Differential role of FL-BID and t-BID during verotoxin-1-induced apoptosis in Burkitt's lymphoma cells

Funding Information: Acknowledgements We thank Yann Lécluse (Imaging and Cytometry Platform, Institut Gustave Roussy) for expert technical assistance in performing flow cytometry analyses. We are very grateful to Hana Raslova and Julie Rivière (INSERM UMR 1170, Institut Gustave-Roussy) for their help in producing lentivirus and to Evelyne May and Martine Raphaël (UMR 8126 CNRS) for helpful discussions and suggestions. This work was supported by grants from the Fondation de France 2014 00047509 (JW), the Ligue National Contre le Cancer (doctoral fellowship to JD) and the Fondation ARC (doctoral fellowship to EH).Peer reviewe

Gb3/CD77 receptor : VT-1 apoptotic signaling pathway analysis in Burkitt lymphoma cells and research of endogens ligands

Le glycolipide Gb3/CD77 qui est fortement exprimé en surface des cellules de lymphome de Burkitt (LB) est le récepteur d’une toxine bactérienne la Vérotoxine-1 (VT-1). Notre équipe a montré précédemment que, dans les cellules de LB, la VT-1 induit une cascade apoptotique mettant en jeu les caspases et la mitochondrie. Mon travail a consisté à poursuivre l’analyse des bases moléculaires de ce processus, notamment en m’intéressant au rôle de la protéine pro-apoptotique Bid. Bid est un membre de la famille Bcl-2 qui est clivé par la caspase-8 au cours de l’apoptose et dont la forme tronquée (t-Bid) se relocalise à la mitochondrie. Grâce à l’utilisation de clones cellulaires de LB où Bid a été inhibée puis réexprimée sous forme non clivable et d’un inhibiteur de la caspase-8, nous avons montré que lors de l’apoptose induite par la VT1 : 1) la protéine entière Bid (full-length Bid ou FL-Bid) contrôle l’activation de protéines pro-apoptotiques Bax et de Bak ; 2) t-Bid et FL-Bid sont, toutes les deux, impliquées dans la libération des protéines pro-apoptotiques (cytochrome c et Smac/DIABLO) de l’espace intermembranaire de la mitochondrie vers le cytosol ; 3) FL-Bid contrôle l’homodimérisation de Bax et de Bak qui contribuerait à la libération initiale du cytochrome c et de Smac/DIABLO alors que t-Bid est nécessaire à l’hétérodimérisation de Bax et Bak qui permettrait l’amplification de cette libération. L’ensemble de ces résultats montre donc une coopération fonctionnelle entre Bax et Bak au cours de l’apoptose induite par la VT-1 et surtout met en évidence que l’activation de la voie caspase-8/t-Bid n’est absolument pas requise pour initier la mort cellulaire.Gb3/CD77 est aussi exprimé à la surface de certains lymphocytes B normaux, où il constitue un marqueur de différenciation mais sa fonction endogène reste encore indéterminée. Une deuxième partie de mon travail a consisté à essayer d’identifier le ligand physiologique de Gb3/CD77 pour comprendre son rôle biologique. Grâce à une analyse en spectrométrie de masse, nous avons identifié deux protéines potentiellement partenaires de Gb3/CD77 : la galectine-7 et la protéine S100A11.The Gb3/CD77 glycolipid, which is strongly expressed in Burkitt's lymphoma (BL) cells, is a receptor for the bacterial toxin Verotoxin-1 (VT-1). Previously, our group has shown that VT-1 induces an apoptotic pathway in BL cells which is dependent on caspases and mitochondria. Here, we provide new insights into this pathway. A pro-apoptotic member in the Bcl-2 family, Bid is cleaved by caspase-8 and its truncated form t-Bid is translocated to mitochondria. Using LB cell clones where Bid was inhibited prior to being reexpressed as a non-cleavable mutated form (BID D59A) and a caspase-8 inhibitor to explore VT-1-induced apoptosis, we showed that 1) the full length Bid (FL-Bid) controls the activation of pro-apoptotic proteins Bax and Bak; 2) Both t-Bid and FL-Bid are involved in the release of pro-apoptotic proteins (cytochrome c and Smac/DIABLO) from the mitochondrial intermembrane space to the cytosol; 3) FL-Bid controls the homo-oligomerization of both Bax and Bak, likely contributing to the initial release of cytochrome c and Smac/DIABLO while t-Bid is needed for their hetero-oligomerization followed by amplification of the release. Together, these results reveal a functional cooperation between Bax and Bak during VT-1-induced apoptosis and, most importantly, that activation of caspase-8 and t-Bid is not required to induce the onset of cell death. Gb3/CD77 is also expressed in a proportion of normal B-lymphocytes where it constitutes a differentiation marker but whose function remains uncharacterized. In an effort to look for physiological ligands, we have used a biochemical approach followed by mass spectrometry analysis. Two proteins have been identified as potentially Gb3/CD77 partners, namely galectin-7 and protein S100A11

Analyse des voies d'apoptose induites par la toxine bactérienne la vérotoxine-1 dans les cellules de lymphome de Burkitt

La vérotoxine-1 (VT-1) est une toxine de la famille des Shiga-toxines qui produite par différentes souches d E.coli, et qui a por récepteur le glycolipide (Gb3). Gb3 est fortement exprimé à la surface des cellules de lymphome de Burkitt (LB), une cascade apoptotique mettant en jeu les caspases et la mitochondrie. Afin de caractériser plus précisément les voies de signalisation impliquées dans cette apoptose, nous nous sommes intéressées aux rôles de protéines pro-apoptotiques de la famille de Bcl-2 : Bid et Bax. Nous avons montré, dans notre modèle, que l activation de Bax (changement de conformation et relocalisation mitochondriale) est importante pour la relocalisation des protéines cytochrome c et Smac de l espace intermembranaire mitochondrial vers le cytosol. Cette activation, partiellement contrôlée par Bid/tBid, est impliquée dans le déclenchement de l apoptose induite par VT-1. Bid/tBid sont aussi cruciaux pour la formation d hétérodimères de Bax/Bak dont la fonction précise reste à déterminer. Par ailleurs, nos résultats montrent que la surexpression de Bcl-2 et l inhibition de Bid emêchent le clivage de la caspase-8 et de Bid, ce qui suggère que la caspase-8 est activée en amont et en aval (boucle d amplification) de la mitochondrie.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

The BCL‐2 family protein inhibitor ABT‐737 as an additional tool for the treatment of EBV‐associated post‐transplant lymphoproliferative disorders

International audiencePost-transplant lymphoproliferative disorders (PTLD) and Burkitt's lymphoma (BL) are B-cell malignancies strongly associated with Epstein-Barr virus (EBV) infection. In these lymphoproliferative disorders, EBV infection induces an increase in the expression of the anti-apoptotic protein BCL-2. Given its chemoprotective effect, BCL-2 constitutes an attractive target for new therapeutic strategies for EBV-positive B-cell malignancies. Here, we show that ABT-737, a small inhibitor of BCL-2, BCL-X(L), and BCL-w, strongly induced apoptosis in vitro in EBV-positive lymphoblastoid cell lines (which is a model for PTLD), whereas BL was less sensitive. ABT-737 reduced tumor growth and increased the overall survival of mice in a xenograft model of PTLD but had no effect on BL xenograft mice. ABT-737 combined with a low dose of cyclophosphamide, a major component of the conventional CHOP chemotherapy regimen for BL patients, reduced tumor growth during treatment but failed to improve the overall survival of BL xenograft mice. By contrast, the combination of ABT-737 and rituximab, one of the main options for the treatment of PTLD, was highly efficient and induced approximately 70% remission in PTLD xenograft mice. These results suggest that the use of agents targeting BCL-2, either alone or in combination with other conventional drugs, represents a novel promising approach for post-transplant EBV-positive B lymphoproliferative disorders

Verotoxin-1-Induced ER Stress Triggers Apoptotic or Survival Pathways in Burkitt Lymphoma Cells

Shiga toxins (Stxs) expressed by the enterohaemorrhagic Escherichia coli and enteric Shigella dysenteriae 1 pathogens are protein synthesis inhibitors. Stxs have been shown to induce apoptosis via the activation of extrinsic and intrinsic pathways in many cell types (epithelial, endothelial, and B cells) but the link between the protein synthesis inhibition and caspase activation is still unclear. Endoplasmic reticulum (ER) stress induced by the inhibition of protein synthesis may be this missing link. Here, we show that the treatment of Burkitt lymphoma (BL) cells with verotoxin-1 (VT-1 or Stx1) consistently induced the ER stress response by activation of IRE1 and ATF6—two ER stress sensors—followed by increased expression of the transcription factor C/REB homologous protein (CHOP). However, our data suggest that, although ER stress is systematically induced by VT-1 in BL cells, its role in cell death appears to be cell specific and can be the opposite: ER stress may enhance VT-1-induced apoptosis through CHOP or play a protective role through ER-phagy, depending on the cell line. Several engineered Stxs are currently under investigation as potential anti-cancer agents. Our results suggest that a better understanding of the signaling pathways induced by Stxs is needed before using them in the clinic

Metabolic Activation of Prasugrel: Nature of the Two Competitive Pathways Resulting in the Opening of Its Thiophene Ring

The mechanism generally admitted for the bioactivation of the antithrombotic prodrug, prasugrel, <b>1c</b>, is its two-step enzymatic conversion into a biologically active thiol metabolite. The first step is an esterase-catalyzed hydrolysis of its acetate function leading to a thiolactone metabolite <b>2c</b>. The second step was described as a cytochrome P450 (P450)-dependent oxidative opening of the thiolactone ring of <b>2c</b>, with intermediate formation of a reactive sulfenic acid metabolite that is eventually reduced to the corresponding active thiol <b>3c</b>. This article describes a detailed study of the metabolism of <b>1c</b> by human liver microsomes and human sera, with an analysis by HPLC-MS under conditions allowing a complete separation of the thiol metabolite isomers, after derivatization with 3′-methoxy phenacyl bromide. It shows that there are two competing metabolic pathways for the opening of the <b>2c</b> thiolactone ring. The major one, which was previously described, results from a P450- and NADPH-dependent redox bioactivation of <b>2c</b> and leads to <b>3c</b>, two previously reported thiol diastereomers bearing an exocyclic double bond. It occurs with NADPH-supplemented human liver microsomes but not with human sera. The second one results from a hydrolysis of <b>2c</b> and leads to an isomer of <b>3c</b>, <b>3c endo</b>, in which the double bond has migrated from an exocyclic to an endocyclic position in the piperidine ring. It occurs both with human liver microsomes and human sera, and does not require NADPH. However, it requires Ca²⁺ and is inhibited by paraoxon, which suggests that it is catalyzed by a thioesterase such as PON-1. Chemical experiments have confirmed that hydrolytic opening of thiolactone <b>2c</b> exclusively leads to derivatives of the endo thiol isomer <b>3c endo</b>