Apogossypolone联合神经酰胺诱导鼻咽癌CNE-2细胞凋亡与自噬

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

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。

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 tech- nique is also evaluated in this study. Methods： ApoG2 and ceramide of different concentrations were applied, individually or simultane- ously, 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 con- ducted 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 au- tophagy were determined by flow cytometry （FCM）. The expressions of Bcl-2 and Beclinl proteins were analyzed by Western blot. Re- suits： 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〈l）. 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 frag- mentation 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 in- tensity of autophagy when treated with both drugs. The differences were statistically significant between the single and combined appli- cation groups （Fapop,os,=106.72, Papop,osi=0.000; Fapopois=140.77, Papopto=0.000）. Western blot analysis showed that Bcl-2 protein expression was downregulated with statistically significant differences between the two groups （F=I 11.071, P〈0.001）. By contrast, Beclinl expres- sion increased in the combined therapy group compared with the other groups. Statistically significant differences were found amongthe 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 re- lated to the downregulation of Bcl-2 expression and the upregulation of Beclinl expression.