Shah Ami N, Summy Justin M, Zhang Jing, Park Serk In, Parikh Nila U, Gallick Gary E
Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA.
Ann Surg Oncol. 2007 Dec;14(12):3629-37. doi: 10.1245/s10434-007-9583-5. Epub 2007 Oct 2.
Pancreatic cancer is an exceptionally lethal disease with an annual mortality nearly equivalent to its annual incidence. This dismal rate of survival is due to several factors including late presentation with locally advanced, unresectable tumors, early metastatic disease, and rapidly arising chemoresistance. To study the mechanisms of chemoresistance in pancreatic cancer we developed two gemcitabine-resistant pancreatic cancer cell lines.
Resistant cells were obtained by culturing L3.6pl and AsPC-1 cells in serially increasing concentrations of gemcitabine. Stable cultures were obtained that were 40- to 50-fold increased in resistance relative to parental cells. Immunofluorescent staining was performed to examine changes in beta-catenin and E-cadherin localization. Protein expression was determined by immunoblotting. Migration and invasion were determined by modified Boyden chamber assays. Fluorescence-activated cell sorting (FACS) analyses were performed to examine stem cell markers.
Gemcitabine-resistant cells underwent distinct morphological changes, including spindle-shaped morphology, appearance of pseudopodia, and reduced adhesion characteristic of transformed fibroblasts. Gemcitabine-resistant cells were more invasive and migratory. Gemcitabine-resistant cells were increased in vimentin and decreased in E-cadherin expression. Immunofluorescence and immunoblotting revealed increased nuclear localization of total beta-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells were activated in the receptor protein tyrosine kinase, c-Met and increased in expression of the stem cell markers CD (cluster of differentiation)24, CD44, and epithelial-specific antigen (ESA).
Gemcitabine-resistant pancreatic tumor cells are associated with EMT, a more-aggressive and invasive phenotype in numerous solid tumors. The increased phosphorylation of c-Met may also be related to chemoresistance and EMT and presents as an attractive adjunctive chemotherapeutic target in pancreatic cancer.
胰腺癌是一种极具致死性的疾病，其年死亡率几乎等同于年发病率。这种令人沮丧的生存率归因于多种因素，包括出现局部晚期、无法切除的肿瘤时已属晚期、早期发生转移以及迅速产生的化疗耐药性。为了研究胰腺癌化疗耐药的机制，我们建立了两种吉西他滨耐药的胰腺癌细胞系。
通过在浓度不断递增的吉西他滨中培养L3.6pl和AsPC-1细胞来获得耐药细胞。获得了稳定的培养物，其耐药性相对于亲本细胞提高了40至50倍。进行免疫荧光染色以检查β-连环蛋白和E-钙黏蛋白定位的变化。通过免疫印迹法测定蛋白质表达。通过改良的博伊登室试验测定迁移和侵袭能力。进行荧光激活细胞分选（FACS）分析以检测干细胞标志物。
吉西他滨耐药细胞出现了明显的形态学变化，包括纺锤形形态、伪足的出现以及转化成纤维细胞特有的黏附性降低。吉西他滨耐药细胞更具侵袭性和迁移性。吉西他滨耐药细胞中波形蛋白增加，E-钙黏蛋白表达减少。免疫荧光和免疫印迹显示总β-连环蛋白的核定位增加。这些改变是上皮-间质转化（EMT）的特征。耐药细胞中的受体蛋白酪氨酸激酶c-Met被激活，干细胞标志物CD（分化簇）24、CD44和上皮特异性抗原（ESA）的表达增加。
吉西他滨耐药的胰腺肿瘤细胞与EMT相关，EMT在众多实体瘤中是一种更具侵袭性的表型。c-Met磷酸化增加也可能与化疗耐药和EMT有关，并成为胰腺癌中一个有吸引力的辅助化疗靶点。
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