中国肿瘤临床
中國腫瘤臨床
중국종류림상
CHINESE JOURNAL OF CLINICAL ONCOLOGY
2013年
6期
312-314
,共3页
石丰榕%汪森明%贺蛟%罗皓%钟梅%吴丹心%朱震威
石豐榕%汪森明%賀蛟%囉皓%鐘梅%吳丹心%硃震威
석봉용%왕삼명%하교%라호%종매%오단심%주진위
鼻咽癌%Apogossypolone%神经酰胺%凋亡%自噬
鼻嚥癌%Apogossypolone%神經酰胺%凋亡%自噬
비인암%Apogossypolone%신경선알%조망%자서
nasopharyngeal carcinoma%apogossypolone%ceramide%apoptosis%autophagy
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.
目的:探討棉酚衍生物Apogossypolone(ApoG2)聯閤神經酰胺體外抑製鼻嚥癌CNE-2細胞增殖,併初步探討其可能機製.方法:CCK-8測定不同濃度ApoG2和神經酰胺單藥毒性及聯閤應用對CNE-2細胞的抑製作用,計算CDI判定藥物聯閤效果.Hoechst33258染色觀察細胞凋亡,吖啶橙(AO)染色、透射電鏡觀察自噬形態學變化,FCM檢測凋亡率與自噬熒光彊度.Western Blot檢測Bcl-2、Beclin1蛋白錶達.結果:CCK-8檢測髮現ApoG2和神經酰胺單獨應用時,隨藥物濃度增加,對CNE-2細胞生長的抑製作用也增加;低濃度兩藥聯閤作用能協同增彊單藥抑製鼻嚥癌細胞CNE-2細胞生長(CDI<1).Hoechst33258染色顯示聯閤用藥後齣現更多的覈固縮和碎裂等凋亡現象;吖啶橙染色顯示聯閤用藥後產生更多的亮紅色痠性自噬泡.透射電鏡觀察到聯閤用藥後細胞內大空泡及膜性雙層結構增多.FCM檢測聯閤用藥組細胞凋亡率和自噬率均較單獨處理組升高,差異具有統計學意義(F凋亡=106.72,P凋亡<0.001,F自噬=140.77,P自噬<0.001).Western Blot檢測髮現聯閤用藥組Bcl-2蛋白錶達較單藥處理組降低(F=111.071,P<0.001),Beclin1蛋白錶達較單獨處理組升高(F=62.271,P<0.001).結論:低濃度ApoG2與神經酰胺聯閤共同誘導細胞凋亡與自噬,協同抑製鼻嚥癌細胞生長,其作用機製可能與下調Bcl-2和上調Beclin1的錶達有關.
목적:탐토면분연생물Apogossypolone(ApoG2)연합신경선알체외억제비인암CNE-2세포증식,병초보탐토기가능궤제.방법:CCK-8측정불동농도ApoG2화신경선알단약독성급연합응용대CNE-2세포적억제작용,계산CDI판정약물연합효과.Hoechst33258염색관찰세포조망,아정등(AO)염색、투사전경관찰자서형태학변화,FCM검측조망솔여자서형광강도.Western Blot검측Bcl-2、Beclin1단백표체.결과:CCK-8검측발현ApoG2화신경선알단독응용시,수약물농도증가,대CNE-2세포생장적억제작용야증가;저농도량약연합작용능협동증강단약억제비인암세포CNE-2세포생장(CDI<1).Hoechst33258염색현시연합용약후출현경다적핵고축화쇄렬등조망현상;아정등염색현시연합용약후산생경다적량홍색산성자서포.투사전경관찰도연합용약후세포내대공포급막성쌍층결구증다.FCM검측연합용약조세포조망솔화자서솔균교단독처리조승고,차이구유통계학의의(F조망=106.72,P조망<0.001,F자서=140.77,P자서<0.001).Western Blot검측발현연합용약조Bcl-2단백표체교단약처리조강저(F=111.071,P<0.001),Beclin1단백표체교단독처리조승고(F=62.271,P<0.001).결론:저농도ApoG2여신경선알연합공동유도세포조망여자서,협동억제비인암세포생장,기작용궤제가능여하조Bcl-2화상조Beclin1적표체유관.
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 Beclin1 proteins were analyzed by Western blot. Re-sults: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 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 (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 expres-sion 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 re-lated to the downregulation of Bcl-2 expression and the upregulation of Beclin1 expression.