萬 琴,王順金

(南昌大學(xué)附屬第二醫(yī)院腫瘤科,江西南昌 330006)

培美曲塞對(duì)SKP2蛋白表達(dá)的影響及意義

萬 琴,王順金△

(南昌大學(xué)附屬第二醫(yī)院腫瘤科,江西南昌 330006)

目的探討S期激酶相關(guān)蛋白2(SKP2)在培美曲塞(PMX)處理后肺腺癌細(xì)胞A549中表達(dá)的改變及意義?方法根據(jù)PMX的濃度設(shè)1個(gè)對(duì)照組和4個(gè)實(shí)驗(yàn)組,分別為:0μmol/L組(A 組)?0.01μmol/L組(B組)?0.1μmol/L組(C組)?1μmol/L組(D組)?10μmol/L組(E組),采用甲基噻唑基四唑(MTT)法檢測(cè)A549細(xì)胞的增殖;利用流式細(xì)胞技術(shù)分析A549細(xì)胞周期的分布;通過Western blot法檢測(cè)A549細(xì)胞SKP2蛋白表達(dá)?結(jié)果PMX明顯抑制A549細(xì)胞增殖,以10μmol/L?72h作用抑制率最高(P0.01);與A組相比C?D?E組S期細(xì)胞顯著增多(P0.05);各組SKP2蛋白相對(duì)表達(dá)水平差異有統(tǒng)計(jì)學(xué)意義(P0.01);與對(duì)照組相比,實(shí)驗(yàn)組SKP2蛋白相對(duì)表達(dá)水平顯著增加(P0.01)?結(jié)論P(yáng)MX能明顯抑制A549細(xì)胞增殖且將其阻滯于S期,PMX處理后的A549細(xì)胞中SKP2蛋白表達(dá)上調(diào),這可能影響PMX對(duì)肺腺癌的化療療效?

肺腫瘤;細(xì)胞周期;S期激酶相關(guān)蛋白質(zhì)類;培美曲塞

S期激酶相關(guān)蛋白2(S-phase kinase-associated protein 2,SKP2)是SCF泛素連接酶復(fù)合體(Skp1-Cullin-1-F-box)的特異性底物識(shí)別亞單位,大量研究已證實(shí)SKP2基因是一種潛在的癌基因,主要存在于惡變細(xì)胞的S期,許多腫瘤細(xì)胞高表達(dá)或普遍表達(dá)SKP2蛋白?SKP2的表達(dá)與非小細(xì)胞肺癌(nonsmall cell lung cancer,NSCLC)的發(fā)生?發(fā)展密切相關(guān)[1],并且是NSCLC的一個(gè)獨(dú)立的預(yù)后指標(biāo)[2-3]?SKP2表達(dá)下調(diào)很可能有利于阻止腫瘤增長,被認(rèn)為是NSCLC分子水平的一個(gè)新治療靶點(diǎn)?培美曲塞(pemetrexed,PMX)是一種新的多靶點(diǎn)葉酸抑制劑,通過抑制葉酸依賴性酶,干擾胸腺嘧啶和嘌呤的合成,達(dá)到抗腫瘤的目的?PMX因有效?低毒等優(yōu)點(diǎn)在NSCLC一線和二線治療中的地位已經(jīng)確立,并在維持治療中顯著延長患者總生存期,有關(guān)PMX的研究已經(jīng)成為腫瘤學(xué)界的一個(gè)熱點(diǎn)?Wu等[4]研究發(fā)現(xiàn)PMX處理后A549細(xì)胞中P27表達(dá)上調(diào),且在0～10μmol/L范圍內(nèi),具有濃度依賴性?SKP2能介導(dǎo)P27多聚泛素化蛋白水解,有關(guān)PMX對(duì)SKP2蛋白表達(dá)的影響國內(nèi)外尚未見報(bào)道?為此,本組檢測(cè)了PMX對(duì)A549細(xì)胞周期及SKP2蛋白表達(dá)的影響,以探討SKP2在PMX處理后的A549細(xì)胞中表達(dá)的改變及意義?

1 材料與方法

1.1 材料 (1)細(xì)胞株:人肺腺癌細(xì)胞系A(chǔ)549細(xì)胞由南昌大學(xué)第二附屬醫(yī)院分子醫(yī)學(xué)中心保存?(2)主要試劑:PMX(江蘇豪森,批號(hào)061101,含量102.2%)?DMEM 培養(yǎng)基?胰蛋白酶(北京Solarbio公司)?胎牛血清(美國Hyclone公司)?二甲基亞砜(美國Ameresco公司)?蛋白提取試劑盒(北京普利萊基因技術(shù)有限公司)?四甲基偶氮唑鹽(methyl thiazolyl tetrazolium,MTT;上海普飛生物技術(shù)有限公司)?酶標(biāo)儀(芬蘭Lab systems公司)?流式細(xì)胞儀(BD公司)?SKP2抗體(Santa Cruz公司)?山羊抗兔IgG抗體(中杉金橋公司)?免疫印跡配膠?電泳?電轉(zhuǎn)裝置(Bio-Rad公司)?

1.2 方法

1.2.1 細(xì)胞培養(yǎng) A549細(xì)胞用DMEM培養(yǎng)基(含10%胎牛血清?100U/mL青霉素和100U/mL鏈霉素),在5%CO2?37℃的條件下常規(guī)培養(yǎng)傳代?

1.2.2 MTT檢測(cè)PMX對(duì)A549細(xì)胞的抑制率 取對(duì)數(shù)生長期細(xì)胞,制成細(xì)胞懸液,按每孔5×103/200μL,接種于3塊96孔培養(yǎng)板中,24h后加入PMX,濃度分別為:0?0.01?0.1?1?10μmol/L?3塊板分別作用24?48?72h后加20μL濃度為5g/L的 MTT,37℃?5%CO2條件下孵育4h,吸出培養(yǎng)液,加二甲基亞砜150μL/孔,避光條件下置于水平搖床上搖10min,

1.3 統(tǒng)計(jì)學(xué)處理 用SPSS17.0軟件進(jìn)行數(shù)據(jù)分析,計(jì)量資料用±s表示,行單因素方差分析?檢驗(yàn)水準(zhǔn)α=0.05?使MTT晶體溶解,經(jīng)酶標(biāo)儀490nm波長檢測(cè)光密度值(optical density,OD)?按以下公式計(jì)算各組抑制率(inhibition rate,IR):抑制率IR(%)=(對(duì)照組OD均值-實(shí)驗(yàn)組OD均值)/對(duì)照組OD均值×100%?實(shí)驗(yàn)重復(fù)3次?

1.2.3 流式細(xì)胞周期技術(shù) 將細(xì)胞接種于6孔板,貼壁后加不同濃度PMX,24h后收集細(xì)胞,制成單細(xì)胞懸液,磷酸鹽緩沖液(phosphate buffer saline,PBS)洗2次,離心,棄上清液,加PBS重懸細(xì)胞,加-20℃預(yù)冷的75%乙醇并振蕩,4℃固定1h,離心,棄冰乙醇,PBS沖洗,棄上清液,加入含碘化吡啶和無DNA酶污染的PBS染色液,4℃避光靜置1h,使用流式細(xì)胞檢測(cè)儀檢測(cè)G1?S?G2各期細(xì)胞所占的百分率?重復(fù)3次?

1.2.4 Western blot法檢測(cè)SKP2的表達(dá) 收集處于對(duì)數(shù)生長期的A549細(xì)胞,根據(jù)細(xì)胞計(jì)數(shù)結(jié)果加入含10%胎牛血清的完全培養(yǎng)基,調(diào)整細(xì)胞濃度為5×105個(gè)/mL,接種于培養(yǎng)瓶,貼壁后加入不同濃度PMX,放入5%CO2孵箱中孵育48h后收集細(xì)胞,PBS洗滌2次,提取總蛋白,二喹啉甲酸(bicinchoninic acid,BCA)法測(cè)定蛋白濃度?取等量蛋白進(jìn)行10%十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳,電濕轉(zhuǎn)法將凝膠上蛋白轉(zhuǎn)至聚偏氟乙烯膜上?5%脫脂奶粉室溫封閉2h?一抗孵育4℃過夜?二抗室溫孵育2h,暗室曝光?重復(fù)3次?

2 結(jié) 果

2.1 MTT比色法檢測(cè)PMX對(duì)A549細(xì)胞增殖的抑制情況見表1?由表1可見,PMX對(duì)A549細(xì)胞具有抑制作用,同一時(shí)間不同濃度PMX組之間的細(xì)胞增殖抑制率以10μM濃度組最高,同一濃度組分別作用24h?48h?72h后,以72h抑制率最高?

表1 不同濃度PMX對(duì)A549細(xì)胞增殖的抑制率(±s,%,n=3)

表1 不同濃度PMX對(duì)A549細(xì)胞增殖的抑制率(±s,%,n=3)

*:P 0.01,與同時(shí)間組比較;#:P 0.01,與同濃度組比較?

PMX(μmol/L)24h 48h 72h 0.01 7.19±3.31 10.93±7.31 21.88±10.23 0.1 17.07±7.89 18.53±7.33 27.39±7.66 1 28.03±7.39* 29.62±10.47* 44.34±7.04*10 58.63±0.86* 68.24±4.29*# 78.61±3.66*#

2.2 PMX使A549細(xì)胞周期阻滯在S期 不同濃度(0.01?0.1?1?10μM)的PMX處理A549細(xì)胞48h后,細(xì)胞周期被阻滯于S期?見表2?封4圖1?

表2 流式細(xì)胞儀檢測(cè)細(xì)胞周期分布的結(jié)果(±s,%,n=3)

表2 流式細(xì)胞儀檢測(cè)細(xì)胞周期分布的結(jié)果(±s,%,n=3)

*:P0.05,#:P 0.01,與同周期0μmol/L組比較?

細(xì)胞周期 0μmol/L 0.01μmol/L 0.1μmol/L 1μmol/L 10μmol/L 75 54.31±2.54 S 17.39±4.21 21.57±3.53 26.95±4.71* 34.76±3.85# 42.47±2.96#G2 5.39±3.69 10.09±7.88 9.33±2.44 5.88±3.91 3.G1 77.22±3.64 68.34±10.71 63.72±6.86 59.36±4.22±1.85

2.3 PMX處理A549細(xì)胞后SKP2蛋白表達(dá)升高 以目標(biāo)蛋白灰度值與β-actin蛋白灰度值之比表示蛋白相對(duì)表達(dá)水平,各組的蛋白相對(duì)表達(dá)水平分別為:A組0.38±0.03?B組0.79±0.02?C組0.84±0.02?D組0.80±0.05?E組0.96±0.03,差異有統(tǒng)計(jì)學(xué)意義(P0.01);與 A組相比,B?C?D?E組蛋白相對(duì)表達(dá)水平顯著增加(P0.01)?PMX處理后SKP2蛋白表達(dá)升高,且在一定的濃度范圍內(nèi),表達(dá)量隨著PMX濃度的增加蛋白表達(dá)量也增加?見封4圖2?

3 討 論

SKP2位于人類第5號(hào)染色體短臂上(5p13),主要存在于惡變細(xì)胞的S期,能特異性地識(shí)別磷酸化的細(xì)胞周期蛋白(cyclin)如:p27kip1?p130?p21wafl?p53?E2F?cyclin D1?cyclin E?cyclin A?cyclin B等依賴性激酶抑制劑,并參與這些蛋白的降解,導(dǎo)致細(xì)胞周期抑制物減少,細(xì)胞過度增殖,促進(jìn)惡性腫瘤的發(fā)生?發(fā)展?SKP2在許多腫瘤細(xì)胞中高表達(dá)或普遍表達(dá),在高表達(dá)的腫瘤中,通過下調(diào)SKP2的表達(dá)可能抑制腫瘤的增長[5]?Kudo等[6]將SKP2siRNA質(zhì)粒轉(zhuǎn)染于口腔鱗癌細(xì)胞后發(fā)現(xiàn),SKP2沉默會(huì)導(dǎo)致P27kip1表達(dá)的增加,抑制口腔鱗癌細(xì)胞的增殖?同樣,Lee與 McCormick[7]通過干擾惡性腦膠質(zhì)瘤細(xì)胞株T98G中SKP2的表達(dá)可以促進(jìn)T98G凋亡,推斷SKP2可作為癌基因治療的一個(gè)靶點(diǎn)?SKP2在NSCLC中也普遍表達(dá),并在NSCLC的發(fā)生?發(fā)展中起重要作用,下調(diào)SKP2會(huì)導(dǎo)致 NSCLC細(xì)胞的凋亡[8]?Jiang等[9]將SKP2-siR-NAs轉(zhuǎn)染于3株肺癌細(xì)胞株后SKP2蛋白表達(dá)降低,P27蛋白表達(dá)升高,其中肺腺癌細(xì)胞A549細(xì)胞轉(zhuǎn)染SKP2-siRNAs后,細(xì)胞增殖率降低12%,凋亡率升高36%?因而,本組認(rèn)為剔除SKP2基因的過度拷貝或抑制SKP2蛋白的表達(dá),將成為治療惡性腫瘤的一條新路徑?

PMX是一種新的多靶點(diǎn)葉酸抑制劑,通過抑制胸腺嘧啶核苷酸合酶?二氫葉酸還原酶和甘氨酰胺核苷酸轉(zhuǎn)甲基酶,影響DNA和RNA的合成,使細(xì)胞停滯于S期,從而促進(jìn)腫瘤細(xì)胞的凋亡達(dá)到抗腫瘤的作用?在NSCLC中,PMX主要用于治療局部晚期或轉(zhuǎn)移性NSCLC(不包括大多數(shù)的鱗狀細(xì)胞癌),它對(duì)肺腺癌及大細(xì)胞癌的療效遠(yuǎn)遠(yuǎn)優(yōu)于鱗狀細(xì)胞癌?因此,本研究選用PMX敏感的肺腺癌細(xì)胞A549細(xì)胞為實(shí)驗(yàn)對(duì)象,通過MTT證實(shí)PMX對(duì)A549細(xì)胞具有明顯抑制作用,且在一定濃度范圍內(nèi),抑制作用隨著濃度增加而增強(qiáng),以10μmol/L的抑制作用最明顯?通過流式細(xì)胞周期技術(shù)分析發(fā)現(xiàn),PMX主要使A549細(xì)胞阻滯于S期,這與 Wouters等[10]的文獻(xiàn)報(bào)道相符?

本研究旨在探討NSCLC治療中常用新藥PMX對(duì)SKP2蛋白表達(dá)的影響及意義,以了解PMX是否能通過對(duì)SKP2的下調(diào)進(jìn)一步促進(jìn)腫瘤細(xì)胞的抑制?本實(shí)驗(yàn)通過Wes tern blot法表明PMX處理后的A549細(xì)胞中SKP2表達(dá)并未下調(diào),相反,實(shí)驗(yàn)組SKP2蛋白表達(dá)量明顯高于對(duì)照組,且隨著藥物濃度的增加,其表達(dá)量也增加?SKP2表達(dá)上調(diào)可能促進(jìn)腫瘤細(xì)胞的增殖,然而MTT法顯示PMX對(duì)A549細(xì)胞具有明顯的抑制作用,本組推斷雖然PMX處理后細(xì)胞SKP2表達(dá)升高,但是SKP2的升高引起的細(xì)胞增殖不足以抵抗PMX對(duì)肺腺癌細(xì)胞的抑制作用,所以總體表現(xiàn)為抑制?SKP2表達(dá)的增加是PMX直接作用的結(jié)果還是對(duì)細(xì)胞周期調(diào)節(jié)后的表現(xiàn),其具體機(jī)制還不清楚,需要作進(jìn)一步研究?再者,SKP2是癌基因,SKP2蛋白表達(dá)上調(diào)是否與PMX治療的療效及預(yù)后有關(guān)目前尚不知曉,這必須引起重視并進(jìn)行更深入的研究?

SKP2也是一個(gè)重要的預(yù)后評(píng)價(jià)指標(biāo),SKP2在卵巢癌?軟組織肉瘤?口腔鱗癌?胃癌和直腸癌中的表達(dá)與預(yù)后相關(guān)都見諸報(bào)道?人們發(fā)現(xiàn)SKP2高表達(dá)患者預(yù)后差,SKP2的表達(dá)就像N分期一樣是總生存率的一個(gè)獨(dú)立預(yù)測(cè)指標(biāo)?Osoegawa等[11]通過多因素分析顯示SKP2是NSCLC的一個(gè)獨(dú)立預(yù)后因子,SKP2高表達(dá)的NSCLC患者預(yù)后不良?SKP2是否是PMX治療肺腺癌的一個(gè)預(yù)后指標(biāo)也有待進(jìn)一步的研究?

總之,本實(shí)驗(yàn)再次肯定了PMX對(duì)肺腺癌細(xì)胞的抑制作用以及S期阻滯;本研究發(fā)現(xiàn)PMX處理后肺腺癌細(xì)胞A549細(xì)胞中SKP2蛋白表達(dá)升高,其具體機(jī)制目前尚不清楚,但SKP2是癌基因,其表達(dá)上調(diào)可能影響PMX的化療療效及預(yù)后?同時(shí),進(jìn)一步探討PMX與特異性下調(diào)SKP2表達(dá)的藥物聯(lián)用后療效是否會(huì)增加將為今后臨床更好地使用PMX以及提高肺腺癌患者的化療療效提供重要的理論基礎(chǔ)?

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Impact of Pemetrexed on expression of SKP2 in adenocarcinoma of lung cell lines A549 cellandits significance

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ObjectiveTo investigate the expression variation and significance of s-phase kinase-associated protein 2(SKP2)in lung adenocarcinoma A549cells after the treatment of Pemetrexed(PMX).MethodsOn the basis of the concentration of PMX,we set one control group and four experimental groups:0μM(group A),0.01μM(group B),0.1μM(group C),1μM(group D),10μM(group E).The growth of A549was tested by MTT assay.The effect of PMX on the cell cycle phase distribution of A549was analyzed by flow cytometry.The expression of SKP2protein of A549cells was detected by Western blot.ResultsMTT assay showed that PMX could significantly inhibit the growth of A549cells,and the highest inhibitory rate was(78.61±3.66)%at the concentration of 10μM when A549cells were cultured for 72h(P0.01).Meanwhile,S-phase cells in the group C,D and E was more than those in the group A(P0.05,P0.01,P0.01).The difference of SKP protein expression among various groups was significant(P0.01),and compared with the control group,SKP2protein expression was significantly increased in the experimental groups(P0.01).ConclusionPMX could inhibit the growth of A549cells and arrest them in S-phase.The expression of SKP2 protein in PMX-treated A549cells is upregulated,which might influence the chemotherapy effect of PMX.

lung neoplasms;cell cycle;S-phase kinase-associated proteins;pemertrexed

10.3969/j.issn.1671-8348.2012.16.020

A

1671-8348(2012)16-1614-03

△通訊作者,Tel:13970018719;E-mail:wangshunjin1950@163.com?

2011-11-08

2011-12-22)

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