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

Kyoung Soo Park; Sa Min Hong; Yoko Iizuka; Chan Yun Kim; Gong Je Seong

doi:10.3341/jkos.2014.55.7.1077

Journal List > J Korean Ophthalmol Soc > v.55(7) > 1009732

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Park, Hong, Iizuka, Kim, and Seong: Cytotoxicities and Anti-Fibrotic Effects of Pirfenidone and Mitomycin C on Human Fibroblasts

Original Article

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

Published online: 30 August 2014

DOI: https://doi.org/10.3341/jkos.2014.55.7.1077

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

Kyoung Soo Park, MD¹, Sa Min Hong, MD², Yoko Iizuka, MD, PhD², Chan Yun Kim, MD², Gong Je Seong, MD^2,

¹Department of Ophthalmology, Siloam Eye Hospital, Seoul, Korea

²The Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea

Address reprint requests to Gong Je Seong, MD, Department of Ophthalmology, Gangnam Severance Hospital, #211 Eonju-ro, Gangnam-gu, Seoul 135-720, Korea, Tel: 82-2-2019-3441, Fax: 82-2-3463-1049 E-mail: gjseong@yuhs.ac

Received 13 December 2013 Revised 4 February 2014 Accepted 9 June 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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)-β1, different concentrations of mitomycin C, and pirfenidone solutions in order to evaluate cytotoxicity. Expressions of fibronectin, collagen type 1, α smooth muscle, and β-actin were evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot in mitomycin C solutions at concentrations of 4 μg/mL and 20 μg/mL, and in pirfenidone solutions at 250 μg/mL and 500 μg/mL.

Results

In comparison to cell cultures exposed to TGF-β1 solutions, cytotoxicities were increased in solutions of mitomycin C at 4 μg/mL, 20 μg/mL, 40 μg/mL and pirfenidone at 500 μg/mL, 750 μg/mL, 1,000 μg/mL (p < 0.05, Mann Whitney U-test). The results of real-time RT-PCR show that expressions of fibronectin, collagen type 1, and α smooth muscle were significantly more decreased in all concentrations of mitomycin C and pirfenidone compared to those in TGF-β1 solution. In western blot analysis, expressions of fibronectin and α smooth muscle were decreased in all concentrations of mitomycin C and pirfenidone compared to TGF-β1 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.

Keywords: Anti-fibrotic effect, Cytotoxicity, Mitomycin C, Pirfenidone

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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.

Table 1.

Primer sequences for real time reverse transcription polymerase chain reaction (RT-PCR)

Gene name	Sequence
Fibronectin	F 5’- CCG TGG GCA ACT CTG TC-3’
Fibronectin	R 5’- TGC GGC AGT TGT CAC AG-3’
Collagen type 1	F 5’- CAC CAA TCA CCT GCG GTA CAG AA-3’
Collagen type 1	R 5’- CAG ATC ACG TCA TCG CAC AAC-3’
α smooth muscle	F 5’- GTG TTA TGT AGC TCT GGA CTT TG AAAA-3’
α smooth muscle	R 5’- GGC AGC GGA AAC GTT CAT T-3’
β-actin	F 5’- GCG GGA AAT CGT GCG TGA CAT T-3’
β-actin	R 5’- GAT GGA GTT GAA GGT AGT TTC GTG-3’

F = forward; R = reverse.

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