徐成坤，陽(yáng)波，曹建國(guó)

(1.南華大學(xué)附屬第一醫(yī)院藥劑科，衡陽(yáng) 421001;2.湖南師范大學(xué)醫(yī)學(xué)院藥理教研室，長(zhǎng)沙 410000)

白楊素誘導(dǎo)肝癌Hep3B細(xì)胞凋亡機(jī)制研究*

徐成坤1，陽(yáng)波1，曹建國(guó)2

(1.南華大學(xué)附屬第一醫(yī)院藥劑科，衡陽(yáng) 421001;2.湖南師范大學(xué)醫(yī)學(xué)院藥理教研室，長(zhǎng)沙 410000)

目的 研究白楊素誘導(dǎo)人肝癌Hep 3B細(xì)胞凋亡是否涉及激活ROS/ASK1/JNK/Bim信號(hào)通路。方法流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡,siRNA轉(zhuǎn)染敲除細(xì)胞凋亡信號(hào)調(diào)節(jié)激酶1(ASK1)及Bim基因,SP600125抑制Jun氨基末端激酶(JNK),N-乙酰-L型半胱氨酸(NAC)清除活性氧(ROS),Western blot檢測(cè)蛋白表達(dá)。結(jié)果白楊素顯著誘導(dǎo)Hep 3B細(xì)胞凋亡,同時(shí)上調(diào)細(xì)胞p-ASK1、p-JNK1/2及Bim水平。NAC或SP600125預(yù)處理以及ASK1或Bim特異性siRNA轉(zhuǎn)染均能有效拮抗白楊素誘導(dǎo)的細(xì)胞凋亡。NAC預(yù)處理能抑制白楊素活化ASK1,NAC預(yù)孵育或ASK1特異性siRNA轉(zhuǎn)染能抑制白楊素活化JNK,NAC預(yù)孵育、ASK1特異性siRNA轉(zhuǎn)染或SP600125預(yù)處理均能拮抗白楊素上調(diào)Bim蛋白表達(dá)效應(yīng)。結(jié)論白楊素可能通過(guò)刺激ROS的生成激活A(yù)SK1,導(dǎo)致JNK活化,進(jìn)而上調(diào)Bim表達(dá),并最終促進(jìn)Hep 3B細(xì)胞凋亡。

白楊素；肝癌；細(xì)胞凋亡；細(xì)胞凋亡信號(hào)調(diào)節(jié)激酶1

白楊素(5,7-二羥基黃酮)是從紫葳科植物木蝴蝶中提取的一種對(duì)多種腫瘤細(xì)胞具有抑制作用的黃酮類(lèi)化合物。楊小紅等[1]研究發(fā)現(xiàn),白楊素通過(guò)活性氧(reactive oxygen species,ROS)的產(chǎn)生和持續(xù)的c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)激活誘導(dǎo)人肝癌細(xì)胞凋亡。然而,白楊素誘導(dǎo)肝癌細(xì)胞凋亡的精確分子機(jī)制尚未完全闡明。細(xì)胞凋亡信號(hào)調(diào)節(jié)激酶1(apoptosis signal-regulating kinase 1,ASK1)是一種多功能絲氨酸/蘇氨酸蛋白激酶,參與多種生物學(xué)過(guò)程,包括細(xì)胞分化和凋亡[2-3]。已有研究報(bào)道ASK1能被包括ROS在內(nèi)的許多刺激信號(hào)活化[4]。然而,ASK1在白楊素誘導(dǎo)肝細(xì)胞癌細(xì)胞凋亡中的作用仍然不清楚。Bim是一種促凋亡分子,是Bcl-2家族中僅含BH3結(jié)構(gòu)域的新成員,Bim通過(guò)與Bcl-2和Bcl-XL結(jié)合,從而解除二者抑制凋亡作用,或者通過(guò)與Bax和Bak結(jié)合,促進(jìn)細(xì)胞凋亡。同時(shí),Bim表達(dá)水平能被JNK激酶激活c-Jun正調(diào)節(jié)[5-6],筆者在本研究中探討ROS/ASK1/JNK/Bim信號(hào)途徑在白楊素誘導(dǎo)肝癌Hep 3B細(xì)胞凋亡中所起的作用,為進(jìn)一步闡明白楊素抗腫瘤作用的分子機(jī)制提供理論和實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 細(xì)胞培養(yǎng)及試劑 用添加10%小牛血清、2 mmol·L-1谷氨酰胺、100 mg·L-1青霉素和100 mg·L-1鏈霉素的達(dá)爾伯克改良伊格爾培養(yǎng)基(Dulbecco's modified eagle medium,DMEM)培養(yǎng)基培養(yǎng)人肝癌Hep 3B細(xì)胞系細(xì)胞(中國(guó)典型培養(yǎng)物保藏中心,中國(guó)武漢),并置于5%二氧化碳(CO2)、37℃增濕環(huán)境中孵育。白楊素購(gòu)自美國(guó)Sigma化學(xué)公司。用二甲基亞砜(dimethyl sulfoxide,DMSO)作為溶劑溶解白楊素,培養(yǎng)體系中DMSO的最高終濃度為0.1%。DMEM培養(yǎng)基,小牛血清(calf serum,CS),青霉素/鏈霉素和OptiMEM,脂質(zhì)體plusTM試劑購(gòu)自美國(guó)Invitrogen公司。JNK1的磷酸化1/2(Thr183/Tyr185)和JNK1的特異性抗體分別購(gòu)自美國(guó)New England Biolabs公司。磷酸化的ASK1,ASK1和Bim的特異性抗體,辣根過(guò)氧化物酶標(biāo)記的抗小鼠和抗兔IgG抗體購(gòu)自美國(guó)Santa Cruz生物技術(shù)公司。增強(qiáng)化學(xué)發(fā)光檢測(cè)試劑購(gòu)自美國(guó)PerkinElmer Life Sciences公司。十二烷基硫酸鈉聚丙烯酰胺凝膠電泳的所有材料來(lái)自美國(guó)Bio-Rad公司。N-乙酰-L-半胱氨酸(N-acetyl cysteine, NAC)、碘化丙啶(propidium iodide,PI)、胃蛋白酶抑制劑、亮肽素、其他化學(xué)品,獲自美國(guó)Sigma公司。

1.2 流式細(xì)胞術(shù)分析 細(xì)胞培養(yǎng)在6孔細(xì)胞培養(yǎng)板中。細(xì)胞融合后,用不同的siRNA轉(zhuǎn)染細(xì)胞48 h或使用特異性抑制劑預(yù)處理1 h,白楊素(40 μmol·L-1)處理24 h。收集細(xì)胞,用磷酸鹽緩沖液(phosphate buffered solution,PBS),氯化鈉137 mmol·L-1,氯化鉀2.7 mmol·L-1,磷酸氫二鈉4.3 mmol·L-1和磷酸二氫鉀1.5 mmol·L-1,pH7.4)洗滌細(xì)胞2次,并重新置于冰冷的70%乙醇中-20℃過(guò)夜。用含50 mmol·L-1磷酸氫二鈉、25 mmol·L-1檸檬酸和0.1%Triton X-100的0.4 mL磷酸鹽-檸檬酸緩沖液(pH7.8)洗滌細(xì)胞2次,并隨之用含0.5%Triton X-100,10 mmol·L-1哌嗪-1,4-二乙磺酸(piperazine-1,4-bisethanesulfonic acid,PIPES),100 mmol·L-1氯化鈉,2 mmol·L-1氯化鎂,0.1 U·mL-1RNase A,and 25 μg·mL-1PI的1.5 mL PI染色緩沖液在流式細(xì)胞分析前避光染色30 min。使用流式細(xì)胞儀(美國(guó)Becton Dickinson公司)和Cellquest程序(美國(guó)Pharmingen BD Bioscience公司)進(jìn)行DNA含量分析。

1.3 小干擾RNA轉(zhuǎn)染 為了瞬時(shí)siRNA轉(zhuǎn)染,細(xì)胞以1×106個(gè)·mL-1接種于6孔細(xì)胞培養(yǎng)板上并培養(yǎng)過(guò)夜。翌日,用包含每孔100 ng的ASK1和Bim特異性siRNA或非特異性對(duì)照小干擾RNA(siRNA,美國(guó)Santa Cruz生物技術(shù)公司)的脂質(zhì)體plusTM試劑轉(zhuǎn)染細(xì)胞。siRNA寡核苷酸的序列為非特異性對(duì)照siRNA(5′-GACGCGATCAGAGAGTAAT-3′),ASK1特異性siRNA(5′-GGTGGCACAAGCAAGTTCT-3′)和Bim特異性siRNA(5′-GATCCGTTCTGAGTGTGACCGAGA-3′)。用每種siRNA轉(zhuǎn)染細(xì)胞并孵育48 h。通過(guò)抗ASK1和Bim抗體的免疫印跡分析確定對(duì)ASK1和Bim蛋白表達(dá)的干擾效果。

1.4 蛋白質(zhì)免疫印跡分析 蛋白質(zhì)免疫印跡按照YANG等[1]報(bào)道的方法完成。抗JNK1、磷酸化JNK1/2、磷酸化ASK1、ASK1、Bim和β-actin抗體作為一抗。使用電化學(xué)發(fā)光(electrochemiluminescence,ECL)高級(jí)蛋白質(zhì)印跡分析系統(tǒng)(美國(guó)Amersham Pharmacia Biotech公司)檢測(cè)信號(hào)。

1.5 統(tǒng)計(jì)學(xué)方法 實(shí)驗(yàn)數(shù)據(jù)錄入SPSS15.0版for windows evaluation軟件,建立數(shù)據(jù)庫(kù),各組實(shí)驗(yàn)數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(±s)表示,用One Way ANOVA方式行方差分析。對(duì)照組均數(shù)與實(shí)驗(yàn)組均數(shù)間的比較用LSD法,P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。檢驗(yàn)水準(zhǔn)α=0.05。

2 結(jié)果

2.1 白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡涉及激活A(yù)SK1流式細(xì)胞術(shù)分析結(jié)果顯示,siRNA敲除ASK1可有效減弱40 μmol·L-1白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡作用(圖1A)。免疫印跡檢測(cè)ASK1磷酸化蛋白水平,發(fā)現(xiàn)40 μmol·L-1白楊素處理的Hep 3B細(xì)胞中ASK1磷酸化蛋白水平增加(圖1B)。提示ASK1活化可能參與了白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡。

圖1 白楊素誘導(dǎo)肝癌Hep 3B細(xì)胞凋亡涉及上調(diào)ASK1 (n=3)A.ASK1特異性小干擾RNA抑制白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡率(Sub-G1 population)增高效應(yīng)。與對(duì)照組比較,*1P＜0.01,*3P＜0.05;與白楊素+siRNA ASK1干預(yù)組比較,*2P＜0.05;B.Western blot分析白楊素對(duì)磷酸化ASK1(p-ASK1)、總ASK1(t-ASK1)蛋白表達(dá)(β-actin為內(nèi)參)Fig.1 Chrysin-induced apoptosis of hepatocellular carcinoma Hep 3B cells involving upregulation of ASK1(n=3)A.Inhibition of ASK1 specific small interfering RNA on chrysin-induced apoptosis rate(Sub-G1 population)of Hep 3B cells. Compared with the control group,*1P＜0.01,*3P＜0.05;compared with chrysin plus ASK1 iRNA intervention group,*2P＜0.05 0.05;B.Western blot analysis on the effect of chrysin on the expression of phosphorylation ASK1(p-ASK1),total ASK1(t-ASK1)protein(β-actin as reference)

2.2 白楊素活化ASK1涉及ROS形成 流式細(xì)胞計(jì)數(shù)分析顯示,抗氧化劑NAC預(yù)處理Hep 3B細(xì)胞可顯著抑制白楊素誘導(dǎo)細(xì)胞凋亡作用(圖2A)。此外,免疫印跡分析表明,10 mmol·L-1NAC預(yù)處理抑制白楊素增加ASK1磷酸化水平作用(圖2B)。提示白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡可能通過(guò)產(chǎn)生ROS,繼而活化ASK1所介導(dǎo)。

2.3 白楊素誘導(dǎo)Hep 3B細(xì)胞JNK活化涉及ROS和ASK1 免疫印跡分析表明,白楊素可增高JNK1/2蛋白磷酸化水平,而對(duì)其總蛋白水平無(wú)影響(圖3A),再次證實(shí)白楊素通過(guò)活化JNK方式介導(dǎo)Hep 3B細(xì)胞凋亡[1]。而NAC預(yù)處理可拮抗白楊素激活JNK(圖3A)。同樣,用ASK1特異性siRNA預(yù)處理也可拮抗白楊素激活JNK效應(yīng)(圖3B)。因此,這些發(fā)現(xiàn)提示,白楊素誘導(dǎo)Hep 3B細(xì)胞JNK1/2活化需要ROS-ASK1級(jí)聯(lián)介導(dǎo)。

圖2 白楊素激活Hep 3B細(xì)胞ASK1涉及ROS形成(±s,n=3)A.NAC抑制白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡率(Sub-G1 population)增高作用。與對(duì)照組比較,*1P＜0.01,*3P＜0.05;與Chrysin+NAC組比較,*2P＜0.05;B.Western blot分析NAC對(duì)chrysin活化ASK1的影響(β-actin為內(nèi)參)Fig.2 Chrysin-induced ASK1 activation in Hep 3B cells involving ROS formation(±s,n=3)A.Inhibition of NAC on chrysin-induced apoptosis rate(Sub-G1 population)of Hep 3B cells.Compared with the control group,*1P＜0.01,*3P＜0.05;compared with chrysin plus NAC group,*2P＜0.05;B.Western blot analysis on the effect of NAC on ASK1 activation by chrysin(β-actin as reference)

2.4 白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡涉及上調(diào)Bim 流式細(xì)胞術(shù)分析顯示,siRNA敲除Bim顯著抑制白楊素誘導(dǎo)細(xì)胞凋亡作用(圖4A)。免疫印跡分析表明,白楊素上調(diào)Bim蛋白表達(dá),NAC、ASK1特異性siRNA及JNK抑制劑SP600125具有拮抗白楊素上調(diào)Bim蛋白表達(dá)作用(圖4B)。提示白楊素誘導(dǎo)Hep 3B細(xì)胞Bim蛋白表達(dá)涉及ROS-ASK1-JNK級(jí)聯(lián)。

圖3 白楊素激活Hep 3B細(xì)胞JNK涉及ROS形成和激活A(yù)SK1A.Western blot分析NAC抑制白楊素活化JNK1/2作用; B.Western blot分析ASK1特異性小干擾RNA(si RNA)對(duì)白楊素活化JNK1/2作用的影響Fig.3 Chrysin-induced JNK activation in Hep 3B cells involving ROS formation and ASK1 activationA.Western blot analysis on the inhibition of NAC on JNK1/2 activation by chrysin;B.Western blot analysis on the effect of specific small interfering RNA of ASK1 on JNK1/2 activation by chrysin

圖4 白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡涉及上調(diào)Bim(n=3)A.Bim特異性小干擾RNA(si RNA)拮抗白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡率(Sub-G1 population)增高作用,與對(duì)照組比較,*1P＜0.01,*3P＜0.05;與Chrysin+siRNA Bim組比較,*2P＜0.05;B.Western blot分析NAC、siRNA ASK1、JNK特異性阻斷劑SP600125對(duì)chrysin誘導(dǎo)Bim蛋白表達(dá)的影響Fig.4 Chrysin-induced apoptosis of Hep 3B cells involving upregulation of Bim(n=3)A.Inhibition of specific small interfering RNA of Bim(SiRNA)on chrysin-induced apoptosis rate(Sub-G1 population)of Hep 3B cells.Compared with the control group,*1P＜0.01,*3P＜0.05;compared with chrysin plus Bim iRNA group,*2P＜0.05; B.Western blot analysis on the effect of NAC,ASK1 siRNA and SP600125,a specific blocker of JNK on chrysin-induced the expression of Bim protein

3 討論

本研究結(jié)果顯示,白楊素通過(guò)激活ROS-ASK1-JNK信號(hào)級(jí)聯(lián)上調(diào)Bim蛋白表達(dá),誘導(dǎo)Hep 3B細(xì)胞凋亡。

活化的ASK1通過(guò)刺激下游信號(hào)事件,包括MKK4/7和激活JNK,在調(diào)節(jié)各種生物學(xué)過(guò)程包括細(xì)胞分化和凋亡中扮演重要角色[7]。JNK被認(rèn)為是ASK1激活后發(fā)揮作用的靶基因[8]。然而,ASK1-JNK信號(hào)通路是否參與白楊素誘導(dǎo)肝癌細(xì)胞凋亡還沒(méi)有闡明。在本實(shí)驗(yàn)中筆者發(fā)現(xiàn),白楊素處理Hep 3B細(xì)胞導(dǎo)致ASK1、JNK依次激活,而且ASK1特異性siRNA和JNK抑制劑(SP600125)有效地拮抗白楊素誘導(dǎo)細(xì)胞凋亡。ASK1特異性siRNA抑制白楊素誘導(dǎo)JNK1/2活化。此外,白楊素誘導(dǎo)Bim蛋白表達(dá)的作用亦被ASK1特異性siRNA和JNK抑制劑所抑制。這些結(jié)果顯示白楊素可能通過(guò)活化ASK1激活JNK,而JNK激酶活化后激活c-Jun上調(diào)Bim蛋白表達(dá),并最終引起Hep 3B細(xì)胞凋亡。

許多研究顯示,ROS可能通過(guò)氧化TRX和GRX活化ASK1[2-3]。最近的報(bào)道顯示,ASK1通過(guò)減少967絲氨酸磷酸化同時(shí)于抑制蛋白解離增加來(lái)響應(yīng)氧化應(yīng)激如過(guò)氧化氫(H2O2),導(dǎo)致細(xì)胞凋亡功能增強(qiáng)[9]。在本實(shí)驗(yàn)中,筆者發(fā)現(xiàn)NAC抑制白楊素誘導(dǎo)ASK1活化、JNK活化、Bim蛋白表達(dá)和細(xì)胞凋亡。這些發(fā)現(xiàn)顯示ROS可能在白楊素誘導(dǎo)ASK1活化和后續(xù)的信號(hào)事件中起關(guān)鍵作用。

Bcl-2家族蛋白通過(guò)抗凋亡和促凋亡因子調(diào)節(jié)線粒體觸發(fā)凋亡途徑,決定細(xì)胞的生或死。Bim作為Bcl-2家族的促凋亡成員,通過(guò)破壞線粒體完整性導(dǎo)致細(xì)胞凋亡。有研究表明,Bim基因表達(dá)能響應(yīng)選擇性的應(yīng)激信號(hào)[10]。在本研究中,筆者發(fā)現(xiàn)白楊素誘導(dǎo)Bim表達(dá)并且Bim特異性siRNA拮抗白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡,這些結(jié)果提示Bim表達(dá)與白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡可能呈因果關(guān)系。此外,抗氧化劑NAC、JNK抑制劑(SP600125)和ASK1特異性siRNA抑制白楊素誘導(dǎo)Bim表達(dá)。因此,筆者認(rèn)為白楊素通過(guò)激活ROS-ASK1-JNK信號(hào)級(jí)聯(lián),上調(diào)Bim表達(dá),并最終誘導(dǎo)Hep 3B細(xì)胞凋亡。最近的報(bào)道顯示,除了Bim外,其他Bcl-2家族成員也參與白楊素誘導(dǎo)癌細(xì)胞凋亡過(guò)程[11]。這些發(fā)現(xiàn),至少部分說(shuō)明,為什么阻斷ASK1信號(hào)級(jí)聯(lián)沒(méi)能完全消除白楊素誘導(dǎo)Hep 3B細(xì)胞凋亡效應(yīng)。

總之,本實(shí)驗(yàn)結(jié)果首次證明白楊素誘導(dǎo)人肝癌Hep3B細(xì)胞凋亡的分子機(jī)制涉及激活ROS-ASK1-JNK信號(hào)級(jí)聯(lián)促進(jìn)Bim蛋白表達(dá)。

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DOI 10.3870/yydb.2014.03.006

Investigation of Mechanism Underlying Chrysin-induced Apoptosis in Hepatocellular Carcinoma Hep3B Cells

XU Cheng-Kun1,YANG bo1,CAO Jian Guo2
(1.Pharmaceutical Preparation Section,the First Affiliated Hospital,the University of South China,Hengyang 421001,China;2.Hunan Normal University School of Medicine Pharmacology Teaching and Research Section,Changsha 410000,China)

Objective To investigate whether the apoptosis induced by chrysin in hepatocellular carcinoma Hep 3B is involved in activation of ROS/ASK1/JNK/Bim signal pathway.MethodsApoptosis was examined using flow cytometry. Apoptosis signal-regulating kinase 1(ASK1)and Bim genes were knocked out by small interfering RNA(siRNA)transfection. Reactive oxygen species(ROS)and c-Jun N-terminal kinase(JNK)activity were suppressed by their specific inhibitorsN-acetyl-L-cysteine(NAC)and SP600125,respectively.The protein expression was detected by western blot analysis.ResultsChrysin significantly induced apoptotic cell death and was able to up-regulate the levels of p-ASK1,p-JNK1/2 and Bim in Hep 3B cells.However,the apoptosis induced by chrysin would be effectively suppressed by pretreatment with NAC or SP600125 and transfected with ASK1-specific siRNA or Bim-specific siRNA.Accordingly,pretreatment with NAC could suppress chrysinmediated ASK1 activation;Pretreatment with NAC or transfected with ASK1-specific siRNA could suppress JNK activation;and pretreatment with NAC or SP600125 and transfected with ASK1-specific siRNA could suppress chrysin-induced Bim expression.ConclusionChrysin might activate ASK1 through ROS production to cause JNK activation,which in turn induces Bim expression,and ultimately results in Hep 3B cell apoptosis.

Chrysin;Hepatocellular carcinoma;Apoptosis;Apoptosis signal-regulating kinasel

R286;R965

A

1004-0781(2014)03-0295-04

2013-08-05

2013-09-10

*國(guó)家自然科學(xué)基金資助項(xiàng)目(NO.09A054)

徐成坤(1963-),男,湖南邵陽(yáng)人,副主任藥師,主要從事醫(yī)院藥學(xué)管理與臨床藥學(xué)工作。電話:0734-8578926,E-mail：ckx1881@163.com。