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

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.

Keywords

• rna-activated protein kinase-like endoplasmic reticulum kinase pathway
• pyropheophorbide-a methyl ester
• photodynamic therapy
• human osteosarcoma hos cells