Inhibition Of PERK Pathway Enhances Sensitivity Of Human Osteosarcoma HOS Cells Induced To Pyropheophorbide-a Methyl Ester-mediated Photodynamic Therapy

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-Ⅰand P62）were investigated using Western blotting;the changes in the expression of p-PERK was also examined using immunofluorescence assay.The morphology of Endoplasmic reticulum and Autophagosomes were observed by means of a transmission electron microscope.The cell apoptotic rates and Ca²⁺concentration 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-Ⅰexpression 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-Ⅰand obviously lowered apoptotic rate and expression levels of PERK,Cleaved caspase-3 and Cleaved PARP.Pretreatment of the cells with siRNAp21 enhanced the effects of MPPa-PDT to increase the expression levels of Cleaved caspase-3 and apoptosis rate despite of suppressing the expression of P21.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.