Inhibition of PERK pathway enhances sensitivity of human osteosarcoma HOS cells induced to pyropheophorbide-a methyl ester-mediated photodynamic therapy

Shenxi ZHONG; Yunsheng OU; Yanyang CHEN; Haoyang YU; Qiang ZUO

doi:10.16016/j.1000-5404.201808197

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Inhibition of PERK pathway enhances sensitivity of human osteosarcoma HOS cells induced to pyropheophorbide-a methyl ester-mediated photodynamic therapy

Basic Medicine | 更新时间：2026-01-09

- Inhibition of PERK pathway enhances sensitivity of human osteosarcoma HOS cells induced to pyropheophorbide-a methyl ester-mediated photodynamic therapy
- Journal of Third Military Medical University Vol. 41, Issue 2, Pages: 100-109(2019)
- 作者机构：
  
  400016 重庆，重庆医科大学附属第一医院骨科
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation(81572634)
- DOI：10.16016/j.1000-5404.201808197
  CLC： R345;R73-36;R738.1
- Received：30 August 2018，
  
  Revised：2018-11-08，
  
  Published：30 January 2019
- 稿件说明：
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Shenxi ZHONG, Yunsheng OU, Yanyang CHEN, et al. Inhibition of PERK pathway enhances sensitivity of human osteosarcoma HOS cells induced to pyropheophorbide-a methyl ester-mediated photodynamic therapy[J]. Journal of Third Military Medical University, 2019, 41(2): 100-109.
DOI：

Shenxi ZHONG, Yunsheng OU, Yanyang CHEN, et al. Inhibition of PERK pathway enhances sensitivity of human osteosarcoma HOS cells induced to pyropheophorbide-a methyl ester-mediated photodynamic therapy[J]. Journal of Third Military Medical University, 2019, 41(2): 100-109. DOI： 10.16016/j.1000-5404.201808197.

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(

0.05)，而P62、PERK表达降低(

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(

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(

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(

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.

关键词

Keywords

references

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