Evaluation du potentiel de lectines végétales dans le ciblage de médicaments anticancéreux : application à la photochimiothérapie

La photochimiothérapie (ou PCT) peut être utilisée pour le traitement de tumeurs solides (thérapie photodynamique ou PDT) ou pour le traitement ex vivo de différentes pathologies hématologiques (photochimiothérapie extracorporelle ou ECP), comme par exemple le lymphome de Sézary. Le développement de la PCT en oncologie demeure limité en raison de la faible sélectivité des photosensibilisants pour les cellules tumorales. Afin d'augmenter la biodisponibilité des photosensibilisants vis-à-vis des cellules cancéreuses, nous proposons d'utiliser des lectines végétales comme molécule vectrice. A côté des anticorps ou des peptides ciblant les cellules tumorales, les lectines peuvent être une approche complémentaire. En effet, certaines lectines reconnaissent des épitopes glycaniques préférentiellement exprimés par les cellules tumorales. De plus, certaines d'entre elles présentent des effets immunomodulateurs et/ou toxiques, induisant des effets anticancéreux directs (toxicité) ou indirects (immuno-modulation). Deux lectines extraites de l'écorce du mûrier noir Morus nigra semblent être particulièrement intéressantes. Il s'agit de Morniga G (MorG) et de Morniga M (MorM). Ces deux lectines présentent des spécificités glycaniques différentes. Alors que MorM est spécifique du mannose, MorG est spécifique du galactose. Cependant, des lectines présentant une spécificité de reconnaissance monosaccharidique identique peuvent interagir avec des oligosaccharides très différents et induire des effets biologiques distincts. Ainsi, la reconnaissance glycanique médiée par MorM pourrait être sensible à des altérations fines de la N-glycosylation. De même, MorG présente une forte affinité pour certains antigènes O-glycaniques associés aux tumeurs, tels que les antigènes T (CD176; Gal1-3GalNAc1-O-Ser/Thr) et Tn (CD175; GalNAc1-O-Ser/Thr). Dans un premier temps, les effets biologiques de ces deux lectines ont été évalués sur des lymphocytes sains et leucémiques. Elles présentent des effets immunomodulateurs sur les lymphocytes sains et induisent un effet toxique à plus forte dose sur les cellules leucémiques Jurkat. MorM pourrait interagir, en partie, avec la N-glycosylation de certains récepteurs de mort tels que Fas, tandis que MorG pourrait interagir avec la O-glycosylation d'autres récepteurs de mort comme TRAIL-R. Ainsi, ces deux lectines induiraient une mort cellulaire caspase-dépendante en activant des récepteurs de mort différents. De plus, après interaction avec les glycoconjugués membranaires, elles sont rapidement endocytées, ce qui peut, à des concentrations non toxiques, en faire des vecteurs intéressants pour faciliter la pénétration intracellulaire de médicaments. Dans un second temps, un conjugué covalent de la lectine Morniga G et de la porphyrine [5-(4-(5-carboxy-1-butoxy)-phényl)-10,15,20-tris(4-N-méthyl)-pyridiniumyl)] (TrMPyP) a été préparé et caractérisé. La phototoxicité du conjugué TrMPyP-MorG a été évaluée contre la lignée leucémique lymphoïde Jurkat exprimant fortement l'antigène Tn. Le conjugué (1) est internalisé rapidement (5 min) par les cellules Jurkat, (2) augmente très fortement l'activité phototoxique de TrMPyP (DL50 de TrMPyP-MorG = 5 nM ; DL50 de TrMPyP = 15 µM), par l'intermédiaire d'un processus dépendant de la O-glycosylation, (3) induit une mort cellulaire majoritairement caspase indépendante, (4) est capable d'éliminer spécifiquement les cellules leucémiques T Jurkat (Tn-positives) mélangées à des lymphocytes T sains (Tn-négatifs), en préservant le potentiel fonctionnel des lymphocytes sains. De façon intéressante, après irradiation, le conjugué TrMPyP-MorG est capable d'entraîner un effet phototoxique sur différentes lignées cellulaires leucémiques d'origine myéloïde ou lymphoïde et sur des cellules tumorales fraîches provenant de patients atteints de leucémies lymphoïdes aigues ou chroniques. Enfin, des photosensibilisants (activables dans le rouge) plus adaptés au traitement PDT de tumeurs solides ont été greffés sur la lectine MorG. Leur phototoxicité a été étudiée in vitro sur le modèle de cellules leucémiques Jurkat. Les présentes observations démontrent la capacité des lectines végétales à cibler des altérations de la glycosylation à la surface de cellules tumorales et à augmenter l'activité phototoxiques des photosensibilisants dans le cadre de la photochimiothérapie, et en particulier de la photochimiothérapie extracorporelle des leucémies.Photochemotherapy is used both for solid tumors and in extracorporeal treatment of various hematologic disorders, such as Sezary lymphoma. Nevertheless, its development in oncology remains limited, because of the low selectivity of photosensitizers. To enhance photosensitizer selectivity towards human tumor cells, a plant lectin was used as a carrier to target tumor O-glycosylation alterations. Beside monoclonal antibodies or targeting peptides, plant lectins might be a complementary approach. Some lectins have the capability to specifically interact with tumor-associated carbohydrate antigens (TACAs). Morevover, some of them induce immunomodulatory and anticancer effects. Anticancer effects can be resume to indirect effects (such as anticancer immune activation) or direct effects (such cytotoxicity on tumor cells). Two Jacalin-Related Lectins (JRL) extracted from Morus nigra seem to be interesting for biomedical applications. Morniga G (MorG) is a galactose-specific lectin, and Morniga M (MorM) is mannose-specific. Although, MorM could interact with N-linked glycans, MorG could interact with O-linked glycans. Thus, MorG seems to be highly specific for TACAs T (CD176; Gal1-3GalNAc1-O-Ser/Thr) and Tn (CD175; GalNAc1-O-Ser/Thr). These differential monosaccharidic-binding properties could induce different biological effects. First, we have investigated biological effects of the two JRLs on healthy and leukemic lymphocytes. MorG and MorM induce immunomodulatory effects on healthy lymphocytes and cytotoxic effects at higher doses on Jurkat leukemic cells. MorM could interact with N-linked death receptors, such as Fas, and MorG could bind to O-linked death receptors, such as TRAIL-R. So, these lectins might induce caspase-dependent apoptotic process by activating different death receptors-mediated signaling. Thus, after membrane interaction, these lectins are rapidly endocytosed by cells, which at low doses, make them interesting targeting molecules to improve medicines efficacy. On the other hand, a white-light activatable porphyrin, [5-(4-(5-carboxy-1-butoxy)-phenyl)-10,15,20-tris(4N-methyl)-pyridiniumyl)-porphyrin] (TrMPyP), was covalently linked to MorG. The conjugate was molecularly and biologically characterized. Its cytotoxicity was evaluated after photochemotherapy, using human lymphoid leukemia cells as a tumor cell model. Using Tn-positive (Jurkat lymphoid leukemia) cells, the conjugate (with a 1:1 photosensitizer:lectin ratio) (i) was quickly (5 min) taken-up; (ii) dramatically increased cytotoxicity upon irradiation (LD50 = 5 nM) as compared to free photosensitizer (LD50 = 15 µM), through an O-glycan-dependent process; (iii) induced caspase-independent cell death; (iv) specifically purged tumor cells from a 1:1 mixture of Jurkat leukemia (Tn-positive) and healthy (Tn-negative) lymphocytes, preserving the activation potential of healthy lymphocytes. Finally, the effectiveness of the conjugate-mediated killing on various leukemic cell lines as well as human primary acute and chronic lymphoid leukemia cells correlates with the ability of the lectin to bind the cell surface antigens. Lastly, different PS that are more adapted to solid tumors PDT treatment, i.e. activatable in the red wavelengths, were covalently linked to MorG. Their phototoxic activities were tested in vitro against Jurkat cell line. These conjugates were able to kill Jurkat cells when irradiated by red light. The present observations demonstrate the potential of plant lectins to target glycan alterations on tumor cells and their ability to improve PS endocytosis and effectiveness in the field of extracorporeal photochemotherapy of leukemias

Targeting of T/Tn Antigens with a Plant Lectin to Kill Human Leukemia Cells by Photochemotherapy

Photochemotherapy is used both for solid tumors and in extracorporeal treatment of various hematologic disorders. Nevertheless, its development in oncology remains limited, because of the low selectivity of photosensitizers (PS) towards human tumor cells. To enhance PS efficiency, we recently covalently linked a porphyrin (TrMPyP) to a plant lectin (Morniga G), known to recognize with high affinity tumor-associated T and Tn antigens. The conjugation allowed a quick uptake of PS by Tn-positive Jurkat leukemia cells and efficient PS-induced phototoxicity. The present study was performed: (i) to evaluate the targeting potential of the conjugate towards tumor and normal cells and its phototoxicity on various leukemia cells, (ii) to investigate the mechanism of conjugate-mediated cell death. The conjugate: (i) strongly increased (×1000) the PS phototoxicity towards leukemic Jurkat T cells through an O-glycan-dependent process; (ii) specifically purged tumor cells from a 1∶1 mixture of Jurkat leukemia (Tn-positive) and healthy (Tn-negative) lymphocytes, preserving the activation potential of healthy lymphocytes; (iii) was effective against various leukemic cell lines with distinct phenotypes, as well as fresh human primary acute and chronic lymphoid leukemia cells; (iv) induced mostly a caspase-independent cell death, which might be an advantage as tumor cells often resist caspase-dependent cell death. Altogether, the present observations suggest that conjugation with plant lectins can allow targeting of photosensitizers towards aberrant glycosylation of tumor cells, e.g. to purge leukemia cells from blood and to preserve the normal leukocytes in extracorporeal photochemotherapy

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy

Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors

Évaluation du potentiel de lectines végétales dans le ciblage de médicaments anticancéreux (application à la photochimiothérapie)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Targeting Glycosylation Aberrations to Improve the Efficiency of Cancer Phototherapy

International audienceThe use of photodynamic therapy in cancer still remains limited, partly because of the lack of photosensitizer (PS) specificity for the cancerous tissues. Various molecular tools are available to increase PS efficiency by targeting the cancer cell molecular alterations. Most strategies use the protein-protein interactions, e.g. monoclonal antibodies directed toward tumor antigens, such as HER2 or EGFR. An alternative could be the targeting of the tumor glycosylation aberrations, e.g. T/Tn antigens that are truncated O-glycans over-expressed in numerous tumors. Thus, to achieve an effective targeting, PS can be conjugated to molecules that specifically recognize the Oglycosylation aberrations at the cancer cell surface

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy

International audienceAberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors

Single Event Transient Compact Model for FDSOI MOSFETs Taking Bipolar Amplification and Circuit Level Arbitrary Generation Into Account

International audienceSingle Event Transients (SET) are ionizing particles induced current pulses which are able to generate soft errors in CMOS circuits. In Silicon-on-Insulator (SOI) technologies, bipolar amplification phenomena is more significant due to presence of the Burried Oxide (BOX), which is detrimental to soft errors sensitivity. State of the art FDSOI SET models account for bipolar amplification through a dynamic pre-factor. This approach is mainly empirical and not compact. In this work, we propose a SET compact model for FDSOI MOSFETs including a physical modeling of bipolar amplification. Results are validated through TCAD simulations. A circuit level approach is proposed considering arbitrary generation within functional SRAM cell. This approach allows more realistic Single Event Upset (SEU) prediction and we show how circuit level generation can influence SEU prediction

L'infirmier en réanimation

International audienc

L'infirmier en réanimation

International audienc

Compact Modelling of Single Event Transient in Bulk MOSFET for SPICE: Application to Elementary Circuit

International audienceSingle Event Transients (SET) are important issues concerning reliability of CMOS circuits. They lead to occurrence of soft errors in integrated circuits, such as Single Event Upset (SEU) which consists in unexpected bit state switch in SRAM cells [1,2]. We can find models which describe SET in literature [1, 5] but they are not compact (i e. physical model implemented in Verilog-A). In previous work [6], we proposed a theoretical SET model but the implementation in Verilog-A was still challenging. Here, we describe the implementation in Verilog-A of this model and use it through standard SPICE simulations to study the effect of SET on SRAM cell and shift register