Cathepsin L upregulation-induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells

Abstract

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.

Figure 1

Paclitaxel or cisplatin induces CTSL protein (26 kDa) expression in A549 cells. (A) A549 cells were treated with different concentrations of PTX for 12, 24, and 48 h. Western blotting was performed to detect the expression of CTSL protein. (B) A549 cells were treated with different concentrations of DDP for 12, 24, and 48 h, and the expression level of CTSL was determined by Western blotting. (C) Western blot analysis was performed to determine the expression levels of CTSL in A549, A549/DDP, and A549/TAX cells. (D and F) Western blot and immunofluorescence analyses were performed to determine the expression level of CTSL in A549/DDP and A549/TAX cells transfected with CTSL siRNAs targeting the human CTSL sequence or the control siRNA. (E and G) Western blot and immunofluorescence analyses were performed to determine the expression level of CTSL in A549 cells transfected with LV-Over-CTSL, which targets the human CTSL sequence or the LV-Vector. At least three independent experiments were performed.

Figure 2

CTSL regulates cisplatin and paclitaxel resistance in A549 cells. (A) Effect of CTSL inhibition on the sensitivity of A549/DDP to cisplatin. (C) Effect of CTSL inhibition on the sensitivity of A549/TAX to paclitaxel. (B and D) Effect of CTSL overexpression on the sensitivity of A549 cells to cisplatin. (E and F) Effect of CTSL overexpression on the sensitivity of A549 cells to paclitaxel. Mean±SD. n=3. ^*P<0.05, ^**P<0.01 compared with the control. ^##P<0.01 compared with the A549-Vector group.

Figure 3

EMT was induced in A549/DDP and A549/TAX cells. (A) Cell morphology was observed by microscopy. Images were captured using a 10x objective lens. (B) Representative phalloidin staining to observe the actin cytoskeleton in A549, A549/DDP, and A549/TAX cells. Cell spreading was analyzed after plating on collagen-coated slides. At the indicated time points, the cells were fixed, stained with phalloidin, and visualized by fluorescence microscopy. Images were captured using a 63x objective lens. (C) The cell migration ability was evaluated by the wound-healing assay. Images were captured using a 4x objective lens. (D) The cell invasion ability was evaluated using a Transwell assay. Images were captured using a 10x objective lens. (E) E-cadherin, N-cadherin, cytokeratin-18, and vimentin were assessed by Western blot analysis. (F) E-cadherin, N-cadherin, and vimentin were subjected to immunofluorescence microscopy. Images were captured using a 63x objective lens. Means±SD. n=3. ^*P<0.05, ^**P<0.01 compared with the control.

Figure 4

CTSL inhibition regulates cisplatin or paclitaxel resistance by reversing EMT in A549/DDP and A549/TAX cells. (A) Effect of CTSL inhibition on cell migration. Images were captured using a 4× objective lens. (B) Effect of CTSL inhibition on cell invasion. Images were captured using a 10× objective lens. (C) Effect of CTSL inhibition on E-cadherin, N-cadherin, cytokeratin-18, and Vimentin were measured by Western blotting. (D) Effect of CTSL inhibition on the actin cytoskeleton, E-cadherin, N-cadherin, and vimentin. F-actin, E-cadherin, N-cadherin, and vimentin were subjected to immunofluorescence microscopy. Images were captured using a 63× objective lens. Means±SD. n=3. ^*P<0.05, ^**P<0.01 compared with the control.

Figure 5A–5D

CTSL overexpression induces EMT in A549 cells. (A) CTSL overexpression induces morphological changes in A549 cells. (B) Effect of CTSL overexpression on cell migration. Images were captured using a 4x objective lens. (C) Effect of CTSL overexpression on cell invasion. Images were captured using a 10× objective lens. (D) Effects of CTSL overexpression on E-cadherin, N-cadherin, cytokeratin-18, and vimentin measured by Western blotting. Means±SD. n=3. ^*P<0.05, ^**P<0.01 compared with the control.

Figure 5E

CTSL overexpression induces EMT in A549 cells. (E) Effect of CTSL inhibition on actin cytoskeleton, E-cadherin, N-cadherin, and vimentin. F-actin, E-cadherin, N-cadherin, and Vimentin were subjected to immunofluorescence microscopy. Images were captured using a 63× objective lens.

Figure 6

CTSL modulates the transcription and expression of EMT-associated transcription factors. (A) The levels of Snail and Slug in A549/DDP and A549/TAX cells were detected by Western blot analysis. (B) Effect of siCTSL transfection on Snail and Slug. (C) Effect of overexpressed CTSL on Snail and Slug. (D) Immunofluorescence was performed to examine the co-localization of CTSL and Snail in A549 cells. (E) The levels of ZEB1 and ZEB2 in A549/DDP and A549/TAX cells were analyzed by q-PCR, and GAPDH served as an internal normalization control. (F) Effect of siCTSL transfection on ZEB1 and ZEB2. (G) Effect of CTSL overexpression on ZEB1 and ZEB2. Means±SD. n=3. ^*P<0.05, ^**P<0.01 compared with the control.

Figure 7A–7E

CTSL overexpression attenuates the sensitivity of A549 cells to paclitaxel in vivo. Vector control or CTSL-overexpressing A549 cells were injected into nude mice, which were treated with the indicated concentrations of paclitaxel five times after two weeks. (A) The tumor size was measured using a Vernier caliper. (B) Body weight was determined using an electronic balance. (C) At the end of the treatment, the tumors were removed from the nude mice. (D, E) Tumor lysates were resolved by SDS-polyacrylamide gel electrophoresis and Western blot analysis using anti-CTSL, anti-E-cadherin, anti-N-cadherin, anti-cytokeratin 18, or anti-vimentin antibodies. β-Actin was used as a loading control. Means±SD. n=3. ^*P<0.05, ^**P<0.01 compared with the control.

Figure 7F

CTSL overexpression attenuates the sensitivity of A549 cells to paclitaxel in vivo. Vector control or CTSL-overexpressing A549 cells were injected into nude mice, which were treated with the indicated concentrations of paclitaxel five times after two weeks. (F) Immunohistochemistry was performed to detect the expression of CTSL, N-cadherin, Snail, and Slug. Photographs were obtained using a 20× objective lens.

Figure 8

Influence of CTSL on drug resistance in A549 cells.