)圖3A，表明胃癌細(xì)胞中二氫青蒿素聯(lián)合5-氟尿嘧啶誘導(dǎo)的JNK的活化受ROS的調(diào)控。然而，流式細(xì)胞實(shí)驗(yàn)結(jié)果顯示，SP600125不能明顯影響二氫青蒿素聯(lián)合5-氟尿嘧啶誘導(dǎo)的ROS的產(chǎn)生，見(jiàn)圖3B，表明胃癌細(xì)胞中JNK是ROS途徑的下游信號(hào)分子。MTT實(shí)驗(yàn)結(jié)果顯示SP600125和NAC均能抑制二氫青蒿素聯(lián)合5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性(P<0.05)，見(jiàn)圖3C，表明二氫青蒿素聯(lián)合5-氟尿嘧啶通過(guò)ROS/JNK通路誘導(dǎo)BGC-823細(xì)胞發(fā)生死亡。Western blot和流式細(xì)胞實(shí)驗(yàn)結(jié)果顯示SP600125和NAC均能抑制二氫青蒿素聯(lián)合5-氟尿嘧啶誘導(dǎo)的caspase-9和caspase-3的活化(P<0.05)和凋亡的發(fā)生(P<0.05)，見(jiàn)圖3D、E，表明二氫青蒿素聯(lián)合5-氟尿嘧啶通過(guò)ROS/JNK通路誘導(dǎo)BGC-823細(xì)胞發(fā)生caspase依賴的凋亡。

討 論

青蒿素是臨床上非常重要的抗瘧藥。近年來(lái)的研究發(fā)現(xiàn)青蒿素及其衍生物還具有一定的抗腫瘤作用，其中，二氫青蒿素的生物活性最強(qiáng)。如文獻(xiàn)報(bào)道二氫青蒿素能抑制鼻咽癌細(xì)胞的增殖并促進(jìn)腫瘤細(xì)胞的凋亡和細(xì)胞周期的阻滯[9]；又如二氫青蒿素還能抑制肺癌的發(fā)展和血管生成[13]。然而，二氫青蒿素是否對(duì)胃癌的化療有輔助治療作用，至今還未充分報(bào)道。5-氟尿嘧啶是治療消化道腫瘤的一線化療藥物，研究表明，5-氟尿嘧啶能通過(guò)誘導(dǎo)ROS的產(chǎn)生誘導(dǎo)腫瘤細(xì)胞發(fā)生線粒體途徑的凋亡[14-15]。然而5-氟尿嘧啶的長(zhǎng)期使用容易使腫瘤細(xì)胞發(fā)生耐藥性，降低其療效。在本研究中，作者通過(guò)實(shí)驗(yàn)發(fā)現(xiàn)二氫青蒿素輔助治療能明顯增強(qiáng)5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性，表明二氫青蒿素對(duì)胃癌的5-氟尿嘧啶化療有協(xié)同效應(yīng)。

Figure 1. Dihydroartemisinin (DHA) enhanced the antitumor effect of 5-FU on BGC-823 cells by down-regulating the expression of SIRT1. A: DHA increased the cytotoxicity of 5-FU to the BGC-823 cells; B: DHA down-regulated the expression of SIRT1 in the BGC-823 cells; C: transfection with SIRT1 plasmid inhibited the cell death of BGC-823 cells co-treated with 5-FU and DHA. Mean±SD.n=3.*P<0.05vscontrol group;#P<0.05vs5-FU+DHA group.

圖1二氫青蒿素通過(guò)下調(diào)SIRT1的表達(dá)增強(qiáng)5-氟尿嘧啶對(duì)BGC-823細(xì)胞的殺傷活性

SIRT1是一種組蛋白脫乙酰酶。研究表明SIRT1在多種腫瘤中發(fā)揮促癌作用，它的過(guò)表達(dá)能促進(jìn)骨肉瘤、胰腺癌和結(jié)直腸癌等腫瘤細(xì)胞的增殖和轉(zhuǎn)移[16-18]，并且腫瘤患者的SIRT1水平與其預(yù)后成反比[19]。更為重要的是，SIRT1的過(guò)表達(dá)還能導(dǎo)致腫瘤的化療抵抗，在一些腫瘤細(xì)胞中對(duì)SIRT1進(jìn)行基因沉默能明顯增強(qiáng)其對(duì)化療的敏感性[20-21]，表明SIRT1可能是提高化療療效的重要靶點(diǎn)。

Figure 2. Dihydroartemisinin (DHA) promoted the generation of ROS induced by 5-FU in the BGC-823 cells by down-regulating the expression of SIRT1. A: DHA up-regulated the expression of NADPH oxidase p47 subunit in the BGC-823 cells; B: DHA increased the production of ROS in 5-FU-treated BGC-823 cells; C: NAC inhibited the death of BGC-823 cells induced by DHA and 5-FU co-treatment； D: DHA enhanced the activation of caspase-9 and caspase-3 in BGC-823 cells treated with 5-FU. Mean±SD.n=3.△P<0.05vscontrol group；*P<0.05vs5-FU group;#P<0.05vs5-FU+DHA group.

圖2二氫青蒿素通過(guò)下調(diào)SIRT1的表達(dá)促進(jìn)5-氟尿嘧啶對(duì)BGC-823細(xì)胞ROS的產(chǎn)生

SIRT1在腫瘤細(xì)胞中發(fā)揮抗氧化作用，過(guò)表達(dá)的SIRT1能明顯抑制NADPH氧化酶并增加超氧化物歧化酶等抗氧化酶類的表達(dá)，從而清除腫瘤細(xì)胞中的ROS使細(xì)胞逃避線粒體途徑的凋亡[22-24]。在ROS依賴的凋亡途徑中，JNK是激活細(xì)胞凋亡的重要下游分子,ROS通過(guò)激活A(yù)SK1蛋白的磷酸化誘導(dǎo)JNK的活化[25-26]。JNK的活化能增加腫瘤細(xì)胞中促凋亡蛋白的表達(dá)并抑制Bcl-2抗凋亡蛋白的功能，從而使細(xì)胞進(jìn)入凋亡程序[27]。這些研究提示了SIRT1的高表達(dá)能通過(guò)抑制NADPH氧化酶/ROS/ASK1/JNK通路降低腫瘤細(xì)胞對(duì)凋亡信號(hào)的敏感性。本研究的結(jié)果表明二氫青蒿素能明顯降低胃癌細(xì)胞中SIRT1蛋白的表達(dá)并增加其下游NADPH氧化酶的蛋白水平，從而使胃癌細(xì)胞在5-氟尿嘧啶的治療下能產(chǎn)生更多的ROS，進(jìn)而誘導(dǎo)下游分子ASK1和JNK的磷酸化，使胃癌細(xì)胞發(fā)生caspase依賴的凋亡。

綜上所述，本研究證明了二氫青蒿素能顯著增強(qiáng)5-氟尿嘧啶的抗胃癌活性。這些研究為降低化療藥物的劑量并提高腫瘤細(xì)胞對(duì)化療的敏感性提供了新的策略和思路。

[1] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013[J]. CA Cancer J Clin, 2013, 63(1):11-30.

[2] Garattini SK, Basile D, Cattaneo M, et al. Molecular classifications of gastric cancers: novel insights and possible future applications[J]. World J Gastrointest Oncol, 2017, 9(5):194-208.

Figure 3. Dihydroartemisinin (DHA) promoted 5-FU-induced apoptosis of the BGC-823 cells through ROS/ASK1/JNK signaling pathway. A: DHA promoted the phosphorylation of ASK1 and JNK in 5-FU-treated BGC-823 cells; B: SP600125 failed to inhibit the ROS production in the BGC-823 cells co-treated with 5-FU and DHA; C: both SP600125 and NAC inhibited the death of BGC-823 cells co-treated with 5-FU and DHA; D: both SP600125 and NAC inhibited the activation of caspase-9 and caspase-3 in BGC-823 cells co-treated with 5-FU and DHA; E: both SP600125 and NAC inhibited the apoptosis of BGC-823 cells co-treated with 5-FU and DHA. Mean±SD.n=3.*P<0.05vscontrol group;#P<0.05vs5-FU+DHA group.

圖3二氫青蒿素通過(guò)ROS/ASK1/JNK途徑促進(jìn)5-氟尿嘧啶對(duì)BGC-823細(xì)胞凋亡的誘導(dǎo)

[3] Sun Z, Yue L, Shen, Z, et al. Downregulation of NPM expression by Her-2 reduces resistance of gastric cancer to oxaliplatin[J]. Oncol Lett, 2017, 13(4):2377-2384.

[4] Jiao S, Li N, Cai S, et al. Inhibition of CYFIP2 promotes gastric cancer cell proliferation and chemoresistance to 5-fluorouracil through activation of the Akt signaling pathway[J]. Oncol Lett, 2017, 13(4):2133-2140.

[5] 劉 宇， 高 東， 靳廣毅， 等. 阿司匹林與氟尿嘧啶協(xié)同抑制結(jié)腸癌細(xì)胞生長(zhǎng)增殖的機(jī)制研究[J]. 中國(guó)病理生理雜志, 2014, 30(6):988-993.

[6] Ghiringhelli F, Apetoh L. Enhancing the anticancer effects of 5-fluorouracil: current challenges and future perspectives[J]. Biomed J, 2015, 38(2):111-116.

[7] Lin X, Tian L, Wang L, et al. Antitumor effects and the underlying mechanism of licochalcone A combined with 5-fluorouracil in gastric cancer cells[J]. Oncol Lett, 2017, 13(3):1695-1701.

[8] Kang Y, Hu W, Bai X, et al. Curcumin sensitizes human gastric cancer cells to 5-fluorouracil through inhibition of the NFκB survival-signaling pathway[J]. Onco Targets Ther, 2016, 9:7373-7384.

[9] Huang Z, Huang X, Jiang D, et al. Dihydroartemisinin inhibits cell proliferation by induced G1 arrest and apoptosis in human nasopharyngealcarcinoma cells[J]. J Cancer Res Ther, 2016, 12(1):244-247.

[10] Li Y, Wang Y, Kong R, et al. Dihydroartemisinin suppresses pancreatic cancer cells via a microRNA-mRNA regulatory network[J]. Oncotarget, 2016, 7(38):62460-62473.

[11] Jiang K, Wang W, Jin X, et al. Silibinin, a natural flavonoid, induces autophagy via ROS-dependent mitochondrial dysfunction and loss of ATP involving BNIP3 in human MCF7 breast cancer cells[J]. Oncol Rep, 2015, 33(6):2711-2718.

[12] Li HY, Zhang J, Sun LL, et al. Celastrol induces apoptosis and autophagy via the ROS/JNK signaling pathway in human osteosarcoma cells: aninvitroandinvivostudy[J]. Cell Death Dis, 2015, 6:e1604.

[13] Shen R, Li J, Ye D, et al. Combination of onconase and dihydroartemisinin synergistically suppresses growth and angiogenesis of non-small-cell lung carcinoma and malignant mesothelioma[J]. Acta Biochim Biophys Sin (Shanghai), 2016, 48(10):894-901.

[14] Zou X, Liang J, Sun J, et al. Allicin sensitizes hepatocellular cancer cells to anti-tumor activity of 5-fluorouracil through ROS-mediated mitochondrial pathway[J]. J Pharmacol Sci, 2016, 131(4):233-240.

[15] Su J, Cheng H, Zhang D, et al. Synergistic effects of 5-fluorouracil and gambogenic acid on A549 cells: activation of cell death caused by apoptotic and necroptotic mechanisms via the ROS-mitochondria pathway[J]. Biol Pharm Bull, 2014, 37(8):1259-1268.

[16] Zhang N, Xie T, Xian M, et al. SIRT1 promotes metastasis of human osteosarcoma cells[J]. Oncotarget, 2016, 7(48):79654-79669.

[17] Li S, Hong H, Li H, et al. SIRT 1 overexpression is associated with metastasis of pancreatic ductal adenocarcinoma (PDAC) and promotes migration and growth of PDAC cells[J]. Med Sci Monit, 2016, 22:1593-1600.

[18] Cheng F, Su L, Yao C, et al. SIRT1 promotes epithelial-mesenchymal transition and metastasis in colorectal cancer by regulating Fra-1 expression[J]. Cancer Lett, 2016, 375(2):274-283.

[19] Zhang S, Huang S, Deng C, et al. Co-ordinated overexpression of SIRT1 and STAT3 is associated with poor survival outcome in gastric cancer patients[J]. Oncotarget, 2017, 8(12):18848-18860.

[20] Shuang T, Wang M, Zhou Y, et al. Over-expression of Sirt1 contributes to chemoresistance and indicates poor prognosis in serous epithelial ovarian cancer (EOC)[J]. Med Oncol, 2015, 32(12):260.

[21] Zhang T, Rong N, Chen J, et al. SIRT1 expression is associated with the chemotherapy response and prognosis of patients with advanced NSCLC[J]. PLoS One, 2013, 8(11):e79162.

[22] Cheng Y, Takeuchi H, Sonobe Y, et al. Sirtuin 1 attenuates oxidative stress via upregulation of superoxide dismutase 2 and catalase in astrocytes[J]. J Neuroimmunol， 2014, 269(1-2):38-43.

[23] Tang Y, Xu J, Qu W, et al. Resveratrol reduces vascular cell senescence through attenuation of oxidative stress by SIRT1/NADPH oxidase-dependent mechanisms[J]. J Nutr Biochem, 2012, 23(11):1410-1416.

[24] Jian KL, Zhang C, Kong LY, et al. Eucalrobusone C suppresses cell proliferation and induces ROS-dependent mitochondrial apoptosis via the p38 MAPK pathway in hepatocellular carcinoma cells[J]. Phytomedicine, 2017, 25:71-82.

[25] Zhang L, Fang Y, Xu XF, et al. Moscatilin induces apoptosis of pancreatic cancer cells via reactive oxygen species and the JNK/SAPK pathway[J]. Mol Med Rep, 2017, 15(3):1195-1203.

[26] Liang T, Zhang X, Xue W, et al. Curcumin induced human gastric cancer BGC-823 cells apoptosis by ROS-mediated ASK1-MKK4-JNK stress signaling pathway[J]. Int J Mol Sci, 2014, 15(9):15754-15765.

[27] Xiang Y, Ye W, Huang C, et al. Brusatol inhibits growth and induces apoptosis in pancreatic cancer cells via JNK/p38 MAPK/NF-κB/Stat3/Bcl-2 signaling pathway[J]. Biochem Biophys Res Commun, 2017, 487(4):820-826.

Dihydroartemisinin enhances antitumor effect of 5-fluorouracil against gastric cancer by down-regulating SIRT1 expression

WAN Bin1, CAO Heng-bin1, YU Gen-hua2

(1DepartmentofPharmacy，2DepartmentofInterventionalRadiology,HuzhouCentralHospital,Huzhou313000,China.E-mail:mysucess@163.com)

AIM: To investigate the effect of dihydroartemisinin (DHA) adjuvant treatment on enhancing the antitumor effect of 5-fluorouracil (5-FU) against gastric cancer.METHODSThe gastric cancer BGC-823 cells were divided into control group, DHA group, 5-FU group, 5-FU+DHA group and 5-FU+DHA+SIRT1 plasmid group. The viability of BGC-823 cells treated with DHA and 5-FU was measured by MTT assay. The expression of SIRT1 and NADPH oxidase, activation of caspase-9 and caspase-3, and phosphorylation of ASK1 and JNK in the BGC-823 cells treated with DHA and 5-FU were determined by Western blot. The production of ROS and the apoptosis of the BGC-823 cells treated with DHA and 5-FU were analyzed by flow cytometry.RESULTSDihydroartemisinin significantly inhibited the expression of SIRT1 and increased NADPH oxidase protein level (P<0.05). DHA increased the sensitivity of BGC-823 cells to 5-FU, thus decreasing the IC50of 5-FU to the gastric cancer cells. However, transfection with SIRT1 plasmid decreased the cytotoxicity of DHA and 5-FU co-treatment to the BGC-823 cells. DHA promoted the production of ROS and phosphorylation of ASK1 and JNK induced by 5-FU in the BGC-823 cells (P<0.05). However, ROS scavengerN-acetylcysteine (NAC) or JNK specific inhibitor SP600125 inhibited the cell death and activation of caspase-9 and caspase-3 induced by DHA and 5-FU co-treatment (P<0.05). In addition, NAC significantly inhibited the phosphorylation of JNK in the BGC-823 cells co-treated with DHA and 5-FU. However, treatment with SP600125 did not influence the ROS production in the BGC-823 cells, indicating that JNK was the downstream target of ROS pathway.CONCLUSIONCombination of DHA with 5-FU induces caspase-dependent apoptosis in gastric cancer cells through the SIRT1/NADPH oxidase/ROS/JNK signaling pathway.

Dihydroartemisinin; SIRT1 protein; 5-fluorouracil; BGC-823 cells; Reactive oxygen species; JNK signaling pathway

1000- 4718(2017)12- 2195- 07

2017- 06- 06

2017- 07- 22

△通訊作者 Tel: 0572-2555806； E-mail： mysucess@163.com

R735.7； R285.5

A

10.3969/j.issn.1000- 4718.2017.12.013

(責(zé)任編輯： 林白霜， 羅 森)