張大田,石建國(guó),張 強(qiáng),劉 巖

(遼寧醫(yī)學(xué)院附屬第一醫(yī)院泌尿外科,遼寧錦州 121001)

sirtinol對(duì)前列腺癌DU145細(xì)胞周期的影響及其機(jī)制

張大田,石建國(guó),張 強(qiáng),劉 巖

(遼寧醫(yī)學(xué)院附屬第一醫(yī)院泌尿外科,遼寧錦州 121001)

目的:觀察沉默信息調(diào)節(jié)因子1(SIRT1)抑制劑sirtinol對(duì)前列腺癌DU145細(xì)胞生長(zhǎng)增殖、細(xì)胞周期進(jìn)程和細(xì)胞周期正性調(diào)節(jié)蛋白Cyclin D1、CDK4及p Rb表達(dá)的影響,探討SIRT1在前列腺癌發(fā)生中的可能作用機(jī)制。方法:取處于對(duì)數(shù)生長(zhǎng)期DU145細(xì)胞隨機(jī)分為對(duì)照組(DMSO組)和sirtinol組(終濃度分別為10、25和50μmol·L－1),MTT法測(cè)定各組DU145細(xì)胞生長(zhǎng)抑制率,RT-PCR和Western blotting法檢測(cè)DU145細(xì)胞中SIRT1 m RNA和蛋白表達(dá)水平,流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期進(jìn)程,Western blotting法檢測(cè)DU145細(xì)胞中細(xì)胞周期蛋白Cyclin D1、CDK4和p Rb的表達(dá)水平。結(jié)果:與對(duì)照組比較,各濃度sirtinol組DU145細(xì)胞生長(zhǎng)抑制率明顯升高(P＜0.01),G1期細(xì)胞明顯增多(P＜0.01),且呈濃度依賴性。與對(duì)照組比較,各濃度sirtinol組DU145細(xì)胞中SIRT1 m RNA和蛋白表達(dá)水平明顯降低(P＜0.01),Cyclin D1和p Rb蛋白表達(dá)水平降低(P＜0.01),CDK4蛋白表達(dá)無(wú)明顯變化(P＞0.05)。結(jié)論:下調(diào)SIRT1表達(dá)可以抑制前列腺癌DU145細(xì)胞的增殖和阻滯細(xì)胞周期進(jìn)程,其機(jī)制可能與改變細(xì)胞周期蛋白CyclinD1和p Rb的表達(dá)有關(guān)。

sirtinol;沉默信息調(diào)節(jié)因子1;前列腺腫瘤;DU145細(xì)胞

隨著人口老齡化進(jìn)程加快,我國(guó)前列腺癌發(fā)病率逐年增加,但前列腺癌發(fā)生和發(fā)展的分子機(jī)制尚不清楚,年齡、種族、飲食和雄激素的分泌及代謝異常等均是腫瘤發(fā)生的危險(xiǎn)因素。前列腺癌的發(fā)病與年齡有密切關(guān)聯(lián),隨著人類(lèi)預(yù)期生存年齡的增加,前列腺癌的發(fā)病率明顯增加[1-2],因此尋找新的分子生物學(xué)標(biāo)記物和信號(hào)通路,明確前列腺癌的發(fā)生與年齡的關(guān)系,可為前列腺癌的診斷和治療提供新的理論基礎(chǔ)和實(shí)驗(yàn)依據(jù)。沉默信息調(diào)節(jié)因子1 (silent information regulator 1,SIRT1)屬于sirtuin家族,與酵母SIR2高度同源,為煙酰胺腺嘌呤二核苷酸(NAD＋)依賴性的組蛋白去乙?；?。SIRT1在DNA修復(fù)、細(xì)胞分化、炎癥和細(xì)胞凋亡等方面發(fā)揮重要作用[3-5]。有研究[6-8]顯示:腫瘤細(xì)胞中內(nèi)源性SIRT1的表達(dá)高于正常細(xì)胞,推測(cè)SIRT1作為癌基因參與腫瘤的發(fā)生和發(fā)展。而另一方面,最近有研究[9]報(bào)道:SIRT1在胃癌細(xì)胞中表達(dá)下調(diào),并通過(guò)NF-κB/Cyclin D1信號(hào)通路導(dǎo)致細(xì)胞G1期阻滯,從而提示SIRT1在胃癌的發(fā)生發(fā)展中可能發(fā)揮抑癌作用。國(guó)內(nèi)外對(duì)SIRT1在前列腺癌中的具體作用報(bào)道[10-11]也不一致。本研究選用不同濃度SIRT1抑制劑sirtinol作用于前列腺癌DU145細(xì)胞株,旨在探討下調(diào)SIRT1表達(dá)對(duì)人前列腺癌細(xì)胞株細(xì)胞周期阻滯和細(xì)胞周期蛋白表達(dá)的影響,初步闡明前列腺癌發(fā)生發(fā)展的可能機(jī)制, 為SIRT1作為前列腺癌的治療靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 主要試劑與儀器sirtinol(S8825,HPLC≥98%,美國(guó)Sigma公司),DMEM培養(yǎng)液(美國(guó)Corning公司),SIRT1(鼠單克隆抗體,美國(guó)Merck Millipore公司),Cyclin D1(兔單克隆抗體,美國(guó)CST公司),CDK4(兔多克隆抗體,美國(guó)Abcam公司),p Rb抗體(山羊多克隆抗體)和β-actin抗體(兔多克隆抗體)(美國(guó)Santacruz公司),HRP標(biāo)記的山羊抗兔、抗鼠IgG(北京中杉金橋生物技術(shù)有限公司),ECL化學(xué)發(fā)光試劑盒(美國(guó)Piece Biotechnology公司),MTT、Western blotting細(xì)胞裂解液和BCA蛋白濃度測(cè)定試劑盒(北京碧云天生物技術(shù)公司),RNA PCR Kit (AMV)Ver 3.0試劑盒(日本Takara公司)。BB16UV CO2培養(yǎng)箱(德國(guó)Heraeus公司),PCR擴(kuò)增儀(美國(guó)Eppendorf公司),水浴式電轉(zhuǎn)印槽DYY-Ⅲ40B型(北京六一儀器廠),凝膠自動(dòng)成像儀GDS8000和電泳儀(美國(guó)Bio-Rad公司), Sunrise自動(dòng)酶標(biāo)檢測(cè)儀(美國(guó)Tecan公司),流式細(xì)胞儀(美國(guó)貝克曼庫(kù)爾特有限公司)。

1.2 細(xì)胞培養(yǎng)及實(shí)驗(yàn)分組人前列腺癌DU145細(xì)胞購(gòu)自上海生命科學(xué)院細(xì)胞和生物化學(xué)研究所,培養(yǎng)于含10%胎牛血清、100 U·m L－1青霉素和100 mg·L－1鏈霉素的DMEM培養(yǎng)基中,在飽和濕度、37℃、5%CO2孵箱中培養(yǎng),當(dāng)細(xì)胞匯合至培養(yǎng)瓶底面積約80%時(shí)以0.25%胰蛋白酶消化傳代。當(dāng)細(xì)胞處于對(duì)數(shù)生長(zhǎng)期時(shí),以1×105m L－1濃度接種于25 cm2的培養(yǎng)瓶或96孔培養(yǎng)板中。24或48 h待細(xì)胞貼壁后給藥,sirtinol組終濃度分別為10、25和50μmol·L－1,對(duì)照組加入5μL DMSO(DMSO體積分?jǐn)?shù)小于0.1%,該濃度對(duì)細(xì)胞生長(zhǎng)無(wú)影響)。

1.3 MTT法檢測(cè)細(xì)胞生長(zhǎng)抑制率取對(duì)數(shù)生長(zhǎng)期DU145細(xì)胞,以1×105m L－1接種于96孔板上,37℃培養(yǎng)箱中培養(yǎng)24 h后,棄上清液,分別加入不同濃度的sirtinol,各濃度組6個(gè)復(fù)孔。繼續(xù)培養(yǎng)24或48 h,加入20μL MTT(5 g·L－1)孵育4 h。棄去培養(yǎng)液,加入150μL二甲基亞砜,振蕩15 min,使沉淀物充分溶解。于酶標(biāo)儀490 nm處測(cè)吸光度(A)值,計(jì)算細(xì)胞生長(zhǎng)抑制率。細(xì)胞生長(zhǎng)抑制率＝(1－加藥組A值/對(duì)照組A值)× 100%。

1.4 流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期進(jìn)程將DU145細(xì)胞株接種于25 cm2培養(yǎng)瓶中,待細(xì)胞貼壁后加入不同濃度的sirtinol分別作用48 h,以胰酶-EDTA消化,采用5 m L培養(yǎng)液重懸細(xì)胞至單細(xì)胞懸液, 1 000 r·min－1離心5 min,用4℃預(yù)冷的PBS吹打成單細(xì)胞懸液,離心棄上清。加入5 m L冷PBS再離心1次,棄去上清。在避光條件下加入1 m L PI染液重懸細(xì)胞至單細(xì)胞懸液,室溫孵育30 min,采用流式細(xì)胞儀檢測(cè),利用CellQuest分析軟件分析各組G1和S期DU145細(xì)胞所占百分率。

1.5 RT-PCR方法檢測(cè)SIRT1 m RNA表達(dá)水平采用Trizol一步法提取經(jīng)不同濃度sirtinol處理48 h的DU145細(xì)胞總RNA,1%瓊脂糖凝膠電泳檢測(cè)RNA的完整性,采用核酸蛋白測(cè)定儀測(cè)定RNA濃度,當(dāng)A260/A280比值達(dá)1.9～2.0時(shí)可用于逆轉(zhuǎn)錄反應(yīng)。用RNA PCR(AMV)Ver 3.0試劑盒,按說(shuō)明書(shū)操作。反轉(zhuǎn)錄條件:42℃、30 min, 99℃、5 min,5℃、5 min。PCR引物序列: SIRT1,5′-TCAGTGTCATGGTTCCTTTGC-3′, 5′-AATCTGCTCCTTTGCCACTCT-3′,820 bp; GAPDH,5′-CGGAGTCAACGGATTTGGTCGTAT-3′,5′-CGGAGTCAACGGATTTGGTCGTAT-3′,306 bp;PCR反應(yīng)條件:94℃預(yù)變性3 min;94℃、30 s,57℃、30 s,72℃、1 min, 30個(gè)循環(huán);72℃終延伸5 min。取PCR產(chǎn)物10μL進(jìn)行瓊脂糖凝膠電泳并拍照,測(cè)量A值,m RNA表達(dá)水平以每一樣品A值與GAPDH A值比值表示。

1.6 Western blotting法檢測(cè)SIRT1、Cyclin D1、CDK4和p Rb的蛋白表達(dá)蛋白裂解液提取加藥處理48 h后的各組細(xì)胞總蛋白,采用BCA法測(cè)定蛋白濃度,－20℃或－80℃保存?zhèn)溆?。電泳?加入5×SDS樣品緩沖液,100℃煮沸5 min,離心后上樣,10%SDS-PAGE電泳分離,然后將蛋白轉(zhuǎn)至硝酸纖維素膜上,之后用含5%BSA的TBS (p H 7.4)將濾膜于室溫?fù)u動(dòng)溫育2 h進(jìn)行封閉,封閉后的濾膜再分別與SIRT1(稀釋比1∶1 000), Cyclin D1、CDK4、p Rb抗體(稀釋比1∶200)及β-actin(稀釋比1∶1 000),4℃溫育過(guò)夜。經(jīng)TTBS洗滌后,HRP偶聯(lián)的IgG作為二抗(稀釋比1∶5 000)室溫溫育2 h,洗膜后,采用ECL發(fā)光法顯色。采用Image J軟件分析密度,計(jì)算A值。蛋白表達(dá)水平以蛋白A值與β-actin A值比值表示。

1.7 統(tǒng)計(jì)學(xué)分析應(yīng)用GraphPad Prism 5軟件進(jìn)行統(tǒng)計(jì)學(xué)分析。各組細(xì)胞生長(zhǎng)抑制率、G1和S期細(xì)胞百分率及m RNA和蛋白表達(dá)水平均以±s表示,兩兩比較采用t檢驗(yàn)。

2 結(jié)果

2.1 各組DU145細(xì)胞生長(zhǎng)抑制率與對(duì)照組比較,各濃度(10、25和50μmol·L－1)sirtinol組DU145細(xì)胞生長(zhǎng)抑制率明顯升高(P＜0.01),且隨sirtinol濃度的增加而升高,呈明顯的濃度-效應(yīng)關(guān)系。見(jiàn)表1。

2.2 各組DU145細(xì)胞的細(xì)胞周期與對(duì)照組比較,不同濃度sirtinol組S期DU145細(xì)胞百分率降低(P＜0.01),隨著藥物濃度的增加,G1期細(xì)胞百分率升高(P＜0.01),且呈濃度效應(yīng)關(guān)系。見(jiàn)表2。

表1 各組DU145細(xì)胞生長(zhǎng)抑制率Tab.1 Inhibitory rates of growth of DU145 cells in various groups(n＝6,±s,η/%)

表1 各組DU145細(xì)胞生長(zhǎng)抑制率Tab.1 Inhibitory rates of growth of DU145 cells in various groups(n＝6,±s,η/%)

?P＜0.01 compared with control group.

Group Inhiibitory rate of growth (t/h) 24 48 Control 4.62±1.21 4.83±1.24 Sirtinol(μmol·L－1) 10 12.61±2.52?14.70±2.71?25 20.64±3.63?22.93±3.98?50 41.51±5.76?49.33±5.86?

表2 各組DU145細(xì)胞各周期細(xì)胞百分率Tab.2 Percentages of DU145 cells at each cell cycle in various groups(n＝6,±s,η/%)

表2 各組DU145細(xì)胞各周期細(xì)胞百分率Tab.2 Percentages of DU145 cells at each cell cycle in various groups(n＝6,±s,η/%)

?P＜0.01 compared with control group.

Group Percentage of DU145 cells G1S Control 35.59±1.83 43.10±1.42 Sirtinol(μmol·L－1) 10 40.30±1.96?38.31±2.17?25 53.42±3.78?33.78±2.91?50 65.38±3.78?22.23±2.52?

2.3 各組DU145細(xì)胞中SIRT1 mRNA和蛋白表達(dá)水平與對(duì)照組比較,不同濃度sirtinol組DU145細(xì)胞SIRT1 m RNA和蛋白表達(dá)水平明顯降低(P＜0.01),且隨sirtinol濃度增加其表達(dá)水平逐漸降低。見(jiàn)表3。各組細(xì)胞中SIRT1 m RNA和蛋白表達(dá)電泳圖見(jiàn)圖1。

表3 各組DU145細(xì)胞中SIRT1 m RNA和蛋白表達(dá)水平Tab.3 Expression levels of SIRT1 mRNA and protein in DU145 cells in various groups(n＝6,±s)

表3 各組DU145細(xì)胞中SIRT1 m RNA和蛋白表達(dá)水平Tab.3 Expression levels of SIRT1 mRNA and protein in DU145 cells in various groups(n＝6,±s)

?P＜0.01 compared with control group.

Group SIRT1/GAPDH SIRT1/β-actin Control 0.028±0.006 0.085±0.009 Sirtinol(μmol·L－1) 10 0.019±0.005?0.073±0.007?25 0.017±0.006?0.038±0.005?50 0.008±0.003?0.012±0.005?

圖1 各組DU145細(xì)胞中SIRT1 m RNA(A)和蛋白(B)表達(dá)電泳圖Fig.1 Electrophoregram of expressions of SIRT1 mRNA (A)and protein(B)in DU145 cells in various groupsLane 1:Control group;Lane 2－4:10,25,and 50μmol·L－1sirtinol groups.

2.4 各組DU145細(xì)胞中Cyclin D1、CDK4和p Rb蛋白表達(dá)水平與對(duì)照組比較,不同濃度sirtinol組DU145細(xì)胞中Cyclin D1和p Rb蛋白表達(dá)水平明顯降低(P＜0.01),且隨sirtinol濃度增加其表達(dá)水平逐漸降低(P＜0.01),但CDK4蛋白表達(dá)水平無(wú)明顯變化(P＞0.05)。見(jiàn)圖2和表4。

表4 各組DU145細(xì)胞中Cyclin D1、CDK4和pRb蛋白表達(dá)水平Tab.4 Expression levels of Cyclin D1,CDK4,and pRb proteins in DU145 cells in various groups(±s)

表4 各組DU145細(xì)胞中Cyclin D1、CDK4和pRb蛋白表達(dá)水平Tab.4 Expression levels of Cyclin D1,CDK4,and pRb proteins in DU145 cells in various groups(±s)

?P＜0.01 compared with control group.

Group Cyclin D1/β-actin CDK4/β-actin p Rb/β-actin Control 1.191±0.023 0.371±0.021 1.082±0.072 Sirtinol(μmol·L－1) 10 0.053±0.011?0.375±0.033 0.022±0.008?25 0.040±0.009?0.367±0.018 0.018±0.006?50 0.023±0.007?0.379±0.041 0.013±0.005?

3 討論

腫瘤的發(fā)生發(fā)展是多因素、多階段、多步驟和逐漸演變的過(guò)程,許多因素調(diào)控眾多的分子表達(dá)變化,形成了一個(gè)復(fù)雜的信號(hào)通路。有研究[5]表明: SIRT1在這一進(jìn)程中扮演了重要角色。為了研究SIRT1在前列腺癌發(fā)生發(fā)展中的作用,本文作者首先使用不同濃度的SIRT1抑制劑sirtinol分別作用于前列腺癌DU145細(xì)胞24和48 h,MTT測(cè)定結(jié)果表明:sirtinol(10～50μmol·L－1)能抑制DU145細(xì)胞的增生,并且具有濃度和時(shí)間依賴性,說(shuō)明sirtinol具有抑制細(xì)胞增殖作用,與Lee等[12]的報(bào)道結(jié)果一致。同時(shí)本研究結(jié)果顯示:不同濃度sirtinol處理DU145細(xì)胞后,隨著sirtinol濃度的增加,SIRT1 m RNA和蛋白表達(dá)水平逐漸降低,說(shuō)明sirtinol可以改變內(nèi)源性SIRT1的表達(dá)和活性。細(xì)胞周期進(jìn)展是反映細(xì)胞增殖速度的一個(gè)重要指標(biāo)。為了研究?jī)?nèi)源性SIRT1對(duì)細(xì)胞周期的影響,本文作者利用不同濃度sirtinol處理DU145細(xì)胞48 h后改變細(xì)胞內(nèi)源性SIRT1的活性,并利用流式細(xì)胞術(shù)分析細(xì)胞周期,結(jié)果表明:與對(duì)照組比較,sirtinol處理后G1期細(xì)胞百分率增加,細(xì)胞周期阻滯在G1期。為了探討SIRT1影響細(xì)胞周期進(jìn)展可能的分子機(jī)制,本文作者檢測(cè)了細(xì)胞周期正性關(guān)鍵調(diào)控分子Cyclin D1、CDK4和p Rb的表達(dá)變化。細(xì)胞周期蛋白和細(xì)胞周期蛋白依賴性激酶(CDK)是調(diào)控細(xì)胞進(jìn)展的2類(lèi)關(guān)鍵分子[13], Cyclin D1是第一個(gè)被發(fā)現(xiàn)的細(xì)胞周期調(diào)控分子,在外界信號(hào)刺激下,Cyclin D1與CDK4結(jié)合形成具有活性的異源二聚體,使視網(wǎng)膜母細(xì)胞瘤蛋白(retinoblastoma,Rb)磷酸化而失活,推動(dòng)細(xì)胞周期由G1期向S期進(jìn)展[14-15]。Western blotting法檢測(cè)結(jié)果提示:下調(diào)SIRT1表達(dá)可以抑制Cyclin D1和p Rb表達(dá),但不引起CDK4的表達(dá)變化。

綜上所述,本研究證實(shí)下調(diào)SIRT1表達(dá)和活性可以抑制前列腺癌細(xì)胞的體外增殖和阻滯細(xì)胞周期進(jìn)程,其機(jī)制可能與改變細(xì)胞周期蛋白Cyclin D1和p Rb的表達(dá)有關(guān)。本研究結(jié)果提示SIRT1在前列腺癌的發(fā)生發(fā)展中可能扮演癌基因的角色。有關(guān)SIRT1在前列腺癌發(fā)生發(fā)展中的分子途徑及信號(hào)通路尚需深入研究。

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Influence of sirtinol in cell cycle of prostate cancer DU145 cells and its mechanism

ZHANG Da-tian,SHI Jian-guo,ZHANG Qiang,LIU Yan
(Department of Urinary Surgery,First Affiliated Hospital,Liaoning Medical University,Jinzhou 121001,China)

ObjectiveTo observe the influence of sirtinol,a silent information regulator 1(SIRT1)inhibitor,in the cell proliferation,cell cycle progression and the expression levels of positive regulator proteins of the cell cycle including Cyclin D1,CDK4 and pRb in prostate cancer DU145 cells,and to explore the possible mechanism of SIRT1 in occurrence of prostagte cancer.MethodsThe DU145 cells at logarithmic growth phase were cultured in vitro and divided into control group(DMSO)and different doses(10,25,50μmol·L－1)of sirtinol groups.The inhibitory rate of growth of DU145 cells was detected with MTT method,the SIRT1 m RNA and protein expression levels were determined by RT-PCR and Western blotting method,and the cell cycle was measured by flow cytometry.The Cyclin D1,CDK4 and p Rb protein expression levels were examined by Western blotting method.ResultsCompared with control group,the inhibitory rates of growth of the DU145 cells in different doses of sirtinol groups were increased markedly in a dose-dependent manner(P＜0.01),and the flow cytometry analysis result showed the DU145 cells at G1phase were increased(P＜0.01).Compared with control group,the expression levels of SIRT1 mRNA and protein in DU145 cells in different doses of sirtinol groups were decreased significantly (P＜0.01);the expression levels of Cyclin D1 and p Rb proteins were decreased(P＜0.01),whereas the expression levels of CDK4 had no change(P＞0.05).ConclusionSIRT1 inhibition by sirtinol can inhibit the cellgrowth of prostate cancer DU145 cells in a dose-dependent manner and arrest the cell cycle progression,and its mechanism may be related to decreasing the CyclinD1 and p Rb protein expressions.

sirtinol;silent information regulator 1;prostate neoplasms;DU145 cells

R737.25

A

2014-02-14

遼寧省科技廳科學(xué)計(jì)劃項(xiàng)目資助課題(2011225015)

張大田(1969－),男,吉林省通化市人,副主任醫(yī)師,醫(yī)學(xué)碩士,主要從事泌尿系統(tǒng)腫瘤的生物學(xué)研究。

劉 巖(Tel:0416-419763,E-mail:liuyanforest＠163.com)

1671-587Ⅹ(2014)05-0967-05

10.13481/j.1671-587x.20140512