摘要
目的:探讨p62在喉癌细胞Hep-2化疗耐药中的作用及潜在的作用机制。方法:实时荧光定量聚合酶链式反应(RT-q PCR)及Western blot法检测喉癌耐药细胞Hep-2/5-FU及其亲本细胞Hep-2中p62的表达水平。在Hep-2/5-FU细胞中转染p62 siRNA敲减p62的表达,采用CCK-8法和流式细胞术检测细胞生存率及细胞凋亡状态;检测细胞中丙二醛(malondialdehyde,MDA)的含量及超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的活性来反映细胞氧化应激水平。Western blot检测细胞凋亡相关调控因子Bcl-2、Bax、caspase-8/cleaved caspase-8、caspase-3/cleaved caspase-3的蛋白水平及抗氧化通路Keap1/Nrf2的活性。结果:耐药细胞株Hep-2/5-FU中p62的mRNA及蛋白表达水平均明显高于亲本细胞株Hep-2;并且在亲本细胞株Hep-2中,p62和Nrf2的蛋白表达水平随着顺铂的浓度增加不断升高。沉默p62可抑制喉癌耐药细胞Hep-2/5-FU的生存,促进其凋亡,上调MDA的含量,降低SOD和GSH-Px的活性,同时上调Bax、cleaved caspase-8、cleaved caspase-3和Keap1的蛋白水平,下调Bcl-2、Nrf2及HO-1的蛋白表达。结论:喉癌耐药细胞Hep-2/5-FU中沉默p62可恢复细胞对5-FU的敏感性,其机制可能与抑制Keap1/Nrf2信号通路的活化、调控细胞内氧化应激反应及细胞凋亡有关。
AIM:To investigate the effects of p62 on drug resistance of human laryngocarcinoma cell line Hep-2.METHODS:The abundance of p62 in Hep-2/5-FU and Hep-2 cells was measured by RT-qPCR and Western blot.Af-ter silencing of p62 with p62 siRNA in the Hep-2/5-FU cells,the cell viability and cell apoptosis were determined by CCK-8 assay and flow cytometry.The levels of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxi-dase(GSH-Px)were measured to reflect the status of oxidative stress in the cells.The protein levels of apoptosis-related molecules Bel-2,Bax,caspase-8/cleaved caspase-8 and caspase-3/cleaved caspase-3,and the activity of anti-oxidative stress pathway-related proteins Keapl/Nrf2 were measured by Western blot.R E SU L T S:The expression of p62 at both mRNA and protein levels was significantly up-regulated in the Hep-2/5-FU cells.The expression of p62 and NrG increased in a dose-dependent manner in the Hep-2 cells.Knockdown of p62 inhibited the viability and promoted the apoptosis of the Hep-2/5-FU cells.Increased content of MDA,and suppressed activity of SOD and GSH-Px were also observed.Further-more,knockdown of p62 up-regulated the protein levels of Bax,cleaved caspase-8,cleaved caspase-3 and K e apl,but down-regulated the protein levels of Bel-2,Nrf2 and HO-1.CONCLUSION:Knockdown of p62 increases the sensitivity of Hep-2/5-FU cells to 5-FU exposure.The mechanism may be related to the inhibition of Keapl/Nrf2 pathway and the modulation of oxidative stress and cell apoptosis.
作者
廖贵华
黄文峰
LIAO Gui-hua;HUANG Wen-feng(Department of ENT&HN Surgery,Yichang Hospital of TCM,College of Clinical Medicine of TCM,China Three Gorges University,Yichang 443003,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2017年第6期1031-1037,共7页
Chinese Journal of Pathophysiology
基金
宜昌市大学科学研究与计划项目(No.A201230234)
关键词
P62
喉癌
氧化应激
细胞凋亡
p62
Laryngeal neoplasms
Oxidative stress
Apoptosis
