【作者】 吴焕文；

【导师】 刘彤华； 梁智勇；

【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 虽然过去数十年来胰腺导管腺癌(Pancreatic ductal adenocarcinoma,PDAC)的基础与临床研究取得了丰硕的成果,但是胰腺癌治疗仍是世界性的医学难题。胰腺癌对放化疗高度耐药是其预后不佳的主要原因之一。大量临床研究表明即使采用健择(gemcitabine, Gem)与其它化疗药物联用的各种治疗方案也不大可能使得胰腺癌的预后得到明显改善,而且还往往伴随着更大的毒性作用。因此,新的治疗策略的探索就显得非常的必要,靶向性治疗与传统化疗的联合应用可能是有效且可行的解决方案。对于胰腺癌来说,开发出与Gem联合应用的靶向药物是目前研究的重点与热点。作为一种非受体酪氨酸激酶,粘着斑激酶(focal adhesion kinases, FAK)与肿瘤发生发展及放化疗耐药密切相关。作为细胞与细胞外基质相互作用的关键性介导因子,在肿瘤细胞中,FAK还可能参与细胞粘附介导的耐药(celladhesion-mediated drug resistance, CAM-DR)肿瘤中FAK功能调节的一种主要模式为磷酸化,FAK磷酸化与CAM-DR及胰腺癌耐药的关系及机制仍不明确。在本研究中,我们首先探讨了胰腺癌细胞系中组成性及层粘连蛋白(laminin, LN)诱导的FAK磷酸化水平与Gem耐药程度间的相关性,进而我们构建FAK特异性RNA干扰(RNA interference, RNAi)载体及FAK显性负变异体粘着斑激酶相关非激酶(FAK-related-non-kinase, FRNK)表达载体(前者抑制FAK表达和磷酸化,后者仅抑制FAK磷酸化而不影响表达),筛选稳转胰腺癌细胞系,检测这两类载体对Gem诱发的细胞毒及凋亡作用的影响并进行初步的机制探讨,试图寻找到能与Gem有效联合治疗胰腺癌的靶点。本研究主要进行了以下几方面的工作：组成性的FAK酪氨酸397(Tyrosine397,Y397)位点磷酸化水平与胰腺癌细胞系对Gem的内在性耐药程度有关1.检测四种胰腺癌细胞系(BxPC-3、AsPC-1、MiaPaCa-2与Panc-1)中组成性FAK表达与Y397位点磷酸化水平；2.检测上述四种胰腺癌细胞系对Gem及5-氟尿嘧啶(5-fluorouracil,5-FU)的耐药程度,计算出半数抑制浓度(50% inhibitory concentration, IC50)值；3. Spearman相关分析组成性的FAK蛋白或磷酸化水平与胰腺癌细胞系对Gem或5-FU的内在性耐药程度之间的相关性。二、构建FAK干扰及FRNK表达重组质粒载体,建立稳转细胞系1.根据invitrogen公司提供的网上设计工具,设计了两对针对FAK的干扰序列,连接进入干扰载体(FAK RNAil,FAK RNAi2)后,测序鉴定。2.以pRKvsv-FRNK为模板,经高保真PCR扩增出FRNK cDNA序列,连接进入pcDNA3.1载体后,测序鉴定。3.选择组成性pFAK(pY397)水平最高的Panc-1细胞系及组成性pFAK(pY397)水平最低的AsPC-1细胞系,用构建好的载体瞬时转染这两种细胞系,鉴定所构建载体的干扰或表达效果,进而筛选出稳定转染细胞系应用进一步的研究。三、重组质粒FAK RNAi2和pcDNA3.1-FRNK对Panc-1细胞中FAK及其下游激酶Akt与ERK1／2表达与磷酸化的影响1.重组质粒FAK RNAi2可以有效地降低胰腺癌细胞系Panc-1细胞中FAKmRNA和蛋白的水平,抑制FAK Tyr397位点及Akt磷酸化。2.重组质粒pcDNA3.1-FRNK可以有效抑制FAK Tyr397位点及Akt磷酸化,但不影响FAK的表达水平。四、FAK RNAi2和pcDNA3.1-FRNK均可以降低Panc-1对Gem的内在性耐药1. MTT实验检测细胞毒性,结果显示,相对于未转染组和对照载体转染组,在重组质粒FAK RNAi2和pcDNA3.1-FRNK稳转细胞系中,Gem的细胞毒性均显著增强,差异具有统计学意义。2.克隆形成结果显示,相对于未转染组和对照载体转染组, FAK RNAi2和pcDNA3.1-FRNK稳转组细胞Gem处理后的克隆形成能力均显著下降,差异具有统计学意义。3. Heochst方法检测细胞凋亡时细胞核形态的改变,结果显示,相对于未转染组和对照载体转染组,FAK RNAi2和pcDNA3.1-FRNK稳转组中Gem诱导的细胞凋亡均显著增多。4. Annexin V-FITC/PI法检测细胞的早期与晚期凋亡。结果显示,FAK RNAi2和pcDNA3.1-FRNK稳转组中Gem诱导的凋亡细胞比例均显著增加,与未转染组和对照载体转染组相比具有统计学差异。5. Western blot检测细胞内caspase-3的剪切状态。结果显示,相对于未转染组和对照载体转染组,FAK RNAi2和pcDNA3.1-FRNK稳转组细胞Gem处理后的caspase-3剪切片段均显著增加。6. Western blot检测细胞内凋亡相关蛋白的表达与磷酸化水平。结果显示,相对于未转染组和对照载体转染组,pcDNA3.1-FRNK稳转细胞系的细胞凋亡抑制蛋白survivin含量下降,pBAD(pS136)水平下降。五、LN对Gem诱导的AsPC-1细胞毒性与凋亡效应影响及LN对凋亡相关蛋白表达的影响1.MTT、实验检测细胞毒性,结果显示,相对于plastic组,在LN作用的AsPC-1细胞中,Gem的细胞毒性显著降低。2.克隆形成结果显示, LN组AsPC-1细胞在接受Gem处理用后的克隆形成能力明显增强,差异具有统计学意义。3. Heochst方法检测细胞凋亡时细胞核形态的改变,结果显示,LN组AsPC-1细胞中,Gem诱导的细胞凋亡显著降低。4. Annexin V-FITC/PI法检测细胞的早期与晚期凋亡。结果显示,LN作用的AsPC-1细胞中,Gem诱导的凋亡细胞比例显著降低,与plastic组相比具有统计学差异。5. Western blot检测细胞内caspase-3的活化情况。结果显示,LN组AsPC-1细胞在接受Gem处理后活化的caspase-3显著增加。6. Western blot检测细胞内凋亡相关蛋白的表达与磷酸化水平。结果显示,相对于plastic组, LN组AsPC-1细胞的survivin蛋白表达及pBAD(ps136)蛋白水平升高。六、LN对AsPC-1细胞FAK及其下游激酶的表达及磷酸化水平的影响1.LN能够时间依赖性上调AsPC-1细胞内FAK Tyr397位点的磷酸化水平,而不影响FAK表达。2.LN能够时间依赖性上调AsPC-1细胞内Akt的磷酸化水平,但是其对ERKl／2磷酸化水平及Akt、ERKl／2表达水平均无明显影响。七、重组质粒载体对LN诱导的AsPC-1细胞FAK及Akt表达及磷酸化水平的影响1.重组质粒FAK RNAi2可以有效减少胰腺癌细胞系AsPC-1中FAK mRNA和蛋白的水平,抑制LN诱导的FAK Tyr397位点及Akt磷酸化。2.pcDNA3.1-FRNK可以有效抑制LN诱导的FAK Tyr397位点及Akt磷酸化,但不影响FAK的表达水平。八、FAK RNAi2与pcDNA3.1-FRNK重组质粒对LN介导的AsPC-1细胞对Gem内诱导细胞凋亡能力的影响1. Heochst方法检测细胞凋亡时细胞核形态的改变,结果显示,LN作用时,相对于未转染组和对照载体转染组,FAK RNAi2与pcDNA3.1-FRNK稳转AsPC-1细胞系中Gem诱导的细胞凋亡均显著增多。2. Annexin V-FITC/PI法检测细胞的早期与晚期凋亡。结果显示,LN作用时,FAK RNAi2和pcDNA3.1-FRNK稳转细胞系中Gem诱导的凋亡细胞比例均显著增加,与未转染组和对照载体转染组相比具有统计学差异。而在无LN作用时,与未转染组和对照载体转染组相比,FAK RNAi2和pcDNA3.1-FRNK稳转细胞系中Gem诱导的凋亡细胞比例无显著改变。3. Western blot检测细胞内caspase-3的活化状态。结果显示,LN作用时,相对于未转染组和对照载体转染组,FAK RNAi2和pcDNA3.1-FRNK稳转细胞系在接受Gem处理后活化的caspase-3均显著增加。4. Western blot检测细胞内凋亡相关蛋白的表达与磷酸化水平。结果显示,LN作用时,相对于未转染组和对照载体转染组,pcDNA3.1-FRNK稳转细胞系的细胞凋亡抑制蛋白survivin含量下降,pBAD(pS136)水平下降。总之,本研究认为,组成性及LN诱导的FAK磷酸化参与介导了胰腺癌细胞系对Gem的内在性耐药；其机制与Akt通路活化及Bad Ser-136位点磷酸化与survivin表达水平改变有关。这些结果提示,FAK可能成为胰腺癌治疗的一个重要靶点,能够增加胰腺癌对Gem的敏感性；pFAK(pY397)是胰腺癌内在性耐药的重要介导因子,可能成为预测与Gem联用的FAK靶向治疗疗效的重要指标。更多还原

【Abstract】 Treatment of pancreatic cancer remains to be an unsolved problem. One of the major reasons for poor prognosis of pancreatic cancer is its highly resistance to currently available agents including gemcitabine which is considered as the most effective first-line drug for the treatment of advanced pancreatic cancer. Gemcitabine alone and gemcitabine-based combination chemotherapy are likely to be accompanied by much higher toxicity and not likely to achieve great success according to clinical trials.Target therapy is emerging as a new era for tumor therapy. As for pancreatic cancer, a combination of conventional chemotherapies with target therapy targeted at the molecular changes in pancreatic cancer seems to be the most promising way to go. Links between tyrosine kinases and pancreatic cancer chemoresistance have attracted more and more attention in recent years, and target therapy agaist tyrosine kinases increases the effectiveness of radiation and cytotoxic drugs in the treatment of pancreatic cancer.In recent years, non-receptor tyrosine kinase focal adhesion kinase (FAK), a central molecule in extracellular matrix (ECM)/integrin-mediated signaling, has been accepted as a determinant of chemoresistance and ECM proteins including laminin (LN) have also been thought to be associated with the intrinsic chemoresistance to different drugs in a viariety of cancers, which is named as cell adhesion-mediated drug resistance (CAM-DR). Besides the regulation of FAK expression, another well-understood mode of FAK regulation in cancer cells is phosphorylation.To determine the roles of FAK and LN in the intrinsic chemoresistance of pancreatic cancer and whether these effects are mediated by constitutive and induced FAK phosphorylation and subsequent downstream signal pathway, the following aspects were studied:1. The relationship between constitutive FAK expression or phosphorylation level and the intrinsic resistance to gemcitabine (Gem) or 5-fluorouracil (5-FU) in four pancreatic cancer cell lines(BxPC-3, AsPC-1, MiaPaCa-2 and Panc-1).2. Construction of recombinant plasmids, including FAK RNAi plasmid (FAK RNAi1 and FAK RNAi2), and FRNK expression plasmid (pcDNA3.1-FRNK) Selection of stable clones.3. Effect of FAK RNAi and FRNK overexpression on the expression and phosphorylation of FAK and its downstream signaling kinases (Akt and ERK1/2) in Panc-1 cells.4. Effects of FAK RNAi and FRNK overexpression on Gem-induced cytotoxicity and apoptosis and apoptosis associated proteins in Panc-1 cells5. Effect of LN on FAK phosphorylation and its downstream signaling kinases (Akt and ERK1/2) activity in AsPC-1 cells6. Effect of FAK RNAi and FRNK overexpression on the expression and phosphorylation of FAK and its downstream signaling kinase Akt in AsPC-1 cells.7. Effect of LN on Gem-induced cytotoxicity and apoptosis and apoptosis associated proteins expression in AsPC-1 cells.8. Effects of FAK RNAi and FRNK overexpression on LN mediated apoptosis resistance to gemcitabine in AsPC-1 cells.The results showed that:1. In pancreatic cancer cell lines (BxPC-3, AsPC-1, MiaPaCa-2 and Panc-1), the level of constitutive phosphorylation of FAK at Tyr-397 is correlated with the extent of intrinsic resistance to Gem.2. In Panc-1 cells, FAK RNAi inhibits FAK both at mRNA and protein levels, and suppresses constitutive FAK and Akt phosphorylation, while FRNK overexpression suppresses constitutive FAK and Akt phosphorylation without total FAK expression alteration. 3. In Panc-1 cells, specific inhibition of constitutive FAK phosphorylation by either FAK RNAi or FRNK overexpression renders Panc-1 cells more sensitive to Gem-induced cytotoxicity and apoptosis and causes a decrease in the expression of survivin and the phosphorylation of Bad at serl36.4. In AsPC-1 cells. LN reduces Gem-induced cytotoxicity and apoptosis, and increases the expression of survivin and the phosphorylation of Bad at serl36.5. In AsPC-1 cells. LN induces FAK and Akt activation in a time-dependent manner.6. In AsPC-1 cells, specific inhibition of constitutive FAK phosphorylation by either FAK RNAi or FRNK overexpression leads to the inhibition of FAK and Akt activation stimulated by LN.7. In AsPC-1 cells, when plated on plastic, FAK RNAi or FRNK overexpression has few effects on Gem-induced cytotoxicity and apoptosis; However, FAK RNAi or FRNK overexpression enhances Gem-induced cytotoxicity and apoptosis in AsPC-1 cells plated on LN.Conclusion:In conclusion, our research demonstrates that both constitutive and LN-induced phosphorylation of FAK contributes to increased intrinsic chemoresistance to Gem in pancreatic cancer cell lines. FAK RNAi and FRNK overexpression both can render pancreatic cancer cells more sensitive to Gem-induced cytotoxicity and apoptosis. These effects are partly due to the regulation of Akt and Bad phosphorylation and survivm expression.更多还原