Tuberc Respir Dis.  2019 Apr;82(2):133-142. 10.4046/trd.2017.0124.

Bleomycin Inhibits Proliferation via Schlafen-Mediated Cell Cycle Arrest in Mouse Alveolar Epithelial Cells

Affiliations
  • 1Department of Thoracic and Cardiovascular Surgery, Kangwon National University School of Medicine, Chuncheon, Korea. seran@kangwon.ac.kr
  • 2Department of Internal Medicine, Kangwon National University School of Medicine, Chuncheon, Korea.
  • 3Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Korea.
  • 4Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Korea.
  • 5Department of Medical Environmental Biology and Tropical Medicine, Kangwon National University School of Medicine, Chuncheon, Korea.

Abstract

BACKGROUND
Idiopathic pulmonary fibrosis involves irreversible alveolar destruction. Although alveolar epithelial type II cells are key functional participants within the lung parenchyma, how epithelial cells are affected upon bleomycin (BLM) exposure remains unknown. In this study, we determined whether BLM could induce cell cycle arrest via regulation of Schlafen (SLFN) family genes, a group of cell cycle regulators known to mediate growth-inhibitory responses and apoptosis in alveolar epithelial type II cells.
METHODS
Mouse AE II cell line MLE-12 were exposed to 1-10 µg/mL BLM and 0.01-100 µM baicalein (Bai), a G1/G2 cell cycle inhibitor, for 24 hours. Cell viability and levels of pro-inflammatory cytokines were analyzed by MTT and enzyme-linked immunosorbent assay, respectively. Apoptosis-related gene expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Cellular morphology was determined after DAPI and Hoechst 33258 staining. To verify cell cycle arrest, propidium iodide (PI) staining was performed for MLE-12 after exposure to BLM.
RESULTS
BLM decreased the proliferation of MLE-12 cells. However, it significantly increased expression levels of interleukin 6, tumor necrosis factor α, and transforming growth factor β1. Based on Hoechst 33258 staining, BLM induced condensation of nuclear and fragmentation. Based on DAPI and PI staining, BLM significantly increased the size of nuclei and induced G2/M phase cell cycle arrest. Results of qRT-PCR analysis revealed that BLM increased mRNA levels of BAX but decreased those of Bcl2. In addition, BLM/Bai increased mRNA levels of p53, p21, SLFN1, 2, 4 of Schlafen family.
CONCLUSION
BLM exposure affects pulmonary epithelial type II cells, resulting in decreased proliferation possibly through apoptotic and cell cycle arrest associated signaling.

Keyword

Idiopathic Pulmonary Fibrosis; Alveolar Epithelial Cells; Cell Cycle Arrest; Schlafen; Bleomycin

MeSH Terms

Animals
Apoptosis
Bisbenzimidazole
Bleomycin*
Cell Cycle Checkpoints*
Cell Cycle*
Cell Line
Cell Survival
Cytokines
Enzyme-Linked Immunosorbent Assay
Epithelial Cells*
Gene Expression
Genes, vif
Humans
Idiopathic Pulmonary Fibrosis
Interleukin-6
Lung
Mice*
Propidium
RNA, Messenger
Transforming Growth Factors
Tumor Necrosis Factor-alpha
Bisbenzimidazole
Bleomycin
Cytokines
Interleukin-6
Propidium
RNA, Messenger
Transforming Growth Factors
Tumor Necrosis Factor-alpha

Figure

  • Figure 1 In the MLE-12 cells was evaluated toxicity effects of by bleomycin (BLM). MLE-12 cells were treated BLM (1–10 µg/mL) for 24 hours. (A) MLE-12 cells were induced morphological change by BLM. Scale bars=100 µm. (B) MLE-12 cells were treated with BLM and determined cell viability using MTT assay. (C) Expression of alveolar epithelial cell marker, surfactant protein C (SPC) was measured by quantitative real-time reverse transcription-polymerase chain reaction. The graph was measured inflammatory cytokines of tumor necrosis factor α (TNF-α) (D), interleukin-6 (IL-6) (E), and transforming growth factor β1 (TGF-β1) (F) in cell culture supernatant in enzyme-linked immunosorbent assay. *p<0.05, **p<0.01, ***p<0.001.

  • Figure 2 The MLE-12 cells induced apoptosis by bleomycin (BLM). (A) MLE-12 cells were stained with Hoechst 33258 staining after BLM treatment for 24 hours. Scale bars=100 µm. Expression of cell apoptosis marker, BAX (B) and Bcl2 (C) was measured by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). BAX/Bcl2 ratio (D) was divided (B, C) that measured by qRT-PCR. *p<0.05, **p<0.01, ***p<0.001.

  • Figure 3 Evaluation of nuclear enlargement and cell cycle arrest induced in MLE-12 cells by bleomycin (BLM). (A) The image was obtained using DAPI staining in MLE-12 cells after BLM treatment. Scale bars=200 µm. (B) Propidium iodide (PI) staining was performed for cell cycle arrest in MLE-12 cells after BLM treatment. (C) Statistical analysis was each percentage of the field in the samples. *p<0.05.

  • Figure 4 Gene expression associated with cell cycle of bleomycin (BLM) treatment in MLE-12 cells. The MLE-12 cells were treated with BLM (1–10 µg/mL) for 24 hours. Measured the p21 (A), p53 (B), and proliferating cell nuclear antigen (Pcna ) (C) at the mRNA levels, which were associated with cell cycle. BLM exposed MLE-12 cells were measured expression of Slfn1 (D), Slfn2 (E), and Slfn4 (F) by quantitative real-time reverse transcription-polymerase chain reaction analysis.

  • Figure 5 The evaluation of baicalein (Bai) affected MLE-12 cells. The MLE-12 cells were treated with Bai (0.01–100 µM) for 24 hours. (A) The cell viability was measured using MTT assay. Measured the p21 (B) and p53 (C) at the mRNA levels, which were associated with cell cycle. Bai exposed MLE-12 cells were measured expression of Slfn1 (D), Slfn2 (E), and Slfn4 (F) by quantitative real-time reverse transcription-polymerase chain reaction analysis. **p<0.01, ***p<0.001.


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