Cathepsin L Mediated Drug Resistance By Promoting Epithelial-Mesenchymal Transition In Human Lung Adenocarcinoma A549 Cells

Objective: To explore the role of Cathepsin L(CTSL) in regulating drug resistance and the underlying molecular mechanisms.Methods: A549 cells,A549/TAX(Paclitaxel-resistant A549 cells) and A549/DDP(Cisplatin-resistant A549 cells) were introduced to performing the following study. We silenced CTSL by transfected A549/DDP and A549/TAX cells with CTSL siRNA and overexpressed CTSL by infected A549 cells with lentivirus, respectively. MTT and colony formation assay were performed to detect the change in drug resistance of cells to cisplatin or paclitaxel after silence or overexpression CTSL; FITC-Phalloidin fluorescent staining and immunofluorescence assay were adopted to observe the actin remodeling of A549 cells; The ability of cell migration was measured in vitro by wound healing assayand cell invasion was detected by transwell invasion assay; Western Blot and immunofluorescence were performed to detect the expression of CTSL, epithelial mesenchymal markers(E-cadherin, cytoskeratin 18, vimentin and N-cadherin);Western Blot or q-PCR assay were introduced to determine the expression of EMT-associated transcription factors(snail, slug, ZEB1 and ZEB2). Overexpressing CTSL cells were inoculated subcutaneously into nude mice, tumor size or body weight was measured by vernier caliper or electronic balance; Western blot and immunohistochemistry were adopted to determine the protein level of CTSL, EMT-associated protein and transcription factors.Results: CTSL was induced after treated with cisplatin or paclitaxel in A549 cells, and CSTL protein level was significantly elevated in drug-resistant lung cancer cell lines. In addition, compared with the normal A549 cells, the drug-resistant lung cancer cells underwent morphologic changes and cellular cytoskeleton remodels, and the ability of cell invasion and migration were enhanced; meanwhile, the expression of the epithelial markers were decreased and the mesenchymal markers were increased in drug-resistant lung cancer cells; furthermore, silencing CSTL in A549/DDP and A549/PTX cells significantly decreased the lamellipodia and stress fibers concomitant with attenuated the ability of cell invasion and migration; simultaneously, the epithelial markers expression were increased and the mesenchymal markers expression were decreased. In contrast, overexpression of CSTL cytoskeleton remodeling, and the ability of cell invasion and migration was enhanced. The forced expression of CSTL decreased the expression of the epithelial markers but increased the mesenchymal markers in A549 cells. Besides, EMT-inducing transcription factors(Snail, Slug, ZEB1 and ZEB2) were high expression in A549/TAX and A549/DDP cells, and CSTL silencing reduced the expression of transcription factors. Overexpression of CSTL in A549 cells increased the expression of Snail, Slug, ZEB1 and ZEB2. Moreover, overexpression of CTSL a significantly reduced the sensitivity of A549 cells to PTX in vivo. The expression of CTSL, EMT-associated proteins and transcription factors were consistent with in vitro.Conclusion: These results suggest that CSTL regulate cisplatin and paclitaxel resistance via transcription factors-mediated EMT in A549 cells. Our findings demonstrate CSTL is a critical regulator in tumor drug resistance and may be an important target for overcoming chemotherapy resistance.