Effect of Mitomycin C and 5-Fluorouracil on Cultured Human Nasal Mucosa Fibroblasts

Keun Hae Kim; In Taek Kim

doi:10.3341/jkos.2011.52.2.233

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Kim and Kim: Effect of Mitomycin C and 5-Fluorouracil on Cultured Human Nasal Mucosa Fibroblasts

Original Article

J Korean Ophthalmol Soc 2011;52(2):233-240.

Published online: 7 September 2011

DOI: https://doi.org/10.3341/jkos.2011.52.2.233

Effect of Mitomycin C and 5-Fluorouracil on Cultured Human Nasal Mucosa Fibroblasts

Keun Hae Kim, MD¹, In Taek Kim, MD²

¹Department of Ophthalmology, Catholic University of Daegu School of Medicine, Daegu, Korea

²Department of Ophthalmology, Kyungpook National University School of Medicine, Daegu, Korea

Address reprint requests to Keun Hae Kim, MD Department of Ophthalmology, Daegu Catholic University Medical Center #3056-6 Daemyeong 4-dong, Nam-gu, Daegu 705-718, Korea Tel: 82-53-650-4148, Fax: 82-53-627-0133, E-mail: kimkh@cu.ac.kr

Received 27 December 20105 January 2011 Accepted 26 January 2011

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

To investigate the effects of two antimetabolites, mitomycin C (MMC) and 5-fluorouracil (5-FU), on proliferation of cultured human nasal mucosa fibroblasts.

Methods

Human nasal mucosa fibroblasts were primarily cultured, and exposed to various concentrations of MMC and 5-FU for 5 minutes. Control fibroblasts were exposed to only DMEM media without the drugs. Effect of drugs on cell morphology was observed by phase-contrast microscopy. Cell viability and apoptosis were measured using MTT [3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay and Acridine orange/Hoechst (AO/HO) staining, respectively.

Results

In both experimental groups exposed to MMC and 5-FU, fibroblasts maintained standard spindle shape. The MTT assay showed that both MMC and 5-FU inhibited fibroblast proliferation in a dose dependent manner. AO/HO staining showed apoptotic cells in both experimental groups.

Conclusions

Both MMC and 5-FU have an antiproliferative effect on fibroblasts in vitro at least through induction of apoptosis. Therefore, adjuvant use of either MMC or 5-FU during endonasal dacryocystorhinostomy may improve the clinical outcome by inhibiting proliferation of the nasal mucosa.

Keywords: 5-fluorouracil, Mitomycin C, Nasal mucosa fibroblast

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Figure 1.

The morphology of cultured human nasal mucosa fibroblasts under phase-contrast microscope. Fibroblasts were treated with various concentrations of MMC for 5 minutes and then incubated. Images are representatives of day 1 of culture. The viable cells were spindle-shaped at all concentrations of MMC. Magnification, ×100. MMC = mitomycin C.

Figure 2.

The morphology of cultured human nasal mucosa fibroblasts under phase-contrast microscope. Fibroblasts were treated with various concentrations of 5-FU for 5 minutes and then incubated. Images are representatives of day 1 of culture. The viable cells were spindle-shaped at all concentrations of 5-FU. Magnification, ×100. 5-FU=5-fluorouracil.

Figure 3.

The viability of the cultured human nasal mucosa fibroblasts treated with various concentrations of MMC. Fibroblasts were treated for 5 minutes with MMC (0.01, 0.1, 0.2, 0.4 mg/ml), washed and further incubated for 24 hr. Optical density (OD) of the fibroblasts was measured using MTT assay. Growth rates (%) were calculated as [100 × (OD values of drug-treated cells/OD values of non-treated cells)]. Values and bars represent means of growth rates and the SD, respectively. ∗ p<0.05 compared to non-treated control. MMC = mitomycin C.

Figure 4.

The viability of the cultured human nasal mucosa fibroblasts treated with various concentrations of 5-FU. Fibroblasts were treated for 5 minutes with 5-FU (0.5, 5, 10, 25 mg/ml), washed and further incubated for 24 hr. Optical density (OD) of the fibroblasts was measured using MTT assay. Growth rates (%) were calculated as [100 × (OD values of drug-treat-ed cells/OD values of non-treated cells)]. Values and bars represent means of growth rates and the SD, respectively. ∗ p<0.05 compared to non-treated control. 5-FU = 5-fluorouracil.

Figure 5.

Acridine orange/Hoechst (AO/HO) staining of human nasal mucosa fibroblasts after treatment with MMC and 5-FU. Fibroblasts were not-treated (A), or treated with 0.4 mg/ml of MMC (B) and 25 mg/ml of 5-FU (C) for 5 minutes, washed and further incubated for 3 hr. The cells were then stained with AO/HO to identify apoptosis as shown in the Methods. (A) Normal control cells. Arrow indicates blue nuclei and brilliant red cytoplasm. (B) MMC-treated cells. The apoptotic cell shows orange-red nuclei, becoming indistinguishable from the cytoplasm. (C) 5-FU-treated cells. The apoptotic cell shows orange-red nuclei, becoming indistinguishable from the cytoplasm. Magnification, ×400. MMC = mitomycin C, 5-FU = 5-fluorouracil.

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