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As the understanding of cancer grows, new therapies have been proposed to improve the well-known limitations of current therapies, whose efficiency relies mostly on early detection, surgery and chemotherapy. Mesenchymal stem cells (MSCs) have been introduced as a promissory and effective therapy. This fact is due to several useful features of MSCs, such as their accessibility and easy culture and expansion in vitro, and their remarkable ability for 'homing' towards tumors, allowing MSCs to exert their anticancer effects directly into tumors. Additionally, MSCs offer the practicability of being genetically engineered to carry anticancer genes, increasing their specificity and efficacy for fighting tumors. In the present study, the antitumoral efficacy and post-implant survival of mice bearing lymphomas implanted intratumorally were determined using mouse bone marrow-derived (BM)-MSCs transduced with soluble TRAIL (sTRAIL), full length TRAIL (flTRAIL), or interferon β (IFNβ), naïve BM-MSCs, or combinations of these. The percentage of surviving mice was determined once all not-implanted mice succumbed. It was found that the percentage of surviving mice implanted with the combination of MSCs-sTRAIL and MSCs-IFN-β was 62.5%. Lymphoma model achieved 100% fatality in the non-treated group by day 41. On the other hand, the percentage of surviving mice implanted with MSCs-sTRAIL was 50% and with MSCs-INFβ 25%. All the aforementioned differences were statistically significant (P<0.05). In conclusion, all implants exhibited tumor size reduction, growth delay, or apparent tumor clearance. MSCs proved to be effective anti-lymphoma agents; additionally, the combination of soluble TRAIL and IFN-β resulted in the most effective antitumor and life enlarging treatment, showing an additive antitumoral effect compared with individual treatments. [ABSTRACT FROM AUTHOR]

The immune status of the tumor microenvironment is a key indicator determining the antitumor effect of immunotherapy. Oncolytic viruses directly target tumor cells or indirectly modulate the tumor microenvironment (TME) especially when properly armed. It was previously demonstrated that conditionally replicating adenovirus serotype 5 (CRAd5) encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) had outstanding antitumor effects in different human cancer cells xenograft models; however, its antitumor immune mechanism has not been evaluated in immunocompetent preclinical mouse models. We first explored the antitumor activity of CRAd5-TRAIL in several murine tumor models and found that the expression of TRAIL induced increases or activation in tumor-infiltrating T cells. To further improve the antitumor effects, mouse CD40 ligand (mCD40L) as an immune activator expressed by recombinant Ad5 vector was firstly used in combination with CRAd5-TRAIL for tumor immunotherapy. Both in vitro and in vivo studies demonstrated that mCD40L effectively activated dendritic cells (DCs), B cells, and tumor-infiltrating T cells, and also promoted tumor cell apoptosis by increasing the expression of TRAIL receptors, thereby significantly enhancing the antitumor activity of oncolytic adenoviruses in CT26 and B16 tumor-bearing models. Although affected by the restriction of oncolytic adenovirus replication in mouse cells, the combination treatment failed to completely eliminate tumor cells, our research still provided a promising strategy for oncolytic adenovirus-mediated solid tumor immunotherapy. [ABSTRACT FROM AUTHOR]

It has been postulated that there is a link between age related degenerative diseases and cancer. The TNF-related apoptosis-inducing ligand (TRAIL) has been shown to selectively kill tumor cells by binding to pro-apoptotic and anti-apoptotic receptors. Our aim was to study the levels of anti-apoptotic receptor (DcR1) in age related macular degeneration (AMD) and controls. AMD patients (115) were classified into two groups: Dry and Wet AMD. Wet AMDs were further classified into occult, predominant classic and minimal classic. 61 healthy individuals were recruited as normal controls. After normalization with total protein, DcR1 levels were analyzed by ELISA. Mann Whitney U-statistic was used for analysis of DcR1 ELISA results. We have observed DcR1 levels in serum sample which were significantly lower in AMD patients as compared to controls (p = 0.001). On the other hand, we did not find difference in DcR1 levels between wet and dry AMD. The present study defines the plausible role of DcR1 in AMD pathology signifying a new therapeutic target for AMD. [ABSTRACT FROM AUTHOR]

Abstract: The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors. [Copyright &y& Elsevier]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in Escherichia coli as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells. [ABSTRACT FROM AUTHOR]

Background: Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro. Methods & Findings: Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production Conclusions: This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease. Author Summary: Dengue is an important endemic tropical disease to which there are no specific therapeutics or approved vaccines. Currently several aspects of pathophysiology remain incompletely understood. A crucial cellular population for viral infections, the plasmacytoid dendritic cells (pDCs) was analyzed in this study. The authors found an in vivo association between the activation state of pDCs and the disease outcome. Membrane TNF-related apoptosis inducing ligand (TRAIL) expressing pDCs, representing activated pDCs, were found in higher frequency in milder cases of dengue than severe cases or healthy individuals. Detection of antiviral cytokine interferon-alpha (IFN-α) and soluble TRAIL positively correlated with pDC activation. Dengue virus (DENV) serotype-2 was able to directly activate pDCs in vitro. Under DENV stimulation TRAIL was relocalized from intracellular to pDC plasma membrane and IFN-α was highly produced. The authors suggest an endocytosis-dependent pathway for DENV-induced pDC activation. It is also highlighted here a role for exogenous IFN-α and pDCs in reducing viral replication in monocytes, one of DENV main target cells. These findings may contribute in the future to the establishment of good prognostic immune responses together with clinical manifestations/warning signs. [ABSTRACT FROM AUTHOR]

Allogeneic stem cell transplantation (allo-SCT) is a potentially curative therapy for chronic myeloid leukemia and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia, and the graft-vs-leukemia (GVL) effect can eradicate residual leukemia after allo-SCT. Ph(+) leukemia cells frequently express death-inducing receptors (DR4 and DR5) for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is one of the cytotoxic ligands expressed on cytotoxic T cells and natural killer cells mediating the GVL effect. Here we demonstrate that imatinib specifically downregulated DR4 and DR5 expression in cell lines and clinical samples of Ph(+) leukemia. Second-generation tyrosine kinase inhibitors (dasatinib and nilotinib) and short hairpin RNA against bcr-abl also downregulated DR4 and DR5 expression in Ph(+) leukemia cells, and transfection of bcr-abl into a Ph(−) leukemia cell line induced DR4 and DR5 expression, which was abrogated by imatinib treatment. Accordingly, Ph(+) leukemia cells that had been pretreated with imatinib showed resistance to the pro-apoptotic activity of recombinant human soluble TRAIL. These observations demonstrate that BCR-ABL is critically involved in the leukemia-specific expression of DR4 and DR5 and in the susceptibility of Ph(+) leukemia to TRAIL-mediated anti-leukemic activity, providing new insight into the mechanisms of the tumor-specific cytotoxic activities of TRAIL. [ABSTRACT FROM AUTHOR]

Abstract: The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4+ T cells is not known. Here we investigated whether primary CD4+ T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4+ T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images. [Copyright &y& Elsevier]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that kills various tumor cells without damaging normal tissues. However, many cancers remain resistant to TRAIL. To overcome TRAIL resistance, combination therapies using sensitizers of the TRAIL pathway would be an efficacious approach. To investigate potential sensitizers of TRAIL-induced apoptosis, we used TRAIL-resistant human T cell leukemia virus type 1 (HTLV-1)-associated adult T cell leukemia/lymphoma (ATL) cells as a model system. So far, HTLV-1-associated ATL is incurable by presently known therapies. Here, we show that wogonin and the structurally related natural flavones apigenin and chrysin break TRAIL resistance in HTLV-1-associated ATL by transcriptional down-regulation of c-FLIP, a key inhibitor of death receptor signaling, and by up-regulation of TRAIL receptor 2 (TRAIL-R2). This effect is mediated through transcriptional inhibition of the p53 antagonist murine double minute 2 (Mdm2), leading to an increase in p53 levels and, consequently, to up-regulation of the p53 target gene TRAIL-R2. We also show that these flavones can sensitize to TNFa- and CD95-mediated cell death. Furthermore, we show that wogonin, apigenin, and chrysin also enhance TRAIL-mediated apoptosis in other human cancer cell lines including breast cancer cell line MDA-MB-231, colon cancer cell line HT-29, hepatocellular carcinoma cell line HepG2, melanoma cell line SK-MEL-37, and pancreatic carcinoma cell line Capan-1 by the same mechanism. Thus, our study suggests the potential use of these flavones as an adjuvant for TRAIL-mediated anticancer therapy. [ABSTRACT FROM AUTHOR]

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed as a potent tool to trigger apoptosis in cancer therapy. However, since ∼60% of tumour cell lines and most primary cancers are resistant to TRAIL-induced apoptosis, several combined therapy approaches aimed to sensitize cells to TRAIL have been developed. One of the major targets of these approaches are cFLIP proteins as they interfere with the initiation of apoptosis induction by TRAIL, are over-expressed in many cancers and their down-regulation enhances TRAIL sensitivity. Although, DNA-damaging agents such as 5-fluorouracil (5-FU), etoposide and adriamycin have been successfully employed due to their ability to trigger cFLIPL and cFLIPs down-regulation the molecular mechanisms underneath their action have been only partially elucidated. We have recently identified ataxia telangiectasia mutated (ATM) as a modulator of cFLIPL and cFLIPS protein levels in the DNA damage response. Here, we provide genetic evidence that ATM kinase activity is required to trigger 5-FU- and neocarzinostatin-dependent cFLIPL and cFLIPS down-regulation, which in turn sensitize hepatocellular carcinoma (HCC) cell lines to TRAIL. ATM activity triggers cFLIP proteins down-regulation in HCC cells independently on p53 and enhances cFLIPL ubiquitination in response to DNA damage. Therefore, we propose that ATM kinase mediates the interplay between DNA damage and death receptor signalling and suggest that expression of catalytically competent ATM in tumour cells may play a key role for successful combinatorial use of TRAIL receptor agonists and DNA-damaging drugs in cancer therapy. [ABSTRACT FROM PUBLISHER]

Cutaneous leishmaniasis (CL) is caused by Leishmania infection of dermal macrophages and is associated with chronic inflammation of the skin. L. aethiopica infection displays two clinical manifestations, firstly ulcerative disease, correlated to a relatively low parasite load in the skin, and secondly non-ulcerative disease in which massive parasite infiltration of the dermis occurs in the absence of ulceration of epidermis. Skin ulceration is linked to a vigorous local inflammatory response within the skin towards infected macrophages. Fas ligand (FasL) and Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expressing cells are present in dermis in ulcerative CL and both death ligands cause apoptosis of keratinocytes in the context of Leishmania infection. In the present report we show a differential expression of FasL and TRAIL in ulcerative and non-ulcerative disease caused by L. aethiopica. In vitro experiments confirmed direct FasL- and TRAIL-induced killing of human keratinocytes in the context of Leishmania-induced inflammatory microenvironment. Systemic neutralisation of FasL and TRAIL reduced ulceration in a model of murine Leishmania infection with no effect on parasitic loads or dissemination. Interestingly, FasL neutralisation reduced neutrophil infiltration into the skin during established infection, suggesting an additional proinflammatory role of FasL in addition to direct keratinocyte killing in the context of parasite-induced skin inflammation. FasL signalling resulting in recruitment of activated neutrophils into dermis may lead to destruction of the basal membrane and thus allow direct FasL mediated killing of exposed keratinocytes in vivo. Based on our results we suggest that therapeutic inhibition of FasL and TRAIL could limit skin pathology during CL. Author Summary: Cutaneous leishmaniases are associated with parasite-induced inflammatory lesions of the skin. The degree of clinical pathology is not associated with parasitic burden; on the contrary, ulcerative lesions are associated with low infectious load, and non-ulcerative lesions are associated with an abundant parasite infiltration. Leishmania are intracellular parasites in mammalian hosts and reside in macrophages in the deep layers of the skin, the dermis. The exact mechanism of ulceration in CL is not known and Leishmania parasites do not directly induce destruction of keratinocytes in the most superficial layer of the skin, the epidermis. In this study we investigated if ulcerated lesions were associated with higher expression of FasL- and TRAIL-induced cell-death of keratinocytes. We found a higher expression of FasL and TRAIL in human skin samples from ulcerative as compared to non-ulcerative leishmaniasis. In a mouse model of ulcerative leishmaniasis neutralisation of FasL and TRAIL reduced ulceration. We suggest that FasL and TRAIL participate in the ulcer formation during leishmaniasis both as a chemoattractant of activated neutrophils leading to tissue destruction and through direct killing of keratinocytes. Possible approaches to use this concept in therapeutical interventions with the aim to reduce immunopathology associated with leishmaniasis are discussed. [ABSTRACT FROM AUTHOR]

MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies. [ABSTRACT FROM AUTHOR]

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent inducer of tumor cell apoptosis, but concerns of considerable liver toxicity limit its uses in human cancer therapy. Here, we show that i.v. injected Escherichia coli DH5α (E. coli DH5α) specifically replicates in solid tumors and metastases in live animals. E. coli DH5α does not enter tumor cells and suits for being the vector for soluble TRAIL (sTRAIL), which induces apoptosis by activating cell-surface death receptors. With the high ‘tumor-targeting’ nature, we demonstrate that intratumoral (i.t.) and intravenous injection of sTRAIL-expressing E. coli DH5α results in the tumor-targeted release of biologically active molecules, which leads to a dramatic reduction in the tumor growth rate and the prolonged survival of tumor-bearing mice. TRAIL delivery by E. coli DH5α did not cause any detectable toxicity to any organs, suggesting that E. coli DH5α-delivered sTRAIL protein therapy may provide a feasible and effective form of treatment for solid tumors. [ABSTRACT FROM AUTHOR]

Abstract: Background: This study was designed to evaluate the biological significance of simultaneous changes in the circulating levels of osteoprotegerin (OPG) and TNF-related apoptosis inducing ligand (TRAIL) in patients with coronary artery disease (CAD), and, in particular, with acute myocardial infarction (AMI). Methods: Total levels of OPG and TRAIL were measured by ELISA in patients with AMI (n =113), unstable angina (UA, n =21) and healthy controls (n =120). Results: Since OPG was elevated during the acute phase (first 12–24–48h) after AMI and in patients with UA with respect to healthy controls, while TRAIL was decreased in acute AMI patients, CAD patients were characterized by an increased OPG/TRAIL ratio. Moreover, the OPG/TRAIL ratio was significantly (p <0.05) higher in the acute AMI patients who developed heart failure (HF) than in those who did not develop HF in the follow-up. Conclusions: An impaired OPG/TRAIL ratio after AMI is related to a higher risk of HF. [Copyright &y& Elsevier]

Whether celastrol, a triterpene from traditional Chinese medicine, can modulate the anticancer effects of TRAIL, the cytokine that is currently in clinical trial, was investigated. As indicated by assays that measure plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3, celastrol potentiated the TRAIL-induced apoptosis in human breast cancer cells, and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that the triterpene down-regulated the expression of cell survival proteins including cFLIP, lAP-1, Bcl-2, Bcl-xL, survivin, and XIAP and up-regulated Bax expression. In addition, we found that celastrol induced the cell surface expression of both the TRAIL receptors DR4 and DR5. This increase in receptors was noted in a wide variety of cancer cells including breast, lung, colorectal, prostate, esophageal, and pancreatic cancer cells, and myeloid and leukemia cells. Gene silencing of the death receptor abolished the effect of celastrol on TRAIL-induced apoptosis. Induction of the death receptor by the triterpenoid was found to be p53-independent but required the induction of CAAT/enhancer-binding protein homologous protein (CHOP), inasmuch as gene silencing of CHOP abolished the induction of DR5 expression by celastrol and associated enhancement of TRAIL-induced apoptosis. We found that celastrol also induced reactive oxygen species (ROS) generation, and ROS sequestration inhibited celastrol-induced expression of CHOP and DR5, and consequent sensitization to TRAIL. Overall, our results demonstrate that celastrol can potentiate the apoptotic effects of TRAIL through down-regulation of cell survival proteins and up-regulation of death receptors via the ROS-mediated up-regulation of CHOP pathway. [ABSTRACT FROM AUTHOR]

[Display omitted] Tumor necrosis factor-related apoptosis ligand (TRAIL) is a promising antitumor agent candidate for its selective proapoptotic activity to various tumor cells. However, TRAIL showed limited efficacy in clinical trials despite of good tolerability. One important reason might be attributed to the poor tumor-homing ability of TRAIL. Herein, we designed an EGFR-targeting TRAIL (Z-TRAIL) by genetically fusing an EGFR-antagonistic affibody (Z EGFR:1907) to the N-terminus of TRAIL. Z-TRAIL was produced as a soluble protein with high yield in E. coli and it maintained the trimeric state of active TRAIL. Under the EGFR-binding mediated by Z EGFR:1907 , Z-TRAIL showed a ∼5 to 20-fold enhancement of cytotoxicity compared to TRAIL on tumor cells in vitro. Furthermore, fusion to Z EGFR:1907 endowed TRAIL with a ∼1.8-fold increase of tumor uptake and a dramatical stronger apoptosis-inducing ability in the mice bearing EGFR-overexpressing A431 tumor xenografts. More importantly, Z-TRAIL exhibited significantly enhanced antitumor efficacy against whether EGFR high-expressing or low-expressing tumors than TRAIL in vivo. In addition, repeated injection of high-dose Z-TRAIL did not show obvious acute toxicity in mice. These results demonstrated that the newly engineered Z-TRAIL might be a promising agent for targeted therapy of EGFR-expressing tumors. [ABSTRACT FROM AUTHOR]

Breast cancer is the most common malignancy affecting women. Although the prognosis generally is good, a substantial number of patients still suffer from relapse, emphasizing the need for novel treatments. Smac mimetics were developed to facilitate cell death by blocking inhibitor of apoptosis proteins (IAPs). It has been suggested that TNF-related apoptosis inducing ligand (TRAIL) can be used together with Smac mimetics to induce cancer cell death. Cell viability was studied with Trypan blue staining and Annexin V assay, siRNA was used to downregulate specific proteins, protein levels were estimated with Western blot, and mRNA levels were analyzed with qPCR, microarray and RNA-seq. For global expression, groups were compared with principal component analysis and the limma package in R. Gene enrichment was analyzed with Fisher's test. For other experiments, significance of difference was tested by one-way ANOVA, followed by Tukey's HSD test. The combination of Smac mimetic LCL-161 and TRAIL induces an irreversible change in phenotype, but not cell death, of luminal MCF-7 breast cancer cells. The cells become small and circular and dissociate from each other and the effect could not be reversed by returning the cells to regular growth medium. The morphology change could be prevented by caspase inhibition using z-VAD-FMK and downregulation of caspase-8. Caspase-7 is also indicated to be of importance since downregulation of this caspase resulted in fewer morphologically changed cells. Enrichment analyses of changes in global gene expression demonstrated that genes associated with estrogen receptor (ER) signaling are downregulated, whereas nuclear factor kappa B- (NF-κB) and interferon- (IFN) driven genes are upregulated in altered cells. However, inhibition of these pathways did not influence the change in morphology. Induction of IFN-induced genes were potentiated but NF-ĸB-driven genes were slightly suppressed by caspase inhibition. The results demonstrate that LCL-161 and TRAIL can irreversibly alter the MCF-7 breast cancer cell phenotype. However, the changes in morphology and global gene expression are mediated via separate pathways. • LCL-161 and TRAIL induce an irreversible morphological change of MCF-7 cells. • The change in morphology is dependent on caspase-8. • There is a sustained change in expression of genes influenced by IFN, NF-κB and ER. • Changes in morphology and gene expression are mediated via different pathways. [ABSTRACT FROM AUTHOR]

Abstract: TRAIL has been suggested to induce the cell death in various tumor cells but not in normal cells; however, several studies have provided the evidence that TRAIL can induce the cell death in some normal cells including human normal hepatocytes, suggesting that TRAIL may show hepatic toxicity in human. In this study, we designed a pro-form of TRAIL (sTRAIL:IL-18) in that soluble TRAIL (sTRAIL) is fused to IL-18, and a matrix metalloproteinases (MMPs) cleavage site is introduced at the connecting site. We showed that sTRAIL:IL-18 has significantly diminished the killing activity in HeLa cells but regains the activity by releasing the free sTRAIL through MMP-2-mediated cleavage. In addition, the killing activity of sTRAIL:IL-18 was significantly increased in HeLa cells when active MMP-2 was produced by TNF-α. Taken together, the data suggested that the sTRAIL:IL-18 can be reactivated at the specialized areas where MMPs are pathologically produced. [Copyright &y& Elsevier]

TRAIL was a tumor-specific protein in development as a novel anticancer therapeutic agent. Generally, when expressed in recombinant Escherichia coli, TRAIL protein was prone to form inclusion bodies. In this study, coexpression of human TRAIL protein and protein isoaspartate methyltranferase (PIMT) from E. coli on plasmid pBV–TRAIL–PCM in E. coli C600 was investigated to overcome the difficulties in soluble expression. The results showed that this PIMT coexpression strategy exerted a positive effect on the TRAIL protein expression in recombinant E. coli, which led to a mean increase in the intracellular concentration of soluble and total protein of TRAIL by 1.57-fold and 1.33-fold, respectively. At the same time, results also suggested that PIMT was a prospective partner for soluble expression of TRAIL protein. [ABSTRACT FROM AUTHOR]

Abstract: We evaluated the ability of α-tocopheryl succinate (α-TOS) to sensitise TRAIL-resistant malignant mesothelioma (MM) cells to TRAIL-induced apoptosis. We show that α-TOS activates expression of DR4/DR5 in a p53-dependent manner and re-establishes sensitivity of resistant MM cells to TRAIL-mediated apoptosis, as documented in p53wt MM cells but not in their p53null counterparts. MM cells selected for TRAIL resistance expressed low cell surface levels of DR4 and DR5. Treatment with sub-lethal doses of α-TOS restored expression of DR4 and DR5. The ability of α-TOS to modulate expression of pro-apoptotic genes may play a role in sensitisation of tumour cells to immunological stimuli. [Copyright &y& Elsevier]

Human renal carcinoma cells (RCCs) were sensitized to the apoptotic effects of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), by treatment with cycloheximide (CHX). In contrast to a previous study, a rapid and dramatic decrease in levels of cellular FLICE (Fas-associated death domain-like IL-1ß-converting enzyme) inhibitory protein (cFLIP) following cycloheximide treatment was observed in all RCCs studied. The unambiguous detection of this decrease in cFLIP was dependent on the quality of the particular antibody preparation used to detect cFLIP. Cycloheximide treatment caused no major change in levels of pro-caspase-8 or cell surface expression of TRAIL receptors. Therefore, cycloheximide treatment resulted in an increase in the pro-caspase-8 to cFLIP ratio, which correlated with sensitization to TRAIL-mediated apoptosis. Furthermore, treatment of human RCCs with small interfering oligoribonucleotides (siRNA) for cFLIP caused a reduction of cFLIP protein and sensitized cells to TRAIL-mediated apoptosis. We concluded that in the presence of an intact TRAIL signaling pathway, a significant reduction of cFLIP alone is sufficient to sensitize human RCCs to TRAIL apoptosis. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor α family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein αB-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type αB-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of αB-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type αB-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation αB-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that αB-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of αB-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer. [ABSTRACT FROM AUTHOR]

In many mammalian cell types, engagement of the TRAIL/Apo2L death receptors DR4 and DR5 alters mitochondrial physiology, thereby promoting the release of pro-apoptotic proteins normally contained within this organelle. A contemporary view of this process is that in so-called type II cells death receptor-activated caspase-8 cleaves the Bcl-2 family member Bid, which generates a truncated Bid fragment that collaborates with Bax, another Bcl-2 relative, to promote the release of mitochondrial factors necessary for activation of executioner caspases and apoptosis. Here we show that in some type II cells caspase-2 is necessary for optimal TRAIL-mediated cleavage of Bid. Downregulation of caspase-2 using RNA interference significantly inhibited TRAIL-induced apoptosis. Analysis of the TRAIL proteolytic cascade following gene silencing of specific pathway components revealed that caspase-2 is necessary for efficient cleavage of Bid; however, caspase-2 proteolytic processing, which occurs downstream of Bax, is not necessary for its role in Bid cleavage. [ABSTRACT FROM AUTHOR]

The TNF-related apoptosis-inducing ligand (TRAIL) is an immunological inducer of apoptosis selectively killing many, but not all, cancer cells. Malignant mesothelioma (MM) is fatal neoplasia with no current treatment, most likely due to high resistance of MM cells towards inducers of apoptosis, including TRAIL. We studied whether inhibition of histone deacetylase (HDAC), recently shown to sensitize malignant cells to a variety of apoptogenic substances, renders MM cells susceptible to TRAIL. Indeed, sub-apoptotic doses of the HDAC inhibitor suberohydroxamic acid (SBHA) sensitized MM cells to TRAIL apoptosis. Of the apoptotic mediators tested, the anti-apoptotic protein Bcl-xL was strongly down-regulated by combined treatment of the cells with SBHA and TRAIL but not by the HDAC inhibitor alone, while little or no change in the expression of other Bcl-2 family members highly expressed in MM cells, including Mcl-1 and Bax, was observed. Our data suggest a cross-talk between HDAC inhibition and TRAIL that results in modulation of expression of specific apoptotic mediators, and point to the potential of their combinatorial use in treatment of TRAIL-resistant neoplastic disease. [Copyright &y& Elsevier]

We demonstrate that PS-341, a small molecule inhibitor of the proteasome, markedly sensitizes resistant prostate, colon, and bladder cancer cells to TNF-like apoptosis-inducing ligand (TRAIL)-induced apoptosis irrespective of Bcl-xL overexpression. PS-341 treatment by itself does not affect the levels of Bax, Bak, caspases 3 and 8, c-Flip or FADD, but elevates levels of TRAIL receptors DR4 and DR5. This increase in receptor protein levels is associated with the ubiquitination of the DR5 protein. When PS-341 is combined with TRAIL, the levels of activated caspase 8 and cleaved Bid are substantially increased. In Bax-negative TRAIL-resistant HC-4 colon cancer cells, the combination of PS-341 and TRAIL overcomes the block to activation of the mitochondrial pathway and causes SMAC and cytochrome c release followed by apoptosis. Similarly, murine embryonic fibroblasts lacking Bax undergo apoptosis when exposed to the combination of PS-341 and TRAIL; however, fibroblasts lacking Bak are significantly resistant. Taken together, these findings indicate that PS-341 enhances TRAIL-induced apoptosis by increasing the cleavage of caspase 8, causing Bak-dependent release of mitochondrial proapoptotic proteins.Oncogene (2003) 22, 4953-4963. doi:10.1038/sj.onc.1206656 [ABSTRACT FROM AUTHOR]

Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis. TRAIL induces apoptosis via death receptors (DR4 and DR5) in a wide variety of tumor cells but not in normal cells. The objectives of this study are to investigate the intracellular mechanisms by which TRAIL induces apoptosis. The death receptor Fas, upon ligand binding, trimerizes and recruits the adaptor protein FADD through the cytoplasmic death domain of Fas. FADD then binds and activates procaspase-8. It is unclear whether FADD is required for TRAIL-induced apoptosis. Here we show that the signaling complex of DR4/DR5 is assembled in response to TRAIL binding. FADD and caspase-8, but not caspase-10, are recruited to the receptor, and cells deficient in either FADD or caspase-8 blocked TRAIL-induced apoptosis. In addition, TRAIL initiates the activation of caspases, the loss of mitochondrial transmembrane potential (Δψm), the cleavage of BID, and the redistribution of mitochondrial cytochrome c. Treatment of Jurkat cells with cyclosporin A delayed TRAIL-induced Δψm, caspase-3 activation and apoptosis. Similarly, Overexpression of Bcl-2 or Bcl-XL delayed, but did not inhibit, TRAIL-induced Δψm and apoptosis. In contrast, XIAP, cowpox virus CrmA and baculovirus p35 inhibited TRAIL-induced apoptosis. These data suggest that death receptors (DR4 and DR5) and Fas receptors induced apoptosis through identical signaling pathway, and TRAIL-induced apoptosis via both mitochondrial-dependent and -independent pathways. Oncogene (2001) 20, 2122–2133. [ABSTRACT FROM AUTHOR]

CD95-L, TNF-α and TRAIL are death-inducing ligands (DILs) which may signal apoptosis via crosslinking of their cognate receptors. The present study shows that treatment of cells with agonistic mAB αAPO-1 (CD95), recombinant TRAIL or TNF-α leads to enhanced mRNA and protein expression of each DIL with concomitant death in target cells. Immunoprecipitation of CD95-L protein from supernatant as well as neutralizing antibodies suggest DIL proteins to be cooperatively acting mediators of these cytotoxic activity. Autoamplification of the death signal was blocked in cells with a defect in apoptosis signaling either due to a dysfunctional FADD molecule or to the failure to activate JNK/SAPKs. Phosphorylation and enhanced binding of cJun and ATF-2 to DIL promoters suggest JNK/SAPKs as activators of these transcription factors following death receptor triggering. In consequence, autocrine production of DILs allows the spread of death signals to sensitive target cells. Oncogene (2000) 19, 4255–4262 [ABSTRACT FROM AUTHOR]

Simple Summary: The mechanisms of leukemic cell resistance to antitumor immunity remains a topical issue. In this work, we found an increase in TRAIL-resistance of human acute myeloid leukemia cells THP-1 in high-density populations in vitro. The results obtained show that a macrophage-like phenotype of the acute myeloid leukemia cells, caused by stressful conditions in high-density culture, can increaser resistance to TRAIL-induced apoptosis, while retaining proliferative potential. The mechanism of the increase in TRAIL-resistance can be related to a decrease in the expression of death receptors DR4 and DR5. The possible realization of these events in vivo may be the reason for tumor progression. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a highly selective and promising anticancer agent due to its specific apoptosis-inducing effect on tumor cells, rather than most normal cells. TRAIL is currently under investigation for use in the treatment of leukemia. However, the resistance of leukemic cells to TRAIL-induced apoptosis may limit its efficacy. The mechanisms of leukemic cell resistance to antitumor immunity remains a topical issue. In this work, we have found an increase in the resistance to TRAIL-induced cell death in human leukemia THP-1 cells, which was caused by differentiation into a macrophage-like phenotype in high-density culture in vitro. Stressful conditions, manifested by the inhibition of cell growth and the activation of cell death in high-density culture of THP-1 cells, induced the appearance of cells adhered to culture dishes. The THP-1ad cell line was derived by selection of these adhered cells. The genetic study, using STR and aCGH assays, has shown that THP-1ad cells were derived from THP-1 cells due to mutagenesis. The THP-1ad cells possessed high proliferative potential and a macrophage-like immunophenotype. The adhesion of THP-1ad cells to the extracellular matrix was mediated by αVβ5 integrin. The cytokine production, as well as the rise of intracellular ROS and NO activities by LPS in THP-1ad cell culture, were characteristic of macrophage-like cells. The THP-1ad cells were found to appear to increase in resistance to TRAIL-induced cell death in comparison with THP-1 cells. The mechanism of the increase in TRAIL-resistance can be related to a decrease in the expression of death receptors DR4 and DR5 on the THP-1ad cells. Thus, the macrophage-like phenotype formation with the maintenance of a high proliferative potential of leukemic cells, caused by stress conditions in high-density cell cultures in vitro, can induce an increase in resistance to TRAIL-induced cell death due to the loss of DR4 and DR5 receptors. The possible realization of these events in vivo may be the reason for tumor progression. [ABSTRACT FROM AUTHOR]

Neuroblastoma is a metastatic brain tumor particularly common in children. The cure rate is below 50% for patients of high-risk condition. Novel therapeutic agents and approaches are needed to improve the cure rate. Tumor necrosis factor-related and apoptosis-inducing ligand (TRAIL) is a promising proapoptotic factor that rapidly induces apoptosis preferentially in transformed and cancerous cells. Unfortunately, the common TRAIL resistance in cancers has hampered the clinical application of the ligand. Previously we prepared a novel TRAIL-armed ER derived nanosomal agent (ERN-T) that overcomes TRAIL resistance in some cancer lines when combined with a synthetic antagonist of inhibitors of apoptosis proteins (IAPs), AZD5582. However, how AZD5582 sensitizes cancer cells to ERN-T remains not well understood. In this study we continued to test the therapeutic efficacy of the combinatory therapy of ERN-T and AZD5582 on neuroblastoma, aiming to reveal the molecular mechanism underlying the synergism between AZD5582 and ERN-T. The obtained data revealed that ERN-Ts overcame TRAIL resistance and showed significant cytotoxicity on the resistant neuroblastoma line SH-SH5Y when combined with AZD5582 whilst sparing normal cells. The combination of low doses of ERN-Ts and AZD5582 induced intensive apoptosis in SH-SY5Y but not in normal skin fibroblasts (NSFs). Importantly we discovered that TRAIL sensitization in SH-SY5Y was associated with the concomitant downregulation of antiapoptotic factors cFLIP, MCL-1 and IAPs and upregulation of proapoptotic protein BAX and the death receptor 5 (DR5) by the cotreatment of ERN-T and AZD5582. In vivo study demonstrated that the combination of ERN-T and AZD5582 constituted a highly effective and safe therapy for subcutaneous SH-SY5Y xenograft neuroblastoma in nude mice. In conclusion, we identified that the concomitant regulation of both antiapoptotic and proapoptotic factors and DR5 is an essential molecular mechanism for overcoming TRAIL resistance in SH-SY5Y and the combination of ERN-T and AZD5582 potentially constitutes a novel therapeutic strategy, which is highly effective and safe for neuroblastoma. [ABSTRACT FROM AUTHOR]

Expression of Concern: Tumor Associated Macrophages Protect Colon Cancer Cells from TRAIL-Induced Apoptosis through IL-1 - Dependent Stabilization of Snail in Tumor Cells Reference 1 Kaler P, Galea V, Augenlicht L, Klampfer L (2010) Tumor Associated Macrophages Protect Colon Cancer Cells from TRAIL-Induced Apoptosis through IL-1 - Dependent Stabilization of Snail in Tumor Cells. After publication of this article [[1]], concerns were raised about some of the western blots in Figs 6, 7 and 8. [Extracted from the article]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills various cancer cell types, but also leads to the activation of signaling pathways that favor resistance to cell death. Here, we investigated the as yet unknown roles of calcium signaling and autophagy regulatory proteins during TRAIL-induced cell death in leukemia cells. Taking advantage of the Gene Expression Profiling Interactive Analysis (GEPIA) project, we first found that leukemia patients present a unique TRAIL receptor gene expression pattern that may reflect their resistance to TRAIL. The exposure of NB4 acute promyelocytic leukemia cells to TRAIL induces intracellular Ca2+ influx through a calcium release-activated channel (CRAC)-dependent mechanism, leading to an anti-apoptotic response. Mechanistically, we showed that upon TRAIL treatment, two autophagy proteins, ATG7 and p62/SQSTM1, are recruited to the death-inducing signaling complex (DISC) and are essential for TRAIL-induced Ca2+ influx and cell death. Importantly, the treatment of NB4 cells with all-trans retinoic acid (ATRA) led to the upregulation of p62/SQSTM1 and caspase-8 and, when added prior to TRAIL stimulation, significantly enhanced DISC formation and the apoptosis induced by TRAIL. In addition to uncovering new pleiotropic roles for autophagy proteins in controlling the calcium response and apoptosis triggered by TRAIL, our results point to novel therapeutic strategies for sensitizing leukemia cells to TRAIL. [ABSTRACT FROM AUTHOR]

A lucanthone, one of the family of thioxanthenones, has been reported for its inhibitory effects of apurinic endonuclease-1 and autophagy. In this study, we investigated whether lucanthone could enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in various cancer cells. Combined treatment with lucanthone and TRAIL significantly induced apoptosis in human renal carcinoma (Caki and ACHN), prostate carcinoma (PC3), and lung carcinoma (A549) cells. However, combined treatment did not induce apoptosis in normal mouse kidney cells (TCMK-1) and normal human skin fibroblast (HSF). Lucanthone downregulated protein expression of deubiquitinase DUB3, and a decreased expression level of DUB3 markedly led to enhance TRAIL-induced apoptosis. Ectopic expression of DUB3 inhibited combined treatment with lucanthone and TRAIL-induced apoptosis. Moreover, lucanthone increased expression level of DR5 mRNA via downregulation of miR-216a-5p. Transfection of miR-216a-5p mimics suppressed the lucanthone-induced DR5 upregulation. Taken together, these results provide the first evidence that lucanthone enhances TRAIL-induced apoptosis through DR5 upregulation by downregulation of miR-216a-5p and DUB3-dependent Mcl-1 downregulation in human renal carcinoma cells. [ABSTRACT FROM AUTHOR]

The article presents a study on effects of tumor necrosis facto related apoptosis inducing ligand (TRAIL) on HUVECs from women affected with gestational diabetes. Topics include information on development of cardiovascular and metabolic diseases in women; TRAIL is a multifunctional cytokine that signals through a complex receptor system; and HUVECs chronically exposed to hyperglycemia and to a pro-inflammatory environment during pregnancy to identify TRAIL effects.

Recent studies have suggested that TNF-related apoptosis-inducing ligand (TRAIL) is associated with mortality in sepsis, possibly through necroptosis. The objective of this study was to analyze the association between the plasma level of TRAIL and sepsis severity and outcomes. Furthermore, the plasma level of TRAIL was compared to that of receptor-interacting protein kinase-3 (RIPK3), a key executor of necroptosis, to identify any correlation between TRAIL and necroptosis. Plasma levels of TRAIL and RIPK3 from consecutively enrolled critically ill patients were measured by ELISA. Of 190 study patients, 59 (31.1%) and 84 (44.2%) patients were diagnosed with sepsis and septic shock, respectively. There was a trend of decreased plasma level of TRAIL across the control, sepsis, and septic shock groups. For 143 patients with sepsis, patients with low plasma TRAIL were more likely to have septic shock and higher SAPS3 and SOFA scores. However, no difference in 28-day and 90-day mortalities was observed between the two groups. The plasma level of TRAIL was inversely associated with RIPK3 in patients with sepsis. Plasma levels of TRAIL increased over time on days three and seven, and were inversely associated with sepsis severity and RIPK3 level, but not with mortality. [ABSTRACT FROM AUTHOR]

Discusses the discovery by Avi Ashhenazi et al at the biotechnology firm Genentech Inc. and Vishva Dixit of the University of Michigan Medical School regarding two new receptor proteins for the protein TRAIL. Details about the discovery; The previous discovery that tumor cells are far more susceptible than healthy ones to another suicide signal.