化香树果序醇提物诱导人鼻咽癌CNE1/2细胞发生巨泡式死亡的机制  

Mechanism for ethanol extract of infructescence of Platycarya strobilacea induced methuosis in human nasopharyngeal carcinoma CNE1/2 cells

作　　者：涂维 刘金坤[2] 沈鸿贵 莫惠婷 蔡红兵[1,3] TU Wei;LIU Jinkun;SHEN Honggui;MO Huiting;CAI Hongbing(College of Traditional ChineseMedicine,Southern Medical University,Guangzhou 510315,China;Chongging Key Laboratory of Traditional Chinese Medicine,Chongqing Hospital of Traditional Chinese Medicine,Chongqing 400021,China;Department of Oncology,Integrated Traditional Chinese and Westem Medicine Hospital of Southern Medical University,Guangzhou 510315,China)

机构地区：[1]南方医科大学中医药学院,广东广州510315 [2]重庆市中医院重点实验室,重庆400021 [3]南方医科大学中西医结合医院肿瘤科,广东广州510315

出　　处：《暨南大学学报（自然科学与医学版）》2021年第5期478-489,共12页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：广东省自然科学基金项目(2016A030313724)。

摘　　要：目的:研究化香树果序醇提物(EPS)诱导鼻咽癌细胞CNE1/2发生巨泡式死亡的机制及对荷鼻咽癌转移瘤裸鼠的抑瘤作用.方法:采用MTT实验及平板克隆形成实验检测EPS对CNE1/2细胞活性及增殖的影响.设计荧光示踪实验,设置空白组、荧光黄处理组、荧光黄+EPS处理组、荧光黄+EPS+细胞松弛剂处理组,在荧光显微镜下观察细胞的形态变化并采用流式细胞分析仪分析细胞荧光值.蛋白免疫印迹法检测c-fos、ARF6、DUSP1、p-DUSP1、ERK1/2、p-ERK1/2等蛋白表达水平.使用小分子c-fos抑制剂T-5224及DUSP1抑制剂BCI分别处理细胞并孵育24 h后观察细胞形态的变化,进一步在倒置显微镜下观察EPS处理过表达ARF6及c-fos的鼻咽癌细胞后细胞的形态变化,并在蛋白水平进行验证.最后通过荷鼻咽癌移植瘤裸鼠模型,研究EPS在体内的抑瘤作用,瘤组织免疫组化研究相关蛋白在瘤组织中的表达差异.结果:EPS能明显抑制CNE1/2细胞的活性及增殖(P<0.05).荧光黄+EPS处理组与荧光黄+EPS+细胞松弛剂组相比,结果无统计学差异,使用细胞松弛剂并不减少细胞对荧光示踪剂的摄取.与对照组相比,p-ERK1/2、c-fos、p-DUSP1蛋白表达明显下降.使用c-fos及DUSP1抑制剂后,巨泡式死亡现象消失,过表达ARF6及c-fos也能逆转液泡在细胞内集聚.体内实验显示EPS能抑制荷鼻咽癌移植瘤裸鼠肿瘤的生长,实验组瘤组织中c-fos、p-DUSP1表达与对照组相比下降,与体外细胞实验结果一致.结论:EPS在体内外均有明显抗肿瘤作用,并通过抑制ARF6/ERK1/2/c-fos通路和DUSP1磷酸化诱导人鼻咽癌CNE1/2细胞发生巨泡式死亡.Objective:To study the mechanism of ethanol extract of inflorescence of Platycarya strobilacea(EPS)inducing Methuosis of nasopharyngeal carcinoma cell line CNE1/2 and its antitumor effect on nude mice bearing nasopharyngeal carcinoma metastasis.Methods:MTT assay and plate clone formation test were used to detect the effect of EPS on the activity and proliferation of CNE1/2 cells.Fluorescence tracing experiment was designed.Blank group,fluorescein treatment group,fluorescein+EPS treatment group and fluorescein+EPS+cell relaxant group were set up.The morphological changes of cells were observed under fluorescence microscope,and the fluorescence value of cells was analyzed by Flow cytometry.The expression levels of c-fos,ARF6,DUSP1,p-DUSP1,ERK1/2 and p-ERK1/2 were detected by Western blot.The cells were treated with small molecule c-fos inhibitor t-5224 and DUSP1 inhibitor BCI respectively and incubated for 24 hours.The morphological changes of nasopharyngeal carcinoma cells expressing ARF6 and c-fos were observed under inverted microscope,and verified at protein level.Finally,the antitumor effect of EPS in vivo and the expression of related proteins in tumor tissue were studied by immunohistochemistry.Results:EPS could significantly inhibit the activity and proliferation of CNE1/2 cells(P<0.05).There was no significant difference between LY+EPS group and LY+EPS+cell relaxant group.The use of cell relaxant did not reduce the uptake of fluorescent tracer.Compared with the control group,the expression of c-fos,c-myc and ARF6 mRNA was significantly different.Western blotting verified that the protein expression of p-ERK1/2,c-fos,p-DUSP1 protein decreased.After using c-fos and DUSP1 inhibitors,the phenomenon of giant bubble death disappeared.Overexpression of ARF6 and c-fos also reversed vacuole aggregation.EPS can inhibit the growth of transplanted NPC in nude mice,which was consistent with the results of cell experiment in vitro.Conclusion:EPS has obvious anti-tumor effect in vivo and in vitro,and induces CNE1/2 cells

关 键 词：化香树果序醇提物 巨泡式死亡 ARF6 C-FOS DUSP1 

分 类 号：R739.6[医药卫生—肿瘤]