賴文升，史紫君，萬(wàn)曉蕾，薛晶，邵根寶，鄒圣強(qiáng)

(1江蘇大學(xué)醫(yī)學(xué)院，江蘇鎮(zhèn)江212013；2江蘇大學(xué)附屬鎮(zhèn)江三院)

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曲古抑菌素A對(duì)卵巢癌細(xì)胞分化、增殖的影響及其機(jī)制

賴文升1，史紫君1，萬(wàn)曉蕾1，薛晶1，邵根寶1，鄒圣強(qiáng)2

(1江蘇大學(xué)醫(yī)學(xué)院，江蘇鎮(zhèn)江212013；2江蘇大學(xué)附屬鎮(zhèn)江三院)

目的觀察曲古抑菌素A(TSA)對(duì)卵巢癌細(xì)胞分化、增殖及細(xì)胞周期的影響，并探討其機(jī)制。方法培養(yǎng)卵巢癌上皮細(xì)胞系HO8910，將細(xì)胞分為A、B、C、D組，分別加入0、100、200、400 nmol/L TSA。分別于培養(yǎng)24、48、72 h觀察A、C組細(xì)胞形態(tài)變化；于培養(yǎng)24 h后采用Western blotting法檢測(cè)四組細(xì)胞中的Y染色體上的性別決定區(qū)盒2(SOX-2)、八聚體結(jié)合轉(zhuǎn)錄因子4(OCT-4)和叉頭樣轉(zhuǎn)錄因子A2(FOXA-2)。分別采用MTT法及EdU染色法檢測(cè)細(xì)胞增殖能力，流式細(xì)胞術(shù)檢測(cè)不同周期細(xì)胞。采用Western blotting法檢測(cè)各組細(xì)胞中的Cyclin D1和p21Cip1蛋白。結(jié)果A組細(xì)胞多為類圓形或橢圓形，外表規(guī)整，成簇生長(zhǎng)。C組細(xì)胞發(fā)生形態(tài)轉(zhuǎn)變，細(xì)胞密度有所下降。與A組相比，B、C、D組細(xì)胞中SOX-2、OCT-4表達(dá)下調(diào)，F(xiàn)OXA-2表達(dá)升高(P均<0.05)。與A組相比，B、C、D組細(xì)胞增殖率降低，G0/G1期細(xì)胞比例增高，S期細(xì)胞比例減小，Cyclin D1蛋白表達(dá)下調(diào)，p21Cip1蛋白表達(dá)上調(diào)(P均<0.05)。結(jié)論TSA可誘導(dǎo)卵巢癌HO8910細(xì)胞分化，抑制細(xì)胞增殖，其機(jī)制可能與調(diào)節(jié)細(xì)胞分化相關(guān)蛋白及細(xì)胞周期相關(guān)蛋白表達(dá)有關(guān)。

曲古抑菌素A；卵巢癌；細(xì)胞分化；細(xì)胞增殖；Y染色體上的性別決定區(qū)盒2;八聚體結(jié)合轉(zhuǎn)錄因子4；叉頭樣轉(zhuǎn)錄因子A2；細(xì)胞周期蛋白D1；細(xì)胞周期蛋白依賴性激酶抑制劑

上皮性卵巢癌(EOC)是最常見(jiàn)的婦科惡性腫瘤之一[1]，70%的患者接受一線化療后18個(gè)月內(nèi)復(fù)發(fā)[2,3]，晚期患者5年生存率較低(僅為30.6%)[4]，因此迫切需要制定有效的化療策略。分化治療作為一種新的化療策略，通過(guò)藥物誘導(dǎo)惡性腫瘤細(xì)胞分化成非惡性細(xì)胞甚至正常細(xì)胞，具有巨大的潛力[5～7]。組蛋白乙?；揎椇腿ヒ阴；揎検潜碛^遺傳調(diào)控的重要組成部分。組蛋白乙酰轉(zhuǎn)移酶(HAT)和去乙?；?HDAC)活性失衡將導(dǎo)致細(xì)胞周期、分化和凋亡等相關(guān)基因異常轉(zhuǎn)錄[8]，而HDAC抑制劑能夠逆轉(zhuǎn)這一作用，并促進(jìn)細(xì)胞分化。曲古抑菌素A(TSA)是近年來(lái)備受關(guān)注的一種異羥肟酸型HDAC抑制劑。關(guān)于TSA與卵巢癌的關(guān)系研究主要集中在TSA的促凋亡和抑制遷移作用，但對(duì)TSA的促分化作用較少涉及。本研究觀察了TSA對(duì)卵巢癌細(xì)胞分化、增殖及細(xì)胞周期的影響，并探討其機(jī)制，為卵巢癌的分化治療提供理論依據(jù)。

1　材料與方法

1.1細(xì)胞培養(yǎng)與分組卵巢癌細(xì)胞系HO8910由江蘇大學(xué)邵啟祥教授實(shí)驗(yàn)室贈(zèng)予，培養(yǎng)于含有10% FBS和抗生素(青霉素100 U/mL、鏈霉素100 mg/mL)的DMEM中，并置于37 ℃、5% CO2培養(yǎng)箱。當(dāng)細(xì)胞融合度至80%時(shí)，以2×105/mL接種于培養(yǎng)皿中培養(yǎng)過(guò)夜。將細(xì)胞分為A、B、C、D四組，分別加入0、100、200、400 nmol/L TSA。

1.2細(xì)胞形態(tài)觀察分別于培養(yǎng)24、48、72 h用倒置顯微鏡觀察A、C組細(xì)胞形態(tài)并用CCD相機(jī)拍照。

1.3細(xì)胞分化相關(guān)蛋白檢測(cè)四組培養(yǎng)24 h后，采用Western blotting法檢測(cè)分化相關(guān)蛋白Y染色體上的性別決定區(qū)盒2(SOX-2)、八聚體結(jié)合轉(zhuǎn)錄因子4(OCT-4)和叉頭樣轉(zhuǎn)錄因子A2(FOXA-2)。提取各組細(xì)胞總蛋白，經(jīng)8%～12% SDS-PAGE凝膠分離和電轉(zhuǎn)移至PVDF膜，轉(zhuǎn)膜后由含5%脫脂奶粉的TBST室溫封閉1 h，一抗孵育液4 ℃過(guò)夜，TBST洗膜3次，二抗室溫孵育1 h，再用TBST洗膜3次，最后用ECL曝光液在化學(xué)發(fā)光檢測(cè)器中曝光并拍照。用Image Pro Plus軟件測(cè)定條帶灰度值，然后計(jì)算目的蛋白條帶灰度值與內(nèi)參照條帶灰度值的比值，表示蛋白相對(duì)表達(dá)量。

1.4細(xì)胞增殖能力檢測(cè)①M(fèi)TT法：四組分別于培養(yǎng)24、48、72 h，將20 μL的MTT加入到96孔板中，4 h后吸除培養(yǎng)基，加入150 μL DMSO，在搖床上搖15 min以溶解甲臜，用酶標(biāo)儀測(cè)定490 nm波長(zhǎng)處光密度值(OD值)。細(xì)胞增殖率=OD實(shí)驗(yàn)組/OD對(duì)照組×100%。②EdU法：取A、C組細(xì)胞，培養(yǎng)48 h后將50 mmol/L EdU添加到培養(yǎng)基中，孵育2 h，再加入DAPI進(jìn)行細(xì)胞核染色，由奧林巴斯激光掃描顯微鏡系統(tǒng)拍攝照片；照片中綠色熒光為正在進(jìn)行DNA復(fù)制的細(xì)胞核，對(duì)綠色熒光細(xì)胞核進(jìn)行計(jì)數(shù)。

1.5不同周期細(xì)胞檢測(cè)四組培養(yǎng)48 h后，胰酶消化細(xì)胞，PBS洗滌2遍，將細(xì)胞與DNA結(jié)合染料PI(50 g/mL)和RNase(1 mg/mL)在37 ℃恒溫箱中避光孵育30 min，采用流式細(xì)胞儀分析。

1.6細(xì)胞周期相關(guān)蛋白檢測(cè)各組細(xì)胞培養(yǎng)48 h后，采用Western blotting法檢測(cè)G1細(xì)胞周期調(diào)控蛋白Cyclin D1和細(xì)胞周期蛋白依賴性激酶抑制劑p21Cip1。方法參考“1.2.2”。

2　結(jié)果

2.1各組細(xì)胞形態(tài)改變A組細(xì)胞多為類圓形或橢圓形，外表規(guī)整，成簇生長(zhǎng)。C組細(xì)胞發(fā)生類圓形—星狀—梭形轉(zhuǎn)變，細(xì)胞密度也有所下降。詳見(jiàn)圖1。

圖1　A、C組不同培養(yǎng)時(shí)間細(xì)胞形態(tài)學(xué)變化(100×)

2.2各組細(xì)胞分化相關(guān)蛋白表達(dá)比較Western blotting檢測(cè)顯示，TSA明顯下調(diào)SOX-2和OCT-4的蛋白表達(dá)水平，而誘導(dǎo)FOXA-2表達(dá)上調(diào)，且呈濃度依賴性。詳見(jiàn)表1。

表1　各組細(xì)胞中SOX-2、OCT-4、FOXA2表達(dá)比較

注：與A組相比，*P<0.05，**P<0.01。

2.3各組細(xì)胞增殖率比較MTT結(jié)果顯示，B、C、D組細(xì)胞增殖率低于A組，且隨著TSA作用濃度增大、時(shí)間延長(zhǎng)，細(xì)胞增殖率逐漸降低(P均<0.05)。見(jiàn)表2。EdU染色結(jié)果顯示，C組綠色熒光細(xì)胞核數(shù)較A組減少(見(jiàn)圖2)。說(shuō)明TSA可抑制HO8910細(xì)胞增殖，而非誘導(dǎo)細(xì)胞凋亡。

表2　各組細(xì)胞增殖率比較

注：與A組相比，*P<0.05，**P<0.01。

注：A為A組；C為C組。

圖2 EdU染色后A、C組細(xì)胞增殖情況

2.4各組不同周期細(xì)胞分布比較與A組相比，B、C、D組G0/G1期細(xì)胞比例增高，S期細(xì)胞比例減小(P均<0.01)。見(jiàn)表3。

表3　各組不同周期細(xì)胞分布比較±s)

注：與A組相比，*P<0.01。

2.5各組細(xì)胞周期相關(guān)蛋白表達(dá)比較A、B、C、D組p21Cip1蛋白相對(duì)表達(dá)量分別為0.12±0.01、0.13±0.01、0.22±0.03、0.36±0.01，Cyclin D1蛋白相對(duì)表達(dá)量分別為0.68±0.04、0.47±0.05、0.21±0.02、0.16±0.01。與A組相比，B、C、D組細(xì)胞Cyclin D1蛋白表達(dá)下調(diào)，p21Cip1蛋白表達(dá)上調(diào)(P均<0.05)。

3　討論

與基因突變或缺失相比，表觀遺傳修飾是可逆的，并不引起基因序列變化，因此成為化療相關(guān)研究的熱點(diǎn)[9]。HDAC抑制劑可通過(guò)上調(diào)組蛋白乙?；綇亩T導(dǎo)細(xì)胞發(fā)生分化和(或)凋亡[10]?，F(xiàn)已證明許多HDAC抑制劑具有誘導(dǎo)細(xì)胞分化的能力[11]。我們利用人類卵巢癌細(xì)胞系HO8910研究TSA對(duì)惡性腫瘤細(xì)胞分化的作用，結(jié)果顯示，A組細(xì)胞多為類圓形或橢圓形，外表規(guī)整，成簇生長(zhǎng)；C組細(xì)胞發(fā)生類圓形—星狀—梭形轉(zhuǎn)變，細(xì)胞密度也有所下降，提示TSA可誘導(dǎo)HO8910細(xì)胞發(fā)生形態(tài)變化。

卵巢癌患者預(yù)后差的一部分原因可能是腫瘤干細(xì)胞的存在[12]。腫瘤干細(xì)胞具有自我更新和分化能力，在腫瘤發(fā)生、化療耐藥、腫瘤復(fù)發(fā)和轉(zhuǎn)移過(guò)程中發(fā)揮重要作用[12,13]。SOX-2、OCT-4、FOXA-2等轉(zhuǎn)錄因子可協(xié)同調(diào)節(jié)干細(xì)胞自我更新和分化所需的基因及信號(hào)通路。據(jù)報(bào)道，SOX-2高表達(dá)可阻止人多能性NT2/D1細(xì)胞向神經(jīng)元或向膠質(zhì)細(xì)胞分化，成熟神經(jīng)元中SOX-2的表達(dá)水平較低[14]。OCT-4表達(dá)上調(diào)也與某些低分化的惡性腫瘤有關(guān)。FOXA-2高表達(dá)則可通過(guò)提高組蛋白H3乙酰化水平從而誘導(dǎo)干細(xì)胞向多巴胺能神經(jīng)元分化[15]。與高分化腫瘤相比，低分化腫瘤組織中更易檢測(cè)到高度表達(dá)的SOX-2、OCT-4蛋白，后兩者在癌旁組織及良性腫瘤組織中不表達(dá)[16]。本研究將SOX-2、OCT-4和FOXA-2作為反映卵巢癌細(xì)胞分化程度的指標(biāo)，結(jié)果顯示，與A組相比，B、C、D組SOX-2、OCT-4蛋白表達(dá)下調(diào)，F(xiàn)OXA-2蛋白表達(dá)增高，提示TSA可通過(guò)調(diào)節(jié)細(xì)胞分化相關(guān)基因表達(dá)誘導(dǎo)卵巢癌細(xì)胞分化，即下調(diào)SOX-2、OCT-4蛋白表達(dá)，上調(diào)FOXA-2蛋白表達(dá)。推測(cè)TSA可抑制HDAC活性，通過(guò)提高組蛋白乙?；狡茐碾姾善胶鉅顟B(tài)，從而打開(kāi)DNA雙鏈，促進(jìn)細(xì)胞譜系特異性基因的轉(zhuǎn)錄激活。具體機(jī)制仍需進(jìn)一步研究。

細(xì)胞增殖與分化是由細(xì)胞周期中的G1期或G1/S期所控制的[17～19]。目前已知HDAC抑制劑誘導(dǎo)分化常伴隨特定基因表達(dá)水平改變，如p21Cip1、Cyclin D1。這些基因表達(dá)水平改變可導(dǎo)致細(xì)胞增殖抑制和細(xì)胞周期阻滯。在參與G1期調(diào)控的細(xì)胞周期蛋白中，Cyclin D1與腫瘤發(fā)生關(guān)系最密切。Cyclin D1與CDK4的結(jié)合為G1期向S期過(guò)渡所必需，CDK抑制劑p21Cip1是細(xì)胞周期進(jìn)程中的負(fù)性調(diào)節(jié)因子，可抑制G1期向S期過(guò)渡。本研究結(jié)果顯示，與A組相比，B、C、D組細(xì)胞增殖率降低，G0/G1期細(xì)胞比例增高，S期細(xì)胞比例減小，Cyclin D1蛋白表達(dá)下調(diào)，p21Cip1蛋白表達(dá)上調(diào)，提示TSA能誘導(dǎo)細(xì)胞增殖抑制并使細(xì)胞周期阻滯，可能通過(guò)調(diào)控p21Cip1和Cyclin D1表達(dá)發(fā)揮上述作用。

綜上所述，TSA可誘導(dǎo)卵巢癌細(xì)胞分化，抑制細(xì)胞增殖，其機(jī)制可能與調(diào)節(jié)細(xì)胞分化相關(guān)蛋白及細(xì)胞周期相關(guān)蛋白表達(dá)有關(guān)。

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Effect of TSA on differentiation and proliferation of ovarian cancer cells and its mechanism

LAI Wensheng1,SHI zijun,WAN Xiaolei,XUE Jing,SHAO Genbao,ZOU Shengqiang

(1 Jiangsu University,Zhenjiang 212013,China)

ObjectiveTo observe the effect of trichostatin A (TSA) on the differentiation,proliferation and cell cycle of ovarian cancer cells and to investigate its mechanism.MethodsEpithelial ovarian cancer cell line HO8910 was cultured and divided into groups A,B,C and D,respectively,which were added with 0,100,200 and 400 nmol/L TSA.Cell morphology of groups A and C were observed at 24 h,48 h and 72 h after culture.The expression of sex-determining region Y chromosome cassette-2 (SOX-2),octamer binding transcription factor 4 (OCT-4) and fork-like transcription factor A2 (FOXA2) in the four groups was detected by using Western blotting 24 hours after culture.The cell proliferation was detected by MTT and EdU staining,distribution of cell cycle was detected by flow cytometry and the expression of Cyclin D1 and p21Cip1protein was detected by Western blotting.ResultsCells in the group A were mostly round or oval,looked neat and grew in clusters.While cell morphology in the group C changed and cell density decreased.Compared with group A,the expression of SOX-2 and OCT-4 in the groups B,C and D were decreased,FOXA-2 expression was increased (all P<0.05).Compared with group A,the cell proliferation rates were decreased,the proportion of cells in G0/G1phase was increased,the proportion of cells in S phase was decreased,the expression of Cyclin D1 was decreased and the expression of p21Cip1protein in the groups B,C and D was increased (all P<0.05).ConclusionTSA can induce ovarian cancer cell differentiation and inhibit the cell proliferation,which may be related to the regulation of cell differentiation-related and cell cycle-related proteins.

trichostatin A; ovarian carcinoma; cell differentiation,cell proliferation; sex-determining region Y chromosome cassette-2; octamer binding transcription factor 4; fork-like transcription factor A2; Cyclin D1; cyclin-dependent kinase inhibitor

國(guó)家自然科學(xué)基金資助項(xiàng)目(81170573)。

賴文升(1986-)，男，在讀研究生，主要研究方向?yàn)槟[瘤學(xué)。E-mail：405560193@qq.com

簡(jiǎn)介：鄒圣強(qiáng)(1968-)，男，主任醫(yī)師，主要研究方向?yàn)橹匕Y醫(yī)學(xué)。E-mail：1210xyz@163.com

10.3969/j.issn.1002-266X.2016.19.003

R737.31

A

1002-266X(2016)19-0008-04

2015-11-25)