AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine

International audienceAGuIX® are sub-5 nm nanoparticles made of a polysiloxane matrix and gadolinium chelates. This nanoparticle has been recently accepted in clinical trials in association with radiotherapy. This review will summarize the principal preclinical results that have led to first in man administration. No evidence of toxicity has been observed during regulatory toxicity tests on two animal species (rodents and monkeys). Biodistributions on different animal models have shown passive uptake in tumours due to enhanced permeability and retention effect combined with renal elimination of the nanoparticles after intravenous administration. High radiosensitizing effect has been observed with different types of irradiations in vitro and in vivo on a large number of cancer types (brain, lung, melanoma, head and neck…). The review concludes with the second generation of AGuIX nanoparticles and the first preliminary results on human

Transient Alteration of Cellular Redox Buffering before Irradiation Triggers Apoptosis in Head and Neck Carcinoma Stem and Non-Stem Cells

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive and recurrent malignancy owing to intrinsic radioresistance and lack of induction of apoptosis. The major focus of this work was to design a transient glutathione depleting strategy during the course of irradiation of HNSCC in order to overcome their radioresistance associated with redox adaptation. Methodology/Principal Findings: Treatment of SQ20B cells with dimethylfumarate (DMF), a GSH-depleting agent, and L-Buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis 4 h before a 10 Gy irradiation led to the lowering of the endogenous GSH content to less than 10 % of that in control cells and to the triggering of radiation-induced apoptotic cell death. The sequence of biochemical events after GSH depletion and irradiation included ASK-1 followed by JNK activation which resulted in the triggering of the intrinsic apoptotic pathway through Bax translocation to mitochondria. Conclusions: This transient GSH depletion also triggered radiation-induced cell death in SQ20B stem cells, a key event to overcome locoregional recurrence of HNSCC. Finally, our in vivo data highlight the relevance for further clinical trials o

Implications des Microdomaines Rafts de la Membrane Plasmique dans la Résistance aux Radiations Ionisantes d un modèle cellulaire de Carcinome Epidermoïde de la Tête et du Cou

The tumoral sensitivity to ionizing radiation results from the balance between the mechanisms of agression leading to cell death and the mechanisms of intracellular repair and survival. Apart from the radiation-induced damage of DNA, early events at the plasma membrane represent a determining phase in the radiation-induced apoptosis. In a model of HNSCC, we demonstrated the fundamental role of raft microdomains in the transduction of signaling pathways leading to radiation-induced apoptosis of radiosensitive SCC61 cells. By contrast, a defect of structural rearrangement of the rafts was observed in SQ20B cells, a result that could partly explain their radio-resistance. A pharmacological modulation of the rafts with methyl-b-cyclodextrin was carried out, leading to apoptosis of SCC61 cells involving the Fas pathway but activating the survival EGF-R pathway in SQ20B cells, both in a ligand-independent mannerLa sensibilité tumorale aux radiations ionisantes résulte de l équilibre entre les mécanismes d agression conduisant à la mort cellulaire et les mécanismes de réparation et de survie cellulaires. En plus des dommages radio-induits de l ADN, les événements précoces au niveau de la membrane plasmique représentent une étape déterminante dans l apoptose radio-induite. A partir de lignées tumorales issues de carcinomes ORL, nous avons démontré que le déclenchement de la voie de signalisation conduisant à l apoptose radio-induite est lié à un réarrangement des microdomaines rafts dans les cellules radiosensibles SCC61. Ce réarrangement structural des rafts fait défaut dans la lignée SQ20B, ce qui pourrait en partie expliquer leur radiorésistance. La modulation pharmacologique des rafts par la cyclodextrine a permis de déclencher l entrée en apoptose des cellules SCC61 en impliquant la voie Fas et d activer la voie de survie EGF-R dans les cellules SQ20B et ceci en absence de tout ligandLYON1-BU.Sciences (692662101) / SudocSudocFranceF

The BH3-mimetic ABT-737 Reverses the Intrinsic Radioresistance of Head and Neck Squamous Carcinoma Stem and Non-stem Cells

International audienceIntrinsic radioresistance of cancer cells remains a fundamental barrier to obtain the maximal efficacy of radiotherapy (RT) for the treatment of HNSCC. This resistance to RT is due, in large part, to aberrant inhibition of apoptosis in tumor cells. A number of signaling proteins and pathways that contribute to cellular survival are known to be overexpressed and/or hyperactivated in HNSCC. Among these, some members of the Blc-2 protein family, such as Bcl-2 and Bcl-XL, contribute this resistance by inhibiting the intrinsic mitochondrial pathway. ABT-737 is a rationally designed small molecule that binds with high affinity to Bcl-2 and Bcl-XL and antagonizes their anti-apoptotic function, thereby inducing apoptosis in many cancer cell types. Although ABT-737 has been shown to make HNSCC cells more susceptible to conventional chemotherapy, no prior study investigating the effect of ABT-737 in combination with radiotherapy has been undertaken for the treatment of HNSCC

Rôle des effets non-ciblés dans la réponse des cellules de cancer ORL à la radiothérapie

International audienceObjectifs : Évaluer in vitro, la contribution des effets ciblés et non-ciblés (effet bystander) des rayonnements ionisants ainsi que les mécanismes moléculaires impliqués.Méthodes : 2 lignées cellulaires de cancers ORL ayant des radiosensibilités opposées ont été utilisées. Les effets non-ciblés ont été évalués en utilisant un protocole de transfert de milieu. Celui-ci consiste à transférer le milieu des cellules irradiées (cellules donneuses) 2 heures après irradiation sur des cellules non irradiées (cellules receveuses). Les effets cytotoxiques produits tels que les lésions de l’ADN et la mort cellulaire ont été évalués.Résultats : Les cellules donneuses SCC61 présentent une forte diminution de la survie cellulaire en réponse à l’irradiation. Lorsque le milieu de ces cellules irradiées est transféré à des cellules SCC61 receveuses, une cytotoxicité significative est également observée, témoignant de la présence des effets non-ciblés. La présence de lésions de l’ADN dans les cellules receveuses, mesurées par la formation de cassures double brin (foci H2AX) et des micronoyaux, a permis de confirmer la présence d’effets non-ciblés. Le transfert de milieu entre les cellules SCC61 et les cellules radiorésistantes SQ20B a permis d’établir que les cellules SQ20B pouvaient transmettre la signalisation bystander mais qu’elles ne pouvaient pas répondre aux signaux des cellules radiosensibles.La présence de radeaux lipidiques, plateformes enrichies en céramide, jouent un rôle essentiel dans les effets non ciblés. En effet, dans les cellules SCC61, l’inhibition de la formation des radeaux lipidiques avec du MBCD (méthyl-béta-cyclodextrine) annule l’effet bystander.Conclusions : Ces résultats permettent d’établir que les effets non-ciblés contribuent à la mort cellulaire des cellules radiosensibles alors que leur absence réduirait l’efficacité d’un traitement par radiothérapie. De plus, la membrane cellulaire jouerait un rôle majeur dans la transmission de ces effets

Involvement of ceramide enriched domains in the targeted and non-targeted effects of HNSCC cancer cells

International audienceHypothesis: We investigated the relative contribution of targeted and non-targeted effects in the response of human head and neck cancer (HNSCC) cells to photon irradiation as well as the molecular mechanisms involved. Methods: The SCC61 (radiosensitive) and SQ20B (radioresistant) cell lines were used. Targeted effects were measured in cells exposed to increased radiation doses (donor cells) while non-targeted effects were investigated in recipient cells. Recipient cells consisted of cells not exposed to X-rays, but grown in medium previously incubated for 2h with donor cells (conditioned medium). DNA double strand breaks (DNA DSBs) were measured in donor and recipient cells, using immunofluorescent detection of gamma-H2AX

Diversity and complexity of ceramide generation after exposure of jurkat leukemia cells to irradiation.

International audiencePURPOSE: To define which intracellular pools of sphingomyelin and ceramide are involved in the triggering of apoptosis of Jurkat leukemia cells in response to gamma-ray exposure. METHODS AND MATERIALS: We examined the kinetics of ceramide generation at the whole-cell level and in different subcellular compartments (plasma membrane rafts, mitochondria, and endoplasmic reticulum) after irradiation with photons. Ceramide was measured by high-performance liquid chromatography or after pulse labeling experiments, and the presence of sphingomyelinase within mitochondria was assessed by electron microscopy. RESULTS: Irradiation of Jurkat leukemia cells resulted in the sequential triggering of sphingomyelin hydrolysis, followed by de novo synthesis that led to a late ceramide response (from 24 h) correlated with the triggering of apoptosis. At the subcellular level, pulse-label experiments, using [(3)H]-palmitate as a precursor, strengthened the involvement of the radiation-induced sphingomyelin breakdown and revealed a very early peak (15 min) of ceramide in plasma membrane rafts. A second peak in mitochondria was measured 4 h after irradiation, resulting from an increase of the sphingomyelin content relating to the targeting of acid sphingomyelinase toward this organelle. CONCLUSION: These data confirm that ceramide is a major determinant in the triggering of radiation-induced apoptosis and highlight the complexity of the sequential compartment-specific ceramide-mediated response of Jurkat leukemia cells to gamma-rays

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

International audienceDespite advances in the understanding of head and neck squamous cell carcinomas (HNSCC) progression, the five-year survival rate remains low due to local recurrence and distant metastasis. One hypothesis to explain this recurrence is the presence of cancer stem-like cells (CSCs) that present inherent chemo- and radio-resistance. In order to develop new therapeutic strategies, it is necessary to have experimental models that validate the effectiveness of targeted treatments and therefore to have reliable methods for the identification and isolation of CSCs. To this end, we present a protocol for the isolation of CSCs from human HNSCC cell lines that relies on the combination of two successive cell sortings performed by fluorescence activated cell sorting (FACS). The first one is based on the property of CSCs to overexpress ATP-Binding Cassette (ABC) transporter proteins and thus exclude, among others, vital DNA dyes such as Hoechst 33342. The cells sorted with this method are identified as a "side population" (SP). As the SP cells represent a low percentage (<5%) of parental cells, a growing phase is necessary in order to increase their number before the second cell sorting. The next step allows for the selection of cells that possess two other HNSCC stem cell characteristics i.e. high expression level of the cell surface marker CD44 (CD44high) and the over-expression of aldehyde dehydrogenase (ALDHhigh). Since the use of a single marker has numerous limitations and pitfalls for the isolation of CSCs, the combination of SP, CD44 and ALDH markers will provide a useful tool to isolate CSCs for further analytical and functional assays requiring viable cells. The stem-like characteristics of CSCs was finally validated in vitro by the formation of tumorispheres and the expression of β-catenin

Ceramide-Enriched Membrane Domains Contribute to Targeted and Nontargeted Effects of Radiation through Modulation of PI3K/AKT Signaling in HNSCC Cells

International audienceWe investigated the potential involvement of ceramide-enriched membrane domains in radiation-induced targeted and nontargeted effects using head and neck squamous cell carcinoma with opposite radiosensitivities. In radiosensitive SCC61 cells, the proportion of targeted effects was 34% and nontargeted effects killed 32% of cells. In contrast, only targeted effects (30%) are involved in the overall death of radioresistant SQ20B cells. We then demonstrated in SCC61 cells that nontargeted cell response was driven by the formation of the radiation-induced ceramide-enriched domain. By contrast, the existence of these platforms in SQ20B cells confers a permissive region for phosphatidylinositol-3-kinase (PI3K)/AKT activation. The disruption of lipid raft results in strong inhibition of PI3K/AKT signaling, leading to radiosensitization and apparition of nontargeted effects. These results suggest that ceramide-enriched platforms play a significant role in targeted and nontargeted effects during radiotherapy and that drugs modulating cholesterol levels may be a good alternative for improving radiotherapy effectivenes