orobol and acetyl-aspartyl-glutamyl-valyl-aspartal

One of the mechanisms of cisplatin cell cytotoxicity is the mitochondria-associated induction of apoptosis. The morphological or functional change of mitochondria in cisplatin-resistant cells has already been reported. Herein we present additional data describing the mitochondrial genomic and functional changes in cisplatin- resistant cells. Cisplatin increased the level of apoptotic cells in cisplatin-sensitive human ovarian carcinoma OV 2008 and C13 cells by 3.90+/-1.01 (SD; N=3) (p<0.01)-fold and 2.03+/-0.20 (SD; N=3) (p<0.01)-fold compared to the basal apoptotic level. This indicates a lower level induction of apoptosis by 50% in cisplatin-resistant OV 2008/C13 *5.25 variant (C13) cells. In both cell types, cisplatin cytotoxicity is mostly inhibited by the caspase-9 inhibitor as well as the caspase-3 inhibitor, Ac-DEVD-CHO, suggesting that the mitochondrial downstream event was functioning well in both the C13 cells and in OV 2008 cells. Mitochondrial transmembrane potential (DeltaPsim) determined by flow cytometry using DiOC6-stained cells revealed a significant depolarization of C13 cells as compared to OV 2008 cells. Treatment of these cells with cisplatin or hydrogen peroxide induces complete mitochondrial DNA damage in OV 2008 cells, while only partial DNA-destruction is observed in C13 cells, strongly suggesting that mitochondria are resistant to cisplatin and oxidative stress response. Continuous oxygen consumption of these cells monitored by a multi-channel dissolved oxygen meter is 1.70-fold higher in OV 2008 cells than C13 cells and the oxygen consumption was decreased by 30% in C13 cells, suggesting mitochondrial respiratory malfunction in these cells. The hypothesis generated here is that mitochondrial DNA resistance to cisplatin and oxidative stress response might be one of the main characteristics concerning the lower level of apoptosis induced by cisplatin. However, the mechanism by which the mitochondrial DNA encoded molecule is involved in cisplatin resistance remains to be determined.

Topics: Apoptosis; Caspase Inhibitors; Cell Line, Tumor; Cisplatin; Cystadenocarcinoma, Serous; DNA, Mitochondrial; Drug Interactions; Drug Resistance, Neoplasm; Female; Flavonoids; Humans; Membrane Potential, Mitochondrial; Mitochondria; Oligopeptides; Ovarian Neoplasms; Oxygen Consumption; Protein Kinase C

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.. 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.. These results indicate that orobol produced DDP sensitivity in human ovarian carcinoma cells by inducing apoptosis through the MT-dependent signaling pathway.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Caspase 3; Caspase Inhibitors; Cisplatin; Cystadenocarcinoma, Serous; Cytochrome c Group; Drug Synergism; Female; Flavonoids; Humans; Microscopy, Confocal; Microscopy, Electron; Mitochondria; Oligopeptides; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Rhodamine 123; Tumor Cells, Cultured