Wnt/β-catenin信号通路与胰腺癌吉西他滨耐药关系的初步研究

【作者】 刘伯南；

【导师】 赵玉沛； 张太平； 廖泉；

【作者基本信息】 中国协和医科大学 ， 普通外科， 2010， 博士

【摘要】 1背景和目的胰腺癌是最常见的恶性肿瘤之一,在美国与癌症相关的死亡原因位居第位四。早期胰腺癌患者通常采用手术治疗、辅助化疗或放疗,或联合治疗。手术切除是治疗胰腺癌的唯一机会,但是,只有不到20%的胰腺癌患者适合手术治疗,因为胰腺癌通常在发现时已处于进展期。吉西他滨是目前治疗胰腺癌的一线药物,治疗局部进展期胰腺癌时单药有效率仅为17.3%,无进展生存期为3.7个月；联合奥沙利铂的有效率为26.8%,无进展生存期为5.8个月。吉西他滨耐药可以是原发的或继发的,这是在化疗失败的最可能的原因之一。吉西他滨有一个复杂的代谢途径,很多机制均可以促进吉西他滨的细胞毒性和／或化疗耐药。Wnt信号通路已被报告将在癌症发展和耐药参与了白血病和肝癌。迄今为止,关于与Wnt信号和吉西他滨耐药性的关系研究很少。在此研究的第一和第二部分中,我们研究了典型信号通路之间的Wnt和吉西他滨耐药的相关性。在第三部分,我们使用流式细胞仪检测了胰腺癌细胞株相关干预前后肿瘤干细胞表面标志的比例分布。2主要试剂吉西他滨(200mg/支),Wnt3a和Dkk-1(10gg,R&D)3统计学分析报告基因和MTT结果两两组间比较采用单因素方差,后续检验采用q检验。相关性检验使用Pearson检验。当p<0.05时认为有统计学显著性差异。4实验方法和结果在本研究的第一部分,四株胰腺癌细胞SW1990，PANC-1,T3M4和BxPC-3等用于MTT法检测吉西他滨的耐药性,以半数抑制浓度或IC50表示；使用荧光素酶报告基因检测Wnt信号通路活性。结果显示,SW1990对吉西他滨耐药性最强,伴随着Wnt信号通路异常活化。对这四株细胞进行Pearson相关性检验分析可得经典Wnt信号通路与吉西他滨耐药呈非线性正相关。在本研究的第二部分,我们选择了SW1990为研究对象。分别用Wnt信号通路的激动剂rWnt3a和抑制剂rDkk-1处理SW1990细胞,发现rWnt3a和rDkk-1分别能有效地刺激或抑制Wnt信号通路的活性。rWnt3a对SW1990的生长和吉西他滨耐药都有促进作用,而rDkk-1则对SW1990的生长和耐药均无统计学显著性差异。在本研究的第三部分,我们运用流式细胞仪检测CD133、CD44、CD24和EpCAM在SW1990,PANC-1,T3M4和SW1990/Gem(SW1990耐吉西他滨细胞株)等四株胰腺癌细胞株中的表达。并且检测用吉西他滨、rWnt3a和rDkk-1对SW1990细胞干预后细胞表面CD133的表达。结果显示SW1990和SW1990/Gem中CD133的阳性表达率分别为5.7%和37.8%,SW1990吉西他滨处理三天CD133的阳性表达率为5.6%；PANC-1口T3M4中CD133无表达；CD44+CD24+EpCAM+细胞的比例在各株细胞间差异较大；使用rWnt3a和rDkk.1处理SW1990后,CD133+细胞的比例可分别上升(13.4%)和下降(3.0%)。5结论胰腺癌细胞株之间对吉西他的滨耐药性差异很大,可分为两组：耐药组(SW1990、PANC-1)和敏感组(T3M4、BxPC-3):Wnt信号通路的高水平激活在胰腺癌细胞株中不是普遍现象；胰腺癌细胞株对吉西他滨的耐药性与经典的Wnt信号通路活性之间呈非线性正相关；rWnt3a和rDkk-1可有效地调节Wnt信号通路活性,rWnt3a上调信号可介导细胞对吉西他滨耐药性增强；胰腺癌细胞株中SW1990细胞低表达CD133,PANC-1和T3M4细胞株不表达CD133。更多还原

【Abstract】 BACKGROUNDPancreatic cancer is the fourth most common malignancy and the fourth leading cause of cancer-related death in the United States. Surgical resection offers the only chance of real cure; however, less than 20% of patients with pancreatic cancer are candidates for surgery because the disease is usually detected only in its later stages. Single-agent Gemcitabine is the standard therapy for advanced pancreatic cancer with low response rate of 17.3% and short progression-free survival of 3.7 month. Cellular resistance to gemcitabine can be intrinsic or acquired during gemcitabine treatment, which is the most possible reason for failure in chemotherapy. Gemcitabine has a complex pathway of metabolism, and there are many mechanisms that can contribute to gemcitabine cytotoxicity and/or chemoresistance. Wnt signaling pathway has been reported to be involved in cancer development and chemoresistance in leukemia and hepatocellular carcinoma. To date, there’s few study on the relationship between Wnt signaling and gemcitabine resistance. In PartⅠandⅡof this preliminary study, the relevance between the canonical Wnt signaling pathway and gemcitabine resistance was studied. In partⅢ, by FCM analysis we determined the percentage of CD133+and CD44+CD24+EpCAM+cells in pancreatic cell lines either with or without treatment.CHEMICALSGemcitabine was a commercial prodcut from Eli Lilly Pharmaceuticals. Recombinant Wnt3a and Dkk-1 were both from R & D.STATISTICAL ANALYSESData from reporter and MTT assays was analyzed using one-way ANOVA followed by Newman-Keul’s multiple comparison test. P-values less than or equal to 0.05 were considered significant.METHODS AND RESULTSIn PartⅠof this study, four pancreatic cancer cell lines, SW1990, PANC-1, T3M4 and BxPC-3, were used for assessing the resistance against gemcitabine and the Wnt activity. The relative cytotoxicity of gemcitabine in each cell line was assessed with a MTT assay, in which cells were exposed to increasing concentrations of gemcitabine. Chemosensitivity was expressed as the drug concentration that inhibited cell proliferation by 50%(IC50 values). Reporter assays were used for Wnt activity, in which TOPflash or FOPflash, constructed in Dr. Moon R.T.’s Laboratory, was co-transfected into cells with renilla luciferase as internal control. Western blotting was performed for determining active P-catenin in cells. The results showed that SW1990 had the highest IC50 and Wnt activity, followed by PANC-1, T3M4 and BxPC-3, accordingly. Non-linear positive correlation was determined by Pearson’s test between the canonical Wnt signaling pathway and gemcitabine resistance.Next in Part II, we chose SW1990 for alternating the Wnt signaling pathway activity. rWnt3a was used as agonist and rDkk-1 as antagonist. The results revealed that rWnt3a could enhance the Wnt activity, but rDkk-1 couldn’t. The IC50 increased with significant difference after Wnt3a treatment, while IC50 slightly fell down without significant difference after rDkk-1 treatment, indicating that up-regulation of Wnt signaling could mediate gemcitabine resistance.In Part III, by FCM analysis we determined percentage of CD133+and CD44+CD24+EpCAM+cells in four pancreatic cancer cell lines (SW1990, PANC-1, T3M4 and SW1990/Gem). SW1990/Gem was a SW1990-derived cell line established after exposure to various concentrations of gemcitabine. The results showed that both SW1990 and SW1990/Gem had CD133 expression on cell surface, the proportion of which was 5.7% and 37.8%, respectively. PANC-1 and T3M4 had not any CD133 expression on cell surface. CD44+CD24+EpCAM+proportion was various in different cell lines, ranging from 10.9% to 72.1%. Here, we also detected Cd133 expression in SW1990 with additional temporary treatment by rWnt3a, rDkk-1 or gemcitabine, with CD133+fraction of 13.4%,3.0% and 5.6%, respectively.CONCLUSIONPancreatic cancer cell lines, SW1990, PANC-1, T3M4 and BxPC-3, have various IC50 value against gemcitabine, by which they can be simply divided into two groups: chemoresistant group (SW1990, PANC-1) and chemosensitive group (T3M4, BxPC-3). Activation of Wnt signaling pathway was not common in pancreatic cancer cell lines. Non-linear positive correlation was determined by Pearson’s test between the canonical Wnt signaling pathway and gemcitabine resistance.rWnt3a and rDkk-1 are valid to regulate the Wnt activity. Up-regulation of Wnt signaling by rWnt3a can mediate gemcitabine resistance.There is a small fraction, or even no fraction, of CD133+cells in pancreatic cancer cell lines. CD 133 expression may be associated with gemcitabine resistance and Wnt signaling pathway activity.更多还原