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).
BACKGROUND
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 microM 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 live-dead 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 microM H(2)O(2) and TGF-beta2. A quantitative detection of histone-associated DNA fragmentation by ELISA was performed.


RESULTS
Minocycline concentrations from 1-50 microM showed no toxic effects on CECs. Pre-treatment with 10-40 microM minocycline led to an increase in viability after H(2)O(2) treatment. In addition, minocycline pre-treatment attenuated the increase of histone-associated DNA fragmentation after treatment with H(2)O(2) and TGF-beta2 significantly. When CECs were treated with minocycline and then consecutively with H(2)O(2) or TGF-beta2, RT-PCR and western blot analysis yielded an overexpression of Bcl-2 and XIAP.


CONCLUSIONS
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.
Kernt
Marcus
M
Department of Ophthalmology, Ludwig-Maximilians-Universität, Munich, Germany. marcus.kernt@med.uni-muenchen.de
Hirneiss
C
C
Neubauer
A S
AS
Kampik
A
A
D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008911:Minocycline / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000096:biosynthesis* / Q000235:genetics
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D008911:Minocycline