【作者】 史鹏程；

【导师】 徐兵；

【作者基本信息】 南方医科大学 ， 血液病学， 2010， 硕士

【摘要】 背景急性白血病(AL)是我国十大恶性肿瘤之一,是青少年和35岁以下成年人发病率最高的恶性肿瘤；近年来随着室内外环境污染等因素的影响,其发病率逐年上升,严重影响人民健康,其中急性髓系白血病(AML)是成人AL最常见的类型。尽管随着化疗方案的不断改善,AML完全缓解(CR)率可达70%左右,但大部分患者会出现复发,一旦复发,再次缓解率低,预后差,难治复发是目前导致AML治疗失败的最根本原因。化疗是治疗AML的首选方法,目前临床上影响AML化疗效果进一步提高主要存在两个障碍：一是白血病细胞对化疗药物的不敏感或获得性的耐药；二是化疗药物对一些重要的组织和器官存在的非特异性的毒性,限制化疗药物有效剂量的提高。由于化疗导致的系统性毒性和药物耐药是白血病化疗成功的主要障碍,而新的分子靶向药物也存在价格昂贵和耐药问题,从传统价廉药物中开发具有抗白血病活性并有可能增强传统化疗药物效果的“老药新用”途径越来越引起重视。Disulfiram (DS)是临床应用超过60年的抗酗酒药物,安全性好,价格低廉。研究发现DS单药可以诱导一系列实体瘤细胞的凋亡及抑制其增殖,但是关于其对白血病细胞的作用尚存争议。作为二价金属离子螯合剂,DS能够够强烈螯合Cu离子形成DS/Cu复合物,且DS/Cu复合物可显著提高DS诱导实体瘤和白血病细胞凋亡的作用,在杀伤肿瘤细胞的作用中DS和Cu是相辅相成的。c-jun N端激酶(c-jun N-terminal kinase,JNK)家族是MAPK(丝裂原激活的蛋白激酶)家族中重要的一员,JNK是一种特异性磷酸化核内转录因子c-jun的激酶,通过使c-jun第63、73位丝氨酸双磷酸化,提高其转录活性,从而提高激活蛋白(AP-1)的转录活性并抑制泛素介导的c-jun降解,在细胞生长、癌基因转化、细胞分化和凋亡等发挥重要作用。近来研究发现JNK/c-Jun通路在AL的发病机制中也占有重要地位,与AL的发生、发展及耐药机制密切相关。研究表明JNK/c-Jun通路活化失败是AML治疗中蒽环类药物耐药的可能机制之一,而JNK/c-Jun通路活化可以通过诱导凋亡逆转白血病和实体瘤细胞耐药。因此JNK/c-Jun通路失活可能是同多药耐药(MDR)相关的另外一个重要机制并且可能是逆转耐药的一个作用靶点。国外研究报道显示DS/Cu复合物可以增强实体瘤细胞对化疗药物的敏感性,但是它的作用靶点和分子机理尚存争议。DS/Cu复合物联合其他传统化疗药物有望一方面提高化疗药物对肿瘤细胞的杀伤作用,减少耐药发生；另一方面降低化疗药物的使用剂量,从而减少化疗药物对人体的毒性。目前关于DS/Cu复合物对耐药白血病细胞的作用研究尚未见文献报道,本课题旨在探讨DS/Cu复合物能否增强阿霉素(Dox)对人急性髓系白血病多药耐药细胞株HL60/Dox的敏感性及其与JNK/c-Jun通路的关系。研究目的白血病细胞对化疗耐药是导致白血病治疗失败的根本原因,逆转白血病细胞耐药是提高白血病疗效的重要手段。本课题以耐阿霉素(Dox)的人急性髓系白血病多药耐药细胞株HL60/Dox为研究对象,探讨DS/Cu能否在体外增强耐药HL60/Dox细胞对阿霉素的敏感性,并进一步从JNK/c-Jun通路深入探讨其潜在的分子机制。方法1、细胞株：耐阿霉素的人急性髓系白血病细胞株HL60/Dox及其野生型HL60细胞株。2、MTT法检测阿霉素对耐药HL60/Dox和野生型HL60细胞的体外增殖抑制作用,评价HL60/Dox细胞的耐药性。3、MTT法检测不同浓度DS联合Cu离子(1μM)作用24和48小时后对HL60/Dox细胞的体外增殖抑制作用。4、MTT法评价无明显增殖抑制作用的IC20浓度的DS/Cu复合物处理HL60/Dox细胞24小时后能否增强HL60/Dox细胞对阿霉素的敏感性。5、Annexin V-FITC/PI双染检测阿霉素、DS/Cu单药组、DS/Cu联合阿霉素组及空白对照组作用HL60/Dox 24小时后凋亡细胞比例；为明确DS/Cu增强HL60/Dox细胞对阿霉素的敏感性是否同JNK/c-Jun通路相关,应用JNK/c-Jun通路抑制剂Sp600125(20uM)对HL60/Dox细胞预处理2小时,然后给予上述各组相同的处理,并进行凋亡细胞比例检测。6、倒置显微镜下观察上述各个处理组作用24小时后HL60/Dox细胞形态学变化。7、Western Bloting检测上述各个处理组作用24小时后HL60/Dox细胞内JNK,磷酸化JNK(p-JNK)及其下游分子c-Jun、磷酸化c-Jun (p-c-Jun)和Bcl-2蛋白表达变化。8、采用SPSS 13.0软件进行统计学分析,两组间均数的比较使用独立样本t检验方法；配对计量资料的比较采用配对样本t检验；多组间均数比较使用单因素方差分析方法进行统计,在方差分析显著的情况下使用Bonferroni(方差齐性)进行多重比较；若方差不齐则采用近似F检验(如Welch方法)代替方差分析后采用Dunnett T3方法进行多重比较；计量资料用x±s表示,检验水准为α=0.05,双侧检验。结果1、阿霉素对野生型HL60和耐药HL60/Dox细胞的增殖抑制作用：MTT结果显示阿霉素作用野生型HL60和耐药HL60/Dox细胞24小时的IC50值分别为0.107±0.017μg/ml、7.69±1.87μg/ml,阿霉素对HL60/Dox细胞的IC50值高于野生型HL60细胞,耐药倍数为71.87,差异有统计学意义(t=7.000,p=0.002),提示HL60/Dox细胞对阿霉素高度耐药。2、DS/Cu对HL60/Dox细胞的增殖抑制作用：根据预试验结果,以Cu离子浓度1μM为联合浓度,应用MTT法观察不同浓度DS联合Cu离子对HL60/Dox细胞的增殖抑制作用,结果显示DS/Cu对HL60/Dox细胞有明显的增殖抑制作用,随着作用时间和浓度的增加,其IC50值和细胞存活率随之下降。DS/Cu作用HL60/Dox细胞48h后的IC50值(0.439±0.108μM)低于24小时(1.11±0.025μM),差异具有统计学意义(t=14.114,p=0.005)。3、DS/Cu增强阿霉素对HL60/Dox细胞的增殖抑制作用：根据MTT结果,我们选择对HL60/Dox细胞无明显增殖抑制作用的IC20浓度的DS (0.63μM)作为联合浓度,Cu离子浓度仍为1μM。DS/Cu联合不同浓度阿霉素处理24h后,阿霉素对HL60/Dox的IC50值从7.69±1.87μg/ml降到0.48±0.021μg/ml,逆转倍数达15.95倍,差异具有统计学意义(t=6.654,p=0.003)。4、DS/Cu增强阿霉素对HL60/Dox细胞的诱导凋亡作用：根据MTT和预实验结果,选取IC20浓度的DS/Cu(DS:0.63μM,Cu离子:1μM)、1.25μg/ml的阿霉素单药及上述浓度的DS/Cu联合阿霉素作用于HL60／Dox细胞24h观察细胞凋亡比例的变化。结果显示阿霉素和DS/Cu单药组凋亡细胞比例仅仅较空白对照组略有增高,且差异无统计学意义(F=92.326,p>0.05),而DS/Cu联合阿霉素组凋亡细胞比例高达73.24±10.58%,较DS/Cu、阿霉素单药组及空白对照组均显著增多,是阿霉素单药组的11.46倍,差异有统计学意义(F=92.326,p<0.05)。我们进一步用JNK/c-Jun通路抑制剂Sp600125(20uM)对HL60/Dox细胞预处理2小时,然后予上述各组相同的处理,结果显示Sp600125将DS/Cu联合阿霉素组凋亡细胞比例从73.24±10.58%降至40.70±6.03%,差异有统计学意义(t=6.218,p=0.025)。5、不同处理组作用24h后HL60/Dox细胞形态学的变化：上述浓度的各个处理组作用HL60/Dox细胞24小时后,倒置显微镜下(放大倍数40×10)观察细胞形态学的变化。结果显示阿霉素单药组细胞凋亡特征不明显,DS/Cu单药组可见个别凋亡细胞,而DS/Cu联合阿霉素组出现明显的细胞凋亡(如染色质的凝聚、细胞出现空泡)甚至细胞死亡现象。而加入JNK/c-Jun通路抑制剂Sp600125后,阿霉素联合DS/Cu组细胞凋亡程度较加入Sp600125前受抑。6、Western blotting检测JNK/c-Jun通路蛋白表达变化：上述各个处理组作用HL60/Dox细胞24小时后,Western Bloting结果显示DS/Cu联合阿霉素组HL60/Dox细胞内磷酸化JNK(p-JNK)及其下游分子磷酸化c-Jun (p-c-Jun)和c-Jun表达较其它组显著增加,DS/Cu和阿霉素单药组蛋白分子表达没有明显变化；而加入JNK/c-Jun通路抑制剂SP600125后,Western Bloting显示DS/Cu联合阿霉素组磷酸化JNK(p-JNK)、磷酸化c-Jun (p-c-Jun)和c-Jun表达均较加入抑制剂前受到明显抑制,总JNK蛋白表达无变化。7、Western blotting检测Bcl-2抗凋亡蛋白表达变化：上述浓度的阿霉素、DS／Cu单药组、DS/Cu联合阿霉素组及空白对照组作用24小时后,Western Bloting检测抗凋亡蛋白Bcl-2表达情况,结果显示DS/Cu联合阿霉素组HL60/Dox细胞内Bcl-2蛋白表达较空白对照组受抑,而阿霉素单药组Bcl-2蛋白表达较空白对照组略有增高。结论1、DS/Cu复合物本身对HL60/Dox细胞具有一定的抑制增殖和诱导凋亡作用,无明显抑制增殖作用IC20浓度的DS/Cu可以通过抑制增殖和诱导凋亡提高阿霉素对HL60/Dox细胞的毒性作用。2、DS/Cu复合物联合阿霉素能够增加HL60/Dox细胞内磷酸化JNK(p-JNK)、磷酸化c-Jun (p-c-Jun)和c-Jun表达水平,而加入JNK/c-Jun通路抑制剂SP600125后不仅凋亡细胞比例下降,而且上述蛋白表达较加入抑制剂前均明显受抑,提示活化JNK/c-Jun通路是DS/Cu增强阿霉素对HL60/Dox细胞毒性作用的重要机制之一。3、抑制抗凋亡蛋白Bcl-2表达可能是DS/Cu增强阿霉素对HL60/Dox细胞毒性作用的另外一个重要机制。更多还原

【Abstract】 BackgroundAcute leukemia (AL) is one of the top ten malignant tumors in china and occupyies the highest incidence of malignancy among teenagers and adults younger than 35 years old.With the aggravated environmental pollution in recent years,the incidence of AL increased year by year and has severely threatened people’s health.Acute myeloid leukemia (AML) is the most common type of adult AL.With the improvement of chemotherapy regimens,70% of the AML patients can achieve complete remission(CR).However, relaps would occur in most of the CR patients thereby accompanying with a poorer prognosis.So relaps and resistance to chemotherapy is currently the major obstacles for successful treatment of patients with acute myeloid leukemia.Chemotherapy remains the first treatment choice for AML patients,however, the clinical success of AL chemotherapy has been seriously hindered by de novo and acquired chemoresistance of leukemia cells and the severe non-specific toxicity of high dose chemotherapy to vital tissues and organs.Due partly to the disappointing results correlated with chemoresistance and the non-specific side effects induced by high-dose chemotherapy, current research efforts are aimed at the identification of novel chemosensitizers which would target anti-apoptotic factors and improve therapeutic index of conventional anticancer drugs without further putting on patient’s medication burden.Disulfiram (DS), a member of the dithiocarbamate family capable of binding copper (Cu), is an anti-alcoholism drug used in the clinic with high safety and low toxicity for over 60 years. Various studies have shown that DS have anticancer effects on several types of solid cancers,but it is controversial about the cytotoxicity of DS to leukemia cells. As a divalent metal ion chelator, DS strongly chelates Cu and the formed DS/Cu complex significantly enhanced the cytotoxicity of DS alone to solid cancers and leukemia cells. There exists an coordination effect between DS and Cu in killing cancer cells.c-Jun NH2-terminal kinase (JNK) is a member of the mitogen-activated protein (MAP) kinase family and is activated by a variety of extracellular stimuli through a MAP kinase cascade consisting of JNK kinases (JNKK1/MKK4/SEK1 and JNKK2/MKK7) and multiple MAP kinase kinase kinases. Activated JNK, in turn, phosphorylates and activates c-Jun, a major component of the transcription factor AP-1, as well as other targets, and play a critical role in diverse cellular processes including regulation of cell proliferation, differentiation and apoptosis. Recent studies have demonstrated that JNK inactivation is a possible mechanism of inducing anthracycline resistance in AML. Certain agents have also been shown to induce apoptosis in leukemia cells by activating the JNK pathway, thereby suggesting that JNK inactivation may be another important mechanism correlated with multidrug resistance(MDR) and may be a target for reversal of chemoresistance.Despite DS/Cu was previously reported to sensitize chemotherapeutic agents to several types of solid cancers, the exact targets and molecular mechanism are still unknown.Morever,the effect of DS/Cu to drug resistant leukemia cells has not been literated. In this study, we investigated the in vitro chemosentisizing effect of DS/Cu on the cytotoxicity of doxorubicin(Dox) in HL60/Dox cells. We also examined DS/Cu-mediated changes in the JNK pathways after treatment with Dox.The aims of this project are set as follows.1. To determine the In vitro chemosentisizing effect of DS/Cu on the cytotoxicity of Dox in HL60/Dox cells.2. To investigate DS/Cu-mediated changes in the JNK/c-Jun pathway after treatment in combination with Dox.Methods:1. Cell lines:Dox resistant human acute myeloid leukemia cell line HL60/Dox and the wild-type cell line HL60.2. MTT analysis of cytotoxicity of Dox to HL60 and HL60/Dox cells.3. MTT analysis of cytotoxicity of serial concentrations of DS plus Cu (1μM) to HL60/Dox cells at 24h and 48h.4. MTT analysis of enhancing effect of～IC20 concentration of DS/Cu complex on the cytotoxicity of Dox in HL60/Dox cells at 24h.5. Flow cytometric analysis of the apoptotic HL60/Dox cells after treatment with DS/Cu complex,Dox or DS/Cu plus Dox for 24h.To vertify whether the in vitro chemosentisizing effect of DS/Cu on the cytotoxicity of Dox in HL60/Dox cell line was correlated to JNK/c-Jun pathways,HL60/Dox cells were firstly pretreated with 20μM of JNK/c-Jun pathway inhibitor SP600125 (Sigma, USA) for 2h, and then exposed to various treatments for 24h as described previously for Flow cytometric analysis.6. Morphological observation of HL60/Dox cells after exposure to indicated treatment as described above for 24h.7. Western bloting analysis of JNK,p-JNK,c-Jun,p-c-Jun and Bcl-2 protein expression in HL60/Dox cells after different treatment as described above.8. The statistical analyses were performed with the statistical software package SPSS 13.0. Student’s t-test was used to compare IC50 values of two independent groups,Paried-Samples T Test was used to compare the apoptotic population of HL60/Dox cells between groups with or without Sp600125.One-Way ANOVA was used to compare the difference of apoptotic population of HL60/Dox cells and Bonferroni was used to do multiple comparison when the variance was homogenous,if not,Dunnett’s T3 was employed.A value of *P<0.05 was accepted as an indication of statistical significance. Results represent the mean±SEM of at least three independent experiments.Results1. MTT analysis showed the acquired Dox-resistant cell line (HL60/Dox; IC50=7.69±1.87) was highly resistant to Dox (71.87-fold) compared to the wild-type HL60 cells (IC50=0.107±0.017) (t=7.000,p=0.002)2. The HL60/Dox cells were exposed to serial concentrations of DS in combination with CuCl2 (1μM) for 24 h and 48 h and analysed using the MTT technique. DS/Cu complex demonstrated high toxicity to HL60/Dox cell line with IC50=1.11±0.025μM and 0.439±0.108μM at 24 h and 48 h, respectively.The cytotoxicity of DS/Cu complex in HL60/Dox cell line enhanced accompanying with prolonged time and increased concentration. The IC50 value of DS/Cu at 48 h was significantly lower than that at 24h incubation(t=14.114,p=0.005).3. Furthermore, to examine if DS/Cu complex can enhance the cytotoxicity of Dox in the HL60/Dox cell line, the cells were exposed to increasing concentrations of Dox in combination with the IC20 concentration of DS/Cu complex (0.63μM) in 1μM solution of CuCl2, to simulate the in vivo cell environment, for 24 h. The effect of exposing the HL60/Dox leukaemia cells to various concentrations of Dox in combination with DS/Cu complex is represented on the dose-effect curves.When combined with DS/Cu complex, the cytotoxicity of Dox in HL60/Dox cells (IC50=0.48±0.21μg/ml) was seen to be very significantly enhanced (15.95-fold;t=6.654,p=0.003), compared with Dox alone (IC50=7.69±1.87μg/ml)4. To investigate the effect of apoptosis induction with combination of Dox and DS/Cu complex in HL60/Dox leukaemic cells, the cells were exposed to Dox (1.25μg/ml),DS/Cu(DS:0.63μM, CuCl2:1μM) alone and Dox plus DS/Cu for 24 h. The cells were harvested and their DNA contents stained with PI and Annexin V are analysed by the flow cytometry technique.Treatment of the cell line with DS/Cu or Dox alone showed no significant increase in apoptotic population compared with the control cells(F=92.326, p>0.05). When treated with the combination of Dox and DS/Cu complex, a 11.46-fold increase in the population of apoptotic cells was observed compared with Dox alone.(F=92.326, p<0.05). For pharmacological inhibition study, HL60/Dox cells were pretreated with the JNK inhibitor SP600125 (20μM, sigma, USA) for 2 hour, and subsequently exposed to different treatment for 24 hours for for Flow cytometric analysis.SP600125 obviously decrease DS/Cu + Dox induced apoptosis of HL60/Dox cells from 73.24±10.58% to 40.70±6:03%(t=6.218,p=0.025).5. HL60/Dox Cells treated with DS/Cu or Dox alone did not show any visible apoptosis characters after 24 hours treatment. Whereas, apoptotic morphology appeared under DS/Cu plus Dox treatment. Apoptosis is characterized by marked changes in cellular morphology, including chromatin condensation, membrane blebbing, nuclear breakdown, and the appearance of membrane-associated apoptotic bodies, internucleosomal DNA fragmentation.Notabley,the apoptosis characters of HL60/Dox cell exposed to DS/Cu plus Dox were inbited with addition of JNK inhibitor SP600125.6. Western blot results showed markedly increase in phosphorylation of JNK and c-Jun in HL60/Dox cells treated with a combination of Dox and DS/Cu complex for 24h, while no obvious increase was noticed with Dox alone and DS/Cu complex alone. Although there was no change in the constitutive expression of JNK, expression of c-Jun was also noticeably increased with the combination therapy. As a control, HL60/Dox cells pretreated with 20μM of JNK inhibitor SP600125 for 2h were exposed to various treatments for 24 h and analysed by Western blotting. As expected, SP600125 inhibited the Dox plus DS/Cu complex-induced phosphorylation of JNK, and c-Jun and the increased expression of c-Jun.7. HL60/Dox cells were were exposed to Dox (1.25μg/ml),DS/Cu(DS:0.63μM, CuCl2:1μM) alone and Dox plus DS/Cu for 24 h.Western blotting analysis demonstrated Bcl-2 expression increased in HL60/Dox cells treated with Dox alone and decreased significantly in the presence of DS/Cu complex compared with control cells.Conclution1. DS/Cu alone was cytotoxicity to HL60/Dox cells,and-IC20 concentration of DS/Cu can significantly enhance the cytotoxicity of Dox to HL60/Dox cells by inhibiting proliferation and inducing apoptosis.2. DS/Cu plus Dox can sigificantly induced phosphorylation of JNK, c-Jun and the expression of c-Jun in HL60/Dox cells, while JNK inhibitor Sp600125 not only decreased the apoptotic population of HL60/Dox but also markedly inhitbited the protein expression of JNK/c-Jun pathway,indicating JNK/c-Jun pathway is responsible for the chemosentisizing effect of DS/Cu on the cytotoxicity of Dox in HL60/Dox cells.3. Treatment of the HL60/Dox cells with the combination of Dox and DS/Cu resulted in the inhibition of the constitutive expression of Bcl-2.Inhibition of Bcl-2 activity may be another important mechanism through which DS/Cu can enhance the cytotoxicity of Dox to HL60/Dox cells.更多还原