钠钙交换体抑制剂苄普地尔对黑色素瘤细胞增殖、迁移及凋亡的影响  

Effect of the Na+/Ca2+exchanger inhibitor bepridil on the proliferation,migration and apoptosis of melanoma cells

作　　者：田翠翠 石浩泽 陈浩 Tian Cuicui;Shi Haoze;Chen Hao(Hospital for Skin Diseases,Institute of Dermatology,Chinese Academy of Medical Sciences&Peking Union Medical College,Nanjing 210042,China;Department of Pathology,Hospital of Dermatology,Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing 210042,China)

机构地区：[1]北京协和医学院,中国医学科学院,皮肤病医院皮肤病研究所,南京210042 [2]中国医学科学院,北京协和医学院皮肤病医院病理科,南京210042

出　　处：《中华皮肤科杂志》2024年第6期530-538,共9页Chinese Journal of Dermatology

基　　金：国家重点研发计划项目(2022YFC3601800);江苏省自然科学基金(BK20211026);江苏省卫健委医学科研立项项目(M2021045);中国医学科学院医学与健康科技创新工程临床转化专项(2023‐I2M‐C&T‐B‐110)。

摘　　要：目的探讨钠钙交换体(NCX)抑制剂苄普地尔对黑色素瘤细胞增殖、迁移及凋亡的影响及可能的调控机制。方法收集2023年1-12月就诊于中国医学科学院皮肤病医院且组织病理学诊断为色素痣及黑色素瘤患者的组织蜡块各3份,采用免疫组化染色检测组织中NCX1的表达,Western印迹验证NCX1在原代黑色素细胞及黑色素瘤细胞系A375、A875、SK-MEL-28、M14、MV3及SK-MEL-5细胞中的表达情况。采用细胞计数试剂盒8(CCK8)检测不同浓度苄普地尔对黑色素瘤细胞活力的影响,绘制增殖曲线并计算半数抑制浓度(IC50)。采用IC50浓度苄普地尔处理A375及SK-MEL-28细胞后,使用Fluo-4钙离子检测试剂盒检测细胞内钙离子水平,Transwell法和流式细胞仪分别检测黑色素瘤细胞A375、SK-MEL-28及A2058细胞迁移能力及凋亡情况。通过转录组测序、基因本体论(GO)富集分析和京都基因与基因组百科全书(KEGG)分析苄普地尔处理对A375细胞中基因表达及通路的影响。采用流式细胞仪检测苄普地尔处理后黑色素瘤细胞内活性氧含量,Western印迹检测内质网应激及线粒体凋亡通路相关分子的表达。两组间比较采用t检验。结果免疫组化检测显示,黑色素瘤组织中NCX1的表达(0.320±0.020)高于色素痣组织(0.235±0.008,t=4.04,P=0.016);Western印迹实验显示,各黑色素瘤细胞系中NCX1蛋白条带灰度均深于原代黑色素细胞中NCX1蛋白条带。CCK8实验显示,随苄普地尔浓度增加黑色素瘤细胞活力逐渐降低。采用IC50浓度(25μmol/L)的苄普地尔处理后,A375和SK-MEL-28细胞荧光强度(64.82±2.98、75.84±2.07)均高于相应对照组(37.10±2.33、66.54±1.47,均P<0.05),A375、SK-MEL-28和A2058细胞的迁移能力(103.00±9.07、67.33±7.22、61.33±1.76)均低于相应对照组(400.00±25.17、276.70±14.63、116.00±10.69,均P<0.05),其细胞凋亡率(5.72%±0.06%、13.58%±0.86%、25.76%±1.95%)均高于相应对照组(3.99%±0.50%、6.47%Objective To elucidate the effect of bepridil,an Na+/Ca2+exchanger(NCX)inhibitor,on the proliferation,migration and apoptosis of melanoma cells,and to explore their potential underlying mechanisms.Methods Six paraffin‐embedded tissue specimens were collected from 3 patients histopathologically diagnosed with melanocytic nevi and 3 patients histopathologically diagnosed with melanoma in the Hospital for Skin Diseases,Chinese Academy of Medical Sciences from January to December 2023.NCX1 expression in tissues was determined by immunohistochemical staining,and Western blot analysis was performed to verify the expression of NCX1 in primary melanocytes and melanoma cell lines A375,A875,SKMEL‐28,M14,MV3,and SK‐MEL‐5.Cell counting kit 8(CCK8)assay was conducted to evaluate the effect of bepridil at different concentrations on the viability of melanoma cells,and the proliferation curve was drawn to calculate the half inhibitory concentration(IC50)of bepridil.Some melanoma cells were then treated with bepridil at IC50(bepridil groups),and cells treated with dimethyl sulfoxide‐containing media served as control groups.Intracellular Ca2+levels were assessed in A375 and SK-MEL-28 cells using the Fluo-4 calcium assay kit,the migration and apoptosis of A375,SK‐MEL‐28,and A2058 cells were estimated by Transwell assay and flow cytometry respectively.The effects of bepridil treatment on gene expression and pathways in A375 cells were evaluated by transcriptome sequencing,Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.The content of reactive oxygen species(ROS)in melanoma cells after the bepridil treatment was detected by flow cytometry,and the expression of endoplasmic reticulum stress-and mitochondrial apoptotic pathway‐related molecules was determined by Western blot analysis.Comparisons between two groups were performed by t test.Results Immunohistochemical assay showed that the expression of NCX1 was significantly higher in the melanoma tissues(0.320±0.0

关 键 词：黑色素瘤 细胞增殖 转录组 内质网应激 钠钙交换体 细胞迁移 苄普地尔 

分 类 号：R739.5[医药卫生—肿瘤]