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B. U. Go, H. C. Oh, D. M. Shin, W. J. Jung, H. B. Ahn, W. C. Park, S. H. Rho; The Experimental Study for Rabbit Conjunctival Epithelial Toxicity on Co-Treatment With Mitomycin-C and Histone Deacetylase Inhibitor(SAHA). Invest. Ophthalmol. Vis. Sci. 2009;50(13):461.
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© ARVO (1962-2015); The Authors (2016-present)
To determined the effect of the co-treatment of the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA) with Mitomycin-C (MMC) in rabbits on their cultured conjuntival epithelial cells. This study based on already published research that co-treatment of SAHA and MMC may be effectively inhibit the proliferation of subconjunctival fibroblasts after glaucoma filteration surgery.
Epithelial cells harvested from the conjunctival tissues of rabbits were cultured in vitro. The determination of experimental groups and the drug concentration followed those methods used in previous experiment. Cultured cell categorized into four groups, and then were given treatment each in BSS, MMC 20µg/ml, SAHA 35µM, a mixture of MMC 20µg/ml and SAHA 35µM for 30-minute. Then, cell viability was measured through trypan blue exclusion, and Hoechst 33342 stain, cell cycle analysis, as well as the measurement of Mitochondrial membrane potential, reactive oxygen species, was performed . Moreover, evaluation of cytotoxicity was conducted through LDH assay. The results were analyzed and the effect of co-treatment of MMC and SAHA on conjunctival epithelial cells was determined.
At the concentration level which shown the 50% cell viability rate for the subconjunctival fibroblasts, the viability rate of conjunctival epithelial cells was to be high at over 80% on the co-trearment group. In the meantime, In the co-trearment group, some nuclear change was shown like as condensation, cleavage, and a cell cycle analysis indicated a high percentage of Sub-G1 cell group compared with the control group. However, which was not a significant level if influences of necrotic cell occurred due to manipulation were excluded. Also, on the co-trearment group, there was little impact on mitochondria, and the incidence of ROS was not observed. In the LDH assay for measuring cell toxicity, the co-treatment group; compared with the control group; showed somewhat enhanced LDH activity (%), which, however, was not deemed to be a high toxicity when we consider the results of positive control.
This study revealed that co-treatment of MMC and SAHA may not induced apoptosis in the cultured conjunctival epithelium. Therefore, the co-treatment of the SAHA with low levels MMC not induced complications can be used as an antimetabolite to effectively inhibit the proliferation of fibroblasts in the glaucoma filteration surgery while minimizing the toxicity to conjunctival epithelium.
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