Ceramide-induced cell death in lens epithelial cells.
OBJECTIVE
To determine whether ceramide treatment contributes to reduced cell viability, increased apoptosis, caspase activation, and reactive oxygen species generation in lens epithelial cells.


METHODS
Cell viability was determined by the 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic cell death was determined by 4,6 diamidino-2-phenylindole (DAPI) nuclear staining. Quantitative DNA fragmentation was determined by specific determination of cytosolic mononucleosomes and oligonucleosome-bound DNA. Caspase-3/7 activation was determined by using the Apo-ONE Assay. Detection of reactive oxygen species was achieved by a carboxy-2,7-dichlorofluorescein diacetate (carboxy-H2DCFDA) staining method and lipid peroxidation assay.


RESULTS
C2-ceramide and C6-ceramide reduced primary bovine lens epithelial cell and human lens epithelial cell survival in a dose- and time-dependent manner. The effect of ceramide on cell viability was specific since C2-dihydroceramide, a chemically similar ceramide lacking four to five double-bonds, did not adversely affect lens epithelial cell viability. Release of endogenous natural ceramides by treatment of lens epithelial cells with bacterial sphingomyelinase reduced cell viability. Ceramide-induced apoptosis in lens epithelial cells was determined by nuclear appearance and DNA fragmentation. Apoptosis was induced by exogenous C2-ceramide in a dose-dependent and time-dependent manner and ceramide-mediated apoptosis of lens epithelial cells was associated with caspase-3/7 activation. C2-ceramide treatment resulted in reactive oxygen species generation.


CONCLUSIONS
These results suggest that ceramide reduced cell viability and increased apoptosis in a dose-dependent and time-dependent manner in lens epithelial cells. Ceramide-induced oxidative stress suggests that age-related cataracts may be modulated by ceramide levels in the lens.
Samadi
Abbas
A
Department of Biochemistry, A.T. Still University of Medical Sciences, Kirksville College of Osteopathic Medicine, Kirksville, MO 63501, USA. asamadi@atsu.edu
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002518:Ceramides / Q000008:administration & dosage / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology*; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D013110:Sphingosine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors
D002518:Ceramides; C064769:N-acetylsphingosine; D017382:Reactive Oxygen Species; C101954:N-caproylsphingosine; D020169:Caspases; D013110:Sphingosine