异硫氰酸苄酯通过激活p53和AMPK-FOXO1a信号通路诱导宫颈癌细胞周期阻滞和凋亡

Benzyl isothiocyanate induces cell cycle arrest and apoptosis in cervical cancerthrough activation of p53 and AMPK-FOXO1a signaling pathways

目的 研究异硫氰酸苄酯(benzyl isothiocyanate, BITC)对小鼠U14宫颈癌细胞增殖的影响,基于转录组数据分析探讨细胞毒性机制。方法 MTT法检测BITC对U14细胞活性的影响,Hochest 33258和荧光倒置显微镜检测细胞核形态变化,流式细胞术检测细胞周期和凋亡,基于Illumina Novaseq 6000测序平台建立BITC(20μmol·L^(-1))给药前后U14细胞转录组数据库,使用生物信息学分析手段对差异基因功能和信号通路进行富集分析。qRT-PCR和Western blot验证信号通路关键分子表达变化。结果 BITC呈时间和浓度依赖性抑制U14细胞的活性,阻滞细胞在G_(0)/G_(1)期,诱导了细胞凋亡,上调了Bax、CytC、cleaved caspase-9/3、下调了Bcl-2、cleaved caspase-7的表达量;通过转录组分析,差异基因主要富集在细胞周期、FOXO、p53、细胞凋亡等信号通路,qRT-PCR或Western blot验证p53、p21、Ire1a、Atf4、CHOP、AMPK、FOXO1a等分子表达上调,Cyclin D3,Cyclin E,Cdk2分子表达下调。结论 BITC阻滞U14细胞周期、通过caspase-3线粒体依赖途径和内质网应激途径诱导细胞凋亡,其机制与p53和AMPK-FOXO1a信号通路有密切关系。

Aim To investigate the effect of benzyl isothiocyanate(BITC)on the proliferation of mouse U14 cervical cancer cells and to explore the mechanism of cytotoxicity based on transcriptomic data analysis.Methods The effect of BITC on U14 cell activity was detected by MTT,nuclear morphological changes were observed by Hochest 33258 and fluorescent inverted microscope,cell cycle and apoptosis were determined by flow cytometry,and the transcriptome database of U14 cells before and after BITC(20μmol·L^(-1))treatment was established based on Illumina Novaseq 6000 sequencing platform.The expression of key molecules in the signaling pathway was verified by qRT-PCR and Western blot.Results BITC inhibited the activity of U14 cells in a time-and concentration-dependent manner,blocked cells in G_(0)/G_(1) phase,induced apoptosis,and up-regulated the expression of Bax,cytC,cleaved caspase-9/3,and down-regulated Bcl-2 and cleaved caspase-7;By transcriptome analysis,the differential genes were mainly enriched in cell cycle,FOXO,p53,apoptosis and other signaling pathways.qRT-PCR or Western blot verified that the expression of p53,p21,Ire1a,Atf4,CHOP,AMPK,and FOXO1a molecules were improved and Cyclin D3,Cyclin E,and Cdk2 molecules were reduced.Conclusions BITC blocks U14 cell cycle,induces apoptosis through caspase-3 mitochondria-dependent pathway and endoplasmic reticulum stress pathway.The mechanism is closely related to p53 and AMPK-FOXO1a signaling pathway.