Yang Anthony D, Fan Fan, Camp E Ramsay, van Buren George, Liu Wenbiao, Somcio Ray, Gray Michael J, Cheng Haiyun, Hoff Paulo M, Ellis Lee M
Department of Surgical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77230-1402, USA.
Clin Cancer Res. 2006 Jul 15;12(14 Pt 1):4147-53. doi: 10.1158/1078-0432.CCR-06-0038.
Epithelial-to-mesenchymal transition (EMT) is a process whereby cells acquire molecular alterations that facilitate cell motility and invasion. In preliminary studies, we observed that oxaliplatin-resistant (OxR) colorectal cancer (CRC) cells underwent morphologic changes suggestive of a migratory phenotype, leading us to hypothesize that OxR CRC cells undergo EMT.
The human CRC cell lines KM12L4 and HT29 were exposed to increasing doses of oxaliplatin to establish stable cell lines resistant to oxaliplatin. Migration and invasion were assessed by modified Boyden chamber assays. Morphologic and molecular changes characteristic of EMT were determined by immunofluorescence staining and Western blot analyses.
The OxR cells showed phenotypic changes consistent with EMT: spindle-cell shape, loss of polarity, intercellular separation, and pseudopodia formation. KM12L4 and HT29 OxR cells exhibited an approximately 8- to 15-fold increase in migrating and invading cells, respectively (P < 0.005 for both). Immunofluorescence staining of OxR cells revealed translocation of E-cadherin and beta-catenin from their usual membrane-bound complex to the cytoplasm and nucleus, respectively. The OxR cells also had decreased expression of the epithelial adhesion molecules E-cadherin and plakoglobin and an increase in the mesenchymal marker vimentin. The KM12L4 OxR cells exhibited increased nuclear expression of Snail, an EMT-regulatory transcription factor, whereas the HT29 OxR cells exhibited an increase in nuclear expression of the EMT-associated transcription factor nuclear factor kappaB.
We hypothesize that induction of EMT may contribute to the decreased efficacy of therapy in chemoresistant CRC, as the tumor cells switch from a proliferative to invasive phenotype. Further understanding of the mechanisms of chemoresistance in CRC will enable improvements in chemotherapy for metastatic disease.
上皮-间质转化（EMT）是一个细胞获得促进细胞运动和侵袭的分子改变的过程。在初步研究中，我们观察到奥沙利铂耐药（OxR）的结直肠癌（CRC）细胞发生了提示迁移表型的形态学变化，这使我们推测OxR CRC细胞会经历EMT。
将人CRC细胞系KM12L4和HT29暴露于递增剂量的奥沙利铂中，以建立对奥沙利铂耐药的稳定细胞系。通过改良的Boyden小室试验评估迁移和侵袭能力。通过免疫荧光染色和蛋白质印迹分析确定EMT的形态学和分子变化特征。
OxR细胞表现出与EMT一致的表型变化：梭形细胞形态、极性丧失、细胞间分离和伪足形成。KM12L4和HT29 OxR细胞的迁移和侵袭细胞分别增加了约8至15倍（两者P均<0.005）。OxR细胞的免疫荧光染色显示，E-钙黏蛋白和β-连环蛋白分别从其通常的膜结合复合物转位至细胞质和细胞核。OxR细胞中上皮黏附分子E-钙黏蛋白和桥粒斑蛋白的表达也降低，间充质标志物波形蛋白增加。KM12L4 OxR细胞中EMT调节转录因子Snail的核表达增加，而HT29 OxR细胞中EMT相关转录因子核因子κB的核表达增加。
我们推测EMT的诱导可能导致化疗耐药的CRC治疗效果下降，因为肿瘤细胞从增殖表型转变为侵袭表型。进一步了解CRC化疗耐药机制将有助于改善转移性疾病的化疗。
