王霞 王暉 蔣楠 梁三紅 呂文 張嘯 張?bào)泺P

·論著·

MEK通路抑制劑對(duì)人胰腺癌細(xì)胞生長(zhǎng)及細(xì)胞周期相關(guān)抑癌基因表達(dá)的影響

王霞 王暉 蔣楠 梁三紅 呂文 張嘯 張?bào)泺P

目的觀察細(xì)胞外信號(hào)調(diào)節(jié)激酶信號(hào)通路(MEK)抑制劑對(duì)人胰腺癌細(xì)胞生長(zhǎng)及細(xì)胞周期相關(guān)的抑癌基因表達(dá)的影響。方法培養(yǎng)人胰腺癌細(xì)胞系CFPAC1、PANC1和MiaPaCa2，應(yīng)用50 μmol/L的MEK抑制劑PD98059處理細(xì)胞24 h，四甲基偶氮唑藍(lán)(MTT)法檢測(cè)細(xì)胞增殖，流式細(xì)胞儀分析細(xì)胞周期，實(shí)時(shí)定量PCR法檢測(cè)p16INK4a、p21WAF1和p27KIP1mRNA的表達(dá)，蛋白質(zhì)印跡法檢測(cè)DNA甲基化酶(Dnmt)1、3a和3b表達(dá)，甲基化特異性PCR(MSP)分析p16INK4a基因啟動(dòng)子甲基化狀況。結(jié)果PD98059處理24 h 后，CFPAC1、PANC1和MiaPaCa2細(xì)胞的增殖抑制率分別為69%、78%和45%； G0/G1期細(xì)胞比例分別從(68.21±0.73)%、(56.54±0.68)%、(54.89±0.79)%增加到(80.37±0.65)%、(72.05±0.52)%、(79.21±0.93)%(P值均<0.05)；S期和G2/M期細(xì)胞比例相應(yīng)減少。PD98059處理后，CFPAC1、PANC1細(xì)胞p27KIP1、p21WAF1和p16INK4amRNA表達(dá)增加， Dnmt1和Dnmt3b蛋白表達(dá)減少；p16INK4a啟動(dòng)子甲基化狀態(tài)被去除。而MiaPaCa2細(xì)胞僅p27KIP1mRNA表達(dá)增加；p21WAF1、p16INK4amRNA和Dnmt表達(dá)均無(wú)明顯變化。結(jié)論MEK通路抑制劑可能通過(guò)下調(diào)DNA甲基化酶、上調(diào)細(xì)胞周期相關(guān)抑癌基因表達(dá)而抑制胰腺癌細(xì)胞周期進(jìn)展和細(xì)胞增殖。

胰腺； 腫瘤細(xì)胞系； MEK抑制劑； 細(xì)胞周期； DNA甲基化酶； 抑癌基因

胰腺癌中RAS基因突變率高達(dá)70%～90%，可引起細(xì)胞外信號(hào)調(diào)節(jié)激酶信號(hào)通路(MEK)轉(zhuǎn)導(dǎo)異常，癌基因激活，抑癌基因失活。因此抑制MEK信號(hào)通路是抗腫瘤治療的潛在靶點(diǎn)[1-2]。本實(shí)驗(yàn)應(yīng)用MEK抑制劑PD98059干預(yù)人胰腺癌細(xì)胞株，觀察其對(duì)細(xì)胞增殖、細(xì)胞周期及相關(guān)抑癌基因表達(dá)的影響，探討其分子機(jī)制。

材料與方法

一、細(xì)胞生長(zhǎng)及增殖抑制率檢測(cè)

人胰腺癌細(xì)胞系CFPAC1、PANC1和MiaPaCa2均購(gòu)自中科院上海細(xì)胞生物學(xué)研究所。收集對(duì)數(shù)生長(zhǎng)期細(xì)胞，制備成5×104/ml的單細(xì)胞懸液，以1×105個(gè)細(xì)胞/孔接種于6孔板，分為2組，分別加入含終濃度為50 μmol/L的MEK抑制劑PD98059(美國(guó)Sigma公司，藥物組)和700 nmol/L DMSO(對(duì)照組)的培養(yǎng)液，培養(yǎng)后第2、4、6天隨機(jī)取3孔采用血細(xì)胞計(jì)數(shù)器計(jì)數(shù)細(xì)胞，繪制生長(zhǎng)曲線。另取5×104/ml的單細(xì)胞懸液接種于96孔培養(yǎng)板，100 μl/孔，給予PD98059或DMSO培養(yǎng)24 h后各孔加入濃度為5 mg/ml的MTT 10 μl，37℃孵育4 h，再加入100 μl DMSO，酶標(biāo)儀測(cè)定各孔570 nm處吸光度(A)值。細(xì)胞生長(zhǎng)抑制率(%)=(1-藥物組A570值/對(duì)照組A570值)×100%。每組5個(gè)復(fù)孔，實(shí)驗(yàn)重復(fù)3次。

二、細(xì)胞周期檢測(cè)

取對(duì)數(shù)生長(zhǎng)期細(xì)胞，應(yīng)用無(wú)血清RPMI 1640培養(yǎng)基培養(yǎng)16～24 h以同步化。分別用PD98059或DMSO干預(yù)24 h，收集各組細(xì)胞，制成1×106/ml的單細(xì)胞懸液。取2 ml細(xì)胞懸液用70%冷乙醇固定。去乙醇，加入含1% RNase的Tris-HCl緩沖液孵育10 min，用碘化丙啶染色，流式細(xì)胞儀檢測(cè)細(xì)胞周期。實(shí)驗(yàn)重復(fù)3次。

三、p16INK4a、p21WAF1和p27KIP1mRNA檢測(cè)

取干預(yù)24 h的各組細(xì)胞， 應(yīng)用Trizol法提取細(xì)胞總RNA。先逆轉(zhuǎn)錄成cDNA，然后行實(shí)時(shí)熒光定量PCR檢測(cè)p21WAF1、p16INK4a和p27KIP1mRNA表達(dá)。各引物和探針序列以及基因登序號(hào)同參考文獻(xiàn)[3]。探針和引物由上海基康生物公司合成。反應(yīng)條件：93℃ 15 s，60℃ 1 min，40個(gè)循環(huán)。每個(gè)樣品設(shè)3個(gè)復(fù)孔。通過(guò)PCR儀自帶軟件獲取Ct值。以β-actin作為內(nèi)參照，對(duì)照組細(xì)胞Ct值為基準(zhǔn)，計(jì)算PD98059組細(xì)胞mRNA的表達(dá)倍數(shù)(N)。N=2-(藥物△Ct-對(duì)照△Ct)，藥物△Ct-對(duì)照△Ct=(藥物Ct-β-actin Ct)-(對(duì)照Ct-β-actin Ct)。N≥3為表達(dá)增強(qiáng)，N≤0.33為表達(dá)減弱[4]。

四、DNA甲基化酶(Dnmt)檢測(cè)

將各組細(xì)胞置裂解緩沖液[20 mmol/L Tris-HCl(pH7.7)、250 mmol/L NaCl、2 mmol/L EDTA、0.5%NP-40、10%甘油、20 mmol/L β-甘油磷酸、1 mmol/L Na3VO4]裂解30 min，離心后取上清。取100 μg 蛋白行蛋白質(zhì)印跡術(shù)，以GAPDH為內(nèi)參照?？笵nmt1、3a和3b抗體(BD公司和Sant Crue公司)1∶500稀釋?zhuān)?HRP標(biāo)記二抗1∶1000稀釋?zhuān)詈驟CL發(fā)光。每組實(shí)驗(yàn)重復(fù)3 次。

五、DNA甲基化狀態(tài)分析

取各組細(xì)胞DNA，按照Gysin等[5]方法以亞硫酸氫鈉處理DNA，采用甲基化特異性PCR分析p16INK4a基因啟動(dòng)子區(qū)甲基化狀態(tài)。引物序列及反應(yīng)條件參考文獻(xiàn)[6]，由上海生工公司合成。

六、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、胰腺癌細(xì)胞生長(zhǎng)、增殖和細(xì)胞周期的變化

PD98059可明顯抑制人胰腺癌細(xì)胞系CFPAC1、PANC1和MiaPaCa2細(xì)胞的生長(zhǎng)(圖1)。藥物干預(yù)24 h后，CFPAC1、PANC1和MiaPaCa2細(xì)胞的增殖抑制率分別為69%、78%和45%。3株細(xì)胞系的G0/G1期細(xì)胞比例均明顯升高，而S期細(xì)胞數(shù)比例降低(表1)。

圖1 PD98059對(duì)人胰腺癌細(xì)胞生長(zhǎng)抑制作用

細(xì)胞系PD98059干預(yù)組G0/G1SG2/MDMSO對(duì)照組G0/G1SG2/MCFPAC180.37±0.65a6.92±0.14a12.54±0.2368.21±0.7313.46±0.3118.52±0.33PANC172.05±0.52a5.13±0.09a24.82±0.3956.54±0.6814.31±0.2929.15±0.41MiaPaCa279.21±0.93a7.35±0.32a13.45±0.4154.89±0.7925.13±0.1711.03±0.26

注：與對(duì)照組比較,aP<0.05

二、細(xì)胞周期相關(guān)抑癌基因的mRNA表達(dá)

PD98059干預(yù)后， CFPAC1、PANC1細(xì)胞p27KIP1、p21WAF1、p16INK4amRNA的表達(dá)均增強(qiáng)；MiaPaCa2細(xì)胞僅p27KIP1mRNA表達(dá)增強(qiáng)，而p21WAF1和p16INK4amRNA表達(dá)無(wú)明顯變化(表2)。

表2 PD98059干預(yù)后抑癌基因mRNA表達(dá)的變化

三、抑癌基因甲基化狀態(tài)和Dnmt蛋白表達(dá)的變化

CFPAC1和PANC1細(xì)胞p16INK4a基因啟動(dòng)子呈高甲基化狀態(tài)(圖2)，PD98059干預(yù)后呈去甲基化狀態(tài)；Dnmt1和Dnmt3b蛋白表達(dá)減少。MiaPaCa2細(xì)胞Dnmt表達(dá)無(wú)明顯變化(圖3)。

圖2 p16INK4a基因啟動(dòng)子甲基化狀態(tài)

圖3 3株胰腺癌細(xì)胞Dnmt蛋白表達(dá)的變化

討 論

胰腺癌中廣泛存在RAS和(或)RAF基因的突變，導(dǎo)致MEK信號(hào)通路的異常激活、細(xì)胞惡性轉(zhuǎn)化和增殖過(guò)度，CpG島甲基化異常[4-5]。MEK抑制劑PD98059是非底物依賴(lài)性競(jìng)爭(zhēng)的蛋白激酶抑制物，對(duì)ATP無(wú)影響，具有抗腫瘤作用[3]，但國(guó)內(nèi)關(guān)于此類(lèi)藥物對(duì)人胰腺癌的作用報(bào)道較少。本實(shí)驗(yàn)結(jié)果顯示，PD98059可抑制人胰腺癌細(xì)胞系CFPAC1、PANC1和MiaPaCa2的生長(zhǎng)，阻滯細(xì)胞周期從G0/G1期向S期轉(zhuǎn)化，并上調(diào)細(xì)胞周期相關(guān)基因p27KIP1、p21WAF1、p16INK4amRNA的表達(dá)，降低DNA甲基化酶活性，其中p16INK4a基因的恢復(fù)表達(dá)與DNA去甲基化有關(guān)。與Gysin等[5]應(yīng)用MEK抑制劑U0216阻滯胰腺癌細(xì)胞于G0/G1期、上調(diào)p27KIP1mRNA 表達(dá)的結(jié)果一致。

抑癌基因的失活與基因突變及DNA甲基化異常密切相關(guān)。隨著表觀遺傳學(xué)的深入研究，發(fā)現(xiàn)人類(lèi)多種腫瘤存在抑癌基因CpG島甲基化異常，其中p16INK4a、cyclin D2、RASSF1A、pp ENK等基因的高甲基化在胰腺癌發(fā)展中起重要作用[5,7]。最近研究[8-10]表明，持續(xù)活化MEK信號(hào)通路介導(dǎo)Par-4、p16INK4a和p21WAF1等基因啟動(dòng)子區(qū)高甲基化與抑制其轉(zhuǎn)錄有關(guān)。本結(jié)果顯示，p16INK4a在CFPAC1、PANC1細(xì)胞系呈高甲基化狀態(tài)，PD98059干預(yù)后p16INK4a基因呈去甲基化狀態(tài)，同時(shí)Dnmt的活性顯著減低，表明MEK抑制劑能降低RAS活化的RAF/MEK/ERK通路活性，下調(diào)Dnmt表達(dá)，導(dǎo)致抑癌基因的去甲基化。

但是，本實(shí)驗(yàn)在3株細(xì)胞系中均未擴(kuò)增出p21WAF1和p27KIP1甲基化條帶，在MiaPaCa2細(xì)胞亦未擴(kuò)增出p16INK4a甲基化條帶，提示PD98059上調(diào)胰腺癌細(xì)胞系p21WAF1和p27KIP1mRNA的表達(dá)與DNA去甲基化無(wú)明顯關(guān)系，且p16INK4a基因啟動(dòng)子區(qū)甲基化亦并非存在于所有的胰腺癌細(xì)胞，這一現(xiàn)象有待于進(jìn)一步探討。

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2010-08-27)

(本文編輯：呂芳萍)

EffectsofMEKsignalinginhibitoronthegrowthofhumanpacreaticcancercelllinesandtheexpressionofcellcycleassociatedgenes

WANGXia,WANGHui,JIANGNan,LIANGSan-hong,LüWen,ZHANGXiao,ZHANGXiao-feng.

FirstPeople′sHospitalofHangzhou,Hangzhou310006,China

WANGXia,Email:wang78xia@hotmail.com

ObjectiveTo examine the effects of the MEK inhibitor on human pancreatic cancer cells, and to explore the molecular mechanisms.MethodsHuman pancreatic cancer cell lines CFPANC1, PANC1 and MiaPaCa2 were treated with MEK inhibitor PD98059 or DMSO, the sensitivity was analyzed by an MTT assay, and cell cycle distribution was evaluated by flow cytometry(FCM), The transcriptional level and protein expression of tumor suppressor genes were detected by real-time RT-PCR and western blot respectively. DNA methyltransferase (Dnmt)1, 3a and 3b were also assayed by western blot，The methylation status of the promoter of the p16INK4A gene was assayed by methylation-specific PCR (MSP).ResultsPD98059 inhibited to various degrees the growth of three pancreatic cancer cell lines, accompanied by G0-G1 cell cycle arrest. PD98059 up-regulated the expression of p16INK4a, p21WAF1, p27KIP1mRNA, demethylated the hypermethylation status of p16INK4agene promoter, and decreased Dnmt1 and Dnmt3b in CFPANC1 and PANC1 cell lines. PD98059 only increased the expression of p27KIP1, while the changes of p16INK4a, p21WAF1and Dnmt were less marked in MiaPaCa2 cell line.ConclusionsMEK inhibitor PD98059 down-regulate the activation of Dnmt and up-regulate tumor supress genes, along with the inhibition of cell proliferation and cell cycle progression.

Pancreas; Tunor cell line; MEK inhibitor; Cell cycle; DNA methyltransferase; Tumor suppressor genes

10.3760/cma.j.issn.1674-1935.2011.04.009

國(guó)家自然科學(xué)青年基金(81001078)

310006 杭州，杭州市第一人民醫(yī)院消化內(nèi)科

王霞，Email：wang78xia@hotmail.com