花姜酮通过免疫原性死亡影响黏液表皮样癌的机制研究  

Mechanism of zerumbone affecting mucoepidermoid carcinoma by immunogenic cell death

作　　者：苏凤芹 苏慧 吴慧炜 邢淑华 SU Feng-qin;SU Hui;WU Hui-wei;XING Shu-hua(Department of Pharmacy,People′s Hospital of Ji′nan City,Shandong Province,Ji′nan 271199,China)

机构地区：[1]济南市人民医院药剂科,山东济南271199

出　　处：《河北医科大学学报》2023年第7期767-772,共6页Journal of Hebei Medical University

基　　金：山东省优秀中青年科学家科研奖励基金(BS2020SW409)。

摘　　要：目的 探究花姜酮(zerumbone,Zer)治疗黏液表皮样癌(mucoepidermoid carcinoma,MEC)的机制研究,以期为临床上开发MEC靶向治疗药物提供新的思路以及理论依据。方法 购入MEC细胞系H292及H3118,通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑[3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide,MTT]检测出Zer对MEC细胞系的IC50;在IC50的浓度下,进一步通过集落形成实验检测细胞增殖,利用流式细胞术检测细胞凋亡及表面钙网蛋白(calreticulin,CRT)蛋白水平,Western blot检测细胞高迁移率族蛋白B1(high mobility group protein B1,HMGB1)表达水平,观察Zer是否可以影响MEC的免疫原性死亡;最后为探寻Zer可能的分子调控机制,采取pcDNA3.1-STAT3上调MEC细胞中信号转导因子和转录激活因子3(signal transducer and activator of transcription 3,STAT3)的水平,通过实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)检测转染效率及qRT-PCR、Western blot检测内质网应激相关因子活化的转录因子4(activating transcription factor 4,ATF4)、ATF3及转录因子同源蛋白(C/EBP-homologous protein,CHOP)的表达水平,三磷酸腺苷(adenosine triphosphate,ATP)测定试剂盒检测培养基中ATP含量,MEC。结果 Zer组细胞凋亡率高于Control组(P<0.05),Zer组H3118细胞上清液中HMGB1蛋白含量高于Control组(P<0.05),Zer组H3118细胞中ATP含量高于Control组(P<0.05),Zer组细胞内质网应激相关基因ATF4、ATF3及CHOP的蛋白和mRNA表达水平均高于Control组(P<0.05),Zer组的MEC细胞中STAT3 mRNA水平低于Control组(P<0.05),Zer+oe-STAT3组细胞内质网应激相关基因ATF4、ATF3及CHOP的蛋白和mRNA表达水平均低于Zer+oe-NC组(P<0.05)。结论 Zer介导STAT3促进MEC细胞内质网应激由此诱导MEC细胞免疫原性死亡。Objective To explore the mechanism of zerumbone(Zer)in the treatment of mucoepidermoid carcinoma(MEC),in order to provide new ideas and theoretical basis for the development of MEC-related targeted drugs in clinical practice.Methods MEC cell lines H292 and H3118 were purchased,and the IC50 of Zer on MEC cell lines was detected by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Cell proliferation was detected by colony formation assay,and cell apoptosis and surface calreticulin(CRT)protein levels were detected by flow cytometry.The expression level of highmobility group protein B1(HMGB1)was detected by Western blot,to observe whether Zer could affect the immunogenic cell death(ICD)of MEC.Finally,to explore the possible molecular regulatory mechanism of Zer,the level of signal transducer and activator of transcription 3(STAT3)in MEC cells was up-regulated by pcDNA3.1-STAT3,and the transfection efficiency was detected by qRT-PCR.The expression levels of endoplasmic reticulum stress-related factors activating transcription factor 4(ATF4),ATF3 and C/EBP-homologousprotein(CHOP)were detected by real-time quantitative PCR(qRT-PCR)and Western blot.The adenosine triphosphate(ATP)level in the medium was detected by ATP assay kit.Results The apoptosis rate of cells in the Zer group was higher than that in the control group(P<0.05),and the HMGB1 protein level in the supernatant of H3118 cells in the Zer group was higher than that in the control group(P<0.05).The ATP level in H3118 cells in the Zer group was higher than that in the control group(P<0.05),and the protein and mRNA expression levels of endoplasmic reticulum stress-related genes ATF4,ATF3,and CHOP in the Zer group were higher than those in the control group(P<0.05).The STAT3 mRNA level in MECs in the Zer group was lower than that in the control group(P<0.05),and the protein and mRNA expression levels of endoplasmic reticulum stress-related genes ATF4,ATF3 and CHOP in the Zer+oe STAT3 group were lower than those in the Zer+oe NC group(P<0.

关 键 词：癌 黏液表皮样 内质网应激 免疫原性死亡 

分 类 号：R730.26[医药卫生—肿瘤]