title: Cathepsin L upregulation-induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
authors.forename: Mei-Ling
authors.initials: ML
authors.lastname: Han
authors.affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
authors.forename: Yi-Fan
authors.initials: YF
authors.lastname: Zhao
authors.affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.|Department of Pharmacy, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China.
authors.forename: Cai-Hong
authors.initials: CH
authors.lastname: Tan
authors.affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
authors.forename: Ya-Jie
authors.initials: YJ
authors.lastname: Xiong
authors.affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
authors.forename: Wen-Juan
authors.initials: WJ
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, School of Pharmacy, Nantong University, Nantong 226001, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Wu
authors.affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Fei
authors.affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
authors.forename: Long
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China.
authors.forename: Zhong-Qin
authors.initials: ZQ
authors.lastname: Liang
mesh_terms: D000072283:A549 Cells; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D056668:Cathepsin L / Q000378:metabolism*; D002945:Cisplatin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D017239:Paclitaxel / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D056668:Cathepsin L; D017239:Paclitaxel; D002945:Cisplatin