TRAIL-induced apoptosis: between tumor therapy and immunopathology

The death ligand members of the tumor necrosis factor (TNF) family are potent inducers of apoptosis in a variety of cell types. In particular, TNF-related apoptosis-inducing ligand (TRAIL) has recently received much scientific and commercial attention because of its potent tumor cell-killing activity while leaving normal untransformed cells mostly unaffected. Furthermore, TRAIL strongly synergizes with conventional chemotherapeutic drugs in inducing tumor cell apoptosis, making it a most promising candidate for future cancer therapy. Increasing evidence indicates, however, that TRAIL may also induce or modulate apoptosis in primary cells. A particular concern is the potential side effect of TRAIL-based tumor therapies in the liver. In this review we summarize some of the recent findings on the role of TRAIL in tumor cell and hepatocyte apoptosis.

Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24.

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.

Total Tumor Volume and Alpha Fetoprotein for selection of transplant candidates with hepatocellular carcinoma: A prospective validation.

The selection of liver transplant candidates with hepatocellular carcinoma (HCC) is currently validated based on Milan criteria. The use of extended criteria has remained a matter of debate, mainly because of the absence of prospective validation. The present prospective study recruited patients according to the previously proposed Total Tumor Volume (TTV ≤115 cm(3) )/alpha fetoprotein (AFP ≤400 ng/ml) score. Patients with AFP >400 ng/ml were excluded, and as such the Milan group was modified to include only patients with AFP <400 ng/ml; these patients were compared to patients beyond Milan, but within TTV/AFP. From January 2007 to March 2013, 233 patients with HCC were listed for liver transplantation. Of them, 195 patients were within Milan, and 38 beyond Milan but within TTV/AFP. The average follow-up from listing was 33,9 ±24,9 months. The risk of drop-out was higher for patients beyond Milan but within TTV/AFP (16/38, 42,1%), than for patients within Milan (49/195, 25,1%, p=0,033). In parallel, intent-to-treat survival from listing was lower in the patients beyond Milan (53,8% vs. 71,6% at four years, p<0,001). After a median waiting time of 8 months, 166 patients were transplanted, 134 patients within Milan criteria, and 32 beyond Milan but within TTV/AFP. They demonstrated acceptable and similar recurrence rates (4,5% vs. 9,4%, p=0,138) and post-transplant survivals (78,7% vs. 74,6% at four years, p=0,932). CONCLUSION Based on the present prospective study, HCC liver transplant candidate selection could be expanded to the TTV (≤115 cm(3) )/AFP (≤400 ng/ml) criteria in centers with at least 8-month waiting time. An increased risk of drop-out on the waiting list can be expected but with equivalent and satisfactory post-transplant survival. This article is protected by copyright. All rights reserved.

Left atrial remodeling, early repolarization pattern, and inflammatory cytokines in professional soccer players

OBJECTIVES: Although regular physical exercise clearly reduces cardiovascular morbidity risk, long-term endurance sports practice has been recognized as a risk factor for atrial fibrillation (AF). However, the mechanisms how endurance sports can lead to AF are not yet clear. The aim of our present study was to investigate the influence of long-term endurance training on vagal tone, atrial size, and inflammatory profile in professional elite soccer players. METHODS: A total of 25 professional major league soccer players (mean age 24+/-4 years) and 20 sedentary controls (mean age 26+/-3 years) were included in the study and consecutively examined. All subjects underwent a sports cardiology check-up with physical examination, electrocardiography, echocardiography, exercise testing on a bicycle ergometer, and laboratory analysis [standard laboratory and cytokine profile: interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, IL-8, IL-10]. RESULTS: Athletes were divided into two groups according to presence or absence of an early repolarization (ER) pattern, defined as a ST-segment elevation at the J-point (STE) >/=0.1mm in 2 leads. Athletes with an ER pattern showed significantly lower heart rate and an increased E/e' ratio compared to athletes without an ER pattern. STE significantly correlated with E/e' ratio as well as with left atrial (LA) volume. The pro-inflammatory cytokines IL-6, IL-8, TNF-alpha as well as the anti-inflammatory cytokine IL-10 were significantly elevated in all soccer players. However, athletes with an ER pattern had significantly higher IL-6 plasma levels than athletes without ER pattern. Furthermore, athletes with "high" level IL-6 had significantly larger LA volumes than players with "low" level IL-6. CONCLUSIONS: Athletes with an ER pattern had significantly higher E/e' ratios, reflecting higher atrial filling pressures, higher LA volume, and higher IL-6 plasma levels. All these factors may contribute to atrial remodeling over time and thus increase the risk of AF in long-term endurance sports.

A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance.

UNLABELLED Patients carrying very rare loss-of-function mutations in interleukin-1 receptor-associated kinase 4 (IRAK4), a critical signaling mediator in Toll-like receptor signaling, are severely immunodeficient, highlighting the paramount role of IRAK kinases in innate immunity. We discovered a comparatively frequent coding variant of the enigmatic human IRAK2, L392V (rs3844283), which is found homozygously in ∼15% of Caucasians, to be associated with a reduced ability to induce interferon-alpha in primary human plasmacytoid dendritic cells in response to hepatitis C virus (HCV). Cytokine production in response to purified Toll-like receptor agonists was also impaired. Additionally, rs3844283 was epidemiologically associated with a chronic course of HCV infection in two independent HCV cohorts and emerged as an independent predictor of chronic HCV disease. Mechanistically, IRAK2 L392V showed intact binding to, but impaired ubiquitination of, tumor necrosis factor receptor-associated factor 6, a vital step in signal transduction. CONCLUSION Our study highlights IRAK2 and its genetic variants as critical factors and potentially novel biomarkers for human antiviral innate immunity.

The transcription factor nuclear factor kappa B (NFkappaB) maintains TRAIL resistance in human pancreatic cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF family of cytokines that induces apoptosis in a variety of tumor cells while sparing normal cells. However, many human cancer cell lines display resistance to TRAIL-induced apoptosis and the mechanisms contributing to resistance remain controversial. Previous studies have demonstrated that the dimeric transcription factor Nuclear Factor kappa B (NFκB) is constitutively active in a majority of human pancreatic cancer cell lines and primary tumors, and although its role in tumor progression remains unclear it has been suggested that NFκB contributes to TRAIL resistance. Based on this, I examined the effects of NFκB inhibitors on TRAIL sensitivity in a panel of nine pancreatic cancer cell lines. I show here that inhibitors of NFκB, including two inhibitors of the proteasome (bortezomib (Velcade™, PS-341) and NPI-0052), a small molecule inhibitor of IKK (PS1145), and a novel synthetic diterpene NIK inhibitor (NPI-1342) reverse TRAIL resistance in pancreatic cancer cell lines. Further analysis revealed that the expression of the anti-apoptosic proteins BclXL and XIAP was significantly decreased following exposure to these inhibitors alone and in combination with TRAIL. Additionally, treatment with NPI0052 and TRAIL significantly reduced tumor burden relative to the control tumors in an L3.6pl orthotopic pancreatic xenograft model. This was associated with a significant decrease in proliferation and an increase in caspase 3 and 8 cleavage. Combination therapy employing PS1145 or NPI-1342 in combination with TRAIL also resulted in a significant reduction in tumor burden compared to either agent alone in a Panc1 orthotopic xenograft model. My studies show that combination therapy with inhibitors of NFκB alone and TRAIL is effective in pre-clinical models of pancreatic cancer and suggests that the approach should be evaluated in patients. ^

The role of transforming growth factor beta signaling in cardiac hypertrophy and fibrosis in heart failure

Objective. To determine whether transforming growth factor beta (TGF-β) receptor blockade using an oral antagonist has an effect on cardiac myocyte size in the hearts of transgenic mice with a heart failure phenotype. ^ Methods. In this pilot experimental study, cardiac tissue sections from the hearts of transgenic mice overexpressing tumor necrosis factor (MHCsTNF mice) having a phenotype of heart failure and wild-type mice, treated with an orally available TGF-β receptor antagonist were stained with wheat germ agglutinin to delineate the myocyte cell membrane and imaged using fluorescence microscopy. Using MetaVue software, the cardiac myocyte circumference was traced and the cross sectional area (CSA) of individual myocytes were measured. Measurements were repeated at the epicardial, mid-myocardial and endocardial levels to ensure adequate sampling and to minimize the effect of regional variations in myocyte size. ANOVA testing with post-hoc pairwise comparisons was done to assess any difference between the drug-treated and diluent-treated groups. ^ Results. There were no statistically significant differences in the average myocyte CSA measured at the epicardial, mid-myocardial or endocardial levels between diluent treated littermate control mice, drug treated normal mice, diluent-treated transgenic mice and drug-treated transgenic mice. There was no difference between the average pan-myocardial cross sectional area between any of the four groups mentioned above. ^ Conclusions. TGF-β receptor blockade using oral TGF-β receptor antagonist does not alter myocyte size in MHCsTNF mice that have a phenotype of heart failure. ^

Role of p38 mitogen-activated protein kinase in HIV type 1 production in vitro

The proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) promote HIV type 1 viral replication in vitro. In the present studies, HIV production was increased in the macrophagic U1 cell line expressing the HIV genome after exposure to IL-1β, osmotic stress, or surface adhesion, suggesting a confluence of signaling pathways for proinflammatory cytokines and cell stressors. The p38 mitogen-activated protein kinase (MAPK) mediates both cytokine and stress responses; thus the role of this kinase in HIV production was investigated. HIV production as measured by p24 antigen correlated with changes in the expression of a specific (non-alpha) isoform of p38 MAPK. In the presence of a specific p38 MAPK inhibitor (p38 inh), IL-1β-induced HIV production was suppressed by more than 90% and IL-1β-induced IL-8 production was suppressed completely, both with IC50 of 0.01 μM. p38 inhibition blocked cell-associated p24 antigen and secreted virus to a similar extent. The p38 inh also decreased constitutive HIV production in freshly infected peripheral blood mononuclear cells by up to 50% (P < 0.05). Interruption of p38 MAPK activity represents a viable target for inhibition of HIV.

Gene therapy in allergic encephalomyelitis using myelin basic protein-specific T cells engineered to express latent transforming growth factor-β1

A myelin basic protein (MBP)-specific BALB/c T helper 1 (Th1) clone was transduced with cDNA for murine latent transforming growth factor-β1 (TGF-β1) by coculture with fibroblasts producing a genetically engineered retrovirus. When SJL x BALB/c F1 mice, immunized 12–15 days earlier with proteolipid protein in complete Freund’s adjuvant, were injected with 3 × 106 cells from MBP-activated untransduced cloned Th1 cells, the severity of experimental allergic encephalomyelitis (EAE) was slightly increased. In contrast, MBP-activated (but not resting) latent TGF-β1-transduced T cells significantly delayed and ameliorated EAE development. This protective effect was negated by simultaneously injected anti-TGF-β1. The transduced cells secreted 2–4 ng/ml of latent TGF-β1 into their culture medium, whereas control cells secreted barely detectable amounts. mRNA profiles for tumor necrosis factor, lymphotoxin, and interferon-γ were similar before and after transduction; interleukin-4 and -10 were absent. TGF-β1-transduced and antigen-activated BALB/c Th1 clones, specific for hemocyanin or ovalbumin, did not ameliorate EAE. Spinal cords from mice, taken 12 days after receiving TGF-β1-transduced, antigen-activated cells, contained detectable amounts of TGF-β1 cDNA. We conclude that latent TGF-β1-transduced, self-reactive T cell clones may be useful in the therapy of autoimmune diseases.

High-affinity binding of bioactive glycosylation-inhibiting factor to antigen-primed T cells and natural killer cells

High-affinity binding was demonstrated between suppressor-T-cell-derived bioactive glycosylation-inhibiting factor (GIF) and helper T hybridomas and natural killer cell line cells. Inactive GIF present in cytosol of suppressor T cells and Escherichia coli-derived recombinant human GIF (rhGIF) failed to bind to these cells. However, affinity of rhGIF for the target cells was generated by replacement of Cys-57 in the sequence with Ala or of Asn-106 with Ser or binding of 5-thio-2-nitrobenzoic acid to Cys-60 in the molecule. Such mutations and the chemical modification of rhGIF synergistically increased the affinity of GIF molecules for the target cells. The results indicated that receptors on the target cells recognize conformational structures of bioactive GIF. Equilibrium dissociation constant (Kd) of the specific binding between bioactive rGIF derivatives and high-affinity receptors was 10–100 pM. Receptors for bioactive GIF derivatives were detected on Th1 and Th2 T helper clones and natural killer NK1.1+ cells in normal spleen but not on naive T or B cells. Neither the inactive rGIF nor bioactive rGIF derivatives bound to macrophage and monocyte lines or induced macrophages for tumor necrosis factor α production.

Cytokine-mediated enhancement of epidermal growth factor receptor expression provides an immunological approach to the therapy of pancreatic cancer

The increased expression of epidermal growth factor receptor induced by tumor necrosis factor α renders pancreatic cancer cells more susceptible to antibody-dependent cellular cytotoxicity by a mAb specific for this receptor. Laboratory studies with athymic mice bearing xenografts of human pancreatic cancer cells demonstrated a cytokine-induced ability of the mAb to cause significant tumor regression. In a phase I/II clinical trial, 26 patients with unresectable pancreatic cancer were enrolled into three cohorts receiving variable amounts of the antibody together with a constant amount of tumor necrosis factor α. With increasing doses of antibody, the growth of the primary tumor was significantly inhibited. This was reflected by a longer median survival, with one complete remission lasting for 3 years obtained with the highest dose of antibody employed. Thus, a combination of the cytokine, tumor necrosis factor α, with a mAb to the epidermal growth factor receptor offers a potentially useful approach for the treatment of pancreatic cancer.

Radiation-induced Release of Transforming Growth Factor α Activates the Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase Pathway in Carcinoma Cells, Leading to Increased Proliferation and Protection from Radiation-induced Cell Death

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0–10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90–240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor α receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor α (TGFα) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFα cleavage 120–180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFα. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFα. Neutralization of TGFα function by an anti-TGFα antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFα–EGFR–MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.

MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration

Exposure of eukaryotic cells to extracellular stimuli results in activation of mitogen-activated protein kinase (MAPK) cascades composed of MAPKs, MAPK kinases (MAP2Ks), and MAPK kinase kinases (MAP3Ks). Mammals possess a large number of MAP3Ks, many of which can activate the c-Jun N-terminal kinase (JNK) MAPK cascade when overexpressed, but whose biological function is poorly understood. We examined the function of the MAP3K MEK kinase 1 (MEKK1) in proinflammatory signaling. Using MEKK1-deficient embryonic stem cells prepared by gene targeting, we find that, in addition to its function in JNK activation by growth factors, MEKK1 is required for JNK activation by diverse proinflammatory stimuli, including tumor necrosis factor α, IL-1, double-stranded RNA, and lipopolysaccharide. MEKK1 is also essential for induction of embryonic stem cell migration by serum factors, but is not required for activation of other MAPKs or the IκB kinase signaling cascade.

Glucocorticoid-mediated repression of nuclear factor-κBdependent transcription involves direct interference with transactivation

Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-κB. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IκB molecule, which in turn would bind and remove activated, DNA-bound NF-κB complexes in the cell nucleus. Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-κB-dependent transcription, without changing the expression level of IκB. Neither the mRNA of IκB nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells. The DNA-binding activity of induced NF-κB also remained unchanged after stimulation of cells with DEX. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-κB. Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX. Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-κB p65 subunit.