Critical role for mitochondrial oxidative phosphorylation in the activation of tumor suppressors Bax and Bak.
BACKGROUND
Activation of Bax and Bak, which act to permeabilize the mitochondrial membrane, is an essential step in the cell death response and therefore in the suppression of tumorigenesis. However, the mechanisms that regulate activation are poorly understood.


METHODS
Bax and Bak activation (conformational change and dimerization) was monitored in Rat-1 fibroblasts and human cancer cells subjected to endoplasmic reticulum (ER) stress, DNA damage, or tumor necrosis factor-alpha (TNF-alpha) treatment. Pharmacologic inhibitors of reactive oxygen species production, electron transport in the respiratory chain, oxidative phosphorylation, and appropriate controls were used to identify potential modes by which Bax and Bak activation and the cell death response are controlled. The oligomerization state of Bax and Bak was determined by cross-linking and subsequent immunoblot analysis; Bax conformational change was analyzed by immunoprecipitation and immunoblotting with an antibody specific for the active conformation. Cell death was evaluated by dye exclusion.


RESULTS
In both fibroblasts and human cancer cells subjected to cell death stimuli, inhibition of oxidative phosphorylation by use of antimycin A or oligomycin prevented ER stress-, DNA damage-, and TNF-alpha-induced Bax and Bak activation and cell death (UV-induced Rat-1 cell death at 15 hours: control, mean = 33.6%, 95% confidence interval [CI] = 18.8% to 48.4%; antimycin A, mean = 10.0%, 95% CI = 0% to 21.7%; oligomycin, mean = 13.1%, 95% CI = 5.7% to 20.5%; tunicamycin-induced MCF-7 cell death at 9 hours: control, mean = 29.2%, 95% CI = 21.6% to 36.8%; antimycin A, mean = 15.3%, 95% CI = 0.8% to 29.8%; oligomycin, mean = 11.5%, 95% CI = 3.9% to 19.1%; TNF-alpha-induced MCF-7 cell death at 6 hours: control, mean = 24.0%, 95% CI = 12.6% to 35.4%; antimycin A, mean = 8.9%, 95% CI = 3.9% to 13.9%; oligomycin, mean = 13.3%, 95% CI = 10.4% to 16.2%). Increasing and decreasing glycolytic adenosine triphosphate production, by adding glucose and 2-deoxy-D-glucose to the cell growth medium, respectively, neither reversed nor recapitulated, respectively, the effect of compromised oxidative phosphorylation on Bax and Bak activation.


CONCLUSIONS
Oxidative phosphorylation is required for the activation of Bax and Bak and cell death triggered by disparate death stimuli. The reliance of tumor cells on glycolysis in preference to oxidative phosphorylation even under normoxic conditions (Warburg effect) may therefore be a potential means by which these cells evade programmed cell death.
Tomiyama
Arata
A
Second Department of Neurosurgery, Toho University School of Medicine, Tokyo, Japan.
Serizawa
Shinobu
S
Tachibana
Ken
K
Sakurada
Kaori
K
Samejima
Hirotsugu
H
Kuchino
Yoshiyuki
Y
Kitanaka
Chifumi
C
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D015153:Blotting, Western; D002458:Cell Fractionation; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D016001:Confidence Intervals; D003432:Cross-Linking Reagents; D004249:DNA Damage; D019281:Dimerization; D004721:Endoplasmic Reticulum; D005347:Fibroblasts / Q000378:metabolism; D006019:Glycolysis; D006801:Humans; D047468:Immunoprecipitation; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D024101:Mitochondrial Proteins / Q000378:metabolism*; D010085:Oxidative Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D011487:Protein Conformation; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology; D014467:Ultraviolet Therapy; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
D003432:Cross-Linking Reagents; D024101:Mitochondrial Proteins; D017382:Reactive Oxygen Species; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D014415:Tunicamycin