Cathepsin L upregulation-induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells
Cathepsin L upregulation-induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells
复制标题
组织蛋白酶 L 上调诱导的 EMT 表型与 A549 细胞中顺铂或紫杉醇耐药性的获得相关
DOI：
10.1038/aps.2016.93
复制
发表时间：
2016-12-01
影响因子：
8.2
通讯作者：
Liang, Zhong-qin
中科院分区：
文献类型：
--
作者：
Han, Mei-ling;Zhao, Yi-fan;Liang, Zhong-qin
目的：组织蛋白酶L（CTSL）是一种溶酶体酸性半胱氨酸蛋白酶，在肿瘤转移和化疗耐药中起重要作用。方法：以人肺癌A549细胞、A549/PTX（紫杉醇耐药）细胞和A549/DDP（顺铂耐药）细胞为研究对象，研究CTSL对人肺癌A549细胞化疗耐药性的影响。MTT法和集落形成实验检测细胞对顺铂和紫杉醇的耐药性。用FITC-Phalloidin荧光染色或免疫荧光法观察肌动蛋白重构。伤口愈合试验或Transwell试验用于评估迁移或侵袭能力。采用免疫印迹和免疫荧光法检测CTSL和上皮及间充质标志物的表达。用Western blotting或q-PCR检测EMT相关转录因子的表达。结果：顺铂或紫杉醇（10-80 ng/mL）均可诱导A549细胞表达CTSL。A549/PTX和A549/DDP细胞CTSL水平明显高于A549细胞。CTSL沉默可逆转A549/DDP和A549/TAX细胞的耐药性，而CTSL过表达可降低A549细胞对顺铂或紫杉醇的敏感性。此外，A549/DDP和A549/TAX细胞发生形态和细胞骨架变化，细胞侵袭和迁移能力增加，伴有上皮标记物（E-cadherin和cytokeratin-18）表达减少和间充质标记物（N-cadherin和vimentin）表达增加，以及EMT相关转录因子Snail、Slug、ZEB 1和ZEB 2表达上调。CTSL基因沉默可逆转A549/DDP和A549/TAX细胞的EMT，而CTSL基因过表达可诱导A549细胞的EMT。结论：CTSL上调A549细胞的EMT表达与A549细胞对顺铂和紫杉醇的耐药有关。因此，CTSL介导的EMT可被用作增强顺铂或紫杉醇对肺癌和其他类型的恶性肿瘤的功效的靶点。
Aim:Cathepsin L (CTSL), a lysosomal acid cysteine protease, is known to play important roles in tumor metastasis and chemotherapy resistance. In this study we investigated the molecular mechanisms underlying the regulation of chemoresistance by CTSL in human lung cancer cells.Methods:Human lung cancer A549 cells, A549/PTX (paclitaxel-resistant) cells and A549/DDP (cisplatin-resistant) cells were tested. The resistance to cisplatin or paclitaxel was detected using MTT and the colony-formation assays. Actin remodeling was observed with FITC-Phalloidin fluorescent staining or immunofluorescence. A wound-healing assay or Transwell assay was used to assess the migration or invasion ability. The expression of CTSL and epithelial and mesenchymal markers was analyzed with Western blotting and immunofluorescence. The expression of EMT-associated transcription factors was measured with Western blotting or q-PCR. BALB/c nude mice were implanted subcutaneously with A549 cells overexpressing CTSL, and the mice were administered paclitaxel (10, 15 mg/kg, ip) every 3 d for 5 times.Results:Cisplatin or paclitaxel treatment (10–80 ng/mL) induced CTSL expression in A549 cells. CTSL levels were much higher in A549/PTX and A549/DDP cells than in A549 cells. Silencing of CTSL reversed the chemoresistance in A549/DDP and A549/TAX cells, whereas overexpression of CTSL attenuated the sensitivity of A549 cells to cisplatin or paclitaxel. Furthermore, A549/DDP and A549/TAX cells underwent morphological and cytoskeletal changes with increased cell invasion and migration abilities, accompanied by decreased expression of epithelial markers (E-cadherin and cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin and vimentin), as well as upregulation of EMT-associated transcription factors Snail, Slug, ZEB1 and ZEB2. Silencing of CTSL reversed EMT in A549/DDP and A549/TAX cells; In contrast, overexpression of CTSL induced EMT in A549 cells. In xenograft nude mouse model, the mice implanted with A549 cells overexpressing CTSL exhibited significantly reduced sensitivity to paclitaxel treatment, and increased expression of EMT-associated proteins and transcription factors in tumor tissues.Conclusion:Cisplatin and paclitaxel resistance is associated with CTSL upregulation-induced EMT in A549 cells. Thus, CTSL-mediated EMT may be exploited as a target to enhance the efficacy of cisplatin or paclitaxel against lung cancer and other types of malignancies.