吳乾波， 張 敏， 陶志華

(1浙江大學(xué)醫(yī)學(xué)院附屬第二醫(yī)院檢驗(yàn)科， 2浙江省血液中心獻(xiàn)服二科， 3浙江省立同德醫(yī)院檢驗(yàn)科，浙江 杭州 310000)

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歐前胡素通過(guò)下調(diào)c-met的表達(dá)提高肺癌CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性

吳乾波1,2， 張 敏3▲， 陶志華1△

(1浙江大學(xué)醫(yī)學(xué)院附屬第二醫(yī)院檢驗(yàn)科，2浙江省血液中心獻(xiàn)服二科，3浙江省立同德醫(yī)院檢驗(yàn)科，浙江 杭州 310000)

目的： 研究PC9 CD133+細(xì)胞亞群對(duì)吉非替尼的耐藥性并探討歐前胡素提高吉非替尼抗肺癌活性的機(jī)制。方法: MTT法檢測(cè)PC9細(xì)胞在吉非替尼和歐前胡素處理下的細(xì)胞活力。Western blot實(shí)驗(yàn)檢測(cè)吉非替尼和歐前胡素對(duì)PC9細(xì)胞c-met表達(dá)水平、caspases 活化水平及表皮生長(zhǎng)因子受體(EGFR)、PI3K、AKT磷酸化水平的影響。流式細(xì)胞術(shù)檢測(cè)歐前胡素和吉非替尼對(duì)PC9細(xì)胞系的CD133+細(xì)胞亞群種群比例的影響及PC9細(xì)胞在二者處理下的凋亡率。結(jié)果: PC9 CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性顯著低于PC9 CD133-細(xì)胞亞群。吉非替尼能顯著抑制PC9 CD133-細(xì)胞亞群EGFR/PI3K/AKT的活化，但對(duì)PC9 CD133+細(xì)胞亞群該通路的影響不大。吉非替尼單獨(dú)處理能提高PC9細(xì)胞系中CD133+細(xì)胞亞群的比例，然而聯(lián)用歐前胡素后PC9 CD133+細(xì)胞亞群的種群比例顯著下降。Western blot實(shí)驗(yàn)表明歐前胡素能顯著降低PC9 CD133+細(xì)胞亞群的c-met蛋白表達(dá)水平，表明c-met是歐前胡素的治療靶點(diǎn)。MTT、Western blot、流式細(xì)胞術(shù)實(shí)驗(yàn)結(jié)果表明在PC9 CD133+細(xì)胞亞群中，歐前胡素通過(guò)抑制c-met的表達(dá)提高吉非替尼對(duì)PI3K/AKT的抑制作用，從而誘導(dǎo)PC9 CD133+細(xì)胞亞群發(fā)生caspases活化和凋亡。結(jié)論: 歐前胡素通過(guò)下調(diào)c-met的表達(dá)提高肺癌CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性，兩者存在協(xié)同抗腫瘤效應(yīng)。

歐前胡素； c-met； 吉非替尼； PC9 CD133+細(xì)胞亞群； PI3K/AKT信號(hào)通路

非小細(xì)胞肺癌(nonsmall-cell lung cancer，NSCLC)是一種發(fā)病率和死亡率都非常高的惡性腫瘤。盡管現(xiàn)在惡性腫瘤的治療手段有了很大的發(fā)展，化療在NSCLC的治療過(guò)程中仍是不可替代的重要方法，然而隨著化療藥物的反復(fù)使用，NSCLC細(xì)胞往往能產(chǎn)生獲得性藥物抵抗，從而降低化療的效果[1-2]。近些年來(lái)的研究表明在腫瘤組織中存在著一組名為腫瘤干細(xì)胞的細(xì)胞群，與化療的獲得性抵抗密切相關(guān)[3]。肺癌組織細(xì)胞中的CD133+的腫瘤干細(xì)胞被報(bào)道能引起化療的失敗和腫瘤的復(fù)發(fā)[4]，因此腫瘤干細(xì)胞已經(jīng)成為肺癌治療的靶點(diǎn)。

吉非替尼屬于一種表皮生長(zhǎng)因子受體(epidermal growth factor receptor, EGFR)酪氨酸激酶抑制劑，廣泛應(yīng)用于肺癌患者的治療，由于它有很好的靶向性，因此吉非替尼目前已經(jīng)成為治療非小細(xì)胞肺癌的一線藥物[5]。然而隨著吉非替尼的持續(xù)使用，肺癌細(xì)胞對(duì)吉非替尼的敏感性會(huì)逐漸降低。鑒于肺癌干細(xì)胞的耐藥性是造成吉非替尼抵抗的重要因素[6]，因此靶向于肺癌干細(xì)胞的輔助治療是提高吉非替尼療效的有效方法。

歐前胡素是從白芷根中提取的香豆素類天然活性物質(zhì)，據(jù)報(bào)道有一定的抗腫瘤效應(yīng)，并且能提高化療藥物對(duì)腫瘤細(xì)胞的殺傷活性[7-8]，然而其對(duì)腫瘤干細(xì)胞的生物作用至今卻仍很少報(bào)道。本研究的目的在于探討歐前胡素是否能調(diào)節(jié)非小細(xì)胞肺癌CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性。

材 料 和 方 法

1 細(xì)胞

人非小細(xì)胞肺癌細(xì)胞系PC9購(gòu)于ATCC。PC9 CD133+細(xì)胞亞群用流式細(xì)胞儀進(jìn)行分選，文獻(xiàn)報(bào)道CD133+的肺癌細(xì)胞即為肺癌腫瘤干細(xì)胞，而CD133-細(xì)胞則為肺癌非干細(xì)胞[9]。細(xì)胞用含10%胎牛血清的DMEM培養(yǎng)基在37 ℃恒溫培養(yǎng)箱中通入5% CO2培養(yǎng)。

2 試劑

吉非替尼、歐前胡素、噻唑藍(lán)(MTT)、凋亡檢測(cè)試劑盒購(gòu)于Sigma-Aldrich；DMEM培養(yǎng)基購(gòu)于Gibco；蛋白提取液、抗c-met、磷酸化EGFR、磷酸化PI3K、磷酸化AKT、cleaved caspase-9、cleaved caspase-7、cleaved caspase-3和β-actin抗體購(gòu)于CST；ECL試劑盒購(gòu)于Pierce；抗CD133-FITC熒光抗體購(gòu)于BD；pcDNA3.1、Lipofectamine 2000購(gòu)于Invitrogen。

3 方法

3.1 吉非替尼半數(shù)有效濃度(IC50)的測(cè)定 將PC9 CD133+細(xì)胞亞群和PC9 CD133-細(xì)胞亞群按每孔5×103個(gè)接種在96孔板上孵育過(guò)夜，用濃度為0～0.3 μmol/L的吉非替尼處理腫瘤細(xì)胞48 h，之后加入20 μL MTT (5 g/L)于37 ℃恒溫培養(yǎng)箱中培養(yǎng)4 h，移除孔內(nèi)培養(yǎng)基，加入100 μL二甲基亞砜，于570 nm波長(zhǎng)下測(cè)定吸光度(A)值。細(xì)胞活力結(jié)果用吉非替尼處理組與對(duì)照組的A值比值表示。繪制細(xì)胞活力-吉非替尼濃度曲線，根據(jù)曲線計(jì)算IC50。

3.2 PC9 CD133+細(xì)胞亞群所占比例測(cè)定 將PC9細(xì)胞系用0.04 μmol/L吉非替尼和10 μmol/L歐前胡素處理48 h，之后在細(xì)胞培養(yǎng)基中加入抗CD133抗體在暗處孵育20 min后將細(xì)胞用流式細(xì)胞儀進(jìn)行分析，計(jì)算PC9 CD133+細(xì)胞所占總的PC9細(xì)胞的比例。

3.3 c-met重組質(zhì)粒的構(gòu)建和轉(zhuǎn)染 將c-met基因的cDNA全長(zhǎng)序列(Gene ID: NM_001324401)以分子克隆的方法與pcDNA3.1連接后構(gòu)建成c-met重組真核表達(dá)質(zhì)粒[9]。使用Lipofectamine 2000按照試劑操作說(shuō)明書(shū)步驟將2 mg/L c-met質(zhì)粒轉(zhuǎn)染入PC9 CD133+細(xì)胞亞群中。

3.4 歐前胡素聯(lián)合吉非替尼對(duì)PC9 CD133+細(xì)胞亞群的殺傷活性 將PC9 CD133+細(xì)胞亞群按每孔5×103個(gè)接種在96孔板上孵育過(guò)夜，用2 mg/L c-met質(zhì)粒轉(zhuǎn)染24 h后加入0.04 μmol/L吉非替尼和10 μmol/L歐前胡素處理48 h，之后再加入20 mL MTT(5 g/L)于37 ℃恒溫培養(yǎng)箱中培養(yǎng)4 h，移除孔內(nèi)培養(yǎng)基，加入100 μL二甲基亞砜，570 nm波長(zhǎng)下測(cè)定A值。細(xì)胞活力結(jié)果用藥物處理組與對(duì)照組的A值比值表示。

3.5 Western blot實(shí)驗(yàn) PC9 CD133+細(xì)胞亞群用2 mg/L c-met質(zhì)粒轉(zhuǎn)染24 h后用0.04 μmol/L吉非替尼和10 μmol/L歐前胡素處理細(xì)胞48 h，之后用蛋白提取液提取腫瘤細(xì)胞中的總蛋白質(zhì)。將等量的總蛋白質(zhì)用12.5% SDS-PAGE分離。分離完畢后通過(guò)電轉(zhuǎn)方法將蛋白質(zhì)從分離膠轉(zhuǎn)到PVDF膜上，用抗c-met、磷酸化EGFR、磷酸化PI3K、磷酸化AKT、cleaved caspase-9、cleaved caspase-7、cleaved caspase-3和β-actin 兔抗人抗體孵育過(guò)夜，之后再用帶辣根過(guò)氧化物酶的 Ⅱ 抗孵育2 h，蛋白條帶用ECL試劑盒顯色發(fā)光。

3.6 細(xì)胞凋亡實(shí)驗(yàn) PC9 CD133+細(xì)胞亞群用2 mg/L c-met質(zhì)粒轉(zhuǎn)染24 h后用0.04 μmol/L吉非替尼和10 μmol/L歐前胡素處理細(xì)胞48 h，之后按照凋亡試劑盒說(shuō)明書(shū)步驟將碘化丙啶和Annexin V加入細(xì)胞中孵育20 min，采用流式細(xì)胞術(shù)檢測(cè)腫瘤細(xì)胞的凋亡。

4 統(tǒng)計(jì)學(xué)處理

用SPSS 14.0統(tǒng)計(jì)分析軟件進(jìn)行數(shù)據(jù)處理。所有實(shí)驗(yàn)重復(fù)3次，實(shí)驗(yàn)數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示。多組間均數(shù)的比較采用單因素方差分析(one-way ANOVA)，兩組間均數(shù)的比較采用 Student’st檢驗(yàn)，以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

結(jié) 果

1 PC9 CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性顯著低于PC9 CD133-細(xì)胞亞群

MTT實(shí)驗(yàn)結(jié)果顯示吉非替尼對(duì)PC9 CD133+細(xì)胞亞群的殺傷力顯著低于PC9 CD133-細(xì)胞亞群，并且PC9 CD133+細(xì)胞亞群的吉非替尼IC50顯著高于PC9 CD133-細(xì)胞亞群，這些結(jié)果表明PC9 CD133+細(xì)胞亞群對(duì)吉非替尼存在抵抗性。Western blot實(shí)驗(yàn)結(jié)果顯示吉非替尼能顯著抑制PC9 CD133-細(xì)胞亞群的EGFR、PI3K、AKT的磷酸化，然而吉非替尼僅能抑制PC9 CD133+細(xì)胞亞群EGFR的活化，而對(duì)其下游PI3K和AKT磷酸化的抑制效果不明顯，表明PC9 CD133+細(xì)胞亞群PI3K/AKT的活化不依賴于EGFR的磷酸化，這可能是PC9 CD133+細(xì)胞亞群對(duì)吉非替尼不敏感的分子機(jī)制。另外，流式細(xì)胞實(shí)驗(yàn)結(jié)果顯示在吉非替尼處理下，PC9 CD133+細(xì)胞亞群的凋亡率顯著低于PC9 CD133-細(xì)胞亞群，表明PC9 CD133+細(xì)胞亞群對(duì)吉非替尼誘導(dǎo)的凋亡信號(hào)不敏感，見(jiàn)圖1。

2 歐前胡素增強(qiáng)PC9 CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性

流式細(xì)胞實(shí)驗(yàn)結(jié)果顯示吉非替尼單獨(dú)治療能顯著提高PC9細(xì)胞系中CD133+細(xì)胞亞群的比例，然而聯(lián)用歐前胡素后，PC9 CD133+細(xì)胞亞群的比例顯著下降。另外，MTT實(shí)驗(yàn)結(jié)果顯示聯(lián)用歐前胡素能顯著提高吉非替尼對(duì)PC9 CD133+細(xì)胞亞群的殺傷力，這些結(jié)果表明聯(lián)用歐前胡素能顯著提高PC9 CD133+細(xì)胞亞群對(duì)吉非替尼誘導(dǎo)的細(xì)胞死亡的敏感性，兩者存在協(xié)同效應(yīng)，見(jiàn)圖2。

3 歐前胡素通過(guò)下調(diào)c-met的表達(dá)提高PC9 CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性

Western blot實(shí)驗(yàn)結(jié)果顯示PC9 CD133+細(xì)胞亞群中的c-met表達(dá)水平顯著高于PC9 CD133-細(xì)胞亞群，提示c-met可能在PC9 CD133+細(xì)胞亞群中發(fā)揮重要作用。另外，歐前胡素能顯著降低PC9 CD133+細(xì)胞亞群中c-met的表達(dá)水平，而吉非替尼對(duì)c-met的表達(dá)無(wú)明顯影響，提示歐前胡素可能以c-met為靶點(diǎn)增強(qiáng)PC9 CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性。MTT實(shí)驗(yàn)結(jié)果顯示在PC9 CD133+細(xì)胞亞群中轉(zhuǎn)染c-met表達(dá)質(zhì)粒后，歐前胡素聯(lián)合吉非替尼對(duì)PC9 CD133+細(xì)胞亞群的殺傷力受到顯著抑制，表明歐前胡素與吉非替尼的聯(lián)合殺傷效應(yīng)依賴于c-met的下調(diào)。Western blot實(shí)驗(yàn)結(jié)果顯示歐前胡素聯(lián)合吉非替尼能顯著抑制PC9 CD133+細(xì)胞亞群的EGFR、PI3K、AKT的磷酸化水平，然而轉(zhuǎn)染c-met質(zhì)粒后，兩者聯(lián)合對(duì)PI3K和AKT磷酸化的抑制作用喪失，表明c-met是PI3K/AKT分子的上游信號(hào)，且PC9 CD133+細(xì)胞亞群通過(guò)過(guò)表達(dá)c-met使PI3K/AKT分子持續(xù)活化。流式細(xì)胞實(shí)驗(yàn)和Western blot實(shí)驗(yàn)結(jié)果顯示轉(zhuǎn)染c-met質(zhì)粒能顯著抑制歐前胡素對(duì)吉非替尼誘導(dǎo)PC9 CD133+細(xì)胞亞群的凋亡和caspase活化的促進(jìn)作用，表明歐前胡素通過(guò)調(diào)控c-met提高PC9 CD133+細(xì)胞亞群對(duì)吉非替尼依賴的凋亡信號(hào)通路的敏感性，見(jiàn)圖3。

討 論

歐前胡素是一種天然藥物，具有毒性低，副作用小的優(yōu)點(diǎn)，文獻(xiàn)表明歐前胡素的輔助治療能有效提高化療藥物對(duì)腫瘤細(xì)胞的殺傷力，因此有良好的應(yīng)用前景[10]。然而，歐前胡素是否能在腫瘤干細(xì)胞中發(fā)揮良好的輔助治療作用，至今還未有充分研究。近期的研究表明腫瘤干細(xì)胞對(duì)藥物治療的低敏感性是造成腫瘤治療失敗的重要原因[11]。研究報(bào)道在肺癌中，CD133可作為腫瘤干細(xì)胞的表面標(biāo)志[9]。而在本研究中，當(dāng)用吉非替尼直接處理非小細(xì)胞肺癌細(xì)胞后發(fā)現(xiàn)，PC9細(xì)胞系中CD133+細(xì)胞亞群比例顯著提高，這可能是因?yàn)镻C9 CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性顯著低于PC9 CD133-細(xì)胞亞群，從而導(dǎo)致PC9 CD133+細(xì)胞亞群在吉非替尼的處理下存活下來(lái)從而引起所占比例的升高。當(dāng)用歐前胡素進(jìn)行聯(lián)合治療后，由于PC9 CD133+細(xì)胞亞群在歐前胡素的作用下對(duì)吉非替尼的敏感性顯著提高，從而導(dǎo)致CD133+細(xì)胞亞群的占比顯著下降。本研究結(jié)果表明歐前胡素能以CD133+肺癌細(xì)胞為靶點(diǎn)提高吉非替尼對(duì)非小細(xì)胞肺癌的治療效果。

Figure 1.The sensitivity of the PC9 CD133+cell subsets to gefitinib was significantly lower than the PC9 CD133-cell subsets. A: the cytotoxicity of gefitinib to PC9 CD133+cell subsets was significantly lower than the PC9 CD133-cell subsets; B: the IC50of gefitinib to PC9 CD133+cell subsets was significantly higher than the PC9 CD133-cell subsets; C: gefitinib failed to inhibit the phosphorylation of PI3K/AKT in the PC9 CD133+cell subsets; D: PC9 CD133+cell subsets showed low sensitivity to the gefitinib-induced apoptosis. Mean±SD.n=3.*P<0.05vsPC9 CD133-;#P<0.05vsPC9 CD133--control;△P<0.05 PC9 CD133+-control.

圖1 PC9 CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性顯著低于PC9 CD133-細(xì)胞亞群

研究表明，眾多腫瘤類型(特別是非小細(xì)胞肺癌)細(xì)胞表面的EGFR會(huì)發(fā)生過(guò)表達(dá)或突變。這些EGFR突變型的肺癌細(xì)胞(如PC9)對(duì)吉非替尼等表皮生長(zhǎng)因子受體酪氨酸激酶抑制劑表現(xiàn)為高度敏感，因此吉非替尼目前是治療非小細(xì)胞肺癌的一線藥物[12-13]。在EGFR信號(hào)通路中，活化的EGFR能導(dǎo)致PI3K及其下游分子AKT的磷酸化使之發(fā)生活化。腫瘤細(xì)胞中AKT的持續(xù)活化又能通過(guò)激活下游分子促進(jìn)細(xì)胞的增殖和存活，同時(shí)抑制凋亡的發(fā)生[14-15]。然而在本研究中，實(shí)驗(yàn)結(jié)果顯示吉非替尼雖然能抑制PC9 CD133+細(xì)胞亞群的EGFR信號(hào)，但卻不能抑制這些細(xì)胞中PI3K/AKT的活化，從而導(dǎo)致肺癌CD133+細(xì)胞亞群對(duì)吉非替尼誘導(dǎo)的凋亡途徑產(chǎn)生抵抗。因此本研究的結(jié)果表明非小細(xì)胞肺癌CD133+細(xì)胞可能通過(guò)另外一種信號(hào)通路維持細(xì)胞中PI3K/AKT的活化。

Figure 2.Imperatorin enhanced the sensitivity of the PC9 CD133+cell subsets to gefitinib. A: imperatorin significantly inhibited the enrichment of the PC9 CD133+cell subsets population induced by gefitinib; B: imperatorin enhanced the cytotoxicity of gefitinib to the PC9 CD133+cell subsets. Mean±SD.n=3.*P<0.05vscontrol;#P<0.05vsgefitinib.

圖2 歐前胡素增強(qiáng)PC9 CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性

Figure 3. Imperatorin increased the sensitivity of the lung cancer CD133+cell subsets to gefitinib by down-regulating the expression of c-met. A: the expression of c-met was up-regulated in the PC9 CD133+cell subsets; B: imperatorin inhibited the expression of c-met in the PC9 CD133+cell subsets; C: transfection of c-met plasmid inhibited the cell death of PC9 CD133+cell subsets induced by the combination of imperatorin with gefitinib; D: transfection of c-met plasmid inhibited the phosphorylation of PI3K and AKT in the PC9 CD133+cell subsets induced by the combination of imperatorin with gefitinib; E: transfection of c-met plasmid inhibited the apoptosis of PC9 CD133+cell subsets induced by the combination of imperatorin with gefitinib; F: transfection of c-met plasmid inhibited the activation of caspases in the PC9 CD133+cell subsets induced by the combination of imperatorin with gefitinib. Mean±SD.n=3.△P<0.05vsPC9 CD133-;*P<0.05vscontrol;#P<0.05vsimperatorin+gefitinib.

圖3 歐前胡素通過(guò)下調(diào)c-met的表達(dá)提高肺癌CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性

c-met是肝細(xì)胞生長(zhǎng)因子(hepatocyte growth factor，HGF)的受體，有報(bào)道指出HGF/c-met途徑能激活腫瘤細(xì)胞中包括PI3K/AKT在內(nèi)的多種促進(jìn)腫瘤存活和生長(zhǎng)的信號(hào)通路[16-17]。此外，近期的研究表明c-met的過(guò)度活化是誘導(dǎo)肺癌細(xì)胞產(chǎn)生對(duì)吉非替尼獲得性耐藥的重要機(jī)制，腫瘤細(xì)胞中過(guò)表達(dá)的c-met同樣能顯著激活PI3K/AKT分子而不依賴于EGFR的活化[18]。因此針對(duì)c-met及其下游信號(hào)分子的靶向治療已成為增強(qiáng)抗腫瘤藥物療效的重要策略。本研究的結(jié)果表明c-met蛋白在肺癌CD133+細(xì)胞亞群中發(fā)生過(guò)表達(dá)。當(dāng)用歐前胡素對(duì)肺癌CD133+細(xì)胞亞群進(jìn)行處理后，細(xì)胞中c-met的表達(dá)水平顯著降低，使c-met/PI3K/AKT通路受到抑制，從而使肺癌CD133+細(xì)胞亞群中PI3K/AKT的活化恢復(fù)對(duì)EGFR信號(hào)的依賴性。因此，當(dāng)用歐前胡素和吉非替尼對(duì)肺癌CD133+細(xì)胞亞群進(jìn)行聯(lián)合治療時(shí)，腫瘤細(xì)胞的PI3K/AKT受到顯著抑制，進(jìn)而誘導(dǎo)CD133+肺癌細(xì)胞發(fā)生caspase的活化和凋亡的發(fā)生。

綜上所述，本研究證明了歐前胡素通過(guò)抑制c-met的表達(dá)使肺癌CD133+細(xì)胞亞群中的PI3K/AKT恢復(fù)對(duì)EGFR信號(hào)的依賴性，從而提高肺癌CD133+細(xì)胞亞群對(duì)吉非替尼的敏感性。這些研究為吉非替尼等EGFR酪氨酸激酶抑制劑的化療增效提供了新的策略和思路。

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(責(zé)任編輯： 林白霜， 余小慧)

Imperatorin increased sensitivity of lung cancer CD133+cell subsets to gefitinib by down-regulating c-met expression

WU Qian-bo1,2, ZHANG Min3, TAO Zhi-hua1

(1DepartmentofClinicalLaboratory,TheSecondAffiliatedHospital,MedicalCollegeofZhejiangUniversity,2DepartmentofBloodDonationService,BloodCenterofZhejiangProvince,3DepartmentofClinicalLaboratory,TongdeHospitalofZhejiangProvince,Hangzhou310000,China.E-mail:zhejiangwqb2@163.com)

AIM: To investigate the role of imperatorin in reversing the resistance of the PC9 CD133+cell subsets to gefitinib. METHODS: MTT assay was performed to evaluate the viability of PC9 cells treated with imperatorin and gefitinib. The expression of c-met, activation of caspases and phosphorylation of epidermal growth factor receptor (EGFR), PI3K and AKT in the PC9 cells treated with imperatorin and gefitinib were determined by Western blot. The percentage of CD133+cell subsets population and the apoptotic rate of the PC9 cells treated with imperatorin and gefitinib were analyzed by flow cytometry. RESULTS: The sensitivity of the PC9 CD133+cell subsets to gefitinib was significantly lower than that of the PC9 CD133-cell subsets. Treatment with gefitinib alone significantly inhibited the protein levels of EGFR/PI3K/AKT in the PC9 CD133-cell subsets but not the PC9 CD133+cell subsets. Treatment with gefitinib alone increased the percentage of CD133+cell subsets population in the PC9 cells. However, combination of gefitinib with imperatorin significantly inhibited the enrichment of CD133+cell subsets population. Imperatorin down-regulated c-met expression, suggesting the c-met was the target of imperatorin in the PC9 CD133+cell subsets. The results of MTT assay, Western blot analysis and flow cytometry indicated that imperatorin increased the gefitinib induced inhibition of PI3K/AKT protein levels by down-regulating the expression of c-met, which subsequently induced the cleavage of caspases and apoptosis in the PC9 CD133+cell subsets.CONCLUSION: Imperatorin increases the sensitivity of lung cancer CD133+cell subsets to gefitinib by down-regulating the expression of c-met, and the synergistic anti-tumor effect exists between imperatorin and gefitinib.

Imperatorin; c-met; Gefitinib; PC9 CD133+cell subsets; PI3K/AKT pathway

1000- 4718(2017)01- 0046- 07

2016- 09- 14

2016- 11- 12

R730.23; R735.7

A

10.3969/j.issn.1000- 4718.2017.01.008

雜志網(wǎng)址： http://www.cjpp.net

△通訊作者 Tel: 0571-89972218; E-mail: zhejiangwqb2@163.com

▲并列第1作者