背景:C末端四肽Lys-Asp-Glu-Leu受体(KDELR)是调节ER应激(ERS)反应的跨膜蛋白,增长,分化,和免疫反应。KDELR2与胶质母细胞瘤肿瘤发生的促进之间存在关联。本研究的目的是探讨KDELR2在神经胶质瘤和替莫唑胺(TMZ)化疗反应中的作用机制。
方法:用免疫组织化学方法检测KDELR2在胶质瘤组织和细胞中的表达,Westernblot和RT-qPCR分析。然后通过CCK8,集落形成证明KDELR2的作用,流式细胞术和Hoschest33258测定。基因(ATF4,ATF6,PERK,eIF2-α,通过RT-qPCR评估U373细胞中的GRP78和CHOP)。基因的蛋白质表达(cleavedcaspase3,caspase3,cleavedPARP,PARP,Bax,Bcl-2JNK,p-JNK,p38,p-p38,ATF4,ATF6,XBP-1s,PERK,p-PERK,GRP78和CHOP)通过蛋白质印迹测定法测量。
结果:高级别胶质瘤组织中KDELR2的表达上调。KDELR2敲低抑制细胞增殖,但增加细胞凋亡。Further,KDELR2的敲除还激活了ER应激(ERS)依赖性CHOP途径,并导致磷酸化c-JunN末端激酶(JNK)和p38水平升高。此外,KDELR2敲低和TMZ应用的组合通过ERS依赖性CHOP和JNK/p38途径在U373细胞中显示出协同的细胞毒性作用。
结论:KDELR2敲低诱导细胞凋亡并使胶质瘤细胞对TMZ敏感,这是由ERS依赖性CHOP和JNK/p38途径介导的。
BACKGROUND: The C-terminal tetrapeptide Lys-Asp-Glu-Leu receptors (KDELRs) are transmembrane proteins that regulate ER stress (ERS) response, growth, differentiation, and immune responses. There is an association between KDELR2and promotion of glioblastoma tumorigenesis. The aim of the present study was to explore the functional mechanism of KDELR2 in glioma and during response to chemotherapy to temozolomide (TMZ).
METHODS: The expression of KDELR2 in glioma tissues and cells was evaluated by immunohistochemistry, western blot and RT-qPCR assay. Then role of KDELR2 was demonstrated by CCK8, colony formation, flow cytometry and Hochest 33258 assays. The expression of genes (ATF4, ATF6, PERK, eIF2-α, GRP78 and CHOP) in U373 cells was evaluated by RT-qPCR. The protein expression of genes (cleaved caspase 3, caspase 3, cleaved PARP, PARP, Bax, Bcl-2, JNK, p-JNK, p38, p-p38, ATF4, ATF6, XBP-1s, PERK, p-PERK, GRP78 and CHOP) was measured by western blot assay.
RESULTS: The expression of KDELR2 was upregulated in high-grade gliomas tissues. KDELR2 knockdown suppressed cell proliferation but increased cell apoptosis. Further, Knockdown of KDELR2 also activated the ER stress (ERS)-dependent CHOP pathway, and resulted in increased levels of phosphorylated c-Jun N-terminal kinase (JNK) and p38. Moreover, the combination of KDELR2 knockdown and TMZ application showed a synergistic cytotoxic effect in U373 cells through the ERS-dependent CHOP and JNK/p38 pathways.
CONCLUSIONS: KDELR2 knockdown induces apoptosis and sensitizes glioma cells to TMZ, which is mediated by the ERS-dependent CHOP and JNK/p38 pathways.