化疗药物诱导的Notch-1及其配体表达上调在人胃癌细胞多药耐药中的作用和机制研究

It　has been suggested that long term adminstration of chemtherapeutic drugs may promote epithelial-mesenchymal transition(EMT) in tumor cells, and the mesenchymal-like cells in tumor are more resistant to chemotherapeutic drugs than those of epithelial-like cells. It is also well-accepted that Notch signal pathway plays as a key regulator in EMT. Our update research data shows that chemotherapeutic drugs up-regulate the expression of Notch-1 and its ligands (Jagged-1 and Jagged-2),promote the epithelial-mesenchymal transition(EMT)in gastric cancer cells. Therefore, we hypothesize that in gastric cancer cells, chemotherapeutic drugs activate the Notch signal and promote EMT through upregulation of Notch-1 and its ligands; the activated Notch then improve the expression and activity of ATP-binding cassette (ABC) transporter through increased expression of EMT associated transcriptional factors, and finally cause the multi-drug resistance and metastasis. The purpose of this work is to comfirm the above hypothesis by means of in vitro and in vivo studies. This work will be very helpful to clarify the potential mechanism of multi-drug resistance to chemotherapeutic drugs in gastric cancer cells.

最新研究显示长期使用化疗药物可以使肿瘤细胞发生上皮细胞间叶样表型转化（EMT），间叶样细胞较之上皮样细胞有更强的化疗抵抗能力。在EMT过程中，Notch信号通路被认为起着重要的调节作用。本课题组的前期研究发现化疗药物可以诱导胃癌细胞Notch-1及其配体Jagged-1,Jagged-2的表达增加，并促使胃癌细胞发生EMT。据此，我们推测化疗药物可能通过激活Notch-1信号通路，上调Notch的下游靶蛋白－EMT相关转录因子的表达和活性、增加三磷酸腺苷结合盒（ATP-binding cassette, ABC）转运体的表达，使胃癌细胞发生EMT、产生多药耐药性并易于转移。本研究拟应用分子和细胞生物学技术在细胞和动物模型中对上述推论加以证实。如能证实上述推论将非常有助于阐明胃癌细胞化疗多药耐药的机制，为克服化疗耐药提供理论依据。

目的：在EMT 过程中，Notch 信号通路被认为起着重要的调节作用。本课题组的前期研究发现化疗药物可以诱导胃癌细胞Notch-1 及其配体Jagged-1,Jagged-2 的表达增加，并促使胃癌细胞发生EMT。观察Notch-1在吉西他滨(Gem)诱导的人胰腺癌PANC-1细胞上皮-间充质转化(EMT)中的作用。并研究Notch1在胃肠道肿瘤侵袭转移机制中的作用。方法：体外培养PANC-1细胞并诱导细胞EMT，Western blot实验和RT-PCR检测Notch-1、E-cadherin、Vimentin、相关转录因子的表达；Transwell小室检测具有EMT改变的PANC-1细胞迁移和侵袭能力。用Nocth1-siRNA转染胃癌、结肠癌细胞，检测它们的侵袭转移能力，并检测成束蛋白（Fascin1）表达情况，并进一步用ChIP实验、EMSA实验证实Notch1调控Fascin1机制。结果：Gem诱导PANC-1细胞EMT最适药物浓度为10 nmol/L，最适时间为9 d。Western blot及RT-PCR检测parental组和Gem(+)组Notch-1、E-cadherin、Vimentin、slug、snail、twist、ZEBl的表达意义(P<0.05)。PANC-1细胞EMT改变后侵袭能力明显增强。在胃癌细胞中Notch1或者Fascin1敲低后均可导致细胞的侵袭能力下降，在结肠癌细胞中Notch1敲低后可导致细胞的侵袭与迁移能力下降，且下降趋势可被Fascin1逆转，且Notch1正向调节Fascin1表达，ChIP和EMSA实验证实了Notch1在转录水平调节Fascin1表达。结论：Gem可诱导PANC-1细胞EMT，Notch-1可能是该过程的重要信号通路。Notch1通过正向调节Fascin1的表达进一步调节胃癌及结肠癌细胞的侵袭与迁移能力，我们的研究结果说明Notch1/RBP-Jk/Fascin1轴可能是胃肠道肿瘤治疗的分子靶点。