Apogossypolone聯(lián)合神經(jīng)酰胺誘導(dǎo)鼻咽癌CNE-2細(xì)胞凋亡與自噬*

石豐榕 汪森明 賀 蛟 羅 皓 鐘 梅 吳丹心 朱震威

Apogossypolone聯(lián)合神經(jīng)酰胺誘導(dǎo)鼻咽癌CNE-2細(xì)胞凋亡與自噬*

石豐榕 汪森明 賀 蛟 羅 皓 鐘 梅 吳丹心 朱震威

目的：探討棉酚衍生物Apogossypolone（ApoG2）聯(lián)合神經(jīng)酰胺體外抑制鼻咽癌CNE-2細(xì)胞增殖，并初步探討其可能機(jī)制。方法：CCK-8測定不同濃度ApoG2和神經(jīng)酰胺單藥毒性及聯(lián)合應(yīng)用對CNE-2細(xì)胞的抑制作用，計算CDI判定藥物聯(lián)合效果。Hoechst33258染色觀察細(xì)胞凋亡，吖啶橙（AO）染色、透射電鏡觀察自噬形態(tài)學(xué)變化，F(xiàn)CM檢測凋亡率與自噬熒光強度。Western Blot檢測Bcl-2、Beclin1蛋白表達(dá)。結(jié)果：CCK-8檢測發(fā)現(xiàn)ApoG2和神經(jīng)酰胺單獨應(yīng)用時，隨藥物濃度增加，對CNE-2細(xì)胞生長的抑制作用也增加；低濃度兩藥聯(lián)合作用能協(xié)同增強單藥抑制鼻咽癌細(xì)胞CNE-2細(xì)胞生長（CDI＜1）。Hoechst33258染色顯示聯(lián)合用藥后出現(xiàn)更多的核固縮和碎裂等凋亡現(xiàn)象；吖啶橙染色顯示聯(lián)合用藥后產(chǎn)生更多的亮紅色酸性自噬泡。透射電鏡觀察到聯(lián)合用藥后細(xì)胞內(nèi)大空泡及膜性雙層結(jié)構(gòu)增多。FCM檢測聯(lián)合用藥組細(xì)胞凋亡率和自噬率均較單獨處理組升高，差異具有統(tǒng)計學(xué)意義（F凋亡=106.72，P凋亡＜0.001，F(xiàn)自噬=140.77，P自噬＜0.001）。Western Blot檢測發(fā)現(xiàn)聯(lián)合用藥組Bcl-2蛋白表達(dá)較單藥處理組降低（F=111.071，P＜0.001），Beclin1蛋白表達(dá)較單獨處理組升高（F=62.271，P＜0.001）。結(jié)論：低濃度ApoG2與神經(jīng)酰胺聯(lián)合共同誘導(dǎo)細(xì)胞凋亡與自噬，協(xié)同抑制鼻咽癌細(xì)胞生長，其作用機(jī)制可能與下調(diào)Bcl-2和上調(diào)Beclin1的表達(dá)有關(guān)。

鼻咽癌 Apogossypolone 神經(jīng)酰胺 凋亡 自噬

鼻咽癌在我國兩廣地區(qū)高發(fā)，初診時晚期患者約占70%以上［1］。早期采用單純放療，晚期采用放化聯(lián)合治療［2］。放射治療失敗的原因主要是遠(yuǎn)處轉(zhuǎn)移和局部復(fù)發(fā)［3］。Apogossypolone（ApoG2）是棉酚的一種新型衍生物，作為一種Bcl-2小分子抑制劑，毒副作用小，患者耐受性好，對胰腺癌［4］、前列腺癌［5］、乳腺癌［6］等多種腫瘤有抑制作用。神經(jīng)酰胺是細(xì)胞膜神經(jīng)鞘磷脂水解產(chǎn)生的一種脂質(zhì)分子，在促進(jìn)細(xì)胞死亡中有重要的生物學(xué)活性。ApoG2和神經(jīng)酰胺毒副作用均較小，兩者聯(lián)合應(yīng)用在國內(nèi)外鮮見報道。本研究主要探討兩種靶向藥物ApoG2聯(lián)合神經(jīng)酰胺體外作用對人鼻咽癌CNE-2細(xì)胞生長抑制，并初步探討其可能機(jī)制。

1 材料與方法

1.1 材料與主要試劑

鼻咽癌CNE-2細(xì)胞株由南方醫(yī)科大學(xué)腫瘤研究所凍存。ApoG2由美國密歇根大學(xué)醫(yī)學(xué)院腫瘤中心徐梁教授惠贈。神經(jīng)酰胺（N-acetyl-D-sphingosine，ceramide）購于Sigma公司。

1.2 CCK-8檢測藥物對細(xì)胞的生長抑制作用

按每孔5×103個細(xì)胞接種于96孔培養(yǎng)板，培養(yǎng)箱中培養(yǎng)24 h，棄廢液，設(shè)ApoG2、神經(jīng)酰胺藥物濃度均為5、10、20、40、60、80 μmol/L，兩藥聯(lián)合作用時保持兩藥終濃度不變；對照組為0.1%DMSO培養(yǎng)液，空白對照組不加細(xì)胞懸液只加培養(yǎng)液，每組4個平行孔，加培養(yǎng)液100 μL。繼續(xù)培養(yǎng)48 h，棄廢液，每孔加100 μL新培養(yǎng)液及CCK-8液10 μL，繼續(xù)孵育1 h。酶標(biāo)儀檢測450 nm波長下各孔OD值，實驗重復(fù)3次，分別計算各組的增殖抑制率，抑制率（%）=1-（OD實驗組-OD空白組）/（OD對照組-OD空白組）×100%。CDI［7］=AB/（A×B）。AB為聯(lián)合作用組吸光度值與對照組吸光度的比值；A、B是單藥作用組的吸光度值與對照組吸光度的比值。當(dāng)CDI＜1時，兩藥有協(xié)同作用；CDI=1時兩藥作用相加；CDI＞1時，兩藥拮抗。

1.3 Hoechst33258染色觀察細(xì)胞凋亡及吖啶橙（AO）染色、透射電鏡觀察細(xì)胞自噬形態(tài)學(xué)變化

消化傳代細(xì)胞，待細(xì)胞生長至50%～70%時，分為對照組（0.1%DMSO），ApoG2藥物組（20 μmol/L），神經(jīng)酰胺藥物組（20 μmol/L），聯(lián)合用藥組（ApoG2、神經(jīng)酰胺均為20 μmol/L）。培養(yǎng)48h后棄廢液，加Hoechst 33258染色液，避光染色5 min，熒光顯微鏡下觀察細(xì)胞凋亡形態(tài)。

按上述分組處理細(xì)胞，加終濃度為1 mg/L的AO避光作用15 min，PBS洗滌3遍，熒光顯微鏡下觀察細(xì)胞自噬形態(tài)。收集按上述分組處理的細(xì)胞1×106個，離心，去上清，固定，送電鏡室進(jìn)行常規(guī)切片，透射電鏡下觀察細(xì)胞超微結(jié)構(gòu)。

1.4 FCM檢測細(xì)胞凋亡率及自噬熒光強度

按上述分組處理細(xì)胞，各收集5×105個細(xì)胞，加入400 μL的Binding Buffer及5 μL Annexin V-FITC混勻后，再加入5 μL碘化丙啶（propidium iodide，PI）混勻；避光反應(yīng)10 min，上機(jī)檢測細(xì)胞凋亡率。細(xì)胞分組處理后，各收集5×105個細(xì)胞，加1mg/L的吖啶橙避光作用15 min，PBS洗滌3次，上機(jī)檢測自噬熒光強度，用FL1-Height和FL3-Height熒光通道分別代表綠色熒光和紅色熒光，實驗重復(fù)3次。

1.5 Western Blot檢測Bcl-2、Beclin1蛋白表達(dá)

按上述分組處理細(xì)胞。收集總蛋白，BCA法測蛋白濃度，12%SDS-PAGE膠上電泳，15V、60 min半干轉(zhuǎn)膜儀轉(zhuǎn)至PVDF，5%脫脂牛奶封閉1 h。洗膜后，加入Bcl-2多克隆抗體（1：500稀釋）、Beclin1多克隆抗體（1：500稀釋）4℃過夜。洗膜后，二抗（1：2 000稀釋）孵育1 h。加化學(xué)發(fā)光劑ECL，暗室曝光，顯影、定影后掃描，用Image J圖像分析系統(tǒng)分析結(jié)果。蛋白的相對表達(dá)量為目的蛋白與內(nèi)參β-actin的灰度值之比。實驗重復(fù)3次。

1.6 統(tǒng)計學(xué)方法

采用SPSS 13.0統(tǒng)計學(xué)軟件進(jìn)行數(shù)據(jù)處理，計量數(shù)據(jù)采用±s表示，組間差異采用單因素方差分析，P＜0.05為差異有統(tǒng)計學(xué)意義。

2 結(jié)果

2.1 CCK-8檢測藥物對細(xì)胞的生長抑制作用

ApoG2與神經(jīng)酰胺單獨作用CNE-2細(xì)胞48h，計算各濃度抑制率及CDI（表1），差異具有統(tǒng)計學(xué)意義（FApoG2=611.533，PApoG2＜0.001；Fceramide=495.730，Pceramide＜0.001），聯(lián)合用藥組抑制率隨著兩者藥物濃度的增加逐漸增加，兩者之間存在交互效應(yīng)（F=13.290，P＜0.001）。當(dāng)藥物濃度低于40 μmol/L時，CDI＜1，兩者發(fā)揮協(xié)同效應(yīng)；當(dāng)藥物濃度為60、80 μmol/L時，CDI＞1，兩藥作用相加甚至拮抗。作用48h后ApoG2與神經(jīng)酰胺IC50值分別為49.20 μmol/L和62.04 μmol/L。測的敏感性，所以建議實驗室通過大樣本的檢測，建立合理的人附睪分泌蛋白生物參考區(qū)間，以提高檢測的準(zhǔn)確性。

本研究分析了電化學(xué)發(fā)光法和酶免法兩種檢測方法的差異，結(jié)果示同一樣本ECLIA法檢測值高于ELISA法檢測值。通過比較兩種方法鑒別診斷卵巢癌的ROC-AUC，得知ECLIA法的曲線下面積更大，說明ECLIA法的診斷準(zhǔn)確性高于ELISA法。

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（2012-12-28收稿）（2013-02-15修回）

Inhibitory action of apogossypolone combined with ceramide on the induction of apoptosis and autophagy in nasopharyngeal carcinoma cell line CNE-2

Fengrong SHI,Senming WANG,Jiao HE,Hao LUO,Mei ZHONG,Danxin WU,Zhenwei ZHU

Senming WANG.E-mail:wsenming@126.com
Department of Oncology,Zhujiang Hospital of Southern Medical University,Guangzhou 510282 China

Objective:This study investigates the in vitro inhibitory action of apogossypolone,a gossypol derivative(ApoG2),combined with ceramide on cell proliferation in human nasopharyngeal cancer cell line CNE-2.The possible mechanism of this technique is also evaluated in this study.Methods:ApoG2 and ceramide of different concentrations were applied,individually or simultaneously,to human nasopharyngeal cancer CNE-2 cells.The cell counting kit-8(CCK-8)method was used to determine the cytotoxicity and assay the synergetic effect by calculating the value of the coefficient of drug interaction(CDI).Hoechst-33258 staining was conducted to observe morphological changes in the cell nucleus.Acridine-orange(AO)staining and transmission electron microscopy(TEM)were employed to observe the morphological alterations in autophagic cells.The apoptosis rate and fluorescence intensity of autophagy were determined by flow cytometry(FCM).The expressions of Bcl-2 and Beclin1 proteins were analyzed by Western blot.Results:The CCK-8 assay showed that the inhibitory action of ApoG2 and ceramide was enhanced with increasing drug concentrations,considering the drugs were used alone.With the conjunctive use of ApoG2 and ceramide both under low concentrations,the action would be synergistic(CDI＜1).Compared with the control group,Hoechst-33258 staining demonstrated the occurrence of apoptosis in the CNE-2 cells treated with ApoG2 or ceramide,or both.However,the morphological changes in the nuclear condensation and fragmentation in CNE-2 cells treated by both drugs were most significant.AO staining revealed more bright red acidic vesicular organelles in the combination group.An increase in the number of large vacuoles and double-layered membrane structure was observed under TEM in the combination group.Compared with the other groups,the FCM assay showed increased apoptosis rate and fluorescence intensity of autophagy when treated with both drugs.The differences were statistically significant between the single and combined application groups(Fapoptosis=106.72,Papoptosis=0.000;Fapoptosis=140.77,Papoptosis=0.000).Western blot analysis showed that Bcl-2 protein expression was downregulated with statistically significant differences between the two groups(F=111.071,P＜0.001).By contrast,Beclin1 expression increased in the combined therapy group compared with the other groups.Statistically significant differences were found among the groups(F=62.271,P＜0.001).Conclusion:The combined application of ApoG2 and ceramide at lower concentrations promotes apoptosis and autophagy,and synergistically inhibits the proliferation of human nasopharyngeal carcinoma cells.Such effects may be related to the downregulation of Bcl-2 expression and the upregulation of Beclin1 expression.

nasopharyngeal carcinoma,apogossypolone,ceramide,apoptosis,autophagy

10.3969/j.issn.1000-8179.2013.06.003

南方醫(yī)科大學(xué)珠江醫(yī)院腫瘤中心（廣州市510282）

*本文課題受廣州市科技計劃項目（編號：2011y2-00019-3）資助

汪森明 wsenming@126.com

This work was financially supported by the Guangzhou Science and Technology Planning Project(Grant No.2011y2-00019-3)

（本文編輯：周曉穎）