J Korean Ophthalmol Soc.  2011 Oct;52(10):1215-1221. 10.3341/jkos.2011.52.10.1215.

Effect of Mitomycin C, Dexamethasone, and Cyclosporine A 0.05% on the Proliferation of Human Corneal Keratocytes

• Shin JH
• Kim SJ
• Lee JE
• Lee JS

• KMID: 2215005
• DOI: http://doi.org/10.3341/jkos.2011.52.10.1215

Abstract

PURPOSE
To investigate the biologic effect of mitomycin C, dexamethasone and cyclosporine A 0.05% on cultured human keratocytes in vitro.
METHODS
Human corneal keratocytes were exposed to a concentration of mitomycin C (0.05%), dexamethasone (0.05%) and cyclosporine A (0.05%) for a period of 3, 5, and 10 minutes. MTT-based colorimetric assay was performed to assess the metabolic activity of cellular proliferation and the concentration of type I procollagen COOH-terminal peptide (PIP) and laminin were measured. Cell damage was determined by using the lactate dehydrogenase (LDH) assay. Apoptotic response was evaluated utilizing flow cytometric analysis with Annexin V and propiodium iodide.
RESULTS
The inhibitory effect of cellular proliferation and cytotoxicity in cultured human keratocytes showed a time-dependent response in all drugs. The production of PIP and laminin showed a time-dependent response in cultured cells. Apoptosis was observed in flow cytometry after being treated with mitomycin C, dexamethasone and cyclosporine A. Cyclosporin A resulted in less apoptosis of keratocytes than mitomycin C and dexamethasone.
CONCLUSIONS
The apoptotic response of mitomycin C, dexamethasone and cyclosporine A is associated with the inhibitory effect of human corneal keratocyte proliferation. To decrease corneal opacity, mitomycin C and dexamethasone were more effective than cyclosporine A in the present study. Additionally, a high concentration of cyclosporine A greater than 0.05% is necessary to lower corneal opacity.

Keyword

Cyclosporine; Dexamethasone; Keratocyte; Mitomycin C

MeSH Terms

Annexin A5
Apoptosis
Cell Proliferation
Cells, Cultured
Collagen Type I
Corneal Keratocytes
Corneal Opacity
Cyclosporine
Dexamethasone
Flow Cytometry
Humans
L-Lactate Dehydrogenase
Laminin
Mitomycin
Annexin A5
Collagen Type I
Cyclosporine
Dexamethasone
L-Lactate Dehydrogenase
Laminin
Mitomycin

Figure

• 

  Figure 1. The absorption rate of the water-insolubale formazan dye in cultured human keratocytes exposed in the four eye drops by a scanning spectrometer (ELISA reader). The longer the exposure duration, the more decreased the metabolic activity of keratocytes in all drugs. After 10 minutes of exposure, the metabolic activity of keratocytes in all drugs was decreased more significantly than control (p = 0.03). Mytomicin-C (MMC) showed most prominent inhibitory effect for cellular metabolic activity, and cyclosporine showed minimal inhibitory effect, but the values were not statistically significant.

• 

  Figure 2. PIP titers of cultured human keratocytes in three eye drops. The concentration of PIP was decreased, the longer the exposure duration in all drugs. Mytomicin-C (MMC) and dexamethasone showed more prominent inhibitory effect for PIP level than cyclosporine, but the values were not statistically significant. PIP = procollagen type I COOH-terminal peptide.

• 

  Figure 3. Laminin titers of cultured human keratocytes exposed in three eye drops, Mytomicin-C (MMC), dexamethasone and cyclosporine. The concentration of laminin was decreased, the longer the exposure time in all drugs. MMC and dexamethasone showed more prominent inhibitory effect for PIP level than cyclosporine, but the values were not statistically significant. PIP = procollagen type I COOH-terminal peptide.

• 

  Figure 4. LDH activity of cultured human keratocytes exposed in three eye drops, mitomycin-C (0.05%), dexamethasone (0.05%) and cyclosporine A (0.05%). The lactate dehydrogenase (LDH) titers of cultured human keratocytes showed a kind of time dependent response. The LDH titers of MMC and dexamethasone were higher values than that of cyclosporine at 10 minutues, but the values were not statistically significant. MMC = mitomycin-C.

• 

  Figure 5. Flow cytometric analysis of apoptotic cells using Annexin V FITC after being treated treating with MMC (A), dexamethasone (B) and cyclosporine A (C). In all cases, apoptosis showed a time-dependent response. Cyclosporine A results in less apoptosis of keartocyte than dexamethasone and MMC. MMC = mitomycin-C; FITC = fluorescein isothiocyanate.

Reference

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