Analysis of the effect of activated MEK-ERK signaling in the chemoresistance-basal research to the molecular targeting therapy

本研究制备了表达IEC-6大鼠肠上皮细胞（IEC-caMEK细胞）的caMEK。用不同浓度的CPT-11处理细胞，WST法测定其抑制浓度为50% （IC_<50>）。用DNA染色和DNA片段定量分析评估细胞凋亡。western blot检测蛋白表达。我们还研究了环氧化酶-2 （COX-2）在CPT-11诱导的细胞系统凋亡中的作用。与空载体转染的细胞相比，IEC-caMEK细胞在血清饥饿或CPT-11处理后具有存活优势。在此条件下，IEC-caMEK细胞的凋亡明显受到抑制。Western blot分析显示，IEC-caMEK细胞中Bcl-2、Bcl-xL、Mcl-1、COX-2的表达增加，Bak的表达降低。COX-2选择性抑制剂NS398改善了IEC-caMEK细胞在cpt -11诱导的凋亡中的抗凋亡性质。MEK激活通过cox -2依赖机制抑制cpt -11诱导的正常肠上皮细胞凋亡。因此，MEK-ERK信号可能与癌细胞的耐药性质有关。COX-2也可能在这一过程中发挥重要作用。

For the study, caMEK expressing IEC-6 rat intestinal epithelial cells (IEC-caMEK cells) were generated. Cells were treated with various concentrations of CPT-11, and the inhibitory concentration of 50% (IC_<50>) was determined by WST assay. Apoptosis was evaluated with DNA staining and quantitative analysis of fragmented DNA. Protein expressions were determined by western blot analysis. We also examined the role of cyclooxygenase-2 (COX-2) in CPT-11 induced apoptosis in the cell systems. IEC-caMEK cells possessed survival advantages following serum starvation, or following treatment with CPT-11 compared to empty vector transfected cells. In the conditions, apoptosis was remarkably suppressed in IEC-caMEK cells. Western blot analysis revealed that increased expression of Bcl-2, Bcl-xL, Mcl-1, COX-2, and decreased expression of Bak in IEC-caMEK cells. The COX-2 selective inhibitor, NS398, ameliorated antiapoptotic nature of IEC-caMEK cells in the CPT-11-induced apoptosis. MEK activation led to suppress CPT-11-induced apoptosis in normal intestinal epithelial cells via COX-2-dependent mechanism. Therefore, the MEK-ERK signaling may contribute to drug resistant nature of cancer cells. COX-2 may also play an important role in this process.