cytochrome-c-t and nimesulide

Nimesulide, a popular nonsteroidal anti-inflammatory drug, has been associated with serious hepatotoxicity. Reactive oxygen species (ROS) and mitochondrial perturbations have been implicated in drug induced hepatotoxicity, although their role in the pathway needs exploration. Study was undertaken to elucidate the effect of Fumaria parviflora Lam. (Fp) on nimesulide induced cell death in primary rat hepatocyte cultures. Fp extract treated cells showed increased viability as compared to nimesulide stressed cells as assessed by MTT assay. LDH leakage increased significantly at 500microM nimesulide, and the data suggested that apoptosis was the predominant mechanism responsible for cell death. Nimesulide induced apoptosis was further confirmed by DNA fragmentation and chromatin condensation. Nimesulide exposure increased intracellular ROS, translocation of Bax and Bcl2 followed by mitochondrial depolarization and cytochrome c (Cyt c) release along with caspase-9/-3 activity confirming involvement of mitochondria in nimesulide induced apoptosis. Events like membrane depolarization of mitochondria, expression of Bax, Bcl2, externalization of phosphatidyl serine are substantially reversed by the pre-treatment of Fp extract. Thus, the study indicates that Fp extract modulates critical events regulating pro and anti-apoptotic proteins in mitochondria dependent apoptosis induced by nimesulide.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cells, Cultured; Chromatin Assembly and Disassembly; Cyclooxygenase 2; Cytochromes c; DNA Fragmentation; Fumaria; Genes, bcl-2; Glutathione; Hepatocytes; Lipid Peroxidation; Male; Membrane Potential, Mitochondrial; Mitochondria, Liver; Plant Extracts; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sulfonamides

Epidemiological and animal model studies have suggested that non-steroidal anti-inflammatory drugs (NSAIDs) can act as chemopreventive agents. The cyclooxygenase-2 (COX-2) inhibitor nimesulide shows anti-cancer effect in different type of cancers. In the current study, five breast carcinoma cell lines were used to explore the anti-cancer mechanisms of a nimesulide derivative compound 76. The compound dose dependently suppressed SKBR-3, BT474 and MDA-MB-453 breast cancer cell proliferation with IC(50) of 0.9microM, 2.2microM and 4.0microM, respectively. However, it needs much higher concentrations to inhibit MCF-7 and MDA-MB-231 breast cancer cell growth with IC(50) at 22.1microM and 19.6microM, respectively. Further investigation reveals that compound 76 induced apoptosis in SKBR-3 and BT474 cells. Since these cells are Her2 overexpressing cells, the Her2 intracellular signaling pathways were examined after the treatment. There was no significant changing of kinase activity. However, the cytochrome c release assay indicated that the apoptosis induced by the compound was mediated by the mitochondria. These results suggest that compound 76 selectively induce apoptosis in Her2 overexpressing breast cancer cells through the mitochondria, and could be used as a lead to design more potent derivatives.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2 Inhibitors; Cytochromes c; Female; Humans; Inhibitory Concentration 50; Mitochondria; Receptor, ErbB-2; Sulfonamides

Nimesulide, a preferential COX-2 inhibitor, has been associated with rare idiosyncratic hepatotoxicity. The underlying mechanisms of liver injury are unknown, but experimental evidence has identified oxidative stress as a potential hazard and mitochondria as a target. The aim of this study was to explore whether genetic mitochondrial abnormalities, resulting in impaired mitochondrial function and mildly increased oxidative stress, might sensitize mice to the hepatic adverse effects of nimesulide. We used heterozygous superoxide dismutase 2 (Sod2(+/-)) mice as a model, as these mice develop clinically silent mitochondrial stress but otherwise appear normal. Nimesulide was administered for 4 weeks (10 mg/kg, ip, bid), at a dose equivalent to human therapeutic dosage. We found that the drug potentiated hepatic mitochondrial oxidative injury (decreased aconitase activity, increased protein carbonyls) in Sod2(+/-), but not wild-type, mice. Furthermore, the nimesulide-treated mutant mice exhibited increased hepatic cytosolic levels of cytochrome c and caspase-3 activity, as well as increased numbers of apoptotic hepatocytes. Finally, nimesulide in vitro caused a concentration-dependent net increase in superoxide anion in mitochondria from Sod2(+/-), but not Sod2(+/+) mice. In conclusion, repeated administration of nimesulide can superimpose an oxidant stress, potentiate mitochondrial damage, and activate proapoptotic factors in mice with genetically compromised mitochondrial function.

Topics: Aconitate Hydratase; Adenosine Triphosphate; Animals; Apoptosis; Caspases; Cytochromes c; Cytosol; Female; Glutathione; Heterozygote; In Situ Nick-End Labeling; Male; Mice; Mice, Knockout; Mitochondria, Liver; Oxidative Stress; Sulfonamides; Superoxide Dismutase; Superoxides