王玖 羅鵬 張磊 鄭新瑞 戴舒惠 楊?lèi)偡?饒維 彭程 李娟 馬文科 費(fèi)舟
(第四軍醫(yī)大學(xué)西京醫(yī)院神經(jīng)外科,陜西 西安 710032)
·腦膠質(zhì)細(xì)胞瘤研究·
甘草查爾酮A對(duì)膠質(zhì)瘤的增殖抑制及促凋亡作用機(jī)制研究
王玖 羅鵬 張磊 鄭新瑞 戴舒惠 楊?lèi)偡?饒維 彭程 李娟 馬文科 費(fèi)舟*
(第四軍醫(yī)大學(xué)西京醫(yī)院神經(jīng)外科,陜西 西安 710032)
目的探討甘草查爾酮A(LCA)對(duì)人膠質(zhì)瘤U87、U251細(xì)胞的增殖抑制和促凋亡作用及其機(jī)制研究。方法通過(guò)四甲基偶氮唑藍(lán)(MTT)比色法檢測(cè)細(xì)胞增殖抑制作用,并計(jì)算出半數(shù)抑制濃度(IC50),按IC50和100 μM不同濃度進(jìn)行分組。采用倒置顯微鏡觀察細(xì)胞形態(tài),流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡以及Western Blot從蛋白水平檢測(cè)凋亡相關(guān)分子的改變情況。結(jié)果MTT顯示LCA對(duì)人膠質(zhì)瘤U87、U251細(xì)胞具有明顯的增殖抑制作用,呈濃度及時(shí)間依賴性,LCA對(duì)U87、U251細(xì)胞48 h的IC50值分別為61.54 μM和53.02 μM,倒置顯微鏡下觀察細(xì)胞密度減少,形態(tài)發(fā)生明顯改變。流式細(xì)胞術(shù)結(jié)果顯示,LCA可促進(jìn)膠質(zhì)瘤U87、U251的細(xì)胞凋亡(Plt;0.01)。Western Blot結(jié)果顯示,LCA干預(yù)后可顯著降低膠質(zhì)瘤U87、U251細(xì)胞內(nèi)Bcl-2的表達(dá)(Plt;0.01),相反,增加Bax的表達(dá)(Plt;0.05),呈濃度依賴性。結(jié)論LCA對(duì)膠質(zhì)瘤U87、U251細(xì)胞具有明顯的增殖抑制作用,可能通過(guò)內(nèi)源性凋亡途徑誘導(dǎo)其凋亡。
膠質(zhì)細(xì)胞瘤; 甘草查爾酮A; 增殖抑制; 凋亡
膠質(zhì)瘤是原發(fā)性腦腫瘤中的一種常見(jiàn)類(lèi)型,包括星形細(xì)胞瘤,少突膠質(zhì)細(xì)胞瘤和室管膜瘤。根據(jù)世界衛(wèi)生組織(World Health Organization, WHO)標(biāo)準(zhǔn),膠質(zhì)瘤分為4個(gè)等級(jí),包括毛細(xì)胞性星形細(xì)胞瘤(I級(jí)),彌散性星形細(xì)胞瘤(II級(jí)),間變性星形細(xì)胞瘤(III級(jí))以及多形性膠質(zhì)母細(xì)胞瘤(glioblastoma multiform, GBM;IV級(jí))[1]?,F(xiàn)今,在治療膠質(zhì)瘤方面,手術(shù)聯(lián)合放、化療仍是標(biāo)準(zhǔn)的治療方案,因?yàn)閱我皇中g(shù)很難完全切除腫瘤,且復(fù)發(fā)率高,必須輔以術(shù)后放、化療殺傷殘余腫瘤細(xì)胞,然而,膠質(zhì)瘤的化療依然存在許多局限性。因此,急需革新治療理念,研發(fā)新型藥物來(lái)有效殺傷膠質(zhì)瘤細(xì)胞,降低復(fù)發(fā)率及死亡率,提高生活質(zhì)量以及改善預(yù)后等。目前,中草藥是研發(fā)新藥的主要來(lái)源,也是現(xiàn)代醫(yī)學(xué)中治療疾病的首選之一,研究中草藥抗腫瘤作用的活性成分以及具有副作用小等優(yōu)勢(shì)為治療腫瘤提供了新思路與新方法[2]。甘草是非常有名的傳統(tǒng)中藥材(traditional Chinese medicine, TCM)之一,早在公元前2100年就有記載,其含有多達(dá)20種三萜類(lèi)化合物以及將近300種黃酮類(lèi)化合物等[3]。甘草根部的主要活性提取物-查爾酮,具有多種藥理學(xué)作用[4],還有抑制細(xì)胞增殖,誘導(dǎo)周期停滯及誘發(fā)腫瘤細(xì)胞凋亡等特性[5]。甘草查爾酮A(licochalcone A, LCA)是查爾酮的主要活性成分,具有抗炎、抗菌、抗病毒、抗氧化、抗瘧疾、抗血管生成、抗腫瘤及抑制腫瘤遠(yuǎn)處轉(zhuǎn)移等特性[6]。此研究中,我們探討LCA對(duì)人膠質(zhì)瘤U87、U251細(xì)胞的增殖抑制以及闡明其促凋亡的潛在分子機(jī)制。
一、主要試劑及抗體
人源性膠質(zhì)瘤細(xì)胞系U87、U251購(gòu)自于美國(guó)標(biāo)準(zhǔn)細(xì)胞庫(kù)(American type culture collection,ATCC)。LCA藥粉(分子式:C21H22O4,分子量:338.40)購(gòu)自于Sigma公司(產(chǎn)品貨號(hào)為68783),純度≥96.0%。用二甲基亞砜(dimethyl sulphoxide, DMSO)溶解配成100 mmol/L儲(chǔ)存液(所含DMSO濃度均lt;1‰,無(wú)細(xì)胞毒性),置于-20 ℃凍存。杜伯改良的依格培養(yǎng)基(Dulbecco's modified Eagle's medium, DMEM)、雙抗(100 IU/mL青霉素、100 μg/mL鏈霉素)和胰蛋白酶由Gibco公司提供,新生胎牛血清由杭州四季青公司提供。兔源性Bcl-2、兔源性Bax,辣根過(guò)氧化物酶標(biāo)記山羊抗兔二抗,均購(gòu)于CST公司。四甲基偶氮唑藍(lán)(methyl thiazolyl terrazaium, MTT)、流式檢測(cè)試劑盒為Sigma公司產(chǎn)品。
二、細(xì)胞培養(yǎng)
膠質(zhì)瘤U87、U251細(xì)胞常規(guī)置于于含有10%胎牛血清及1%雙抗的DMEM中, 37℃、5%CO2的孵箱中培養(yǎng),每1~2 d更換一次培養(yǎng)基。當(dāng)細(xì)胞生長(zhǎng)密度達(dá)到70%~85%時(shí),用0.25%胰蛋白酶消化,按1 ∶2或1 ∶3比例傳代,繼續(xù)培養(yǎng)。
三、細(xì)胞活力
取對(duì)數(shù)期U87、U251細(xì)胞,經(jīng)0.25%胰酶消化后,通過(guò)細(xì)胞計(jì)數(shù)將濃度調(diào)整至2×105個(gè)/mL接種于96孔板,每孔體積100 μL,過(guò)夜貼壁。將LCA加入DMEM中配制不同濃度的藥物,使其終濃度為20、40、60、80、100、120 μM,每個(gè)濃度設(shè)6個(gè)復(fù)孔,空白對(duì)照組沒(méi)有細(xì)胞,對(duì)照組為正常細(xì)胞但未加LCA處理,其他處理與藥物組相同。37 ℃、5%CO2的孵箱培養(yǎng)24或48 h后,每孔加入20 μL 5 g/L MTT,繼續(xù)培養(yǎng)4 h,棄上清,每孔加入150 μL DMSO,震蕩10 min。酶標(biāo)儀檢測(cè)490 nm波長(zhǎng)處各孔吸光度(optical density, OD)值。按照公式計(jì)算抑制率:抑制率=(1-實(shí)驗(yàn)組OD/對(duì)照組OD)×100%。
四、倒置顯微鏡觀察LCA處理U87、U251細(xì)胞后的形態(tài)改變
取對(duì)數(shù)期U87、U251細(xì)胞傳代培養(yǎng),貼壁后更換為含有終濃度為IC50及100 μM LCA的DMEM,作用24 h后,在顯微鏡下觀察細(xì)胞的變化情況。
五、流式細(xì)胞儀檢測(cè)細(xì)胞凋亡
將U87、U251細(xì)胞按密度為2×105個(gè)/mL接種于6孔板,培養(yǎng)過(guò)夜后,換成含有終濃度為IC50及100 μM LCA的DMEM繼續(xù)培養(yǎng)24 h,消化收集細(xì)胞置于離心管中,并用磷酸鹽緩沖液(phosphate-buffered solution, PBS)洗滌。用配置好的1×結(jié)合緩沖液(1×binding buffer)洗滌重懸細(xì)胞,將密度調(diào)整為1×106~5×106個(gè)/mL,取100 μL細(xì)胞懸液,加入5 μL膜聯(lián)蛋白-V(Annexin-V)標(biāo)記的異硫氰酸熒光素(fluoresein isothiocyanate, FITC)反應(yīng)液后室溫避光孵育15~20 min,1×binding buffer 洗滌重懸細(xì)胞后再加入5 μL碘化丙啶(propidium iodide, PI)反應(yīng)液,混勻室溫孵育10 min,上機(jī)待測(cè)。
六、蛋白質(zhì)免疫印跡檢測(cè)(Western Blot)
取對(duì)數(shù)期U87、U251細(xì)胞,分別設(shè)DMSO對(duì)照組和IC50、100 μM濃度組進(jìn)行藥物干預(yù),作用24 h后,小心吸凈培養(yǎng)皿中培養(yǎng)液,用4 ℃預(yù)冷PBS洗1~2遍,然后加入含有1%蛋白酶抑制劑的裂解液充分裂解細(xì)胞,12000 r/min 離心20 min后吸上清提取蛋白。用二喹啉甲酸(bicinchoninic acid, BCA)試劑盒以570 nm于酶標(biāo)儀測(cè)定蛋白含量,12%十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(sodium dodecyl sulphate-polyacry lamidegel electrophoresis, SDS-PAGE)分離,上樣量為20 μg,再轉(zhuǎn)移至硝酸纖維素膜上,用5%脫脂牛奶室溫封閉2 h,用Bcl-2一抗(1 ∶1000)、Bax一抗(1 ∶1000)和β-actin(1 ∶4000)一抗4 ℃孵育過(guò)夜。用配置好的含0.1%Tween20的PBS(phosphate-buffered solution tween, PBST)充分洗膜,二抗室溫孵育1 h,再用PBST洗膜后發(fā)光顯色,用Gel-pro軟件分析各條帶灰度值與相應(yīng)β-actin的灰度值的比值作統(tǒng)計(jì)分析。
七、統(tǒng)計(jì)學(xué)方法
一、LCA對(duì)U87、U251細(xì)胞的增殖抑制
通過(guò)MTT實(shí)驗(yàn)測(cè)定LCA的細(xì)胞毒性,數(shù)據(jù)表明,不同濃度LCA(20、40、60、80、100或120 μM)對(duì)U87、U251細(xì)胞的增殖抑制作用具有顯著的濃度及時(shí)間依賴性(圖1)。LCA作用后顯著抑制兩種細(xì)胞系的生長(zhǎng),降低生存力。藥物處理48 h后,60、100 μM濃度LCA對(duì)U87、U251細(xì)胞的活性抑制率分別為66.57%和66.31%及94.23%和95.10%,與正常組相比,存在統(tǒng)計(jì)學(xué)差異(Plt;0.01)。通過(guò)SPSS 19.0分析得出LCA對(duì)U87、U251細(xì)胞48 h的半數(shù)抑制濃度(half maximal inhibitory concentration, IC50)分別為61.54 μM和53.02 μM。
二、倒置顯微鏡觀察細(xì)胞形態(tài)
顯微鏡下觀察對(duì)照組細(xì)胞生長(zhǎng)狀況良好,輪廓清晰,觸角豐滿,U87細(xì)胞呈梭形,U251細(xì)胞呈不規(guī)則多角形。IC50和100 μM濃度LCA作用U87、U251細(xì)胞24 h后,呈現(xiàn)濃度依賴性的形態(tài)改變,細(xì)胞皺縮,密度減少,呈圓形貼壁生長(zhǎng),輪廓不規(guī)則,邊緣圓鈍,胞內(nèi)可見(jiàn)顆粒樣物質(zhì) (圖2)。
三、LCA促進(jìn)膠質(zhì)瘤U87、U251細(xì)胞凋亡
流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡,結(jié)果表明,IC50和100 μM濃度LCA處理U87、U251細(xì)胞24 h后,活細(xì)胞百分率減少,凋亡細(xì)胞百分率明顯增加,呈濃度依賴性改變。與對(duì)照組相比,100 μM濃度組U87和U251細(xì)胞凋亡率分別為25.48%±1.65%和25.63%±0.93%,顯著高于對(duì)照組的1.87%±0.31%和2.85%±0.25%,差異有統(tǒng)計(jì)學(xué)意義(Plt;0.01, 圖3)。
四、LCA對(duì)Bcl-2和Bax蛋白表達(dá)的影響
IC50和100 μM濃度LCA處理U87、U251細(xì)胞24 h后,Western Blot檢測(cè)凋亡相關(guān)蛋白Bcl-2和Bax的表達(dá)變化。結(jié)果提示,隨著LCA藥物濃度的增加,U87和U251細(xì)胞Bcl-2蛋白表達(dá)量顯著降低,相反,Bax蛋白表達(dá)量增加。MTT、流式細(xì)胞術(shù)和Western結(jié)果共同提示,LCA可通過(guò)促凋亡效應(yīng)殺傷膠質(zhì)瘤細(xì)胞,調(diào)控Bcl-2和Bax蛋白可能是其潛在分子機(jī)制 (圖4)。
圖1 不同濃度LCA作用不同時(shí)間對(duì)U87、U251細(xì)胞的增殖抑制作用(n=3)
Fig 1 Effects of LCA on the inhibition of U87 and U251 cells with different concentrations for 24 h and 48 h (n=3)
A: Effects of LCA on the inhibition of U87 glioma cells with different concentrations for 24 h and 48 h; B: Effects of LCA on the inhibition of U251 glioma cells with different concentrations for 24 h and 48 h
aPlt;0.01,vscontrol group.
圖2 不同濃度LCA作用24 h對(duì)U87、U251細(xì)胞的形態(tài)改變(×200)
Fig 2 The effect of LCA on morphology of U87 and U251 cells for 24 h (×200 )
A: U251 control group under inverted microscopy; B: U87 control group under inverted microscopy; C: U251 cells showed typical morphological change after treatment with LCA IC50 for 24 h; D: U87 cells showed typical morphological change after treatment with LCA IC50 for 24 h; E: U251 cells showed typical morphological change after treatment with LCA 100 μM for 24 h (×200); F: U87 cells showed typical morphological change after treatment with LCA 100 μM for 24 h.
圖3 LCA誘導(dǎo)膠質(zhì)瘤U87、U251的細(xì)胞凋亡(n=3)
Fig 3 Detection of cellular apoptosis in U87 and U251 cells after treatment with LCA (n=3)
A: Cellular apoptosis in different groups were detected by flow cytometry; B:Quantity of cellular apoptosis detected by flow cytometry
aPlt;0.01,vsU87-Con;bPlt;0.01,vsU251-Con.
圖4 LCA對(duì)膠質(zhì)瘤U87、U251細(xì)胞Bcl-2和Bax蛋白表達(dá)的影響
Fig 4 Effect of LCA on expression of Bcl-2 and Bax in U87 and U251 cells
A: Bcl-2 and Bax were detected by Western Blot after treatment of LCA in U251 cells; B: Quantity of Bcl-2 and Bax detected by Western Blot in U251 cells; C: Bcl-2 and Bax were detected by Western Blot after treatment of LCA in U87 cells; D: Quantity of Bcl-2 and Bax detected by Western Blot in U87 cells
aPlt;0.01,vsBcl-2-Con;bPlt;0.01,vsBax-Con;cPlt;0.05,vsBax-Con.
膠質(zhì)瘤是致死率較高的類(lèi)型之一,其來(lái)源于神經(jīng)膠質(zhì)前體細(xì)胞,在組織及分子結(jié)構(gòu)中異質(zhì)性高,占所有原發(fā)性腦腫瘤的27%以及占所有原發(fā)性惡性腦腫瘤的80%。治療方面,人工合成化療藥存在許多局限性,比如,經(jīng)典的系統(tǒng)給藥途徑很難在CNS以及腫瘤位點(diǎn)達(dá)到有效治療濃度;顯著的骨髓抑制等副作用。盡管手術(shù)及放、化療等均有較大改進(jìn),但膠質(zhì)瘤治療的有效率仍不盡如人意。
目前,中草藥為研究熱點(diǎn),因?yàn)橹胁菟幹泻旋嫶蟮奶烊换瘜W(xué)基團(tuán),可作用于腫瘤發(fā)生、發(fā)展的任一階段;與人工化療藥相比毒副作用小,療效肯定。甘草是中國(guó)傳統(tǒng)中藥材,其中,LCA是甘草的主要活性提取成分,可通過(guò)不同機(jī)制殺傷多種腫瘤細(xì)胞,例如膀胱癌、胃癌、肝癌等。本研究采用LCA干預(yù)膠質(zhì)瘤U87、U251細(xì)胞,探討LCA是否具有增殖抑制、促凋亡等作用以及研究可能存在的潛在分子機(jī)制。
將濃度為100 μM的LCA處理U87、U251細(xì)胞48 h后抑制率分別為94.23%和95.10%,呈濃度及時(shí)間依賴性,表明LCA對(duì)膠質(zhì)瘤細(xì)胞具有明顯的增殖抑制作用。Zeng等[7]的研究闡明在人口腔鱗癌SCC-25細(xì)胞系中,LCA的IC50值約300 μM,明顯高于本實(shí)驗(yàn)中的IC50值,說(shuō)明針對(duì)不同腫瘤細(xì)胞,LCA的IC50值不同。例如,Xiao等[8]的研究表明在胃癌MKN-28、AGS、MKN-45細(xì)胞系中,LCA的IC50值為40 μM;另外,在人膀胱癌T24細(xì)胞系中,LCA的IC50值為60 μM[9]。因此,以上結(jié)論說(shuō)明細(xì)胞對(duì)藥物的敏感性決定該藥物的IC50值。
通過(guò)MTT、細(xì)胞形態(tài)學(xué)及流式細(xì)胞術(shù)等實(shí)驗(yàn)初步證明LCA可顯著抑制膠質(zhì)瘤U87、U251的細(xì)胞增殖并促進(jìn)其凋亡,呈現(xiàn)濃度及時(shí)間依賴性。有研究表明,LCA可激活線粒體依賴的內(nèi)源性細(xì)胞凋亡,下調(diào)抗凋亡蛋白Bcl-2和Bcl-xL的蛋白表達(dá)量,顯著上調(diào)促凋亡蛋白Bax和BAD的蛋白表達(dá)量,而且,Bcl-2和Bax在內(nèi)源性凋亡通路中起重要作用,因?yàn)锽cl-2和Bax的平衡決定細(xì)胞存活或凋亡[10]。因此本實(shí)驗(yàn)通過(guò)檢測(cè)Bcl-2和Bax的表達(dá)變化,進(jìn)一步闡明LCA促凋亡作用的分子機(jī)制,Western Blot結(jié)果表明,隨著LCA藥物濃度的增加,抗凋亡蛋白Bcl-2表達(dá)量降低,相反,促凋亡蛋白Bax表達(dá)量逐漸增加,說(shuō)明LCA通過(guò)調(diào)控Bcl-2和Bax等促使膠質(zhì)瘤U87、U251細(xì)胞凋亡,發(fā)揮其抗腫瘤作用。
初步研究證實(shí)LCA對(duì)U87、U251細(xì)胞具有明顯的增殖抑制和促凋亡作用,該機(jī)制可能與激活線粒體依賴的內(nèi)源性細(xì)胞凋亡通路相關(guān),但針對(duì)藥物作用靶點(diǎn)以及通路相關(guān)機(jī)制等還需更深入的研究,為臨床應(yīng)用奠定堅(jiān)實(shí)基礎(chǔ)。
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LicochalconeAinhibitsproliferationandinducescellapoptosisinhumanglioblastomacelllines
WANGJiu,LUOPeng,ZHANGLei,ZHENGXinrui,DAIShuhui,YANGYuefan,RAOWei,PENGCheng,LIJuan,MAWenke,FEIZhou
DepartmentofNeurosurgery,XijingHospital,FourthMilitaryMedicalUniversity,Xi'an710032, China
ObjectiveThe goal of this study is to investigate the cytotoxic effects of licochalcone A on the human glioblastoma cell proliferation and apoptosis and to identify the underlying molecular mechanism.MethodsThe growth-inhibiting effect of licochalcone A(LCA) in the human glioblastoma cells were detected by methyl thiazolyl tetrazolium (MTT) assay and calculated the half maximal inhibitory concentration (IC50), according to different concentrations grouping IC50 or 100 μM. The cell morphological changes were observed under inverted microscope, the cell apoptosis was examined by the flow cytometry and the protein levels of apoptosis-related molecules were detected by Western Blot.ResultsThe results of MTT revealed that LCA could significantly inhibit U87 and U251 glioma cells proliferation in a dose- and time-dependent manner. The inhibitory concentrations of LCA for U87 and U251 glioma cells at 48 h were 61.54 μm and 53.02 μm, respectively. The density and morphology of the U87 and U251 glioma cells were remarkably changed under inverted microscope. Results of flow cytometry showed that LCA could induce apoptosis in U87 and U251 glioma cells (Plt;0.01). Furthermore, LCA significantly reduced the level of B-cell lymphoma-2(Bcl-2) (Plt;0.01), while increased the levels of Bcl2-Associated X(Bax) (Plt;0.05) in a dose-dependent manner.ConclusionLCA could significantly inhibit the cell proliferation of the human glioblastoma cells, subsequently triggering the mitochondrial apoptotic pathway.
Glioma; Licochalcone A; Inhibiting proliferation; Apoptosis
1671-2897(2017)16-025-05
R 739
A
國(guó)家自然科學(xué)基金重點(diǎn)項(xiàng)目資助項(xiàng)目(81430043);“十二五”國(guó)家科技支撐計(jì)劃基金資助項(xiàng)目(2012BAI11B02)
王玖,碩士研究生,E-mail: doctoralixwj@126.com
*通訊作者: 費(fèi)舟,教授、主任醫(yī)師,博士生導(dǎo)師,E-mail: feizhou@fmmu.edu.cn
2016-08-20;
2016-10-24)