Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study

Abstract

Objective: To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell), particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast.

Methods: The normal dermal fibroblast and HaCat cell were cultured in vitro. Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution, and serum-free culture medium was used as control. The cellular morphology change, growth characteristics, cell proliferation, and apoptosis were observed at different intervals. For the fibroblasts, the mRNA expression changes of transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), procollagen I, and III were detected by reverse transcription polymerase chain reaction (RT-PCR).

Results: The cultured normal human skin fibroblast and HaCat cell grew exponentially. A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells. Cell morphology changed, cell density decreased, and the growth curves were without an exponential phase. The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml. Meanwhile, 5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis. The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05). A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-β1, procollagen I and III, and a marked increase in the mRNA production of bFGF.

Conclusions: Mitomycin can inhibit fibroblast proliferation, induce fibroblast apoptosis, and regulate intracellular protein expression on mRNA levels. In addition, mitomycin can inhibit HaCat cell proliferation, so epithelial cell needs more protecting to avoid mitomycin's side effect when it is applied clinically.

Fig. 1

Microscopic analyses of control and treated fibroblasts stained by AO (a) Control on Day 3; (b) Mitomycin 0.04 mg/ml treated on Day 3; (c) Mitomycin 0.4 mg/ml treated on Day 3

Fig. 2

Microscopic analyses of control and treated HaCat cell stained by AO (a) Control on Day 3; (b) Mitomycin 0.04 mg/ml treated on Day 3; (c) Mitomycin 0.4 mg/ml treated on Day 3

Fig. 3

Growth curve of fibroblasts after mitomycin exposure

Fig. 4

Growth curve of HaCat cells after mitomycin exposure

Fig. 5

Inhibition rate of fibroblast after the exposure to mitomycin

Fig. 6

Inhibition rate of HaCat cell after the exposure to mitomycin

Fig. 7

Different apoptosis stage of fibrosis after the exposure to mitomycin The cell white arrow pointed was normal; the cell green arrow pointed was in the early stage of apoptosis; the cell yellow arrow pointed was in the middle stage; the cell red arrow pointed was in the late stage

Fig. 8

Apoptosis rate of fibroblast after mitomycin application Apoptosis rates of fibroblasts exposed to mitomycin were much higher than the control (p<0.05). However, there was no statistically significant difference of apoptosis rate between 0.4 and 0.04 mg/ml groups

Fig. 9

Analyses of TGF-β1 mRNA expression and β-actin using RT-PCR Each mRNA value is corrected for β-actin mRNA value (TGF-β1/β-actin mRNA ratio)

Fig. 10

Analyses of bFGF mRNA expression and β-actin using RT-PCR Each mRNA value is corrected for β-actin mRNA value (bFGF/β-actin mRNA ratio)

Fig. 11

Analyses of procollagen I mRNA expression and β-actin using RT-PCR Each mRNA value is corrected for β-actin mRNA value (procollagen I/β-actin mRNA ratio)

Fig. 12

Analyses of procollagen III mRNA expression and β-actin using RT-PCR Each mRNA value is corrected for β-actin mRNA value (procollagen III/β-actin mRNA ratio)