bongkrekic-acid and Neuroblastoma

Cell models of neurodegenerative diseases (NDD) can involve expression of mutant nuclear genes associated with Mendelian forms of the diseases or effects of toxins believed to replicate essential disease features. Death produced by exposing neural cells to methylpyridinium ion (MPP(+)) or neurotoxic beta amyloid (BA) peptides is frequently used to study features of the sporadic, most prevalent forms of Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. We examined in replicating SH-SY5Y human neuroblastoma cells the release of cytochrome C into cytoplasm, activation of caspases 9 and 3, and loss of calcein retention as markers of the "mitochondrial" pathway of cell death. Exposure to 5 mM MPP(+), which induces apoptotic cell death within 18-24 hr, released cytochrome C within 4 hr, activated caspases 9 and 3, and reduced calcein accumulation. BA 25-35 peptide produced more rapid and greater elevations of caspase 3 activity; no effects were observed with the nontoxic BA 35-25 reverse sequence. The dependence on mitochondrial transition pore (MTP) activity of MPP(+)-induced caspase activations was demonstrated by preincubation with bongkreckic acid, which blocked elevations of caspases 9 and 3. Stereoisomers of pramipexole (PPX), a free radical scavenger and inhibitor of MTP opening, inhibited caspase activation (MPP(+) and BA) and restored calcein accumulation (MPP(+)). Our results demonstrate that MPP(+) and BA can induce cell death through MTP-dependent activation of caspase cascades. PPX stereoisomers interfere with activation of these cell death pathways and may be useful clinically as neuroprotectants in PD and AD and related diseases.

Topics: 1-Methyl-4-phenylpyridinium; Alzheimer Disease; Amyloid beta-Peptides; Anti-Bacterial Agents; Aristolochic Acids; Benzothiazoles; Bongkrekic Acid; Brain; Caspase Inhibitors; Caspases; Cell Death; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Humans; Neuroblastoma; Neurodegenerative Diseases; Neurons; Parkinson Disease; Peptide Fragments; Phenanthrenes; Pramipexole; Thiazoles; Tumor Cells, Cultured

We have attempted to elucidate the precise mechanism of nitric oxide (NO)-induced apoptotic neuronal cell death. Enzymatic cleavages of DEVD-AFC, VDVAD-AFC, and LEHD-AFC (specific substrates for caspase-3-like protease (caspase-3 and -7), caspase-2, and caspase-9, respectively) were observed by treatment with NO. Western blot analysis showed that pro-forms of caspase-2, -3, -6, and -7 are decreased during apoptosis. Interestingly, Ac-DEVD-CHO, a caspase-3-like protease inhibitor, blocked not only the decreases in caspase-2 and -7, but also the formation of p17 from p20 in caspase-3 induced by NO, suggesting that caspase-3 exists upstream of caspase-2 and -7. Bongkrekic acid, a potent inhibitor of mitochondrial permeability transition, specifically blocked both the loss of mitochondrial membrane potential and subsequent DNA fragmentation in response to NO. Thus, NO results in neuronal apoptosis through the sequential loss of mitochondrial membrane potential, caspase activation, and degradation of inhibitor of caspase-activated DNase (CAD) (CAD activation).

Topics: Apoptosis; Bongkrekic Acid; Caspase 2; Caspase 3; Caspase 7; Caspase 9; Caspases; Coumarins; DNA Fragmentation; Enzyme Activation; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Neuroblastoma; Nitric Oxide; Oligopeptides; Serine Proteinase Inhibitors; Substrate Specificity; Tumor Cells, Cultured

Different classes of anticancer drugs may trigger apoptosis by acting on different subcellular targets and by activating distinct signaling pathways. Here, we report that betulinic acid (BetA) is a prototype cytotoxic agent that triggers apoptosis by a direct effect on mitochondria. In isolated mitochondria, BetA directly induces loss of transmembrane potential independent of a benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone-inhibitable caspase. This is inhibited by bongkrekic acid, an agent that stabilizes the permeability transition pore complex. Mitochondria undergoing BetA-induced permeability transition mediate cleavage of caspase-8 (FLICE/MACH/Mch5) and caspase-3 (CPP32/Yama) in a cell-free system. Soluble factors such as cytochrome c or apoptosis-inducing factor released from BetA-treated mitochondria are sufficient for cleavage of caspases and nuclear fragmentation. Addition of cytochrome c to cytosolic extracts results in cleavage of caspase-3, but not of caspase-8. However, supernatants of mitochondria, which have undergone permeability transition, and partially purified apoptosis-inducing factor activate both caspase-8 and caspase-3 in cytosolic extracts and suffice to activate recombinant caspase-8. These findings show that induction of mitochondrial permeability transition alone is sufficient to trigger the full apoptosis program and that some cytotoxic drugs such as BetA may induce apoptosis via a direct effect on mitochondria.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-X Protein; Betulinic Acid; Bongkrekic Acid; Caspase 3; Caspase 8; Caspase 9; Caspases; Cytochrome c Group; DNA Fragmentation; Humans; Intracellular Membranes; Kinetics; Membrane Potentials; Mitochondria; Neuroblastoma; Pentacyclic Triterpenes; Permeability; Proto-Oncogene Proteins c-bcl-2; Triterpenes; Tumor Cells, Cultured