【作者】 高峰；

【导师】 孙开来；

【作者基本信息】 中国医科大学 ， 遗传学， 2018， 博士

【摘要】 目的:肿瘤化疗失败的主要原因是肿瘤细胞多药耐药性（multidrug resistance,MDR）的产生,即肿瘤细胞一旦对某种药物产生耐受,对其它结构各异、作用机制不同的化疗药物也产生交叉耐受,从而使肿瘤治疗的效果得不到显著提高,这一现象给临床治疗带来极大的障碍。MDR有多种机制,P糖蛋白（P-glycoprotein,P-gp）过表达是肿瘤细胞多药耐药的主要原因。P-gp由MDR1基因编码,又称ABCB1,是ATP结合盒（ATP-binding cassette,ABC）转运输蛋白家族成员之一,可通过ATP依赖的药物运输泵机制将细胞毒性试剂从细胞内运送到细胞膜外。白血病治疗主要依靠联合化疗和骨髓移植,临床上成年急性髓性白血病（acute myeloid leukemia,AML）患者中60-80%常规化疗后可以完全缓解,随着年龄增长复发/难治患者比例明显升高,且有研究表明复发难治患者白血病细胞P-gp表达明显高于治疗敏感组。P–gp在CD34⁺干细胞和恶性白血病细胞中表达,随着白血病细胞的分化,P–gp表达呈下降趋势,但CD34⁺表达与白血病细胞P-gp表达及功能的相关性还不十分清楚。P-gp表达被认为是临床急性髓性白血病不良预后的独立因素,且已经开展应用P-gp抑制剂逆转耐药的辅助治疗,但效果不佳。三氧化二砷（As₂O₃）是具有2000多年历史的传统中药,1985年被用于急性早幼粒细胞白血病（acute promyelocytic leukemia,APL）治疗,目前As₂O₃在APL和一些实体瘤的治疗上都取得了一定的疗效。近来发现砷剂有抑制P-gp表达的作用,但目前关于三氧化二砷逆转多药耐药的作用机制仍不清楚。研究发现各种细胞压力如DNA损伤、血清饥饿等都可使MDR1发生活化,MDR1基因上游启动子区缺乏一般编码蛋白基因具有的TATA盒,而是具有与NF-Y和Sp家族转录因子结合的反向的CCAAT和GC富集区,这些转录因子蛋白可募集组蛋白乙酰转移酶启动周围组蛋白乙酰化,使染色质重塑,从而激活MDR1启动子,有研究报道表皮生长因子（EGF）、肿瘤坏死因子TNF-α、阿霉素、核因子κB（NF-κB）等各种活化信号都可刺激MDR1表达。Mathas等研究表明NF-κB除了可诱导抗凋亡基因表达而产生抗凋亡作用以外,还可直接诱导P-gp表达。Bentires-Alj等证实MDR1基因启动子区域的第一内含子内存在一个NF-κB结合序列,并且NF-κB能够激活连接MDR1启动子的报告基因转录,这提示As₂O₃可能通过抑制NF-κB来诱导细胞凋亡和抑制MDR1的表达,从而产生逆转耐药的作用。因此,本研究拟通过对AML患者白血病细胞P-gp表达、功能及其与CD34表达相关关系的分析和通过体内、体外实验检测As₂O₃对P-gp表达的影响,以及As₂O₃是否通过NF-κB途径影响MDR1启动子活性等进行分析,探讨As₂O₃逆转AML白血病细胞耐药机制,为AML临床化疗方案的制定提供依据。方法:本研究以85例临床AML患者、白血病药物敏感细胞系K562/S和耐药细胞系K562/D为研究对象,用免疫细胞化学方法检测AML患者白血病细胞P-gp表达和CD34表达,用流式细胞术检测P-gp功能,分析了CD34表达、P-gp表达及功能与临床急性髓性白血病病人耐药的相关关系;为证明As₂O₃是否具有逆转P-gp表达及耐药功能的作用,分别采用对P-gp表达阳性的3例复发/难治患者使用As₂O₃进行药物治疗,As₂O₃10mg每日一次静脉滴注,持续治疗4天;分离9例P-gp表达阳性患者外周血单个核细胞进行体外培养,用终浓度为1μM的As₂O₃处理,用Real-time PCR方法检测用药前后原代培养的急性髓性白血病细胞和K562/D细胞的MDR1表达变化;K 562/D细胞以相同方式进行药物处理,用流式细胞术检测As₂O₃作用前后K562/D细胞总P-gp表达及药物释放功能的变化;为进一步分析As₂O₃逆转耐药的机制,用EMSA方法体外鉴定MDR1启动子区NF-κB反应元件,并通过Western印迹杂交实验检测As₂O₃对K562/D细胞NF-κB亚基p65、抑制因子IκB和磷酸化的IκB表达水平的影响;并通过构建MDR1启动子萤光素酶报告系统分析了NF-κB刺激因子TNF–α对MDR1启动子活性的调节作用和As₂O₃对TNF–α诱导的MDR1启动子活性的调节作用,并进一步构建野生型、突变型IκB表达载体和p65siRNA,通过萤光素酶报告基因检测系统鉴定NF-κB反应元件在As₂O₃对MDR1启动子活性调节中的作用。结果:免疫细胞化学实验显示63例初治组中23例P-gp⁺_表达,占36.5%;22例复发/难治组中11例P-gp⁺_表达,占50.0%,两组无显著差异（P>0.05）。63例初治组中P-gp⁺_功能18例,占28.6%,22例复发/难治组中P-gp⁺_功能13例,占59.1%,复发/难治组高于初治组（P<0.05）;63例初治组中20例CD34⁺,占31.7%;22例复发/难治组中13例CD34⁺,占59.1%,高于初治组（P<0.05）。CD34表达与P-gp表达呈正相关（P=0.0001.r=0.483）;CD34表达与P-gp功能也呈正相关（P=0.0001,r=0.550）;3、63例初治AML患者按年龄分为<50岁和≥50岁的年龄分组,46例<50岁初治组中8例CD34⁺（17.4%）,12例P-gp⁺_表达（26.1%）,9例P-gp⁺_功能（19.6%）;17例（≥50岁）初治组中11例CD34⁺（64.7%）,11例P-gp⁺_表达（64.7%）,9例P-gp⁺_功能（52.9%）,≥50岁组均高于<50岁组（P<0.05）;Real-time RT-PCR结果显示体内和原代培养急性髓性白血病细胞和耐药细胞系经As₂O₃作用后总MDR1表达下调;流式细胞术检测K562/D细胞经As₂O₃作用后P-gp表达及药物释放功能明显下降;EMSA实验在体外证明了MDR1启动子区+561⁺571为NF-κB反应元件;Western印迹杂交结果显示As₂O₃使K562/D细胞IκB降解减少,磷酸化IκB水平降低,核NF-κB p65减少;应用萤光素酶检测系统证明了TNF–α对MDR1启动子有正调控作用,As₂O₃可抑制TNF–α诱导的MDR1启动子活性;萤光素酶报告基因系统证明细胞转染野生型和突变型IκB表达载体后As₂O₃通过NF-κB反应元件对TNF–α诱导的MDR1启动子活性的抑制减弱,后者比前者减弱程度小;萤光素酶报告基因检测系统证明细胞转染p65siRNA后As₂O₃通过NF-κB反应元件对TNF–α诱导的MDR1启动子活性抑制明显减弱。结论:1、MDR1高表达是临床急性髓性白血病病人耐药的主要原因。2、As₂O₃可使K562/D细胞和临床急性髓性白血病细胞MDR1表达下调。3、As₂O₃可通过NF-κB通路逆转P-gp调节的白血病细胞耐药,MDR1基因上游存在一段NF-κB特异结合序列,NF-κB可通过与该序列结合调节MDR1基因表达,As₂O₃可能通过抑制磷酸化酶活性来抑制IκB磷酸化,进而抑制NF-κB活性,抑制MDR1基因表达,逆转白血病细胞耐药。更多还原

【Abstract】 Objective:The main cause for the failure of clinical cancer chemotherapy is the formation of multidrug resistance（MDR）of tumor cells,which means tumor cells once is exposed to a cytotoxic agent they will develop cross-resistance to a range of structurally and functionally unrelated compounds.There is a variety of mechanisms on MDR in resistant cell and overexpression of P-glycoprotein（P-gp）is one of the main mechanisms involved in the development of MDR.P-gp is encoded by the MDR1 gene,also referred to as ABCB1,which is one of ATP-binding cassette（ABC）transporters family.ABCB1efficiently removes cytotoxic drugs from the intracellular environment by drug-efflux pump through an ATP-dependent mechanism.Therefore it is possible to counteract some cases of MDR by inhibiting P-gp in cancer.Leukemia treatment depends mainly on the chemotherapy and bone marrow transplantation.Clinically,60-80%of acute myeloid leukemia（AML）patientsexperiencecompleteremissionfollowingroutine chemotherapy treatment,however,numerous patients relapse.Tumor cell resistance to chemotherapy is therefore a more common phenomena in leukemia treatment.P-gp expression was significantly higher in leukemia cells of relapsed or refractory patients than that in treatment-sensitive patients of AML.The P-gp overexpression is also considered as the main reason on acute myeloid leukemia cell to MDR.It is reported that the proportion of relapsed/refractory patients increases with age.During the differentiation of leukemia cells,CD34 is expressed in AML malignant cells,while the expression P-gp is decreased.Whether CD34 levels correlate with the expression or function of P-gp remains unclear.The competition inhibitors to P-gp has been used as the adjuvant treatment for clinical AML to reversal drug resistance,but the results are not very good.Arsenic trioxide（As₂O₃）has been used over the last centuries as a form of traditional herbal medicine.applied to the treatment of acute promyelocytic leukemia（APL）and some solid tumor,the treatment has achieved some effect.Recently it was found that arsenic trioxide inhibits P-gp expression of leukemia cells.Up to date,the mechanism on which As₂O₃ reverses P-gp-mediated MDR is not clear.The upstream region of the MDR1 gene promoter lacks the TATA box commonly found in many protein-coding genes;however,it does contain a reverse CCAAT and GC-rich region that can bind with NF-Y and Sp family transcription factors,which recruit histone acetyltransferase to initiate histone acetylation,chromatin remodeling,thus,activating the MDR1 promoter.Induction of apoptosis is considered to be the mechanism by which As₂O₃ acts as a chemotherapeutic agent.Mathas et al found that As₂O₃ inhibits the activity of nuclear factor-κB（NF-κB）and induces apoptosis.NF-κB also induces the expression of P-gp in addition to inducing the expression of antiapoptotic genes.Bentires-Alj et al demonstrated that the promoter of MDR1contained an NF-κB binding sequence that activates transcription of an MDR1promoter-driven reporter gene.These findings support the notion that As₂O₃ has an effect on the reversal of drug resistance by inhibition of NF-κB,consequently inducing apoptosis and expression of MDR1.In this research,we used As₂O₃ as a treatment of reversal MDR and studied the effect of As₂O₃ on P-gp expression in human leukemia cells.We identified that the influence of NF-κB signal passway on P-gp promoter activity may be one of the mechanisms by which As₂O₃ regulates P-gp expression.Furthermore,we probed the role of As₂O₃ in the regulation of P-gp by NF-κB.Methods:In this research,85 cases of acute myeloid leukemia,human leukemia cell lines,sensitive cell line K562/S and the resistant cell line K562/D were used as research objects.Immuno cell chemistry was used to test P-gp and CD₃₄4 expression in leukemic cells from peripheral blood or bone marrow of leukemia patients.Flow cytometry was performed to test P-gp function.Real-time reverse transcription-polymerase chain reaction（RT-PCR）was used to test the changes of MDR1 expression leval in P-gp expression positive leukemic cells of 3 relapsed/refractory patiants and primary cultured cells of 9 AML patiants and drug resistant cell line K562/D treated with As₂O₃.Flow cytometry was used to test P-gp expression and functional changes in K562/D cells treated with As₂O₃.EMSA was carried out to identify the NF-κB responsive elements within MDR1 promoter.Western blot was performed to test the influence of As₂O₃ on NF-κB p65 subunit,IκB and phosphorylated IκB expression levels.MDR1promoter luciferase reporter gene was constructed.Luciferase assay was performed to detect the role of NF-κB element in the regulation of TNF-αinduced MDR1promoter activity by As₂O₃.After cells were transfected with wild-type,mutant construct of IκB expression plasmids and p65siRNA,luciferase assay were carried out to analyze the role of NF-κB element in the regulation of TNF-αinduced MDR1promoter activity by As₂O₃.Results:1.Immunocytochemistry results showed 36.5%and 50.0%P-gp expressions in63 patients with newly treatment group and in 22 patients with relapsed/refractory group,respectively.No significant difference（P>0.05）between the two groups was found.P-gp function positive was 28.6%and 59.1%in the two groups,respectively,and there was a significant difference between the two groups（P<0.05）.P-gp expression level and function were positive correlated（P=0.0001,r=0.579）.2.Immunocytochemistry results showed 31.7%and 59.1%CD₃₄4 expressions in 63 patients with newly diagnosed group and in 22 patients with relapsed/refractory group,respectivelly（P<0.05）.CD₃₄4 expression and P-gp expression levels were positively correlated（P=0.0001.r=0.483）;CD₃₄4 expression levels and P-gp function were positively correlated（P=0.0001,r=0.550）.3.In the 46 cases（<50 years）of initial treatment group,17.4%CD₃₄4 expression was positive,26.1%P-gp expression positive and 19.6%P-gp function positive;In the 17 patients（≥50 years）of initial treatment group,64.7%CD₃₄4 was positive,64.7%P-gp positive,52.9%function P-gp positive,which were higher than those in<50 years group（P<0.05）.4.Real-time RT-PCR results showed that As₂O₃ reduced overall MDR1 expression in leukemia cells in vivo,primary cultured acute myeloid leukemia cells and K562/D cells in vitro.5.Results from flow cytometry assy showed that As₂O₃ reduced P-gp expression and function of drug release in K562/D cells.6.EMSA result demonstrated that the position+561⁺571 of the MDR1 promoter was an NF-κB responsive element.7.Western blot results showed that As₂O₃ decreased IκB degradation,down-regulated phosphorylated IκB levels and reduced nuclear NF-κB p65 protein in K562/D cells.8.Results from luciferase assay showed that TNF-αpositively regulated the activity of MDR1 promoter and As₂O₃inhibited TNF-α-induced activity of MDR1 promoter.9.Results from luciferase assay showed that the activity of MDR1 promoter inhibited by As₂O₃,which was regulated by NF-κB response element and cited by TNF-α,significantly decreased after cell transfected with wild-type and mutant IκB expression plasmids.The decreased activity of the former was less than that transfed with mutant IκB plasmid.10.Luciferase assy resluts showed the activity of MDR1 promoter inhibited by As₂O₃ which was regulated by NF-κB response element and cited by TNF-α.was significantly decreased after cell transfection with p65siRNA.Conclusions:1.P-gp overexpression is the main reason in clinical drug resistance in acute myeloid leukemia patients.2.As₂O₃ inhibites MDR1 expression in K562/D cells and clinical acute myeloid leukemia cells.3.As₂O₃ reverses P-gp-mediated leukemia cell resistance thorough NF-κB pathway.The position+561⁺571 of the MDR1 promoter was an NF-κB responsive element.As₂O₃ inhibits IκB phosphorylation probably through inhibiting the phosphorylase activity,resulting in the inhibition of NF-κB activity and MDR1 gene expression leading to the reversal of the drug resistance in leukemia cells.更多还原