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

对于该研究，产生了表达caMEK的IEC-6大鼠肠上皮细胞（IEC-caMEK细胞）。用不同浓度的CPT-11处理细胞，并通过WST测定法测定50%抑制浓度（IC_<50>）。DNA染色和DNA片段定量分析检测细胞凋亡。蛋白质表达通过蛋白质印迹分析测定。我们还研究了环氧化酶-2（考克斯-2）在CPT-11诱导的细胞凋亡中的作用。与空载体转染的细胞相比，IEC-caMEK细胞在血清饥饿后或用CPT-11处理后具有存活优势。在该条件下，IEC-caMEK细胞的凋亡被显著抑制。Western blot分析显示，Bcl-2、Bcl-xL、Mcl-1、考克斯-2表达增加，巴克蛋白表达减少。考克斯-2选择性抑制剂NS 398改善了CPT-11诱导的IEC-caMEK细胞凋亡中的抗凋亡性质。MEK激活通过考克斯-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信号传导可能有助于癌细胞的耐药性。考克斯-2在此过程中可能也发挥了重要作用。

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.