Redox Regulation of Apoptosis by Members of the TNF Superfamily (original) (raw)
Cancer Letters, 2002
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves as an extracellular signal triggering apoptosis in tumor cells. However, the molecular mechanisms leading to the apoptosis are largely unknown. To characterize the molecular events involved in TRAIL-induced apoptosis, we examined the association of reactive oxygen species (ROS) in human adenocarcinoma HeLa cells. In this study, we show strong ROS accumulation upon TRAIL induction, with activation of caspases, followed by apoptosis. The pre-treatment with g-glutamylcysteinylglycine or estrogen, both effective antioxidants, significantly attenuated TRAIL-induced apoptosis through the reduction of ROS accumulation and diminished caspases activity. Furthermore, zVAD-fmk, an inhibitor of pan-caspase, effectively inhibited the activation of caspases and prevented apoptosis by TRAIL, although TRAIL-induced ROS generation was not attenuated. These data indicate that ROS may play a role as an upstream mediator of caspases. Taken together, our results suggest that oxidative stress mediates TRAIL-induced apoptosis in HeLa cells.
TNF-Induced Signaling in Apoptosis
1999
Out of the almost 17 members of the TNF superfamily, TNF is probably the most potent inducer of apoptosis. TNF activates both cell-survival and cell-death mechanisms simultaneously. Activation of NF-kB-dependent genes regulates the survival and proliferative effects pf TNF, whereas activation of caspases regulates the apoptotic effects. TNF-induced apoptosis is mediated primarily through the activation of type I receptors, the death domain of which recruits more than a dozen different signaling proteins, which together are considered part of an apoptotic cascade. This cascade does not, however, account for the role of reactive oxygen intermediates, ceramide, phospholipases, and serine proteases which are also inplicated in TNF-induced apoptosis. This cascade also does not explain how type II TNF receptors which lack the death domain, induce apoptosis. Nevertheless, this review of apoptosis signaling will be limited to those proteins that makeup the cascade.
The American Journal of Pathology, 2000
Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours ϳ50% of the cells had died. Exposure of the cells to TNF؉Act D did not block NFB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant ␣-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF؉Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF؉Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF؉Act D causes apoptosis be-cause Act D blocks the transcription of genes required for antioxidant defenses.
TNF-α activates at least two apoptotic signaling cascades
Oncogene, 1998
Apoptosis, the process whereby cells activate an intrinsic death program, can be induced in HeLa cells by TNF-a treatment. The aims of the present study were (i) to examine the precise role and the origin of Reactive Oxygen Species (ROS) in the TNF-a-induced programmed cell death, (ii) to characterize and order the morphological and mitochondrial changes associated with this process and (iii) to link these events with the activation of caspases. Analyses were performed on TNF-a-treated cells in the presence of an anti-oxidant, or of a general caspase inhibitor. To assess the role of mitochondria in the cell death signal transduction, these studies were also realized on HeLa-variant cell lines lacking functional mitochondrial respiratory chain. We show that at least two separate signaling cascades, both mediated by Z-VAD-sensitive caspase(s), contribute to the TNF-a-induced apoptosis of HeLa cells. One signaling pathway involves an early mitochondriadependent ROS production, the other being ROSindependent.
Free Radical Biology and Medicine, 2009
Fas-mediated caspase-dependent cell apoptosis has been well investigated. However, recent studies have shown that Fas can induce nonapoptotic caspase-independent cell death (CICD) when caspase activity is inhibited. Currently, the molecular mechanism of this alternative cell death mediated by Fas remains unclear. In this study, we investigated the signaling pathway of Fas-induced CICD in mouse embryonic fibroblasts (MEFs) whose caspase function was disrupted by the pan-caspase inhibitor Z-VAD-FMK and its coupling to inflammatory responses. Our results revealed that receptor-interacting protein 1 and tumor necrosis factor receptor-associated factor 2 play important roles in FasL-induced CICD. This death is associated with intracellular reactive oxygen species (ROS) production from mitochondria, as a ROS scavenger (BHA), antioxidants (trolox, NAC), and a mitochondrial respiratory chain uncoupler (rotenone) could prevent this event. Furthermore, delayed and sustained JNK activation, mitochondrial membrane potential breakdown, and loss of intracellular GSH were observed. In addition to CICD, FasL also induces cyclooxygenase-2 and MIP-2 gene upregulation, and both responses are attributed to ROS-dependent JNK activation. Taken together, these results demonstrate alternative signaling pathways of Fas upon caspase inhibition in MEFs that are unrelated to the classical apoptotic pathway, but steer cells toward necrosis and an inflammatory response through ROS production.
TNFα and reactive oxygen species in necrotic cell death
Cell Research, 2008
Death receptors, including the TNF receptor-1 (TNF-RI), have been shown to be able to initiate caspase-independent cell death. This form of "necrotic cell death" appears to be dependent on the generation of reactive oxygen species. Recent data have indicated that superoxide generation is dependent on the activation of NADPH oxidases, which form a complex with the adaptor molecules RIP1 and TRADD. The mechanism of superoxide generation further establishes RIP1 as the central molecule in ROS production and cell death initiated by TNFα and other death receptors. A role for the sustained JNK activation in necrotic cell death is also suggested. The sensitization of virus-infected cells to TNFα indicates that necrotic cell death may represent an alternative cell death pathway for clearance of infected cells.
More Than One Way to Die: Methods to Determine TNF-Induced Apoptosis and Necrosis
Tumor Necrosis Factor, 2004
In most cellular systems tumor necrosis factor (TNF) induces apoptotic cell death. However, in some particular cell lines, such as the L929sA fibrosarcoma, TNF induces necrotic cell death. This effect is not the result of an inability to die apoptotically, because triggering of Fas in L929sAhFas cells leads to apoptosis. Moreover, TNFR-1-induced necrosis can be reverted to apoptosis when cells are pretreated with geldanamycin, an Hsp90 inhibitor. In contrast, addition of caspase-inhibitors (zVAD-fmk) prevents Fas-induced apoptosis and switches it to necrosis. These results demonstrate that depending on the cellular context, the same stimulus can induce either apoptosis or necrosis. Apoptosis and necrosis are clearly distinguished by their morphology, although in the absence of phagocytosis, the late stage of apoptosis is associated with secondary necrotic cell death, which is hard to distinguish from necrotic cell death. Necrosis is described mostly in negative terms as cell death that is characterized by the absence of apoptotic parameters, such as caspase activation, cytochrome c release, and DNA fragmentation. Here we describe a selection of techniques used to distinguish both modes of TNFR-1induced cell death, namely apoptotic or necrotic cell death.
Biochemical Journal, 2005
ROS (reactive oxygen species) from mitochondrial and non-mitochondrial sources have been implicated in TNFα (tumour necrosis factor α)-mediated signalling. In the present study, a new class of specific mitochondria-targeted antioxidants were used to explore directly the role of mitochondrial ROS in TNF-induced apoptosis. MitoVit E {[2-(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)ethyl]triphenylphosphonium bromide} (vitamin E attached to a lipophilic cation that facilitates accumulation of the antioxidant in the mitochondrial matrix) enhanced TNF-induced apoptosis of U937 cells. In time course analyses, cleavage and activation of caspase 8 in response to TNF were not affected by MitoVit E, whereas the activation of caspase 3 was significantly increased. Furthermore, there was an increased cleavage of the proapoptotic Bcl-2 family member Bid and an increased release of cytochrome c from mitochondria, in cells treated with TNF in the presence of MitoVit E. We conside...
American Journal of Physiology Gastrointestinal and Liver Physiology, 2008
viously we have shown that both Rac1 and c-Jun NH2-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor (TNF)-␣/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Rac1-mediated ROS production is involved in TNF-␣-induced apoptosis. In this study, we showed that TNF-␣/CHX-induced ROS production and hydrogen peroxide (H2O2)-induced oxidative stress increased apoptosis. Inhibition of Rac1 by a specific inhibitor NSC23766 prevented TNF-␣-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Rac1 by expression of dominant negative Rac1 decreased TNF-␣-induced mitochondrial ROS production. Moreover, TNF-␣-induced cytosolic ROS production was inhibited by Rac1 inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-␣induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Rac1 or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-␣-induced apoptosis requires Rac1-dependent ROS production in intestinal epithelial cells. intestinal epithelial cells-6; N17Rac1; diphenyleneiodonium; JNK1/2; oxidative stress REACTIVE OXYGEN SPECIES, (ROS), namely H 2 O 2 , O 2 •Ϫ , and OH Ϫ , are important mediators in cellular signal transduction cascades regulating proliferation (8), apoptosis (4, 23), and migration (28). Exposure to stimuli such as cytokines or growth factors increases intracellular ROS in a variety of cells including fibroblasts (52), endothelial cells (35), cardiac myocytes (48), and epithelial cells (5), suggesting that these highly reactive molecules play crucial roles in cellular signaling. Tumor necrosis factor-␣ (TNF-␣), a pleiotropic cytokine produced by many cells and originally identified by its cytotoxic effects, induces cell death in some types of cells, and it also elicits a wide range of physiological responses, such as inflammation, cell proliferation, and differentiation. Recent accumulating evidence has demonstrated that ROS are key mediators of many cellular responses to TNF-␣ such as apoptosis (1), transcriptional factor activation (15), calcium spark activation (7), and insulin signaling (20).