Generation of reactive oxygen species is an early event in dolichyl phosphate-induced apoptosis.
The mechanism of induction of apoptosis by dolichyl phosphate (Dol-P) was investigated in U937 cells. Studies using isolated mitochondria revealed that the respiratory complex II activity was almost completely inhibited by 20 microg/ml of Dol-P but not by the same concentration of dolichol. Activities of complex I and III were also inhibited by Dol-P, but nearly 50% of activity still remained at 20 microg/ml. Dol-P induced release of cytochrome-c from the isolated mitochondria. Fluorometric microtiter plate assay revealed that generation of reactive oxygen species (ROS) increased in a time-dependent manner. Flow cytometric analysis also indicated that Dol-P caused loss of mitochondrial membrane potential (Deltapsi(m)) and increased ROS generation. The addition of the antioxidant pyrrolidine dithiocarbamate (PDTC) significantly inhibited Dol-P-induced ROS generation and activation of caspase-3. A specific inhibitor of respiratory complex II, thenoyltrifluoroacetone (TTFA), increased ROS generation, potentially mimicking the consequence of inhibition of electron flow at complex II by Dol-P in U937 cells. Electron microscopy revealed that mitochondria became swollen and spherical in shape by the treatment with Dol-P. Neither the tyrosine kinase inhibitor k252a nor mitogen activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitors PD98059 and U0126 inhibited the Dol-P-induced apoptosis. Together, these results suggest that the direct disruption of mitochondrial respiratory complexes and the consequent ROS generation play a critical role in the initiation of Dol-P-induced apoptosis.
Yokoyama
Yoshiko
Y
Division of Molecular Epidemiology, Kobe University Graduate School of Medicine, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
Nohara
Kazunari
K
Okubo
Tomoko
T
Kano
Itsu
I
Akagawa
Keisuke
K
Kano
Kazutaka
K
D017209:Apoptosis / Q000187:drug effects*; D016227:Benzoquinones / Q000633:toxicity; D053148:Caspase 3 / Q000378:metabolism; D061945:Caspase Inhibitors; D045744:Cell Line, Tumor; D019069:Cell Respiration / Q000187:drug effects; D048430:Cell Shape; D045304:Cytochromes c / Q000378:metabolism; D004288:Dolichol Phosphates / Q000494:pharmacology*; D004286:Dolichols / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047029:Lactams, Macrocyclic / Q000633:toxicity; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D011759:Pyrrolidines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D017828:Rifabutin / Q000031:analogs & derivatives; D013859:Thiocarbamates / Q000494:pharmacology; D013997:Time Factors
D016227:Benzoquinones; D061945:Caspase Inhibitors; D004288:Dolichol Phosphates; D004286:Dolichols; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D011759:Pyrrolidines; D017382:Reactive Oxygen Species; D013859:Thiocarbamates; C028306:dolichol monophosphate; D017828:Rifabutin; C020972:pyrrolidine dithiocarbamic acid; C020754:herbimycin; D045304:Cytochromes c; D020928:Mitogen-Activated Protein Kinases; D053148:Caspase 3