Apogossypolone联合神经酰胺诱导鼻咽癌CNE-2细胞凋亡与自噬*

石丰榕 汪森明 贺 蛟 罗 皓 钟 梅 吴丹心 朱震威

Apogossypolone联合神经酰胺诱导鼻咽癌CNE-2细胞凋亡与自噬*

石丰榕 汪森明 贺 蛟 罗 皓 钟 梅 吴丹心 朱震威

目的：探讨棉酚衍生物Apogossypolone（ApoG2）联合神经酰胺体外抑制鼻咽癌CNE-2细胞增殖，并初步探讨其可能机制。方法：CCK-8测定不同浓度ApoG2和神经酰胺单药毒性及联合应用对CNE-2细胞的抑制作用，计算CDI判定药物联合效果。Hoechst33258染色观察细胞凋亡，吖啶橙（AO）染色、透射电镜观察自噬形态学变化，FCM检测凋亡率与自噬荧光强度。Western Blot检测Bcl-2、Beclin1蛋白表达。结果：CCK-8检测发现ApoG2和神经酰胺单独应用时，随药物浓度增加，对CNE-2细胞生长的抑制作用也增加；低浓度两药联合作用能协同增强单药抑制鼻咽癌细胞CNE-2细胞生长（CDI＜1）。Hoechst33258染色显示联合用药后出现更多的核固缩和碎裂等凋亡现象；吖啶橙染色显示联合用药后产生更多的亮红色酸性自噬泡。透射电镜观察到联合用药后细胞内大空泡及膜性双层结构增多。FCM检测联合用药组细胞凋亡率和自噬率均较单独处理组升高，差异具有统计学意义（F凋亡=106.72，P凋亡＜0.001，F自噬=140.77，P自噬＜0.001）。Western Blot检测发现联合用药组Bcl-2蛋白表达较单药处理组降低（F=111.071，P＜0.001），Beclin1蛋白表达较单独处理组升高（F=62.271，P＜0.001）。结论：低浓度ApoG2与神经酰胺联合共同诱导细胞凋亡与自噬，协同抑制鼻咽癌细胞生长，其作用机制可能与下调Bcl-2和上调Beclin1的表达有关。

鼻咽癌 Apogossypolone 神经酰胺 凋亡 自噬

鼻咽癌在我国两广地区高发，初诊时晚期患者约占70%以上［1］。早期采用单纯放疗，晚期采用放化联合治疗［2］。放射治疗失败的原因主要是远处转移和局部复发［3］。Apogossypolone（ApoG2）是棉酚的一种新型衍生物，作为一种Bcl-2小分子抑制剂，毒副作用小，患者耐受性好，对胰腺癌［4］、前列腺癌［5］、乳腺癌［6］等多种肿瘤有抑制作用。神经酰胺是细胞膜神经鞘磷脂水解产生的一种脂质分子，在促进细胞死亡中有重要的生物学活性。ApoG2和神经酰胺毒副作用均较小，两者联合应用在国内外鲜见报道。本研究主要探讨两种靶向药物ApoG2联合神经酰胺体外作用对人鼻咽癌CNE-2细胞生长抑制，并初步探讨其可能机制。

1 材料与方法

1.1 材料与主要试剂

鼻咽癌CNE-2细胞株由南方医科大学肿瘤研究所冻存。ApoG2由美国密歇根大学医学院肿瘤中心徐梁教授惠赠。神经酰胺（N-acetyl-D-sphingosine，ceramide）购于Sigma公司。

1.2 CCK-8检测药物对细胞的生长抑制作用

按每孔5×103个细胞接种于96孔培养板，培养箱中培养24 h，弃废液，设ApoG2、神经酰胺药物浓度均为5、10、20、40、60、80 μmol/L，两药联合作用时保持两药终浓度不变；对照组为0.1%DMSO培养液，空白对照组不加细胞悬液只加培养液，每组4个平行孔，加培养液100 μL。继续培养48 h，弃废液，每孔加100 μL新培养液及CCK-8液10 μL，继续孵育1 h。酶标仪检测450 nm波长下各孔OD值，实验重复3次，分别计算各组的增殖抑制率，抑制率（%）=1-（OD实验组-OD空白组）/（OD对照组-OD空白组）×100%。CDI［7］=AB/（A×B）。AB为联合作用组吸光度值与对照组吸光度的比值；A、B是单药作用组的吸光度值与对照组吸光度的比值。当CDI＜1时，两药有协同作用；CDI=1时两药作用相加；CDI＞1时，两药拮抗。

1.3 Hoechst33258染色观察细胞凋亡及吖啶橙（AO）染色、透射电镜观察细胞自噬形态学变化

消化传代细胞，待细胞生长至50%～70%时，分为对照组（0.1%DMSO），ApoG2药物组（20 μmol/L），神经酰胺药物组（20 μmol/L），联合用药组（ApoG2、神经酰胺均为20 μmol/L）。培养48h后弃废液，加Hoechst 33258染色液，避光染色5 min，荧光显微镜下观察细胞凋亡形态。

按上述分组处理细胞，加终浓度为1 mg/L的AO避光作用15 min，PBS洗涤3遍，荧光显微镜下观察细胞自噬形态。收集按上述分组处理的细胞1×106个，离心，去上清，固定，送电镜室进行常规切片，透射电镜下观察细胞超微结构。

1.4 FCM检测细胞凋亡率及自噬荧光强度

按上述分组处理细胞，各收集5×105个细胞，加入400 μL的Binding Buffer及5 μL Annexin V-FITC混匀后，再加入5 μL碘化丙啶（propidium iodide，PI）混匀；避光反应10 min，上机检测细胞凋亡率。细胞分组处理后，各收集5×105个细胞，加1mg/L的吖啶橙避光作用15 min，PBS洗涤3次，上机检测自噬荧光强度，用FL1-Height和FL3-Height荧光通道分别代表绿色荧光和红色荧光，实验重复3次。

1.5 Western Blot检测Bcl-2、Beclin1蛋白表达

按上述分组处理细胞。收集总蛋白，BCA法测蛋白浓度，12%SDS-PAGE胶上电泳，15V、60 min半干转膜仪转至PVDF，5%脱脂牛奶封闭1 h。洗膜后，加入Bcl-2多克隆抗体（1：500稀释）、Beclin1多克隆抗体（1：500稀释）4℃过夜。洗膜后，二抗（1：2 000稀释）孵育1 h。加化学发光剂ECL，暗室曝光，显影、定影后扫描，用Image J图像分析系统分析结果。蛋白的相对表达量为目的蛋白与内参β-actin的灰度值之比。实验重复3次。

1.6 统计学方法

采用SPSS 13.0统计学软件进行数据处理，计量数据采用±s表示，组间差异采用单因素方差分析，P＜0.05为差异有统计学意义。

2 结果

2.1 CCK-8检测药物对细胞的生长抑制作用

ApoG2与神经酰胺单独作用CNE-2细胞48h，计算各浓度抑制率及CDI（表1），差异具有统计学意义（FApoG2=611.533，PApoG2＜0.001；Fceramide=495.730，Pceramide＜0.001），联合用药组抑制率随着两者药物浓度的增加逐渐增加，两者之间存在交互效应（F=13.290，P＜0.001）。当药物浓度低于40 μmol/L时，CDI＜1，两者发挥协同效应；当药物浓度为60、80 μmol/L时，CDI＞1，两药作用相加甚至拮抗。作用48h后ApoG2与神经酰胺IC50值分别为49.20 μmol/L和62.04 μmol/L。测的敏感性，所以建议实验室通过大样本的检测，建立合理的人附睾分泌蛋白生物参考区间，以提高检测的准确性。

本研究分析了电化学发光法和酶免法两种检测方法的差异，结果示同一样本ECLIA法检测值高于ELISA法检测值。通过比较两种方法鉴别诊断卵巢癌的ROC-AUC，得知ECLIA法的曲线下面积更大，说明ECLIA法的诊断准确性高于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

南方医科大学珠江医院肿瘤中心（广州市510282）

*本文课题受广州市科技计划项目（编号：2011y2-00019-3）资助

汪森明 wsenming@126.com

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

（本文编辑：周晓颖）