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
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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MPPaPDT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Cleavedcaspase3、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Cleavedcaspase3、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。
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 MPPaPDT.Methods We first observed the apoptotic morphology of human osteosarcoma HOS cells after MPPaPDT 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 MPPaPDT 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.
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 MPPaPDT.Methods We first observed the apoptotic morphology of human osteosarcoma HOS cells after MPPaPDT 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 MPPaPDT 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.
引文
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[19]CHEN J,ZHANG Z Q,SONG J,et al.18β-glycyrrhetinicacid-mediated unfolded protein response induces autophagy and apoptosis in hepatocellular carcinoma[J].Sci Rep,2018,8(1):9365.DOI:10.1038/s41598-018-27142-5.
[20]RODVOLD J J,CHIU K T,HIRAMATSU N,et al.Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells[J].Sci Signal,2017,10(482):eaah7177.DOI:10.1126/scisignal.aah7177.
[21]ZHU Z C,LIU J W,LI K,et al.KPNB1 inhibition disrupts proteostasis and triggers unfolded protein response-mediated apoptosis in glioblastoma cells[J].Oncogene,2018,37(22):2936-2952.DOI:10.1038/s41388-018-0180-9.
[22]FRACCHIOLLA D,SAWA-MAKARSKA J,MARTENS S.Beyond Atg8 binding:The role of AIM/LIR motifs in autophagy[J].Autophagy,2017,13(5):978-979.DOI:10.1080/15548627.2016.1277311.
[23]ZHANG Y,QU X,JIANG L.An oasis in the desert of cancer chemotherapeutic resistance:The enlightenment from reciprocal crosstalk between signaling pathways of UPR and autophagy in cancers[J].Biomed Pharmacother,2017,92:972-981.DOI:10.1016/j.biopha.2017.05.132.
[24]JI G R,YU N C,XUE X,et al.PERK-mediated autophagy in osteosarcoma cells resists ER stress-induced cell apoptosis[J].Int J Biol Sci,2015,11(7):803-812.DOI:10.7150/ijbs.11100.
[25]SHI D,NIU P,HENG X,et al.Autophagy induced by cardamonin is associated with m TORC1 inhibition in SKOV3cells[J].Pharmacol Rep,2018,70(5):908-916.DOI:10.1016/j.pharep.2018.04.005.
[26]WANG J,HUANG S,TIAN R,et al.The protective autophagy activated by GANT-61 in MYCN amplified neuroblastoma cells is mediated by PERK[J].Oncotarget,2018,9(18):14413-14427.DOI:10.18632/oncotarget.24214.
[27]MA X H,PIAO S F,DEY S,et al.Targeting ER stressinduced autophagy overcomes BRAF inhibitor resistance in melanoma[J].J Clin Invest,2014,124(3):1406-1417.DOI:10.1172/JCI70454.
[28]FUSAKIO M E,WILLY J A,WANG Y,et al.Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver[J].Mol Biol Cell,2016,27(9):1536-1551.DOI:10.1091/mbc.E16-01-0039.
[29]MELBER A,HAYNES C M.UPRmt regulation and output:a stress response mediated by mitochondrial-nuclear communication[J].Cell Res,2018,28(3):281-295.DOI:10.1038/cr.2018.16.
[30]ROZPEDEK W,PYTEL D,MUCHA B,et al.The role of the PERK/e IF2α/ATF4/CHOP signaling pathway in tumor progression during endoplasmic reticulum stress[J].Curr Mol Med,2016,16(6):533-544.DOI:10.2174/1566524016666160523143937.
[2]黄志鹏,宋科官.骨肉瘤的诊断及治疗进展[J].国际骨科学杂志,2018,39(3):150-153.DOI:10.396/j.issn.1673-7083.2018.03.007.HUANG Z P,SONG K G.Progress in the diagnosis and treatment of osteosarcoma[J].Int J Orthop,2018,39(3):150-153.DOI:10.396/j.issn.1673-7083.2018.03.007.
[3]孙旭,孟宪瑛,王瑶琪,等.光动力疗法的抗肿瘤作用及其机制的研究进展[J].吉林大学学报(医学版),2018,44(1):200-204.DOI:10.13481/j.1671-587 x.20180140.SUN X,MENG X Y,WANG Y Q,et al.Research progress in anti-tumor effect and mechanism of photodynamic therapy[J].J Jilin Univ(Med Ed),2018,44(1):200-204.DOI:10.13481/j.1671-587 x.20180140.
[4]IURLARO R,MUOZ-PINEDO C.Cell death induced by endoplasmic reticulum stress[J].FEBS J,2016,283(14):2640-2652.DOI:10.1111/febs.13598.
[5]CORAZZARI M,GAGLIARDI M,FIMIA G M,et al.Endoplasmic reticulum stress,unfolded protein response,and cancer cell fate[J].Front oncol,2017,7:78.DOI:10.3389/fonc.2017.00078.
[6]TAALAB Y M,IBRAHIM N,MAHER A,et al.Mechanisms of disordered neurodegenerative function:concepts and facts about the different roles of the protein kinase RNA-like endoplasmic reticulum kinase(PERK)[J].Rev Neurosci,2018,29(4):387-415.DOI:10.1515/revneuro-2017-0071.
[7]陶勇,黄秋,欧云生,等.焦脱镁叶绿酸-a甲酯介导的光动力疗法诱导人骨肉瘤MG63细胞凋亡的研究[J].中国修复重建外科杂志,2016,30(6):669-674.DOI:0.7507/1002-1892.20160136.TAO Y,HUANG Q,OU Y S,et al.Apoptosis in human osteosarcoma cell line mg63 induced by pyropheophorbide-a methyl ester-mediated photodynamictherapy[J].Chin J Repar Reconstr Surg,2016,30(6):669-674.DOI:0.7507/1002-1892.20160136.
[8]HUANG Q,OU Y S,TAO Y,et al.Apoptosis and autophagy induced by pyropheophorbide-a methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells[J].Apoptosis,2016,21(6):749-760.DOI:10.1007/s10495-016-1243-4.
[9]LI K T,CHEN Q,WANG D W,et al.Mitochondrial pathway and endoplasmic reticulum stress participate in the photosensitizing effectiveness of AE-PDT in MG63 cells[J].Cancer Med,2016,5(11):3186-3193.DOI:10.1002/cam4.895.
[10]LIN S,YANG L,SHI H,et al.Endoplasmic reticulumtargeting photosensitizer hypericin confers chemosensitization towards oxaliplatin through inducing pro-death autophagy[J].Int J Biochem Cell Biol,2017,87:54-68.DOI:10.1016/j.biocel.2017.04.001.
[11]黄秋,涂平华,欧云生,等.MPPa-PDT诱导人骨巨细胞瘤细胞自噬[J].第三军医大学学报,2015,37(17):1755-1760.DOI:10.16016/j.1000-5404.201412133.HUANG Q,TU P H,OU Y S,et al.MPPa-PDT induces autophagy in human bone giant cell tumor cell line SGC-0404[J].J Third Mil Med Univ,2015,37(17):1755-1760.DOI:10.16016/j.1000-5404.201412133.
[12]TAO Y,OU Y,YIN H,et al.Establishment and characterization of human osteosarcoma cells resistant to pyropheophorbide-a methyl ester-mediated photodynamic therapy[J].Int J Oncol,2017,51(5):1427-1438.DOI:10.3892/ijo.2017.4136.
[13]QIAN G,WANG L,ZHENG X,et al.Deactivation of cisplatin-resistant human lung/ovary cancer cells with pyropheophorbide-a methyl ester-photodynamic therapy[J].Cancer Biol Ther,2017,18(12):984-989.DOI:10.1080/15384047.2017.1385683.
[14]TU P H,HUANG W J,WU Z L,et al.Induction of cell death by pyropheophorbide-a methyl ester-mediated photodynamic therapy in lung cancer A549 cells[J].Cancer Med,2017,6(3):631-639.DOI:10.1002/cam4.1012.
[15]SINGH S S,VATS S,CHIA A Y,et al.Dual role of autophagy in hallmarks of cancer[J].Oncogene,2018,37(9):1142-1158.DOI:10.1038/s41388-017-0046-6.
[16]HAN X B,LI H X,JIANG Y Q,et al.Upconversion nanoparticle-mediated photodynamic therapy induces autophagy and cholesterol efflux of macrophage-derived foam cells via ROS generation[J].Cell Death Dis,2017,8(6):e2864.DOI:10.1038/cddis.2017.242.
[17]DOMNGUEZ-MARTN E,HERNNDEZ-ELVIRA M,VINCENT O,et al.Unfolding the endoplasmic reticulum of a social amoeba:Dictyostelium discoideum as a new model for the study of endoplasmic reticulum stress[J].Cells,2018,7(6):56.DOI:10.3390/cells7060056.
[18]MA Y,HENDERSHOT L M.The role of the unfolded protein response in tumour development:friend or foe?[J].Nat Rev Cancer,2004,4(12):966-977.DOI:10.1038/nrc1505.
[19]CHEN J,ZHANG Z Q,SONG J,et al.18β-glycyrrhetinicacid-mediated unfolded protein response induces autophagy and apoptosis in hepatocellular carcinoma[J].Sci Rep,2018,8(1):9365.DOI:10.1038/s41598-018-27142-5.
[20]RODVOLD J J,CHIU K T,HIRAMATSU N,et al.Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells[J].Sci Signal,2017,10(482):eaah7177.DOI:10.1126/scisignal.aah7177.
[21]ZHU Z C,LIU J W,LI K,et al.KPNB1 inhibition disrupts proteostasis and triggers unfolded protein response-mediated apoptosis in glioblastoma cells[J].Oncogene,2018,37(22):2936-2952.DOI:10.1038/s41388-018-0180-9.
[22]FRACCHIOLLA D,SAWA-MAKARSKA J,MARTENS S.Beyond Atg8 binding:The role of AIM/LIR motifs in autophagy[J].Autophagy,2017,13(5):978-979.DOI:10.1080/15548627.2016.1277311.
[23]ZHANG Y,QU X,JIANG L.An oasis in the desert of cancer chemotherapeutic resistance:The enlightenment from reciprocal crosstalk between signaling pathways of UPR and autophagy in cancers[J].Biomed Pharmacother,2017,92:972-981.DOI:10.1016/j.biopha.2017.05.132.
[24]JI G R,YU N C,XUE X,et al.PERK-mediated autophagy in osteosarcoma cells resists ER stress-induced cell apoptosis[J].Int J Biol Sci,2015,11(7):803-812.DOI:10.7150/ijbs.11100.
[25]SHI D,NIU P,HENG X,et al.Autophagy induced by cardamonin is associated with m TORC1 inhibition in SKOV3cells[J].Pharmacol Rep,2018,70(5):908-916.DOI:10.1016/j.pharep.2018.04.005.
[26]WANG J,HUANG S,TIAN R,et al.The protective autophagy activated by GANT-61 in MYCN amplified neuroblastoma cells is mediated by PERK[J].Oncotarget,2018,9(18):14413-14427.DOI:10.18632/oncotarget.24214.
[27]MA X H,PIAO S F,DEY S,et al.Targeting ER stressinduced autophagy overcomes BRAF inhibitor resistance in melanoma[J].J Clin Invest,2014,124(3):1406-1417.DOI:10.1172/JCI70454.
[28]FUSAKIO M E,WILLY J A,WANG Y,et al.Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver[J].Mol Biol Cell,2016,27(9):1536-1551.DOI:10.1091/mbc.E16-01-0039.
[29]MELBER A,HAYNES C M.UPRmt regulation and output:a stress response mediated by mitochondrial-nuclear communication[J].Cell Res,2018,28(3):281-295.DOI:10.1038/cr.2018.16.
[30]ROZPEDEK W,PYTEL D,MUCHA B,et al.The role of the PERK/e IF2α/ATF4/CHOP signaling pathway in tumor progression during endoplasmic reticulum stress[J].Curr Mol Med,2016,16(6):533-544.DOI:10.2174/1566524016666160523143937.