漢防己甲素衍生物P-42對(duì)人乳腺癌細(xì)胞增殖、凋亡的影響及機(jī)制探討

晏文濤，潘衛(wèi)東，劉柏岑，吳翱蘭，劉金河，劉杰麟

(貴州醫(yī)科大學(xué),貴陽(yáng)550025)

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·論著·

漢防己甲素衍生物P-42對(duì)人乳腺癌細(xì)胞增殖、凋亡的影響及機(jī)制探討

晏文濤，潘衛(wèi)東，劉柏岑，吳翱蘭，劉金河，劉杰麟

(貴州醫(yī)科大學(xué),貴陽(yáng)550025)

目的觀察漢防己甲素(TET)衍生物P-42對(duì)人乳腺癌細(xì)胞株MDA-MB-435增殖、凋亡的影響，并探討其機(jī)制。方法收集對(duì)數(shù)生長(zhǎng)期MDA-MB-435細(xì)胞，觀察組分別加入不同濃度P-42，空白對(duì)照組加入DMEM培養(yǎng)液，培養(yǎng)24 h后，采用MTT法測(cè)算細(xì)胞增殖抑制率，流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率。收集對(duì)數(shù)生長(zhǎng)期MDA-MB-435細(xì)胞，觀察組分別加入終濃度2、10 μmmol/L的P-42，對(duì)照組加入DMEM培養(yǎng)液，另設(shè)正常乳腺細(xì)胞MCF-10a為空白組，采用Western blot法檢測(cè)各組細(xì)胞布盧姆綜合征(BLM)蛋白。結(jié)果當(dāng)P-42 濃度在5、20、80 μmmol/L時(shí)，MDA-MB-435細(xì)胞增殖抑制率分別為76.00%、84.77%和85.53%，20、80 μmmol/L時(shí)細(xì)胞增殖抑制率均高于5 μmmol/L時(shí)，P均<0.05；80 μmmol/L時(shí)細(xì)胞增殖抑制率高于20 μmmol/L時(shí)，但差異無(wú)統(tǒng)計(jì)學(xué)意義。觀察組P-42 濃度在 2、10 μmmol/L時(shí)，細(xì)胞早期凋亡率分別為39.47%、87.95%，晚期凋亡率分別為2.73%、3.82%，對(duì)照組分別為1.40%、0.90%，組間比較，P均<0.05。觀察組P-42 濃度在 2、10 μmmol/L時(shí)， BLM蛋白的相對(duì)表達(dá)量為31.57±7.80、33.82±8.61，對(duì)照組和空白組BLM蛋白相對(duì)表達(dá)量分別為12.73±1.14、19.12±1.58，組間比較，P均<0.05。 結(jié)論TET衍生物P-42可抑制MDA-MB-435細(xì)胞增殖，誘導(dǎo)細(xì)胞凋亡，其機(jī)制可能與促進(jìn)BLM蛋白表達(dá)有關(guān)。

漢防己甲素；乳腺癌；細(xì)胞增殖；細(xì)胞凋亡；布盧姆綜合征基因

乳腺癌是女性常見(jiàn)的惡性腫瘤，病死率較高[1]。細(xì)胞增殖與凋亡的失衡在乳腺癌的發(fā)生發(fā)展中起重要作用[2]。布盧姆綜合征(BLM)基因是Recq DNA解螺旋酶家族成員之一，其缺乏或突變可導(dǎo)致細(xì)胞發(fā)生癌變或凋亡[3]。漢防己甲素(TET)是一種提取自防己科植物粉防己根部的中藥成分[4]，屬于雙芐基異喹啉類生物堿，具有止痛、抗高血壓、抗風(fēng)濕、抗炎癥和抗腫瘤等效果;TET有多種衍生物，如P-36、P-39、P-40、P-41、P-42、P-44、P-47、P-49、P-51、P-53。本課題組前期研究發(fā)現(xiàn)P-42抗腫瘤活性較好，但其具體機(jī)制尚不明確。2015年5～12月，我們觀察了P-42對(duì)人乳腺癌細(xì)胞株MDA-MB-435增殖及凋亡的影響，并探討其可能的作用機(jī)制。現(xiàn)報(bào)告如下。

1　材料與方法

1.1材料MDA-MB-435細(xì)胞來(lái)自貴州醫(yī)科大學(xué)組織工程與干細(xì)胞實(shí)驗(yàn)中心細(xì)胞庫(kù)，培養(yǎng)條件：高糖DMEM基礎(chǔ)培養(yǎng)基加入10%胎牛血清(FBS)、1%青-鏈霉素、0.2 mol/L谷氨酰胺和0.2 U/mL的胰島素。P-42由貴州省中科院天然藥物化學(xué)重點(diǎn)實(shí)驗(yàn)室提供；高糖DMEM培養(yǎng)基、DMEM/F12(1∶1)培養(yǎng)基、0.25%胰蛋白酶、青-鏈霉素(Hyclone)；FBS(杭州四季青)、Annexin Ⅴ-FITC/PI雙染凋亡檢測(cè)試劑盒(上海貝博)；RIPA強(qiáng)裂解液、BCA蛋白濃度測(cè)定試劑盒(江蘇碧云天)，兔抗人BLM多克隆抗體和HRP偶聯(lián)山羊抗兔IgG(北京中杉金橋)；美國(guó)BioTek公司ELx800通用酶標(biāo)儀、Beckman公司FC500MCL/MPL流式細(xì)胞分析儀。

1.2MDA-MB-435培養(yǎng)及傳代MDA-MB-435培養(yǎng)條件：高糖DMEM基礎(chǔ)培養(yǎng)基加入10%FBS、1%青-鏈霉素、0.2 mol/L谷氨酰胺和0.2 U/mL的胰島素。置于5%CO2、37 ℃恒溫細(xì)胞培養(yǎng)箱中單層傳代培養(yǎng)。

1.3MDA-MB-435增殖觀察采用MTT法。取對(duì)數(shù)生長(zhǎng)期細(xì)胞， PBS洗滌1～2遍，0.25%胰酶消化，10% FBS的培養(yǎng)基終止消化，1 500 r/min離心3 min，8×103/孔接種于96孔板，分為4組，每組6個(gè)復(fù)孔，分別加入終濃度5、20、80 μmmol/L的P-42干預(yù)48 h，另設(shè)空白對(duì)照組(加入DMEM培養(yǎng)液)。取上清液，加入含MTT 50 μL的培養(yǎng)基，37 ℃孵育4 h，每孔加入150 μL的DMSO，采用全自動(dòng)酶標(biāo)儀測(cè)定490 nm波長(zhǎng)處各孔光密度(OD)值，重復(fù)3次，取平均值，計(jì)算各組細(xì)胞增殖抑制率。細(xì)胞增殖抑制率=(對(duì)照組OD值-觀察組OD值)/對(duì)照組OD值×100%。

1.4MDA-MB-435凋亡觀察采用流式細(xì)胞儀。取對(duì)數(shù)生長(zhǎng)期細(xì)胞接種于96孔板，CO2培養(yǎng)箱中培養(yǎng)24 h，分為2組，每組6個(gè)復(fù)孔，分別加入終濃度為2、10 μmmol/L P-42干預(yù)16 h，同時(shí)設(shè)立對(duì)照組(加入DMEM培養(yǎng)液)，加入 Annexin V-FITC和PI染色，采用流式細(xì)胞儀檢測(cè)各組細(xì)胞早期和晚期細(xì)胞凋亡情況，計(jì)算細(xì)胞凋亡率。

1.5BLM蛋白檢測(cè) 采用Western blot法。取對(duì)數(shù)生長(zhǎng)期細(xì)胞，分別用終濃度為2、10 μmmol/L P-42和DMEM培養(yǎng)液(對(duì)照組)培養(yǎng)24 h,另設(shè)正常乳腺細(xì)胞MCF-10a為空白組。PBS洗滌2次，采用BCA試劑盒提取總蛋白， SDS-PAGE電泳，分離，采用半干轉(zhuǎn)膜法將蛋白轉(zhuǎn)印至PVDF膜，將PVDF膜放入含5%脫脂奶粉的TBST緩沖液中，室溫封閉2 h；加入一抗，4 ℃孵育過(guò)夜，充分洗膜，辣根過(guò)氧化物酶標(biāo)記二抗37 ℃孵育1 h，充分洗膜，ECL顯色。結(jié)果經(jīng)成像系統(tǒng)掃描條帶，以β-actin為內(nèi)參，以目的蛋白與內(nèi)參灰度比值來(lái)表示目的蛋白的相對(duì)表達(dá)量。

2　結(jié)果

2.1P-42對(duì)MDA-MB-435增殖的影響 當(dāng)P-42 濃度分別在5、20、80 μmmol/L時(shí)，MDA-MB-435細(xì)胞增殖抑制率分別76.00%、84.77%和85.53%。P-42濃度為20、80 μmmol/L時(shí)細(xì)胞增殖抑制率均高于5 μmmol/L時(shí)，P均<0.05；20、80 μmmol/L時(shí)細(xì)胞增殖抑制率間差異無(wú)統(tǒng)計(jì)學(xué)意義。

2.2P-42對(duì)MDA-MB-435細(xì)胞凋亡的影響觀察組P-42 濃度為2、10 μmmol/L時(shí)，細(xì)胞早期凋亡率分別為39.47%、87.95%，對(duì)照組為1.40%，組間比較，P均<0.05。觀察組P-42濃度為2、10 μmmol/L時(shí)，細(xì)胞晚期凋亡率分別為2.73%、3.82%，對(duì)照組為0.90%，組間比較，P均<0.05。

2.3P-42對(duì)MDA-MB-435中BLM蛋白表達(dá)的影響觀察組P-42 濃度為2、10 μmmol/L時(shí)， BLM蛋白相對(duì)表達(dá)量分別為31.57±7.80、33.82±8.61，對(duì)照組和空白組BLM蛋白相對(duì)表達(dá)量分別為12.73±1.14、19.12±1.58，組間比較，P均<0.05。

3　討論

乳腺癌的病因和發(fā)病機(jī)制目前尚不明確。BLM作為一種解旋酶在維持基因組穩(wěn)定方面發(fā)揮至關(guān)重要的作用[3]，主要參與DNA損傷修復(fù)、基因重組和染色體錯(cuò)配等。BLM作為一種分子發(fā)動(dòng)機(jī)，可以將腺苷三磷酸水解產(chǎn)生的化學(xué)能量轉(zhuǎn)化為機(jī)械能，進(jìn)而促使其本身以“蠕動(dòng)”和“滾動(dòng)”兩種模式沿DNA分子運(yùn)動(dòng)，解開(kāi)雙鏈DNA[5]；其具有3′～5′解旋解鏈活性，能夠解旋多種類型的雙鏈DNA，包括3′末端雙鏈DNA、G-四鏈體DNA、bubble型DNA、復(fù)制叉DNA、D型環(huán)狀DNA及Holiday結(jié)構(gòu)DNA[6]。BLM是細(xì)胞內(nèi)重要的DNA損傷修復(fù)酶，無(wú)論是基因復(fù)制過(guò)程中隨機(jī)發(fā)生的DNA錯(cuò)配，或是機(jī)械性、物理性、生物性等因素引起的細(xì)胞內(nèi)DNA錯(cuò)配及損傷[5]，BLM均可通過(guò)代償性增多發(fā)揮修復(fù)錯(cuò)配及損傷DNA的效應(yīng)，以維持細(xì)胞內(nèi)正常代謝及功能。

TET作為中國(guó)傳統(tǒng)的中藥有效成分，可用于治療關(guān)節(jié)炎、心律不齊、炎癥反應(yīng)、矽肺病、腫瘤等多種疾病[7]，其抗腫瘤機(jī)制主要通過(guò)介導(dǎo)凋亡[7]、阻斷Ca2+通道[8]、引起自噬并調(diào)節(jié)細(xì)胞內(nèi)活性氧分子[9]等途徑抑制和殺傷腫瘤細(xì)胞。Bai等[4]研究發(fā)現(xiàn),TET能通過(guò)誘導(dǎo)凋亡抑制結(jié)腸癌細(xì)胞異種移植后的腫瘤生長(zhǎng)。Li 等[8]研究表明，TET抗腫瘤效應(yīng)的一種機(jī)制是作為一種非選擇性的Ca2+通道阻斷劑和Ca2+調(diào)節(jié)蛋白拮抗劑發(fā)揮作用。研究發(fā)現(xiàn)，高濃度的TET能誘導(dǎo)肝癌細(xì)胞凋亡，低濃度的TET能誘導(dǎo)肝癌細(xì)胞自噬，且TET聯(lián)合其他化療藥物能發(fā)揮更明顯的抗腫瘤效應(yīng)[10～12]。本研究結(jié)果發(fā)現(xiàn)，隨P-42濃度升高細(xì)胞增殖抑制率逐漸升高。據(jù)文獻(xiàn)[13,14]報(bào)道，順鉑抑制多種腫瘤細(xì)胞的半數(shù)抑制濃度(IC50)為10～30 μmmol/L。本實(shí)驗(yàn)前期研究中發(fā)現(xiàn)P-42對(duì)MDA-MB-435的 IC50為2 μmmol/L左右，抑制效果明顯優(yōu)于順鉑。當(dāng)P-42 濃度在 2、10 μmmol/L時(shí)，MDA-MB-435細(xì)胞早期凋亡率分別為39.47%、87.95%，對(duì)照組早期凋亡率為1.40%，組間比較，P均<0.05。P-42 濃度在 2、10 μmmol/L時(shí)，MDA-MB-435晚期期凋亡率分別為2.73%、3.82%，對(duì)照組晚期凋亡率為0.90%，組間比較，P均<0.05。由于衍生物P-42的強(qiáng)抑制活性，在2、10、20 μmmol的P-42作用下，細(xì)胞均無(wú)完整的形態(tài)，且大部分細(xì)胞已被裂解殺死或呈皺縮形態(tài)異；藥物作用24 h，細(xì)胞在不同藥物濃度間活性表現(xiàn)出明顯差異，隨著藥物濃度的增加，細(xì)胞死亡數(shù)呈增多趨勢(shì)，差異有統(tǒng)計(jì)學(xué)意義。本研究結(jié)果顯示，觀察組BLM蛋白的相對(duì)表達(dá)量高于對(duì)照組、空白組。推測(cè)可能是由于P-42可穿過(guò)細(xì)胞膜進(jìn)入細(xì)胞內(nèi)部引起DNA損傷[15,16]，同時(shí)細(xì)胞內(nèi)部啟動(dòng)DNA修復(fù)程序，代償性合成更多DNA修復(fù)酶BLM來(lái)修復(fù)損傷的DNA，以維持基因組穩(wěn)定和正常核酸代謝[17～19]。

綜上所述，TET衍生物P-42可抑制MDA-MB-435的增殖，誘導(dǎo)其凋亡，其機(jī)制可能與促進(jìn)BLM表達(dá)有關(guān)，但其具體作用機(jī)制尚有待于進(jìn)一步研究。

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Effects of tetrandrine derivatrve P-42 on proliferation and apoptosis of human breast cancer cells and its mechanism

YANWentao,PANWeidong,LIUBocen,WUAolan,LIUJinhe,LIUJielin

(GuizhouMedicalUniversity,Guiyang550025,China)

ObjectiveTo observe the effects of tetrandrine (TET) derivative P-42 on the proliferation and apoptosis of human breast cancer cell line MDA-MB-435 and to investigate its mechanism.MethodsMDA-MB-435 cells in the logarithmic phase were collected, and then the observation group was treated with different concentrations of P-42, and the control group was added with DMEM nutrient solution. After 24 hours, MTT assay was used to measure the proliferation inhibition rate of MDA-MB-435 cells and flow cytometry was applied to analyze the apoptosis rate. MDA-MB-435 cells in the logarithmic phase were collected, and then the observation group was treated with 2 and 10 μmmol/L P-42, and the control group was added with DMEM nutrient solution, meanwhile, the normal breast cells MCF-10a were taken as the blank group. Bloom syndrome (BLM) protein in each group was detected Western blotting. ResultsWhen the concentrations of P-42 were 5, 20 and 80 μmmol/L, the proliferation inhibition rates of MDA-MB-435 cells were 76.00%, 84.77% and 85.53%, respectively. The proliferation inhibition rates of MDA-MB-435 cells treated with 20 and 80 μmmol/L P-42 were higher than that treated with 5 μmmol/L P-42, allP<0.05. The proliferation inhibition rate of MDA-MB-435 cells treated with 80 μmmol/L P-42 were higher than that treated with 20 μmmol/L P-42, allP<0.05. When the concentrations of P-42 were 2 and 10 μmmol/L, the early apoptosis rates of MDA-MB-435 cells were 39.47% and 87.95%, and that in the control group was 1.4%, allP<0.05. When the concentrations of P-42 were 2 and 10 μmmol/L, the late-stage apoptosis rates of MDA-MB-435 cells were 2.73% and 3.82%, and that in the control group was 0.90%, allP<0.05.When the concentrations of P-42 were 2 and 10 μmmol/L, the relative expression of BLM protein was 31.57±7.80 and 33.82±8.61, and that in the control group and blank group was 12.73±1.14 and 19.12±1.58, allP<0.05.ConclusionTetrandrine derivative P-42 can inhibit the proliferation and induce apoptosis of MDA-MB-435 cells, adn the underlying mechanism may be associated with the up-regulation of BLM expression.

tetrandrine; breast carcinoma; cell proliferation; apoptosis; Bloom syndrome gene

國(guó)家自然科學(xué)基金資助項(xiàng)目(81360349)。

晏文濤(1989-)，男，碩士，主要研究方向?yàn)榭鼓[瘤藥物篩選及抑瘤機(jī)制研究。E-mail： 714419389@qq.com

簡(jiǎn)介：劉杰麟(1963-)，男，博士，教授，主要研究方向?yàn)樾》肿犹烊换钚晕镔|(zhì)抑制RecQ家族解旋酶等腫瘤相關(guān)基因。E-mail：779713773@qq.com

10.3969/j.issn.1002-266X.2016.18.001

R392-33

A

1002-266X(2016)18-0001-04

2016-01-01)