Tumor Necrosis Factor-related Apoptosis-inducing Ligand Protein Expression Research Articles

EL4-derived Exosomes Carry Functional TNF-related Apoptosis-inducing Ligand that are Able to Induce Apoptosis and Necrosis in the Target Cells.

Exosomes released by tumor cells play critical roles in tumor progression, immune cell suppression, and cancer metastasis. The aim of the present study was to investigate whether the exosomes released by EL4 cells carry a functional TNF-related apoptosis-inducing ligand (TRAIL) molecule. Exosomes were harvested from the supernatants of EL4 cell culture, and the shape, size, and identity of EL4-derived exosomes were evaluated by utilizing scanning electron microscopy, dynamic light scattering, and dot-blot method. The expression of mRNA and TRAIL protein in EL4 cells and EL4-exosomes were investigated using real-time PCR method and dot-blot analysis. Moreover, the effects of EL4-derived exosomes on cell death in a TRAIL-sensitive cell line (4T1) were studied by using flow cytometry (annexin V/propidium iodide (PI) staining) and fluorescent microscopy analyses (acridine orange/ethidium bromide staining). The results showed that EL4 cells continuously and without the need for stimulation, produce exosomes that carry TRAIL protein. In addition, EL4-derived exosomes were capable to induce apoptosis as well as necrosis in 4T1 cells. It was ultimately revealed that EL4 cells express TRAIL protein and release exosomes containing functional TRAIL. Moreover, the released exosomes were able to induce apoptosis and necrosis in a TRAIL-sensitive cell line. Further studies are needed to reveal the potential roles of tumor-derived exosomes in the pathogenesis of cancers.

HIV elite control is associated with reduced TRAILshort expression.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) dependent apoptosis has been implicated in CD4 T-cell death and immunologic control of HIV-1 infection. We have described a splice variant called TRAILshort, which is a dominant negative ligand that antagonizes TRAIL-induced cell death in the context of HIV-1 infection. HIV-1 elite controllers naturally control viral replication for largely unknown reasons. Since enhanced death of infected cells might be responsible, as might occur in situations of low (or inhibited) TRAILshort, we tested whether there was an association between elite controller status and reduced levels of TRAILshort expression. Cohort study comparing TRAILshort and full length TRAIL expression between HIV-1 elite controllers and viremic progressors from two independent populations. TRAILshort and TRAIL gene expression in peripheral blood mononuclear cells (PBMCs) was determined by RNA-seq. TRAILshort and TRAIL protein expression in plasma was determined by antibody bead array and proximity extension assay respectively. HIV-1 elite controllers expressed less TRAILshort transcripts in PBMCs (P = 0.002) and less TRAILshort protein in plasma (P < 0.001) than viremic progressors. Reduced TRAILshort expression in PBMCs and plasma is associated with HIV-1 elite controller status.

Positive acceleration adaptive training attenuates gastric ischemia-reperfusion injury through COX-2 and PGE2 expression.

The mechanism involved in the effects of positive acceleration adaptive training (PAAT) on gastric ischemia-reperfusion injury (GI-RI) has not been fully characterized. The aim of the present study was to investigate the effects of PAAT in attenuating GI-RI in a rat model. The inflammatory factor and caspase-3 levels were measured using ELISA kits. A western blot assay was used to analyze tumor necrosis factor-α (TNF)-α, tumor necrosis factor receptor 1 (TNFR1), tumor necrosis factor-related apoptosis inducing ligand (TRAIL), death receptor (DR) 4, DR5, cyclooxygenase (COX)-2, COX-1 and prostaglandin E2 (PGE2) protein expression levels. It was revealed that PAAT could alleviate GI-RI and inflammatory factor levels in a rat model. PAAT suppressed TNF-α and TNFR1 protein expression levels, inhibited TRAIL, DR4, DR5, COX-2 and PGE2 protein expression levels; however, it did not have an effect on COX-1 protein expression in the model of GI-RI. The data indicated that the effects of PAAT attenuated GI-RI through the downregulation of COX-2 and PGE2 expression.

Gene Expression of Molecules Regulating Apoptotic Pathways in Glioblastoma Multiforme Treated with Umbilical Cord Stem Cell Conditioned Medium.

BackgroundGlioblastoma multiforme (GBM) is the most malignant primary brain tumour and there is no definite cure. It has been suggested that there are significant interactions among mesenchymal stem cells (MSCs), their released factors and tumour cells that ultimately determine GBM’s growth pattern. This study aims to analyse the expression of molecules involved in GBM cell apoptotic pathways following treatment with the MSC secretome.MethodsA conditioned medium of umbilical cord-derived MSCs (UCMSC-CM) was generated by culturing the cells on serum-free αMEM for 24 h. Following this, human GBM T98G cells were treated with UCMSC-CM for 24 h. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was then performed to measure the mRNA expression of survivin, caspase-9, TNF-related apoptosis-inducing ligand (TRAIL), DR4 and DcR1.ResultsmRNA expression of caspase-9 in CM-treated T98G cells increased 1.6-fold (P = 0.017), whereas mRNA expression of survivin increased 3.5-fold (P = 0.002). On the other hand, TRAIL protein expression was upregulated (1.2-fold), whereas mRNA expression was downregulated (0.4-fold), in CM-treated cells. Moreover, there was an increase in the mRNA expression of both DR4 (3.5-fold) and DcR1 (1,368.5-fold) in CM-treated cells.ConclusionThe UCMSC-CM was able to regulate the expression of molecules involved in GBM cell apoptotic pathways. However, the expression of anti-apoptotic molecules was more upregulated than that of pro-apoptotic molecules.

Delivery of TRAIL-expressing plasmid DNA to cancer cells in vitro and in vivo using aminoglycoside-derived polymers.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand that can preferentially induce apoptosis in cancer cells over normal cells. The transmembrane form of TRAIL has been shown to elicit much stronger activity than its soluble counterpart but delivery is a potential challenge. Here, we investigated the potential of aminoglycoside-derived polymers to enhance delivery of a plasmid (pEF-TRAIL) that expresses the transmembrane form of TRAIL in order to determine the effect on cell death in vitro and tumor growth in vivo. Transgene delivery efficacy and toxicity of aminoglycoside-derived polymers was first evaluated using a GFP-expressing plasmid (pEF-GFP) at different plasmid amounts and plasmid : polymer ratios in UMUC3 bladder cancer and HeLa cervical cancer cells. Delivery of the TRAIL plasmid using aminoglycoside-derived polymers resulted in up to 60% cell death in UMUC3 and HeLa cells; TRAIL protein expression was confirmed using Western blots. TRAIL plasmid delivery resulted in a decrease in cellular procaspase-8 and an increase in TRAIL receptor DR5 levels, suggesting a role for the death receptor and caspase cascade in TRAIL-mediated apoptosis. The TRAIL plasmid did not cause cell death in normal human or mouse fibroblasts. The in vivo delivery of the TRAIL plasmid using a paromomycin-derived polymer resulted in significant reduction in tumor burden and increased survival in tumor-bearing live mice.

TRAIL mediates and sustains constitutive NF-κB activation in LGL leukemia

AbstractLarge granular lymphocyte (LGL) leukemia results from clonal expansion of CD3+ cytotoxic T lymphocytes or CD3− natural killer (NK) cells. Chronic antigen stimulation is postulated to promote long-term survival of LGL leukemia cells through constitutive activation of multiple survival pathways, resulting in global dysregulation of apoptosis and resistance to activation-induced cell death. We reported previously that nuclear factor κB (NF-κB) is a central regulator of the survival network for leukemic LGL. However, the mechanisms that trigger constitutive activation of NF-κB in LGL leukemia remain undefined. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis in tumor cells but can also activate NF-κB through interaction with TRAIL receptors 1, 2, and 4 (also known as DR4, DR5, and DcR2, respectively). The role of TRAIL has not been studied in LGL leukemia. In this study, we hypothesized that TRAIL interaction with DcR2 contributes to NF-κB activation in LGL leukemia. We observed upregulated TRAIL messenger RNA and protein expression in LGL leukemia cells with elevated levels of soluble TRAIL protein in LGL leukemia patient sera. We also found that DcR2 is the predominant TRAIL receptor in LGL leukemia cells. We demonstrated that TRAIL-induced activation of DcR2 led to increased NF-κB activation in leukemic LGL. Conversely, interruption of TRAIL-DcR2 signaling led to decreased NF-κB activation. Finally, a potential therapeutic application of proteasome inhibitors (bortezomib and ixazomib), which are known to inhibit NF-κB, was identified through their ability to decrease proliferation and increase apoptosis in LGL leukemia cell lines and primary patient cells.

Arsenic trioxide and sorafenib combination therapy for human hepatocellular carcinoma functions via up-regulation of TNF-related apoptosis-inducing ligand.

The survival benefits of sorafenib treatment for patients with hepatocellular carcinoma (HCC) are limited due to drug resistance and side effects. Therefore, combinations of sorafenib with other low toxicity drugs, including arsenic trioxide (As2O3) require investigation. The present study aimed to evaluate the potency of apoptosis-induction by As2O3/sorafenib treatment in HCC cell lines, Huh7, 97H and freshly-isolated HCC cells, and also to elucidate the underlying mechanism. A total of 10 patients with HCC were enrolled in the present study. Freshly-isolated HCC cells were purified from HCC tissues collected at surgery. HCC-cell apoptosis was measured by flow cytometry using proprium iodide/Annexin-V staining. The impacts of As2O3 and/or sorafenib on Huh7, 97H and fresh-isolated HCC-cell proliferation were evaluated by Cell Counting Kit-8 assay. The expression of TNF-related apoptosis-inducing ligand (TRAIL) was determined by reverse transcription-quantitative polymerase chain reaction and western blotting. The downregulation of TRAIL protein expression was achieved using small interfering RNA. The combination of As2O3 and sorafenib had anti-proliferative and pro-apoptotic effects in the liver cancer cell line, Huh7, via increased expression of TRAIL, but not in 97H cells. TRAIL-knockdown increased the drug-resistance of Huh7 cells. Freshly-isolated HCC cells were more sensitive to the As2O3 and sorafenib combination than the single drug treatments. Overall, the combination of As2O3 and sorafenib demonstrated potent anti-tumor activity in Huh7 and freshly-isolated HCC cells via a TRAIL-dependent pathway. This may be a potential therapeutic approach for advanced HCC treatment.

Altered expression of mir-222 and mir-25 influences diverse gene expression changes in transformed normal and anaplastic thyroidcells, and impacts on MEK and TRAIL protein expression.

Thyroid cancer is the most common endocrine malignancy and accounts for the majority of endocrine cancer-related deaths each year. Our group and others have previously demonstrated dysfunctional microRNA (miRNA or miR) expression in the context of thyroid cancer. The objective of the present study was to investigate the impact of synthetic manipulation of expression of miR-25 and miR-222 in benign and malignant thyroid cells. miR-25 and miR-222 expression was upregulated in 8505C (an anaplastic thyroid cell line) and Nthy-ori (a SV40-immortalised thyroid cell line) cells, respectively. A transcriptomics-based approach was utilised to identify targets of the two miRNAs and real-time PCR and western blotting were used to validate a subset of the targets. Almost 100 mRNAs of diverse functions were found to be either directly or indirectly targeted by both miR-222 and miR-25 [fold change ≥2, false discovery rate (FDR) ≤0.05]. Gene ontology analysis showed the miR-25 gene target list to be significantly enriched for genes involved in cell adhesion. Fluidigm real-time PCR technologies were used to validate the downregulation of 23 and 22 genes in response to miR-25 and miR-222 overexpression, respectively. The reduction of the expression of two miR-25 protein targets, TNF-related apoptosis-inducing ligand (TRAIL) and mitogen-activated protein kinase kinase 4 (MEK4), was also validated. Manipulating the expression of both miR-222 and miR-25 influenced diverse gene expression changes in thyroid cells. Increased expression of miR-25 reduced MEK4 and TRAIL protein expression, and cell adhesion and apoptosis are important aspects of miR-25 functioning in thyroid cells.

Adipose‑derived mesenchymal stem cell‑facilitated TRAIL expression in melanoma treatment invitro.

Adipose-derived stem cells (ADSCs) may be useful as an efficient vehicle in cell-based gene therapy of human diseases due to their ability to migrate to disease lesions. This study investigated the ability of ADSC-harbored human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cDNA to facilitate TRAIL expression and induce A375 melanoma cell apoptosis as observed using a Transwell co-culture system. A cell migration assay was used to observe ADSC migration ability. In addition, TRAIL protein expression was successfully detected by western blot analysis in ADSCs after stable transfection of TRAIL cDNA. The Transwell co-culture system data showed that TRAIL-ADSCs could induce A375 cell apoptosis in a dose-dependent manner. At the gene level, the killing activity of TRAIL-ADSCs was associated with activation of caspase-4 and caspase-8. Collectively, the data from the current study provides preclinical support of ADSC-facilitated TRAIL expression in the treatment of melanoma. Further investigation is required to evaluate and confirm the in vivo ability of TRAIL-ADSCs in therapy of melanoma in animal models.

17β-Estradiol inhibits TNF-α-induced proliferation and migration of vascular smooth muscle cells via suppression of TRAIL

Atherosclerosis is an inflammatory disease and involves migration of vascular smooth muscle cells (VSMCs). Estrogen inhibits VSMCs migration, while the underlying mechanism remains to be revealed. Recent years, there is emerging evidence showing that TNF-related apoptosis-inducing ligand (TRAIL) increases proliferation and migration of VSMCs. In this study, we investigated the regulatory effect of estrogen on TRAIL expression in VSMCs. TNF-α greatly enhanced TRAIL protein expression and stimulated VSMCs proliferation and migration. This effect was partially inhibited by the addition of TRAIL neutralizing antibody, suggesting that TRAIL is important in TNF-α-induced migration. 17β-estradiol (E2) inhibited TRAIL expression under TNF-α stimulation in a time- and concentration-dependent manner. This effect was was mimicked by ERα agonist 4′,4″,4‴-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), but not ERβ agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN), indicating that ERα is involved in this action. TNF-α led to nuclear factor kappa B (NF-κB) p65 phosphorylation and the inhibitor pyrrolidine dithiocarbama (PDTC) inhibited TRAIL expression, suggesting that NF-κB signaling is crucial for TARIL production. E2 suppressed p65 phosphorylation in VSMCs and the overexpression of p65 subunit reversed the inhibitory effect of E2 on TRAIL expression and cell proliferation and migration. Taken together, our results indicate that E2 inhibits VSMCs proliferation and migration by downregulation of TRAIL expression via suppression of NF-κB pathway.

Oxaliplatin immuno hybrid nanoparticles for active targeting: an approach for enhanced apoptotic activity and drug delivery to colorectal tumors

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) proved to be a promising new target for colorectal cancer treatment. Elevated expression of TRAIL protein in tumor cells distinguishes it from healthy cells, thereby delivering the drug at the specific site. Here, we formulated oxaliplatin immunohybrid nanoparticles (OIHNPs) to deliver oxaliplatin and anti-TRAIL for colorectal cancer treatment in xenograft tumor models. The polymeric chitosan layer binds to the lipid film with the mixture of phospholipids by an ultra sound method followed by conjugating with thiolated antibody using DSPE-PEG-mal3400, resulting in the formation of OIHNPs. The polymer layer helps in more encapsulation of the drug (71 ± 0.09%) with appreciable particle size (95 ± 0.01 nm), and lipid layer prevents degradation of the drug in serum by preventing nanoparticle aggregation. OIHNPs have shown a 4-fold decrease in the IC50 value compared to oxaliplatin in HT-29 cells by the MTT assay. These immuno-nanoparticles represent the successful uptake and internalization of oxaliplatin in HT-29 cells rather than in MCF-7 cells determined by triple fluorescence method. Apoptotic activity in vitro of OIHNPs was determined by the change in the mitochondria membrane potential that further elevates its anti-tumor property. Furthermore, the conjugated nanoparticles can effectively deliver the drug to the tumor sites, which can be attributed to its ability in reducing tumor mass and tumor volume in xenograft tumor models in vivo along with sustaining its release in vitro. These findings indicated that the oxaliplatin immuno-hybrid nanoparticles would be a promising nano-sized active targeted formulation for colorectal-tumor targeted therapy.

The expression of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)protein and it's receptor 3,4 in colorectal cancer tissue and its significance

Objective To investigate the potential roles of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)signaling system in the pathogenesis of colorectal cancer. Methods Immunohistochemistry techniques were used,the TRAIL and its receptor(TRAILR3,TRAILR4)protein were analyzed in both 41 colorectal cancer samples(colorectal cancer group)and normal samples beside cancer tissue(normal group). Results The levels of TRAIL and TRAILR3 protein expression in the colorectal cancer group were significantly lower than those in the normal group[(0.237±0.036)vs.(0.289±0.069);(0.226±0.052)vs.(0.281±0.068),t=4.125,4.025,all P 0.05).The expression levels of them in the colorectal cancer group with poorl differentiation were notably lower than those with high-moderate differentiation[(0.205±0.021)vs(0.245±0.034);(0.185±0.032)vs(0.236±0.051),t=4.025,2.664,P 0.05]. Moreover, the positive rate and expression levels of TRAILR4 protein was non-statistic significant difference between the two groups[93%vs 98%;(0.196±0.085)vs(0.219±0.061),χ2=1.051,t=1.353,all P>0.05],and it was also non-significantly correlated with cancer cell differentiation and lymph nodes metastasis[(0.176±0.052)vs(0.201± 0.091);(0.194±0.054)vs(0.197±0.100),t=0.667,0.448,all P>0.05]. Conclusion The levels of TRAIL and TRAILR3 expression are attenuated at colorectal cancer tissue. The expression of them are correlated with cancer cell differentiation grade. These findings indicate that TRAIL system may be associated with the malignant phenotype in colorectal cancer. Key words: Colorectal Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Signal Transduction

Conditionally replicating adenovirus combined with gene-targeted radiotherapy induces apoptosis via TRAIL death receptors in MDA-MB-231 cells

Malignant tumors are usually treated using monotherapies, which are not always effective. Therefore, combination therapies have gained increasing attention. The aims of this study were to investigate the effects of conditionally replicating adenovirus (CRAd) in combination with X-ray irradiation on the proliferation and apoptosis of MDA-MB-231 cells, as well as to determine the molecular mechanisms involved. MDA-MB-231 cells were treated simultaneously with CRAd and X-ray irradiation. Then, cell viability was measured using Cell Counting Kit-8. Cell apoptosis was assessed by flow cytometry with Annexin V and propidium iodide (PI) double-staining. The expression of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), death receptor 5 (DR5), caspase-3 and caspase-8 mRNA was detected by quantitative polymerase chain reaction. The expression of TRAIL, DR5, caspase-3 and caspase-8 protein was measured by enzyme-linked immunosorbent assay (ELISA) and western blotting, respectively. The results showed that CRAd, in combination with irradiation, inhibited cell proliferation, promoted cell apoptosis and significantly increased the expression of TRAIL, DR5, caspase-3 and caspase-8 mRNA and proteins in MDA-MB-231 cells. Therefore, three aspects, including the targeted killing effect of CRAd, the apoptosis-promoting role of TRAIL and the direct killing effect of ionizing radiation to MDA-MB-231 cells, contribute to the mechanisms of CRAd in combination with irradiation to inhibit the proliferation of MDA-MB-231 cells. The pro-apoptotic effect may involve the interaction between TRAIL, DR5, caspase-3 and caspase-8.

Abstract 4886: PTHrP sensitizes breast cancer cells to TRAIL-induced apoptosis

Abstract Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that has been implicated in breast tumor biology. Breast cancers commonly express PTHrP, and when such tumors are grown in bone in murine models, PTHrP results in enhanced osteolysis. Whilst clinical studies have determined that PTHrP expression by primary breast cancers was an independent predictor of improved prognosis (Henderson et al., 2006). Thus, the action of PTHrP upon breast cancers directly remains to be elucidated. The aim of this study was to assess the interaction of PTHrP with molecules associated with cell survival and cell death pathways, such as Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces apoptosis in a wide range of transformed cells but not in normal cells. To understand the role of PTHrP in relation to TRAIL in breast cancer cells, PTHrP was overexpressed in two breast cancer cell lines; MDA-MB-231 (TRAIL-sensitive) and MCF-7 (TRAIL-resistant), and functional assays were performed. Over-expression of PTHrP in both MCF-7 and MDA-MB-231 cells enhanced cell cycle progression and cell growth rate in comparison to parental and vector controls. However, treatment of PTHrP overexpressing breast cancer cells with TRAIL resulted in an enhancement of TRAIL-induced apoptosis in the TRAIL sensitive cell line, MDA-MB-231, and a sensitisation to TRAIL-induced apoptosis in the TRAIL-resistant cell line, MCF-7. Western blot showed decreased levels of Caspase-10, Caspase-9, Caspase-6, Capase-7 precursor proteins and increased PARP cleavage levels in PTHrP overexpressing cells treated with TRAIL when compared to controls. There was no change observed in levels of Caspase-8 percursor protein, suggesting that the apoptosis signal is acting via the intrinsic pathway in PTHrP overexpressing cells. To determine TRAIL sensitivity in MCF-7 cells overexpressing PTHrP, TRAIL receptor mRNA and protein expression was assessed by qRT-PCR and FACS analysis. TRAIL receptors; DR4, DR5, DcR1 and DcR2, mRNA expression was not altered in PTHrP overexpressing cells. However, flow cytometric analysis demonstrated an increase in cell surface expression of both death receptors (DR4 and DR5) in cells overexpressing PTHrP. To determine whether TRAIL-induced apoptosis in PTHrP overexpressing cells was signaling either through DR4, DR5 or a combination of both death receptors, antagonistic antibodies against DR4 or DR5 were used. Pre-incubation of PTHrP overexpressing cells with DR5 antagonistic antibody attenuated TRAIL-induced apoptosis. This demonstrated that TRAIL preferentially signals through DR5 in the PTHrP overexpressing cells to activate the intrinsic apoptotic pathway. In conclusion, these results demonstrate a new function for PTHrP in breast cancer and may provide utility when TRAIL is used as a treatment for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4886. doi:1538-7445.AM2012-4886

Hypoxia-Ischemia Upregulates TRAIL and TRAIL Receptors in the Immature Rat Brain

The immature brain is susceptible to inflammatory injury induced by hypoxia-ischemia (HI) or infection, which causes serious neurodevelopmental disabilities in the survivors of preterm births. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (death receptor DR4/5 and decoy receptor DcR1/2) were reported to mediate various neuroinflammatory responses. However, little information is available regarding the role of TRAIL and its receptors in the immature brain after HI. The purpose of this study was to evaluate the expression of TRAIL and its receptors in the immature brain after HI and relate this expression to neurological function. We performed right common carotid artery ligation followed by hypoxia (6% O₂, 37°C) for 2.5 h to induce HI in postnatal day 3 rats. The distribution of TRAIL and its receptors, caspase-3 and CD68-labeled microglia/macrophages was evaluated 24 h after HI by immunostaining. The protein and mRNA expression of TRAIL and DR5 was measured by Western blot and real-time PCR, respectively. Delayed neuronal loss was evaluated by NeuN and Nissl staining 7 days after HI. Furthermore, neurological deficits were evaluated by a righting reflex test, time of eye opening and T-maze test. The expression of TRAIL, DR5 and DcR1/2 receptors and caspase-3 was more pronounced in the ipsilateral hemisphere compared with the contralateral part and the control group 24 h after HI. DR5/active caspase-3 double-positive cells were observed at 24 h after HI in the ipsilateral hemisphere but not in the contralateral hemisphere. The TRAIL and CD68 double-labeled cells were more pronounced in the ipsilateral cortical regions compared with the corresponding regions of the contralateral part. HI also resulted in a significant increase in TRAIL and DR5 protein and mRNA expression at 24 h, which corresponded to neuronal cell loss 7 days after HI. Furthermore, the HI group displayed impaired neurobehavioral development compared with the control group (p < 0.05). Altogether our results show that the TNF-α superfamily ligand TRAIL is induced on CD68+ microglia/macrophages after perinatal HI and that one of its receptors, DR5, is induced on neocortical neurons and glial cells. That many DR5+ cells were also caspase-3+ strongly supports the conclusion that these signaling molecules are involved in the delayed loss of neurons in the neocortex and in the neurobehavioral deficits that are often seen after perinatal HI.

TRAIL contributes to the apoptotic effect of 13-cis retinoic acid in human sebaceous gland cells

The full mechanism of action of isotretinoin [13-cis retinoic acid (13-cis RA)] in treating acne is unknown. 13-cis RA induces key genes in sebocytes that are involved in apoptosis, including Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL). In this study, we investigated the role of 13-cis RA-induced TRAIL within SEB-1 sebocytes. Using 13-cis RA and recombinant human TRAIL (rhTRAIL) protein, we assessed induction of TRAIL and apoptosis in SEB-1 sebocytes, normal keratinocytes and patient skin biopsies. Treatment with rhTRAIL protein increased TUNEL-positive staining in SEB-1 sebocytes. TRAIL siRNA significantly decreased the percentage of TUNEL-positive SEB-1 sebocytes in response to 13-cis RA treatment. Furthermore, TRAIL expression increased in the skin of patients with acne after 1 week of isotretinoin therapy compared with baseline. TRAIL expression localized within sebaceous glands. Unlike sebocytes, TRAIL protein expression was not increased in normal human epidermal keratinocytes in response to 13-cis RA, nor did rhTRAIL induce apoptosis in keratinocytes, suggesting that TRAIL is key in the sebocyte-specific apoptotic effects of 13-cis RA. Taken together, our data suggest that TRAIL, like the neutrophil gelatinase-associated lipocalin, is involved in mediating 13-cis RA apoptosis of sebocytes.

Evaluation of TNF superfamily molecules release by neutrophils and B leukemic cells of patients with chronic B – cell lymphocytic leukemia

It was demonstrated that TNF superfamily proteins may affect significantly the time of leukemic cells' survival in the course of B-cell chronic lymphocytic leukemia (B-CLL). The aim of our study was to evaluate the expression and release of BAFF (B-cell activating factor), APRIL (a proliferation-inducing ligand) and TRAIL (TNF-related apoptosis inducing ligand) molecules belonging to the cytokines of the superfamily of the tumor necrosis factor (TNF) by neutrophils (PMNs) and, for comparison, B cells isolated from the blood of patients with B-CLL vs. their concentration in the blood serum. 40 patients suffering from B-CLL and a control group of 15 healthy subjects were included in the study. Cytoplasmic fractions of PMNs and B cells were analyzed with the use of western blotting for the presence of TRAIL, BAFF and APRIL. Soluble TRAIL, BAFF and APRIL in the culture supernatants and the serum were assessed using ELISA kits. PMNs and B cells of patients with B-CLL before treatment demonstrated the statistically significantly higher expression of APRIL and BAFF proteins when compared with the control group of healthy subjects. In contrast, the expression of TRAIL protein in both types of cells of patients was statistically significantly lower than its expression in the control cells. In the supernatants of PMN and B lymphocytes of patients the decreased concentrations of sBAFF, unchanged of APRIL and increased of sTRAIL molecules were demonstrated. The results of studies carried out in patients with B-CLL before treatment indicate that the relations demonstrated between APRIL, BAFF and TRAIL molecules, released by neutrophils and B cells and relations between their concentrations in the serum can significantly influence the development of B-CLL.

Evaluation of TNF Superfamily Molecules Release by Neutrophils and B Leukemic Cells of Patients with Chronic B – Cell Lymphocytic Leukemia

AbstractAbstract 4855It was demonstrated that TNF superfamily proteins may affect significantly the time of leukemic cells' survival in the course of B-cell chronic lymphocytic leukemia (B-CLL).The aim of our study was to evaluate the expression and release of BAFF (B-cell activating factor), APRIL (a proliferation-inducing ligand) and TRAIL (TNF-related apoptosis inducing ligand) molecules belonging to the cytokines of the superfamily of the tumor necrosis factor (TNF) by neutrophils (PMNs) and, for comparison, B cells isolated from the blood of patients with B-CLL vs. their concentration in the blood serum.40 patients suffering from B-CLL and a control group of 15 healthy subjects were included in the study. Cytoplasmic fractions of PMNs and B cells were analyzed with the use of western blotting for the presence of TRAIL, BAFF and APRIL. Soluble TRAIL, BAFF and APRIL in the culture supernatants and the serum were assessed using ELISA kits.PMNs and B cells of patients with B-CLL before treatment demonstrated the statistically significantly higher expression of APRIL and BAFF proteins in comparison in relation to the control group of healthy subjects. In contrast, the expression of TRAIL protein in both types of cells of patients was statistically significantly lower than its expression in the control cells.In the supernatants of PMN and B lymphocytes of patients the decreased concentrations of sBAFF, unchanged of APRIL and increased of sTRAIL molecules were demonstrated.The results of studies carried out in patients with B-CLL before treatment indicate that the relations demonstrated between APRIL, BAFF and TRAIL molecules, released by neutrophils and B cells and relations between their concentrations in the serum can significantly influence the development of B-CLL. Disclosures:No relevant conflicts of interest to declare.

Expression and significance of TRAIL and its receptor DR5 in murine myocardium with acute viral myocarditis

Objective To deterine the expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptor DR5 in murine myocardium with acute viral myocarditis(VM).Methods We builted the model of VM.Eight mice of the VM group and the normal control group were sacrificed on the 7th,10th,14th,21st,28th day after inoculation CVB3 virus.The myocardial histopathological scores were counted.The terminal reansferase-mediated dUPT-biotin nick end-labeling (TUNEL) assays was used to quantified apoptosis rate.The expression of TRAIL and DR5 protein and mRNA were determined by the method of immunohistochemistry and reverse-transcription polymerase chain reaction (RT-PCR).Results The expression of TRAIL and DR5 protein and mRNA were found in myocardium of both the normal control group and the VM group.The expression of TRAIL protein of the VM group(14th) were significantly higher than which in the normal group at the same time and the VM group(7th)(F=9.17,P<0.01).The DR5 protein of the VM group(10th,14th,21st)were significantly higher than which in the normal group at the same time and the VM group(7th)(F=13.32,P<0.01).The expression of TRAIL and DR5 protein were positive correlated with the myocardial histopathological scores and the apoptosis rate.The expression of TRAIL mRNA of the VM group(10th) were significantly higher than which in the normal group at the same time and the VM group(7th)(F=10.86,P<0.01).The DR5 mRNA of the VM group(10th,14th)were significantly higher than which in the normal group at the same time and the VM group(7th)(F=22.75,P<0.01).Conclusion High characteristic expressions of protein/mRNA TRAIL and DR5 were observed in the myocardium of mice with VM.The level was positive correlationed with the account of pathology and the rate of apoptosis.The apoptosis induced by TRAIL and DR5 may participate in the pathophysiological processes of VM. Key words: Coxsackie virus; Myocarditis; Tunor necrosis factor related apoptosis inducing ligand; Death receptor 5; Apoptosis

A Placental Protective Role for Trophoblast-Derived TNF-Related Apoptosis-Inducing Ligand (TRAIL)

Recent studies show that apoptosis, programmed cell death, plays an important role in the normal development of the human placenta and that an altered balance between proliferation and apoptosis of villous trophoblasts is associated with abnormal pregnancies. The TNF-related apoptosis-inducing ligand (TRAIL) is a molecule belonging to TNF superfamily. The role of TRAIL and its Death Receptor 5 (DR5) in regulating villous trophoblast cell turnover in normal and pathologic pregnancies remains to be explored. In order to elucidate the role of TRAIL in the regulation of placental growth, primary cytotrophoblast cells were isolated from normal term placentas ( n = 13) and cultured for 18 and 66 h to generate mononucleate and multinucleate trophoblasts, respectively. The protein expression and localisation of TRAIL and DR5 were determined by Western blotting and immunofluorescence. Secreted sTRAIL was also measured by ELISA. Trophoblast apoptosis was measured by TUNEL in the presence of recombinant TRAIL (rTRAIL), and DR5 relocalisation was assessed by immunostaining after 18 h exposure to TNFα. We demonstrated that TRAIL protein expression and the secretion of soluble TRAIL (sTRAIL) were down-regulated in syncytialised villous trophoblasts and that sTRAIL was independent of biochemical differentiation, as TRAIL-neutralizing antibody (2E5) failed to influence hCG production. TRAIL immunoreactivity was detected in mono- and multinucleated trophoblast cells and localised to the cytoplasm and cellular membranes – more intense staining was associated with apoptotic nuclei. rTRAIL failed to induce apoptosis in trophoblasts cells owing to the nuclear localisation of DR5. However, TNFα treatment caused the redistribution of intracellular DR5 to the cell surface, potentiating apoptotic susceptibly to exogenously administered rTRAIL. These findings highlight a mechanism by which TRAIL and DR5 serve to protective trophoblasts in normal development, but may be activated in conditions of excessive TNFα.