p75NTR對(duì)乳腺癌耐藥細(xì)胞MDA-MB-231/ADR耐藥及細(xì)胞周期的影響

鄧曉芳，徐崗，何利珍

(廣州醫(yī)科大學(xué)附屬腫瘤醫(yī)院內(nèi)二科1、胸外科2、病理科3，廣東廣州510095)

p75NTR對(duì)乳腺癌耐藥細(xì)胞MDA-MB-231/ADR耐藥及細(xì)胞周期的影響

鄧曉芳1，徐崗2，何利珍3

(廣州醫(yī)科大學(xué)附屬腫瘤醫(yī)院內(nèi)二科1、胸外科2、病理科3，廣東廣州510095)

目的探討神經(jīng)營(yíng)養(yǎng)因子受體(p75NTR)對(duì)乳腺癌耐藥細(xì)胞MDA-MB-231/ADR化療耐藥性及細(xì)胞周期的影響。方法采用CCK-8法檢測(cè)轉(zhuǎn)染p75NTR及p75NTR-siRNA后乳腺癌耐藥細(xì)胞系MDA-MB-231/ ADR對(duì)耐藥的影響；FCM檢測(cè)轉(zhuǎn)染p75NTR及p75NTR-siRNA的乳腺癌及耐藥細(xì)胞的細(xì)胞周期分布及細(xì)胞凋亡的影響。結(jié)果過表達(dá)p75NTR可有效增強(qiáng)MDA-MB-231/ADR細(xì)胞對(duì)ADM、GEM和OXA的耐藥性(P＜0.05)；抑制p75NTR的表達(dá)可抑制MDA-MB-231/ADR細(xì)胞對(duì)ADM、GEM和OXA的耐藥性(P＜0.05)。轉(zhuǎn)染pcDNA3.1-p75NTR后，MDA-MB-231/ADR-p75NTR細(xì)胞的細(xì)胞周期阻滯于G0/G1期，G0/G1期細(xì)胞增至(61.12± 1.89)%，S期細(xì)胞減至(32.76±0.23)%，凋亡率減至(16.83±1.29)%，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)；轉(zhuǎn)染p75NTR-siRNA后MDA-MB-231/ADR-p75NTRsi1細(xì)胞的細(xì)胞周期于G0/G1期減至(39.26±1.31)%，S期細(xì)胞增至(52.88±1.74)%，凋亡率增至(21.49±1.41)%,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。結(jié)論過表達(dá)p75NTR能夠使乳腺癌耐藥細(xì)胞MDA-MB-231/ADR細(xì)胞周期阻滯于G0/G1期，促進(jìn)細(xì)胞存活，抑制其凋亡，降低MDA-MB-231/ADR細(xì)胞對(duì)化療藥物的敏感性而促進(jìn)其多藥耐藥性。

乳腺癌；神經(jīng)營(yíng)養(yǎng)因子受體；多藥耐藥；細(xì)胞周期

乳腺癌是女性最常見的惡性腫瘤，在國(guó)內(nèi)發(fā)病率每年增加約3%[1]。提高乳腺癌的治療療效是目前亟待解決的問題之一。特別是三陰型乳腺癌，惡性程度高、進(jìn)展快、侵襲性強(qiáng)，更易復(fù)發(fā)和轉(zhuǎn)移。三陰型乳腺癌的分子靶向治療作為一種新的治療方式正逐漸得到研究和應(yīng)用[2]。神經(jīng)營(yíng)養(yǎng)因子受體(the neurotrophins receptor p75，p75NTR)是神經(jīng)營(yíng)養(yǎng)因子受體，本小組在前期研究中發(fā)現(xiàn)p75NTR的表達(dá)可能與三陰乳腺癌及Luminal B亞型相關(guān)[3]，并利用基因重組技術(shù)，構(gòu)建了p75NTR的正義及反義表達(dá)載體，成功轉(zhuǎn)染至乳腺癌耐藥細(xì)胞MDA-MB-231/ADR中[4]。但p75NTR與乳腺癌耐藥之間的關(guān)系尚未完全明確，本研究擬進(jìn)一步以p75NTR為靶點(diǎn)，對(duì)轉(zhuǎn)染后細(xì)胞進(jìn)行耐藥性、細(xì)胞周期及凋亡等方面的研究，探討其可能影響乳腺癌耐藥細(xì)胞耐藥的部分機(jī)制。

1 材料與方法

1.1 材料乳腺癌細(xì)胞系MDA-MB-231購(gòu)于軍事科學(xué)研究院，乳腺癌耐藥細(xì)胞系MDA-MB-231/ ADR由本所建立；RPMI1640培養(yǎng)基、胎牛血清購(gòu)自Gibco；阿霉素(ADM)購(gòu)自深圳萬樂藥業(yè)公司；吉西他濱(GEM)購(gòu)自江蘇豪森藥業(yè)公司；奧沙利鉑(OXA)購(gòu)自山東齊魯藥業(yè)公司；ECL Western blot檢測(cè)試劑盒購(gòu)自Amersham公司。

1.2 細(xì)胞培養(yǎng)MDA-MB-231細(xì)胞培養(yǎng)于含10%胎牛血清、青霉素(100 μg/mL)、鏈霉素(100 μg/mL)的RPMI1640培養(yǎng)液，MDA-MB-231/ADR在上述培養(yǎng)條件下，加入1.0 μg/mLADM培養(yǎng)，細(xì)胞置于含5% CO2飽和濕度，37℃培養(yǎng)箱中培育。實(shí)驗(yàn)前2周更換為不含ADM的RPMI1640培養(yǎng)液，經(jīng)3～4次傳代后，取對(duì)數(shù)生長(zhǎng)細(xì)胞進(jìn)行實(shí)驗(yàn)。

1.3 CCK-8法檢測(cè)p75NTR對(duì)MDA-MB-231/ ADR細(xì)胞耐藥性的影響取對(duì)數(shù)生長(zhǎng)期的各轉(zhuǎn)染細(xì)胞，制備成單細(xì)胞懸液，記數(shù)細(xì)胞，調(diào)整細(xì)胞密度為6× 104個(gè)/mL，每孔100 μL接種于96孔板中。實(shí)驗(yàn)分成四組：MDA-MB-231/ADR-p75NTR組、MDA-MB-231/ ADR-pcDNA3.1組、MDA-MB-231/ADR-p75NTR-siRNA組、MDA-MB-231ADR-siRNA組；每組設(shè)3個(gè)復(fù)孔，細(xì)胞培養(yǎng)24 h后，分別加入不同濃度GEM(0.02 μg/mL、0.2 μg/mL、2 μg/mL、8 μg/mL、16 μg/mL、32 μg/mL、64 μg/mL、128 μg/mL)、OXA(0.5 μg/mL、5 μg/mL、10 μg/mL、20 μg/mL、40 μg/mL、80 μg/mL、160 μg/mL、240 μg/mL)和ADM(0.2 μg/mL、2 μg/mL、10 μg/mL、50 μg/mL、100、150 μg/mL、200 μg/mL、400 μg/mL)；在培養(yǎng)箱中培養(yǎng)48 h，檢測(cè)各組細(xì)胞的OD值，計(jì)算細(xì)胞增殖抑制率(IR)，IR=(1-藥物組OD值/對(duì)照組OD值)×100%，得到半數(shù)抑制濃度(IC50)值。

1.4 FCM法檢測(cè)p75NTR對(duì)MDA-MB-231/ ADR細(xì)胞周期的影響取對(duì)數(shù)生長(zhǎng)期的各轉(zhuǎn)染細(xì)胞，制備成單細(xì)胞懸液，調(diào)整細(xì)胞密度為6×104個(gè)/mL，接種于6孔板中，2 mL/孔。實(shí)驗(yàn)分組同1.3部分。每組設(shè)3個(gè)復(fù)孔，細(xì)胞培養(yǎng)24 h后，分別加入同一濃度(按1.3的方法獲得的IC50值的濃度)的ADM；繼續(xù)培養(yǎng)48 h，收集細(xì)胞用PBS洗滌3次，上流式細(xì)胞儀檢測(cè)。

1.5 FCM檢測(cè)p75NTR對(duì)MDA-MB-231/ADR細(xì)胞凋亡改變的影響取對(duì)數(shù)生長(zhǎng)期的各轉(zhuǎn)染細(xì)胞，實(shí)驗(yàn)分組同1.3部分。培養(yǎng)、收集、洗滌并重懸細(xì)胞，加入5 μL Annexin VFITC，輕輕振蕩混勻，室溫避光孵育10 min，再加入5 μL PI，室溫避光孵育5 min，輕輕振蕩混勻，上流式細(xì)胞儀檢測(cè)。

1.6 統(tǒng)計(jì)學(xué)方法應(yīng)用SPSS19.0統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)分析，計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差()表示，兩樣本均數(shù)比較采用t檢驗(yàn)，以P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 CCK-8法檢測(cè)p75NTR對(duì)MDA-MB-231/ ADR細(xì)胞耐藥性的參與作用CKK-8檢測(cè)結(jié)果(表1)顯示，MDA-MB-231/ADR對(duì)ADM、GEM和OXA均顯示其耐藥性。轉(zhuǎn)染pcDNA3.1-p75NTR后，MDA-MB-231/ADR-p75NTR、MDA-MB-231-p75NTR細(xì)胞對(duì)三種抗腫瘤藥物的耐藥性增強(qiáng)，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)；轉(zhuǎn)染p75NTR-siRNA后MDA-MB-231/ ADR-p75NTRsi1、MDA-MB-231-p75NTRsi1細(xì)胞對(duì)三種抗腫瘤藥物的敏感性增強(qiáng)，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)；結(jié)果提示過表達(dá)p75NTR可有效增強(qiáng)MDA-MB-231/ADR細(xì)胞對(duì)ADM、GEM和OXA的耐藥性；抑制p75NTR的表達(dá)可抑制MDA-MB-231/ ADR細(xì)胞對(duì)ADM、GEM和OXA的耐藥性。

表1 p75NTR對(duì)乳腺癌耐藥細(xì)胞MDA-MB-231/ADR耐藥的參與作用

表1 p75NTR對(duì)乳腺癌耐藥細(xì)胞MDA-MB-231/ADR耐藥的參與作用

組別孔數(shù)IC50(μg/mL) MDA-MB-231/ADR-p75NTR MDA-MB-231/ADR-pcDNA t值P值MDA-MB-231/ADR-pSilencer MDA-MB-231/ADR-p75NTRsi1 t值P值3 3 3 3 ADM 183.37±4.45 124.28±4.94 8.31＜0.05 124.48±4.35 66.9±3.17 13.15＜0.05 GEM 25.43±2.15 12.13±2.02 9.76＜0.05 13.24±1.86 7.36±1.43 15.48＜0.05 OXA 65.72±1.85 38.45±1.56 11.78＜0.05 40.15±1.73 28.17±1.42 12.86＜0.05

2.2 p75NTR對(duì)MDA-MB-231/ADR細(xì)胞周期影響FCM檢測(cè)結(jié)果(圖1和表2)顯示，轉(zhuǎn)染pcDNA3.1-p75NTR后，MDA-MB-231/ADR-p75NTR細(xì)胞的細(xì)胞周期阻滯于G0/G1期，于G0/G1期增多，S期細(xì)胞減少；轉(zhuǎn)染p75NTR-siRNA后MDA-MB-231/ ADR-p75NTRsi1細(xì)胞組的細(xì)胞周期于G0/G1期減少，S期細(xì)胞增多，兩組間比較差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。

圖1 FCM檢測(cè)p75NTR對(duì)MDA-MB-231/ADR細(xì)胞的細(xì)胞周期分布影響

圖2 FCM檢測(cè)p75NTR對(duì)MDA-MB-231/ADR細(xì)胞的凋亡的影響

表2 p75NTR對(duì)MDA-MB-231/ADR細(xì)胞的周期分布及凋亡的影響

表2 p75NTR對(duì)MDA-MB-231/ADR細(xì)胞的周期分布及凋亡的影響

組別孔數(shù)細(xì)胞周期凋亡S MDA-MB-231/ADR-p75NTR MDA-MB-231/ADR-pcDNA t值P值MDA-MB-231/ADR-pSilencer MDA-MB-231/ADR-p75NTRsi1 t值P值3 3 3 3 G0/G161.12±1.89 51.57±1.28 3.68＜0.05 52.43±1.74 39.26±1.31 11.49＜0.05 32.76±0.23 42.68±1.37 4.51＜0.05 43.19±1.16 52.88±1.74 12.12＜0.05 G2/M 6.12±0.48 5.75±0.88 3.8＜0.05 4.38±0.73 7.86±1.02 16.09＜0.05 16.83±1.29 18.59±1.53 6.59＜0.05 20.57±1.65 21.49±1.41 7.24＜0.05

2.3 p75NTR對(duì)MDA-MB-231/ADR細(xì)胞凋亡的影響p75NTR可影響MDA-MB-231/ADR細(xì)胞凋亡。FCM檢測(cè)結(jié)果(圖2和表2)顯示，轉(zhuǎn)染pcDNA3.1-p75NTR后，MDA-MB-231/ADR-p75NTR細(xì)胞的凋亡率減少，存活率增多；轉(zhuǎn)染p75NTR-siRNA后MDA-MB-231/ADR-p75NTRsi1細(xì)胞組的凋亡率增多，存活率減少，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。

3 討論

p75NTR又名神經(jīng)生長(zhǎng)因子受體(nerve growth factor receptor，NGFR)，定位于人染色體17q12-17q22，包含6個(gè)外顯子，屬于腫瘤壞死因子受體超家族的一員?，F(xiàn)有研究表明p75NTR的表達(dá)有利于促進(jìn)食管癌、乳腺癌細(xì)胞的生長(zhǎng)和強(qiáng)烈抵制抗腫瘤治療[5-6]，也可以作為生存受體促進(jìn)黑色素瘤細(xì)胞的腦轉(zhuǎn)移[7]。p75NTR陽性腫瘤表現(xiàn)了許多三陰型乳腺癌的特征，如高組織分化，高增殖指數(shù)及與肌上皮細(xì)胞標(biāo)志物相關(guān)[8]。三陰型乳腺癌惡性程度高，易轉(zhuǎn)移復(fù)發(fā)，臨床治療效果差，預(yù)后差[9]，推測(cè)可能與此機(jī)制有關(guān)。隨著對(duì)p75NTR研究的深入，發(fā)現(xiàn)多種藥物可通過作用于p75NTR起到調(diào)控腫瘤細(xì)胞耐藥的作用[10-12]。通過用p75NTR的過表達(dá)載體上調(diào)乳腺癌細(xì)胞中p75NTR的表達(dá)；p75NTR特異性siRNA抑制乳腺癌細(xì)胞中p75NTR的表達(dá)，發(fā)現(xiàn)過表達(dá)p75NTR可有效增強(qiáng)MDA-MB-231/ADR細(xì)胞對(duì)ADM、GEM和OXA的耐藥性；抑制p75NTR的表達(dá)可降低MDA-MB-231/ ADR細(xì)胞對(duì)ADM、GEM和OXA的耐藥性。

惡性腫瘤的發(fā)生發(fā)展通常表現(xiàn)為細(xì)胞周期異常、細(xì)胞周期調(diào)控機(jī)制紊亂以及腫瘤細(xì)胞的無限增殖。G1/S期和G2/M期是細(xì)胞周期的關(guān)鍵點(diǎn)。Vrbeke等[13]的研究認(rèn)為p75NTR的過表達(dá)，可上調(diào)p21，而p21是細(xì)胞周期cyclin/CDK抑制劑，抑制細(xì)胞從G1期到S期，使細(xì)胞停留在G0/G1期，處于慢周期或靜息狀態(tài)，對(duì)化療藥物敏感性下降，增加了細(xì)胞的耐藥性。本研究發(fā)現(xiàn)p75NTR對(duì)MDA-MB-231/ADR細(xì)胞周期有一定影響，過表達(dá)p75NTR細(xì)胞可使細(xì)胞周期阻滯于G0/G1期，G0/G1期細(xì)胞比率高于MDA-MB-231/ ADR-pcDNA組，S期細(xì)胞比率亦低于后者；抑制p75NTR表達(dá)后MDA-MB-231/ADR-p75NTRsi1細(xì)胞組的細(xì)胞周期于G0/G1期細(xì)胞比率低于MDA-MB-231/ADR-pSilencer組，S期細(xì)胞比率高于后者。本結(jié)果提示，通過調(diào)節(jié)p75NTR的表達(dá)可影響耐藥細(xì)胞周期進(jìn)程從而在發(fā)病過程中發(fā)揮重要作用，過表達(dá)p75NTR使MDA-MB-231/ADR細(xì)胞周期阻滯于G0/G1期，增殖細(xì)胞不能通過“G1/S”調(diào)節(jié)點(diǎn)，處于慢周期或靜息狀態(tài)的細(xì)胞增多，細(xì)胞增殖延緩，對(duì)化療敏感性下降，增加了細(xì)胞的耐藥性。而抑制p75NTR的表達(dá)使MDA-MB-231/ADR細(xì)胞周期于G0/G1期減少，處于慢周期或靜息狀態(tài)細(xì)胞減少，細(xì)胞增殖加快，對(duì)化療敏感性增加，從而逆轉(zhuǎn)細(xì)胞的耐藥性。

p75NTR可通過減少腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(TRAIL)誘導(dǎo)的PARP和caspase3分解促進(jìn)細(xì)胞的存活，且其促生存作用是獨(dú)立的，與PI3-激酶，MAPK和NF-κB無關(guān)，提示p75NTR可通過多元通路發(fā)揮其促生存作用[14]。本研究FCM檢測(cè)結(jié)果顯示，過表達(dá)p75NTR的MDA-MB-231/ADR-p75NTR細(xì)胞凋亡率明顯低于抑制p75NTR表達(dá)的MDA-MB-231/ ADR-p75NTRsi1細(xì)胞，證實(shí)過表達(dá)p75NTR細(xì)胞更能抵抗凋亡，而抑制p75NTR的表達(dá)使MDA-MB-231/ ADR-p75NTRsi1細(xì)胞的凋亡率增加。誘導(dǎo)細(xì)胞凋亡是絕大多數(shù)抗腫瘤藥物殺傷腫瘤細(xì)胞的共同通路，凋亡受抑可能是腫瘤細(xì)胞產(chǎn)生耐藥的機(jī)制之一，Asakura等[15]認(rèn)為在凋亡過程中Bcl-2/Bax的比值參與調(diào)節(jié)線粒體滲透小孔使細(xì)胞色素C從線粒體釋放到胞液，這與其他關(guān)于多藥耐藥性發(fā)生機(jī)制一樣具有普遍意義。

綜上所述，過表達(dá)p75NTR可有效增強(qiáng)MDA-MB-231/ADR細(xì)胞對(duì)ADM、GEM和OXA的耐藥性，使MDA-MB-231/ADR細(xì)胞周期阻滯于G0/G1期，并更能抵抗細(xì)胞的凋亡，抑制p75NTR表達(dá)則呈相反的效應(yīng)。這為p75NTR增強(qiáng)乳腺癌耐藥細(xì)胞株MDA-MB-231/ADR耐藥的研究提供實(shí)驗(yàn)依據(jù)，但其在細(xì)胞內(nèi)通過何種分子信號(hào)轉(zhuǎn)導(dǎo)通路參與了乳腺癌耐藥細(xì)胞的耐藥機(jī)制還需要進(jìn)一步的研究。

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Effects of neurotrophin receptor p75NTR on drug resistance and cell cycle in breast cancer resistant cell line MDA-MB-231/ADR.

DENG Xiao-fang1,XU Gang2,HE Li-zhen3.Second Department of Internal Medicine1,Department of Thoracic Surgery2,Department of Pathology3,Cancer Center of Guangzhou Medical University,Guangzhou 510095, Guangdong,CHINA

ObjectiveTo investigate the effects of neurotrophin receptor p75NTR on drug resistance and cell cycle in breast cancer resistant cell line MDA-MB-231/ADR.Methods The multi-drug resistance effects of the overexpression and knock-down p75NTR on breast cancer cell line MDA-MB-231/ADR were detected by CCK-8 assay.Flow cytometry(FCM)was used to detect the effects of the overexpression and knock-down of p75NTR on the cell cycles distribution and apoptosis.ResultsThe overexpression of p75NTR can effectively enhance the resistance of MDA-MB-231/ADR cells to Doxorubicin(ADM),Gentamicin(GEM)and Oxacillin(OXA)(P＜0.05).The inhibition of p75NTR expression can increase the sensitivity of MDA-MB-231/ADR cells to ADM,GEM and OXA(P＜0.05).After the transfection of pcDNA3.1-p75NTR,MDA-MB-231/ADR-p75NTR cell cycle arrested in G0/G1phase.The number of cells in G0/G1phase increased to(61.12±1.89)%and those in S phase decreased to(32.76±0.23)%.Additionally,apoptosis rate in S phase decreased to(16.83±1.29)%,P＜0.05.After the transfection of p75NTR-siRNA,the number of cells in G0/G1phase decreased to(39.26±1.31)%and those in S phase increased to(52.88±1.74)%.And apoptosis rate in S phase increased to(21.49±1.41)%(P＜0.05).ConclusionThe overexpression of p75NTR can make MDA-MB-231/ADR cell cycle arrest in G0/G1phase,promote cell survival,inhibit the apoptosis,reduce the sensitivity to chemotherapeutic drugs and promote its multidrug resistance.

Breast cancer;Neurotrophin receptor p75/p75NTR;Multidrug resistance;Cell cycle

10.3969/j.issn.1003-6350.2017.15.002

R737.9

A

1003—6350（2017）15—2414—04

2017-05-17)

廣東省衛(wèi)生計(jì)生委醫(yī)學(xué)科研基金(編號(hào)：B2016070)；廣州醫(yī)科大學(xué)科研資助項(xiàng)目(編號(hào)：2015C42)

鄧曉芳。E-mail：dengxf2046@126.com