Tumour necrosis factor-alpha- vs. growth factor deprivation-promoted cell death: distinct converging pathways

Perturbations of neuronal physiological homeostasis are likely to underscore neuronal demise/impairments that are reportedly associated with aging of the central nervous system and age-related neurodegenerative diseases such as Alzheimer's disease (AD). A number of age- and/or disease-associated neurotoxic events has been described. These include abnormally modified proteins such as beta amyloid and hyper-phosphorylated Tau, cytokines such as tumour necrosis factor-alpha (TNFalpha), high levels of free radicals conducive to oxidative stress, and impaired/decreased neuronal trophic support by neurotrophic factors. Overall, it could be argued that toxic events in the aged brain are either active, such as those due to a direct action of cytokines, or passive, such as those due to lack of growth factor support. It is therefore conceivable that cellular responses to such diverse toxic stimuli are different, suggesting that interventions should be targeted accordingly. In order to begin answering this question, we determined in PC12 cells the time course of activity, in response to TNFalpha (active) or growth factor withdrawal (passive), of protein kinase c-zeta (PKCzeta), nuclear factor kappa B (NFkappaB), caspases 3 and 8, and poly (ADP-ribose) polymerase (PARP), key signal transduction elements associated with modulation of cell death/survival in PC12 cells. We found that the overall activity of PKCzeta, NFkappaB and caspase 8 was significantly different depending on the apoptotic initiator. The pattern of caspase 3 and PARP activity, however, was not statistically different between serum-free- and TNFalpha-induced cell death conditions. This suggests that two distinct cell responses are elicited that converge at caspase 3, which then induces downstream events involved in the execution of a common apoptotic programme. These results contribute to the aim of differentially targeting neuronal death in the aged brain (characterized by neurotrophic factor impairments) or in the diseased brain (e.g. AD, characterized by elevated levels of pro-inflammatory cytokines).     

Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.     

TRAIL-induced apoptosis proceeding from caspase-3-dependent and -independent pathways in distinct HeLa cells

The apoptotic pathway in higher eukaryotes remains controversial with respect to the necessity of activation of caspase-3 in TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-treated cells. In this study, a fluorescence resonance energy transfer (FRET) probe was developed to image the activation of caspase-3 and the related apoptotic pathway in TRAIL-treated cells in real time. Both kinds of apoptotic pathways were observed simultaneously in the same experiment proceeding from activation and non-activation of caspase-3. The total apoptotic rate was 56.08%, the apoptotic rates for activation and non-activation of caspase-3 pathways were 21.5% and 34.58%, respectively, which were examined later for Hoechst 33258 staining and morphological characteristics. The apoptotic rate due to the activation of caspase-3 pathways in TRAIL-treated cells has been independently measured to be around 25.11% by capillary electrophoresis (CE) analysis, which confirmed the apoptotic rate due to activation of caspase-3 pathways as found by FRET analysis. This result also suggests that rest apoptosis is preceded by caspase-3-independent pathways, as CE has the ability to quantitatively detect caspase-dependent apoptosis. The observation of the coexistence of caspase-3-dependent and caspase-3-independent apoptotic pathways in the TRAIL-treated cells was unusual in comparison with the previous reports.     

Phenylarsine oxide interferes with the death inducing signaling complex and inhibits tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis

The mechanism by which tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death is the subject of intense scrutiny due to its preferential targeting of transformed cells for deletion. Based on recent findings that the TRAIL-dependent death inducing signaling complex (DISC) forms and signals at the plasma membrane without being internalized, we investigated the possibility that agents that prevent endocytosis may stabilize the surface bound DISC and thereby enhance TRAIL-dependent signaling. We utilized phenylarsine oxide (PAO), a trivalent arsenical that has been reported to inhibit endocytosis and to induce mitochondrial permeability transition. Therefore PAO could, by two separate and independent activities, enhance TRAIL-induced killing. Paradoxically, we found that rather than synergizing with TRAIL, PAO was an effective inhibitor of TRAIL-induced killing. Recruitment of FADD and caspase-8 to the TRAIL-dependent DISC was diminished in a concentration-dependent manner in cells exposed to PAO. The effects of PAO could not be reversed by washing cells under non-reducing conditions, suggesting covalent linkage of PAO with its cellular target(s); however, 2,3-dimercaptoethanol effectively overcame the inhibitory action of PAO and restored sensitivity to TRAIL-induced apoptosis. PAO inhibited formation of the TRAIL-dependent DISC and therefore prevented all subsequent apoptotic events.     

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)-1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL-1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL-1 receptors [IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII) and IL-1 receptor accessory protein (IL-1RAcP)] on microglia. In the present study we investigated whether microglia express IL-1 receptors and whether they present target or modulatory properties for IL-1 actions. RT-PCR analysis demonstrated lower expression of IL-1RI and higher expression of IL-1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL-1RI, IL-1RII and IL-1RAcP mRNAs, induced the release of IL-1beta, IL-6 and prostaglandin-E2 (PGE2), and activated nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) p38, and extracellular signal-regulated protein kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK) in microglial cultures. In comparison, IL-1beta induced the release of PGE2, IL-6 and activated NF-kappaB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL-1beta also failed to affect LPS-primed microglial cells. Interestingly, a neutralizing antibody to IL-1RII significantly increased the concentration of IL-1beta in the medium of LPS-treated microglia and exacerbated the IL-1beta-induced IL-6 release in mixed glia, providing the first evidence that microglial IL-1RII regulates IL-1beta actions by binding excess levels of this cytokine during brain inflammation.     

Metabolic stress in insulin's target cells leads to ROS accumulation - a hypothetical common pathway causing insulin resistance

The metabolic syndrome is a cluster of cardiovascular risk factors, and visceral adiposity is a central component that is also strongly associated with insulin resistance. Both visceral obesity and insulin resistance are important risk factors for the development of type 2 diabetes. It is likely that adipose tissue, particularly in the intra-abdominal depot, is part of a complex interplay involving several tissues and that dysregulated hormonal, metabolic and neural signalling within and between organs can trigger development of metabolic disease. One attractive hypothesis is that many factors leading to insulin resistance are mediated via the generation of abnormal amounts of reactive oxygen species (ROS). There is much evidence supporting that detrimental effects of glucose, fatty acids, hormones and cytokines leading to insulin resistance can be exerted via such a common pathway. This review paper mainly focuses on metabolic and other 'stress' factors that affect insulin's target cells, in particular adipocytes, and it will highlight oxidative stress as a potential unifying mechanism by which these stress factors promote insulin resistance and the development and progression of type 2 diabetes.     

Cellular FLIPL plays a survival role and regulates morphogenesis in breast epithelial cells

Strong evidences support the inhibitory activity of cellular FLICE-inhibitory protein (FLIP) in the apoptotic signalling by death receptors in tumor cells. However, little is known about the role of FLIP in the regulation of apoptosis in non-transformed cells. In this report, we demonstrate that FLIP_L plays an important role as a survival protein in non-transformed breast epithelial cells. Silencing of FLIP_L by siRNA methodology enhances TRAIL-R2 expression and activates a caspase-dependent cell death process in breast epithelial cells. This cell death requires the expression of TRAIL, TRAIL-R2, FADD and procaspase-8 proteins. A mitochondria-operated apoptotic pathway is partially required for FLIP_L siRNA-induced apoptosis. Interestingly, FLIP_L silencing markedly abrogates formation of acinus-like structures in a three-dimensional basement membrane culture model (3D) of the human mammary MCF-10A cell line through a caspase-8 dependent process. Furthermore, over-expression of FLIP_L in MCF-10A cells delayed lumen formation in 3D cultures. Our results highlight the central role of FLIP in maintaining breast epithelial cell viability and suggest that the mechanisms regulating FLIP levels should be finely controlled to prevent unwanted cell demise.     

Cellular inhibitor of apoptosis 1 (cIAP-1) degradation by caspase 8 during TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent with high selectivity for malignant cells. Many tumors, however, are resistant to TRAIL cytotoxicity. Although cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and -2) are often over-expressed in cancers, their role in mediating TRAIL resistance remains unclear. Here, we demonstrate that TRAIL-induced apoptosis of liver cancer cells is associated with degradation of cIAP-1 and X-linked IAP (XIAP), whereas cIAP-2 remains unchanged. Lower concentrations of TRAIL causing minimal or no apoptosis do not alter cIAP-1 or XIAP protein levels. Silencing of cIAP-1 expression, but not XIAP or cIAP-2, as well as co-treatment with a second mitochondrial activator of caspases (SMAC) mimetic (which results in rapid depletion of cIAP-1), sensitizes the cells to TRAIL. TRAIL-induced loss of cIAP-1 and XIAP requires caspase activity. In particular, caspase 8 knockdown stabilizes both cIAP-1 and XIAP, while caspase 9 knockdown prevents XIAP, but not cIAP-1 degradation. Cell-free experiments confirmed cIAP-1 is a substrate for caspase 8, with likely multiple cleavage sites. These results suggest that TRAIL-mediated apoptosis proceeds through caspase 8-dependent degradation of cIAP-1. Targeted depletion of cIAP-1 by SMAC mimetics in conjunction with TRAIL may be beneficial for the treatment of human hepatobiliary malignancies.     

Rottlerin activates AMPK possibly through LKB1 in vascular cells and tissues

AMP-activated protein kinase (AMPK) is a cellular energy sensor involved in multiple cell signaling pathways that has become an attractive therapeutic target for vascular diseases. It is not clear whether rottlerin, an inhibitor of protein kinase Cδ, activates AMPK in vascular cells and tissues. In the present study, we have examined the effect of rottlerin on AMPK in vascular smooth muscle cells (VSMCs) and isolated rabbit aorta. Rottlerin reduced cellular ATP and activated AMPK in VSMCs and rabbit aorta; however, inhibition of PKCδ by three different methods did not activate AMPK. Both VSMCs and rabbit aorta expressed the upstream AMPK kinase LKB1 protein, and rottlerin-induced AMPK activation was decreased in VSMCs by overexpression of dominant-negative LKB1, suggesting that LKB1 is involved in the upstream regulation of AMPK stimulated by rottlerin. These data suggest for the first time that LKB1 mediates rottlerin-induced activation of AMPK in vascular cells and tissues.     

Divergent effect of proteasome inhibition on interleukin-1beta and tumor necrosis factor alpha signaling in human astroglial cells

Impaired functioning of the proteasome pathway is one of the molecular mechanism underlying neurodegenerative changes in Alzheimer's disease. In this study, we report that dysfunction of the proteasome pathway in astroglial cells leads to decreased survival and dysregulation of chemokines by differential regulation of the nuclear factor kappa B and c-jun N-terminal kinase (JNK) pathways. We further demonstrated that proteasome inhibition augmented interleukin-1 beta- and tumor necrosis factor-alpha-induced activation of the IkappaBalpha kinase and MKK4/JNK/c-Jun pathway along with TAK1 activation. These results suggest that impaired function of the proteasome pathway may potentiate the immuno-pathologic role of secondarily activated astrocytes in the brain.     

AZD9291 promotes autophagy and inhibits PI3K/Akt pathway in NSCLC cancer cells

AZD9291, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is highly selective against EGFR T790M-mutant non–small cell lung cancer (NSCLC). On investigating the growth inhibitory effects of AZD9291 on NSCLC and the underlying mechanism, we found that AZD9291 can trigger autophagy-mediated cell death in both A549 and H1975 cells by increasing the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3) and decreasing the expression of p62. In the presence of the autophagy inhibitor chloroquine, the AZD9291-induced increase in LC3 level was further augmented. AZD9291 decreased the levels of phosphoinositide-3 kinase (PI3K), protein kinase B (Akt), and phosphorylated Akt. AZD9291-induced cell death was enhanced by Akt knockdown, and the levels of both EGFR and phosphorylated EGFR were decreased by AZD9291. AZD9291 was also found to significantly suppress the tumor growth in H1975 xenograft nude mice. Thus, AZD9291 was found to induce autophagy, decrease in EGFR levels, and show a strong inhibitory effect on NSCLC both in vitro and in vivo. Furthermore, the PI3K/Akt signaling pathway was found to play a critical role in AZD9291-induced cell death.     

Brain-derived neurotrophic factor stimulates β-amyloid gene promoter activity by a Ras-dependent/AP-1-independent mechanism in SH-SY5Y neuroblastoma cells

The beta-amyloid peptide, the major component of Alzheimer-associated plaques, derives from a larger beta-amyloid precursor protein (APP), that is expressed in both neural and non-neural cells. Overexpression of APP actively contributes to the development of senile plaques and is considered a risk factor for the disease. APP expression is regulated by a variety of cellular mediators, among them ligands of tyrosine kinase receptors. In this study, we present evidence that brain-derived neurotrophic factor (BDNF) modulates, in a dose- and time-dependent fashion, APP promoter activity in SH-SY5Y neuroblastoma cells transiently expressing the receptor TrkB. The APP promoter contains two potential AP-1 sites, and we examined whether or not protein kinase C (PKC) and the AP-1 sites of the promoter mediate the BDNF-induced stimulation of APP. Stimulation of APP promoter activity by BDNF was not affected by the PKC inhibitor bisindolylmaleimide, or by dominant negative mutants of the AP-1 components Fos and Jun, which, however, blocked the response to phorbol esters. These results suggest that activation of the APP promoter by BDNF is largely independent of PKC and AP-1. In contrast, activated Ras increased APP promoter activity in SH-SY5Y cells, and a dominant negative mutant of Ras abolished BDNF-mediated promoter stimulation. Taken together, our results suggest a mechanism that involves activation of the Ras/MAP kinase signaling pathway, and phosphorylation of as yet unidentified effectors which in turn can activate response elements within the APP promoter.     

p75 neurotrophin receptor is required for constitutive and NGF-induced survival signalling in PC12 cells and rat hippocampal neurones

We have previously shown that nerve growth factor (NGF)-induced activation of nuclear factor-kappaB increased neuronal expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein. In the present study we determined the role of the p75 neurotrophin receptor in constitutive and NGF-induced survival signalling. Treatment of rat pheochromocytoma (PC12) cells with a blocking anti-rat p75 antibody or inhibition of p75 expression by antisense oligonucleotides reduced constitutive and NGF-induced bcl-xL expression. Treatment with the blocking anti-p75 antibody also inhibited NGF-induced activation of the survival kinase Akt. Inhibition of phosphatidylinositol-3-kinase (PI3 kinase) activity or overexpression of a dominant-negative mutant of Akt kinase inhibited NGF-induced nuclear factor-kappaB activation. Activation of Akt kinase by NGF was also observed in PC12nnr5 cells and cultured rat hippocampal neurones which both lack significant TrkA expression. Treatment of hippocampal neurones with the blocking anti-p75 antibody inhibited constitutive and NGF-induced Bcl-xL expression, activation of Akt, and blocked the protective effect of NGF against excitotoxic and apoptotic injury. Our data suggest that the p75 neurotrophin receptor mediates constitutive and NGF-induced survival signalling in PC12 cells and hippocampal neurones, and that these effects are mediated via the PI3-kinase pathway.     

Aplasia Ras homologue member I overexpression induces apoptosis through inhibition of survival pathways in human hepatocellular carcinoma cells in culture and in xenograft

The aim of the present study was to determine the effects of ARHI (aplasia Ras homologue member I; also known as DIRAS3), a member of the Ras superfamily, on HCC (hepatocellular carcinoma) cells and to define the molecular pathways involved. Stable transfection of ARHI into the HCC cell line Hep3B that lacks expression of this gene reduced cell proliferation significantly as compared with the transfection of empty vector (P<0.01). Moreover, the re-expression of ARHI induced significant apoptosis, whereas a few vector transfectants or non-transfected cells displayed apoptosis. Mechanistically, ARHI restoration impeded the activation of both Akt (also called protein kinase B) and NF-κB (nuclear factor κB). In vivo, restoring ARHI also exerted suppressive effects on xenograft tumour growth, which was coupled with increased apoptosis. Together, these results indicate that ARHI has pro-apoptotic effects on HCC cells, which is associated with the inactivation of both Akt and NF-κB survival pathways.