TNF-related apoptosis inducing ligand (TRAIL) and its receptors in tumor surveillance and cancer therapy

TNF-related apoptosis-inducing ligand (TRAIL/APO-2L) is a typical member of the TNF ligand family that induces apoptosis by activating the death receptors TRAIL-R1 and TRAIL-R2. TRAIL has attracted great attention in recent years as a promising anti cancer reagent because recombinant soluble TRAIL derivatives induce apoptosis in a broad range of tumor cells but not or only rarely in non-transformed cells. In this review we will address the putative role of TRAIL in cancer treatment in the light of the emerging importance of TRAIL in tumor surveillance and discuss the molecular basis of the cooperation of TRAIL and chemotherapeutic drugs. In particular, we debate controversial data in the literature concerning the cytotoxicity of different TRAIL derivatives on primary human cells.     

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis is dependent on activation of cysteine and serine proteases

We examined the role of caspases and serine protease(s) in cell death induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). After incubation of adenocarcinoma cells with TRAIL, caspase-3, -8 were activated and the cleavage of Bid induced the release of cytochrome c, from the mitochondria to the cytosol. Tetrapeptide inhibitors of caspase-1, -2, -3, and -8 suppressed DNA fragmentation and attenuated the release of cytochrome c, whereas inhibitors of caspase-5 did not. Interestingly, the general serine protease(s) inhibitor 4-(2-aminoethyl)benzylsulfonyl fluoride (AEBSF) resulted in the arrest of apoptosis. However, the AEBSF did not prevent the release of mitochondrial cytochrome c during TRAIL-induced apoptosis. From these results, we postulate that serine protease(s) may be involved in post-mitochondrial apoptotic events, that lead to the activation of the initiator, caspase-9.     

Death ligand TRAIL induces no apoptosis but inhibits activation of human (auto)antigen-specific T cells

TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis in susceptible cells, which can be both malignant and nontransformed. Despite homologies among the death ligands, there are great differences between the TRAIL system on the one hand and the TNF and CD95 systems on the other hand. In particular, TRAIL-induced apoptosis differs between rodents and man. Studies on animal models of autoimmune diseases suggested an influence of TRAIL on T cell growth and effector functions. Because we previously demonstrated that TRAIL does not induce apoptosis in human (auto)antigen-specific T cells, we now asked whether TRAIL exhibits other immunoregulatory properties in these cells. Active TRAIL inhibited calcium influx through store-operated calcium release-activated calcium channels, IFN-gamma/IL-4 production, and proliferation. These effects were independent of APC, Ag specificity, and Th differentiation, and no differences were detected between healthy donors and multiple sclerosis patients. TRAIL affected neither the expression of the cell cycling inhibitor p27(Kip1) nor the capacity of T cells to produce IL-2 upon Ag rechallenge, indicating that signaling via TRAIL receptor does not induce T cell anergy. Instead, the TRAIL-induced hypoproliferation could be attributed to the down-regulation of the cyclin-dependent kinase 4, indicating a G(1) arrest of the cell cycle. Thus, although it does not contribute to mechanisms of peripheral T cell tolerance such as clonal anergy or deletion by apoptosis, TRAIL can directly inhibit activation of human T cells via blockade of calcium influx.     

Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.     

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

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

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

Contribution of epigenetic silencing of tumor necrosis factor-related apoptosis inducing ligand receptor 1 (DR4) to TRAIL resistance and ovarian cancer

Dysregulation of apoptosis may support tumorigenesis by allowing cells to live beyond their normally intended life span. The various receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are located on chromosome 8p21.2, a region frequently deleted in ovarian cancer. Lack of expression of TRAIL receptor 1 (death receptor 4, DR4) correlates with resistance to TRAIL-induced apoptosis in ovarian cancer cells. Reconstitution of DR4 in the TRAIL-resistant A2780 ovarian cancer cell line was investigated with the demethylating agent 5-aza-2'-deoxycytidine and transient gene transfer. Regulation of other genes in the TRAIL pathway by 5-aza-2'-deoxycytidine was assessed in DNA GeneChip experiments. Primary ovarian cancers were analyzed by methylation-specific PCR and immunohistochemical analysis of a tissue microarray. Regulation of DR4 expression by demethylation or transient transfection is of functional relevance for TRAIL resistance in an ovarian cancer cell line. Hypermethylation of the DR4 promoter could be found in 10 of 36 (27.7%) DNAs isolated from ovarian cancer tissue. In an independent set of 68 ovarian cancer cases, a complete loss or down-regulation of DR4 protein expression was observed 10.3% and 8.8% patients, respectively. A significant (P = 0.019) majority of these patients was below 50 years of age. Our findings show a functional relevance of the level of DR4 expression in ovarian cancer and suggest a substantial contribution of DR4 hypermethylation and consequent loss of DR4 expression to ovarian cancer pathogenesis, particularly in premenopausal patients.     

The anti‐seizure drugs vinpocetine and carbamazepine,but not valproic acid,reduce inflammatory IL‐1β and TNF‐α expression in rat hippocampus

In the present study, the effects of the two classical anti‐epileptic drugs, carbamazepine and valproic acid, and the non‐classical anti‐seizure drug vinpocetine were investigated on the expression of the pro‐inflammatory cytokines IL‐1β and TNF‐α in the hippocampus of rats by PCR or western blot after the administration of one or seven doses. Next, the effects of the anti‐seizure drugs were investigated on the rise in cytokine expression induced by lipopolysaccharides (LPS) inoculation in vivo. To validate our methods, the changes induced by the pro‐convulsive agents 4‐aminopyridine, pentylenetetrazole and pilocarpine were also tested. Finally, the effect of the anti‐seizure drugs on seizures and on the concomitant rise in pro‐inflammatory cytokine expression induced by 4‐aminopyridine was explored. Results show that vinpocetine and carbamazepine reduced the expression of IL‐1β and TNF‐α from basal conditions, and the increase in both pro‐inflammatory cytokines induced by LPS. In contrast, valproic acid failed to reduce both the expression of the cytokines from basal conditions and the rise in IL‐1β and TNF‐α expression induced by LPS. Tonic‐clonic seizures induced either by 4‐aminopyridine, pentylenetetrazole or pilocarpine increased the expression of IL‐1β and TNF‐α markedly. 4‐aminopyridine‐induced changes were reduced by all the tested anti‐seizure drugs, although valproic acid was less effective. We conclude that the anti‐seizure drugs, vinpocetine and carbamazepine, whose mechanisms of action involve a decrease in ion channels permeability, also reduce cerebral inflammation.

     

Hypoxia inhibition of apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Hypoxia is a common environmental stress. Particularly, the center of rapidly growing solid tumors is easily exposed to hypoxic conditions. Thus, tumor cell response to hypoxia plays an important role in tumor progression as well as tumor therapy. However, little is known about hypoxic effect on apoptotic cell death. To examine the effects of hypoxia on TRAIL-induced apoptosis, human lung carcinoma A549 cells were exposed to hypoxia and treated with TRAIL protein. Hypoxia significantly protected A549 cells from apoptosis induced by TRAIL. Western blotting analysis demonstrated that hypoxia increased expression of antiapoptotic proteins such as Bcl-2, Bcl-XL, and IAP family members. The increase of these antiapoptotic molecules is believed to play an hypoxia-mediated protective role in TRAIL-induced apoptosis. Our findings suggest that an increase of antiapoptotic proteins induced by hypoxia may regulate the therapeutic activity of TRAIL protein in cancer therapy.     

IFN-gamma, but not Fas, mediates reduction of allergen-induced mucous cell metaplasia by inducing apoptosis

Inflammatory responses induced by allergen exposure cause mucous cell metaplasia (MCM) by differentiation of existing and proliferating epithelial cells into mucus-storing cells. Airway epithelia have various mechanisms that resolve these changes to form normal airway epithelia. In this report, we first investigated the state of mucous cell metaplasia and the mechanisms by which MCM is reduced despite continued exposures to allergen. After 5 days of allergen exposure, extensive MCM had developed but was reduced when allergen challenge was continued for 15 days. During this exposure period, IL-13 levels decreased and IFN-gamma levels increased in the bronchoalveolar lavage fluid. In contrast, IL-13 levels decreased but IFN-gamma was not detected at any time point during the resolution of MCM following cessation of allergen exposure. Instillation of IFN-gamma but not anti-Fas caused accelerated resolution of MCM and MCM was not resolved in Stat1-deficient mice exposed to allergen for 15 days, confirming that IFN-gamma is crucial for reducing MCM during prolonged exposures to allergen. IFN-gamma but not anti-Fas induced apoptotic cell death in proliferating normal human bronchial epithelial cells and in human bronchial epithelial cells from subjects with asthma. The apoptotic effect of IFN-gamma was caspase dependent and was inhibited by IL-13, indicating that the Th2 milieu in asthmatics may maintain MCM by preventing cell death in metaplastic mucous cells. These studies could be useful in the understanding of deficiencies leading to chronicity in airway changes and designing novel therapies to reverse MCM and airway obstruction in asthmatics.     

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

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

TRAIL mediates apoptosis in cancerous but not normal primary cultured cells of the human reproductive tract

Cancer of the reproductive tract encompasses malignancies of the uterine corpus, cervix, ovary, Fallopian tube, among others and accounts for 15% of female cancer mortalities. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis by binding to death receptors and offers a promising cancer treatment. The goal of this study was to investigate and characterize the effect of TRAIL in endometrial cancer cell lines and normal (non-cancerous) epithelial cells of endometrial origin. We also examined the effect of TRAIL in other primary cultured cancers and normal cells of the human female reproductive tract and evaluated if TRAIL mediated apoptosis correlated with death receptors and decoy receptors 1 and 2. Herein, we demonstrate that TRAIL at concentrations which kill cancerous cells, does not mediate apoptosis or alter cell viability in normal human endometrium, ovary, cervix or Fallopian tube. The partial inhibition by a caspase 9 inhibitor and the total inhibition by a caspase 8 inhibitor demonstrates the dependency on the extrinsic apoptotic pathway. The selective mortality does not correlate with the presence of death or decoy receptors. These results suggest that TRAIL may be an effective treatment for endometrial cancer and other female reproductive cancers, with minimal secondary effects on healthy tissue. This work was supported by a grant from the Wellcome Trust GR071469 (GIO) and the Chilean national science grants FONDECYT 1060495 (GIO) and 1020675 (MC). An erratum to this article is available at .     

Dexamethasone counteracts the immunostimulatory effects of triiodothyronine (T3) on dendritic cells

Glucocorticoids (GCs) are widely used as anti-inflammatory and immunosuppressive agents. Several studies have indicated the important role of dendritic cells (DCs), highly specialized antigen-presenting and immunomodulatory cells, in GC-mediated suppression of adaptive immune responses. Recently, we demonstrated that triiodothyronine (T3) has potent immunostimulatory effects on bone marrow-derived mouse DCs through a mechanism involving T3 binding to cytosolic thyroid hormone receptor (TR) β1, rapid and sustained Akt activation and IL-12 production. Here we explored the impact of GCs on T3-mediated DC maturation and function and the intracellular events underlying these effects. Dexamethasone (Dex), a synthetic GC, potently inhibited T3-induced stimulation of DCs by preventing the augmented expression of maturation markers and the enhanced IL-12 secretion through mechanisms involving the GC receptor. These effects were accompanied by increased IL-10 levels following exposure of T3-conditioned DCs to Dex. Accordingly, Dex inhibited the immunostimulatory capacity of T3-matured DCs on naive T-cell proliferation and IFN-γ production while increased IL-10 synthesis by allogeneic T cell cultures. A mechanistic analysis revealed the ability of Dex to dampen T3 responses through modulation of Akt phosphorylation and cytoplasmic-nuclear shuttling of nuclear factor-κB (NF-κB). In addition, Dex decreased TRβ1 expression in both immature and T3-maturated DCs through mechanisms involving the GC receptor. Thus GCs, which are increased during the resolution of inflammatory responses, counteract the immunostimulatory effects of T3 on DCs and their ability to polarize adaptive immune responses toward a T helper (Th)-1-type through mechanisms involving, at least in part, NF-κB- and TRβ1-dependent pathways. Our data provide an alternative mechanism for the anti-inflammatory effects of GCs with critical implications in immunopathology at the cross-roads of the immune-endocrine circuits.     

Hypericin photo-induced apoptosis involves the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and activation of caspase-8

Hypericin (HYP) is a photosensitizing pigment from Hypericum perforatum that displays cytotoxic effects in neoplastic cell lines. Therefore, HYP is presently under consideration as a new anticancer drug in photodynamic therapy. Here, we investigated the mechanism of action of HYP photo-induced apoptosis of Jurkat cells compared to the cytostatic drug paclitaxel (PXL). Both photoactivated HYP and PXL similarly increased the activity of caspase-8 and caspase-3, and drug-induced apoptosis of Jurkat cells was completely blocked by inhibitors of caspase-8 (Z-IETD-FMK) and caspase-3 (Z-DEVD-FMK). The involvement of death receptors was analyzed using neutralizing monoclonal antibodies against Fas (SM1/23), FasL (NOK-2) and TNF-R1 (MAB225), and a polyclonal rabbit anti-human TNF-related apoptosis-inducing ligand (TRAIL) antiserum. TRAIL antibody blocked TRAIL-induced and HYP photo-induced, but not PXL-induced apoptosis of Jurkat cells. In contrast, PXL-induced, but not HYP-induced apoptosis was blocked by the SM1/23 and NOK-2 antibodies. Anti-TNF-R1 antibody had no effect. These findings suggest that HYP photo-induced apoptosis of Jurkat cells is mediated in part by the TRAIL/TRAIL-receptor system and subsequent activation of upstream caspases.     

Dexamethasone increases the activity but not the amount of choline-phosphate cytidylyltransferase in fetal rat lung

The activity of choline-phosphate cytidylyltransferase is increased by glucocorticoids in late gestation fetal lung in association with increased phosphatidylcholine biosynthesis. Previous indirect data had suggested that the stimulatory effect of the hormone was due to activation of existing enzyme rather than synthesis of new cytidylyltransferase protein. Using a rabbit antibody raised against purified rat liver choline-phosphate cytidylyltransferase, we have now quantitated the amount of the enzyme in fetal rat lung explants cultured with and without dexamethasone. Our results show that the hormone increased the activity of the enzyme but not the amount of cytidylyltransferase protein. Thus the stimulatory effect of dexamethasone on cytidylyltransferase is due to activation of existing enzyme rather than induction of enzyme synthesis.     

The role of NF-kappa B in TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of melanoma cells

Previous studies have shown that activation of NF-kappaB can inhibit apoptosis induced by a number of stimuli. It is also known that TNF-related apoptosis-inducing ligand (TRAIL) can activate NF-kappaB through the death receptors TRAIL-R1 and TRAIL-R2, and decoy receptor TRAIL-R4. In view of these findings, we have investigated the extent to which activation of NF-kappaB may account for the variable responses of melanoma lines to apoptosis induced by TRAIL and other TNF family members. Pretreatment of the melanoma lines with the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (LLnL), which is known to inhibit activation of NF-kappaB, was shown to markedly increase apoptosis in 10 of 12 melanoma lines with death receptors for TRAIL. The specificity of results for inhibition of NF-kappaB activation was supported by an increase of TRAIL-induced apoptosis in melanoma cells transfected with a degradation-resistant IkappaBalpha. Furthermore, studies with NF-kappaB reporter constructs revealed that the resistance of melanoma lines to TRAIL-induced apoptosis was correlated to activation of NF-kappaB in response to TRAIL. TRAIL-resistant sublines that were generated by intermittent exposure to TRAIL were shown to have high levels of activated NF-kappaB, and resistance to TRAIL could be reversed by LLnL and by the superrepressor form of IkappaBalpha. Therefore, these results suggest that activation of NF-kappaB by TRAIL plays an important role in resistance of melanoma cells to TRAIL-induced apoptosis and further suggest that inhibitors of NF-kappaB may be useful adjuncts in clinical use of TRAIL against melanoma.