【作者】 梁赟；

【导师】 赵小英；

【作者基本信息】 浙江大学 ， 内科学， 2010， 博士

【摘要】 多发性骨髓瘤(multiple myeloma, MM)是一种B淋巴细胞的恶性肿瘤,特点是骨髓中浆细胞异常增生并产生单克隆的免疫球蛋白,其发病率占血液系统恶性肿瘤10%,占所有肿瘤死亡率的2%左右。目前骨髓瘤的治疗包括联合化疗,大剂量化疗联合自体干细胞移植以及干扰素,近年来靶向性药物如蛋白酶体抑制剂硼替佐米(Bortezomib),沙利度胺(thalidomide)和来那度胺(lenalidomide)等应用于临床,有效提高了治疗缓解率。但是,骨髓瘤迄今为止仍不可治愈,大部分患者因为对化疗药物耐药或者难以耐受药物的毒副反应而不能维持长期缓解,寻求高效低毒的药物仍是MM研究的重点。小檗胺(Berbamine, BBM)是一种从我国中草药小檗属植物中提取的双苄基异喹啉类生物碱,具有促进造血和调节免疫的功能,目前已广泛应用于肿瘤病人化疗、放疗引起的白细胞减少症的治疗。我们课题组前期的工作已经证实BBM可以明显诱导K562、K562/r、Jurkat、NB4等多种白血病细胞凋亡,而在治疗浓度对正常骨髓细胞的增殖没有明显影响。进一步对K562细胞的研究发现,BBM诱导细胞凋亡的机制涉及到下调K562细胞bcr/abl融合基因和p210蛋白的表达、抑制分子伴侣热休克蛋白90(Hsp90)、下调survivin基因、增加caspase3活性并涉及到多种与细胞增殖分化密切相关的信号通路,这些分子机制的发现提示BBM可能具有更广泛的抗肿瘤谱。同时,小檗胺为一种天然的小分子化合物,其结构式公开,可以对其进行化学结构修饰从而产生系列小檗胺衍生物。本研究首次以人骨髓瘤细胞株为研究对象,探讨小檗胺体外抗骨髓瘤作用及其可能机制,并从小檗胺衍生物中筛选出高效低毒的抗骨髓瘤的先导化合物,进一步从分子水平明确小檗胺衍生物对肿瘤相关信号通路的影响及可能的作用靶位,从而为后续新型抗骨髓瘤药物的研发奠定实验基础。第一部分小檗胺抗骨髓瘤细胞增殖及其机制的研究目的：研究小檗胺对人骨髓瘤细胞株增殖抑制和诱导凋亡的作用并探讨其相关机制。方法：MTT法检测小檗胺单用及联合常规抗骨髓瘤药物(地塞米松,阿霉素,砷剂)对人骨髓瘤细胞株KM3,RPMI8226和U266的增殖抑制作用；Wright-Giemsa染色观察药物作用前后细胞形态学改变；流式细胞术分析细胞周期及凋亡细胞百分比；间接免疫荧法观察BBM干预后p65蛋白改变及亚细胞定位；Western blot检测BBM作用前后p65,IκBα激酶α(IκBαkinase, IKKa), IκBα, p-IκBa, TNFAIP3 (A20), cyclin D1, Bcl-2, BAX, Bcl-XL, Bid及survivin蛋白的表达。结果：BBM可以抑制KM3, RPMI8226和U266细胞的增殖,呈浓度依赖及时间依赖性,48h对三株细胞的IC50值分别为5.091μg/ml,3.83μg/pml,4.71μg／ml,并且低剂量的BBM与常规化疗药物联合48h后可以增加抗肿瘤效应；流式细胞术检测BBM作用后KM3细胞发生G1／S期阻滞,8μg／ml BBM作用36h后KM3细胞凋亡率从对照组的0.54%升至51.83%,并且光镜下可见凋亡特征性形态学改变；8μg/ml BBM作用KM3细胞24h后能明显下调IKKα,p-IκBα,上调A20,抑制骨髓瘤细胞NF-κB活性,下调核内p65表达,继而下调NF-κB下游cyclinDl, Bcl-XL,Bid及survivin等蛋白的表达。结论：小檗胺对人骨髓瘤细胞株具有增殖抑制和诱导凋亡作用,并能增加骨髓瘤细胞对常规化疗药物的敏感性,抑制NF-κB信号通路的活化可能是其抗骨髓瘤细胞增殖的重要机制之一,小檗胺可能成为一种新型的NF-κB抑制剂,其抗骨髓瘤作用值得进一步研究。第二部分小檗胺衍生物抗骨髓瘤细胞增殖及其机制的研究目的：从系列小檗胺衍生物中筛选出更有效的抗骨髓瘤的化合物并探讨其作用的靶位。方法：采用课题组已合成的小檗胺衍生物中筛选出的BBD9#衍生物和BBD24#衍生物,用MTT方法检测衍生物对人骨髓瘤细胞株的增殖抑制作用以及对常规抗骨髓瘤药物(地塞米松,阿霉素,砷剂)的增敏作用,MTT法检测BBD9#对正常造血细胞的毒性作用；流式细胞术分析细胞周期及凋亡细胞百分比；间接免疫荧法观察BBD9#干预后RPMI8226细胞p65蛋白改变及亚细胞定位；Western blot检测BBD9#作用后NF-κB信号通路及上下游相关蛋白的改变,同样方法检测药物作用后ERK,AKT及JNK等信号分子是否存在活化表达。结果：小檗胺衍生物与小檗胺比较具有更强的抗骨髓瘤细胞增殖活性,从中筛选出9#衍生物和24#衍生物作用RPMI8226细胞48h后比较小檗胺效应增加6倍以上。通过MTT方法检测这2种衍生物对3种骨髓瘤细胞株(KM3, RPMI8226, U266)的增殖抑制率发现48h的IC50值在0.65μg/ml～0.81μg／ml之间。低剂量的BBD9#(0.5μg／ml)可以增加KM3, RPMI8226和U266细胞对常规化疗药物的敏感性,而在相同浓度范围内对正常造血细胞没有明显毒性。流式细胞术分析显示BBD9#可以引起细胞周期G1／S期阻滞,继而诱导凋亡,1μg/ml BBD9#作用KM3, RPMI8226和U266细胞36h后凋亡率分别从0.67%,1.12%,1.08%增至34.60%,47.30%,26.48%。进一步研究BBD9#抗骨髓瘤作用的分子靶点,间接免疫荧光可见到胞核内p65蛋白减少,Western blot检测核内p65蛋白表达减少,NF-κB活化关键的上游激酶IKK和p-IκBα受抑,同时NF-κB下游蛋白cyclin D1和survivin的表达减少。对其它相关信号通路的研究发现,BBD9#作用前后p-ERK和p-AKT蛋白表达没有明显改变,但可活化JNK信号通路,p-JNK表达明显增高且其下游c-Jun蛋白表达增高,联合应用JNK特异性抑制剂SP6000125后,BBD9#对骨髓瘤细胞增殖抑制作用部分受阻。结论：小檗胺衍生物与小檗胺比较具有更强的抗骨髓瘤活性,筛选出9#衍生物和24#抗骨髓瘤活性比较小檗胺效应增加。其抗肿瘤作用可能与抑制NF-κB活性表达和活化JNK信号通路相关,这些研究为后续新型抗骨髓瘤药物的研发奠定实验基础。更多还原

【Abstract】 Multiple myeloma (MM) is a B-cell malignancy characterized by abnormal proliferation of plasma cells in bone marrow secreting monoclonal immunoglobulins. MM accounts for 2% of all cancers and more than 10% of all hematologic cancers. Prior treatment options for MM include chemotherapy, stem cell transplantation and immunotherapy with interferon-gamma. Recently, novel agents such as bortezomib, thalidomide and lenalidomide were approved for MM treatment and have improved the complete remission rate and patient outcome. However, MM remains incurable, and development of drug resistance or intolerable toxicity emerges as a major problem especially for patients with refractory or relapsed MM. Therefore, design, identification and validation of novel chemicals with therapeutic potential are clearly needed for MM treatment.Berbamine (BBM), a natural small molecular compound, is distilled from Chinese traditional medicine Berberis amurensis, which has been shown to improve normal hematopoiesis and immune function of cancer patients over the past decades. Our former studies showed that berbamine down-regulates p210 bcr-abl oncoprotein in K562 cells, and induces apoptosis through a caspase-3-dependent pathway. In addition, berbamine was highly effective in suppressing the growth of imatinib-resistant cell line K562 (K562-r) cells, human acute promyelocytic leukemia cell line NB4 cells and T-cell leukemia cell line Jurkat cells. Therefore, berbamine appears to have broad anti-tumor activities with complex mechanism of actions. These previous studies prompted us to explore whether BBM exist anti-proliferation effect on MM cells and develop novel berbamine derivatives (BBD) designed to treat malignant disease.This is the first report to identify the effects of berbamine on MM cells, and a series of new berbamine derivatives were obtained by synthesis. In this series, high to very high activity has been found. The aim of our study is to investigate the target genes and the transduction signaling involved in the effects on MM cells induced by berbamine and berbamine derivatives. Our studies provide enough rational for considering berbamine worthy of more research and designing chemically synthetic compounds based on berbamine may represent a promising strategy to develop novel anti-myeloma drugs.Part 1 Anti-proliferation Effect and Mechanism of Berbamine on Multiple Myeloma CellsObjective:We sought to investigate the effect of berbamine on the growth of human multiple myeloma cells and elucidate the mechanism of its action.Methods:MTT assay was used to determine the inhibitory effect of berbamine alone or combined with chemotherapeutic drugs. Morphological analysis was used to detect apoptosis. Flow cytometry was performed to characterize cell cycle profile upon berbamine treatment. Changes and subcellular localization of p65 protein were observed by fluorescence microscopy. Western blot was used to measure the protein levels of p65, IκB Kinase a (IKKa), IκBa, p-IκBα, TNFAIP3 (A20), cyclinD1, Bcl-2 family proteins and survivin.Results:Berbamine inhibits the proliferation of KM3, RPMI8226 and U266 cells in a dose-and time-dependent manner, and IC50 of 48 hours were 5.09μg/ml,3.83μg/ml and 4.7μg/ml respectively. A remarkable increased antiproliferative activity on MM cells was found in combination of berbamine with dexamethasone (Dex), doxorubicin (Dox) or arsenic trioxide (ATO). Flow cytometric analysis revealed that KM3 cells were arrested at G1/S phase and apoptotic cells increased from 0.54% to 51.83% for 36 hours. Morphological changes of cells undergoing apoptosis were observed under light microscope. Mechanistically,8μg/ml berbamine treatment led to increased expression of A20, down-regulation of IKKa, p-IκBa, and followed by inhibition of p65 nuclear localization. As a result, the expression of NF-κB downstream targets such as cyclinD1, Bcl-xL, Bid and survivin were down-regulated.Conclusion:Berbamine inhibited the proliferation of MM cells and induced G1/S arrest as well as apoptosis. The mechanism of berbamine on MM cells may be through modulating NF-κB activity. Our results suggest that berbamine is a novel inhibitor of NF-κB activity with remarkable anti-myeloma efficacy.Part 2 Anti-proliferation Effect and Mechanism of Berbamine Derivatives Synthesis on Multiple Myeloma CellsObjective:To explore anti-proliferation effect and mechanism of berbamine derivatives Synthesis on Multiple Myeloma Cells.Methods:A series of new berbamine derivatives were obtained by synthesis and the anti-myeloma activities of them against RPMI8226 cells were evaluated. Compounds 9# showed the best activity and had low cytotoxicity on normal blood cells. MTT assay was used to determine the inhibitory effects of BBD9# and BBD24# on KM3, RPMI8226 and U266 cells individually or combined with conventional anti-myeloma drugs. Flow cytometric analysis was performed to evaluate the effects of BBD9#on cell cycle progression and apoptosis. Changes and subcellular localization of p65 protein were observed by fluorescence microscopy. Western blotting was used to evaluate the protein levels of p65 and its up-stream and down-stream events in the NF-kB activation cascade in response to 1μg/m BBD9# reatment and the same method was used to evaluate the activation of ERK, AKT and JNK pathways.Results:BBD9# and BBD24# displayed higher anti-MM activities against RPMI8226 cells. Both of berbamine derivatives inhibit the proliferation of KM3, RPMI8226 and U266 cells in a dose-and time-dependent manner, and IC50 of 48h were 0.65μg/ml to 0.8μg/ml. Low does BBD9# potentiated the cytotoxicity effecs of other chemotherapeutic agents. It induced cell cycle arrest at Gl/S plase, and then triggered MM cells apoptosis by Flow cytometric analysis. After 24 hours treated withμg/ml BBD9#, the apoptotic cells of KM3, RPMI8226 and U266 cells increased from 0.67%, 1.12% and 1.08% to 36.60%,47.30% and 26.48% respectively. Western blot showed the expressions of IKKa and p-IΚBαwere down-regulated and the expression of NF-κB downstream targets such as cyclinDl and survivin proteins were down-regulated. We also found the expressions of p-JNK and c-Jun were increased in different time, whereas had no effect on the activation of ERK and AKT patyways. JNK inhibitor SP600125 partly blocks BBD9#-induced cell death, suggest that the activation of JNK modulated BBD9#-induced apoptosis on RPMI8226 cells.Conclusion:Both BBD9# and BBD24# can improve its cytotoxicity against MM cells significantly. BBD9#can suppress proliferation and induce apoptosis in RPMI8226 cells mediate suppression of the NF-κB pathway and its effect in part through the up-regulateing JNK signaling pathway. Designing chemically synthetic compounds based on berbamine may represent a promising strategy to develop novel anti-myeloma drugs.更多还原