Enhancement of sensitivity to cisplatin by orobol is associated with increased mitochondrial cytochrome c release in human ovarian carcinoma cells.
OBJECTIVE
Based on our previous report showing that orobol, a potent phosphatidylinositol 4-kinase (PI4K) inhibitor, produced cisplatin (DDP) sensitivity, we have determined the mechanism of orobol-sensitization effect.


RESULTS
Orobol produced >2-fold DDP sensitivity in human ovarian carcinoma 2008 cells and its DDP-resistant variant 2008/C13*5.25 cells (C13). Because orobol had no effect on conventional mechanisms such as DDP accumulation or cellular metallothionein and glutathione content, we have focused on the apoptotic signaling pathway. Orobol induced a significant increase in apoptosis in DDP-treated cells, as estimated by frequency of condensed nuclear chromatin with Hoechst 33342 stain, although orobol alone did not have any effect on apoptotic potential. The caspase-3-inhibiting peptide Ac-DEVD-CHO completely inhibited the orobol sensitization effect but did not block DDP cell cytotoxicity per se. Orobol rendered both of these cells resistant to rhodamine 123 (Rh) by more than 2.5-fold, indicating significant decrease of mitochondrial membrane potential (DeltaPsim). Confocal laser microscopy of cells stained with the mitochondria (MT)-specific dye Rh revealed that orobol decreased Rh-fluorescent intensity. Electron microscopy of these cells showed that orobol induced swelling and condensation of MT. Orobol suppressed both naturally expressed and the DDP-induced Bcl-2 expression significantly. Orobol and DDP treatment reduced cytochrome c level in MT determined by Western blot analysis, indicating increased amount of cytochrome c release from MT, whereas orobol alone did not alter the amount of cytochrome c in MT.


CONCLUSIONS
These results indicate that orobol produced DDP sensitivity in human ovarian carcinoma cells by inducing apoptosis through the MT-dependent signaling pathway.
Isonishi
Seiji
S
Department of Obstetrics/Gynecology, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105, Japan.
Saitou
Misato
M
Yasuda
Makoto
M
Ochiai
Kazunori
K
Tanaka
Tadao
T
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3; D061945:Caspase Inhibitors; D002945:Cisplatin / Q000008:administration & dosage / Q000494:pharmacology*; D018284:Cystadenocarcinoma, Serous / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D003574:Cytochrome c Group / Q000378:metabolism*; D004357:Drug Synergism; D005260:Female; D005419:Flavonoids / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D020112:Rhodamine 123 / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D005419:Flavonoids; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D020112:Rhodamine 123; C505407:CASP3 protein, human; D053148:Caspase 3; C059811:orobol; D002945:Cisplatin