Pancreatic cancer cells surviving gemcitabine treatment express markers of stem cell differentiation and epithelial-mesenchymal transition
Pancreatic cancer cells surviving gemcitabine treatment express markers of stem cell differentiation and epithelial-mesenchymal transition
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DOI：
10.3892/ijo.2012.1648
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发表时间：
2012-12-01
影响因子：
5.2
通讯作者：
Neureiter, Daniel
中科院分区：
文献类型：
--
作者：
Quint, Karl;Tonigold, Manuel;Neureiter, Daniel
胰腺癌对标准化疗方案的客观缓解率仍然较低。已在各种实体瘤中鉴定出表达干细胞相关标记物并与放化疗耐药性增加相关的细胞亚群。我们研究了在高浓度吉西他滨治疗后存活的胰腺癌细胞中干细胞基因和上皮间质转化标志物的表达。 Capan-1和Panc-1细胞与1和10μM吉西他滨连续孵育。 1,3和6天后收集存活细胞。通过 qPCR 或免疫细胞化学对 PDX-1、SHH、CD24、CD44、CD133、EpCAM、CBX7、OCT4、SNAIL、SLUG、TWIST、Ki-67、E-钙粘蛋白、β-连环蛋白和波形蛋白的表达进行定量。通过伤口愈合测定评估迁移。 SHH 通过 RNA 干扰被敲除。使用五种原代胰腺癌细胞系来验证 qPCR 结果。吉西他滨孵育 6 天后，所有研究的基因均上调。 Capan-1 中的 OCT4（13.4 倍）、CD24（47.3 倍）和 EpCAM（15.9 倍）以及 Panc-1 细胞中的 PDX-1（13.3 倍）、SHH（24.1 倍）、CD44（17.4 倍）、CD133（20.2 倍）和 SLUG（15.2 倍）的相对表达水平最高。在原代细胞中观察到明显的上调模式。 Panc-1 细胞的迁移增加，E-钙粘蛋白和 β-连环蛋白表达的变化是两种细胞系上皮-间质转化的典型变化。 SHH 敲低将 Capan-1 中的 IC50 从 30.1 nM 降低至 27.6 nM，同时强烈抑制 Panc-1 细胞中的增殖。经过高剂量吉西他滨治疗后存活的细胞表达干细胞基因水平升高，表现出与上皮-间质转化相关的特征并保留其增殖能力。
Objective response rates to standard chemotherapeutic regimens remain low in pancreatic cancer. Subpopulations of cells have been identified in various solid tumors which express stem cell-associated markers and are associated with increased resistance against radiochemotherapy. We investigated the expression of stem cell genes and markers of epithelial-mesenchymal transition in pancreatic cancer cells that survived high concentrations of gemcitabine treatment. Capan-1 and Panc-1 cells were continuously incubated with 1 and 10 mu M gemcitabine. Surviving cells were collected after 1,3 and 6 days. Expression of PDX-1, SHH, CD24, CD44, CD133, EpCAM, CBX7, OCT4, SNAIL, SLUG, TWIST, Ki-67, E-cadherin, beta-catenin and vimentin were quantified by qPCR or immunocytochemistry. Migration was assessed by wound-healing assay. SHH was knocked down using RNA interference. Five primary pancreatic cancer cell lines were used to validate the qPCR results. All investigated genes were upregulated after 6 days of gemcitabine incubation. Highest relative expression levels were observed for OCT4 (13.4-fold), CD24 (47.3-fold) and EpCAM (15.9-fold) in Capan-1 and PDX-1 (13.3-fold), SHH (24.1-fold), CD44 (17.4-fold), CD133 (20.2-fold) and SLUG (15.2-fold) in Panc-1 cells. Distinct upregulation patterns were observed in the primary cells. Migration was increased in Panc-1 cells and changes in the expression of E-cadherin and beta-catenin were typical of epithelial-mesenchymal transition in both cell lines. SHH knockdown reduced IC50 from 30.1 to 27.6 nM in Capan-1 while it strongly inhibited proliferation in Panc-1 cells. Cells surviving high-dose gemcitabine treatment express increased levels of stem cell genes, show characteristics associated with epithelial-mesenchymal transition and retain their proliferative capacity.