郭俊　楊靖　李艷

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·論著·

雙氫青蒿素誘導(dǎo)肝癌細(xì)胞Huh-7凋亡并抑制其遷移的機(jī)制研究

郭俊楊靖李艷

430060 武漢大學(xué)人民醫(yī)院檢驗科(郭俊、李艷)；442000 十堰，湖北醫(yī)藥學(xué)院附屬人民醫(yī)院感染性疾病科(楊靖)

【摘要】目的探討雙氫青蒿素(DHA)能否影響具有突變型p53的人肝癌細(xì)胞株Huh-7增殖、凋亡及遷移等生物學(xué)行為及其作用機(jī)制。方法DHA處理Huh-7細(xì)胞后MTT法檢測增殖抑制率，免疫熒光分析DNA雙鏈損傷情況，WB法測γH2AX蛋白表達(dá)水平，流式細(xì)胞術(shù)檢測細(xì)胞凋亡。細(xì)胞劃痕法檢測DHA對細(xì)胞遷移能力的影響。WB法測細(xì)胞中STAT3蛋白的活性及表達(dá)水平。結(jié)果DHA能抑制Huh-7細(xì)胞的增殖活性，抑制作用呈濃度依賴性。20 μmol/L DHA處理48 h能升高γH2AX表達(dá)水平，損傷Huh-7細(xì)胞DNA雙鏈。DHA處理48 h細(xì)胞的凋亡百分率為(3.15±0.39)%。DHA降低Huh-7細(xì)胞遷移能力，p-STAT3(Y705)表達(dá)水平下降。結(jié)論DHA能造成Huh-7細(xì)胞DNA雙鏈損傷，誘導(dǎo)細(xì)胞凋亡，但此過程與p53活性無關(guān)。可能的機(jī)制是通過降低STAT3磷酸化水平抑制突變型p53的Huh-7細(xì)胞增殖，誘導(dǎo)Huh-7細(xì)胞凋亡和抑制細(xì)胞遷移。

【主題詞】原發(fā)性肝細(xì)胞癌；雙氫青蒿素；細(xì)胞增殖；細(xì)胞凋亡

Fund program: Initial Project for Post-Graduates of Hubei University of Medicine (2015QDJZR08)

原發(fā)性肝細(xì)胞癌(hepatocellular carcinoma, HCC)是常見惡性腫瘤，死亡率高居癌癥的第三位[1]。抗腫瘤藥物耐受及腫瘤細(xì)胞的不斷侵襲和遷移限制了HCC的放療、化療效果。雙氫青蒿素(Dihydroartemisinin，DHA)作為抗瘧疾藥物青蒿素的重要衍生物，能在體內(nèi)外發(fā)揮抗腫瘤作用，極有潛力成為價廉且有效的抗癌藥[1-3]。研究顯示，DHA能通過降低血管內(nèi)皮生長因子及其受體的表達(dá)而抑制大鼠原發(fā)性肝癌及小鼠腫瘤的血管生成，促進(jìn)肝癌凋亡[4,5]。DHA在體外能誘導(dǎo)具有野生型p53的人肝癌SMMC-7721[5]細(xì)胞及HepG2細(xì)胞凋亡[6]。但DHA對p53失活的HCC細(xì)胞生物學(xué)的作用及其機(jī)制方面研究甚少。本研究通過對DHA在體外對具有突變型p53的Huh-7細(xì)胞凋亡及遷移的影響研究，探索DHA在體外對肝癌細(xì)胞Huh-7的生物學(xué)作用機(jī)制，為DHA在肝癌治療中的進(jìn)一步應(yīng)用提供科學(xué)依據(jù)。

1材料與方法

以不同濃度DHA分別處理Huh-7細(xì)胞，用MTT法檢測各組細(xì)胞的增殖抑制率，確定IC50。用此濃度的DHA處理Huh-7細(xì)胞，Etoposide作為DNA雙鏈損傷的陽性質(zhì)控藥物，γH2AX抗體孵育后免疫熒光分析DNA雙鏈損傷情況，Western Blot法檢測γH2AX蛋白表達(dá)水平變化進(jìn)行驗證。采用流式細(xì)胞術(shù)檢測DHA處理后Huh-7細(xì)胞凋亡。細(xì)胞劃痕法檢測DHA對細(xì)胞遷移能力的影響。Western Blot法檢測各組細(xì)胞中STAT3蛋白的活性及表達(dá)。

1.1材料

1.1.1細(xì)胞系：人肝癌細(xì)胞株Huh-7為四川大學(xué)李明遠(yuǎn)教授惠贈。

1.1.2主要試劑和抗體：DHA、EtoposideMTT試劑均購自美國Sigma公司；Annexin V-FITC Apoptosis Kit購自BD Biosciences；兔抗人γH2AX抗體購自北京博奧森生物技術(shù)有限公司；兔抗人p-STAT3(Y705)及STAT3均購自上海生工生物工程股份有限公司，兔抗人β-actin抗體及羊抗兔IgG-Cy3熒光抗體均購自北京中杉金橋生物技術(shù)有限公司。

1.2方法

1.2.1細(xì)胞培養(yǎng)：用含10%小牛血清、1%青鏈霉素雙抗的DMEM(高糖)培養(yǎng)基作為完全DMEM培養(yǎng)基。細(xì)胞接種后置5% CO2孵箱中37 ℃連續(xù)培養(yǎng)。

1.2.2細(xì)胞分組及藥物處理：NC組：20 μmol/L DMSO處理48 h；DHA組：20 μmol/L雙氫青蒿素處理48 h；Eto組：20 μmol/L依托泊苷處理48 h。

1.2.3MTT檢測細(xì)胞體外增殖能力：鋪96孔板(5000～10000個細(xì)胞/100 μl/孔)，37 ℃孵育，過夜；棄培養(yǎng)基，換成含有藥物的含10%小牛血清的DMEM培養(yǎng)基處理48 h，每組3復(fù)孔，同時設(shè)置調(diào)零孔(DMEM、MTT、DMSO)和對照孔(細(xì)胞、DMEM、MTT、DMSO)；在預(yù)訂的時間點(diǎn)每孔加入5 mg/ml 的MTT溶液10 μl(終濃度為0.5% MTT)，繼續(xù)培養(yǎng)4 h；小心吸棄孔內(nèi)培養(yǎng)液，每孔加入150 μl DMSO，置搖床上低速振蕩至結(jié)晶完全溶解；用酶聯(lián)免疫檢測儀測量各孔光密度值(OD490 nm)；細(xì)胞活性按下列公式[77]計算：細(xì)胞活性(%)=(OD490 nm-OD空白)處理組/(OD490 nm-OD空白)對照組×100%。

1.2.4免疫熒光分析DNA雙鏈損傷情況：將細(xì)胞以合適的濃度接種于預(yù)先放有無菌小玻片的24孔板中，培養(yǎng)細(xì)胞至匯合度約80%；藥物處理48 h后，4% PFA固定,0.25% Triton X-100通透；封閉后，與兔抗人γH2AX抗體孵育，再經(jīng)抗兔IgG-Cy3熒光標(biāo)記物的二抗孵育；DAPI封片后，熒光顯微鏡下拍照。

1.2.5Annexin V-FITC/PI雙染檢測凋亡細(xì)胞數(shù)：鋪6孔板(1 × 105個細(xì)胞/孔)，每組3復(fù)孔，37 ℃孵育過夜；藥物處理48 h，用不含EDTA的0.25%胰酶消化處理、離心收集細(xì)胞；經(jīng)冷PBS 洗滌細(xì)胞兩次后，按說明書在細(xì)胞懸浮液(濃度大約為1 ×106cells/ml)中加入Annexin V-FITC及碘化丙啶(propidium iodide，PI)，輕輕混勻后4 ℃ 避光孵育5 min，在1 h內(nèi)用流式細(xì)胞儀檢測；以Annexin V-FITC陽性率作為凋亡細(xì)胞百分率。

1.2.6細(xì)胞劃痕法檢測細(xì)胞遷移能力：用滅菌的10 μl槍頭在單層細(xì)胞上呈一直線劃痕，無血清培養(yǎng)液輕柔洗滌，去除懸浮細(xì)胞，藥物處理24 h。換成完全DMEM培養(yǎng)基，繼續(xù)培養(yǎng)24 h，用倒置相差顯微鏡觀察劃痕之間的距離變化。

1.2.7Western Blot檢測γH2AX、pSTAT3及STAT3蛋白表達(dá)：適量1×SDS上樣緩沖液裂解藥物處理后的Huh-7細(xì)胞，吸取細(xì)胞裂解液于EP管中，98 ℃煮5 ～10 min，離心后上清為蛋白樣品。樣品進(jìn)行SDS-PAGE電泳后將蛋白轉(zhuǎn)至PVDF膜上，5%脫脂牛奶封閉；一抗(1∶500)4 ℃孵育過夜，洗膜后與二抗(1∶5 000)室溫孵育30 min；洗膜后加ECL顯影液，放入凝膠分析系統(tǒng)中曝光，拍照存儲并用Quantity One軟件分析計算各條帶的灰度值，以每組目的蛋白和內(nèi)參蛋白灰度值的比值表示各組蛋白的表達(dá)水平。

1.3統(tǒng)計學(xué)方法各組實驗數(shù)據(jù)用SPSS 16.0統(tǒng)計軟件進(jìn)行分析，采用單因素方差分析(one-way ANOVA)，兩兩比較用turkey檢驗，結(jié)果中數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差的方式表示 (n=3)，P<0.05為差異有統(tǒng)計學(xué)意義。

A 不同濃度DHA處理Huh-7細(xì)胞48 h后的增殖抑制率；B 倒置相差顯微鏡觀察細(xì)胞數(shù)目的減少情況(100×) 圖1　MTT檢測細(xì)胞體外增殖能力A Inhibition of Huh-7 cells proliferation in medium containing DHA in different concentrations after cultured 48 hour;B The changes of Huh-7 cells form observed by upside down and discrepancy microscopeFig.1　Cellular proliferation capacity detection with MTT

A 熒光顯微鏡觀察細(xì)胞爬片中γH2AX表達(dá)情況(紅色熒光，400×)；B Western Blot檢測γH2AX表達(dá)水平變化； C 條帶灰度值用Quantity One軟件分析結(jié)果圖2　DHA導(dǎo)致Huh-7細(xì)胞發(fā)生雙鏈DNA損傷A　Observing the cells climbing to the carry sheet glass under a fluorescent microscope, which are stained with antibody of γH2AX (red fluorescence, 400×);B　The expression levels of γH2AX were detected by Western Blot assay;C　The result of gray-scale value of straps was analyzed by Quantity One softwareFig.2　DHA lead to DNA double-strand break of Huh-7 cell line

2結(jié)果

2.1DHA抑制Huh-7細(xì)胞增殖不同濃度的DHA作用Huh-7細(xì)胞48 h，細(xì)胞增殖活性均被抑制，DHA對Huh-7細(xì)胞增殖活性的抑制作用有劑量依賴性(圖1A)。48 h時，IC50為20 μmol/L，選用20 μmol/L DHA作用48 h作為下述實驗作用濃度和時間。倒置相差顯微鏡觀察，DHA處理后均能減少Huh-7細(xì)胞數(shù)目(圖1B)。

2.2DHA導(dǎo)致Huh-7細(xì)胞發(fā)生雙鏈DNA損傷細(xì)胞爬片用γH2AX抗體染色，實驗組DHA組與陽性對照組Eto組發(fā)生DNA雙鏈損傷的細(xì)胞數(shù)目均增多(圖2A)。與NC組相比，DHA使γH2AX表達(dá)水平升高(4.00±0.30)(圖2)，DHA能誘導(dǎo)Huh-7細(xì)胞發(fā)生DNA雙鏈損傷。

2.3DHA能誘導(dǎo)Huh-7細(xì)胞凋亡Annexin V-FITC/PI雙染后進(jìn)行流式細(xì)胞計數(shù)結(jié)果顯示，Huh-7細(xì)胞的凋亡百分率DHA組(3.15±0.39)及Eto組(9.21±0.41)比NC組(1.03±0.29)均上調(diào)，差異具有統(tǒng)計學(xué)意義(P<0.05，圖3)，DHA能誘導(dǎo)Huh-7細(xì)胞凋亡。

2.4DHA抑制Huh-7細(xì)胞遷移劃痕實驗顯示對照組Huh-7細(xì)胞遷移能力強(qiáng)，而DHA處理后，細(xì)胞向劃痕處爬行的速度明顯慢于對照組和Eto組，DHA使Huh-7細(xì)胞遷移能力降低 (圖4)。

2.5DHA降低了STAT3的磷酸化水平Western blot檢測結(jié)果顯示，Huh-7細(xì)胞存在p-STAT3(Y705)，即STAT3處于活化狀態(tài)(圖5A)。DHA處理不影響STAT3蛋白的表達(dá)，但降低了p-STAT3(Y705)蛋白表達(dá)水平(3.21±0.23)(圖5B)。

A　Annexin V-FITC/PI雙染后流式細(xì)胞術(shù)檢測凋亡細(xì)胞數(shù)目的散點(diǎn)圖；B　凋亡百分率柱狀圖，*表示與NC組比較，P<0.05圖3　DHA處理Huh-7細(xì)胞的凋亡率A　Scatter diagram to inspect the number of apoptotic cells were employed by Flow cytometry with the Annexin V-FITC/PI double staining;B　Histogram to assess the changes of Huh-7 cells apoptosis percentage;*Compared with NC group, P<0.05Fig.3　Apoptosis rate of Huh-7 cells delection with flow cytometer

圖4　倒置相差顯微鏡觀察劃痕之間的距離變化(100×)Fig.4　Cellular migration abilities of Huh-7 cells was detected by scratch test with upside down and discrepancy microscope(100×)

A Western Blot檢測p-STAT3(Tyr705)表達(dá)水平變化；B 條帶灰度值用Quantity One軟件分析結(jié)果圖5　Western blot檢測DHA處理后Huh-7細(xì)胞中STAT3蛋白的活性及表達(dá)A　The expression levels of p-STAT3(Y705)were detected by Western blot assay;B　The results of gray-scale value of straps was analyzed by Quantity One softwareFig.5　Western blot assay was used to examine the expression and the activity of the STAT3 protein

3討論

DNA雙鏈斷裂是機(jī)體最致命的DNA損傷，能激活p53等凋亡通路，但約有50%肝癌存在p53功能的缺失、突變或失活[7]。目前，DHA與肝癌腫瘤細(xì)胞的相互作用機(jī)制尚不明確，Hou等[8]發(fā)現(xiàn)DHA可以通過caspase-3通路誘導(dǎo)Huh-7細(xì)胞和BEL-7404細(xì)胞(突變型p53)發(fā)生凋亡。

STAT3 作為EGFR、IL-6/JAK和PI3K/Akt/mTOR等信號通路匯聚的焦點(diǎn)，參與腫瘤的凋亡、侵襲、轉(zhuǎn)移、血管生成和免疫抑制，是信號轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄激活因子(signal transducers and activators of transcription，STATs)蛋白家族中與腫瘤發(fā)生、發(fā)展關(guān)系最為密切的一個成員。STAT3活性抑制劑能否成為肝癌的治療藥物仍然處在探索研究階段，STAT3失調(diào)與肝癌發(fā)生、發(fā)展及耐藥等相關(guān)[9,10]。降低或抑制STAT3活性，會增敏放化療效果。STAT3 活化的標(biāo)志是705 位的酪氨酸(Tyr705)發(fā)生磷酸化[11]。實驗證實Sorafenib能通過抑制STAT3活性，增敏PLC5、Huh-7、Sk-Hep1及Hep3B細(xì)胞的放療效果[12]。新型組蛋白脫乙?；敢种苿㏄anobinostat(LBH589)能通過降低p-STAT3(Tyr705)的表達(dá)水平誘導(dǎo)肝癌細(xì)胞凋亡[13]。乙?；D(zhuǎn)移酶抑制劑 garcinol能結(jié)合肝癌細(xì)胞中的STAT3的SH2結(jié)構(gòu)域阻止其二聚體化誘導(dǎo)肝癌細(xì)胞凋亡[14]。

Ho等[15]認(rèn)為DHA是具有多靶標(biāo)的藥物，能通過抑制STAT3活性發(fā)揮抗炎、抗腫瘤等多種生物學(xué)作用。本研究發(fā)現(xiàn)DHA能造成Huh-7細(xì)胞DNA雙鏈斷裂，降低p-STAT3(Tyr705)表達(dá)水平；進(jìn)一步誘導(dǎo)Huh-7細(xì)胞凋亡，抑制Huh-7細(xì)胞遷移，且此過程與p53的活性無關(guān)。綜上所述，價廉易于獲取的DHA有望成為治療肝癌的新突破。

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通信作者：李艷， Email: 20090077@hbmu.edu.cn

DOI：10.3760/cma.j.issn.1003-9279.2016.03.005

基金項目：湖北醫(yī)藥學(xué)院博士啟動金(2015QDJZR08)

(收稿日期：2016-04-12)

Molecular mechanism of dihydroartemisinin to induce apoptosis and inhibit migration in human liver cancer cell line Huh-7

GuoJun,YangJing,LiYan

DepartmentofClinicalLaboratory,People’sHospitalofWuhanUniversity,Wuhan430060,China(GuoJ，LiY);DepartmentofInfectiousDisease,People’sHospitalofHubeiUniversityofMedicine,Shiyan442000,China(YangJ)Correspondingauthor:LiYan,Email: 20090077@hbmu.edu.cn

【Abstract】ObjectiveTo investigate the effect and mechanism of DHA on the cell proliferation, apoptosis, and migration in human liver cancer cell line Huh-7 with mutant TP53 in vitro. MethodsMTT assay was used to detect the inhibitory effect. Immunofluorescence assay and WB were applied to detect the expression of γH2AX after treatment of DHA. FCM was employed to assess the apoptosis rate. Scratch method was used to test the cell migration ability. Western blot was used to determine the expression of p-STAT3(Y705). ResultsDHA inhibited the cell proliferation in Huh-7 cells.The results of immunofluorescence assay and WB showed that DHA enhanced the expression level of γH2AX and induced the DNA double-strand break (DSB) in Huh-7 cells. WB showed that DHA downregulated the phosphorylation of STAT3 at the Tyr705. ConclusionsDHA caused the DSB and induced apoptosis in Huh-7 cells regardless of p53 status. DHA inhibited cell proliferation and migration of Huh-7 cells. It is probable that DHA induced apoptosis via downregulation of p-STAT3 (Tyr705).

【Key words】Hepatocellular carcinoma; Dihydroartemisinin; Cell proliferation; Cell apoptosis