Modulation of TRAIL resistance in colon carcinoma cells: Different contributions of DR4 and DR5

<p>Abstract</p> <p>Background</p> <p>rhTRAIL is a therapeutic agent, derived from the TRAIL cytokine, which induces apoptosis in cancer cells by activating the membrane death receptors 4 and 5 (DR4 and DR5). Here, we investigated each receptor's contribution to rhTRAIL sensitivity and rhTRAIL resistance. We assessed whether agonistic DR4 or DR5 antibodies could be used to circumvent rhTRAIL resistance, alone or in combination with various chemotherapies.</p> <p>Methods</p> <p>Our study was performed in an isogenic model comprised of the SW948 human colon carcinoma cell line and its rhTRAIL resistant sub-line SW948-TR. Effects of rhTRAIL and agonistic DR4/DR5 antibodies on cell viability were measured using MTT assays and identification of morphological changes characteristic of apoptosis, after acridine orange staining. Sensitivity to the different death receptor ligands was stimulated using pretreatment with the cytokine IFN-gamma and the proteasome inhibitor MG-132. To investigate the mechanisms underlying the changes in rhTRAIL sensitivity, alterations in expression levels of targets of interest were measured by Western blot analysis. Co-immunoprecipitation was used to determine the composition of the death-inducing signalling complex at the cell membrane.</p> <p>Results</p> <p>SW948 cells were sensitive to all three of the DR-targeting agents tested, although the agonistic DR5 antibody induced only weak caspase 8 cleavage and limited apoptosis. Surprisingly, agonistic DR4 and DR5 antibodies induced equivalent DISC formation and caspase 8 cleavage at the level of their individual receptors, suggesting impairment of further caspase 8 processing upon DR5 stimulation. SW948-TR cells were cross-resistant to all DR-targeting agents as a result of decreased caspase 8 expression levels. Caspase 8 protein expression was restored by MG-132 and IFN-gamma pretreatment, which also re-established sensitivity to rhTRAIL and agonistic DR4 antibody in SW948-TR. Surprisingly, MG-132 but not IFN-gamma could also increase DR5-mediated apoptosis in SW948-TR.</p> <p>Conclusions</p> <p>These results highlight a critical difference between DR4- and DR5-mediated apoptotic signaling modulation, with possible implications for future combinatorial regimens.</p

TRAIL-receptor preferences in pancreatic cancer cells revisited: Both TRAIL-R1 and TRAIL-R2 have a licence to kill

Background TRAIL is a potent and specific inducer of apoptosis in tumour cells and therefore is a possible new cancer treatment. It triggers apoptosis by binding to its cognate, death-inducing receptors, TRAIL-R1 and TRAIL-R2. In order to increase its activity, receptor-specific ligands and agonistic antibodies have been developed and some cancer types, including pancreatic cancer, have been reported to respond preferentially to TRAIL-R1 triggering. The aim of the present study was to examine an array of TRAIL-receptor specific variants on a number of pancreatic cancer cells and test the generality of the concept of TRAIL-R1 preference in these cells. Methods TRAIL-R1 and TRAIL-R2 specific sTRAIL variants were designed and tested on a number of pancreatic cancer cells for their TRAIL-receptor preference. These sTRAIL variants were produced in HEK293 cells and were secreted into the medium. After having measured and normalised the different sTRAIL variant concentrations, they were applied to pancreatic and control cancer cells. Twenty-four hours later apoptosis was measured by DNA hypodiploidy assays. Furthermore, the specificities of the sTRAIL variants were validated in HCT116 cells that were silenced either for TRAIL-R1 or TRAIL-R2. Results Our results show that some pancreatic cancer cells use TRAIL-R1 to induce cell death, whereas other pancreatic carcinoma cells such as AsPC-1 and BxPC-3 cells trigger apoptosis via TRAIL-R2. This observation extended to cells that were naturally TRAIL-resistant and had to be sensitised by silencing of XIAP (Panc1 cells). The measurement of TRAIL-receptor expression by FACS revealed no correlation between receptor preferences and the relative levels of TRAIL-R1 and TRAIL-R2 on the cellular surface. Conclusions These results demonstrate that TRAIL-receptor preferences in pancreatic cancer cells are variable and that predictions according to cancer type are difficult and that determining factors to inform the optimal TRAIL-based treatments still have to be identified

Early growth response-1 is a regulator of DR5-induced apoptosis in colon cancer cells

BACKGROUND: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces tumour cell apoptosis by binding to death receptor 4 (DR4) and DR5. DR4 and DR5 activation however can also induce inflammatory and pro-survival signalling. It is not known how these different cellular responses are regulated and what the individual role of DR4 vs DR5 is in these processes.METHODS: DNA microarray study was carried out to identify genes differentially expressed after DR4 and DR5 activation. RT-PCR and western blotting was used to examine the expression of early growth response gene-1 (Egr-1) and the proteins of the TRAIL signalling pathway. The function of Egr-1 was studied by siRNA-mediated knockdown and overexpression of a dominant-negative version of Egr-1.RESULTS: We show that the immediate early gene, Egr-1, regulates TRAIL sensitivity. Egr-1 is constitutively expressed in colon cancer cells and further induced upon activation of DR4 or DR5. Our results also show that DR4 mediates a type II, mitochondrion-dependent apoptotic pathway, whereas DR5 induces a mitochondrion-independent, type I apoptosis in HCT15 colon carcinoma cells. Egr-1 drives c-FLIP expression and the short splice variant of c-FLIP (c-FLIPS) specifically inhibits DR5 activation.CONCLUSION: Selective knockdown of c-FLIPS sensitises cells to DR5-induced but not DR4-induced apoptosis and Egr-1 exerts an effect as an inhibitor of the DR5-induced apoptotic pathway, possibly by regulating the expression of c-FLIPS. British Journal of Cancer (2010) 102, 754-764. doi:10.1038/sj.bjc.6605545 www.bjcancer.com Published online 19 January 2010 (C) 2010 Cancer Research U

Analysis of Death Receptor 5 and Caspase-8 Expression in Primary and Metastatic Head and Neck Squamous Cell Carcinoma and Their Prognostic Impact

Death receptor 5 (DR5) and caspase-8 are major components in the extrinsic apoptotic pathway. The alterations of the expression of these proteins during the metastasis of head and neck squamous cell carcinoma (HNSCC) and their prognostic impact have not been reported. The present study analyzes the expression of DR5 and caspase-8 by immunohistochemistry (IHC) in primary and metastatic HNSCCs and their impact on patient survival. Tumor samples in this study included 100 primary HNSCC with no evidence of metastasis, 100 primary HNSCC with lymph node metastasis (LNM) and 100 matching LNM. IHC analysis revealed a significant loss or downregulation of DR5 expression in primary tumors with metastasis and their matching LNM compared to primary tumors with no evidence of metastasis. A similar trend was observed in caspase-8 expression although it was not statistically significant. Downregulation of caspase-8 and DR5 expression was significantly correlated with poorly differentiated tumors compared to moderately and well differentiated tumors. Univariate analysis indicates that, in HNSCC with no metastasis, higher expression of caspase-8 significantly correlated with better disease-free survival and overall survival. However, in HNSCC with LNM, higher caspase-8 expression significantly correlated with poorer disease-free survival and overall survival. Similar results were also generated when we combined both DR5 and caspase-8. Taken together, we suggest that both DR5 and caspase-8 are involved in regulation of HNSCC metastasis. Our findings warrant further investigation on the dual role of caspase-8 in cancer development

Death ligands and granulysin: mechanisms of tumor cell death induction and therapeutic opportunities

The immune system plays a key role in cancer immune surveillance to control tumor development. The final goal is recognizing and killing transformed cells and consequently the elimination of the tumor. The main effector cell types exerting cytotoxicity against tumors are natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although the mechanism of activation of NK cells and CTLs are quite different, both cell types share common antitumor effector mechanisms of cytotoxicity which lead to induction of cell death of tumor cells by apoptosis. Among these mechanisms are the death ligand- and granulysin-mediated cell deaths. In this review, we summarize the main concepts of these effector cytotoxic mechanisms against cancer cells, how NK cells and CTLs use them to control tumor development and the therapeutic approaches currently developed based on these molecules.This work was supported by Grants PI13/00416 (L Martinez-Lostao) from Instituto de Salud Carlos III, SAF2010-15341 and SAF2013-48626-C2-1-R (A Anel) from Ministerio de Economía y Competitividad. D Miguel was supported by predoctoral fellowships from Gobierno de Aragón and an EMBO short-term Fellowship and SAW was supported by a predoctoral fellowship from the Government of Yemen.Peer reviewe

Intake of fish and marine n-3 fatty acids in relation to coronary calcification:the Rotterdam Study

Background: Epidemiologic and experimental data suggest a cardioprotective effect of n23 (omega-3) fatty acids from fish [eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA)]. Objective: The objective was to examine the association of fish and EPA plus DHA intakes with coronary calcification in a general older population. Design: Diet was assessed between 1990 and 1993 by using a semiquantitative 170-item food-frequency questionnaire. Coronary calcification was assessed \xe2\x89\x887 y later by electron-beam computed tomography in 1570 asymptomatic cardiac subjects with complete dietary data (44% men, mean age of 64 y). Calcium scores according to Agatston\'s method were divided into \xe2\x89\xa410 (no/minimal coronary calcification), 11-400 (mild/moderate calcification), and >400 (severe calcification). Prevalence ratios (PRs) for mild/moderate and severe calcification were obtained in categories of fish and EPA plus DHA intake. PRs were adjusted for age, sex, body mass index, diabetes mellitus, socioeconomic status, smoking, alcohol intake, physical activity, and dietary factors. Results: Subjects with a fish intake >19 g/d had a significantly lower prevalence of mild/moderate calcification (PR: 0.87; 95% CI: 0.78, 0.98; full model) than did subjects who consumed no fish. Subjects with a high fish intake also had a lower prevalence of severe calcifi-cation (PR: 0.88; 95% CI: 0.74, 1.04), which was borderline statistically significant. EPA plus DHA intake showed no significant associations (PR: 0.93 and 0.97, respectively; P > 0.05). Conclusions: We found a weak inverse association between fish intake and coronary calcification. If confirmed in other population-based studies, more research is warranted to determine which components in fish can inhibit vascular calcification