【作者】 刘坤平；

【导师】 赵彤；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 研究背景与目的结直肠癌是一种常见恶性肿瘤,其发病率呈上升趋势,但治疗效果并未因多年的努力而得到明显改善,其中肿瘤多药耐药是影响肿瘤化疗疗效的一个重要因素。研究表明体内存在的实体肿瘤是一个三维的细胞群集体,其细胞间的相互作用使实体肿瘤耐药机制错综复杂,细胞间信号转导可能参与肿瘤细胞多药耐药的调控,为多药耐药机制的研究带来了新的思路。糖原合成酶激酶-3p(glycogen synthase kinase-3β,GSK-3β)是一种多功能的丝氨酸/苏氨酸激酶,参与细胞增殖、分化和凋亡等多种重要生理过程,已成为备受关注的研究热点。研究表明GSK-3β是细胞内Wnt/β-链接素(β-catenin)、核因子-κB (nuclear factor-κB, NF-κB)及RB/E2F-1等众多信号传导通路的主要调控酶,通过对下游核转录因子的影响参与细胞增殖、凋亡的调控及肿瘤多药耐药的调控,但GSK-3β对肿瘤细胞生物学特性的影响以及GSK-3β能否成为肿瘤治疗的靶点目前仍存有较大争议,而且GSK-3β参与肿瘤多药耐药调控的机制尚不明确。因此,进一步研究GSK-3β在结直肠癌细胞生长及多药耐药机制中的作用具有重要的临床意义。肿瘤细胞多药耐药是指肿瘤细胞在接触某种化学治疗药物后,不仅对该种化疗药物产生耐药,同时对其他结构、功能和作用机制不同的化学药物也产生不同程度交叉耐药的现象。肿瘤多药耐药的形成机制错综复杂,目前比较受关注的耐药机制主要有：(1)调控细胞调亡进程的相关基因或蛋白的改变,导致肿瘤细胞对多种化疗药物诱导细胞凋亡的抵抗或逃避,如β-catenin、P53、Bcl-2等；(2)通过ABC (ATP binding cassette, ABC)跨膜转运蛋白作用增加药物外排,目前发现的ABC转运蛋白家族共有48个基因,与耐药相关的基因有：ABCA2、ABCB1 (P-gp)、ABCC1 (MRP1)、ABCC2(MRP2)、ABCC3、ABCC4、ABCC5、ABCC6、ABCC11、ABCG2,其中ABCB1 (P-gp)及ABCC2 (MRP2)在结直肠组织有较高的表达；(3)激活药物解毒系统,如谷胱甘肽-S-转移酶(glutathione-S-transferase, GST)系统的激活；(4)参与DNA复制、修复的蛋白酶改变,如拓朴异构酶Ⅱ(TOPOⅡ); (5) DNA合成关键酶,如胸苷酸合成酶(thymidylate synthase, TS), TS是DNA合成及代谢过程的关键酶也是结直肠癌基本化疗药物5-Fu的靶酶,TS表达的高低是影响5-Fu化疗敏感性的重要因素；(6)肿瘤干细胞耐药机制,肿瘤干细胞逃避化疗药物及靶向治疗的作用继续生存并可上调ABC转运蛋白的表达。新近在GSK-3β抑制剂与化疗药物联合用药的实验研究中发现GSK-3β抑制剂降低了部分化疗药物诱导细胞凋亡的作用,但机制尚不明确,在结直肠癌GSK-3β抑制剂对基础化疗药物5-Fu诱导的细胞凋亡有何作用尚未知,为了进一步研究GSK-3β对结直肠癌耐药性的影响及其调控机制,我们选择来源于同一结肠腺癌患者的原发灶SW480细胞及淋巴结转移灶SW620细胞作为研究对象,应用一种小分子ATP竞争性GSK-3β抑制剂2’Z,3’E)-6-bromoindirubin-3’-oxime(BIO)作用于结肠癌SW480、SW620细胞,比较BIO作用前后原发及转移二种不同发展阶段的结肠癌细胞多药耐药蛋白P-gp、MRP2、TS及相关调控蛋白β-catenin、Bcl-2、E2F-1蛋白及mRNA含量变化,同期观测BIO对二种肠癌细胞生物学特性及5-Fu诱导细胞凋亡的影响并分析其机制,应用基因芯片及生物信息学分析BIO对二种肠癌细胞多药耐药相关基因表达的影响及调控机制。方法1. GSK-3β抑制剂对原发和转移结肠癌细胞生物学特性的影响应用不同浓度GSK-3β抑制剂BIO作用于结肠癌SW480、SW620细胞,采用倒置显微镜观察BIO作用前后结肠癌SW480、SW620细胞生长情况并检测细胞内ATP浓度；采用流式细胞技术检测细胞周期及凋亡；Western blot检测β-catenin、E2F-1和Bcl-2蛋白表达；免疫细胞化学检测β-catenin、Ki-67及CyclinD1蛋白表达及细胞定位情况；HE染色及电镜观察细胞形态；Tunel染色观察凋亡细胞形态。2. GSK-3β抑制剂对原发和转移结肠癌细胞多药耐药相关蛋白表达的影响采用Western blot检测BIO作用前后结肠癌SW480、SW620细胞P-gp、MRP2、TS蛋白表达；免疫荧光双重标记β-catenin及P-gp、β-catenin及MRP2,共聚焦显微镜观察β-catenin与P-gp、MRP2共定位情况,并进行罗丹明123外排能力检测。3. GSK-3β抑制剂对5-Fu诱导原发和转移结肠癌细胞凋亡的影响采用流式细胞术检测不同浓度5-Fu诱导结肠癌SW480、SW620细胞凋亡情况,筛选适宜的5-Fu药物浓度；再采用流式细胞技术及Tunel染色技术检测5-Fu及5-Fu和BIO联合作用前后结肠癌SW480、SW620细胞凋亡情况。4. GSK-3β抑制剂对原发和转移结肠癌细胞耐药性影响的基因芯片检测及分析采用NimbleGen公司的人类基因组表达谱芯片(NimbleGen Human Gene Expression Microarrays)对BIO作用前后结肠癌SW480、SW620细胞进行基因芯片检测(45,033个基因),进行差异表达基因的筛选,分析多药耐药相关基因表达变化及调控机制。结果1. GSK-3β抑制剂影响原发和转移结肠癌细胞生物学特性GSK-3β抑制剂BIO作用结肠癌SW480、SW620细胞后,细胞内ATP浓度适度升高。在SW480细胞,BIO明显上调β-catenin蛋白表达(F=33.250,P=0.000)、促进β-catenin核移位、下调E2F-1及Bcl-2蛋白表达(F=317.869, P =0.001; F=12.389, P=0.002)、降低细胞凋亡率(F=11.114,P=0.003)、增加Ki-67阳性表达率及S和G2/M期细胞数量,细胞形态呈高增殖状态,Cyclin Dl表达变化不明显。在SW620细胞,BIO明显上调β-catenin及E2F-1蛋白表达(F =19.608 P=0.000; F=22.630, P=0.000)、促进β-catenin核移位、轻度下调Bcl-2蛋白表达、轻度降低细胞凋亡率、增加Ki-67阳性表达率,S和G2/M期细胞数量、细胞形态及Cyclin D1表达变化不明显。2. GSK-3p抑制剂影响原发和转移结肠癌细胞多药耐药相关蛋白的表达GSK-3p抑制剂BIO明显上调结肠癌SW480细胞P-gp、MRP2、TS蛋白表达(F=29.600, P=0.000; F=11.555, P=0.003; F=32.996, P=0.000)、也明显上调SW620细胞P-gp、MRP2、TS蛋白表达(F=26.792, P=0.000; F=17.657, P=0.001; F=92.953, P=0.000),增强SW480和SW620细胞罗丹明123外排能力(P=0.027; P=0.038), P-gp、MRP2与β-catenin有较明显的共定位表达现象。3. GSK-3β抑制剂影响5-Fu诱导的原发和转移结肠癌细胞凋亡筛选出5-Fu作用于结肠癌SW480、SW620细胞的最佳浓度为50μnol/L,在SW480细胞,GSK-3p抑制剂BIO明显降低5-Fu诱导的细胞凋亡(P=0.000),在SW620, GSK-3p抑制剂BIO仅轻度降低5-Fu诱导的细胞凋亡。4. GSK-3β抑制剂对原发和转移结肠癌细胞耐药性影响的基因芯片检测及分析从基因芯片检测结果中筛选出SW620与SW480细胞之间耐药相关差异表达基因有：GSTM1、TOP2A、ABCB7、ABCB7、ABCC2、TOP1、ABCA11 ABCB9。GSK-3β抑制剂BIO作用后,在SW480细胞上调的耐药基因有ABCB1,在SW620细胞上调的耐药基因ABCB1、ABCC2、ABCB10、TAP2、ABCE1。其中ABCB1、ABCC2经Real-time PCR检测验证,GSK-3β抑制剂BIO明显上调SW480细胞ABCB1 mRNA含量(F=6.451、P=0.016),明显上调SW620细胞ABCB1及ABCC2 mRNA含量(F=16.204,P=0.001;F=13.122,P=0.002)。结论1. GSK-3β抑制剂BIO增加了结肠癌SW480和SW620细胞对5-Fu的耐药性,此作用在SW480细胞比SW620细胞明显。2. GSK-3β抑制剂BIO增加结肠癌SW480和SW620细胞耐药性的机制有：(1)凋亡抵抗增强；(2)P-gβ、MRP2转运蛋白增多,功能增强；(3)TS蛋白增多；(4)细胞内ATP含量增多,可能有助于增强ABC转运蛋白的功能。其中凋亡抵抗在SW480高于SW620细胞。3.β-catenin、E2F-1与Bcl-2共同参与了BIO对结肠癌SW480和SW620细胞增殖凋亡及耐药性的调控,β-catenin在GSK-3β调控结肠癌细胞耐药性方面可能起到关键作用,E2F-1在SW480和SW620细胞表达的不同可能是二株细胞耐药性不尽相同的重要原因。4.在SW480和SW620细胞,GSK-3β抑制剂BIO上调P-gp、MRP2转运蛋白与β-catenin上调有关,而TS上调可能通过E2F-1或非E2F-1途径,其机制有待进一步研究。5.结肠癌SW480和SW620细胞之间存在差异表达基因,这些差异是二株细胞对GSK-3β抑制剂及GSK-3β抑制剂联合5-Fu作用产生不同反应的基础。创新点1.证实GSK-3β参与结肠癌细胞耐药性的调控,提出并验证了GSK-3β参与结肠癌细胞耐药性调控的机制,为GSK-3β作为结肠癌耐药靶点的研究提供了重要的理论及实验依据。2.初步验证了β-catenin在结肠癌细胞凋亡及P-gp、MRP2转运蛋白调控中的作用,提出β-catenin在GSK-3β调控结肠癌细胞耐药性方面可能起到关键作用。3.发现GSK-3β抑制剂对原发灶和转移灶结肠癌细胞耐药性及耐药相关调控因子的影响有所不同,尤其是对E2F-1有相反的作用,提示GSK-3β在结肠癌的不同发展时期可能具有不同的功能,为GSK-3β在转移灶癌细胞耐药性方面的研究提供了新的线索。更多还原

【Abstract】 BACKGROUND & OBJECTIVEColorectal cancer (CRC) is a type of common malignant tumor, and it’s incidence rate keeps increasing over the years. The therapeutic efficacy of CRC was not significantly improved in spite of great efferts having been made in many years. One of the main obstacles is the multidrug-resistance (MDR) property of CRC, which leads to the failure of chemotherapy. Researchs show that solid tumor is a three-dimensional cell cluster. The inter-communication among the cells results the complex drug resistance mechanisms. The signal transduction among the cells may directly involve in and contribute to the drug resistance. This may shade some new light on the study of the MDR mechanism.Glycogen synthase kinase 3β(GSK-3β), a multiFunctional serine/threonine kinase which has become a research focus, participates in a variety of important physiological processes, such as intracellular glucose metabolism, cell proliferation, differentiation and apoptosis. Study has shown that GSK-3β, as a major regulating enzyme to Wnt/β- catenin, nuclear factor-KB (NF-κB), RB/E2F-1 and other moleculars in many signaling pathways, participates in regulating cell proliferation, apoptosis and MDR of tumor by influence on downstream nuclear transcription factors. However, the role of GSK-3βand GSK-3βinhibitors, the influence of GSK-3P to the biological properties of tumor cell, and whether GSK-3βis the prime target during the tumor therapy remain controversial. The MDR regulation mechanism of GSK-3βin CRC is not clear. Thus, It’s important to elucidate the role of of GSK-3p and the mechanism of MDR in CRC.MDR refers to the phenomenon that the tumor cells may simultaneously become cross-resistant to a wide variety of chemotherapeutic drug with different structures, Functions and cellular targets, when selected for resistance to a single cytotoxic agent. The mechanism of MDR is very complex. At present, some drug-resistant mechanisms are highly concerned:1) The change of apoptosis regulation gene or protein which results in tumor cells resistance or escape from apoptosis induced by varieties of chemotherapeutic drug, such asβ-catenin, P53, Bcl-2, etc; 2) ATP binding cassette(ABC) transmembrane transporter protein increases drug’s efflux. There are 48 genes have been found in ABC translocator family, the ones which are associated with drug resistance as follow:ABCA2, ABCB1 (P-gp), ABCC1 (MRP1), ABCC2 (MRP2), ABCC3, ABCC4, ABCC5, ABCC6, ABCC11, ABCG2. Among of them, ABCB1 (P-gp) and ABCC2 (MRP2) have higher expression in colorectal tissues; 3) Activating drug’s detoxification systems, such as the activation of glutathione-S-transferase (GST) system; 4) The changing of proteases which participate in DNA replication and repair, such as topoisomeraseⅡ(TOPOⅡ); 5) The changing of the key enzymes of DNA synthesis, such as thymidylate synthase (TS), TS is a key enzyme in the process of DNA synthesis and metabolism and it is a target enzyme of 5-Fu, which is an essential chemotherapy drug for colorectal cancer. The level of TS expression is an important factor in chemosensitivity of 5-Fu; 6) the mechanisms of cancer stem cell resistance to chemotherapy. cancer stem cells could survive cytotoxic or targeted therapies and enhanced ABC transporter protein expression.Recent experimental study on combination therapy of GSK-3βinhibitor with chemotherapy drugs found that GSK-3βinhibitors reduces some cell apoptosis induced by chemotherapy drug, however, the mechanism is still not clear. It has not yet known the effect of GSK-3βinhibitor on apoptosis induced by 5-Fu chemotherapy in colorectal cancer. In order to Further study the effects and regulatory mechanism of GSK-3βin drug resistance of colorectal cancer, the SW480 cells from primary lesion and the SW620 cells from lymph node metastases lesion, which originate from the same patient with colon cancer, were used as the research objects.. A small molecule ATP competitive GSK-3βinhibitor (2’Z,3’E) -6-bromoindirubin-3’-oxime (BIO) was used in colon cancer SW480 and SW620 cells, to compare the variation of multidrug resistence protein P-gp, MRP2, TS and the corresponding regulatory proteinβ-catenin, Bcl-2, E2F-1 in two cells before and after BIO was used. At the same time, the effects and mechanism of BIO on cancer cell’s biological properties and apoptosis induced by 5-Fu were observed. Gene chips, Real-time PCR and Bioinformatics analysis method were applied to Further investigate the affect of BIO on the multidrug resistance-associated gene expression and regulation mechanism in both types of colon cancer cells.METHODS1. The effects of GSK-3βinhibitor BIO on biological properties of primary and metastatic colon cancer cells.The colon cancer SW480 and SW620 cells were treated with BIO in different concentrations. The growth of SW480 and SW620 cells was observed by inverted microscope and intracellular ATP concentration was detected by luminometer. Cell cycle distribution and apoptosis levels were detected by Flow Cytometry and morphology of Apoptosis cell was observated by Tunel staining. The expressions ofβ-catenin, E2F-1 and Bcl-2 protein were detected by Western blot. Theβ-catenin, Ki-67 and Cyclin D1 expression, and cellular localization were detected by Immunocytochemical. Cell morphology of SW480 and SW620 was observated using HE staining and light microscopy and cell ultrastructure was observated by electron microscopy before and after 24h of BIO treatment in different concentration.2. The effects of GSK-3βinhibitor BIO on expression of MDR-associated proteins in primary and metastatic colon cancer cell.The expressions of P-gp, MRP2, TS protein were detected by Western blot in SW480 and SW620 cells before and after 24h of BIO treatment in different concentration. P-catenin and P-gp, P-catenin and MRP2 were stained with double immunofluorescence, and the staining of P-catenin and P-gp, P-catenin and MRP2 were observed by confocal microscope, meanwhile, the ability of rhodamine 123 efflux was detected in SW480 and SW620 cells before and after 24h of BIO treatment.3. The effects of GSK-3P inhibitor BIO on the apoptosis induced by 5-Fu in primary and metastatic colon cancer cellThe Apoptosis induced by 5-Fu with different concentration was detected by flow cytometry in colon cancer SW480 cells and then appropriate 5-Fu concentration was selected. The cell apoptosis induced by 5-Fu and 5-Fu combined with BIO was detected with flow cytometry and Tunel staining in colon cancer cell SW480 and SW620. 4. Gene Chip detection and analysis of the effects of GSK-3βinhibitors BIO on expression of MDR-associated genes in primary and metastatic colon cancer cells Gene expression was detected with NimbleGen Human Gene Expression Microarrays(consist of 45,033 gene) in colon cancer SW480 and SW620 cells before and after BIO treatment. The changing of gene expression was validated by Real-time PCR, and analysed with Bioinformatics.RESULTS1. GSK-3βinhibitor BIO affects the biological characteristics of primary and metastatic colon cancer cellsCompared with those of untreated colon cancer SW480 and SW620 cells, the intracellular ATP concentration is slightly elevated in SW480 and SW620 cells after the GSK-3βinhibitor BIO treatment with different concentration. In SW480 cells, BIO significantly increases the expression ofβ-catenin protein and promotes nuclear translocation ofβ-catenin (F=33.250, P=0.000), and decreases the expressions of E2F-1 and Bcl-2 protein (F=317.869, P=0.001; F=12.389, P=0.002), also decreases apoptosis rate (F=11.114, P=0.003). BIO significantly increases the expression of Ki-67 protein and the cell quantity in S and G2/M phase. The cells showed a high proliferation state, while Cyclin Dl expression change is not obvious. In SW620 cells, BIO significantly increases the expression of P-catenin protein and promotes nuclear translocation of P-catenin protein (F=19.608 P=0.000), increases the expression of E2F-1 protein (F=22.630, P=0.000), slightly decreases the expression of Bcl-2 protein, and moderately decreases the cell apoptosis rate. BIO increases the expression of Ki-67 protein, but the change of cell quantity in S and G2/M phase, cell morphology and Cyclin D1 expression were not obvious. 2. GSK-3βinhibitor BIO affects the expression of MDR-associated protein in primary and metastatic colon cancer cells.GSK-3βinhibitor BIO significantly upregulates the expressions of P-gp, MRP2 and TS protein in SW480 (F=29.600, P=0.000; F=11.555, P=0.003; F=32.996, P=0.000) and SW620 cells (F=26.792, P=0.000; F=17.657, P=0.001; F=92.953, P=0.000), and enhances the efflux ability of of rhodamine 123 in SW480 and SW620 cells (P=0.027; P=0.038). The P-gp, MRP2 andβ-catenin retains obvious co-localization expressing phenomenon in SW480 and SW620 cells.3. GSK-3βinhibitor BIO affects the apoptosis induced by 5-Fu in primary and metastatic colon cancer cellsThe optimum concentration of 5-Fu inducing cell apoptosis in SW480 cells is 50μmol /L. With this concentration, GSK-3βinhibitor BIO reduces apoptosis of SW480 and SW620 which induced by 5-Fu, and this is particularly proninent in SW480 cell (P=0.000)4. Gene Chip detection and analysis of GSK-3P inhibitor BIO affects the expression of MDR-associated genes in primary and metastatic colon cancer cellsThe differential expression genes with drug resistance between SW620 and SW480 cells were as follows:GSTM1, TOP2A, ABCB7, ABCB7, ABCC2, TOP1, ABCA11, ABCB9. The increased expression genes with drug resistance after GSK-3P inhibitor BIO treatment in SW480 cells was as follow:ABCB1; The increased expression genes with drug resistance after GSK-3βinhibitor BIO treatment in SW620 cells were as follows:ABCB1,ABCC2,ABCB10,TAP2,ABCE1. With the detection of Real-time PCR, GSK-3βinhibitor BIO significantly increases mRNA content of ABCB1 in SW480 cells (F=6.45、P=0.016) and increases mRNA content of ABCB1 and ABCC2 in SW620 cells (F=16.204,P=0.001; F=13.122, P=0.002).CONCLUSION1. GSK-3βinhibitor BIO enhance the drug-resistant phenomenon in SW480 and SW620 cells. GSK-3βinhibitor BIO significantly inhibited apoptosis induced by 5-Fu in SW480 cells, whereas the effect was slight in SW620 cells.2. The mechanisms of drug-resistant induced by GSK-3βinhibitor BIO in SW480 and SW620 cells including:(1) The enhancement of apoptosis resistance (2) The enhancement of expression and Function of P-gp and MRP2 transporter protein; (3) The increase of TS protein expression; (4) the increase of intracellular ATP level may enhance the transportion Function of ABC transmembrane transporter protein. The apoptosis resistance induced by GSK-3βinhibitor BIO was obvious in SW480 cells, whereas the effect was slight in SW620 cells.3.β-catenin, E2F-1 and Bcl-2 jointly participate in the cell proliferation, apoptosis and drug-resistant regulation induced by BIO in colon cancer SW480 and SW620 cells, and The P-catenin is probably a key factor in this process. The different expression of E2F-1 in SW480 and SW620 cells is likely one of the mechanisms leading to the different outcome in the two kinds of cells.4. BIO is able to increase the expressions of P-gp, MRP2 transporter protein and TS protein in SW480 and SW620 cells. The increase of P-gp and MRP2 expressions induced by BIO is bound up with the activation ofβ-catenin signal transduction pathways. The Increase of TS is likely related with E2F-1 or non E2F-1 pathways, and the mechanism needs to be made a Further studied.5. There are differences of gene expressions between SW480 cell and SW620 cell, which are the bases to bring different responses to GSK-3βinhibitor BIO and BIO combined with 5-Fu.更多还原