孫 珂， 褚翠英， 鄭夢(mèng)琦， 高 遠(yuǎn)， 齊由坤， 王鳳澤

(泰山醫(yī)學(xué)院生命科學(xué)學(xué)院， 山東 泰安 271016)

CP466722抑制HepG2細(xì)胞增殖并誘導(dǎo)細(xì)胞凋亡*

孫 珂， 褚翠英， 鄭夢(mèng)琦， 高 遠(yuǎn)， 齊由坤， 王鳳澤△

(泰山醫(yī)學(xué)院生命科學(xué)學(xué)院， 山東 泰安 271016)

目的： 研究共濟(jì)失調(diào)毛細(xì)血管擴(kuò)張癥突變蛋白(ATM)抑制劑CP466722對(duì)人肝細(xì)胞癌HepG2細(xì)胞增殖和凋亡的影響及其可能的分子機(jī)制。方法： MTT法檢測(cè)CP466722對(duì)HepG2細(xì)胞活力的抑制作用，細(xì)胞集落形成實(shí)驗(yàn)觀察CP466722對(duì)細(xì)胞增殖的影響，流式細(xì)胞術(shù)分析細(xì)胞周期的變化，TUNEL染色觀察CP466722對(duì)細(xì)胞凋亡誘導(dǎo)作用，Western blotting檢測(cè)細(xì)胞內(nèi)蛋白的表達(dá)水平。結(jié)果： CP466722能夠劑量依賴(lài)性地抑制HepG2細(xì)胞活力與細(xì)胞增殖；HepG2細(xì)胞經(jīng)CP466722作用24 h后，細(xì)胞周期明顯阻滯于G2/M期，同時(shí)磷酸化細(xì)胞分裂周期蛋白2(p-Cdc2)、細(xì)胞分裂周期蛋白25 C (Cdc25C)和磷酸化Cdc25C(p-Cdc25C)的蛋白水平下降，而p27的蛋白表達(dá)則上調(diào)。較高濃度的CP466722誘導(dǎo)HepG2細(xì)胞發(fā)生凋亡，促進(jìn)胱天蛋白酶3(caspase-3)和多腺苷二磷酸核糖聚合酶(PARP)的剪切。此外，CP466722抑制β-聯(lián)蛋白(β-catenin)及其下游因子生存素(survivin)的表達(dá)，上調(diào)p38絲裂原活化蛋白激酶(p38 MAPK)的磷酸化水平。結(jié)論： CP466722抑制HepG2細(xì)胞增殖并誘導(dǎo)細(xì)胞發(fā)生凋亡，其機(jī)制可能與其抑制β-catenin信號(hào)途徑和激活p38 MAPK相關(guān)。

CP466722； 肝細(xì)胞癌； 細(xì)胞增殖； 細(xì)胞凋亡； p38絲裂原活化蛋白激酶； β-聯(lián)蛋白

肝細(xì)胞癌(hepatocellular carcinoma，HCC)是全球最常見(jiàn)惡性腫瘤之一，嚴(yán)重威脅人類(lèi)的生存與健康。中國(guó)是全球HCC發(fā)病率最高的國(guó)家，據(jù)統(tǒng)計(jì)2015年全國(guó)新發(fā)病例約47萬(wàn)，新增死亡病例約42萬(wàn)[1]。雖然手術(shù)治療是HCC的主要治療方法，但由于多數(shù)HCC 診斷時(shí)即為中晚期，使得化學(xué)藥物治療已成為HCC臨床治療的主要手段之一[2-3]。但應(yīng)注意的是，藥物治療的毒副作用以及化療藥物耐藥等復(fù)雜情況限制了治療方案的選擇[4]。因此，提高抗腫瘤藥物的療效及尋找新的化療藥物已成為HCC治療的主要方向之一。

CP466722 (CP)是一種小分子共濟(jì)失調(diào)毛細(xì)血管擴(kuò)張癥突變蛋白(ataxia telangiectasia mutated protein，ATM)的可逆抑制劑，能夠抑制離子輻射誘導(dǎo)的ATM磷酸化激活，從而增加腫瘤細(xì)胞的放療敏感性[5]。本研究以肝癌細(xì)胞HepG2為實(shí)驗(yàn)用細(xì)胞株，研究CP466722對(duì)肝癌細(xì)胞增殖和凋亡的影響，同時(shí)初步探索其抑癌活性的分子機(jī)制，為CP466722的臨床應(yīng)用以及HCC治療提供新思路和實(shí)驗(yàn)依據(jù)。

材 料 和 方 法

1 實(shí)驗(yàn)材料

CP466722購(gòu)自Selleck Chemicals；胎牛血清購(gòu)自Gibco；MTT和抗β-actin抗體購(gòu)自Sigma；TUNEL凋亡檢測(cè)試劑盒購(gòu)自Roche；ECL試劑盒購(gòu)自Thermo Scientific Pierce；抗p-Cdc2(Tyr15)、p-Cdc25C(Ser216)、β-catenin、survivin、caspase-3和PARP抗體購(gòu)自Cell Signaling Technology；抗ERK、p-ERK、p38、p-p38、p27、Cdc2和cyclin B1抗體購(gòu)自Santa Cruz；抗Cdc25C抗體購(gòu)自Abcam； II 抗均購(gòu)自北京中杉金橋。

2 實(shí)驗(yàn)方法

2.1 細(xì)胞培養(yǎng) 肝癌細(xì)胞株HepG2購(gòu)自中科院上海細(xì)胞庫(kù)，用含有10%胎牛血清的DMEM培養(yǎng)基在37 ℃和5% CO2條件下培養(yǎng)。

2.2 細(xì)胞活力測(cè)定 將HepG2細(xì)胞接種于96孔板中，加入不同劑量的CP466722作用24 h。在終止培養(yǎng)前4 h加20 μL (5 g/L)的MTT于各孔中。吸去細(xì)胞培養(yǎng)液并加入150 μL的DMSO，室溫振蕩5～10 min后用酶標(biāo)儀在490 nm波長(zhǎng)下測(cè)定吸光度值，并對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行統(tǒng)計(jì)分析。

2.3 細(xì)胞集落形成實(shí)驗(yàn) 將HepG2細(xì)胞以合適密度接種于6孔培養(yǎng)板中，加入不同濃度CP466722進(jìn)行藥物干預(yù)。10 d后吸出培養(yǎng)基，PBS洗滌，加入無(wú)水甲醇固定細(xì)胞20 min。PBS洗滌細(xì)胞，加入結(jié)晶紫染色10 min，PBS洗滌細(xì)胞，倒置顯微鏡下拍照并計(jì)數(shù)每孔細(xì)胞團(tuán)數(shù)目。

2.4 細(xì)胞周期的檢測(cè) 接種HepG2細(xì)胞于6孔培養(yǎng)板中。待細(xì)胞貼壁后加入不同濃度的CP466722處理細(xì)胞24 h。胰酶消化收集細(xì)胞，800 r/min 離心5 min，PBS洗滌后用70% 預(yù)冷乙醇固定過(guò)夜。離心去除乙醇，加入RNase A于37 ℃作用細(xì)胞30 min，然后用碘化丙啶(propidium iodide，PI)避光染色10 min，流式細(xì)胞術(shù)分析細(xì)胞周期。

2.5 細(xì)胞凋亡的檢測(cè) 經(jīng)CP466722處理24 h的HepG2細(xì)胞用PBS洗滌2次后，于4 ℃固定1 h，接著用0.1% Triton X-100 打孔2 min。然后按照TUNEL凋亡檢測(cè)試劑盒說(shuō)明書(shū)對(duì)細(xì)胞進(jìn)行凋亡染色，最后于倒置熒光顯微鏡下觀察TUNEL染色結(jié)果并拍照。

2.6 Western blotting檢測(cè)蛋白表達(dá) 細(xì)胞用RAPI裂解液(含有蛋白酶抑制劑)冰上裂解20 min，然后4 ℃ 13 000×g離心20 min，收集上清定量。取30～40 μg總蛋白進(jìn)行SDS-PAGE，電轉(zhuǎn)移至PVDF膜上。采用5%脫脂奶粉室溫封閉PVDF膜2 h，接著加入 I 抗室溫孵育3 h，PBS洗滌3次后加入 II 抗，室溫孵育1 h，PBS洗滌3次后ECL顯色試劑盒顯影。實(shí)驗(yàn)重復(fù)3次。

3 統(tǒng)計(jì)學(xué)處理

采用SPSS 16.0 統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示，多組之間比較采用單因素方差分析，應(yīng)用SNK-q檢驗(yàn)進(jìn)行組間兩兩比較，以P<0.05 為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 CP466722降低HepG2細(xì)胞的存活率

MTT實(shí)驗(yàn)的檢測(cè)結(jié)果顯示，HepG2細(xì)胞經(jīng)不同濃度CP466722作用24 h后，細(xì)胞活力與對(duì)照組相比呈下降趨勢(shì)，且具有明顯的濃度依賴(lài)性關(guān)系，表明CP466722能夠劑量依賴(lài)性地降低HepG2細(xì)胞活力,見(jiàn)圖1。

Figure 1.CP466722 reduced the viability of HepG2 cells. The cells were incubated with indicated concentrations of CP466722 for 24 h and then processed for MTT assay. Mean±SD.n=3.*P<0.05vs0 μmol/L.

圖1 CP466722降低HepG2細(xì)胞活力

細(xì)胞集落形成實(shí)驗(yàn)的結(jié)果顯示CP466722對(duì)HepG2細(xì)胞增殖具有抑制作用，CP466722能明顯減少HepG2細(xì)胞克隆的形成，抑制了HepG2細(xì)胞的增殖，見(jiàn)圖2。

Figure 2.CP466722 inhibited the colony formation ability of HepG2 cells. Mean±SD.n=3.*P<0.05vs0 μmol/L.

圖2 CP466722抑制HepG2細(xì)胞增殖

2 CP466722阻滯HepG2細(xì)胞于G2/M期

PI 法檢測(cè)CP466722對(duì)HepG2細(xì)胞周期的影響，結(jié)果顯示CP466722 作用于細(xì)胞24 h后，與對(duì)照組相比，G2/M期細(xì)胞比例明顯升高，而S期的細(xì)胞比例則相應(yīng)減少，提示CP466722能夠阻滯肝癌細(xì)胞于G2/M期，見(jiàn)圖3、表1。

Figure 3.Upon exposure to CP466722 for 24 h, HepG2 cells exhibited G2/M phase arrest.

圖3 CP466733誘導(dǎo)HepG2細(xì)胞發(fā)生G2/M期阻滯

表1 CP466722誘導(dǎo)HepG2細(xì)胞周期阻滯于G2/M期

Table 1.Upon exposure to CP466722 for 24 h, HepG2 cells exhibited G2/M phase arrest (%.Mean±SD.n=3)

CP466722(μmol/L)G0/G1G2/MS038.81±2.445.08±0.6355.96±2.90552.12±2.64*19.93±1.22*27.62±1.17*1044.11±1.8531.94±1.53*23.76±1.09*2034.13±1.2646.82±1.77*19.15±0.85*

*P<0.05vs0 μmol/L.

Western blotting結(jié)果顯示，不同濃度CP466722作用于HepG2 細(xì)胞24 h后， p27的蛋白表達(dá)增加，而p-Cdc2、Cdc25C和p-Cdc25C的蛋白水平降低，cyclin B1的表達(dá)水平未見(jiàn)明顯變化，見(jiàn)圖4。

3 CP466722誘導(dǎo)HepG2細(xì)胞發(fā)生凋亡

采用TUNEL染色法觀察CP466722對(duì)HepG2細(xì)胞凋亡的影響，結(jié)果顯示與對(duì)照組相比，較高濃度的CP466722能夠誘導(dǎo)HepG2細(xì)胞發(fā)生凋亡，見(jiàn)圖5。

Western blotting結(jié)果證實(shí)，CP466722促進(jìn)HepG2細(xì)胞中caspase-3的活性剪切，同時(shí)也增加PARP的切割，表明胱天蛋白酶家族成員的激活可能與CP466722誘導(dǎo)肝癌細(xì)胞凋亡相關(guān)，見(jiàn)圖6。

4 CP466722激活HepG2細(xì)胞中p38 MAPK 的活性

Western blotting結(jié)果顯示，CP466722作用肝癌細(xì)胞后，p38 MAPK的磷酸化水平明顯升高，而ERK的磷酸化未見(jiàn)明顯變化，見(jiàn)圖7。

Figure 4.The effects of CP466722 treatment at different concentrations for 24 h on the protein levels of cell cycle-associated proteins in the HepG2 cells. β-actin served as loading control. Mean±SD.n=3.*P<0.05vs0 μmol/L.

圖4 CP466722對(duì)細(xì)胞周期相關(guān)蛋白水平的影響

Figure 5.HepG2 cells were treated with 20 μmol/L of CP466722 for 24 h, and the apoptosis was evaluated by TUNEL staining (×400). Mean±SD.n=3.*P<0.05vs0 μmol/L.

圖5 CP466722誘導(dǎo)HepG2細(xì)胞發(fā)生凋亡

5 CP466722對(duì)β-catenin信號(hào)通路的抑制作用

如圖8所示，HepG2細(xì)胞經(jīng)CP466722作用24 h后，β-catenin的表達(dá)水平明顯下降，同時(shí)其下游因子survivin的蛋白表達(dá)也受到抑制。

討 論

本研究主要探討CP466722對(duì)肝癌細(xì)胞增殖的抑制活性及細(xì)胞凋亡的誘導(dǎo)作用，同時(shí)闡明其可能的內(nèi)在分子機(jī)理。研究結(jié)果表明，CP466722能夠劑量依賴(lài)性地抑制肝癌細(xì)胞活力和細(xì)胞集落形成能力；較高濃度的CP466722能夠激活與細(xì)胞凋亡密切相關(guān)的caspase-3 發(fā)生活性剪切，促進(jìn)其底物PARP的切割，進(jìn)而誘導(dǎo)HepG2細(xì)胞發(fā)生凋亡。

細(xì)胞周期調(diào)控在腫瘤的發(fā)生和治療中起著重要作用，多種化療藥物均可誘導(dǎo)細(xì)胞發(fā)生G2/M 期阻滯并促進(jìn)細(xì)胞發(fā)生凋亡[6-7]。Cdc2與cyclin B1復(fù)合物是G2/M檢驗(yàn)點(diǎn)的關(guān)鍵調(diào)節(jié)因子，而Cdc25C又是調(diào)節(jié)Cdc2 活性的關(guān)鍵酶，當(dāng)Ser216位點(diǎn)磷酸化時(shí)，Cdc25C與14-3-3蛋白結(jié)合并滯留在胞質(zhì)中，不能進(jìn)入到細(xì)胞核而失去活性[8-9]。本研究發(fā)現(xiàn)，CP466722作用于HepG2細(xì)胞24 h 后，G2/M期的細(xì)胞數(shù)目明顯增多，而處于S期的細(xì)胞比例則相應(yīng)下降，表明CP466722抑制HepG2細(xì)胞增殖與誘導(dǎo)G2/M 期阻滯有密切關(guān)系。Western blot結(jié)果顯示，CP466722對(duì)細(xì)胞周期的調(diào)控可能與其下調(diào)Cdc25C和p-Cdc25C表達(dá)水平進(jìn)而抑制Cdc2的磷酸化相關(guān)，其上調(diào)p27蛋白表達(dá)也可能參與該過(guò)程。

Figure 6.HepG2 cells were exposed to indicate concentrations of CP466722 for 24 h and cleavages of caspase-3 and PARP were estimated by Western blotting. β-actin was used as a loading control. Mean±SD.n=3.*P<0.05vs0 μmol/L.

圖6 CP466722促進(jìn)caspase-3 和PARP的剪切

Figure 7.CP466722 increased the activation of p38 in the HepG2 cells. The cells were exposed to CP466722 for 24 h, and the protein levels of p-ERK1/2, p-p38, ERK1/2 and p38 were identified by Western blotting. Mean±SD.n=3.*P<0.05vs0 μmol/L.

圖7 CP466722 促進(jìn)p38 MAPK磷酸化

Figure 8.CP466722 inhibited the expression of β-catenin and survivin in the HepG2 cells. The cells were exposed to CP466722 for 24 h, and the levels of β-catenin and survivin were measured by Western blotting. Mean±SD.n=3.*P<0.05vs0 μmol/L.

圖8 CP466722抑制β-catenin和survivin的表達(dá)

MAPK 家族蛋白參與調(diào)節(jié)細(xì)胞增殖、凋亡及分化等生理病理過(guò)程，在細(xì)胞的各種生命活動(dòng)中均發(fā)揮著重要作用[10]。因此，本研究檢測(cè)了MAPK家族因子ERK和p38 MAPK是否參與CP466722調(diào)控HepG2細(xì)胞增殖和凋亡的過(guò)程。實(shí)驗(yàn)結(jié)果表明，CP466722能夠促進(jìn)肝癌細(xì)胞中p38 MAPK的磷酸化水平，提示p38 MAPK可能與CP466722的抗腫瘤活性相關(guān)。

此外，CP466722顯著抑制HepG2 細(xì)胞中β-catenin及其下游因子survivin的蛋白水平。Wnt/β-catenin 信號(hào)途徑能夠促進(jìn)腫瘤細(xì)胞的增殖、遷移并抑制細(xì)胞凋亡，該信號(hào)通路的異?；罨c多種腫瘤發(fā)生過(guò)程密切相關(guān)，而抑制該信號(hào)通路則可減緩腫瘤細(xì)胞生長(zhǎng)及誘導(dǎo)腫瘤細(xì)胞發(fā)生凋亡[11-12]。由此推測(cè)，CP466722抑制Wnt/β-catenin 信號(hào)通路可能參與調(diào)控HepG2細(xì)胞增殖和誘導(dǎo)細(xì)胞凋亡的過(guò)程。

綜上所述，CP466722能夠顯著抑制HepG2細(xì)胞增殖，促進(jìn)細(xì)胞發(fā)生G2/M期阻滯，同時(shí)誘導(dǎo)細(xì)胞發(fā)生凋亡。其可能機(jī)制與CP466722 促進(jìn)p38 MAPK的磷酸化及抑制β-catenin和survivin的表達(dá)水平密切相關(guān)。

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(責(zé)任編輯： 林白霜， 羅 森)

CP466722 inhibits proliferation and triggers apoptosis of HepG2 cells

SUN Ke, CHU Cui-ying, ZHENG Meng-qi, GAO Yuan, QI You-kun, WANG Feng-ze

(SchoolofLifeSciences,TaishanMedicalUniversity,Tai’an271016,China.E-mail:wfz221@sina.com)

AIM: To investigate the effect of CP466722, an inhibitor of ataxia telangiectasia mutated protein (ATM), on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells. METHODS: The cell viability was detected by MTT assay. The cell growth inhibition was measured by colony formation assay. The effect of CP466722 on the cell cycle distribution of the HepG2 cells was examined by flow cytometry. The cell apoptosis was analyzed by TUNEL staining. The protein expression was examined by Western blotting. RESULTS: CP466722 inhibited the cell viability and cell proliferation in a dose-dependent manner. In CP466722-treated HepG2 cells, the cell cycle was arrested in G2/M phase, and the protein levels of phosphorylated cell division cycle protein 2 (p-Cdc2), cell division cycle protein 25C (Cdc25C) and phosphorylated Cdc25C (p-Cdc25C) were inhibited, whereas the protein expression of p27 was up-regulated. CP466722 triggered the apoptosis of HepG2 cells through cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP). In addition, CP466722 increased the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and suppressed the expression of β-catenin and survivin in the HepG2 cells. CONCLUSION: CP466722 inhibits the proliferation and induces the apoptosis of HepG2 cells, which may be related to activating p38 MAPK and inhibiting the expression of β-catenin and survivin.

CP466722; Hepatocellular carcinoma; Cell proliferation; Cell apoptosis; p38 mitogen-activated protein kinase; β-catenin

1000- 4718(2017)04- 0655- 06

2016- 11- 08

2016- 12- 19

國(guó)家自然科學(xué)基金資助項(xiàng)目(No. 81272683)； 國(guó)家級(jí)大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目(No. 201510439067； No. 201610439275)

R730.23

A

10.3969/j.issn.1000- 4718.2017.04.013

△通訊作者 Tel: 0538-6231086; E-mail: wfz221@sina.com