劉 磊,方艷秋,齊亞靈,蘆小單,魏海峰,譚 巖

(1.吉林省人民醫(yī)院腫瘤生物治療中心,吉林長(zhǎng)春 130021; 2.佳木斯大學(xué)基礎(chǔ)醫(yī)學(xué)院組織學(xué)與胚胎學(xué)教研室,黑龍江佳木斯 154007)

PI3K特異性抑制劑LY294002增強(qiáng)rsTRAIL蛋白對(duì)A549細(xì)胞的殺傷作用

劉 磊1,方艷秋1,齊亞靈2,蘆小單1,魏海峰1,譚 巖1

(1.吉林省人民醫(yī)院腫瘤生物治療中心,吉林長(zhǎng)春 130021; 2.佳木斯大學(xué)基礎(chǔ)醫(yī)學(xué)院組織學(xué)與胚胎學(xué)教研室,黑龍江佳木斯 154007)

目的:探討磷脂酰肌醇3激酶(PI3K)參與非小細(xì)胞肺癌A549細(xì)胞株抵抗重組可溶性腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(rs TRAIL)蛋白誘導(dǎo)凋亡的機(jī)制,尋找增強(qiáng)rs TRAIL蛋白殺傷非小細(xì)胞肺癌細(xì)胞的新方法。方法:選取對(duì)數(shù)生長(zhǎng)期A549細(xì)胞,隨機(jī)分為rs TRAIL蛋白組和PI3K特異性抑制劑LY294002聯(lián)合rs TRAIL蛋白(LY294002＋rs TRAIL蛋白)組。MTT法檢測(cè)2組A549細(xì)胞生長(zhǎng)抑制率,流式細(xì)胞技術(shù)檢測(cè)2組A549細(xì)胞周期和細(xì)胞凋亡的變化,蛋白質(zhì)印跡法檢測(cè)2組A549細(xì)胞中p-Akt(Ser473)、c-FLIPL、Bcl-2和Bax蛋白表達(dá)水平。結(jié)果:與rs TRAIL蛋白組(5.16%±0.32%)比較,LY294002＋rs TRAIL蛋白組A549細(xì)胞生長(zhǎng)抑制率(74.6%±2.63%)明顯升高(P＜0.05)。與rs TRAIL蛋白組比較,LY294002＋rs TRAIL蛋白組處理2 h后G0/G1期A549細(xì)胞百分比增加(P＜0.05),S期細(xì)胞百分比降低(P＜0.05)。LY294002＋rs TRAIL蛋白組A549細(xì)胞凋亡率為(61.50±3.02)%,顯著高于rs TRAIL蛋白組(3.21%±0.96%)(P＜0.05)。蛋白質(zhì)印跡檢測(cè),與rs TRAIL蛋白組比較,LY294002＋rs TRAIL蛋白組A549細(xì)胞中p-Akt、c-FLIPL和Bcl-2蛋白表達(dá)水平降低(P＜0.05),Bax/Bcl-2比值升高(P＜0.05)。結(jié)論:LY294002能夠抑制PI3K活性,增強(qiáng)rs TRAIL蛋白對(duì)A549細(xì)胞的殺傷作用。

癌,非小細(xì)胞肺;A549細(xì)胞株;腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體;磷脂酰肌醇3激酶;LY294002

肺癌是目前世界上嚴(yán)重威脅人類健康與生命的惡性腫瘤,發(fā)病率呈明顯上升趨勢(shì),全球每年有超過(guò)百萬(wàn)人死于肺癌,其中85%死于非小細(xì)胞肺癌(non-small cell lung cancer,NSCLC)。研究肺癌的發(fā)生、發(fā)展以及轉(zhuǎn)移的分子機(jī)制,探索有效的靶向治療手段具有重要的臨床意義[1]。腫瘤壞死因子相關(guān)的凋亡誘導(dǎo)配體(TNF-related apoptosis inducing ligand,TRAIL)是一種天然的殺傷腫瘤細(xì)胞因子,被證實(shí)是唯一具有對(duì)正常組織細(xì)胞無(wú)毒性的抗腫瘤因子[2]。研究[3]顯示:TRAIL可以抑制體外培養(yǎng)的NSCLC細(xì)胞增殖和SCID小鼠體內(nèi)NSCLC細(xì)胞的生長(zhǎng)。本文作者[4-5]獲得的重組可溶性TRAIL(rs TRAIL)蛋白在抗NSCLC的應(yīng)用研究中展現(xiàn)出了良好的抗腫瘤活性,然而腫瘤細(xì)胞內(nèi)某些蛋白的異常表達(dá)和活化可以抑制TRAIL誘導(dǎo)的腫瘤細(xì)胞凋亡的發(fā)生,影響其在臨床研究中的廣泛應(yīng)用。磷脂酰肌醇3激酶/蛋白酶B(PI3K/Akt)信號(hào)轉(zhuǎn)導(dǎo)通路的活化在人類惡性腫瘤中非常普遍, PI3K/Akt調(diào)控失調(diào)參與NSCLC細(xì)胞存活、增殖、抗凋亡和血管生成的各個(gè)環(huán)節(jié),中斷PI3K/Akt信號(hào)轉(zhuǎn)導(dǎo)通路的轉(zhuǎn)導(dǎo)可以增強(qiáng)藥物對(duì)腫瘤細(xì)胞誘導(dǎo)凋亡的敏感性[6-7]。本研究選用PI3K特異性抑制劑LY294002對(duì)PI3K/Akt信號(hào)轉(zhuǎn)導(dǎo)進(jìn)行干預(yù),觀察參與TRAIL誘導(dǎo)腫瘤細(xì)胞凋亡過(guò)程的c-FLIPL、Bcl-2和Bax蛋白表達(dá)的變化及A549細(xì)胞凋亡和增殖情況,探討PI3K/Akt通路在NSCLC抵抗TRAIL耐藥中的作用。

1 材料與方法

1.1 細(xì)胞和主要試劑A549細(xì)胞由吉林省人民醫(yī)院中心實(shí)驗(yàn)室傳代保存。rs TRAIL蛋白由吉林大學(xué)第一醫(yī)院中心實(shí)驗(yàn)室表達(dá)純化[2]。IMDM培養(yǎng)基、胰蛋白酶(美國(guó)Gibco公司),小牛血清(杭州四季青生物材料公司),四甲基偶氮唑藍(lán)(MTT)(美國(guó)Sigma公司),LY294002(美國(guó)Calbiochem公司),抗p-Akt(Ser473)、抗Bcl-2、抗c-FLIPL、抗β-actin一抗和HRP標(biāo)記抗鼠IgG或抗兔IgG二抗均為美國(guó)Cell Signaling Technology產(chǎn)品,細(xì)胞周期和細(xì)胞凋亡檢測(cè)試劑盒購(gòu)于貝克曼庫(kù)爾特商貿(mào)有限公司。

1.2 MTT法檢測(cè)細(xì)胞生長(zhǎng)抑制率選取對(duì)數(shù)生長(zhǎng)期的A549細(xì)胞,調(diào)濃度為5×105m L－1,接種于96孔板,每孔100μL。細(xì)胞貼壁后分別采用rs TRAIL蛋白(100 mg·L－1)作用12 h和20μmol·L－1LY294002作用不同時(shí)間(2、4、8 和16 h),再應(yīng)用rs TRAIL蛋白(100 mg·L－1)作用12 h,每組設(shè)3個(gè)平行孔,每孔加入新配制的5 g·L－1MTT 20μL,37℃繼續(xù)孵育4 h,棄上清后加150μL DMSO溶解,混勻后在490 nm波長(zhǎng)處測(cè)定吸光度(A)值,計(jì)算細(xì)胞生長(zhǎng)抑制率。細(xì)胞生長(zhǎng)抑制率＝(1－實(shí)驗(yàn)組A值/對(duì)照組A值)× 100%。

1.3 流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期選取對(duì)數(shù)生長(zhǎng)期的A549細(xì)胞,調(diào)整濃度為5×105m L－1,接種于6孔板,每孔1 m L,待其生長(zhǎng)至80%匯合時(shí),采用20μmol·L－1LY294002作用1 h,收取單細(xì)胞懸液,用預(yù)冷的PBS洗滌2次,并用預(yù)冷的乙醇固定于4℃冰箱保存。PI染液(500μL/管),避光靜置30 min后采用流式細(xì)胞儀檢測(cè)細(xì)胞周期,分析各周期細(xì)胞百分率。

1.4 流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡選取對(duì)數(shù)生長(zhǎng)期的A549細(xì)胞,調(diào)整濃度為5×105m L－1,接種于6孔板,每孔1 m L,待其生長(zhǎng)至80%匯合時(shí),采用20μmol·L－1LY294002作用2 h,加入100μg·L－1的rs TRAIL蛋白處理12 h,收取3孔細(xì)胞。同時(shí)以rs TRAIL蛋白單獨(dú)處理組作為對(duì)照組。獲得的單細(xì)胞懸液用PBS洗滌2次, Annexin-Ⅴ/FITC與PI匹配標(biāo)記細(xì)胞,流式細(xì)胞術(shù)測(cè)定細(xì)胞凋亡率。

1.5 Western blotting法檢測(cè)p-Akt、c-FLIPL、Bcl-2和Bax蛋白表達(dá)水平選取對(duì)數(shù)生長(zhǎng)期的A549細(xì)胞,調(diào)整濃度為5×105m L－1,并接種于6孔板,每孔1 m L,待其生長(zhǎng)至80%匯合時(shí), 20μmol·L－1LY294002作用2 h,加入100μg·L－1rs TRAIL蛋白處理12 h,收取3孔細(xì)胞。同時(shí)以rs TRAIL蛋白單獨(dú)處理組做為對(duì)照。收取細(xì)胞并采用裂解液獲取總蛋白,測(cè)定各組細(xì)胞蛋白水平并將蛋白樣品濃度調(diào)整為40 g·L－1。10%SDS聚丙烯酰胺凝膠電泳、轉(zhuǎn)膜2 h,10%脫脂奶封閉作用1 h。棄去封閉液,加入用封閉液稀釋的一抗工作液(抗p-Akt、c-FLIPL、Bcl-2和Bax及抗β-actin抗體),4℃振搖孵育過(guò)夜。TBST洗膜3次,加入1∶3 000稀釋的相應(yīng)二抗,室溫反應(yīng)1 h;TBST洗膜3次,增強(qiáng)化學(xué)發(fā)光試劑顯色,采用BandScan 5.0分析軟件進(jìn)行灰度分析,以目的蛋白條帶灰度值與β-actin蛋白條帶灰度值的比值表示目的蛋白的相對(duì)表達(dá)水平。

1.6 統(tǒng)計(jì)學(xué)分析采用SPSS 10.0統(tǒng)計(jì)學(xué)軟件進(jìn)行統(tǒng)計(jì)處理。細(xì)胞生長(zhǎng)抑制率、各細(xì)胞周期A549細(xì)胞所占百分率、A549細(xì)胞凋亡率及p-Akt、c-FLIPL、Bcl-2和Bax蛋白表達(dá)水平以±s表示,組間比較采用t檢驗(yàn)。

2 結(jié)果

2.1 2組A549細(xì)胞的生長(zhǎng)抑制率100 mg·L－1rs TRAIL作用A549細(xì)胞12 h后,細(xì)胞生長(zhǎng)抑制率為(5.61±0.32)%;20μmol·L－1LY294002分別作用2、4、8和16 h后,再應(yīng)用100 mg·L－1rs TRAIL蛋白作用12 h,A549細(xì)胞的生長(zhǎng)抑制率分別為(74.6±2.63)%、(75.9±2.97)%、(76.7±2.84)%和(78.4±3.12)%;與rs TRAIL蛋白組比較,LY294002＋rs TRAIL蛋白組A549細(xì)胞的生長(zhǎng)抑制率明顯升高(P＜0.05)。

2.2 2組A549細(xì)胞的周期與rs TRAIL蛋白組比較,LY294002＋rs TRAIL蛋白組G1期A549細(xì)胞百分率明顯升高(P＜0.05),S期細(xì)胞百分率明顯降低(P＜0.05)。見表1。

2.3 2組A549細(xì)胞凋亡率LY294002＋rs TRAIL蛋白組A549細(xì)胞凋亡率為(61.5± 3.02)%,明顯高于rs TRAIL蛋白組(3.21%± 0.96%)(P＜0.05)。

表1 流式細(xì)胞術(shù)檢測(cè)2組A549細(xì)胞周期Tab.1 Cell cycle of A549 cells detected by flow cytometry in two groups(±s,η/%)

表1 流式細(xì)胞術(shù)檢測(cè)2組A549細(xì)胞周期Tab.1 Cell cycle of A549 cells detected by flow cytometry in two groups(±s,η/%)

?P＜0.05 compared with control group.

Group Percentage of A549 cells G1G2S rs TRAIL 54.12±2.46 13.54±2.23 32.34±1.75 LY294002＋rsTRAIL 81.74±3.12?7.07±1.59 11.19±1.26?

2.4 2組A549細(xì)胞中p-Akt、c-FLIPL、Bcl-2和Bax蛋白表達(dá)水平與rs TRAIL蛋白組比較, LY294002＋rs TRAIL蛋白組A549細(xì)胞中p-Akt、c-FLIPL和Bcl-2蛋白表達(dá)水平下調(diào),Bax表達(dá)水平無(wú)明顯改變。見圖1?；叶确治鼋Y(jié)果:與rs TRAIL蛋白組比較,LY294002＋rs TRAIL蛋白組p-Akt、c-FLIPL和Bcl-2蛋白表達(dá)水平下降(P＜0.05),Bax/Bcl-2比值升高2.21倍(P＜0.05)。見表2。

圖1 2組A549細(xì)胞中p-Akt、c-FLIPL、Bcl-2和Bax蛋白表達(dá)電泳圖Fig.1 Electrophoregram of expressions of p-Akt,c-FLIPL, Bcl-2 and Bax proteins in A549 cells in two groupsLane 1:LY294002＋rs TRAIL group;Lane 2:rs TRAIL group.

3 討論

目前化療依然是NSCLC治療的主要手段,但應(yīng)用化療治療NSCLC(尤其是晚期患者)已經(jīng)達(dá)到了平臺(tái)期,為了進(jìn)一步提高NSCLC的治療效果,需要探索新的安全有效的臨床用藥。TRAIL能選擇性地殺死腫瘤細(xì)胞,抑制動(dòng)物體內(nèi)移植瘤的形成與生長(zhǎng),而對(duì)大多數(shù)正常組織和細(xì)胞無(wú)明顯毒性,耐藥細(xì)胞株的出現(xiàn)限制了TRAIL抗腫瘤的應(yīng)用范圍[1,4-5]。

表2 各組A549細(xì)胞中p-Akt、c-FLIPL、Bcl-2和Bax蛋白表達(dá)水平Tab.2 Expression levels of p-Akt,c-FLIPL,Bcl-2,and Bax proteins in A549 cells in two groups(±s)

表2 各組A549細(xì)胞中p-Akt、c-FLIPL、Bcl-2和Bax蛋白表達(dá)水平Tab.2 Expression levels of p-Akt,c-FLIPL,Bcl-2,and Bax proteins in A549 cells in two groups(±s)

?P＜0.05 compared with rs TRAIL group.

Goup p-Akt/β-actin c-FLIPL/β-actin Bcl-2/β-actin Bax/β-actin Bax/Bcl-2 rs TRAIL 1.10±0.25 0.79±0.19 0.62±0.14 0.45±0.12 0.73±0.24 LY294002＋rs TRAIL 0.17±0.09?0.21±0.07?0.33±0.10?0.53±0.16 1.61±0.38?

PI3K/Akt通路活化在人類惡性腫瘤中非常普遍,活化的PI3K/Akt通路可以激活下游多條信號(hào)通路的轉(zhuǎn)導(dǎo),促進(jìn)NSCLC的發(fā)展和參與腫瘤的耐藥[8]。LY294002為人工合成的小分子化合物,是PI3K/Akt信號(hào)通路的特異性抑制劑,通過(guò)共價(jià)結(jié)合到PI3K的賴氨酸殘基,靶向抑制PI3K催化亞基p110的活化[9]。本課題組前期研究[4-5]結(jié)果表明:A549細(xì)胞內(nèi)高表達(dá)c-FLIPL蛋白導(dǎo)致的Caspase-8活化抑制是細(xì)胞抵抗rs TRAIL蛋白誘導(dǎo)凋亡的重要原因,Willems等[10]研究發(fā)現(xiàn):p-Akt是導(dǎo)致細(xì)胞內(nèi)c-FLIPL蛋白水平升高的重要原因, Akt活化的特異性抑制劑能夠降低c-FLIPL蛋白水平。本研究結(jié)果顯示:PI3K靶向抑制劑LY290042能夠抑制PI3K的活性從而降低p-Akt蛋白的表達(dá)水平,伴隨著p-Akt蛋白表達(dá)水平的降低,c-FLIPL蛋白表達(dá)水平亦降低,rs TRAIL蛋白聯(lián)合LY290042共同作用于A549細(xì)胞后細(xì)胞凋亡率明顯升高,說(shuō)明PI3K/Akt通路參與調(diào)節(jié)c-FLIPL蛋白對(duì)TRAIL誘導(dǎo)凋亡的抵抗作用。

PI3K/Akt通路是抑制細(xì)胞凋亡的中樞介質(zhì), p-Akt可以磷酸化凋亡蛋白的絲氨酸位點(diǎn),改變其空間構(gòu)象,阻止凋亡蛋白進(jìn)入細(xì)胞核或者促進(jìn)抗凋亡與之結(jié)合,從而抑制細(xì)胞凋亡。研究[11]證實(shí): PI3K/Akt通路的激活可以導(dǎo)致Bcl-2蛋白表達(dá)水平升高,參與阻止TRAIL引起的線粒體細(xì)胞凋亡途徑的激活。本研究結(jié)果顯示:LY290042抑制p-Akt表達(dá)的同時(shí),促進(jìn)了Bcl-2蛋白表達(dá)降低,說(shuō)明LY290042可能促進(jìn)TRAIL激活的線粒體途徑細(xì)胞凋亡能力。有關(guān)前列腺癌、直腸癌、白血病和宮頸癌等的臨床研究[12-13]發(fā)現(xiàn):Bcl-2家族成員的構(gòu)成比例是凋亡調(diào)控的關(guān)鍵因素,尤其是Bax/Bcl-2比值是啟動(dòng)細(xì)胞凋亡的“分子開關(guān)”,直接決定了線粒體外膜各種通道的開放程度。外界因素刺激可以通過(guò)調(diào)節(jié)Bcl-2和Bax 2種調(diào)控因子的平衡改變細(xì)胞的命運(yùn)。盡管Bcl-2和Bax的表達(dá)均可被Akt調(diào)節(jié)[14],但本研究結(jié)果顯示:PI3K/ Akt抑制劑LY290042并未改變A549細(xì)胞中Bax的表達(dá)水平,但可以通過(guò)降低Bcl-2蛋白表達(dá)水平,增加Bax/Bcl-2比值,促進(jìn)細(xì)胞凋亡信號(hào)的啟動(dòng)。

綜上所述,20μmol·L－1LY290042作用2 h可以有效改善凋亡相關(guān)蛋白的表達(dá)水平,從2條凋亡途徑增強(qiáng)TRAIL引起的腫瘤細(xì)胞凋亡作用;同時(shí)LY290042還可以增加G1期A549細(xì)胞的百分比,降低腫瘤細(xì)胞的增殖速度。G1期細(xì)胞的百分比增加可以增強(qiáng)TRAIL誘導(dǎo)腫瘤細(xì)胞凋亡的敏感性[15]。PI3K/Akt可以作為良好的靶點(diǎn)應(yīng)用于NSCLC的治療研究。

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Enhanced effect of rsTRAIL-based therapy for A549 cells by phosphatidylinositol 3′-kinase inhibitor LY294002

LIU Lei1,FANG Yan-qiu1,QI Ya-ling2,LU Xiao-dan1,WEI Hai-feng1,TAN Yan1
(1.Tumor Biological Treatment Center,Jilin Province People’s Hospital,Changchun 130021,China; 2.Department of Histology and Embryology,School of Basic Medical Science,Jiamusi University, Jiamusi 154007,China)

ObjectiveTo explore the potential mechanisms of non-small cell lung carcinoma cells to rs TRAIL protein-induced apoptosis by phosphatidylinositol 3′-kinase(PI3K/Akt)inhibitor LY294002,and to provide new ways to increase killing activities of rs TRAIL protein for non-small cell lung cancer.MethodsThe A549 cells at logarithmic growth phase were selected and randomly divided into rs TRAIL group and LY294002＋rs TRAIL group.The inhibitory rate of growth of the A549 cells was tested by MTT assay.The cell cycle and apoptotic rate were detected by flow cytometry analysis.The expression levels of Ser473 phosphorylated form of Akt(p-Akt),c-FLIPLprotein and Bcl-2 protein in the A549 cells in two groups were analyzed by Western blotting method.ResultsThe inhibitory rate of growth of the A549 cells in LY294002＋rs TRAIL group(74.6%±2.63%)was higher than that in rs TRAIL group(5.61%±0.32%)(P＜0.05).Compared with rs TRAIL group,the percentage of the cells at G0/G1phase in LY294002＋rs TRAIL group was increased(P＜0.05)and the percentage of the cells at S phase was decreased(P＜0.05).The apoptotic rate of the A549 cells in LY294002＋rs TRAIL group(61.5%±3.02%)was higher than that in rs TRAIL group(3.21%±0.96%)(P＜0.05).The Western blotting results showed that the expression levels of p-Akt,c-FLIPL and Bcl-2 proteins in the A549 cells in LY294002＋rs TRAIL group were decreased(P＜0.05)and the ratio of Bax/Bcl-2 was increased(P＜0.05)compared with rs TRAIL group.ConclusionLY294002 can increase the killing activity of rs TRAIL protein in A549 cells by inhibiting the activity of PI3K.

cancer non-small cell lung;A549 cells;TNF-related apoptosis inducing ligand;LY294002;PI3K/Akt

R733.6

A

2014-01-21

吉林省科技廳基礎(chǔ)項(xiàng)目資助課題(201115201);吉林省科技廳重點(diǎn)實(shí)驗(yàn)室項(xiàng)目資助課題(20122113);吉林省衛(wèi)生廳科研基金資助課題(20082036);吉林省科技廳科技創(chuàng)新項(xiàng)目資助課題(20082102)

劉 磊(1978－),男,黑龍江省齊齊哈爾市人,醫(yī)師,醫(yī)學(xué)博士,主要從事腫瘤免疫相關(guān)研究。

方艷秋(Tel:0431-85595659,E-mail:yq.fang＠163.com);齊亞靈(Tel:0454-8618297,E-mail:qiyaling87016＠163.com)

1671-587Ⅹ(2014)05-0972-05

10.13481/j.1671-587x.20140513