Death Receptor 5 Displayed on Extracellular Vesicles Decreases TRAIL Sensitivity of Colon Cancer Cells

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is considered to be a promising antitumor drug because of its selective proapoptotic properties on tumor cells. However, the clinical application of TRAIL is until now limited because of the resistance of several cancer cells, which can occur at various levels in the TRAIL signaling pathway. The role of decoy receptors that can side-track TRAIL, thereby preventing the formation of an activated death receptor, has been extensively studied. In this study, we have focused on extracellular vesicles (EVs) that are known to play a role in cell-to-cell communication and that can be released by donor cells into the medium transferring their components to recipient cells. TRAIL-induced apoptotic signaling is triggered upon the binding of two death receptors, DR4 and DR5. Here, we found that DR5 but not DR4 is present in the conditioned medium (CM)-derived from various cancer cells. Moreover, we observed that DR5 was exposed on EVs and can act as "decoy receptor" for binding to TRAIL. This results in a strongly reduced number of apoptotic cells upon treatment with DR5-specific TRAIL variant DHER in CM. This reduction happened with EVs containing either the long or short isoform of DR5. Taken together, we demonstrated that colon rectal tumor cells can secrete DR5-coated EVs, and this can cause TRAIL resistance. This is to our knowledge a novel finding and provides new insights into understanding TRAIL sensitivity

Death receptor 5 is activated by fucosylation in colon cancer cells

The remarkable pro-apoptotic properties of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) have led to considerable interest in this protein as a potential anticancer therapeutic. However, TRAIL is largely ineffective in inducing apoptosis in certain cancer cells, and the mechanisms underlying this selectivity are unknown. In colon adenocarcinomas, posttranslational modifications including O- and N- glycosylation of death receptors were found to correlate with TRAIL-induced apoptosis. Additionally, mRNA levels of fucosyltransferase 3 (FUT3) and 6 (FUT6) were found to be high in the TRAIL-sensitive colon adenocarcinoma cell line COLO 205. In this study, we use agonistic receptor-specific TRAIL variants to dissect the contribution of FUT3 and FUT6-mediated fucosylation to TRAIL-induced apoptosis via its two death receptors, DR4 and DR5. Triggering of apoptosis by TRAIL revealed that the low FUT3/6-expressing cells DLD-1 and HCT 116 are insensitive to DR5 but not to DR4-mediated apoptosis. By contrast, efficient apoptosis is mediated via both receptors in high FUT3/6-expressing COLO 205 cells. The reconstitution of FUT3/6 expression in DR5-resistant cells completely restored TRAIL sensitivity via this receptor, while only marginally enhancing apoptosis via DR4 at lower TRAIL concentrations. Interestingly, we observed that induction of the salvage pathway by external administration of l-fucose restores DR5-mediated apoptosis in both DLD-1 and HCT 116 cells. Finally, we show that fucosylation influences the ligand-independent receptor association that leads to increased death inducing signalling complex (DISC) formation and caspase-8 activation. Taken together, these results provide evidence for the differential impact of fucosylation on signalling via DR4 or DR5. These findings provide novel opportunities to enhance TRAIL sensitivity in colon adenocarcinoma cells that are highly resistant to DR5-mediated apoptosis

The ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour in humans and is highly resistant to current treatment modalities. We have explored the combined treatment of the endoplasmic reticulum (ER) stress-inducing agent 2,5-dimethyl-celecoxib (DMC) and TNF-related apoptosis-inducing ligand (TRAIL WT) or the DR5-specific TRAIL D269H/E195R variant as a potential new strategy to eradicate GBM cells using TRAIL-resistant and -sensitive GBM cells. GBM cell lines were investigated for their sensitivity to TRAIL, DMC and combination of both agents. Cell viability was measured by MTS assay and apoptosis was assessed by Annexin V/PI and acridine orange staining. Caspase activation and protein expression levels were analysed with Western blotting. Death Receptor (DR) cell surface expression levels were quantified by flow cytometry. DR5 expression was increased in U87 cells by ectopic expression using a retroviral plasmid and survivin expression was silenced using specific siRNAs. We demonstrate that A172 expresses mainly DR5 on the cell surface and that these cells show increased sensitivity for the DR5-specific rhTRAIL D269H/E195R variant. In contrast, U87 cells show low DR cell surface levels and is insensitive via both DR4 and DR5. We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells. The dramatic decrease in cell viability is not accompanied by a correspondent increase in Annexin V/PI or caspase activation typically seen in apoptotic or/and necrotic cells within 24h of treatment. Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells. In A172 cells, sub-toxic concentrations of DMC greatly potentiated TRAIL-induced apoptosis. Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis. Our findings corroborate that DMC is a promising agent against GBM, and uncovers a potential synergistic cooperation with TRAIL in this highly malignant cancer

Regulation of Apoptotic Mediators Reveals Dynamic Responses to Thermal Stress in the Reef Building Coral Acropora millepora

Background: Mass coral bleaching is increasing in scale and frequency across the world's coral reefs and is being driven primarily by increased levels of thermal stress arising from global warming. In order to understand the impacts of projected climate change upon corals reefs, it is important to elucidate the underlying cellular mechanisms that operate during coral bleaching and subsequent mortality. In this respect, increased apoptotic cell death activity is an important cellular process that is associated with the breakdown of the mutualistic symbiosis between the cnidarian host and their dinoflagellate symbionts