Minocycline is cytoprotective in human corneal endothelial cells and induces anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and X-linked inhibitor of apoptosis (XIAP)

Introduction Loss of corneal endothelial cells (CECs) is one major
factor limiting transplant clarity and survival after keratoplasty.
Amongst other factors, apoptosis due to cellular stress is
responsible for these problems. This study investigates the
possible anti-apoptotic and cytoprotective effects of minocycline
on a human corneal endothelial cell line (HCEC-SV40) cultured under
oxidative stress and with transforming growth factor beta
(TGF-beta). Methods CECs were treated with 1-150 mM minocycline.
Cell viability and the median inhibitory concentration (IC(50))
were evaluated after 48 h and after H(2)O(2) treatment (tetrazolium
dye reduction assay and liveedead assay). Expression of B-cell
CLL/lymphoma 2 (Bcl-2) and X-linked inhibitor of apoptosis (XIAP)
and their mRNA were assessed by reverse transcriptase (RT)-PCR and
western blot analysis after treatment with minocycline alone and
consecutive incubation with 200 mM H(2)O(2) and TGF-beta 2. A
quantitative detection of histone-associated DNA fragmentation by
ELISA was performed. Results Minocycline concentrations from 1-50
mM showed no toxic effects on CECs. Pre-treatment with 10-40 mM
minocycline led to an increase in viability after H(2)O(2)
treatment. In addition, minocycline pretreatment attenuated the
increase of histone-associated DNA fragmentation after treatment
with H(2)O(2) and TGF-beta 2 significantly. When CECs were treated
with minocycline and then consecutively with H(2)O(2) or TGF-beta
2, RT-PCR and western blot analysis yielded an overexpression of
Bcl-2 and XIAP. Conclusion In this study minocycline prevented
apoptotic cell death in cultured CECs in vitro. Our results suggest
that minocycline might offer cytoprotective properties that might
help to prevent loss of corneal endothelial cells in vivo.