新型Bcr-Abl和Src/Abl抑制剂抗慢性髓性白血病的实验研究

【作者】 刘鹤；

【导师】 陈晓光； 程桂芳； 李燕；

【作者基本信息】 中国协和医科大学 ， 药理学， 2008， 博士

【摘要】 慢性髓性白血病（chronic myelogenous leukemia,CML）是一种血液系统恶性增生性疾病。以细胞内出现嵌合蛋白Bcr-Abl为主要特征。Bcr-Abl蛋白具有持续活化的酪氨酸激酶活性,激活下游多条信号传导通路,引起疾病的发生。伊马替尼（Imatinib,Gleevec,STI-571）属一种Abl酪氨酸激酶抑制剂,它是第一个小分子靶向性治疗药物。体内、外实验表明,Imatinib通过抑制Bcr-Abl自身磷酸化和底物磷酸化,而抑制细胞增殖和诱导凋亡。Imatinib在临床上用于费城染色体阳性（Philadelphia chromosome positive,Ph⁺）的CML患者。由于其所产生的较好血液学和细胞遗传学缓解（尤其是慢性期CML患者）,Imatinib已经作为新近诊断的CML患者的一线药物。但是Imatinib在治疗CML急变期及Ph⁺的急性淋巴细胞白血病患者时效果却并不理想,而且随着临床的用药,Imatinib逐渐出现耐药。因此,这促使人们寻找新的治疗CML和Imatinib耐药的CML的药物。AMN107属第二代Abl抑制剂,许多临床前研究结果已经显示出非常好的效果。研究表明,在许多Bcr-Abl转化的造血细胞系,AMN107对Bcr-Abl自身磷酸化和增殖的抑制较Imatinib高20-50倍。而且AMN107对很多Imatinib耐药的Bcr-Abl蛋白突变株有很强的抑制作用。目前AMN107正在进行临床试验,但已有些病例出现AMN107耐药,其机制是否与Imatinib耐药相同还在进一步研究中,因此Novartis制药公司又继续研发更强的Abl抑制剂,其中368485就是Novartis制药公司研制的一种新型此类化合物,其药物活性尚没有相关报道。本研究就是探讨一种新型苯胺嘧啶类衍生物FAB107在体内、外对CML细胞系K562细胞和K562耐Imatinib细胞系K562/G3.0的抑制作用。本研究采用MTT方法观察了FAB107与Imatinib和368485相比较对K562和K562/G3.0细胞的生长抑制作用。研究结果表明,FAB107抑制K562和K562/G3.0细胞生长,IC₅₀分别为0.03μmol/L和0.07μmol/L。FAB107对2种细胞的抑制作用强于Imatinib(IC₅₀分别为0.31μmol/L和6.32μmol/L),而与368485(IC₅₀分别为0.02μmol/L和0.082μmol/L)相当。裸鼠中空纤维模型实验进一步证实,FAB107可以抑制K562和K562/G3.0细胞在裸鼠体内的生长。给药7天后,与对照组相比,FAB107的3个剂量组（15μmol/kg、30μmol/kg和45μmol/kg）在体内对K562及K562/G3.0细胞生长均呈剂量依赖性的抑制作用（P＜0.01）,并且抑制强度强于等摩尔浓度的Imatinib,与等摩尔浓度的368485相当。流式细胞术结果表明,FAB107可以剂量依赖性地诱导K562和K562/G3.0细胞凋亡,与等摩尔浓度的368485相当。为了阐明FAB107对K562和K562/G3.0细胞生长抑制作用的机制,我们检测了FAB107对K562和K562/G3.0细胞Bcr-Abl蛋白自身磷酸化的抑制作用。FAB107可以明显抑制K562和K562/G3.0细胞Bcr-Abl蛋白自身磷酸化。其作用优于等摩尔浓度的Imatinib,与等摩尔浓度的368485相当。总而言之,新型苯胺嘧啶类衍生物FAB107在体内外可以明显抑制K562和K562/G3.0细胞的生长,其作用机制可能与抑制Bcr-Abl蛋白自身磷酸化有关。上述研究提示,FAB107将是治疗Imatinib敏感和耐药的CML的较好候选化合物。慢性髓系白血病（chronic myelogenous leukemia,CML）是一种多能造血干细胞恶性克隆增殖性疾病,其恶性克隆标志是费城染色体（Philadelphia chromosome,Ph）及Bcr-Abl融合基因。后者编码产生的Bcr-Abl融合蛋白,即P210^Bcr-Abl蛋白,具有异常增高的蛋白酪氨酸激酶（proteinty rosineki nase,PTK）活性,被认为是导致CML发病的根本原因。Imatinib（又称Gleevec,STI-571）是Bcr-Abl的抑制剂。该药用于CML的治疗以来,取得了显著的临床疗效。但随着它的广泛应用,人们发现部分患者对Imatinib存在原发或继发耐药。如此高度靶向性治疗药物如同其它抗肿瘤化疗药物一样,很快出现了耐药现象,这引起了人们的关注,并成为CML研究的又一热点。目前,关于Imatinib耐药的机制仍不完全清楚,主要包括以下几个观点:首先是血浆蛋白结合;第二是药泵;第三是BCR-ABL激酶突变;第四为不依赖BCR-ABL的耐药机制,如SRC家族;第五为基因扩增。Src激酶参与了Bcr-Abl介导的白血病的发生,并与一些Imatinib耐药病例有关。Dasatinib（Bristol-Myers Squibb,布迈-施贵宝）属噻唑类Src/Abl双靶点抑制剂。2006年6月已通过美国FDA批准上市,用于包括慢性、加速期、原发粒细胞性、原发淋巴细胞性白血病以及耐药的白血病病例（如对Imatinib耐药）在内的各类成人白血病的治疗。本研究试图在体外建立Imatinib耐药CML细胞系,并对其耐药机制进行初步探讨。同时探讨一种新型噻唑类衍生物FB2在体内外对K562细胞和K562耐Imatinib细胞系K562/G5.0的生长抑制作用及其作用机制。一、Imatinib耐药株K562/G5.0细胞系的建立和鉴定采用逐步递增药物浓度的方法培养K562细胞,进行耐药细胞的诱导;采用MTT法检测不同浓度Imatinib对K562和K562/G5.0的细胞毒作用;采用流式细胞术分析K562和K562/G5.0的细胞周期;MTT法检测阿霉素（doxorubicin）对两种细胞的细胞毒作用;测序分析Abl酪氨酸激酶ATP结合区基因点突变;MTT法检测Dasatinib对两种细胞的抑制作用;RT-PCR法检测K562和K562/G5.0细胞LynmRNA的表达;Western blot法检测K562、K562/G3.0和K562/G5.0细胞的C-Abl、P-C-Abl、Lyn和P-Lyn的表达;Western blot法检测Dasatinib对Lyn自身磷酸化的影响。研究结果表明,MTT法检测了Imatinib对K562和K562/G5.0的半数抑制浓度(50%inhibiting concentration,IC₅₀)分别为0.37μmol/L和44.40μmol/L,K562/G5.0的耐药倍数为122.5倍。细胞周期分析表明K562/G5.0耐药细胞中G0/G1期和G2/M期细胞均稍有减少,而S期细胞有所增加。RT-PCR结果表明,MDR-1和BCRP在耐药株中的mRNA表达与K562和KB/V相比都有明显下降,进一步用阿霉素处理两种细胞,发现阿霉素对两种细胞的细胞毒作用相当。Abl激酶ATP结合区基因测序结果表明,两者序列完全一致。进一步我们还发现,耐药株的Lyn mRNA表达水平增高,Lyn和P-Lyn的蛋白表达水平也增高,但是C-Abl和p-C-Abl表达水平下降。同时,我们发现Lyn和P-Lyn的蛋白表达水平随着耐药浓度的增加而增高,而C-Abl和p-C-Abl的表达水平在耐药浓度低的细胞（K562/G3.0）中表达增高,而在耐药浓度高的细胞（K562/G5.0）中表达降低,Src/Abl双靶点抑制剂Dasatinib对K562/G5.0仍具有非常强的抑制作用。二、FB2在体内外对CML和Imatinib耐药的CML细胞的生长抑制作用MTT法检测FB2对K562和K562/G5.0细胞的生长抑制作用;MTT法检测FB2对MDA-MB-231和DU145细胞的生长抑制作用;采用Western blot法检测FB2对K562和K562/G5.0的C-Abl、Lyn和C-Src自身磷酸化水平的抑制作用;采用荷人CML的Imatinib耐药株K562/G5.0细胞的NOD/SCID小鼠,观察FB2对其生存期的影响。研究结果表明,MTT法检测了FB2对K562、K562/G01和K562/G5.0的IC₅₀分别为0.03nmol/L、0.10nmol/L和0.38nmol/L。FB2还可以抑制其它上皮来源的实体肿瘤细胞MDA-MB-231和DU145细胞的生长。FB2可以明显抑制C-Abl、C-Src和Lyn蛋白的自身磷酸化。FB2还可以延长荷CML的Imatinib耐药株K562/G5.0细胞的NOD/SCID小鼠的生存期。总之,本研究在体外成功地建立起Imatinib耐药的K562细胞系,K562/G5.0,耐药浓度为5μmol/L,耐药倍数为122.5倍。K562/G5.0细胞的耐药机制属Bcr-Abl非依赖性,与Lyn激酶过度表达和激活有关。FB2作为一种新型Src/Abl双激酶抑制剂,可能成为治疗CML、Imatinib耐药的CML以及Src激酶过表达的肿瘤的较好候选化合物。更多还原

【Abstract】 Chronic myelogenous leukemia（CML） is a myeloproliferative disorder of hematopoietic stem cells.The pathologic hallmark of this disease is the Philadelphia chromosome（Ph）,which is seen in＞90%of CML patients.The Ph produces Bcr-Abl fusion protein,which displays constitutive tyrosine kinase activity and activates several signaling transduction pathways.The 2-phenylamino-pyrimidine derivative Imatinib（Gleevec,STI571） was developed as the first molecularly targeted therapy that specifically inhibits the Bcr-Abl tyrosine kinase activity in patients with Ph positive（Ph⁺） CML.Imatinib acts by revoking the effects of the Bcr-Abl oncoprotein through inhibition of Bcr-Abl autophosphorylation,inhibition of proliferation,and induction of apoptosis.Due to its excellent hematologic and cytogenetic responses,particularly in patients with chronic phase CML,Imatinib has moved towards first-line treatment for newly diagnosed CML. However,reponses to Imatinib in patients with more advanced disease are generally transient.Nevertheless,resistance to the drug has been frequently reported.As a result,a variety of strategies derived from structural studies of the Abl-Imatinib complex have been developed,resulting in the design of novel Abl inhibitors.Several preclinical studies have shown promising results for second generation Abl inhibitor AMN107.Using numerous Bcr-Abl tranformed hematopoietic cell lines, AMN107 was found to be 20-50 fold more potent compared with Imatinib in reduction of both autophosphorylation and proliferation.Nevertheless,cell lines expressing the Imatinib-resistant Bcr-Abl mutants can be inhibited by AMN107.AMN107 has made its way into clinical trial.Unfortunately,resistance to AMN107 reduced inhibition of cell growth.The molecular mechanisms underlying resistance still need to be determined.In this study,we investigated the activity of a novel phenylamino pyrimidine derivative FAB107,compared with Imatinib and 368485,in both K562 and K562/G3.0 cells.Effects of FAB107 on the growth of K562 and K562/G3.0 cells were analyzed by MTT assay.The growth of K562 and K562/G3.0 cells was inhibited by FAB 107 with the IC₅₀ value of 0.03μmol/L and 0.07μmol/L,respectively.The inhibition was stronger than that of Imatinib(IC₅₀:0.31μmol/L and 6.32μmol/L) and equal to 368485(IC₅₀: 0.02μmol/L and 0.082μmol/L).In vivo result from nude mice bearing hollow fibers with leukemia cells showed that FAB107 inhibited growth of K562 and K562/G3.0 cells in nude mice.The inhibitory effects of FAB107 were in a dose-dependent manner and 15μmol/kg,30μmol/kg and 45μmol/kg on FAB107 had P values less than 0.01,compared with control group.The inhibitory activity was stronger than that of Imatinib and equal to 368485.Flow cytometric assay was perfomed to evaluate the cell cycle and apoptosis. Exposure of K562 and K562/G3.0 cells to FAB107,Imatinib and 368485 resulted in an increase of percentage of apoptotic cells.The potency of inhibition was stronger than that of Imatinib and equral to 368485.To investigate the mechanism of FAB107 on the growth inhibition of K562 and K562/G3.0 cells,we tested the effect of FAB107 on the autophosphorylation to Bcr-Abl in K562 and K562/G3.0 cells.FAB107 inhibited autophorylation of Bcr-Abl kinase more effectively than that of Imatinib in both cell lines,and its potency of inhibition was equal to 368485.In conclusion,FAB107,a novel phenylamino pyrimidine class derivative,inhibited growth of K562 and K562/G3.0 cells in vitro and in vivo,and the probAble mechanisms was inhibition on autophosphorylation to Bcr-Abl kinase.These results suggest that FAB107 is a promising candidate as a novel therapeutic agent for Imatinib-sensitive and Imatinib-resistance CML. Chronic myelogenous leukemia（CML） is a hematopoietic disorder characterizd by the malignant expansion of bone marrow stem cells.Its malignant clonal marker is Philadelphia chromosome（Ph） which harbors the Bcr-Abl fusion gene.The latter encdes a chimeric Bcr-Abl protein,P210^Bcr-Abl,with a deregulated tyrosine kinase activity and identified as having a central role in the pathogenesis of CML.Imatinib（Gleevec,STI-571;Novartis,Inc.） is a tyrosine kinase inhibitor that competitively inhibitis Bcr-Abl kinases.It inhibits proliferation and induces apoptosis in Bcr-Abl positive cells and has shown remarkAble clinical activity in patients with CML. However,a significant proportion of patients treated with Imatinib develop resistance and some of them have primitive resistance,which usually are found in patients treated with other chemotherapeutic drugs.So the potential mechanisms of Imatinib resistance become a new focal point.There are 5 main mechanisms currently known that may result in Imatinib resistance.The first is plasma protein binding.The second mechanism is drug efflux.The third mechanism is the mutation of the Bcr-Abl kinase.The fourth mechanism is independent of Bcr-Abl.The fifth mechanism is gene amplification.Src family kinase activation is involved in leukemia mediated by Bcr-Abl,and may function in cases of resistance to Imatinib.Dasatinib（Bristol-Myers Squibb Inc.） is a thiazole-based dual Src/Abl kinase inhibitor.Dasatinib has recently been approved by U.S.Food and Drug Administration in 2006 for use in patients with CML or Philadelphia chromosome-positive acute lympho-blastic leukemia（ALL） who are unable to tolerate or have not responded to other treatments.To investigate possible mediators of acquired Imatinib resistance,K562 cells of resistant to 5μM Imatinib（K562/G5.0） were cloned and compared with the parental cell population.In this study,We also investigated the activity of a novel thiazole-based derivative FB2,relative to Dasatinib,in both K562 and K562/G5.0 cells.1.The induction of Imatinib resistance cell line（K562/G5.0） and analysis of the induced-resistane mechanisms The wild-type K562 cells were cultured in gradually increased concentrations of Imatinib.The cytotoxic effects of K562 and K562/G5.0 treated with different concentration of Imatinib were analyed by MTT assay and the cell cycle was evaluated by flow cytometric assay.Effects of Imatinib,doxorubicin and Dasatinib on the growth of K562 and K562/G5.0 cells were analyzed by MTT assay.C-Abl,P-C-Abl,Lyn and P-Lyn protein expression,sequence analysis were used to study the potential mechanisms of acquired resistance of K562/G5.0.IC₅₀s（50%inhibiting concentration） of K562 and K562/G5.0 were 0.37μmol/L and 44.40μmol/L respectively by MTT assay and the resistance to Imatinib increased to 122.5 fold.The analysis to the cell cycle in K562/G5.0 cells indicated that the cells of G0/G1 and G2/M phase decreased and the cells of S phase increased.In contrast with K562 and KB/V,the MDR-1 and BCRP mRNA expression in K562/G5.0 cells decreased. It was testified that there was no statistic difference after comparing the cytotoxic effects of K562 and K562/G5.0 treated with different concentration of doxorubicin by MTT assay.No point mutant in the Bcr-Abl ATP-binding site was detected.By comparison with K562,expression of Lyn mRNA in K562/G5.0 cells increased.Interesting,the expression of Lyn and p-Lyn increasd following the increase of the Imatinib resistance fold in K562 resistance cell lines.However,the expression of c-Abl and p-c-Abl increased at lower level in K562/G3.0 cells and decreased at higer resistance fold in K562/G5.0 cells.Furthermore,Dasatinib,the dual Src/Abl tyrosine kinase inhibitor,was still effective in K562/G5.0 cells,with resistance presumed due to expressing a high number of Lyn gene copies and transcripts.2.The inhibition of FB2 on Imatinib-resistant chronic myeloid leukemia in vitro and in vivoThe cytotoxic effects in K562,K562/G5.0,MDA-MB-231 and DU145 treated with different concentrations of FB2 were analyed by MTT assay.Western blot analysis was used to evaluate the effects of FB2 on the inhibitions of c-Abl,c-Src,and Lyn protein autophosphorylation in K562 and K562/G5.0 cells.The efficacy of FB2 was assessed in a nonobese diabetic/severe combined immunodeficient（NOD/SCID） mouse model bearing of Imatinib-resistant CML cells.IC₅₀s of K562,K562/G01 and K562/G5.0 were 0.03nmol/L,0.10nmol/L and 0.38nmol/L respectively by MTT assay.Besides Imatinib-sensitive cell line（K562）,FB2 significantly inhibited the growth of Imatinib-resistant cell lines with different resistance mechanisms（K562/G5.0 and K562/G01）,and decreased the expressions of autophosphorylation of Bcr-Abl,c-Src and Lyn kinases in them.It also inhibited the proliferations of Src overactivated cells DU145 and MDA-MB-231.Furthermore,FB2 potently prolonged the survival time of NOD/SCID mice harbored K562/G5.0 cells.In vitro,we successfully established a resistant cell line（K562/G5.0） with resistance to 5μmol/L and 122.5 fold resistance as compared with that of K562.The potential mechanism of the resistant cell line above established was the Bcr-Abl-independent and Lyn-activated phenotype.Our results also indicated that FB2,an Abl/Src dual tyrosine kinase inhibitor,is a promising candidate for Imatinib-resistant CML and Src overactivated cancer.更多还原