BJcuL, a lectin purified from Bothrops jararacussu venom, induces apoptosis in human gastric carcinoma cells accompanied by inhibition of cell adhesion and actin cytoskeleton disassembly

We show that BJcuL, a lectin purified from Bothrops jararacussu venom, exerts cytotoxic effects to gastric carcinoma cells MKN45 and AGS. This effect was due to the direct interaction with specific glycans on the cells surface and was observed by cell viability decrease, disorganization of actin filaments and apoptosis. In addition, BJcuL was able to reduce tumor cell adhesion to matrigel, what was inhibited by specific carbohydrate or partially inhibited when cells were pre-incubated with matrigel. Our results suggest that BJcuL was able to promote apoptosis in both tumor cells lines and therefore has a prospect for potential use in cancer therapy. (C) 2011 Elsevier Ltd. All rights reserved.CNP

Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

International audienceEzrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type depen-dant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton-and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells

Tissue distribution of quiescin Q6/sulfhydryl oxidase (QSOX) in developing mouse

Quiescin Q6/sulfhydryl oxidases (QSOX) are revisited thiol oxidases considered to be involved in the oxidative protein folding, cell cycle control and extracellular matrix remodeling. They contain thioredoxin domains and introduce disulfide bonds into proteins and peptides, with the concomitant hydrogen peroxide formation, likely altering the redox environment. Since it is known that several developmental processes are regulated by the redox state, here we assessed if QSOX could have a role during mouse fetal development. For this purpose, an anti-recombinant mouse QSOX antibody was produced and characterized. In E-13.5, E-16.5 fetal tissues, QSOX immunostaining was confined to mesoderm- and ectoderm-derived tissues, while in P1 neonatal tissues it was slightly extended to some endoderm-derived tissues. QSOX expression, particularly by epithelial tissues, seemed to be developmentally-regulated, increasing with tissue maturation. QSOX was observed in loose connective tissues in all stages analyzed, intra and possibly extracellularly, in agreement with its putative role in oxidative folding and extracellular matrix remodeling. In conclusion, QSOX is expressed in several tissues during mouse development, but preferentially in those derived from mesoderm and ectoderm, suggesting it could be of relevance during developmental processes

TRAIL receptor gene editing unveils TRAIL-R1 as a master player of apoptosis induced by TRAIL and ER stress

TRAIL induces selective tumor cell death through TRAIL-R1 and TRAIL-R2. Despite the fact that these receptors share high structural homologies, induction of apoptosis upon ER stress, cell autonomous motility and invasion have solely been described to occur through TRAIL-R2. Using the TALEN gene-editing approach, we show that TRAIL-R1 can also induce apoptosis during unresolved unfolded protein response (UPR). Likewise, TRAIL-R1 was found to co-immunoprecipitate with FADD and caspase-8 during ER stress. Its deficiency conferred resistance to apoptosis induced by thaspigargin, tunicamycin or brefeldin A. Our data also demonstrate that tumor cell motility and invasion-induced by TRAIL-R2 is not cell autonomous but induced in a TRAIL-dependant manner. TRAIL-R1, on the other hand, is unable to trigger cell migration owing to its inability to induce an increase in calcium flux. Importantly, all the isogenic cell lines generated in this study revealed that apoptosis induced TRAIL is preferentially induced by TRAIL-R1. Taken together, our results provide novel insights into the physiological functions of TRAIL-R1 and TRAIL-R2 and suggest that targeting TRAIL-R1 for anticancer therapy is likely to be more appropriate owing to its lack of pro-motile signaling capability

Trail receptor gene editing unveils trail-r1 as a master player of apoptosis induced by trail and er stres

TRAIL induces selective tumor cell death through TRAIL-R1 and TRAIL-R2. Despite the fact that these receptors share high structural homologies, induction of apoptosis upon ER stress, cell autonomous motility and invasion have solely been described to occur through TRAIL-R2. Using the TALEN gene-editing approach, we show that TRAIL-R1 can also induce apoptosis during unresolved unfolded protein response (UPR). Likewise, TRAIL-R1 was found to co-immunoprecipitate with FADD and caspase-8 during ER stress. Its deficiency conferred resistance to apoptosis induced by thaspigargin, tunicamycin or brefeldin A. Our data also demonstrate that tumor cell motility and invasion-induced by TRAIL-R2 is not cell autonomous but induced in a TRAIL-dependant manner. TRAIL-R1, on the other hand, is unable to trigger cell migration owing to its inability to induce an increase in calcium flux. Importantly, all the isogenic cell lines generated in this study revealed that apoptosis induced TRAIL is preferentially induced by TRAIL-R1. Taken together, our results provide novel insights into the physiological functions of TRAIL-R1 and TRAIL-R2 and suggest that targeting TRAIL-R1 for anticancer therapy is likely to be more appropriate owing to its lack of pro-motile signaling capability

Ezrin-mediated TRAIL inhibition involves WWOX.

<p>(A) WWOX expression levels in indicated cell lines were analyzed by immunoblot. HSC70 was used here as a loading control. (B) VSV-ezrin WT was expressed ectopically in WWOX-deficient cells SK-HEP-1 and MIA PaCa-2. Expression levels were analysed by immunoblot and apoptosis in the corresponding cell lines after FasL (100 ng/ml) or TRAIL (500 ng/ml) stimulation was analysed by Hoechst staining. (C) S66A and S66D ezrin mutants were expressed in MIA PaCa-2. Expression levels were analysed by immunoblot as above and cell sensitivity to TRAIL-induced cell death was quantified by methylene blue. (D) SW480 cells expressing either ezrin wt or ezrin S66A or S66D were transfected with scramble (Scr) or WWOX (WOXX) si-RNAs for 4 hours and sensitivity to apoptosis induced by TRAIL was quantified by Hoechst staining. Data represent the mean ± SD of at least three different experiments. (**P<0.01; *P<0.05 respective to Scr siRNA tranfected cells; ns stands for not statistically relevant).</p

Ezrin inhibits TRAIL-induced apoptosis downstream of the DISC.

<p>(A) HCT116 or (B) SW480 cells, expressing or not VSV-ezrin were treated for 6 hours with Fas ligand (100 ng/ml) or His-TRAIL (500 ng/ml) or 16 hours with 1μM staurosporin (STS). Apoptosis was quantified by Hoechst staining. Data represent the mean ± SD of at least three different experiments. (*P<0.05; **P<0.01 respective to control cells). Ezrin ectopic expression levels were analyzed by immunoblotting using an anti-VSV antibody in control or ezrin WT-expressing HCT116 and SW480 cells. HSC70 was used as a loading control. (C) HCT116 or (D) SW480 cells, were transfected ezrin or scramble (Scr) siRNAs. 72 h after transfection cells were stimulated for 6 hours with Fas ligand (100 ng/ml) or His-TRAIL (500 ng/ml) and apoptosis was quantified after staining with APO2.7 antibody by flow cytometry. Data represent the mean ± SD of at least three different experiments. (*P<0.05; **P<0.01 respective to control cells). Ezrin expression levels were analyzed by immunoblotting. Actin was used as a loading control. (E) Analysis of TRAIL DISC formation. HCT116 and SW480 cells were stimulated or not with 5 μg/ml Flag-TRAIL cross-linked with 10 μg/ml anti-Flag (M2) antibody. Cells were lysed, and the DISC was immunoprecipitated and analyzed by western blot. One of three independent experiments is shown. (F) HCT116 cells were stimulated or not with 5 μg/ml His-TRAIL for 20 and 60 minutes. After cell lysis, GAPDH antibody was added to the cell lysates and immunoprecipitates were analyzed by western blot.</p