【作者】 周星露；

【导师】 何俏军； 杨波；

【作者基本信息】 浙江大学 ， 药理学， 2011， 硕士

【摘要】 研究目的：硼替佐米(万珂,Bortezomib/velcade)是一种二肽硼酸衍生物,是第一个由美国食品药物管理局(FDA)批准上市的蛋白酶体抑制剂,对多发性骨髓瘤的治疗具有较高的总体有效率和完全缓解率。Bortezomib抗瘤谱广,研究表明Bortezomib能诱导急性髓性白血病细胞(AML)发生凋亡,且单药或联合其他化疗药物治疗白血病均具有一定的临床疗效,但其作用机制阐释尚不明确。随着对细胞分化异常在恶性肿瘤发病学和治疗学上的意义认识的日益加深,肿瘤的分化治疗成为当今肿瘤学研究的热点之一。其中,全反式维甲酸(ATRA)诱导分化治疗急性早幼粒白血病(M3, APL)疗法,已列为诱导缓解APL的首选方案之一,但ATRA耐药、严重毒副反应以及复发等仍是当前迫切需要解决的问题。因此,寻找高效低毒的新型分化诱导剂或寻找有效的药物联用方案是解决上述问题的关键途径。基于此,本研究探讨了Bortezomib单药或联合ATRA在诱导分化治疗AML中的药效及其作用机制。研究内容主要包含了两个部分：(1) Bortezomib对急性髓性白血病细胞的诱导分化作用及其作用机制研究；(2) Bortezomib联合ATRA对急性髓性白血病的协同治疗作用,并探究了维甲酸受体RARα通路在该过程中所发挥的作用。研究方法：1)选用人白血病细胞株HL60和U937为主要研究对象,人原代急性髓性白血病细胞为辅助研究对象,考察了Bortezomib对AML细胞的分化诱导作用：采用台盼蓝染色结合血细胞计数板计数观察细胞增殖情况,描绘细胞增殖曲线；应用吉姆萨染色观察细胞形态学改变；采用硝基四氮唑蓝(NBT)测试细胞还原能力；细胞经CD14-FITC或CD11b-PE抗体孵育后采用流式细胞术检测细胞表面分化抗原CD14或CD11b表达；应用Western blotting检测MAPK家族蛋白表达及活化水平(p-MEK、p-ERK、p-JNK、p-p38、MEK、JNK2、p38),检测分化启动因子STAT1表达及活性水平(p-STAT1 Try701、STAT1);应用Realtime PCR检测细胞STAT1 mRNA表达水平,并利用CHX抑制蛋白合成研究Bortezomib引起的STAT1蛋白上调的原因；用携带shRNA-STAT1的慢病毒载体转染HL60细胞,通过沉默STAT1研究其在Bortezomib诱导的AML细胞分化中的关系。利用MEK特异性抑制剂PD98059、JNK特异性抑制剂SP600125和p38抑制剂SB203580研究MAPK通路在Bortezomib诱导的AML细胞分化中的关系；采用Annexin V-FITC/PI染色结合流式细胞术测定Bortezomib对AML细胞的凋亡率及初步探讨Bortezomib诱导的细胞分化与凋亡的关系。2)选用人白血病细胞株HL60和NB4为主要研究对象,人原代急性髓性白血病细胞为辅助研究对象,考察Bortezomib与ATRA对AML细胞的联合治疗作用：采用台盼蓝染色结合血细胞计数板计数观察细胞增殖情况和细胞存活率,描绘细胞增殖曲线；应用吉姆萨染色观察细胞形态学改变；采用硝基四氮唑蓝(NBT)测试细胞还原能力；细胞经CD11b-PE抗体孵育后采用流式细胞术检测细胞表面分化抗原CDllb表达；采用HL60细胞裸小鼠移植瘤模型考察Bortezomib与ATRA在体内的联合治疗作用；采用免疫组化法检测瘤组织切片C/EBPε的蛋白表达含量；采用TUNEL法考察瘤组织切片中凋亡情况；应用Western blotting检测RARα蛋白表达情况,检测相关分化转录因子的表达(p-STAT1 Try701、STAT1、c-Myc、C/EBPε、p-27),检测MAPK家族蛋白表达情况(p-MEK、p-JNK);应用Realtime PCR检测细胞RARα、STAT1 mRNA表达水平；采用免疫荧光法检测p65和STAT1在细胞内的定位；采用非放射性凝胶迁移法检测STAT1与DNA的结合能力；用慢病毒介导的STAT1 shRNA转染HL60细胞,通过沉默STAT1研究其在Bortezomib与ATRA联用治疗AML中的作用。研究结果：第一部分：Bortezomib通过上调MEK/ERK-JNK p46通路活性,激活STAT1从而诱导AML细胞向单核/巨噬系细胞分化。1) Bortezomib具有诱导人急性髓性白血病细胞向单核/巨噬系细胞分化成熟的能力。用细胞增殖抑制率、细胞形态学、生化指标、分子标志等指标观察Bortezomib对AML细胞的诱导分化成熟能力。结果显示Bortezomib (5 nM)能诱导人急性髓性白血病(AML)细胞向单核／巨噬系细胞分化。Bortezomib (5 nM)可显著抑制HL60和U937细胞增殖,第3天的抑制率分别为44.6%和60.6%。吉姆萨染色结果表明Bortezomib (5 nM)作用72小时后,HL60细胞和U937细胞分化成为幼稚单核细胞或成熟单核细胞,形态学上表现为胞体变小,核／浆比例下降,细胞核固缩,部分呈半月核,细胞中核仁减少或消失。Bortezomib作用后的HL60和U937对NBT的还原能力增强,并呈现剂量依赖性关系。同时Bortezomib诱导的分化抗原CD11b/CD14的表达也呈现剂量依赖性和时间依赖性关系。此外,Bortezomib对人原代急性髓性白血病细胞(M3-AML, APL)增殖也有显著的抑制作用,吉姆萨染色表明Bortezomib具有诱导人原代急性髓性白血病细胞成熟分化的能力。2) Bortezomib诱导AML细胞分化成熟的机制研究Western blotting检测结果显示,Bortezomib能激活MAPK通路(包括MEK/ERK级联反应、JNK p46通路和p38通路),p-MEK、p-ERK、p-JNK p46、p-p38均呈现上调趋势,并呈现时间依赖性关系,而其相应的原型蛋白表达水平不变。当给予MEK特异性抑制剂PD98059或JNK特异性抑制剂SP600125时,Bortezomib引起的MEK、ERK或JNK p46的活化被抑制,同时CDl4和CD11b的表达分别从Bortezomib单药组的27.3%和35.8%下降到对照组水平。此外,PD98059同时抑制了Bortezomib引起的JNK p46的活化,但SP600125对MEK活性无影响。当给与p38特异性抑制剂SB203580时,Bortezomib诱导的CD14和CD11B的表达率为42.6%和56.0%,提示了p38可能对Bortezomib诱导的AML细胞的分化起到负调控作用。以上结果证明了MEK/ERK-JNK通路参与介导Bortezomib诱导的AML细胞分化。Western blotting检测结果表明,Bortezomib诱导AML细胞分化过程中转录因子STAT1的蛋白水平与活性形式pSTAT1 Tyr 701均呈时间依赖性上调。Realtime PCR检测结果表明STAT1 mRNA的表达也呈时间依赖性增加。给与CHX抑制STAT1 mRNA的表达,Bortezomib作用后,STAT1蛋白水平仍有所上调,提示Bortezomib同时抑制了STAT1蛋白的降解。此外我们也观察到STAT1下游基因C/EBPα蛋白的上调。慢病毒介导的shRNA-STAT1显著下调了HL60细胞中的STAT1蛋白水平,Bortezomib作用于shRNA空载转染的HL60细胞72小时后,CD14和CD11B的表达率分别为20.4%和39.6%,而在shRNA-STAT1转染的细胞中,Bortezomib诱导的CD14和CD11b仅为10.1%和19.6%。给与PD98059或SP600125抑制Bortezomib引起的HL60细胞分化的同时,观察到STAT1水平上调被抑制,活性水平被抑制,C/EBPa表达也受到抑制。上述结果表明Bortezomib激活的MEK/ERK-JNK通路可能通过进一步激活分化相关转录因子STAT1诱导AML细胞的分化。3) Bortezomib诱导的AML细胞分化与凋亡为RARα非依赖的过程。Western blotting结果显示,Bortezomib作用于HL60后,维甲酸受体RARa蛋白水平增加,但通过双荧光素酶检测系统检测结果显示,RARa转录活性无显著变化。同时流式细胞术检测结果表明Bortezomib能诱导RARa突变的HL60R细胞分化抗原CD11b的表达及凋亡的发生。以上结果提示Bortezomib诱导AML细胞的分化与凋亡是RARα非依赖的过程。4) Bortezomib诱导AML细胞分化的同时,伴随着AML细胞的凋亡,并初步探讨Bortezomib引起的AML细胞分化与凋亡之间的关系。Annexin V/PI双染流式细胞术检测结果显示,5 nM Bortezomib在诱导白血病细胞分化的同时,伴随了时间依赖性的细胞凋亡。但由MEK/ERK-JNK通路的抑制引起的Bortezomib细胞分化的抑制过程中,Bortezomib引起的细胞凋亡率无显著性变化。由此推测,Bortezomib诱导的AML细胞的分化与凋亡可能是两个相对独立的过程。第二部分：Bortezomib通过抑制ATRA引起的RARa蛋白的降解,协同ATRA诱导AML细胞粒性分化。1) Bortezomib协同ATRA诱导AML细胞粒性分化。MTT检测法显示Bortezomib与ATRA联合作用能协同抑制HL60与NB4细胞的增殖。通过台盼蓝拒染法与血细胞计数板法,计数并绘制Bortezomib与ATRA联合作用后的细胞的增殖曲线和存活率曲线。结果显示Bortezomib(在HL60细胞上选用浓度2.5 nM,在NB4细胞上选用浓度2.5、5 nM)单药作用不影响细胞的存活率和细胞增殖能力,但能显著增强ATRA引起的细胞增殖抑制作用,而这种作用并不是通过诱导细胞死亡实现的。我们从形态学、生化指标及分子标志三方面指标证实了Bortezomib能促进ATRA诱导的HL60、NB4细胞的分化,具体表现为与ATRA单用组相比,药物联用组引起的核分叶比例显著增加,NBT还原能力显著增强,分化抗原CDllb表达量显著增加。在人原代白血病细胞上,ATRA与Bortezomib也具有协同作用。在M3-AML型、AML/ALL混合型、AML-resistance型三个原代病人骨髓中分离得到原代白血病。通过细胞计数或CCK8检测,结果显示两药联合作用后细胞存活率显著低于单用组。在M3-AML型原代病人样本上,Bortezomib增强了ATRA引起的细胞NBT还原能力。2) Bortezomib联合ATRA通过诱导HL60细胞分化,抑制裸小鼠HL60移植瘤的生长。裸小鼠HL60人白血病细胞移植瘤实验结果显示,ATRA (5 mg/kg)组及Bortezomib (0.1 mg/kg)组均表现出一定的抗肿瘤作用,其T/C%值分别为58.0%和62.0%,抑瘤率分别为40.0%和20.0%,联合作用组T/C%值和抑瘤率分别为45%和58.9%,说明Bortezomib与ATRA在体内也有较好抗肿瘤协同作用。通过分离得到瘤组织单细胞悬液,通过流式细胞术检测,显示联用组瘤组织中分化抗原CD11b的表达率为53.1%,显著高于ATRA单用组(40.4%)和Bortezomib单用组(38.6%)。从免疫组化结果也得出类似的结果,在联用组中C/EBPε的表达量明显高于对照组与单药组。此外,采用TUNEL染色检测了瘤组织中的凋亡情况,显示该剂量配比下,瘤组织中几乎检测不到凋亡的发生。3) Bortezomib协同ATRA诱导AML细胞分化的机制研究20S蛋白酶体活性检测结果表明,蛋白酶体活性在ATRA诱导的HL60和NB4细胞分化过程中明显上调,Bortezomib显著抑制了这一上调现象。与之对应的,在ATRA诱导的HL60和NB4细胞分化过程中RARα蛋白经泛素蛋白酶体通路介导呈下调趋势,Bortezomib与ATRA联合作用后,RARα蛋白得以累积,而其mRNA水平无显著性变化。RARα与维甲酸反应元件RARE结合能直接介导转录因子STAT1的表达。ATRA能诱导STAT1的表达,Bortezomib与ATRA联合作用后,STAT1 mRNA水平显著增加。以上结果提示了Bortezomib抑制了ATRA引起的RARα蛋白的降解,增强了RARα转录活性。Western blotting检测瘤组织中RARα蛋白的表达含量,结果显示Bortezomib抑制了ATRA引起的RARα蛋白的降解,联用组中RARα蛋白含量显著高于ATRA单用组。Western blotting结果显示ATRA上调了STAT1蛋白水平和Tyr701位点磷酸化水平,Bortezomib与ATRA联合作用进一步上调STAT1的蛋白水平和活性,而该浓度Bortezomib单独作用对其无显著影响。免疫荧光结果显示合用组细胞核中STAT1的分布显著增加,EMSA检测结果进一步证实提示了STAT1与DNA结合能力显著增加。慢病毒介导的shRNA-STAT1显著下调了HL60细胞中的STAT1蛋白水平,同时部分逆转了Bortezomib对ATRA诱导的细胞分化的协同作用。以上结果显示STAT1参与了Bortezomib协同ATRA诱导细胞分化的过程。免疫荧光结果显示,p65在ATRA单用组、与Bortezomib联用组细胞中的分布无显著性差异。此外,ATRA或Bortezomib单用组中MEK活性增强,但联用组与单用组MEK活性无显著性差别。JNK通路活性在ATRA与Bortezomib联合诱导细胞分化过程中变化不大。以上结果显示了NFκB通路与MEK. JNK通路在Bortezomib协同ATRA诱导细胞分化过程中不起关键性作用。结论：本论文较为系统地研究了Bortezomib诱导或促进ATRA诱导AML细胞分化治疗中的作用,并对其机制进行了初步的探讨。高浓度Bortezomib通过激活MEK/ERK-JNK通路启动转录因子STAT1介导的AML细胞的单核／巨噬系分化。低浓度Bortezomib可以通过抑制ATRA引起的RARa的泛素化降解,提高RARa的转录活性,进而促进了STAT1活性发挥,促进了AML细胞的粒性分化。本研究首次提出了Bortezomib在肿瘤分化诱导治疗中的应用,为全面了解Bortezomib的抗肿瘤机制提供了新的视角,为临床髓性白血病的治疗提供了新的策略和理论指导。更多还原

【Abstract】 Objective:The proteasome inhibitor Bortezomib (Velcade, formerly PS-341) is known to be effective in therapy of various neoplasms and is approved by the FDA for use in multiple myeloma. Clinical trials in acute myeloid leukemia (AML) patients given bortezomib also have revealed a definite clinical activity. Although all of the mechanisms by which this novel drug acts as an antineoplastic agent are not fully understood. All-trans-retinoic acid (ATRA) represents the sole example of clinically useful cytodifferentiating agent. However the therapeutic efficacy of this compound is still burdened by problems such as toxicity and resistance. One possible strategy to increase the therapeutic index of ATRA is the development of ATRA-based pharmacologic combinations that are more powerful and easily tolerated than the individual components. The aim of this study was to investigate the function and mechanisms of Bortezomib-induced differentiation as well as the synergistic activity of Bortezomib on ATRA-induced differentiation of AML cells.Methods:1) Primary patient AML blasts isolated from bone marrow by LSM. For proliferation assay, human myeloid leukemia cells HL60 and U937 were treated with Bortezomib, cell growth curve was performed by counting cells each day. Giemsa staining was performed to evaluate morphologic changes. NBT-reducing activity as a potent differentiation marker of leukemia cells was used and the blue cells were counted by light microscopy for at least 200 cells. Cell-surface markers (CD11b and CD14) were detected by flow cytometry. The expressions of MAPK related proteins (p-MEK, p-ERK, MEK, p-JNK, JNK, p-p38, p38) and transcription factors (STAT1, p-STAT1, C/EBPa) were shown by Western blotting. The transcription level of STAT1 was determined by realtime PCR. Lentivirus-mediated shRNA-STAT1 transfected into HL60 cells to support the concept that the induced differentiation effect of bortezomib is related to the up-regulation of STAT1. Inhibitors (PD98059, SP600125, SB203580 and CHX) were used to further explore the involvement of MAPK pathway and STAT1 in Bortezomib-induced differentiation. The ratio of apoptosis was assessed by annexinⅤ-fluorescein and propidium iodide staining, analyzed by Flow Cytometry. cos7 cells were transiently co-tranfected, with RARE-pGL4-LUC reporter plasmids and with the Renilla-encoding pRL-TK plastmid. Luciferase antivity quantified using the Dual Luciferase Reporter kit.2) MTT method and CCK8 assay were used in proliferation assay to evaluate the inhibition effect of ATRA and Bortezomib on HL60 and NB4 cells or primary patient AML blasts. Cell growth curve was performed by counting cells each day after staining with trypan blue. Giemsa staining was performed to evaluate morphologic changes. NBT-reducing activity was used and the blue cells were counted by light microscopy for at least 200 cells. Cell-surface markers CD11b was detected by flow cytometry.20S proteasome activity was determined by using fluorogenic peptide substrate Suc-LLVY-AMC. The mRNA level of RARαand STAT1 were shown by realtime PCR. Immunofluorescence analyse was used in the detection of STAT1 and p65 distribution. CD11b expression was examined in HL60 cells transfected with lentivirus-mediated shRNA-STAT1. All the proteins expression and activation were measured by western blotting. In vivo antiproliferation of ATRA and Bortezomib was evaluated using human tumor models xenografted HL60 in nude mice, the weight of tumor were measured and the inhibitory rate were calculated. C/EBPεprotein expression were confirmed by immunohistochemistry in HL60 tumors. The radio of apoptosis was assessed by TUNEL staining in HL60 tumors. Results:Part 1 Involvement of MAP kinase in STAT1-mediated differentiation induced by Bortezomib in AML cells.1) Bortezomib inhibits growth and induces cytodifferentiation in AML cell lines and primary patient AML blasts.It was observed that Bortezomib induces morphologic, biochemical and functional changes indicative of macrophage differentiation in HL60 and U937 cells. By the third day, a significant increase in cell growth inhibiton induced by Bortezomib was evident at 5 nM (44.6% of vehicle-treated cells in HL60 cells and 60.6% in U937 cells. HL60, U937 cells and primary patient cells treated with Bortezomib for 3 days underwent striking changes consistent with myeloid maturation with condensation and lobulation of the nucleus, which was assessed by Giemsa staining. Similar results were observed on primary patient cells. Besides, time and dose-dependent effect of Bortezomib were showed on monocytic maturation associated myeloid cell surface markers and NBT reduction assay.2) Bortezomib activates the MEK/ERK cascade, which further up-regulates the expression and activity of the key myeloid transcription factor STAT1, thus promoting other events in myeloid differentiation.MAPK pathways, including MEK/ERK, JNKp46, and p38, were activated during Bortezomib-induced myeloid differentiation of both HL60 cells and U937 cells in a time-dependent manner. PD98059, a specific MEK inhibitor, or SP600125, a specific INK inhibitor was employed to examine the role of MEK signaling in Bortezomib-induced differentiation. Both inhibitors completely suppressed both CD 14 and CDllb expression induced by Bortezomib, in condition of suppression of MEK, ERK or JNKp46 activation. Western blotting results indicated that MEK/ERK cascade activation may be crucial for the induction of the cytodifferentiation effect by the Bortezomib, however it was conjectured that JNK may participate in the process as a downstream kinase. However, activation of p38 might play a negative role in the process of monocytic differentiation of AML cells induced by Bortezomib.Treatment of HL60 cells with Bortezomib led to a significant increase in the amounts of STAT1. as well as the level of phosphorylation of Tyr701. Realtime PCR and the employment of CHX proved that increase of STAT1 accumulation in HL60 cells treated with Bortezomib was caused by the combined effects of its synthesis and degradation inhibition. In addition, an increased level of C/EBPa, a downstream factor of STAT1, was also observed with treatment of Bortezomib. HL60 cells were transduced with lenti viral-vector, expressing shRNA against STAT1 or control vector using established method. The level of STAT1 protein was decreased in cells transfected with STAT1-shRNA, resulting in inhibition of bortezomib-dependent differentiation.We further found that the activation of STAT1 might be mediated by MEK/ERK-JNK, as pre-treatment with PD98059 or SP600125 attenuated the increased level of STAT1 and its phosphorylation form in Bortezomib-treated HL60 cells, which was in consistence with block of differentiation.3) Bortezomib induced differentiation is independent on RARa.An increased level of RARa was observed without the transcription activity. And HL60R went into myeloid maturation with the treatment of Bortezomib.4) Bortezomib-induced differentiation and apoptosis are two independent processes.A time-dependent study showed that Bortezomib at 5 nM induced apoptosis in HL60 or U937 cells. And differentiation and apoptosis induced by Bortezomib seemed to be independent responses, as the rate of apoptosis cells was not declined when Bortezomib-induced differentiation was suppressed.Part 2 Bortezomib enhances the cytodifferentiating properties of ATRA via stabilization of RARa1) Bortezomib enhances terminal differentiation of ATRA-treated AML cells.Bortezomib enhanced the growth-inhibitory and cyto-differentiation effects of ATRA on HL60 and NB4 cells, which were based on inhibition of proliferation, morphologic assessment, differentiation marker CDllb expression, and nitro-blue tetrazolium reduction.Bortezomib enhanced the apoptogenic and cyto-differentiation effect of ATRA on primary patient cells. As for the three leukemic samples tested, one patient sample was derived from a patient with AML-M3 with relapsed leukemia, another one was from a patient with AML/APL, and the third one was from a patient with AML-resistant. Bortezomib and ATRA combination caused a significant decrease in cell viability relative to what is bserved in cultures treated with on drug alone. This was associated with a stronger induction of NBT-reducing activity in combination than in ATRA-differentiation cells.2) The combination of Bortezomib and ATRA inhibits tumor growth via differentiation induction in nude mice implanted with human HL60 xenograftsThe combination therapy of ATRA (5 mg/kg) and Bortezomib (0.1 mg/kg) notably inhibited the tumor growth of the HL60 xenografts, with T/C value 45% and inhibition rate 58.9%, which was prominently stronger than ATRA-administrated group (T/C value:58%, inhibition rate:40.0%), and Bortezomib-treated group (T/C value:62%, inhibition rate:20.0%). The tumor volume of combination group was remarkably decreased from that of vehicle group, and the mono-treated groups. Combination group exerted significantly more potent activities on induction of CD11b expression comparing with the mono-treated groups. And similar observation was obtained on C/EBPεby immunohistochemistry, while no apoptosis was detected after TUNEL staining.3) Mechanism of the enhanced differentiation effect of ATRA in presence of Bortezomib in AML cells.Upregulation of proteasome activity by ATRA was inhibited in presence of Bortezomib. Bortezomib suppressed ATRA-induced UPP-dependent degradation of RARαin HL60 and NB4 cells, but enhanced the transcription of ATRA-responsive genes, represented by STAT1. Western blotting showed the synergistic effect of this combination on the HL60 xenografts might be related to RARαsatabilization.Bortezomib enhanced the ATRA-dependent induction of STAT1, and also in the transcriptional active state, as indicated by the level of phosphorylation of Tyr701 and nuclear translocation. Downregulation of STAT1 expression partially suppressed the potentiates differentiation effect of Bortezomib in HL60 cells transfected with lentivirus-mediated shRNA-STAT1.The progress that Bortezomib synergized ATRA-induced differentiation was NFκB independent, as well as MAPK pathways.Conclusion:In this study, we first report the unexpected pharmacologic effects of Bortezomib in acute myelogenous leukemia cells, which is that Bortezomib induces myeloid differentiation and apoptosis, and also enhaces the cyto-differentiation, and antiproliferative activity of ATRA. These results indicate a potential mechanism of action whereby Bortezomib warrants clinical efficacy for patients with AML and may provide some hints toward developing new treatment approaches for human myeloid leukemia.更多还原