過(guò)表達(dá)表皮生長(zhǎng)因子受體對(duì)人類黑素瘤腦轉(zhuǎn)移瘤細(xì)胞系H1生長(zhǎng)和轉(zhuǎn)移的影響

康晶 Hrvoje Miletic 郭淑蘭

過(guò)表達(dá)表皮生長(zhǎng)因子受體對(duì)人類黑素瘤腦轉(zhuǎn)移瘤細(xì)胞系H1生長(zhǎng)和轉(zhuǎn)移的影響

康晶 Hrvoje Miletic 郭淑蘭

目的探索表皮生長(zhǎng)因子受體(EGFR)在黑素瘤生長(zhǎng)和轉(zhuǎn)移過(guò)程中的作用。方法選用本實(shí)驗(yàn)室已建立的人黑素瘤腦轉(zhuǎn)移瘤細(xì)胞系(H1),通過(guò)慢病毒載體穩(wěn)定轉(zhuǎn)染野生型EGFR(H1EGFRwt),使其呈EGFR過(guò)表達(dá)狀態(tài),對(duì)照組轉(zhuǎn)染綠色熒光蛋白(H1GFP)后分選純化。采用細(xì)胞劃痕實(shí)驗(yàn)對(duì)實(shí)驗(yàn)組H1EGFRwt細(xì)胞和對(duì)照組H1GFP細(xì)胞的遷移能力進(jìn)行分析,結(jié)果以劃痕面積愈合率表示。采用集落形成實(shí)驗(yàn)觀察EGFR對(duì)兩組細(xì)胞獨(dú)立生存能力和集落形成能力的影響,通過(guò)刃天青還原試驗(yàn)評(píng)估集落形成結(jié)果,結(jié)果以兩組細(xì)胞活細(xì)胞數(shù)及其代謝活性評(píng)分表示。Western印跡分析EGFR激活的信號(hào)通路。采用SPSS 20.0軟件進(jìn)行重復(fù)測(cè)量的方差分析比較兩組細(xì)胞劃痕面積愈合率的差異,采用配對(duì)樣本t檢驗(yàn)比較兩組細(xì)胞不同預(yù)處理的代謝評(píng)分差異。結(jié)果流式細(xì)胞儀檢測(cè)顯示,H1細(xì)胞EGFR和GFP轉(zhuǎn)染成功,分選后得到了純化H1EGFRwt和H1GFP細(xì)胞。細(xì)胞劃痕實(shí)驗(yàn)顯示,實(shí)驗(yàn)組H1EGFRwt在給予表皮生長(zhǎng)因子(EGF)刺激的不同時(shí)間點(diǎn)(6、18、24、48 h),劃痕面積愈合率分別為0.145±0.066、0.479±0.096、0.571±0.198、0.672±0.199,對(duì)照組H1GFP在相同條件下分別為0.051±0.036、0.254±0.038、0.303±0.077和0.498±0.111,H1EGFRwt細(xì)胞的愈合程度均高于H1GFP細(xì)胞,兩組比較,F=68.49,df=5,P＜0.05。集落形成實(shí)驗(yàn)證實(shí),H1EGFRwt細(xì)胞的代謝評(píng)分(92 225.2±6 632.1)高于H1GFP細(xì)胞(62 935.7±8 159.2),兩組比較,t=2.26,df=9,P＜0.01。Western印跡結(jié)果顯示,H1EGFRwt細(xì)胞可在EGF的刺激下激活下游信號(hào)磷酸化磷脂酰肌醇特異性磷脂酶C-γ(pPLCγ)、磷酸化信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子5(pSTAT 5)、磷酸化信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子3(pSTAT3),而對(duì)照組未能激活。此外,H1EGFRwt細(xì)胞可在EGF刺激下使磷酸化絲/蘇氨酸蛋白激酶(pAKT)和磷酸化有絲裂原活化蛋白激酶(pMAPK)表達(dá)明顯高于對(duì)照組。結(jié)論EGFR在黑素瘤H1細(xì)胞的轉(zhuǎn)移機(jī)制中發(fā)揮重要作用,并有可能成為黑素瘤轉(zhuǎn)移治療中的靶向目標(biāo)。

黑色素瘤;受體,表皮生長(zhǎng)因子;表皮生長(zhǎng)因子

黑素瘤是一種具有高度侵襲及轉(zhuǎn)移性的惡性腫瘤,目前,首選治療方案仍然是手術(shù)切除,但是治愈率很大程度上要取決于是否能早期診斷[1]。表皮生長(zhǎng)因子受體(epidermal growth factor receptor,EGFR)是一種具有酪氨酸激酶活性的膜受體,可被表皮樣生長(zhǎng)因子如表皮生長(zhǎng)因子(epidermal growth factor,EGF)和轉(zhuǎn)化生長(zhǎng)因子α(transforming growth factor-α,TGF-α)等激活,隸屬于 ErbB 受體家族,這個(gè)家族還包括ErbB2(HER2 or Neu)、ErbB3(HER3)和ErbB4(HER4)三個(gè)成員[2]。EGFR被證實(shí)與很多人類腫瘤的發(fā)生發(fā)展及不良預(yù)后有關(guān)[3],也參與了黑素瘤的發(fā)展與轉(zhuǎn)移。我們通過(guò)創(chuàng)傷愈合實(shí)驗(yàn)和集落形成實(shí)驗(yàn)探索EGFR對(duì)黑素瘤細(xì)胞轉(zhuǎn)移及獨(dú)立存活能力的影響,采用Western印跡研究EGFR激活的信號(hào)通路,尋找有可能成為黑素瘤轉(zhuǎn)移治療中的理想靶點(diǎn)。

材料和方法

一、細(xì)胞培養(yǎng)和試劑

人黑素瘤腦轉(zhuǎn)移瘤細(xì)胞系H1的描述見(jiàn)文獻(xiàn)[4],培養(yǎng)基為DMEM(美國(guó)Sigma公司)并添加8%～10%胎牛血清,320 μg/L L谷氨酰胺,100 U/ml青霉素和100 mg/L鏈霉素。磷酸化絲/蘇氨酸蛋白激酶(pAkt)、磷酸化磷脂酰肌醇特異性磷脂酶C-γ(pPLCγ)、磷酸化信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子 5(pSTAT 5)、磷酸化信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子3(pSTAT 3)、磷酸化有絲裂原活化蛋白激酶(pMAPK)和甘油醛-3-磷酸脫氫酶(GAPDH)一抗(美國(guó)Cell Signaling Technology公司),山羊抗兔二抗(美國(guó)Vector Laboratories公司),綠色熒光蛋白(GFP,美國(guó)Proteintech Group公司),刃天青鈉鹽、低熔點(diǎn)瓊脂糖、十二烷基硫酸鈉-聚丙烯酰胺凝膠(SDS-PAGE)(美國(guó)Sigma公司),磷酸鹽緩沖液(PBS)、EGF(美國(guó)Peprotech公司),Difco Noble瓊脂(美國(guó)BD公司),BCA蛋白測(cè)定試劑盒(美國(guó)Thermo Fisher Scientific公司)。

二、流式細(xì)胞儀檢測(cè)

流式細(xì)胞儀(型號(hào)為Acurri C6)分析H1細(xì)胞中EGFR的表達(dá)。實(shí)驗(yàn)組H1細(xì)胞采用慢病毒載體轉(zhuǎn)染野生型EGFR,對(duì)照組轉(zhuǎn)染GFP。流式細(xì)胞儀(型號(hào):FACS Aria)對(duì)細(xì)胞進(jìn)行純化和分選,得到EGFR過(guò)表達(dá)的H1EGFRwt細(xì)胞和對(duì)照組的H1GFP細(xì)胞。

三、細(xì)胞劃痕實(shí)驗(yàn)

H1EGFRwt和H1GFP細(xì)胞接種在24孔板中并于培養(yǎng)箱中培養(yǎng)24 h,直至細(xì)胞達(dá)100%匯合。然后將細(xì)胞進(jìn)行饑餓處理,無(wú)血清培養(yǎng)基中培養(yǎng)24 h。用微量移液器槍頭沿單層細(xì)胞劃過(guò),PBS洗滌2次以除去細(xì)胞碎片,加入20 μg/L EGF繼續(xù)培養(yǎng)。0、6、18、24和48 h時(shí)在顯微鏡下拍照,通過(guò)Cell Profiler軟件測(cè)量劃痕面積。結(jié)果以劃痕面積愈合率表示,劃痕面積愈合率=(0 h時(shí)劃痕面積-各時(shí)間點(diǎn)的劃痕面積)/0 h時(shí)劃痕面積。

四、集落形成實(shí)驗(yàn)

2.4%Difco Noble瓊脂與細(xì)胞培養(yǎng)液1∶3混合制備底層瓊脂,2.4%低熔點(diǎn)瓊脂糖和預(yù)熱的細(xì)胞培養(yǎng)液同樣1∶3混合制備上層瓊脂。單層細(xì)胞懸液調(diào)整到8×104個(gè)/ml,與制備好的上層瓊脂等體積混合,于37～38℃保存。先以每孔50 μl體積的底層瓊脂加入到96孔板中,每組10個(gè)孔,其上覆蓋同體積的瓊脂糖/細(xì)胞懸液,最后每孔加入100 μl細(xì)胞培養(yǎng)液。將96孔板置于培養(yǎng)箱中培養(yǎng)10 d。10 d后上機(jī)檢測(cè),采用Workout 2.0檢測(cè)其活細(xì)胞數(shù)及代謝活性并進(jìn)行評(píng)分。之后采用刃天青還原試驗(yàn)評(píng)估集落形成結(jié)果,每孔加入0.1 g/L刃天青溶液20 μl,培養(yǎng)箱中4 h孵育后,再次上機(jī)檢測(cè)兩組細(xì)胞的活細(xì)胞數(shù)及代謝活性并進(jìn)行評(píng)分,其代謝評(píng)分用來(lái)反映細(xì)胞集落形成實(shí)驗(yàn)結(jié)果。

五、Western印跡

H1EGFRwt和H1GFP細(xì)胞饑餓培養(yǎng)24 h,加入20 μg/L的EGF[5]刺激細(xì)胞生長(zhǎng)30 min后提取總蛋白,并用BCA蛋白測(cè)定試劑盒檢測(cè)總蛋白濃度。將等量樣品通過(guò)SDS-PAGE凝膠電泳后轉(zhuǎn)膜。分別加入不同濃度一抗4℃孵育過(guò)夜,清洗后二抗室溫孵育1.5 h。GAPDH用作內(nèi)參照。

六、統(tǒng)計(jì)學(xué)處理

采用SPSS 20.0進(jìn)行統(tǒng)計(jì)分析,重復(fù)測(cè)量的方差分析比較兩組細(xì)胞劃痕面積愈合率的差異,配對(duì)樣本t檢驗(yàn)比較兩組細(xì)胞不同預(yù)處理的代謝評(píng)分差異,P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

一、H1細(xì)胞系的EGFR表達(dá)

圖1 H1細(xì)胞轉(zhuǎn)染前后表皮生長(zhǎng)因子受體(EGFR)表達(dá)情況 1a:轉(zhuǎn)染前H1細(xì)胞為EGFR陰性細(xì)胞;1b:EGFR轉(zhuǎn)染后EGFR的陽(yáng)性率高達(dá)98.0%;1c:H1GFP細(xì)胞為EGFR陰性細(xì)胞

圖2 表皮生長(zhǎng)因子(EGF)處理不同時(shí)間對(duì)H1EGFRwt和H1GFP細(xì)胞劃痕面積愈合的影響(×100) 給予EGF刺激時(shí),相同時(shí)間內(nèi)H1EGFRwt細(xì)胞的愈合程度明顯高于H1GFP細(xì)胞;不給予任何處理時(shí),H1EGFRwt細(xì)胞和H1GFP細(xì)胞的愈合程度無(wú)顯著差異

流式細(xì)胞儀檢測(cè)結(jié)果顯示,H1細(xì)胞系為EGFR陰性細(xì)胞系(圖1a),轉(zhuǎn)染后H1細(xì)胞系的EGFR表達(dá)率高達(dá)98.0%(圖1b)。流式細(xì)胞儀分選后可得到純化的EGFR過(guò)表達(dá)的細(xì)胞系H1EGFRwt細(xì)胞。對(duì)照組H1GFP細(xì)胞根據(jù)GFP的綠色熒光,同樣用流式細(xì)胞儀對(duì)其進(jìn)行分選,從而得到純化的H1GFP細(xì)胞,H1GFP細(xì)胞為EGFR陰性細(xì)胞系(圖1c)。

二、細(xì)胞劃痕實(shí)驗(yàn)結(jié)果

結(jié)果顯示,給予實(shí)驗(yàn)組H1EGFRwt細(xì)胞以及對(duì)照組H1GFP細(xì)胞EGF處理時(shí),相同時(shí)間內(nèi)H1EGFRwt細(xì)胞的愈合程度明顯高于H1GFP細(xì)胞;而兩組細(xì)胞不給予任何處理時(shí),愈合程度無(wú)顯著差異(圖2)。為進(jìn)一步確定實(shí)驗(yàn)結(jié)果,分別計(jì)算兩組細(xì)胞不同時(shí)間點(diǎn)的劃痕面積愈合率,根據(jù)不同時(shí)間點(diǎn)的劃痕面積愈合率以及不同的處理因素繪制柱形圖(圖3)。結(jié)果顯示,給予EGF處理時(shí),H1EGFRwt細(xì)胞的劃痕面積愈合率比對(duì)照組H1GFP細(xì)胞高,兩組劃痕愈合率數(shù)據(jù)滿足球形分布假設(shè),差異有統(tǒng)計(jì)學(xué)意義(F=68.49,df=5,P＜0.05);不給予任何處理時(shí),H1EGFRwt細(xì)胞與H1GFP細(xì)胞的劃痕面積愈合率數(shù)據(jù)不滿足球形分布假設(shè),多變量方差分析差異無(wú)統(tǒng)計(jì)學(xué)意義(F=66.64,df=5,P＞0.05)。此外,由圖3還可發(fā)現(xiàn),EGF刺激下在18 h和24 h時(shí),H1EGFRwt細(xì)胞與對(duì)照組H1GFP細(xì)胞相比劃痕面積愈合率差異最顯著。

三、集落形成實(shí)驗(yàn)結(jié)果

結(jié)果顯示,添加刃天青溶液前H1EGFRwt細(xì)胞的活細(xì)胞數(shù)評(píng)分為2018.7±116.2,而H1GFP細(xì)胞為2 069.7±95.0,兩組比較,t=1.32,df=9,P＞0.05。加入刃天青后,實(shí)驗(yàn)組H1EGFRwt細(xì)胞的代謝評(píng)分為92 225.2±6 632.1,對(duì)照組H1GFP細(xì)胞為62 935.7±8 159.2,實(shí)驗(yàn)組代謝評(píng)分顯著高于對(duì)照組(t=7.27,df=9,P＜0.01),表明 H1EGFRwt細(xì)胞與對(duì)照組相比有更高的集落形成能力和獨(dú)立生存能力。

四、Western印跡結(jié)果

H1EGFRwt細(xì) 胞 在 EGF刺 激 下 pPLCγ、pSTAT5、pSTAT3均被激活,而對(duì)照組H1GFP細(xì)胞以及不給予EGF處理時(shí)的兩組細(xì)胞均未被激活;pAkt和pMAPK在有無(wú)EGF刺激時(shí)各組細(xì)胞均可表達(dá),但H1EGFRwt細(xì)胞給予EGF刺激時(shí)表達(dá)明顯增高。見(jiàn)圖4。

討 論

EGFR在人類腫瘤中常常呈現(xiàn)過(guò)表達(dá)狀態(tài)[3],而EGFR的過(guò)表達(dá)狀態(tài)與許多人類腫瘤的進(jìn)展和治療效果不佳有關(guān),已成為預(yù)后不良的標(biāo)志物。Normanno等[6]證實(shí),EGFR在鱗狀細(xì)胞癌中呈過(guò)表達(dá)狀態(tài)。Oh等[7]和Adamczyk等[8]分別發(fā)現(xiàn)宮頸癌和胰腺癌患者血清中EGFR的表達(dá)水平增高。而EGFR在黑素瘤中表達(dá)情況尚有爭(zhēng)議。一方面有研究證實(shí),EGFR對(duì)于人葡萄膜黑素瘤細(xì)胞的增殖存活很重要,但與其表達(dá)水平關(guān)系不大[9];另一方面Hurks等[10]發(fā)現(xiàn),EGFR在人葡萄膜黑素瘤中表達(dá),且其表達(dá)與存活率之間呈負(fù)相關(guān)。此外Udart等[11]在晚期黑素瘤中檢測(cè)到了過(guò)表達(dá)的EGFR。我們的集落形成實(shí)驗(yàn)顯示,EGFR過(guò)表達(dá)的H1EGFRwt細(xì)胞與對(duì)照組H1GFP細(xì)胞相比,H1EGFRwt細(xì)胞的獨(dú)立存活能力以及集落形成能力高于對(duì)照組細(xì)胞,說(shuō)明EGFR過(guò)表達(dá)的H1EGFRwt細(xì)胞更容易存活。

圖3 表皮生長(zhǎng)因子(EGF)處理不同時(shí)間對(duì)H1EGFRwt和H1GFP細(xì)胞劃痕面積愈合程度的影響 EGF處理時(shí)H1EGFRwt細(xì)胞的劃痕面積愈合率比H1GFP細(xì)胞高(F=68.49,df=5,P＜0.05);不給予任何處理時(shí)H1EGFRwt細(xì)胞與H1GFP細(xì)胞的劃痕面積愈合率差異無(wú)統(tǒng)計(jì)學(xué)意義(F =66.64,df=5,P＞0.05),而且EGF刺激下在18 h和24 h時(shí)H1EGFRwt細(xì)胞與對(duì)照組H1GFP細(xì)胞劃痕面積愈合率差異最顯著

圖4 表皮生長(zhǎng)因子(EGF)處理后H1EGFRwt和H1GFP細(xì)胞的信號(hào)激活情況 1:經(jīng)EGF處理的H1GFP細(xì)胞;2:經(jīng)EGF處理的H1EGFRwt細(xì)胞;3:H1GFP細(xì)胞;4:H1EGFRwt細(xì)胞

腫瘤轉(zhuǎn)移是一個(gè)多因素多步驟的復(fù)雜過(guò)程,包括黏附、遷移和侵襲等方面的改變[12]。已有研究證實(shí),EGFR過(guò)表達(dá)與多種腫瘤的生長(zhǎng)和轉(zhuǎn)移機(jī)制有關(guān)[10],但目前對(duì)過(guò)表達(dá)的EGFR參與黑素瘤的發(fā)生發(fā)展及轉(zhuǎn)移的機(jī)制仍不清楚。有研究報(bào)道,在黑素瘤轉(zhuǎn)移瘤中可檢測(cè)到EGFR基因的高頻率表達(dá)[13]。Slominski等[14]發(fā)現(xiàn),惡性黑素瘤中EGFR的表達(dá)增加與腫瘤侵襲和轉(zhuǎn)移的程度之間有相關(guān)性。Huang等[15]也發(fā)現(xiàn),EGFR的過(guò)表達(dá)與人黑素瘤細(xì)胞系在裸小鼠實(shí)驗(yàn)中的自發(fā)轉(zhuǎn)移有相關(guān)性。我們的研究發(fā)現(xiàn),過(guò)表達(dá)的EGFR在EGF的刺激下顯著提高了細(xì)胞的遷移能力(圖2、3),此外,細(xì)胞獨(dú)立存活和集落形成能力的提高也有助于腫瘤細(xì)胞的轉(zhuǎn)移以及轉(zhuǎn)移瘤的存活發(fā)展。

EGFR所參與的信號(hào)轉(zhuǎn)導(dǎo)通路調(diào)控著細(xì)胞的增殖、分化和凋亡,對(duì)腫瘤的發(fā)展以及轉(zhuǎn)移有重要作用。Udart等[11]證實(shí),EGFR活性隨黑素瘤的發(fā)生發(fā)展及轉(zhuǎn)移逐漸增強(qiáng),說(shuō)明EGFR信號(hào)通路與黑素瘤的發(fā)生、發(fā)展以及轉(zhuǎn)移相關(guān)。EGF可結(jié)合其受體,促進(jìn)受體二聚化和自身磷酸化,從而導(dǎo)致各種下游信號(hào)分子如MAPK和PI3K/Akt信號(hào)激活[16],而MAPK和PI3K/Akt信號(hào)通路在腫瘤細(xì)胞侵襲和轉(zhuǎn)移的調(diào)控中起到關(guān)鍵性的作用[17]。除此之外,STAT3的激活在腫瘤的形成和發(fā)展過(guò)程中也起到重要作用[18]。研究表明,在黑素瘤腫瘤模型中靶向抑制STAT3可誘導(dǎo)腫瘤消退[19],抑制血管生成[20],預(yù)防腫瘤轉(zhuǎn)移[21],促進(jìn)免疫應(yīng)答的活化[22-23]。Wellbrock等[24]發(fā)現(xiàn),STAT5過(guò)度表達(dá)可促進(jìn)黑素瘤的發(fā)生發(fā)展,而Wellbrock等[25]在早期還發(fā)現(xiàn),STAT5的激活在黑素細(xì)胞向黑素瘤的轉(zhuǎn)化過(guò)程中發(fā)揮重要作用。Jiang等[26]發(fā)現(xiàn),如果抑制PI3K-Akt和PLCγ-PKC通路可誘導(dǎo)細(xì)胞凋亡,說(shuō)明PI3K-Akt和PLCγ-PKC信號(hào)通路在維持細(xì)胞存活方面發(fā)揮了重要作用。在我們的研究中,H1EGFRwt細(xì)胞在EGF刺激下,pPLCγ、pSTAT5、pSTAT3均被激活,pAkt和 pMAPK在H1EGFRwt細(xì)胞給予EGF刺激時(shí)表達(dá)明顯高于其他對(duì)照組,說(shuō)明EGFR過(guò)表達(dá)的情況下,EGF可以刺激EGFR激活或增強(qiáng)磷酸化信號(hào)Akt、STAT5、STAT3、PLCγ以及MAPK所參與的各條信號(hào)通路,而他們所參與的信號(hào)通路對(duì)腫瘤細(xì)胞侵襲、轉(zhuǎn)移、增殖和存活方面有促進(jìn)作用,促進(jìn)了腫瘤的發(fā)展和轉(zhuǎn)移。

綜上所述,我們的研究證明,EGFR過(guò)表達(dá)可以提高H1細(xì)胞系的獨(dú)立存活能力,在EGF的刺激下可提高H1細(xì)胞系的遷移能力以及激活下游信號(hào)pPLCγ、pSTAT5和 pSTAT3, 并且使 pAkt以及pMAPK表達(dá)顯著增強(qiáng),從而促進(jìn)了黑素瘤細(xì)胞轉(zhuǎn)移的發(fā)生。我們的研究可能為黑素瘤轉(zhuǎn)移的靶向治療提供了一定的理論依據(jù),從而進(jìn)一步支持EGFR有可能成為黑素瘤轉(zhuǎn)移治療的新靶點(diǎn)。

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2014-05-02)

(本文編輯:顏艷)

Effect of overexpression of epidermal growth factor receptor on the growth and migration of a brainmetastatic H1 human melanoma cell line

Kang Jing*，Hrvoje Miletic，Guo Shulan.*Shandong University School of Medicine，Jinan 250012，China

Guo Shulan，Email:guoshulan@medmail.com.cn

ObjectiveTo investigate the role of epidermal growth factor receptor(EGFR)in the growth and migration of melanoma.MethodsA brain-metastatic human melanoma cell line H1，which had been developed in our laboratory，was used in this study.Some H1 melanoma cells were stably transfected with wild-type EGFR via lentiviral vectors to overexpress EGFR(H1EGFRwt)，and some H1 cells transfected with green fluorescent protein(GFP)served as the control group(H1GFP).After transfection，the cells were sorted and purified.Then，scratch wound healing assay was performed to estimate the migratory activity of H1 cells;colony-forming assay was conducted to investigate the effects of EGFR on the survival ability and colony-forming ability of H1 cells，and resazurin reduction test was used to evaluate colony formation results;Western blot was carried out to measure the expressions of signaling pathways activated by EGFR.Statistical analysis was done by repeated measures analysis of variance(ANOVA)for the comparison of wound-area healing rate，and by pairedttest for the comparison of metabolic activity，between the two groups of H1 cells by using SPSS software version 20.0.ResultsFlow cytometry showed that H1 cells were successfully transfected with EGFR and GFP respectively，and purified H1EGFRwt and H1GFP cells were obtained after cell sorting.The wound-area healing rate at 6，18，24 and 48 hours after epidermal growth factor(EGF)stimulation was 0.145±0.066，0.479±0.096，0.571±0.198 and 0.672±0.199 respectively for H1EGFRwt cells，0.051± 0.036，0.254± 0.038，0.303± 0.077 and 0.498 ± 0.111 respectively for H1GFP cells.The degree of wound healing in H1EGFRwt cells was significantly higher than that in H1GFP cells(F=68.49，df=5，P＜0.05).Colony-forming assay revealed that the average metabolic activity score of H1EGFRwt cells was significantly higher than that of H1GFP cells(92 225.2 ± 6 632.1 vs.62 935.7 ± 8 159.2，t=2.26，df=9，P＜0.01).Western blot showed that EGF might induce the phosphorylation of downstream signaling proteins such as phosphatidylinositol-specific phospholipase C-γ (PLCγ)，signal transducer and activator of transcription 5(STAT5)and 3(STAT3)in H1EGFRwt cells，but not in H1GFP cells.In addition，the expressions of phosphorylated serine/threonine protein kinase(pAKT)and mitogen-activated protein kinase(pMAPK)were significantly higher in H1EGFRwt cells than in H1GFP cells after EGF stimulation.Conclusions EGFR may play an important role in the metastasis of H1 melanoma cells，and might serve as a target in the treatment of metastatic melanoma.

Melanoma;Receptor，epidermal growth factor;Epidermal growth factor

10.3760/cma.j.issn.0412-4030.2014.12.013

250012濟(jì)南,山東大學(xué)醫(yī)學(xué)院(康晶);挪威卑爾根大學(xué)生物醫(yī)學(xué)系(Hrvoje Miletic);山東大學(xué)齊魯醫(yī)院皮膚科(郭淑蘭)

郭淑蘭,Email:guoshulan@medmail.com.cn