The mammalian response to stress is complex, often involving multiple signalling pathways that act in concert to influence cell fate. To examine potential interactions between the signalling cascades, we have focused on the effects of a model oxidant stress in a single cell type through an examination of the relative influences of mitogen-activated protein kinases (MAPKs) as well as two proposed apoptosis regulators, nuclear factor kappaB (NF-kappaB) and Bcl-2, in determining cell survival. Treatment of HeLa cells with H2O2 resulted in a time- and dose-dependent induction of apoptosis accompanied by sustained activation of all three MAPK subfamilies: extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38. This H2O2-induced apoptosis was markedly enhanced when ERK2 activation was selectively inhibited by PD098059. Apoptosis decreased when JNK/SAPK activation was inhibited by expression of a dominant negative mutant form of SAPK/ERK kinase 1. Inhibition of the p38 kinase activity with p38-specific inhibitors SB202190 and SB203580 had no effect on cell survival. Because NF-kappaB activation by H2O2 is potentially related to both the ERK and JNK/SAPK signalling pathways, we examined the effects of inhibiting the activation of NF-kappaB; this interference had no effect on the cellular response to H2O2. Overexpression of the anti-apoptotic protein Bcl-2 significantly decreased the apoptosis seen after treatment with H2O2 without altering ERK or JNK/SAPK activities. Our results suggest that ERK and JNK/SAPK act in opposition to influence cell survival in response to oxidative stress, whereas neither p38 nor NF-kappaB affects the outcome. Bcl-2 acts independently and downstream of ERK and JNK/SAPK to enhance the survival of H2O2-treated cells.

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Dopamine (DA) is a neurotransmitter, but it also exerts a neurotoxic effect under certain pathological conditions, including age-related neurodegeneration such as Parkinson's disease. By using both the 293 cell line and primary neonatal rat postmitotic striatal neuron cultures, we show here that DA induces apoptosis in a time-and concentration-dependent manner. Concomitant with the apoptosis, DA activates the JNK pathway, including increases in JNK activity, phosphorylation of c-Jun, and subsequent increase in c-Jun protein. This DA-induced JNK activation precedes apoptosis and is persistently sustained during the process of apoptosis. Transient expression of a dominant negative mutant SEK1(Lys 3 Arg), an upstream kinase of JNK, prevents both DA-induced JNK activation and apoptosis. A dominant negative c-Jun mutant FLAG⌬169 also reduces DA-induced apoptotic cell death. Anti-oxidants N-acetylcysteine and catalase, which serve as scavengers of reactive oxygen species generated by metabolic DA oxidation, effectively block DA-induced JNK activation and subsequent apoptosis. Thus, our data suggest that DA triggers an apoptotic death program through an oxidative stress-involved JNK activation signaling pathway. Given the fact that the anti-oxidative defense system declines during aging, this molecular event may be implicated in the age-related striatal neuronal cell loss and age-related dopaminergic neurodegenerative disorders, such as Parkinson's and Huntington's diseases.

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

Amyloid ␤-peptide (A␤) is implicated as the toxic agent in Alzheimer's disease and is the major component of brain amyloid plaques. In vitro, A␤ causes cell death, but the molecular mechanisms are unclear. We analyzed the early signaling mechanisms involved in A␤ toxicity using the SH-SY5Y neuroblastoma cell line. A␤ caused cell death and induced a 2-to 3-fold activation of JNK. JNK activation and cell death were inhibited by overexpression of a dominant-negative SEK1 (SEK1-AL) construct. Butyrolactone I, a cdk5 inhibitor, had an additional protective effect against A␤ toxicity in these SEK1-AL-expressing cells suggesting that cdk5 and JNK activation independently contributed to this toxicity. A␤ also weakly activated ERK and Akt but had no effect on p38 kinase. Inhibitors of ERK and phosphoinositide 3-kinase (PI3K) pathways did not affect A␤-induced cell death, suggesting that these pathways were not important in A␤ toxicity. Insulin-like growth factor I protected against A␤ toxicity by strongly activating ERK and Akt and blocking JNK activation in a PI3K-dependent manner. Pertussis toxin also blocked A␤-induced cell death and JNK activation suggesting that G i/o proteins were upstream activators of JNK. The results suggest that activation of the JNK pathway and cdk5 may be initial signaling cascades in A␤-induced cell death.

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