Cytokines such as tumor necrosis factor-related apoptosisinducing ligand (TRAIL) can induce apoptosis in colon cancer cells through engagement of death receptors. Nevertheless, evading apoptosis induced by anticancer drugs characterizes many types of cancers. This results in the need for combination therapy. In this study, we have investigated whether the flavonoid quercetin could sensitize human colon adenocarcinoma cell lines to TRAIL-induced apoptosis. We report that quercetin enhanced TRAILinduced apoptosis by causing the redistribution of DR4 and DR5 into lipid rafts. Nystatin, a cholesterol-sequestering agent, prevented quercetin-induced clustering of death receptors and sensitization to TRAIL-induced apoptosis in colon adenocarcinoma cells. In addition, our experiments show that quercetin, in combination with TRAIL, triggered the mitochondrial-dependent death pathway, as shown by Bid cleavage and the release of cytochrome c to the cytosol. Together, our findings propose that quercetin, through its ability to redistribute death receptors at the cell surface, facilitates death-inducing signaling complex formation and activation of caspases in response to death receptor stimulation. Based on these results, this study provides a challenging approach to enhance the efficiency of TRAILbased therapies. [Mol Cancer Ther 2007;6(9):2591-9]
The hypermethylated in cancer 1 (HIC1) gene is epigenetically inactivated in cancer, and in addition, the haploinsufficiency of HIC1 is linked to the development of human Miller-Dieker syndrome. HIC1 encodes a zinc-finger transcription factor that acts as a transcriptional repressor. Additionally, the HIC1 protein oligomerizes via the N-terminal BTB/POZ domain and forms discrete nuclear structures known as HIC1 bodies. Here, we provide evidence that HIC1 antagonizes the TCF/b-catenin-mediated transcription in Wnt-stimulated cells. This appears to be due to the ability of HIC1 to associate with TCF-4 and to recruit TCF-4 and b-catenin to the HIC1 bodies. As a result of the recruitment, both proteins are prevented from association with the TCF-binding elements of the Wnt-responsive genes. These data indicate that the intracellular amounts of HIC1 protein can modulate the level of the transcriptional stimulation of the genes regulated by canonical Wnt/b-catenin signaling.
Death receptor 6 (DR6/TNFRSF21) is a death domain-containing receptor of the TNFR superfamily with an apparent regulatory function in hematopoietic and neuronal cells. In this study we document that DR6 is an extensively posttranslationally modified transmembrane protein and that N- and O-glycosylations of amino acids in its extracellular part are mainly responsible for its approximately 40 kDa mobility shift in SDS polyacrylamide gels. Site-directed mutagenesis confirmed that all six extracellular asparagines are N-glycosylated and that the Ser/Thr/Pro cluster in the "stalk" domain juxtaposed to the cysteine-rich domains (CRDs) is a major site for the likely mucine-type of O-glycosylation. Deletion of the entire linker region between CRDs and the transmembrane domain, spanning over 130 amino acids, severely compromises the plasma membrane localization of DR6 and leads to its intracellular retention. Biosynthetic labeling with radiolabeled palmitate and side-directed mutagenesis also revealed that the membrane-proximal Cys368 in the intracellular part of DR6 is, similarly as cysteines in Fas/CD95 or DR4 ICPs, S-palmitoylated. However, palmitoylation of Cys368 is apparently not required for DR6 targeting into Brij-98 insoluble lipid rafts. In contrast, we show that N-glycosylation of the extracellular part might participate in directing DR6 into these membrane microdomains.
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