热休克蛋白27通过降低内质网应激抑制硼替佐米诱导的骨髓瘤细胞凋亡
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摘要
目的探讨多发性骨髓瘤(MM)细胞热休克蛋白27(HSP27)表达对硼替佐米诱导凋亡的影响及机制。方法人MM细胞株U266细胞暴露于硼替佐米,实时荧光定量PCR检测HSP27mRNA,蛋白质印迹法检测HSP27、磷酸化-HSP27(p-HSP27)、葡萄糖调节蛋白78(GRP78)、磷酸化蛋白激酶R样内质网激酶(p-PERK)、活化的天冬氨酸特异性半胱氨酸蛋白酶3/9(cl-Caspase 3/9)表达。收集12例初治和10例复发MM患者骨髓瘤细胞,荧光原位杂交法检测染色体异常,比较HSP27mRNA和蛋白表达差异。将U266细胞分为观察组和对照组,分别转染HSP27-siRNA和NC-siRNA,流式细胞术检测硼替佐米诱导的细胞凋亡率。慢病毒介导HSP27基因在U266细胞过表达,蛋白质印迹法检测硼替佐米诱导的GRP78和cl-Caspase 3表达。结果硼替佐米暴露显著上调U266细胞HSP27 mRNA和蛋白的表达(P<0.01),上调GRP78和p-PERK表达(P<0.05),活化Caspase-3和Caspase-9(P<0.05);4-苯基丁酸预处理显著抑制硼替佐米诱导的Caspase-3和Caspase-9活化(P<0.01)。复发MM患者HSP27mRNA和蛋白的表达显著高于初治MM患者(P<0.01)。沉默HSP27表达显著增加硼替佐米诱导的U266细胞凋亡(P<0.01)。慢病毒介导HSP27过表达显著抑制硼替佐米诱导的GRP78表达和Caspase-3活化(P<0.01)。结论 HSP27负反馈抑制内质网应激,减少硼替佐米诱导的MM细胞凋亡。
Objective To investigate the effect of heat shock protein 27(HSP27)on bortezomib induced apoptosis of multiple myeloma cells and its mechanism. Methods The human multiple myeloma U266 cells were exposed to bortezomib,and HSP27 mRNA expression was measured by real-time fluorescent quantitative PCR. The expression of HSP27,p-HSP27,GRP78,p-PERK,cl-Caspase 3,and clCaspase 9 were determined by western blot analysis. Myeloma cells from 12 newly treated and 10 relapsed myeloma patients were collected,chromosomal abnormalities were detected by fluorescence in situ hybridization,and expression of HSP27 mRNA and protein were compared. HSP27-siRNA was transfected into U266 cells in observation group and NC-siRNA was transfected in control group,the apoptotic rate induced by bortezomib was detected by flow cytometry. Lentivirus-mediated overexpression of HSP27 gene in U266 cells,bortezomib-induced expression of GRP78,and cl-Caspase-3 were detected by western blotting. Results Bortezomib exposure significantly increased the expression of HSP27 mRNA and protein in U266 cells(P<0.01). Bortezomib exposure significantly increased the expression of GRP78,p-PERK,cl-Caspase-3,and cl-Caspase-9(P<0.05),while pretreatment with 4-phenylbutyric acid attenuated the effect of bortezomib exposure on activation of Caspase-3 and Caspase-9 of U266 cells(P<0.01). The expression of HSP27 mRNA and protein in relapsed multiple myeloma patients were significantly higher than that in newly treated multiple myeloma patients(P<0.01). Silencing the expression of HSP27 significantly increased the apoptosis of U266 cells induced by bortezomib(P<0.01).Lentivirus mediated HSP27 overexpression significantly inhibited bortezomib exposure induced GRP78 and cl-Caspase-3 expression in U266 cells(P<0.01). Conclusion HSP27 negative feedback inhibited endoplasmic reticulum stress and reduced bortezomib induced apoptosis of myeloma cells.
Objective To investigate the effect of heat shock protein 27(HSP27)on bortezomib induced apoptosis of multiple myeloma cells and its mechanism. Methods The human multiple myeloma U266 cells were exposed to bortezomib,and HSP27 mRNA expression was measured by real-time fluorescent quantitative PCR. The expression of HSP27,p-HSP27,GRP78,p-PERK,cl-Caspase 3,and clCaspase 9 were determined by western blot analysis. Myeloma cells from 12 newly treated and 10 relapsed myeloma patients were collected,chromosomal abnormalities were detected by fluorescence in situ hybridization,and expression of HSP27 mRNA and protein were compared. HSP27-siRNA was transfected into U266 cells in observation group and NC-siRNA was transfected in control group,the apoptotic rate induced by bortezomib was detected by flow cytometry. Lentivirus-mediated overexpression of HSP27 gene in U266 cells,bortezomib-induced expression of GRP78,and cl-Caspase-3 were detected by western blotting. Results Bortezomib exposure significantly increased the expression of HSP27 mRNA and protein in U266 cells(P<0.01). Bortezomib exposure significantly increased the expression of GRP78,p-PERK,cl-Caspase-3,and cl-Caspase-9(P<0.05),while pretreatment with 4-phenylbutyric acid attenuated the effect of bortezomib exposure on activation of Caspase-3 and Caspase-9 of U266 cells(P<0.01). The expression of HSP27 mRNA and protein in relapsed multiple myeloma patients were significantly higher than that in newly treated multiple myeloma patients(P<0.01). Silencing the expression of HSP27 significantly increased the apoptosis of U266 cells induced by bortezomib(P<0.01).Lentivirus mediated HSP27 overexpression significantly inhibited bortezomib exposure induced GRP78 and cl-Caspase-3 expression in U266 cells(P<0.01). Conclusion HSP27 negative feedback inhibited endoplasmic reticulum stress and reduced bortezomib induced apoptosis of myeloma cells.
引文
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[16]朱蓉,奚昊,李勇华,等.硼替佐米耐药骨髓瘤细胞株的建立及其差别表达蛋白的飞行时间质谱分析[J].浙江大学学报(医学版),2009,38(5):445-452.
[17]Yasui H,Hideshima T,Ikeda H,et al.BIRB 796enhances cytotoxicity triggered by bortezomib,heat shock protein(Hsp)90inhibitor,and dexamethasone via inhibition of p38mitogen-activated protein kinase/Hsp27pathway in multiple myeloma cell lines and inhibits paracrine tumour growth[J].Br J Haematol,2007,136(3):414-423.
[18]Chauhan D,Li G,Auclair D,et al.2-Methoxyestardiol and bortezomib/proteasome-inhibitor overcome dexamethasoneresistance in multiple myeloma cells by modulating Heat Shock Protein-27[J].Apoptosis,2004,9(2):149-155.
[19]White-Gilbertson S,Hua Y,Liu B.The role of endoplasmic reticulum stress in maintaining and targeting multiple myeloma:a double-edged sword of adaptation and apoptosis[J].Front Genet,2013,4(2):109.
[20]Ron D,Walter P.Signal integration in the endoplasmic reticulum unfolded protein response[J].Nat Rev Mol Cell Biol,2007,8(7):519-529.
[21]Rutkowski D T,Kaufman R J.A trip to the ER:coping with stress[J].Trends Cell Biol,2004,14(1):20-28.
[22]Rao R,Nalluri S,Fiskus W,et al.Role of C/EBP homologous protein(CHOP)in panobinostat-mediated potentiation of bortezomib-induced lethal ER stress in mantle cell lymphoma cells[J].Clinical Cancer Research,2010,16(1):4742-4754.
[23]Lamoureux F,Yin M J,Zoubeidi A,et al.Downregulation of Hsp27using OGX-427induces ER stress and potentiates Hsp90inhibitors to delay castrate resistant prostate cancer[J].European Urology Supplements,2012,11(1):245.
[24]Rocchi P,Beraldi E,Ettinger S,et al.Increased Hsp27after androgen ablation facilitates androgen-independent progression in prostate cancer via signal transducers and activators of transcription 3-mediated suppression of apoptosis[J].Cancer Research,2005,65(23):11083-11093.
[25]Meares G P,Liu Y,Rajbhandari R,et al.PERK-dependent activation of JAK1and STAT3contributes to endoplasmic reticulum stress-induced inflammation[J].Mol Cell Biol,2014,34(20):3911-3925.
[2]Huang Q,Ye J,Chen,W,et al.Heat shock protein 27is over-expressed in tumor tissues and increased in sera of patients with gastric adenocarcinoma[J].Clin Chem Lab Med,2010,48(2):263-269.
[3]Giaginis C,Daskalopoulou S S,Vgenopoulou S,et al.Heat Shock Protein-27,-60and-90expression in gastric cancer:Association with clinicopathological variables and patient survival[J].BMC Gastroenterol,2009,9(1):14.
[4]Tweedle E M,Khattak I,Ang C W,et al.Low molecular weight heat shock protein HSP27is a prognostic indicator in rectal cancer but not colon cancer[J].Gut,2010,59(11):1501-1510.
[5]Andrulis M,Chatterjee M,Jain S,et al.Heat shock protein90alpha and beta are overexpressed in multiple myeloma cells and critically contribute to survival[J].Verh Dtsch Ges Pathol,2007,91(1):330-337.
[6]邹彬镔,郭宁红,石庆之,等.多发性骨髓瘤患者血浆HSP90的表达及其临床意义[J].中国实验血液学杂志,2014,22(5):1326-1330.
[7]中国医师协会血液科医师分会,中华医学会血液学分会,中国医师协会多发性骨髓瘤专业委员会.中国多发性骨髓瘤诊治指南(2017年修订)[J].中华内科杂志,2017,56(11):866-870.
[8]Kouroukis T C,Baldassarre F G,Haynes A E,et al.Bortezomib in multiple myeloma:systematic review and clinical considerations[J].Curr Oncol,2014,21(4):573-603.
[9]Fall D J,Stessman H,Patel S S,et al.Utilization of translational bioinformatics to identify novel biomarkers of bortezomib resistance in multiple myeloma[J].J Cancer,2014,5(9):720-727.
[10]Iida S,Ri M.Determinants of sensitivity to proteasome inhibitors and strategies to overcome acquired resistance to bortezomib in multiple myeloma[J].Rinsho Ketsueki,2014,55(3):304-310.
[11]Ri M.Endoplasmic-reticulum stress pathway-associated mechanisms of action of proteasome inhibitors in multiple myeloma[J].Int J Hematol,2016,104(3):273-280.
[12]Nikesitch N,Lee J M,Ling S,et al.Endoplasmic reticulum stress in the development of multiple myeloma and drug resistance[J].Clinical&Translational Immunology,2018,7(1):e1007.
[13]Liu Y,Chang A.Heat shock response relieves ER stress[J].Embo J,2014,27(7):1049-1059.
[14]Richardson P G,Mitsiades C.Bortezomib:proteasome inhibition as an effective anticancer therapy[J].Future Oncology,2005,1(2):161-167.
[15]Mcconkey D J,Zhu K.Mechanisms of proteasome inhibitor action and resistance in cancer[J].Drug Resist Updat,2008,11(4):164-179.
[16]朱蓉,奚昊,李勇华,等.硼替佐米耐药骨髓瘤细胞株的建立及其差别表达蛋白的飞行时间质谱分析[J].浙江大学学报(医学版),2009,38(5):445-452.
[17]Yasui H,Hideshima T,Ikeda H,et al.BIRB 796enhances cytotoxicity triggered by bortezomib,heat shock protein(Hsp)90inhibitor,and dexamethasone via inhibition of p38mitogen-activated protein kinase/Hsp27pathway in multiple myeloma cell lines and inhibits paracrine tumour growth[J].Br J Haematol,2007,136(3):414-423.
[18]Chauhan D,Li G,Auclair D,et al.2-Methoxyestardiol and bortezomib/proteasome-inhibitor overcome dexamethasoneresistance in multiple myeloma cells by modulating Heat Shock Protein-27[J].Apoptosis,2004,9(2):149-155.
[19]White-Gilbertson S,Hua Y,Liu B.The role of endoplasmic reticulum stress in maintaining and targeting multiple myeloma:a double-edged sword of adaptation and apoptosis[J].Front Genet,2013,4(2):109.
[20]Ron D,Walter P.Signal integration in the endoplasmic reticulum unfolded protein response[J].Nat Rev Mol Cell Biol,2007,8(7):519-529.
[21]Rutkowski D T,Kaufman R J.A trip to the ER:coping with stress[J].Trends Cell Biol,2004,14(1):20-28.
[22]Rao R,Nalluri S,Fiskus W,et al.Role of C/EBP homologous protein(CHOP)in panobinostat-mediated potentiation of bortezomib-induced lethal ER stress in mantle cell lymphoma cells[J].Clinical Cancer Research,2010,16(1):4742-4754.
[23]Lamoureux F,Yin M J,Zoubeidi A,et al.Downregulation of Hsp27using OGX-427induces ER stress and potentiates Hsp90inhibitors to delay castrate resistant prostate cancer[J].European Urology Supplements,2012,11(1):245.
[24]Rocchi P,Beraldi E,Ettinger S,et al.Increased Hsp27after androgen ablation facilitates androgen-independent progression in prostate cancer via signal transducers and activators of transcription 3-mediated suppression of apoptosis[J].Cancer Research,2005,65(23):11083-11093.
[25]Meares G P,Liu Y,Rajbhandari R,et al.PERK-dependent activation of JAK1and STAT3contributes to endoplasmic reticulum stress-induced inflammation[J].Mol Cell Biol,2014,34(20):3911-3925.