沈金花，陳劍霖

(中南民族大學(xué) 生命科學(xué)學(xué)院，醫(yī)學(xué)生物研究所&武陵山區(qū)特色資源植物種質(zhì)保護(hù)與利用湖北省重點(diǎn)實(shí)驗(yàn)室，武漢430074)

Linifanib和Tivozanib對(duì)肺癌細(xì)胞增殖的影響

沈金花，陳劍霖

(中南民族大學(xué) 生命科學(xué)學(xué)院，醫(yī)學(xué)生物研究所&武陵山區(qū)特色資源植物種質(zhì)保護(hù)與利用湖北省重點(diǎn)實(shí)驗(yàn)室，武漢430074)

為探討小分子化合物L(fēng)inifanib和Tivozanib對(duì)肺癌細(xì)胞A549和H358增殖的影響，以不同濃度Linifanib和Tivozanib預(yù)處理細(xì)胞48 h，用MTT法檢測(cè)了細(xì)胞存活率，并通過(guò)流式細(xì)胞儀檢測(cè)了細(xì)胞周期和凋亡情況，用RT-PCR檢測(cè)了腫瘤抑制因子P21的mRNA表達(dá)量.結(jié)果表明：Linfanib和Tivozanib能抑制肺癌細(xì)胞的增殖，呈現(xiàn)劑量依賴(lài)性.Linifanib能誘導(dǎo)細(xì)胞凋亡，同時(shí)引起細(xì)胞周期在G2/M期阻滯.Tivozanib主要引起細(xì)胞周期在G2/M期阻滯.Linifanib和Tivozanib均能引起A549和H358細(xì)胞中P21的mRNA表達(dá)量顯著升高，說(shuō)明Linifanib和Tivozanib能抑制肺癌細(xì)胞A549和H358的增殖.

小分子化合物；肺癌細(xì)胞； 增殖

肺癌是全球范圍內(nèi)發(fā)病率和死亡率最高的疾病，每年約有156萬(wàn)人因肺癌死亡，其中85%均是非小細(xì)胞肺癌.早期患者可以通過(guò)手術(shù)進(jìn)行治療，但由于很多患者確診時(shí)已是晚期，癌細(xì)胞已轉(zhuǎn)移，有近40%的患者已無(wú)法用手術(shù)治療.因此化療是目前治療非小細(xì)胞肺癌的主要手段.但化療毒副作用大，化療的肺癌患者5年存活率較低.因此，積極探索新的治療肺癌的思路對(duì)臨床醫(yī)療具有重要的意義.在腫瘤的生長(zhǎng)過(guò)程中，血管起到了為癌細(xì)胞提供營(yíng)養(yǎng)，促進(jìn)腫瘤生長(zhǎng)和增殖的功能.血管的生長(zhǎng)與血管表皮生長(zhǎng)因子受體(VEGFR)的激活密切相關(guān).因此，抑制VEGFR的激活逐漸成為治療肺癌的新思路.

Linifanib和Tivozanib均為新型的VEGFR的酪氨酸激酶抑制劑，其中Linifanib是氨基吲唑類(lèi)尿素衍生物，對(duì)肝癌、乳腺癌、直腸癌等有明顯的治療效果[1].Tivozanib是Aveo公司和安斯泰來(lái)公司合作開(kāi)發(fā)的高效抑制劑，目前主要用于腎癌的治療[2].

Linifanib和Tivozanib是高效的VEFGR抑制劑，該藥物已用于其他癌癥的治療.但是在肺癌上的應(yīng)用鮮有報(bào)道.有研究表明VEGFR在肺癌細(xì)胞上高表達(dá)[3].故理論上VEGFR抑制劑對(duì)肺癌的增殖有一定的影響.期望本文通過(guò)研究Linifanib和Tivozanib對(duì)非小細(xì)胞肺癌細(xì)胞增殖和凋亡的影響，期望為肺癌的治療提供新的思路.

1 材料與方法

1.1 試劑及樣品

Linifanib 和 Tivozanib購(gòu)自Selleckchem公司；MTT試劑購(gòu)自Promega公司；碘化丙啶(PI)購(gòu)自Biosharp公司；胎牛血清、RPMI-1640以及雙抗均購(gòu)自Hyclone公司.H358細(xì)胞株由武漢大學(xué)張曉東教授贈(zèng)予，A549細(xì)胞株購(gòu)自Procell公司.

1.2 實(shí)驗(yàn)儀器

酶標(biāo)儀(Infinite200型, TECAN公司)，流式細(xì)胞儀(AriaⅢ型, BD公司)，Real-Time PCR System(7500fast型, Applied Biosystems)，核酸定量?jī)x(Nanodrop 2000型, Thermo fisher Scientific公司)，核酸凝膠成像系統(tǒng)(Gel Doc XR+型, BIO-RAD公司)

1.3 細(xì)胞培養(yǎng)

H358和A549細(xì)胞株均用含有10%胎牛血清和1%雙抗的RPMI-1640培養(yǎng)液，于37℃、5% CO2飽和濕度條件下培養(yǎng)[4].

1.4 實(shí)驗(yàn)分組

實(shí)驗(yàn)分為7組：對(duì)照組，Linifanib低、中、高劑量組(2,5,10 μM)，Tivozanib低、中、高劑量組(2,10,20 μM).

1.5 MTT實(shí)驗(yàn)

分別接種3000個(gè)生長(zhǎng)狀態(tài)良好的H358和A549細(xì)胞于96孔板中，細(xì)胞貼壁后按照實(shí)驗(yàn)分組加入不同濃度藥物，在培養(yǎng)箱中培養(yǎng)48 h后向每個(gè)孔加入10 μL MTT試劑，3 h后用酶標(biāo)儀在490 nm波長(zhǎng)下檢測(cè)吸光值.取各平行孔吸光值的平均值，根據(jù)下列公式計(jì)算細(xì)胞存活率：存活率(%)=(實(shí)驗(yàn)組吸光度值/對(duì)照組吸光度值)×100 %(以不含細(xì)胞的培養(yǎng)液為空白組調(diào)零).

1.6 流式細(xì)胞術(shù)

分別接種6×105個(gè)生長(zhǎng)狀態(tài)良好的H358和A549細(xì)胞于60 mm培養(yǎng)皿中.細(xì)胞貼壁后按照實(shí)驗(yàn)分組加入不同濃度藥物.在培養(yǎng)箱中培養(yǎng)48 h后收集細(xì)胞，PBS洗2次后加入70 %乙醇，-20 ℃下固定過(guò)夜后，用PI染色30 min.用流式細(xì)胞儀檢測(cè)細(xì)胞周期.

1.7 逆轉(zhuǎn)錄-鏈?zhǔn)骄酆厦阜磻?yīng)(RT-PCR)

接種6×105個(gè)生長(zhǎng)狀態(tài)良好的H358和A549細(xì)胞于60 mm培養(yǎng)皿中，細(xì)胞貼壁后按照實(shí)驗(yàn)分組加入不同濃度藥物，在培養(yǎng)箱中培養(yǎng)48 h后用Trizol法提取RNA，按RT-PCR試劑盒方法進(jìn)行逆轉(zhuǎn)錄，反應(yīng)結(jié)束后取4 μL的逆轉(zhuǎn)錄產(chǎn)物進(jìn)行RT-PCR反應(yīng)[5].

1.8 統(tǒng)計(jì)分析

采用Excel、CorelDRAW X4和Power Point 2013軟件繪圖，用t-檢驗(yàn)法(student′st-test)對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行顯著性差異比較，P<0.05，具有統(tǒng)計(jì)學(xué)意義.

2 結(jié)果與分析

2.1 Linifanib和Tivozanib對(duì)肺癌細(xì)胞增殖的影響

Linifanib和Tivozanib對(duì)肺癌細(xì)胞增殖的影響結(jié)果見(jiàn)圖1.

**P<0.01,***P<0.001a) Linifanib處理A549細(xì)胞; b) Linifanib處理H358細(xì)胞; c) Tivozanib處理A549細(xì)胞; d) Tivozanib處理H358細(xì)胞圖1 Linifanib和Tivozanib對(duì)A549和H358細(xì)胞增殖的影響Fig.1 Effect of Linifanib and Tivozanib on proliferation of A549 and H358 cells

由圖1可見(jiàn)：2～10 μM Linifanib或2～20 μM Tivozanib處理細(xì)胞后，A549和H358細(xì)胞的增殖均受到抑制，細(xì)胞存活率隨著藥物濃度的升高而降低，且呈現(xiàn)劑量依賴(lài)性，具有統(tǒng)計(jì)學(xué)意義(P<0.05)，說(shuō)明Linifanib和Tivozanib可以抑制A549和H358細(xì)胞的增殖.

2.2 Linifanib和Tivozanib對(duì)肺癌細(xì)胞周期的影響

用不同濃度Linifanib或Tivozanib分別處理A549或H358細(xì)胞后，通過(guò)流式細(xì)胞儀檢測(cè)細(xì)胞周期和凋亡的情況，結(jié)果見(jiàn)圖2.由圖2可見(jiàn)：在一定濃度范圍內(nèi)，隨著Linifanib濃度升高，凋亡細(xì)胞和G2/M期細(xì)胞的比例逐漸升高.隨著Tivozanib濃度升高，G2/M期細(xì)胞的比例逐漸升高.說(shuō)明Linifanib對(duì)肺癌細(xì)胞增殖的影響是通過(guò)誘導(dǎo)細(xì)胞凋亡和細(xì)胞G2/M期阻滯共同引起的.Tivozanib可以通過(guò)引起細(xì)胞G2/M期阻滯進(jìn)而影響細(xì)胞增殖.

2.3 Linifanib和Tivozanib對(duì)肺癌細(xì)胞中P21 mRNA的影響

通過(guò)對(duì)調(diào)控細(xì)胞周期相關(guān)蛋白的mRNA表達(dá)量進(jìn)行檢測(cè)，發(fā)現(xiàn)用不同濃度Linifanib或Tivozanib分別處理A549和H358細(xì)胞后，在一定濃度范圍內(nèi)，隨著藥物濃度升高，實(shí)驗(yàn)組中P21的mRNA水平逐漸升高.而藥物對(duì)Cyclin D1的mRNA表達(dá)沒(méi)有顯著影響.結(jié)果提示Linifanib和Tivozanib引起肺癌細(xì)胞的G2/M期阻滯是因?yàn)镻21表達(dá)量升高，進(jìn)而抑制與G2/M期相關(guān)的周期蛋白激活.

3 討論

目前對(duì)抗癌藥物的開(kāi)發(fā)集中在靶向藥物方面.EGFR是目前廣泛研究的靶點(diǎn)之一.它是肺癌細(xì)胞膜表面高表達(dá)的蛋白，通過(guò)與外界配體結(jié)合，引發(fā)一系列生物學(xué)效應(yīng)，如增殖、凋亡、分化和遷移等[6].因此可能通過(guò)抑制該蛋白的激活可以抑制細(xì)胞生長(zhǎng).現(xiàn)在最常見(jiàn)的治療肺癌的靶向藥物是EGFR酪氨酸激酶抑制劑.該類(lèi)抑制劑在肺癌的治療上取得了不錯(cuò)的臨床效果[7].但是，單一的EGFR酪氨酸激酶抑制劑對(duì)肺癌的治療效果有限，故本研究的靶點(diǎn)是另一個(gè)肺癌細(xì)胞膜表面高表達(dá)的蛋白VEGFR.VEGFR在肝癌、胃癌和結(jié)腸癌等癌癥的治療中已經(jīng)得到普遍關(guān)注，針對(duì)該靶點(diǎn)設(shè)計(jì)的小分子化合物很多，其中如Sorafenib、Bevacizumab和Sunitinib等在癌癥的治療中取得了很好的效果[8,9].鑒于在肺癌細(xì)胞中也存在VEGFR高表達(dá)的現(xiàn)象，推測(cè)相關(guān)的小分子化合物對(duì)治療肺癌也有一定的作用.實(shí)驗(yàn)以肺癌細(xì)胞系H358和A549細(xì)胞為對(duì)象，初步探討了Linifanib和Tivozanib對(duì)肺癌細(xì)胞增殖和凋亡的影響.

研究分別從細(xì)胞水平和分子水平探究了小分子化合物對(duì)肺癌細(xì)胞A549和H358的影響.通過(guò)MTT實(shí)驗(yàn)發(fā)現(xiàn)Linifanib和Tivozanib能降低A549和H358細(xì)胞存活率.流式細(xì)胞術(shù)分析證明Linifanib能誘導(dǎo)細(xì)胞凋亡，同時(shí)引起細(xì)胞周期在G2/M期阻滯，Tivozanib主要引起細(xì)胞周期在G2/M期阻滯.RT-PCR檢測(cè)發(fā)現(xiàn) Linifanib和Tivozanib均可以引起A549和H358細(xì)胞中P21的mRNA表達(dá)量顯著升高.以上結(jié)果說(shuō)明了Linifanib和Tivozanib可以影響肺癌細(xì)胞A549和H358的增殖.

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EffectofLinifanibandTivozanibonProliferationofLungCancerCells

ShenJinhua,ChenJianlin

( Institute for Medical Biology & Hubei Provincial Key Laboratory for Protection and Application of Special Plant Germplasm in Wuling Area of China,College of Life Sciences, South-Central University for Nationalities, Wuhan 430074, China)

To investigate the effect of Linifanib and Tivozanib on the proliferation of lung cancer cell, A549 and H358 cells were treated with different concentrations of these compounds for 48 h and MTT assay was used to detect cell viability. Cell cycle and cell apoptosis were detected by flow cytometry analysis, and the mRNA expression of tumor-inhibiting factor P21 was tested by RT-PCR. The results showed that Linifanib and Tivozanib could inhibit the proliferation of lung cancer cells in a dose-dependent manner. Linifanib could induce cell apoptosis and arrest cell cycle in G2/M phase. Tivozanib could arrest cell cycle in G2/M phase. Meanwhile P21 mRNA expression in A549 and H358 cells increased after the Linifanib and Tivozanib treatment. It was concluded that Linifanib and Tivozaninb could inhibit the proliferation of lung cancer cell A549 and H358.

small molecule compounds; lung cancer cell; proliferation

2016-04-14

沈金花(1975-)，女，教授，博士，研究方向：疾病相關(guān)基因的功能研究，E-mail：shenjinhua2013@163.com

國(guó)家自然科學(xué)基金面上資助項(xiàng)目(31771274)；湖北省自然科學(xué)基金重點(diǎn)資助項(xiàng)目(2012FFA028)

Q291

A

1672-4321(2017)04-0036-04