[1]钟申熹,欧云生,陈艳阳,等.阻滞PERK信号通路增强人骨肉瘤HOS细胞对MPPa-PDT疗法的敏感性 [J].第三军医大学学报,2019,41(02):100-109.
 ZHONG Shenxi,OU Yunsheng,CHEN Yanyang,et al.Inhibition of PERK pathway enhances sensitivity of human osteosarcoma HOS cells induced to pyropheophorbide-a methyl ester-mediated photodynamic therapy[J].J Third Mil Med Univ,2019,41(02):100-109.
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阻滞PERK信号通路增强人骨肉瘤HOS细胞对MPPa-PDT疗法的敏感性 (/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第02期
页码:
100-109
栏目:
基础医学
出版日期:
2019-01-30

文章信息/Info

Title:
Inhibition of PERK pathway enhances sensitivity of human osteosarcoma HOS cells induced to pyropheophorbide-a methyl ester-mediated photodynamic therapy
作者:
钟申熹欧云生陈艳阳余浩洋左强
重庆医科大学附属第一医院骨科
Author(s):
ZHONG Shenxi OU Yunsheng CHEN Yanyang YU Haoyang ZUO Qiang

Department of Orthopedic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China

关键词:
蛋白激酶样内质网激酶通路焦脱镁叶绿酸-a甲酯光动力疗法人骨肉瘤HOS细胞
Keywords:
RNA-activated protein kinase-like endoplasmic reticulum kinase pathway pyropheophorbide-a methyl ester photodynamic therapy human osteosarcoma HOS cells
分类号:
R345; R7336; R738.1
文献标志码:
A
摘要:

目的  观察蛋白激酶样内质网激酶(RNA-activated protein kinase-like endoplasmic reticulum kinase, PERK)通路在焦脱镁叶绿酸-a甲酯介导的光动力疗法(pyropheophorbide-a methyl ester-mediated photodynamic therapy,MPPa-PDT)诱导人骨肉瘤HOS细胞凋亡、自噬中的作用,并探讨阻滞PERK通路增强人骨肉瘤HOS细胞对MPPa-PDT敏感性的影响及机制。方法 MPPa-PDT处理人骨肉瘤HOS细胞后以Hoechst染色观察胞核形态学变化。以空白组、MPPa组、LED组为对照组,以MPPa-PDT处理组(MPPa-PDT)、MPPa-PDT联合PERK通路抑制剂GSK2656157处理组(MPPa-PDT+GSK2656157)、MPPa-PDT联合自噬抑制剂Bafilomycin A1处理组(MPPa+PDT+Bafilomycin A1)为实验组。采用Western blot检测PERK通路相关蛋白PERK、p-PERK、ATF4、CHOP,凋亡相关蛋白Cleaved-caspase3、Cleaved-PARP,自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ、P62的表达,免疫荧光检测p-PERK表达变化,流式细胞仪检测细胞凋亡率。结果 MPPa-PDT诱导人骨肉瘤HOS细胞凋亡、自噬、PERK通路激活,细胞核呈固缩、碎裂等凋亡形态学变化。与空白组、MPPa组、LED组比较,MPPa-PDT组Cleaved-caspase3、Cleaved-PARP、LC3-Ⅱ/LC3-Ⅰ、p-PERK、ATF4、CHOP表达升高,p-PERK荧光强度增强(P<0.05),而P62、PERK表达降低(P<0.05)。与MPPa-PDT组比较,MPPa-PDT+GSK2656157组p-PERK、ATF4、LC3-Ⅱ/LC3-Ⅰ表达降低,p-PERK 荧光强度减弱,PERK、P62、Cleaved-caspase3、Cleaved-PARP表达升高,凋亡增强(P<0.05);与MPPa-PDT组比较,MPPa-PDT+Bafilomycin A1组LC3-Ⅱ/LC3-Ⅰ、P62、Cleavedcaspase3、Cleaved-PARP表达升高,凋亡增强(P<0.05);与MPPa-PDT+GSK2656157组比较,MPPa-PDT+Bafilomycin A1组p-PERK、ATF4、LC3-Ⅱ/LC3-Ⅰ表达升高,PERK、Cleaved-caspase3、Cleaved-PARP表达降低,凋亡减弱(P<0.05)。结论  MPPa-PDT可激活PERK通路,诱导保护性自噬及未折叠蛋白反应,从而促细胞存活。阻滞PERK通路可解除该作用,增强MPPa-PDT对人骨肉瘤HOS细胞的杀伤作用。

Abstract:

Objective    To explore the role of RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) pathway in the crosstalk of apoptosis and autophagy in human osteosarcoma HOS cells induced by pyropheophorbide-a methyl ester-mediated photodynamic therapy (MPPa-PDT) and investigate the mechanism by which PERK pathway inhibition enhances the sensitivity of the cells to MPPa-PDT. Methods   We first observed the apoptotic morphology of human osteosarcoma HOS cells after MPPa-PDT using Hoechst 33258 staining. HOS cells were treated with MPPa-PDT alone or in combination with a PERK inhibitor (GSK2656157) or an autophagy inhibitor (Bafilomycin A1), and the changes in the expression of the proteins related to PERK pathway (PERK, p-PERK, ATF4, and CHOP), apoptosis (Cleaved caspase-3, Cleaved PARP) and autophagy (LC3-Ⅱ/LC3-Ⅰ and P62) were investigated using Western blotting; the changes in the expression of p-PERK was also examined using immunofluorescence assay. The cell apoptotic rates following the treatments were analyzed using flow cytometry. Results MPPa-PDT resulted in typical morphological changes of apoptosis (nuclear pyknosis and fragmentation) in HOS cells, and induced obvious autophagy and activation of PERK pathway. HOS cells treated with MPPa-PDT, compared with the control cells, showed significantly increased expression of Cleaved caspase-3, Cleaved PARP, LC3-Ⅱ/LC3-Ⅰ, p-PERK, ATF4 and CHOP and lowered expression of P62 and PERK; the cells exhibited stronger green fluorescence signals of p-PERK after MPPa-PDT treatment than the control cells. The application of GSK2656157 significantly blocked MPPa-PDT-induced increases in p-PERK, ATF4 and LC3-Ⅱ/LC3-Ⅰ expression levels, while treatment of the cells with GSK2656157 prior to MPPa-PDT enhanced the cell apoptosis and increased the expression levels of PERK, P62, Cleaved caspase-3 and Cleaved PARP. The cells with combined treatment with MPPa-PDT and GSK2656157 showed weaker green fluorescence signal of p-PERK than those with MPPa-PDT alone. Pretreatment of the cells with Bafilomycin A1 augmented the effects of MPPa-PDT to increase the expression levels of LC3-Ⅱ/LC3-Ⅰ, P62, Cleaved caspase-3 and Cleaved PARP and the apoptosis rate. Compared with the cells with combined MPPa-PDT and GSK2656157 treatment, the cells treated with MPPa-PDT and Bafilomycin A1 showed significantly increased levels of p-PERK, ATF4 and LC3-Ⅱ/LC3-Ⅰ and obviously lowered apoptotic rate and expression levels of PERK, Cleaved caspase-3 and Cleaved PARP. Conclusion   The activation of PERK pathway induced by MPPa-PDT may mediate prosurvival autophagy and unfolded protein response, and blocking PERK pathway enhances the killing effect of MPPa-PDT in human osteosarcoma HOS cells.

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更新日期/Last Update: 2019-01-22