TNFα-induced Apoptosis Research Articles

Tumor necrosis factor α sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid.

Regulation of Prostate Cancer Cell Survival by Protein Kinase C∈ Involves Bad Phosphorylation and Modulation of the TNFα/JNK Pathway

Protein kinase Cepsilon (PKCepsilon), a diacyglycerol- and phorbol ester-responsive serine-threonine kinase, has been implicated in mitogenic and survival control, and it is markedly overexpressed in human tumors, including in prostate cancer. Although prostate cancer cells undergo apoptosis in response to phorbol ester stimulation via PKCdelta-mediated release of death factors, the involvement of PKCepsilon in this response is not known. PKCepsilon depletion by RNAi or expression of a dominant negative kinase-dead PKCepsilon mutant potentiated the apoptotic response of PMA and sensitized LNCaP cells to the death receptor ligand TNFalpha. On the other hand, overexpression of PKCepsilon by adenoviral means protected LNCaP cells against apoptotic stimuli. Interestingly, PKCepsilon RNAi depletion significantly enhanced the release of TNFalpha in response to PMA and greatly potentiated JNK activation by this cytokine. Further mechanistic analysis revealed that PMA fails to promote phosphorylation of Bad in Ser(112) in PKCepsilon-depleted LNCaP cells, whereas PKCepsilon overexpression greatly enhanced Bad phosphorylation. This effect was independent of Akt, ERK, or p90Rsk, well established kinases for Ser(112) in Bad. Moreover, expression of a S112A-Bad mutant potentiated PMA-induced apoptosis. Finally, we found that upon activation PKCepsilon accumulated in mitochondrial fractions in LNCaP cells and that Bad was a substrate of PKCepsilon in vitro. Our results established that PKCepsilon modulates survival in prostate cancer cells via multiple pathways.

E3 ubiquitin ligase Mule ubiquitinates Miz1 and is required for TNFα-induced JNK activation

The zinc finger transcription factor Miz1 is a negative regulator of TNFalpha-induced JNK activation and cell death through inhibition of TRAF2 K63-polyubiquitination in a transcription-independent manner. Upon TNFalpha stimulation, Miz1 undergoes K48-linked polyubiquitination and proteasomal degradation, thereby relieving its inhibition. However, the underling regulatory mechanism is not known. Here, we report that HECT-domain-containing Mule is the E3 ligase that catalyzes TNFalpha-induced Miz1 polyubiquitination. Mule is a Miz1-associated protein and catalyzes its K48-linked polyubiquitination. TNFalpha-induced polyubiquitination and degradation of Miz1 were inhibited by silencing of Mule and were promoted by ectopic expression of Mule. The interaction between Mule and Miz1 was promoted by TNFalpha independently of the pox virus and zinc finger domain of Miz1. Silencing of Mule stabilized Miz1, thereby suppressing TNFalpha-induced JNK activation and cell death. Thus, our study reveals a molecular mechanism by which Mule regulates TNFalpha-induced JNK activation and apoptosis by catalyzing the polyubiquitination of Miz1.

TNF-alpha induced PMN apoptosis in whole human blood: Protective effect of SSR180575, a potent and selective peripheral benzodiazepine ligand

The peripheral benzodiazepine receptor (PBR) has been shown to play a key role in the regulation of the mitochondrial process leading to apoptosis. Despite much controversy in the literature on this subject, PBR synthetic ligands (and specifically agonists such as Ro5-4864 and SSR180575) are described as presenting potent anti-apoptotic effect against oxidative stress, TNFα- and tamoxifen-induced apoptosis when the PBR ligand is administrated at a low dose, close to the affinity range of the ligand to its receptor. Such anti-apoptotic activity has already been correlated with a protective effect of PBR ligands against ischemia–reperfusion induced tissue dysfunction. Previously, we had shown that SSR180575 is a specific and high affinity PBR ligand of potential interest in pathological cardiovascular, renal and neurodegenerative indications. Beyond its expression in steroid-producing tissues, heart, liver and kidney, the PBR is also known to be highly expressed in blood cells. In this work, we demonstrate by flow cytometry experiments, that SSR180575, at low concentrations, is able to protect polymorphonuclear leukocytes (PMNs) against TNFα-induced apoptosis in whole blood. Thus, in a new context, SSR180575 again shows potent anti-apoptotic properties. Moreover, TNFα- induced PMN apoptosis appears to be a good surrogate marker for determining SSR180575 blood availability and activity in treated patients.

Synergistic effects of telmisartan and simvastatin on endothelial progenitor cells

Circulating endothelial progenitor cells (EPC) contribute to endothelial replenishment. Telmisartan is an angiotensin-receptor blocker with PPARγ-agonistic properties. PPARγ-agonists and HMG-CoA reductase inhibitors have been shown to enhance EPC number and function. We focused on the effects of telmisartan alone or in combination with simvastatin on EPC. EPC were isolated from healthy human volunteers, cultured and stimulated with telmisartan, simvastatin, or the combination of telmisartan and simvastatin. Telmisartan significantly increased the number of acLDL/lectin double-positive early EPC, the number of colony forming units (EC-CFU) as well as EPC migratory capacity, inhibited TNFα-induced EPC apoptosis and reduced glucose-induced oxidative stress. The telmisartan effect was dose-dependent and could be inhibited by GW9662, indicating a PPARγ-dependent mechanism. The combination of telmisartan and simvastatin led to a significant additive increase in EPC count and function. In wild-type mice, systemic treatment with either telmisartan or simvastatin elevated the number of sca-1/flk-1-positive EPC in bone marrow and peripheral blood, spleen-derived acLDL/lectin double-positive EPC, EPC migration and EC-CFU. Consistent with the in vitro findings, the combination of telmisartan and simvastatin resulted in a further enhancement of EPC counts. Re-endothelialization after carotid injury was significantly enhanced by telmisartan, simvastatin and the combination. Telmisartan increases EPC number and function mediated by a PPARγ-dependent mechanism. This effect is further enhanced by combination with simvastatin, suggesting a synergistic activation of potentially diverse intracellular pathways.

Mir-200c Regulates Induction of Apoptosis through CD95 by Targeting FAP-1

Tumor progression shares many characteristics with the process of epithelial-to-mesenchymal transition (EMT). Cells that have undergone an EMT are known to have an increased resistance to apoptosis. CD95/Fas is an apoptosis-inducing receptor expressed on many tissues and tumor cells. During tumor progression CD95 is frequently downregulated, and tumor cells lose apoptosis sensitivity. miR-200 microRNAs repress both the EMT-inducing ZEB1 and ZEB2 transcription factors. We now demonstrate that miR-200c sensitizes cells to apoptosis mediated by CD95. We have identified the apoptosis inhibitor FAP-1 as a target for miR-200c. FAP-1 was demonstrated to be responsible for the reduced sensitivity to CD95-mediated apoptosis in cells with inhibited miR-200. The identification of FAP-1 as an miR-200c target provides a molecular mechanism to explain both the downregulation of CD95 expression and the reduction in sensitivity of cells to CD95-mediated apoptosis that is observed in the context of reduced miR-200 expression during tumor progression.

Inhibition of nuclear factor-kappa B differentially affects thyroid cancer cell growth, apoptosis, and invasion

BackgroundNuclear factor-κB (NF-κB) is constitutively activated in many cancers and plays a key role in promoting cell proliferation, survival, and invasion. Our understanding of NF-κB signaling in thyroid cancer, however, is limited. In this study, we have investigated the role of NF-κB signaling in thyroid cancer cell proliferation, invasion, and apoptosis using selective genetic inhibition of NF-κB in advanced thyroid cancer cell lines.ResultsThree pharmacologic inhibitors of NF-κB differentially inhibited growth in a panel of advanced thyroid cancer cell lines, suggesting that these NF-κB inhibitors may have off-target effects. We therefore used a selective genetic approach to inhibit NF-κB signaling by overexpression of a dominant-negative IκBα (mIκBα). These studies revealed decreased cell growth in only one of five thyroid cancer cell lines (8505C), which occurred through a block in the S-G2/M transition. Resistance to TNFα-induced apoptosis was observed in all cell lines, likely through an NF-κB-dependent mechanism. Inhibition of NF-κB by mIκBα sensitized a subset of cell lines to TNFα-induced apoptosis. Sensitive cell lines displayed sustained activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway, defining a potential mechanism of response. Finally, NF-κB inhibition by mIκBα expression differentially reduced thyroid cancer cell invasion in these thyroid cancer cell lines. Sensitive cell lines demonstrated approximately a two-fold decrease in invasion, which was associated with differential expression of MMP-13. MMP-9 was reduced by mIκBα expression in all cell lines tested.ConclusionsThese data indicate that selective inhibition of NF-κB represents an attractive therapeutic target for the treatment of advanced thyroid. However, it is apparent that global regulation of thyroid cancer cell growth and invasion is not achieved by NF-κB signaling alone. Instead, our findings suggest that other important molecular processes play a critical role in defining the extent of NF-κB function within cancer cells.

M1967 Inhibition of GSK3 Activity Sensitizes Pancreatic Cancer Cells to TNFα-Induced Apoptosis

M1967 Inhibition of GSK3 Activity Sensitizes Pancreatic Cancer Cells to TNFα-Induced Apoptosis

S1694 Protease-Activated Receptor-2 Reduces TNFα-Induced Apoptosis in Colonic Epithelial Cells

overexpressed Plexin B1 compared to normal pancreas tissues. Endogenous Plexin B1 and Met protein were detected in eight pancreatic cancer cell lines, with a variety of expression levels. Sema 4D stimulation activated Met protein, and significantly induced cell migration. Discussion: Here we show that Sema 4D involves in the regulation of cell motility through its receptor Plexin B1. And the results of immunostaining suggest that Sema 4D could play a role in the interaction between tumor cells and tumor microenvironment in pancreatic cancer tissues. As many attempts are now ongoing to target the tumor stroma, these findings provide a new insight into molecular targeted therapies for pancreatic cancer.

Abstract 1267: The RING domain of TRAF2 inhibits TNFα-induced cell death independent of NF-κB activation

Abstract TNFα is a pleiotropic cytokine that activates both the pro-survival IKK/NF-κB and the pro-apoptotic FADD/caspase-8 pathways. TNF receptor associated factor 2 and 5 (TRAF2/5) contain N-terminal RING domains, and TRAF2/5 RING domain-mediated RIP1 ubiquitination is believed to be essential for NF-κB activation and inhibition of cell death in response to TNFα stimulation. In support of this notion, TRAF2/5 double knockout (TRAF2/5 DKO) cells are sensitive to TNFα-induced apoptosis. In addition, the RING domain-deleted TRAF2 (TRAF2-ΔR) has been widely used in the field as a dominant negative to block TNFα-induced JNK and IKK activation in transient overexpression systems. Here, we report that stable expression of TRAF2-ΔR in TRAF2/5 DKO cells at a physiological level completely restores normal TNFα-induced NF-κB activation, but that it neither restores RIP1 polyubiquitination nor inhibits cell death. We also found that the basal and inducible expression of anti-apoptotic proteins in TRAF2-ΔR-expressing cells is normal, yet these cells remain sensitive to TNFα-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. This suggests that two events are required for the inhibition of TNFα-induced cell death: i) NF-κB-dependent expression of anti-apoptotic proteins such as cIAP1/2 and cFLIP; and ii) TRAF2 RING domain-dependent retention of cIAP1 in the TNFR1 complex. Ablation of either event leads to caspase-8 activation and cell death in response to TNFα stimulation. Our data also suggest that RIP1 ubiquitination is not essential for TNFα-induced NF-κB activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1267.

Maslinic acid potentiates the anti-tumor activity of tumor necrosis factor α by inhibiting NF-κB signaling pathway

BackgroundTumor necrosis factor alpha (TNFα) has been used to treat certain tumors in clinic trials. However, the curative effect of TNFα has been undermined by the induced-NF-κB activation in many types of tumor. Maslinic acid (MA), a pharmacological safe natural product, has been known for its important effects as anti-oxidant, anti-inflammatory, and anti-viral activities. The aim of this study was to determine whether MA potentiates the anti-tumor activity of TNFα though the regulation of NF-κB activation.ResultsIn this study, we demonstrate that MA significantly enhanced TNFα-induced inhibition of pancreatic cancer cell proliferation, invasion, and potentiated TNFα-induced cell apoptosis by suppressing TNFα-induced NF-κB activation in a dose- and time-dependent manner. Addition of MA inhibited TNFα-induced IκBα degradation, p65 phosphorylation, and nuclear translocation. Furthermore, MA decreased the expression levels of NF-κB-regulated genes, including genes involved in tumor cell proliferation (Cyclin D1, COX-2 and c-Myc), apoptosis (Survivin, Bcl-2, Bcl-xl, XIAP, IAP-1), invasion (MMP-9 and ICAM-1), and angiogenesis (VEGF). In athymic nu/nu mouse model, we further demonstrated that MA significantly suppressed pancreatic tumor growth, induced tumor apoptosis, and inhibited NF-κB-regulated anti-apoptotic gene expression, such as Survivin and Bcl-xl.ConclusionsOur data demonstrate that MA can potentiate the anti-tumor activities of TNFα and inhibit pancreatic tumor growth and invasion by activating caspase-dependent apoptotic pathway and by suppressing NF-κB activation and its downstream gene expression. Therefore, MA together with TNFα could be new promising agents in the treatment of pancreatic cancer.

Heat shock protein 70 together with its co-chaperone CHIP inhibits TNF-α induced apoptosis by promoting proteasomal degradation of apoptosis signal-regulating kinase1

Inducible heat shock protein70 (HSP70) is one of the most important HSPs for maintenance of cell integrity during normal cellular growth as well as pathophysiological conditions. Apoptosis signal-regulating kinase (ASK) 1, a mammalian MAPKKK, activates the JNK and p38 pathways. Here we report a novel function of HSP70 in regulating TNF-alpha-induced cell apoptosis. Our study demonstrated that HSP70 physically interacted with ASK1 and promoted the ubiquitin-dependent proteasomal degradation of ASK1. CHIP (carboxyl terminus of the HSC70-interacting protein) which acted as a co-chaperone of HSP70 cooperated with HSP70 in regulating ASK1. We also found that TNF-alpha stimulated HSP70/CHIP/ASK1 association and through cooperating with CHIP, HSP70 inhibits TNF-alpha-induced cell apoptosis both in over-expression and RNAi conditions. Structural analysis indicated that C-terminal domain of HSP70 was necessary for ASK1 degradation, and N- terminal domain of ASK1 was essential for its binding to HSP70. All these findings indicated that HSP70 and CHIP association is important for HSP70 in interacting with ASK1. Through forming the complex of HSP70/CHIP/ASK1, HSP70 promotes ASK1 proteasomal degradation and prevents TNF-alpha-induced cell apoptosis.

Reduced vascular responsiveness to adiponectin in hyperlipidemic rats—mechanisms and significance

Deficiency of adiponectin (APN), an adipocyte-derived vascular protective molecule, contributes to diabetic vascular injury. The current study determined whether obesity/hyperlipidemia may alter the vascular response to APN, and investigated the involved mechanisms and pathologic significance. Adult male Sprague–Dawley rats were fed a regular or high-fat diet (HF) for 4–16 weeks. Circulating APN levels, aortic pAMPK/AMPK, peNOS/eNOS, and APN receptor expression levels were determined. Compared to time-matched animals fed control diet, plasma APN levels in HF-diet animals were significantly increased at 8 weeks, and rapidly declined thereafter. Despite unchanged or elevated circulating APN levels, phosphorylated AMPK and eNOS in vascular tissue were significantly reduced at all observed time points. Recombinant full-length APN (rAPN)-induced AMPK/eNOS phosphorylation and vasodilatation were significantly reduced in 16-week obese/hyperlipidemic aortic segments. Vascular APN receptor 1 (AdipoR1) and receptor 2 (AdipoR2) expression were significantly reduced 16 weeks after HF-diet. Pre-incubation of rAPN with obese/hyperlipidemic plasma, but not with normal plasma, significantly reduced its AMPK and eNOS activation effect, and blunted its protective effect against TNFα-induced HUVEC apoptosis. This study demonstrated for the first time that obesity/hyperlipidemia reduces vascular responsiveness to APN. Modification/inactivation of APN by unidentified factors present in obese/hyperlipidemic plasma, decreased vascular AdipoR1/R2 expression, and reduced circulating APN levels contribute to reduced vascular responsiveness to APN at different stages of the obese condition. Reduced APN bioactivity allows unmitigated TNFα pro-apoptotic and pro-inflammatory actions, contributing to vascular injury in obesity/hyperlipidemia.

Exendin-4 Prevents c-Jun N-Terminal Protein Kinase Activation by Tumor Necrosis Factor-α (TNFα) and Inhibits TNFα-Induced Apoptosis in Insulin-Secreting Cells

Glucagon-like peptide-1 and its analogs may preserve pancreatic beta-cell mass by promoting resistance to cytokine-mediated apoptosis. The mechanisms of TNFalpha-induced apoptosis and of its inhibition by exendin-4 were investigated in insulin-secreting cells. INS-1 and MIN6 insulinoma cells were exposed to 20 ng/ml TNFalpha, with or without pretreatment with 10 nm exendin-4. Treatment with TNFalpha increased c-Jun N-terminal protein kinase (JNK) phosphorylation 2-fold, reduced inhibitor-kappaBalpha (IkappaBalpha) protein content by 50%, induced opposite changes in caspase-3 and Bcl-2 protein content, and increased cellular apoptosis. Moreover, exposure to TNFalpha resulted in increased serine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-2 and reduced basal and insulin-induced Akt phosphorylation. However, in the presence of a JNK inhibitor, TNFalpha-induced apoptosis was diminished and serine phosphorylation of IRS proteins was prevented. When cells were pretreated with exendin-4, TNFalpha-induced JNK and IRS-1/2 serine phosphorylation was markedly reduced, Akt phosphorylation was increased, caspase-3 and Bcl-2 protein levels were restored to normal, and TNFalpha-induced apoptosis was inhibited by 50%. This was associated with a 2-fold increase in IRS-2 expression levels. A similar ability of exendin-4 to prevent TNFalpha-induced JNK phosphorylation was found in isolated pancreatic human islets. The inhibitory effect of exendin-4 on TNFalpha-induced JNK phosphorylation was abrogated in the presence of the protein kinase A inhibitor H89. In conclusion, JNK activation mediates TNFalpha-induced apoptosis and impairment of the IRS/Akt signaling pathway in insulin-secreting cells. By inhibiting JNK phosphorylation in a PKA-dependent manner, exendin-4 counteracts TNFalpha-mediated apoptosis and reverses the inhibitory events in the IRS/Akt pathway, resulting in promotion of cell survival.

Abstract 5136: Reduction in Myocardial Ischemia-reperfusion Injury in Group IV Cytosolic Phospholipase A 2 -deficient Mice Partly Due to Inhibition of TNF α -mediated Pathway to Cell Injury

Type IV cytosolic phospholipase A 2 (cPLA 2 ), which preferentially cleaves arachidonic acid from phospholipids, plays an important role in tissue injury. The downstream signal in response to TNF α , a key mediator of myocardial ischemia reperfusion (I/R) injury, involves cPLA 2 activation. This study examined a pathogenic role of cPLA 2 and its mechanistic link with TNF α in myocardial I/R injury using cPLA 2 α knockout mice (cPLA 2 −/− mice). Methods and Results: The cPLA 2 −/− mice with C57BL/6 background used in this study had comparable heart size, heart rate and blood pressure with the littermate cPLA 2 +/+ (wild-type; WT) mice (male at 10 weeks). In vivo myocardial I/R was created in 10-week-old male mice by 1-hr ligation of the left anterior descending coronary artery, followed by 24 hrs of reperfusion. Expression levels of mRNA and protein of TNF α in the ischemic myocardium were increased from baseline by 5-fold similarly in both cPLA 2 −/− and WT mice. Expression of TNF α receptors was unchanged after I/R in both genotypes. cPLA 2 −/− mice had a 45% reduction in myocardial infarct size and a preservation of LV fractional shortening (25% vs. 15%, respectively) after I/R compared with WT mice. The contents of thromboxane B 2 and leukotriene B 4 , arachidonic acid-derived proinflammatory mediators, in injured myocardium were 40% ~ 50% lower in cPLA 2 −/− mice than WT mice. cPLA 2 −/− mice had a 30% decrease in caspase-3 activity and a 40% decrease in TUNEL-positive apoptotic cells in injured myocardium compared with WT mice. In cultured cardiomyocytes from WT mice, hypoxia-reoxygenation (H/R) induced cPLA 2 activation that was suppressed by preincubation with the TNF α -neutralizing antibody. Incubation of cardiomyocytes with TNF α induced apoptosis, cell damage (MTT assay) and production of thromboxane B 2 and leukotriene B 4 that were attenuated in cPLA 2 −/− cardiomyocytes compared with WT cardiomyocytes. H/R exhibited similar results obtained from TNF α . Preincubation with the TNF α antibody significantly inhibited H/R-induced apoptosis and cell damage in WT cardiomyocytes, while it did not in cPLA 2 −/− cardiomyocytes. Conclusions: Disruption of cPLA 2 attenuated myocardial I/R injury partly through inhibition of TNF α -mediated pathway to cell injury and apoptosis.

Abstract 4912: Multidrug Resistance Associated Protein-1 (MRP-1) Regulates Endothelial Cell Apoptosis

The multidrug resistance associated protein-1 (MRP-1) is the main transporter of oxidized glutathione in endothelial cells, and blockade of MRP-1 prevents endothelial cell dysfunction induced by reactive oxygen species (ROS). We therefore sought to investigate the effect of MRP-1 downregulation on TNF α -induced endothelial cell apoptosis. HAEC’s were either transfected with a siRNA against MRP-1 or a negative control siRNA and treated with TNF α /cycloheximide(CHX ) for 3h or 6h. TNF α /CHX lead to endothelial cell apoptosis as indicated by Annexin V staining (FACS analysis) and increased cleavage of caspase 3 (Western blot). Downregulation of MRP-1 via siRNA significantly diminished Annexin V staining and caspase 3 cleavage. TNF α /CHX augmented endothelial cell superoxide production which was significantly attenuated by siRNA against MRP-1. Further, pre-treatment with the glutathione precursor N-acetylcysteine prevented cleavage of caspase 3. TNF α /CHX mediated activation of caspases 7 and 8 were reduced when MRP-1 was downregulated, while no influence was found on phosphorylation levels of the Fas-Associated protein with Death Domain (FADD). TNF α /CHX induced phosphorylation of p38-, ERK- and JNK-MAP-kinases. While phosphorylation of p38 MAP kinase and ERK was not influenced by MRP-1 downregulation, TNF α /CHX induced JNK phosphorylation was significantly attenuated in HAEC’s transfected with MRP-1 siRNA. These data indicate a major role for MRP-1 in regulation of TNF α /CHX-induced endothelial cell apoptosis via modulation of ROS levels, caspase activation, and JNK phosphorylation.

Inflammatory protein sPLA2-IIA abrogates TNFα-induced apoptosis in human astroglioma cells: Crucial role of ERK

Brain injury induces the expression of well-known cytokines, such as tumor necrosis factor-alpha (TNFα), and other, which functions are less understood, as secreted phospholipase A2 group IIA (sPLA2-IIA). Since in pathological processes, cytokines function coordinately in networks, to further explore the actions of sPLA2-IIA in tumorigenesis, we investigated the effect of sPLA2-IIA in the presence of TNFα in human 1321N1 astrocytoma cells. In these cells, TNFα activates the apoptotic programme that is accompanied of cytoskeleton changes; however, simultaneous treatment with sPLA2-IIA prevents TNFα-mediated apoptosis and reverses the modification of the markers associated to this response. In fact, the mitogenic activity elicited by the phospholipase alone is preserved. This inhibitory effect is not found in other TNFα-mediated responses, even a functional cooperation is observed on COX-2 protein induction. The cross-talk between TNFα and sPLA2-IIA is associated with ERK activity since its pharmacological inhibition attenuates both synergistic and inhibitory responses. We have also observed that upon sPLA2-IIA stimulation, endogenous ERK has the capacity to bind and phosphorylate sequences present within the cytoplasmic domain of TNFR1/CD120a. These findings thus indicate that sPLA2-IIA and TNFα transduction pathways interact to modulate inflammatory responses and provide additional insights about the capacity of sPLA2-IIA to promote apoptosis resistance in astrocytoma cells.

RETRACTED: Identification of Phosphorylated p38 as a Novel DAPK-Interacting Partner during TNFα-Induced Apoptosis in Colorectal Tumor Cells

Death-associated protein kinase (DAPK) is a serine/threonine kinase that contributes to pro-apoptotic signaling on cytokine exposure. The role of DAPK in macrophage-associated tumor cell death is currently unknown. Recently, we suggested a new function for DAPK in the induction of apoptosis during the interaction between colorectal tumor cells and tumor-associated macrophages. Using a cell-culture model with conditioned supernatants of differentiated/activated macrophages (U937) and human HCT116 colorectal tumor cells, we replicated DAPK-associated tumor cell death; this model likely reflects the in vivo tumor setting. In this study, we show that tumor necrosis factor-alpha exposure under conditions of macrophage activation induced DAPK-dependent apoptosis in the colorectal tumor cell line HCT116. Simultaneously, early phosphorylation of p38 mitogen-activated protein kinase (phospho-p38) was observed. We identified the phospho-p38 mitogen-activated protein kinase as a novel interacting protein of DAPK in tumor necrosis factor-alpha-induced apoptosis. The general relevance of this interaction was verified in two colorectal cell lines without functional p53 (ie, HCT116 p53(-/-) and HT29 mutant) and in human colon cancer and ulcerative colitis tissues. Supernatants of freshly isolated human macrophages were also able to induce DAPK and phospho-p38. Our findings highlight the mechanisms that underlie DAPK regulation in tumor cell death evoked by immune cells.

Biological Responses to Arsenic Compounds

Arsenic is a metalloid that generates various biological effects on cells and tissues. Depending on the specific tissue exposed and the time and degree of exposure, diverse responses can be observed. In humans, prolonged and/or high dose exposure to arsenic can have a variety of outcomes, including the development of malignancies, severe gastrointestinal toxicities, diabetes, cardiac arrhythmias, and death. On the other hand, one arsenic derivative, arsenic trioxide (As(2)O(3)), has important antitumor properties. This agent is a potent inducer of antileukemic responses, and it is now approved by the Food and Drug Administration for the treatment of acute promyelocytic leukemia in humans. The promise and therapeutic potential of arsenic and its various derivatives have been exploited for hundreds of years. Remarkably, research focused on the potential use of arsenic compounds in the treatment of human diseases remains highly promising, and it is an area of active investigation. An emerging approach of interest and therapeutic potential involves efforts to target and block cellular pathways activated in a negative feedback manner during treatment of cells with As(2)O(3). Such an approach may ultimately provide the means to selectively enhance the suppressive effects of this agent on malignant cells and render normally resistant tumors sensitive to its antineoplastic properties.

Targeting transcription factor NFκB: comparative analysis of proteasome and IKK inhibitors

Nuclear factor-κB (NF-κB) plays a critical role in cancer development and progression. Thus, the NF-κB signaling pathway provides important targets for cancer chemoprevention and anti-cancer chemotherapy. The central steps in NF-κB activation are phosphorylation and proteasome-dependent degradation of its inhibitory proteins termed IκBs. Consequently, the major pharmacological approaches to target NF-κB include (i) repression of IκB kinases (IKKs) and (ii) blocking the degradation of IκBs by proteasome inhibitors. We quantitatively compared the efficacy of various proteasome inhibitors (MG132, lactacystin and epoxomicin) and IKK inhibitors (BAY 11-7082 and PS1145) to block NF-κB activity induced by TNFα or TPA and to sensitize LNCaP prostate carcinoma cells to apoptosis. Our studies revealed significant differences between these two classes of NF-κB inhibitors. We found that proteasome inhibitors epoxomicin and MG132 attenuated NF-κB induction much more effectively than the IKK inhibitors. Furthermore, in contrast to IKK inhibitors, all studied proteasome inhibitors specifically blocked TPA-induced generation de novo of NF-κB p50 homodimers – (p50/p50). These results suggest that the proteasome plays a dominant role in TPA-induced formation of functional p50 homodimers, while IKK activity is less important for this process. Interestingly, profound attenuation of p50/p50 DNA-binding does not reduce the high potency of proteasome inhibitors to suppress NF-κB-dependent transcription. Finally, proteasome inhibitors were much more effective in sensitizing LNCaP cells to TNFα-induced apoptosis compared to IKK inhibitors at the concentrations when both types of agents similarly attenuated NF-κB activity. We conclude that this remarkable pro-apoptotic potential of proteasome inhibitors is partially mediated through NF-κB-independent mechanism.