顺铂诱导的CNTN-1表达增加在人小细胞肺癌获得性多药耐药中的作用机制研究

In our previous studies,CNTN-1 has been found to be associated with acquired MDR of SCLC induced by DDP,but the mechanism is still unclear.Based on the present study outcomings,we deduced that CNTN-1,the potential downstream effector of FGF10,might play important roles in SCLC MDR by reducing the cell permeability which causes the decreased intake and the decreased concentration of chemotherapeutic drug in cells with the interaction of PTPRD,CTNNAl and other proteins.Therefore, in this proposal,siRNA,Western Blot,Q-PCR,and other methods will be utilized in three different SCLC MDR cells induced by DDP,their progenitor cells and carcinoma xnograft in SCID mice to discover the mechanisms of CNTN-1 involvement in the acquired MDR of SCLC. Additionally,the interacting proteins and regulation mechanism of CNTN-1 in the MDR development of SCLC will be investigated by siRNA,FLAG pull-down,Western Blot,and other methods.Furthermore,Q-PCR and immuno histochemical staining will be used to determine the expression level of CNTN-1,PTPRD, and other interacting-genes in the specimens of SCLC patients to evaluate the relationship between the expression of these genes and the curative effect of chemotherapeutic drugs, and the prognosis of SCLC patients. The successful conduction of the project will not only help us further understand the mechanisms of SCLC acquired MDR to provide novel targets to explore new chemotherapeutic or molecular target drugs for SCLC, but also provide potential biomarkers to predict the curative effect of chemotherapeutic drugs and the prognosis of SCLC patients.

我们前期研究发现，CNTN-1是顺铂诱导的SCLC获得性MDR相关基因，但其作用机制尚不清楚。现有的研究结果提示，作为FGF10潜在的下游效应分子，CNTN-1可能和PTPRD、CTNNAL1等蛋白相互作用，通过降低细胞通透性导致药物摄取减少，从而参与SCLC获得性MDR。基于此，本课题拟以3株SCLC获得性MDR细胞、亲代细胞及其移植瘤SCID小鼠为研究对象，一方面在沉默或上调CNTN-1表达的情况下，检测细胞通透性、胞内顺铂浓度、细胞凋亡等，探讨CNTN-1在SCLC获得性MDR中的作用机制；另一方面拟采用siRNA和FLAG pull-down等探讨CNTN-1相互作用蛋白及调控机制，为研发新的化疗或分子靶向药物提供靶点；最后检测CNTN-1、PTPRD等基因在临床SCLC标本中的表达水平，探讨其表达与SCLC患者化疗疗效及预后的相关性，为SCLC患者化疗方案的制定及其预后提供依据。

顺铂是一种细胞周期非特异性的细胞毒药物，主要通过抑制肿瘤细胞DNA合成、诱导凋亡而发挥作用。由于顺铂不是ABC超家族成员P-gp、MRP1、BCRP1作用的底物，肿瘤对其耐药的机制尚未完全阐明。目前认为损伤修复过程中DNA剪切物的增加、抗凋亡、与调控信号通路相关的调节蛋白表达的改变、药物摄取减少导致细胞内顺铂浓度降低等均有可能与对顺铂的耐药有关。与用阿霉素和足叶乙甙在体外诱导耐药的MDR细胞系不同，用顺铂诱导的MDR细胞系并不过度表达p-gp和MRP，这说明不同化疗药物导致的MDR可能存在不同的机制。此外，肿瘤细胞的异质性也是影响MDR的重要原因。因此，深入研究不同的SCLC细胞系细胞在顺铂诱导MDR前后其共同的差异表达基因，对于阐明SCLC获得性MDR形成机制及其逆转具有重要意义。为了探讨SCLC获得性MDR 机制，我们在已成功建立一株顺铂诱导的SCLC获得性MDR细胞系H446/DDP基础上开展相关研究。本研究结果显示，（1）CNTN-1是顺铂诱导的SCLC获得性MDR相关基因；（2）CNTN-1不仅参与顺铂诱导的SCLC获得性MDR的产生，并且可以促进SCLC细胞迁移和侵袭，从而作为枢纽使得SCLC获得性MDR与迁移和侵袭之间具有一定的内在联系；（3）与肺腺癌不同的是，在SCLC中CNTN-1可能是FGF10而非VEGF-C/Flt-4轴的下游效应分子；（4）CNTN-1可能和PTPR D、CTNNAL1等蛋白相互作用而参与SCLC获得性MDR。