Mitomycin C Induces Autophagy in Human Tracheal Fibroblasts and Suppresses Their Growth

Abstract

Objective: Mitomycin C (MMC) is frequently used to prevent postoperative fibrosis in tracheal stenosis, yet its precise cellular mechanisms remain inadequately understood. This study aimed to elucidate the cytotoxic and autophagic effects of MMC on normal human tracheal fibroblasts (hTF) and human bronchial/tracheal epithelial cells (hTEC) to better understand its potential role in fibrosis regulation.

Methods: hTF and hTEC were exposed to MMC at concentrations of 0.01, 0.1, and 1 μg/mL for 24, 48, and 72 h. Cell proliferation, autophagy induction, and the expression of autophagy-related proteins were assessed using viability assays and Western blot analysis. Additionally, the effects of MMC on cell migration and fibroblast-to-myofibroblast transition were investigated.

Results: MMC partially reduced hTEC proliferation without inducing autophagy. In contrast, MMC significantly suppressed hTF growth in a dose- and time-dependent manner while promoting autophagy. Western blot analysis revealed increased expression of LC3, ATG5, and Rab7 in MMC-treated hTF, along with reduced cyclin D1 levels. Furthermore, MMC attenuated TGFβ-induced αSMA expression in fibroblasts, suggesting an inhibitory effect on fibrosis-related cellular transformation.

Conclusion: These findings indicate that MMC suppresses human tracheal fibroblast proliferation through autophagy-mediated cell death while sparing epithelial cells. This dual effect underscores its potential as a targeted antifibrotic agent for tracheal stenosis management. Further research is needed to optimize MMC's application and elucidate its long-term impact on airway remodeling.

Level of evidence: 5.

Keywords: Mitomycin C; autophagy; bronchial/tracheal epithelial cell; tracheal fibroblast; tracheal stenosis.

FIGURE 1

Mitomycin C suppressed the proliferation of human tracheal epithelial cell (hTEC) and human tracheal fibroblast (hTF). Proliferation of hTEC (1A) and hTF (1B) following MMC treatment was assessed using the CELLOMAX proliferation assay on days 1, 2, and 3. Inhibition rate (%) of hTEC and hTF proliferation was quantified based on absorbance values (1C and 1D). Data are presented as the mean ± SD, with individual data points representing three independent experiments. Statistical significance was determined using One‐way ANOVA followed by Turkey's multiple comparisons test. *p < 0.05, ****p < 0.0001 versus control. OD: optical density.

FIGURE 2

Mitomycin C induces autophagy in hTF. (A, B) Autophagy was assessed using an autophagy detection kit in hTEC and hTF treated with or without mitomycin C (0.1 and 1 μg/mL) for 24 h. Autophagy‐positive cells were stained with green fluorescence and visualized under fluorescence microscopy. Rapamycin (0.5 μM) served as a positive control for autophagy induction. Scale bar: 50 μm. (C, D) Western blot analysis of autophagy‐related protein expression in hTEC and hTF following MMC treatment. The expression levels of LC3‐I, LC3‐II, ATG5, and Rab7 were analyzed in hTEC at 4 and 24 h post‐treatment. In hTF, the protein levels of LC3‐I, LC3‐II, ATG5, Rab7, and cyclin D1 were assessed under similar conditions. GAPDH was used as a loading control. Uncropped Western blot images are provided in Figure S1. Data are presented as mean ± SD from three independent experiments, with individual data points shown. Statistical significance was determined using an unpaired t‐test (*p < 0.05, **p < 0.01, ****p < 0.0001 vs. control).

FIGURE 3

Mitomycin C inhibits hTF migration. (A) Representative bright‐field images illustrating the reduced migration of hTF following a single MMC treatment compared to the control group. (B) The migratory percentage was quantified using ImageJ by measuring the distance covered by cells moving toward the scratched region over 30 h. Data are presented as mean ± SD, with individual data points from three independent experiments. Statistical significance was determined using one‐way ANOVA followed by Tukey's multiple comparisons test (**p < 0.01 vs. control). Scale bar: 50 μm.

FIGURE 4

Mitomycin C reduces the expression of fibrosis‐related proteins in hTF. Western blot analysis was performed to assess the expression levels of collagen‐1, fibronectin, MMP‐2, MMP‐9, and caveolin in hTF following treatment with or without mitomycin C (0.1 and 1 μg/mL) for 4 or 24 h. Data are presented as mean ± SD from three independent experiments. GAPDH was used as an internal control. Uncropped Western blot images are provided in Figure S2. Statistical significance was determined using an unpaired t‐test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control).

FIGURE 5

Mitomycin C inhibits TGFβ‐induced fibroblast‐to‐myofibroblast transformation in hTF. Western blot analysis was performed to assess the expression of αSMA, a myofibroblast marker, in hTF treated with TGFβ (5 ng/mL), mitomycin C (1 μg/mL), or a combination of both for 72 h. Representative blots and densitometric analysis of three independent experiments are presented as mean ± SD. GAPDH was used as a loading control. Uncropped Western blot images are provided in Figure S3. Statistical significance was determined using an unpaired t‐test (*p < 0.05, ****p < 0.0001 vs. control, ^$$$ p < 0.001 vs. TGFβ).