Reactive oxygen species regulate FLICE inhibitory protein (FLIP) and susceptibility to Fas-mediated apoptosis in cardiac myocytes.
OBJECTIVE
Fas ligand (FasL) is a key cytokine which initiates apoptosis when FasL binds to its receptor, Fas. Cardiac myocytes are generally resistant to Fas-induced apoptosis. However, sublethal dose of doxorubicin (Dox) can sensitize cardiac myocytes to Fas-induced apoptosis. We investigated the molecular mechanism by which Dox sensitizes cardiac myocytes to Fas-induced apoptosis. FLICE inhibitory protein (FLIP) is a key molecule for blocking Fas-induced apoptosis by functioning as a caspase-8 dominant negative.


RESULTS
FLIP was constitutively expressed in cultured neonatal rat cardiac myocytes. FLIP protein levels were markedly down-regulated by Dox in a time-dependent and dose-dependent manner. Next, we examined the relation of reactive oxygen species (ROS) by Dox to the expression of FLIP. Both of N-acetylcysteine (NAC) and the combination of superoxide dismutase and catalase restored the decreased FLIP in Dox-treated cardiac myocytes to the basal level. NAC also restored the increased formation of thiobarbituric acid-reactive substance after Dox-treatment. Concurrently, the susceptibility to Fas-mediated apoptosis disappeared with the treatments of the antioxidant agents. Hydrogen peroxide down-regulated FLIP in a dose-dependent fashion and also sensitized cardiac myocytes to Fas-induced apoptosis.


CONCLUSIONS
FLIP, an inhibitor of apoptosis induced by cytokines of TNF family, contributes at least partly to Dox-induced sensitization to Fas-mediated apoptosis in cardiac myocytes. The expression of FLIP in cardiac myocytes is regulated by ROS.
Nitobe
Joji
J
First Department of Internal Medicine, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan. jnitobe@med.id.yamagata-u.ac.jp
Yamaguchi
Seiji
S
Okuyama
Masaki
M
Nozaki
Naoki
N
Sata
Masataka
M
Miyamoto
Takuya
T
Takeishi
Yasuchika
Y
Kubota
Isao
I
Tomoike
Hitonobu
H
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002374:Catalase / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000494:pharmacology; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D013997:Time Factors
D000903:Antibiotics, Antineoplastic; D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; D017392:Thiobarbituric Acid Reactive Substances; D004317:Doxorubicin; D002374:Catalase; D013482:Superoxide Dismutase; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D000111:Acetylcysteine