Salinomycin對乳腺癌阿霉素耐藥細(xì)胞株MCF-7/DOX的增殖抑制作用及機(jī)制

劉 浩，盧敏瑩，賀智敏

(廣州醫(yī)科大學(xué)附屬腫瘤醫(yī)院腫瘤研究所，廣東 廣州 510095)

Salinomycin對乳腺癌阿霉素耐藥細(xì)胞株MCF-7/DOX的增殖抑制作用及機(jī)制

劉 浩，盧敏瑩，賀智敏

(廣州醫(yī)科大學(xué)附屬腫瘤醫(yī)院腫瘤研究所，廣東 廣州 510095)

目的 本研究旨在探討Salinomycin對乳腺癌阿霉素耐藥細(xì)胞株MCF-7/DOX增殖和凋亡的影響及可能作用機(jī)制。方法 MTS實(shí)驗(yàn)檢測Salinomycin對MCF-7/DOX細(xì)胞增殖的影響；Annexin V-FITC/PI染色檢測Salinomycin對MCF-7/DOX耐藥細(xì)胞凋亡的影響；DCFH-DA染色檢測Salinomycin對MCF-7/DOX耐藥細(xì)胞活性氧(reactive oxygen species, ROS)產(chǎn)生的影響；JC-1法測定細(xì)胞線粒體膜電位；Western blot法檢測細(xì)胞凋亡相關(guān)蛋白BAX、BCL-2、caspase-3和caspase-9的表達(dá)變化。結(jié)果 Salinomycin能明顯抑制MCF-7/DOX耐藥細(xì)胞增殖，且具有濃度依賴性；流式分析發(fā)現(xiàn)Salinomycin能夠誘導(dǎo)MCF-7/DOX細(xì)胞凋亡，增加細(xì)胞內(nèi)ROS水平，降低細(xì)胞線粒體膜電位；與對照組相比較，Salinomycin處理明顯抑制BCL-2的表達(dá)，上調(diào)BAX、cleaved caspase-3和cleaved caspase-9的蛋白表達(dá)；抗氧化劑N-acetylcysteine(NAC)則逆轉(zhuǎn)上述作用。結(jié)論 Salinomycin能夠誘導(dǎo)MCF-7/DOX細(xì)胞凋亡，其機(jī)制可能與Salinomycin誘導(dǎo)ROS的產(chǎn)生，激活線粒體凋亡途徑有關(guān)。

乳腺癌；Salinomycin；阿霉素耐藥；細(xì)胞凋亡；活性氧；線粒體膜電位

乳腺癌是女性最常見的惡性腫瘤之一。近年來，我國女性乳腺癌發(fā)病率逐年升高，并有逐步年輕化的趨勢[1]。目前化療仍是乳腺癌臨床治療的主要方法之一。阿霉素(doxorubicin，DOX)是一種周期非特異性廣譜抗癌藥，對各期細(xì)胞均有作用，具有很強(qiáng)的抗癌活性，廣泛應(yīng)用于包括乳腺癌在內(nèi)的臨床各類腫瘤化療中。然而，研究表明乳腺癌細(xì)胞易對阿霉素產(chǎn)生耐藥，導(dǎo)致化療失敗，從而使阿霉素在乳腺癌治療的應(yīng)用中受到很大限制[2]。

Salinomycin是由白色鏈球菌(streptomyces albus)經(jīng)發(fā)酵培養(yǎng)產(chǎn)生的一種一元羧酸聚醚類離子載體型抗生素類，具有殺菌、抑菌的作用[3]。自2009年，Gupta等[4]發(fā)現(xiàn)Salinomycin能選擇性抑制乳腺癌干細(xì)胞以來，Salinomycin的抗腫瘤作用越來越受到關(guān)注。隨后多項(xiàng)研究表明， Salinomycin能夠誘導(dǎo)結(jié)直腸癌[5]、肺癌[6]、前列腺癌[7]等多種腫瘤細(xì)胞的凋亡。此外，研究發(fā)現(xiàn)Salinomycin能夠逆轉(zhuǎn)人白血病KG-1a細(xì)胞的耐藥性[8]，然而相關(guān)機(jī)制還有待進(jìn)一步明確。

本實(shí)驗(yàn)旨在研究Salinomycin對體外培養(yǎng)的乳腺癌阿霉素耐藥細(xì)胞MCF-7/DOX的增殖抑制作用，并對其作用機(jī)制進(jìn)行初步探討。

1 材料與方法

1.1 材料人乳腺癌MCF-7/DOX耐藥細(xì)胞購自中科院上海細(xì)胞庫。DMEM培養(yǎng)基、胎牛血清、胰蛋白酶購自Gibco公司；Salinomycin購自Selleck公司；P-gp、BCL-2、BAX、caspase-3、caspase-9抗體購自CST公司；P-gp，β-actin抗體購自Santa Cruz公司；預(yù)染蛋白質(zhì)Marker購自Fermentas公司；HRP標(biāo)記山羊抗鼠IgG、山羊抗兔IgG購自北京鼎國生物技術(shù)有限公司；NAC、cyclosporine A、Doxorubicin、DCFH-DA、JC-1購自Sigma公司；MTS購自Promega公司；Annexin V-FITC/PI細(xì)胞凋亡檢測試劑盒購自碧云天生物技術(shù)研究所。ECL化學(xué)發(fā)光底物試劑盒購自Pierce公司；Quick Start Bradford蛋白定量試劑購自Bio-Rad公司；其余試劑均為國產(chǎn)分析純。

1.2 細(xì)胞培養(yǎng)人乳腺癌MCF-7/DOX耐藥細(xì)胞用含10%胎牛血清的DMEM培養(yǎng)液，于37℃、5% CO2的培養(yǎng)箱內(nèi)飽和濕度培養(yǎng)。將MCF-7/DOX細(xì)胞暴露于低劑量[1/10的半數(shù)抑制濃度(50% concentration of inhibition, IC50)]的阿霉素中維持細(xì)胞耐藥性。

1.3 MTS法檢測Salinomycin對MCF-7/DOX耐藥細(xì)胞增殖的影響接種5×103個(gè)細(xì)胞每孔至96孔板，培養(yǎng)過夜使細(xì)胞貼壁。向?qū)?yīng)試驗(yàn)孔加入不同濃度的Salinomycin，繼續(xù)培養(yǎng)24、48、72 h，吸去培養(yǎng)基，加入100 μL含0.5 g·L-1MTS的RPMI 1640，繼續(xù)培養(yǎng)4 h。最后用酶標(biāo)儀測定490 nm波長下的光密度(optical density, OD)值，并計(jì)算藥物對細(xì)胞的抑制率。抑制率/%=(1-實(shí)驗(yàn)組OD值/對照組OD值)×100%。以Salinomycin濃度為橫坐標(biāo)，抑制率為縱坐標(biāo)作圖并擬合抑制曲線，并計(jì)算IC50值。

1.4 流式細(xì)胞術(shù)檢測Salinomycin對MCF-7/DOX耐藥細(xì)胞凋亡的影響接種3×105個(gè)細(xì)胞每孔到6孔板中，Salinomycin處理24、48、72 h。分別收集細(xì)胞，按照Annexin V/PI凋亡檢測試劑盒的說明書要求，先后加入Annexin V和PI，避光、室溫孵育10 min后，流式細(xì)胞儀檢測細(xì)胞凋亡。

1.5 蛋白提取和Western blot分析收集細(xì)胞，經(jīng)PBS洗滌后用三去污裂解液[50 mmol·L-1Tris-Cl (pH 8.0)，150 mmol·L-1NaCl，0.2 g·L-1疊氮鈉，100 mg·L-1Aprotin，100 mg·L-1PMSF，1 g·L-1SDS，10 g·L-1NP-40，5 g·L-1去氧膽酸鈉]裂解，離心后收集上清，用Bradford法測定蛋白濃度；等量蛋白樣品經(jīng)10%的SDS-PAGE電泳分離后，轉(zhuǎn)印至PVDF膜上，5%脫脂奶粉室溫封閉2 h，P-gp、BAX、BCL-2、caspase-3、caspase-9、α-actin一抗均以1 ∶1 000比例稀釋，4℃孵育過夜。經(jīng)PBST洗滌后，加入HRP標(biāo)記的特異性二抗以1 ∶5 000比例稀釋，室溫下孵育2 h，最后用ECL化學(xué)發(fā)光試劑對X光片顯影，掃描圖片。

1.6 DCFH-DA染色檢測Salinomycin對MCF-7/DOX耐藥細(xì)胞內(nèi)ROS的影響DCFH-DA可以自由透過細(xì)胞膜，被細(xì)胞內(nèi)部的酯酶水解后得到DCFH，不能自由通過細(xì)胞膜，從而被裝載到細(xì)胞內(nèi)部。DCFH與活性氧反應(yīng)后，被氧化生成熒光物質(zhì)DCF，在485 nm處被激發(fā)，發(fā)射波長為525 nm，產(chǎn)生的熒光信號通過流式細(xì)胞儀檢測。接種3×105個(gè)細(xì)胞每孔到6孔板中，加入不同濃度Salinomycin處理細(xì)胞，12 h后加DCFH-DA探針，37℃下孵育20分鐘，收集細(xì)胞，PBS洗滌2 次，重懸浮于400 μL PBS，迅速進(jìn)行流式細(xì)胞儀檢測，實(shí)驗(yàn)重復(fù)3次。

1.7 JC-1染色檢測Salinomycin對MCF-7/DOX耐藥細(xì)胞線粒體膜電位的影響接種3×105個(gè)細(xì)胞每孔到6孔板中，加入不同濃度Salinomycin處理細(xì)胞24 h，PBS 洗滌2次，按試劑盒說明加入1 mL細(xì)胞培養(yǎng)液和1 mL JC-1染色工作液混勻， 37℃孵育20 min， JC-1染色緩沖液洗滌2次，加入培養(yǎng)液，迅速進(jìn)行流式細(xì)胞儀檢測，JC-1單體的最大激發(fā)波長為514 nm，最大發(fā)射波長為529 nm；JC-1 聚合物的最大激發(fā)波長為585 nm，最大發(fā)射波長為590 nm，實(shí)驗(yàn)重復(fù)3次。

1.8 統(tǒng)計(jì)學(xué)方法采用SPSS 15.0軟件One-way analysis of variance方式進(jìn)行方差分析，兩兩比較采用Student′t檢驗(yàn)。

2 結(jié)果

2.1 Salinomycin抑制乳腺癌MCF-7/DOX耐藥細(xì)胞的增殖采用8個(gè)不同濃度的Salinomycin在3 個(gè)不同時(shí)間點(diǎn)對MCF-7/DOX細(xì)胞進(jìn)行處理，MTS試驗(yàn)檢測Salinomycin對細(xì)胞增殖抑制的量效和時(shí)效關(guān)系，結(jié)果表明，0、1、2、4、8、16、32、64 μmol·L-1Salinomycin作用隨著其濃度的增加和作用時(shí)間的延長，細(xì)胞存活率逐漸降低(Fig 1)。作用24、48、72 h后的IC50分別為(13.27±0.27)、(8.83±0.33)、(3.72±0.15) μmol·L-1。結(jié)果說明Salinomycin明顯抑制MCF-7/DOX耐藥細(xì)胞增殖，且具有濃度和時(shí)間依耐性。

2.2 Salinomycin對乳腺癌MCF-7/DOX耐藥細(xì)胞多藥耐藥蛋白P-糖蛋白(P-gp)表達(dá)和功能的影響采用不同濃度Salinomycin (0、4、8 μmol·L-1)分別處理MCF-7/DOX細(xì)胞48 h，Western blot 檢測Salinomycin對P-gp蛋白表達(dá)的影響，結(jié)果發(fā)現(xiàn)，Salinomycin處理對P-gp蛋白的表達(dá)沒有明顯影響(Fig 2A)。采用流式細(xì)胞儀分析發(fā)現(xiàn)，Salinomycin處理并不影響細(xì)胞內(nèi)藥物的濃度(Fig 2B)。進(jìn)一步采用Salinomycin與P-gp蛋白抑制劑cyclosporine A共處理MCF-7/DOX細(xì)胞，以阿霉素作為對照，MTT實(shí)驗(yàn)結(jié)果顯示,cyclosporine A能增強(qiáng)阿霉素對MCF-7/DOX耐藥細(xì)胞的增值抑制作用，而對Salinomycin的增值抑制效果沒有明顯的影響(Fig 2C)。結(jié)果說明Salinomycin對MCF-7/DOX耐藥細(xì)胞的增值抑制作用可能與P-gp蛋白無關(guān)。

2.3 Salinomycin誘導(dǎo)乳腺癌MCF-7/DOX耐藥細(xì)胞凋亡采用Salinomycin (8 μmol·L-1)分別處理MCF-7/DOX細(xì)胞24、48、72 h，Annexin V-FITC/PI染色，流式細(xì)胞術(shù)檢測Salinomycin對MCF-7/DOX耐藥細(xì)胞凋亡的影響。結(jié)果顯示，隨著時(shí)間的增加，MCF-7/DOX細(xì)胞的凋亡比率明顯增加，24、48、72 h凋亡比率分別為7.5%、28.2%、46.3% (Fig 3)。

Fig 1 Effects of Salinomycin on cell viability of MCF-7/DOX cells

MCF-7/DOX cells were treated with salinomycin (0, 1, 2, 4, 8, 16, 32, 64 μmol·L-1) for 24, 48, 72 h; cell viability was determined by MTS assay. The results shown representative of five independent experiments.*P<0.05vscompared to control group.

2.4 Salinomycin對凋亡相關(guān)蛋白BAX，Bcl-2，caspase-3和caspase-9表達(dá)的影響采用不同濃度Salinomycin(0, 4, 8 μmol·L-1)分別處理MCF-7/DOX細(xì)胞48 h，Western blot檢測Salinomycin對凋亡相關(guān)蛋白BAX、Bcl-2、caspase-3和caspase-9表達(dá)的影響，結(jié)果發(fā)現(xiàn)Salinomycin能明顯抑制Bcl-2的表達(dá)，而促進(jìn)BAX、cleaved caspase-3、cleaved caspase-9的表達(dá)(Fig 4)。

2.5 Salinomycin對乳腺癌MCF-7/DOX耐藥細(xì)胞ROS、線粒體膜電位的影響為了進(jìn)一步探究Salinomycin誘導(dǎo)MCF-7/DOX細(xì)胞凋亡的機(jī)制，采用DCFH-DA熒光探針標(biāo)記流式細(xì)胞儀檢測Salinomycin對細(xì)胞內(nèi)ROS產(chǎn)生的影響。結(jié)果顯示，Salinomycin處理明顯增加細(xì)胞內(nèi)DCFH熒光，并呈現(xiàn)濃度依賴性特點(diǎn)(Fig 5A)。線粒體膜電位的下降是細(xì)胞凋亡早期的一個(gè)標(biāo)志性事件，通過JC-1從紅色熒光到綠色熒光的轉(zhuǎn)變可以檢測到細(xì)胞膜電位的下降。我們發(fā)現(xiàn)Salinomycin處理MCF-7/DOX細(xì)胞24 h 后，線粒體膜電位明顯下降( Fig 5B) 。為進(jìn)一步證實(shí)Salinomycin誘導(dǎo)腫瘤細(xì)胞凋亡中是否與ROS的產(chǎn)生有關(guān)，我們在Salinomycin處理細(xì)胞時(shí)加入抗氧化劑5 mmol·L-1NAC共處理。結(jié)果顯示NAC明顯抑制ROS的產(chǎn)生(Fig 5A)，增加線粒體膜電位(Fig 5B)。NAC處理增加Bcl-2蛋白的表達(dá)，抑制BAX、cleaved caspase-3蛋白的表達(dá)( Fig 5C)。結(jié)果說明ROS的積累在Salinomycin誘導(dǎo)腫瘤細(xì)胞凋亡中起著關(guān)鍵的作用。

Fig 2 Effects of salinomycin on protein expression and function of P-gp in MCF-7/DOX cells

A: MCF-7/DOX cells were treated with salinomycin (0, 4, 8 μmol·L-1) for 48 h; the protein expression level of P-gp was determined by Western blot assay. B: MCF-7/DOX cells were co-treated with salinomycin (8 μmol·L-1) and doxorubicin (10 μmol·L-1) for 48 h, and cellular doxorubicin concentration was determined by flow cytometry. C: MCF-7/DOX cells were co-treated with salinomycin (8 μmol·L-1) and cyclosporine A (5 μmol·L-1) or doxorubicin (10 μmol·L-1) and cyclosporine A (5 μmol·L-1) for 48 h, The cell viability was determined by MTS assay. The results shown representative of three independent experiments.*P<0.05vscompared to control group.

Fig 3 Effects of salinomycin on cell apoptosis of MCF-7/DOX cells

MCF-7/DOX cells were treated with salinomycin (8 μmol·L-1) for 24 h, 48 h, 72 h. The cell apoptosis was determined by Annexin V/PI staining. The dual parameter dot plots combining Annexin V-fluorescein isothiocyanate (FITC) and PI fluorescence showed the viable cell population in the lower left quadrant (Annexin V-/PI-), apoptotic cells in the lower right quadrant (Annexin V+/PI+) and the upper right quadrant (Annexin V+/PI+), and necrotic cells in the upper left quadrant (Annexin V-/PI+). The results shown representative of three independent experiments.

Fig 4 Effects of salinomycin on protein expression of BAX, BCL-2, caspase-3 and caspase-9 of MCF-7/DOX cells

MCF-7/DOX cells were treated with salinomycin (0, 4, 8 μmol·L-1) for 48 h; the protein expression levels of BAX, BCL-2, caspase-3, and caspase-9 were determined by Western blot assay. The results shown representative of three independent experiments.

3 討論

腫瘤耐藥的發(fā)生是多因素、多途徑共同作用的結(jié)果[9]。最新研究表明腫瘤細(xì)胞對凋亡的敏感性是影響化療效果的重要因素之一[10]。細(xì)胞凋亡過程受到抑制時(shí)可導(dǎo)致腫瘤細(xì)胞耐藥的發(fā)生，尋找高效低毒的凋亡誘導(dǎo)劑，或者選擇無交叉耐藥的聯(lián)合化療方案是逆轉(zhuǎn)腫瘤耐藥性的重要策略。近年來，Salinomycin的藥理作用越來越受到關(guān)注。由于Salinomycin被發(fā)現(xiàn)能夠選擇性抑制乳腺癌干細(xì)胞，且其效率比臨床常用化療藥物紫杉醇高出100多倍[4]。一項(xiàng)最新臨床研究發(fā)現(xiàn)，每2 d靜脈給藥200～250 μg·kg-1Salinomycin能有效抑制乳腺癌、頭頸癌轉(zhuǎn)移，且無明顯的毒副作用[11]。因此Salinomycin被認(rèn)為是一類潛在的抗癌藥物[12]。

本實(shí)驗(yàn)首次觀察了Salinomycin對乳腺癌MCF-7/DOX耐藥細(xì)胞增殖的影響，結(jié)果發(fā)現(xiàn)，Salinomycin能有效抑制乳腺癌耐藥細(xì)胞的增殖。腫瘤細(xì)胞增加藥物的外排，減少藥物的吸收是其耐受化療藥物的一種主要手段。已知乳腺癌MCF-7/DOX耐藥細(xì)胞高表達(dá)多藥耐藥蛋白P-gp，抑制P-gp蛋白的表達(dá)能夠增加MCF-7/DOX耐藥細(xì)胞的化療敏感性[13]。Fuchs 等[8]研究發(fā)現(xiàn)，Salinomycin可以抑制P-gp蛋白高表達(dá)的白血病細(xì)胞增殖。我們發(fā)現(xiàn)雖然Salinomycin同樣抑制MCF-7/DOX的增殖，卻對P-gp蛋白的表達(dá)沒有影響，提示Salinomycin可以克服P-gp蛋白引起的促增殖作用，其機(jī)制與P-gp蛋白表達(dá)無關(guān)。

腫瘤細(xì)胞凋亡抵抗是腫瘤化療耐藥的另一重要原因[14]，研究發(fā)現(xiàn)Salinomycin可以克服多種凋亡抵抗促進(jìn)多種腫瘤細(xì)胞凋亡[15]。我們發(fā)現(xiàn)Salinomycin同樣能夠有效誘導(dǎo)乳腺癌MCF-7/DOX耐藥細(xì)胞凋亡，且具有時(shí)間依賴性。文獻(xiàn)報(bào)道[16]，

Fig 5 Effects of salinomycin on ROS production and mitochondrial membrane potential in MCF-7/DOX cells

A: MCF-7/DOX cells were treated with salinomycin (0, 4, 8 μmol·L-1) for 12 h, cellular ROS level was determined by DCFH-DA staining; B: MCF-7/DOX cells were treated with salinomycin (0, 4, 8 μmol·L-1) for 24 h, mitochondrial membrane potential assay kit with JC-1 was used to determine the effects of salinomycin on the change of mitochondrial membrane potential. C: MCF-7/DOX cells were co-treated with salinomycin (8 μmol·L-1) and NAC (5 mmol·L-1) for 48 h, The protein expression levels of BAX, BCL-2, and caspase-3 were determined by Western blot assay. The results shown representative of three independent experiments.

Salinomycin可以通過誘導(dǎo)氧化應(yīng)激反應(yīng)來抑制前列腺癌腫瘤細(xì)胞增殖而且不影響正常前列腺上皮細(xì)胞。Kim等同樣發(fā)現(xiàn)Salinomycin能夠引起腫瘤細(xì)胞氧化應(yīng)激反應(yīng)以及線粒體膜去極化導(dǎo)致細(xì)胞凋亡[7]。證據(jù)表明氧化應(yīng)激適應(yīng)與腫瘤細(xì)胞耐藥性具有相似的機(jī)制[17]，一些抗氧化蛋白(如PrxII、CAT等)的表達(dá)與乳腺癌、膠質(zhì)瘤、頭頸癌等腫瘤細(xì)胞耐藥性成正相關(guān)[18]。Diehn等[19]發(fā)現(xiàn)乳腺癌干細(xì)胞含有較低的ROS水平，而增加細(xì)胞內(nèi)ROS水平能增加乳腺癌干細(xì)胞對放、化療的敏感性[20]。我們發(fā)現(xiàn)Salinomycin處理：(1)明顯增加乳腺癌MCF-7/DOX耐藥細(xì)胞內(nèi)ROS的水平；(2)降低細(xì)胞線粒體膜電位；(3)下調(diào)凋亡抑制蛋白BCL-2的表達(dá)，上調(diào)促凋亡蛋白BAX的表達(dá)；(4)激活caspase-9、caspase-3蛋白。相反，抗氧化劑預(yù)處理則能夠逆轉(zhuǎn)Salinomycin的作用。結(jié)果說明Salinomycin可通過促進(jìn)ROS產(chǎn)生，激活線粒體凋亡途徑而誘導(dǎo)乳腺癌MCF-7/DOX耐藥細(xì)胞的凋亡。

總之，本實(shí)驗(yàn)首次發(fā)現(xiàn)Salinomycin對乳腺癌MCF-7/DOX耐藥細(xì)胞具有很好的增殖抑制效果，其作用機(jī)制可能與誘導(dǎo)ROS產(chǎn)生導(dǎo)致的細(xì)胞凋亡有關(guān)。那么Salinomycin誘導(dǎo)ROS產(chǎn)生的機(jī)制，以及Salinomycin是否在體內(nèi)同樣具有逆轉(zhuǎn)乳腺癌耐藥的作用，還需要進(jìn)一步通過實(shí)驗(yàn)深入研究，以期為臨床聯(lián)合應(yīng)用化療藥物逆轉(zhuǎn)乳腺癌耐藥提供實(shí)驗(yàn)基礎(chǔ)和聯(lián)合用藥策略。

[1] 鄭 瑩, 吳春曉, 張敏璐. 乳腺癌在中國的流行狀況和疾病特征 [J]. 中國癌癥雜志, 2013, 23(8):561-9.

[1] Zheng Y, Wu C X, Zhang M L. The epidemic and characteristics of female breast cancer in china [J].ChinOncol, 2013, 23(8):561-9.

[2] Tacar O, Dass C R. Doxorubicin-induced death in tumour cells and cardiomyocytes: is autophagy the key to improving future clinical outcomes [J]?JPharmPharmacol, 2013, 65(11):1577-89.

[3] Miyazaki Y, Shibuya M, Sugawara H, et al. Salinomycin, a new polyether antibiotic [J].JAntibiot, 1974, 27(11):814-21.

[4] Gupta P B, Onder T T, Jiang G, et al. Identification of selective inhibitors of cancer stem cells by high-throughput screening [J].Cell, 2009, 138(4):645-59.

[5] Dong T T, Zhou H M, Wang L L, et al. Salinomycin selectively targets CD133+ cell subpopulations and decreases malignant traits in colorectal cancer lines [J].AnnalsSurgOncol, 2011, 18(6):1797-804.

[6] Wang Y. Effects of salinomycin on cancer stem cell in human lung adenocarcinoma A549 cells [J].MedChem, 2011, 7(2):106-11.

[7] Kim K Y, Yu S N, Lee S Y, et al. Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization [J].BiochimBiophysResCommun, 2011, 413(1): 80-6.

[8] Fuchs D, Daniel V, Sadeghi M, et al. Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells [J].BiochimBiophysResCommun, 2010, 394(4):1098-104.

[9] Lu H P, Chao C C. Cancer cells acquire resistance to anticancer drugs: an update [J].BiomedJ, 2012, 35(6):464-72.

[10] Sui X, Kong N, Ye L, et al. p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents [J].CancerLett, 2014, 344(2):174-9.

[11] Naujokat C, Steinhart R. Salinomycin as a drug for targeting human cancer stem cells [J].JBiomedBiotechnol, 2012, 2012:950658.

[12] Zhou S, Wang F, Wong E T, et al. Salinomycin: a novel anti-cancer agent with known anti-coccidial activities [J].CurrMedChem, 2013, 20(33):4095-101.

[13] Wind N S, Holen I. Multidrug resistance in breast cancer: from in vitro models to clinical studies [J].IntJBreastCancer, 2011, 2011:967419.

[14] 李 玄，賀曉麗，畢明剛. 內(nèi)質(zhì)網(wǎng)應(yīng)激凋亡通路中的耐藥節(jié)點(diǎn)[J]. 中國藥理學(xué)通報(bào)，2013，29(9)：1192-6.

[14] Li X,He X L,Bi M G. Medicine resistance motecules in the endoplasmic reticulum stress mediated apoptosis pathway[J].ChinPharmBull,2013，29(9)：1192-6.

[15] Fuchs D, Heinold A, Opelz G, et al. Salinomycin induces apoptosis and overcomes apoptosis resistance in human cancer cells [J].BiochemBiophysResCommun, 2009, 390(3):743-9.

[16] Ketola K, Hilvo M, Hy?tyl?inen T, et al. Salinomycin inhibits prostate cancer growth and migration via induction of oxidative stress [J].BrJCancer, 2012, 106(1):99-106.

[17] Pennington J D, Wang T J, Nguyen P, et al. Redox-sensitive signaling factors as a novel molecular targets for cancer therapy [J].DrugResistUpdate, 2005, 8(5): 322-30.

[18] Wang T, Diaz A J, Yen Y. The role of peroxiredoxin II in chemoresistance of breast cancer cells [J].BreastCancer(DoveMedPress), 2014, 6:73-80.

[19] Diehn M, Cho R W, Lobo N A, et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells [J].Nature, 2009, 458(7239):780-3.

[20] Nagano O, Okazaki S,Saya H. Redox regulation in stem-like cancer cells by CD44 variant isoforms [J].Oncogene, 2013, 32(44): 5191-8.

Anti-proliferative effect of salinomycin on doxorubicin- resistant human breast cancer MCF-7/DOX cells

LIU Hao, LU Min-ying, HE Zhi-min

(CancerResearchInstituteandCancerHospital,GuangzhouMedicalUniversity,Guangzhou510095,China)

Aim To investigate the anti-proliferative effect of salinomycin on doxorubicin-resistant human breast cancer MCF-7/DOX cells. Methods MCF-7 and MCF-7/DOX cells were treated or untreated with salinomycin. Cell viability was detected by MTS assay. Cell apoptosis was detected by Annexin V-FITC/PI assay. Reactive oxygen species (ROS) was measured by DCFH-DA staining. Mitochondrial membrane potential was measured by JC-1 assay. The expression of apoptosis related proteins BAX, BCL-2, caspase-3, and caspase-9 were evaluated by Western blot analysis. Results The cell viability was significantly reduced by salinomycin treatment in a dose-dependent manner. The flow cytometry results showed that salinomycin induced MCF-7/DOX cell apoptosis, increased ROS production, and decreased mitochondrial membrane potential. Furthermore, salinomycin decreased the expression of BCL-2, and increased the expression of BAX, cleaved caspase-3, and cleaved caspase-9. Moreover, the antioxidant N-acetylcysteine (NAC) markedly blocked the above effects. Conclusions Our results suggest that salinomycin-induced apoptosis in MCF-7/DOX is associated with induction of ROS production, and activation of mitochondria apoptosis pathway, which may become a potential chemotherapeutic agent for the therapy of doxorubicin resistant breast cancer.

breast cancer; salinomycin; doxorubicin resistance; cell apoptosis; ROS; mitochondrial membrane potential

時(shí)間：2015-3-16 15:41 網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/detail/34.1086.R.20150316.1541.003.html

2014-10-31,

2015-01-04

國家自然科學(xué)基金資助項(xiàng)目(No 81402497)

劉 浩(1981-)，男，博士，助理研究員，研究方向：腫瘤轉(zhuǎn)移、耐藥的分子機(jī)制，Tel：020-66673666，E-mail：haoliu2020@ 163.com； 賀智敏(1957-)，男，博士，教授，博士生導(dǎo)師，研究方向：惡性腫瘤發(fā)病和治療耐受的分子機(jī)制，通訊作者，Tel: 020-66673666，E-mail：hezhimin2005@yahoo.com

10.3969/j.issn.1001-1978.2015.04.022

A

1001-1978(2015)04-0549-06

R329.24；R329.25；R737.902.2；R979.1；R979.14