J Korean Ophthalmol Soc.  2014 Jul;55(7):1077-1083.

Cytotoxicities and Anti-Fibrotic Effects of Pirfenidone and Mitomycin C on Human Fibroblasts

• Park KS
• Hong SM
• Iizuka Y
• Kim CY
• Seong GJ

Abstract

PURPOSE
The cytotoxicities and anti-fibrotic effects of mitomycin C and pirfenidone on human dermal fibroblast were evaluated.
METHODS
Initially, 24-hour cell cultures were exposed to transforming growth factor (TGF)-beta1, different concentrations of mitomycin C, and pirfenidone solutions in order to evaluate cytotoxicity. Expressions of fibronectin, collagen type 1, alpha smooth muscle, and beta-actin were evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot in mitomycin C solutions at concentrations of 4 microg/mL and 20 microg/mL, and in pirfenidone solutions at 250 microg/mL and 500 microg/mL.
RESULTS
In comparison to cell cultures exposed to TGF-beta1 solutions, cytotoxicities were increased in solutions of mitomycin C at 4 microg/mL, 20 microg/mL, 40 microg/mL and pirfenidone at 500 microg/mL, 750 microg/mL, 1,000 microg/mL (p < 0.05, Mann Whitney U-test). The results of real-time RT-PCR show that expressions of fibronectin, collagen type 1, and alpha smooth muscle were significantly more decreased in all concentrations of mitomycin C and pirfenidone compared to those in TGF-beta1 solution. In western blot analysis, expressions of fibronectin and alpha smooth muscle were decreased in all concentrations of mitomycin C and pirfenidone compared to TGF-beta1 solution.
CONCLUSIONS
Both drugs have cytotoxicities and anti-fibrotic effects, but pirfenidone was found to have less cytotoxicity and mitomycin C was found to have more anti-fibrotic effects when compared to each other.

Keyword

Anti-fibrotic effect; Cytotoxicity; Mitomycin C; Pirfenidone

MeSH Terms

Actins
Blotting, Western
Cell Culture Techniques
Collagen
Fibroblasts*
Fibronectins
Humans
Mitomycin*
Muscle, Smooth
Transforming Growth Factor beta1
Transforming Growth Factors
Actins
Collagen
Fibronectins
Mitomycin
Transforming Growth Factor beta1
Transforming Growth Factors

Figure

• 

  Figure 1. Result of lactate dehydrogenase (LDH) assay. M4, M5, M6, P4, P5, P6 solutions increased cytotoxicity significantly (*p < 0.05, Mann Whitney U-test). Sample C: only Dulbecco's modified eagle's medium (DMEM); T-P6: add transforming growth factor (TGF)-βl 5 ng/mL; M1-M6: add Mitomycin C, 0.00004 (Ml), 0.004 (M2), 0.4 (M3), 4 (M4), 20 (M5), 40 μg/mL (M6; P1-P6: add Pirfenidone, 10 (Pl), 100 (P2), 250 (P3), 500 (P4), 750 (P5), 1,000 μg/mL (P6). TGF = transforming growth factor; C = control; T = TGF-β 1; M = mitomycin C; P = pirfenidone.

• 

  Figure 2. Result of real time reverse transcription polymerase chain reaction (RT-PCR). Mitomycin C and pirfenidone solution decreased ribonucleic acid (RNA) expression of fibronectin, collagen I, α smooth muscle, which compared to the only transforming growth factor (TGF)-β1 solution.

• 

  Figure 3. Result of western blot. Mitomycin C and pirfenidone solution decreased protein expression of fibronectin, α smooth muscle, which compared to the only transforming growth factor (TGF)-β1 solution. Protein expression of collagen type I was too weak.

Reference

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