【作者】 高瑛；

【导师】 陈协群；

【作者基本信息】 第四军医大学 ， 内科学， 2009， 博士

【摘要】 背景和目的:多发性骨髓瘤(multiple myeloma,MM)作为一种浆细胞恶性肿瘤,其高蛋白合成与分泌活性极易导致未/错折迭蛋白积聚于内质网腔,故骨髓瘤细胞极易诱发内质网应激(endoplasmic reticulum stress,ERS)。ERS的生化基础称为未折迭蛋白反应(unfolded protein response,URP)。UPR作为一种旨在修复、稳定内质网功能的细胞适应性反应,能通过IRE1α与PERK/eIFα信号通路而调控分子伴侣BiP表达及转录因子XBP-1、CHOP的活化,其中BiP与XBP-1促进细胞生存,而CHOP则介导细胞凋亡。两类信号间的紊乱失衡最终导致细胞死亡。真核细胞适应性反应除了UPR,还有自噬反应(autophagy)。自噬是真核细胞适应营养或生长因子缺乏、低氧、内质网应激等有害刺激,由溶酶体介导的长寿命蛋白或细胞器的降解的一种分解性代谢反应。自噬体形成是自噬的关键步骤,而自噬体形成依赖Atg5及Atg8/LC3等自噬相关蛋白与Beclin1-磷脂酰肌醇三磷酸激酶(PI3K)复合物的综合调控。研究表明,在酵母细胞中UPR既能触发凋亡又能诱发自噬,而在骨髓瘤细胞中,UPR与自噬关系尚不清楚。蛋白酶体抑制剂硼替佐米(Bortezomib)能选择性抑制26S蛋白酶体,对多发性骨髓瘤临床疗效显著,其抗瘤效应可能通过干扰NF-κB信号来实现,然而最近发现NF-κB抑制剂诱发骨髓瘤细胞凋亡的程度却远不及蛋白酶体抑制剂显著。提示NF-κB途径不能完全解释硼替佐米的抗瘤效应。近年报道内质网应激诱导的细胞凋亡为硼替佐米的重要效应机制,但其具体调控过程尚不清楚。考虑到内质网应激与UPR及自噬调控之间的相互关系,以及UPR与自噬在骨髓瘤细胞死亡调控中的作用,我们设想,硼替佐米极可能通过影响UPR与自噬的调控过程而发挥其抗骨髓瘤效应。方法和结果:为了探讨骨髓瘤细胞UPR与自噬之间的关系,我们绘制了经内质网应激剂和/或自噬诱导剂处理骨髓瘤细胞H929和RPMI8226的剂量-效应曲线,确定了所用药物的实验浓度,并采用Western blot检测靶细胞UPR和自噬相关蛋白的表达。结果表明:(1)采用自噬诱导剂雷帕霉素分别处理H929和RPMI8226细胞不同时间后,靶细胞中UPR相关蛋白XBP-1s、XBP-1u、BiP、CHOP和自噬相关蛋白LC-3、Beclin-1、Atg-5、PI3KC3的表达均呈时间依赖性的增加;(2)经标准内质网应激剂BFA或Tg处理不同时间后,H929和RPMI8226细胞凋亡率及上述UPR相关蛋白和自噬相关蛋白的表达均呈时间依赖性的增加;(3)雷帕霉素能干扰BFA或Tg诱导的UPR相关蛋白的表达;(4) BFA或Tg可以干扰雷帕霉素诱发的自噬相关蛋白的表达。以上结果强烈提示,在骨髓瘤细胞中,UPR与自噬反应的调控过程相互依赖。为了进一步探讨硼替佐米是否造成UPR信号紊乱失衡,我们以不同方式处理细胞后,我们采用AnnexinⅤ-FITC/PI双染联合流式细胞术检测靶细胞凋亡率;采用Western blot检测靶细胞UPR相关蛋白的表达。结果表明:(1)经硼替佐米、BFA和Tg处理不同时间后,靶细胞凋亡率及UPR相关蛋白的表达均呈时间依赖性增加;(2)硼替佐米能干扰BFA或Tg诱导的XBP-1s、XBP-1u及BiP蛋白的表达,但却促进BFA或Tg诱导的CHOP蛋白的表达,提示硼替佐米可以引起UPR反应中保护信号的表达与信号输出的紊乱失衡。为了探讨硼替佐米是否影响自噬过程,我们以不同方式处理细胞后,采用电镜技术检测自噬体/自噬溶酶体;采用Western blot检测靶细胞自噬相关蛋白的表达,采用免疫共沉淀检测Beclin-1-PI3KC3复合物的形成。结果表明:(1)硼替佐米处理H929细胞24小时后电镜检测未发现有经典自噬体/自噬溶酶体形成; (2)硼替佐米分别处理H929和RPMI8226细胞不同时间后,靶细胞自噬相关蛋白的表达呈时间依赖性减少;(3)硼替佐米可以干扰自噬诱导剂雷帕霉素诱发的自噬相关蛋白的表达;(4)硼替佐米可以抑制Beclin-1-PI3KC3复合物的形成;(5)活化自噬可以降低硼替佐米的促骨髓瘤细胞凋亡活性,抑制自噬对其活性无明显影响。提示,硼替佐米除了干扰UPR、促进骨髓瘤细胞凋亡外,尚抑制骨髓瘤细胞内源性自噬反应,从而进一步消除了自噬对骨髓瘤细胞的保护作用。结论:1.骨髓瘤细胞中自噬与未折迭蛋白反应的调控过程密切关联;2.蛋白酶体抑制剂硼替佐米虽能诱导骨髓瘤细胞内质网应激,但却干扰了未折迭蛋白反应,致使其保护性成分BiP和XBP-1与促凋亡成份CHOP表达紊乱失衡,进而触发靶细胞凋亡;3.硼替佐米干扰骨髓瘤细胞内源性自噬反应,可能与其抑制Beclin-1/PI3KC3复合体的形成有关;4.硼替佐米除了干扰未折迭蛋白反应、促进骨髓瘤细胞凋亡外,尚能干扰骨髓瘤细胞的内源性自噬反应,从而消除了自噬对骨髓瘤细胞的生存保护作用,这一效应极可能是硼替佐米抗骨髓瘤效应显著优于其它常规化疗药物的重要生物学机制。更多还原

【Abstract】 Background and Objectives:Multiple myeloma(MM) , which produces and secretes abundant immunoglobulins (Igs), is a kind of malignant counterpart of the plasma cells malignacy. Because of heavy load of protein translation and abrupt accumulation of unfoaded or misfoaded protein in endoplasmic reticulum, MM cells are subjected to endoplasmic reticulum stress (ERS). To alleviate ER stress, MM cells activate a signaling pathway called the unfolded protein response (UPR), which aims to reestablish homeostasis by transmitting information to the nucleus about the protein-folding status in the ER lumen, triggering adaptive responses. BiP, XBP-1 and CHOP are activated by IRE1αand PERK/eIFαsignal. BiP and XBP-1 are prosurvival components, and CHOP is proapoptotic one. Disequilibrium between cytoprotective outputs and proapoptotic ones results in cell death. Besides UPR, autophagy is also an adaptive response in eukaryocyte. Autophagy, which mediates mainly the bulk degradation of long-lived cytoplasmic proteins, large protein compounds or organelles by lysosomes, is an adaptive catabiolic reaction when eukaryocytes meet insufficient of nutrients and growth factors, hypoxia, endoplasmic reticulum stress, and other harmful stimuli. The formation of autophagosome, an important procession, depends on not only Atg5-Atg12 and Agt8 (LC3), but also the fuction of Beclin1-PI3K compounds. It was reported that UPR can’t only trigger ERS, but induce autophagy in yeast cells. Whereas it is unclear that the relationship between UPR and autophagy in MM cells.As a new kind of proteasome inhibitor (PI), Bortezomib, a potent and specific 26s proteasome inhibitor has been shown to selectively induce apoptosis of MM cells, has remarkable effect on refractory MM in clinic. It had been constantly thought that PI killed tumor cells by inhibiting nuclear factor (NF)-κB activation. But recently, it was discovered that the apoptotic MM cells induced by the inhibitor of NF-κB were obviously less than those by PI, suggesting that NF-κB inhibitor cannot completely explain the nature of the selectivity of Bortezomib for multiple myeloma cells. Recently, ERS-induced MM cells death is considered as an important mechanism of Bortezomib. However, the mechanism by which this compound acts remains unknown. On account of the relationship of ERS, UPR and autophagy, we assume that Bortezomib maybe affect the signals regulated by UPR and autophagy.Methods and Results:To investigate the relationship between UPR and autophagy induced by ERS in MM cells, we exammed the expression of UPR related proteins and autophagy related ones by western blot. Our data showed: (1) After cells were treated by classical autophagy inducer, Rapamycin, for different time, the apoptotic rates of H929 or RPMI8226 and the expression levels of XBP-1s,XBP-1u,BiP and CHOP rose in time-dependent manner; (2) After cells were treated by classical ERS stressors, BFA or Tg, for different time, the apoptotic rates of H929 or RPMI8226 and the expression levels of UPR related proteins, and autophagy-related rose in time-dependent manner; (3) Rapamycin disrupted the expressions of XBP-1s、XBP-1u and BiP , but promote the expression of CHOP induced by BFA or Tg; (4) BFA or Tg disrupted the expressions of autophagy-related. Above data strongly suggest that there should be close relationship beween UPR and autophagy in MM cells.To further study whether Bortezomib results in disequilibriumin of UPR, annexin V-FITC/PI staining followed by flow cytometry analyzed the apoptotic rates of target cells, and western blot showed the expression of UPR related proteins. Our data showed: (1) After H929 and RPMI8226 cells were treated by the classical endoplasmic reticulum stressor BFA or Tg for different time, the apoptotic rates of target cells and the expression levels of UPR related proteins rose in time-dependent manner. The tendency was coincident with Bortezomib induced; (2) Bortezomib inhibited classic ER stressors BFA or Tg-induced up-regulation of prosuevival UPR components XBP-1s、XBP-1u and BiP and disrupted the proapoptotic outputs CHOP. In other words: Bortezomib induced ERS eventually to apoptosis that was closely associated with the regulatory imbalance between proapoptotic and prosurvival UPR.To investigate whether Bortezomib affects on autophagy procession, western blot, electron microscope and co-immunoprecipitation showed: (1) There had few autophagosomes or autolysosomes induced by Bortezomib; (2) After cells were treated by Bortezomib for different time, the apoptotic rates of H929 or RPMI8226 and the expression levels of autophagy-related proteins decreased in time-dependent manner; (3) Bortezomib disrupted the expressions of autophagy-related proteins induced by Rapamycin; (4) Bortezomib inhibited the formation of Beclin-1-PI3KC3 compounds; (5) Induction autophagy by Rapamycin prompted Bortezomib-induced MM cell apoptosis. Our data inferred that Bortezomib did not only disrupt UPR to induc MM cell apoptosis, but disrupted endogenous autophagy to eliminate the protection mechanisms in MM cells.Conclusions:1. There is close relationship between UPR and regulative procession of autophagy in MM cells.2. Bortezomib could induce ERS, but it induces proapoptotic UPR outputs, such as CHOP, whereas inhibits cytoprotective componets for instance BiP and XBP-1.3. Bortezomib could disrupt endogenous autophagy in MM cells, as maybe involve in inhibition the formation of Beclin-1-PI3KC3 compounds.4. Bortezomib could not only disrupt UPR to induce MM cell apoptosis, but disrupt endogenous autophagy to eliminate the protection mechanisms in MM cells. It is the important mechanism that Bortezomib outweigh to treat multiple myeloma compared with other chemotherapeutics.更多还原