Proapoptotic activity of Ukrain is based on Chelidonium majus L. alkaloids and mediated via a mitochondrial death pathway

BACKGROUND: The anticancer drug Ukrain (NSC-631570) which has been specified by the manufacturer as semisynthetic derivative of the Chelidonium majus L. alkaloid chelidonine and the alkylans thiotepa was reported to exert selective cytotoxic effects on human tumour cell lines in vitro. Few clinical trials suggest beneficial effects in the treatment of human cancer. Aim of the present study was to elucidate the importance of apoptosis induction for the antineoplastic activity of Ukrain, to define the molecular mechanism of its cytotoxic effects and to identify its active constituents by mass spectrometry. METHODS: Apoptosis induction was analysed in a Jurkat T-lymphoma cell model by fluorescence microscopy (chromatin condensation and nuclear fragmentation), flow cytometry (cellular shrinkage, depolarisation of the mitochondrial membrane potential, caspase-activation) and Western blot analysis (caspase-activation). Composition of Ukrain was analysed by mass spectrometry and LC-MS coupling. RESULTS: Ukrain turned out to be a potent inducer of apoptosis. Mechanistic analyses revealed that Ukrain induced depolarisation of the mitochondrial membrane potential and activation of caspases. Lack of caspase-8, expression of cFLIP-L and resistance to death receptor ligand-induced apoptosis failed to inhibit Ukrain-induced apoptosis while lack of FADD caused a delay but not abrogation of Ukrain-induced apoptosis pointing to a death receptor independent signalling pathway. In contrast, the broad spectrum caspase-inhibitor zVAD-fmk blocked Ukrain-induced cell death. Moreover, over-expression of Bcl-2 or Bcl-x(L )and expression of dominant negative caspase-9 partially reduced Ukrain-induced apoptosis pointing to Bcl-2 controlled mitochondrial signalling events. However, mass spectrometric analysis of Ukrain failed to detect the suggested trimeric chelidonine thiophosphortriamide or putative dimeric or monomeric chelidonine thiophosphortriamide intermediates from chemical synthesis. Instead, the Chelidonium majus L. alkaloids chelidonine, sanguinarine, chelerythrine, protopine and allocryptopine were identified as major components of Ukrain. Apart from sanguinarine and chelerythrine, chelidonine turned out to be a potent inducer of apoptosis triggering cell death at concentrations of 0.001 mM, while protopine and allocryptopine were less effective. Similar to Ukrain, apoptosis signalling of chelidonine involved Bcl-2 controlled mitochondrial alterations and caspase-activation. CONCLUSION: The potent proapoptotic effects of Ukrain are not due to the suggested "Ukrain-molecule" but to the cytotoxic efficacy of Chelidonium majus L. alkaloids including chelidonine

In addition to membrane injury, an affinity for melanin might be involved in the high sensitivity of human melanoma cells to hederacolchiside A1

We previously reported that hederacolchiside A1 (Hcol A1), a new oleanene saponin isolated from Hedera colchica Koch (Araliaceae) exhibits a preferential cytotoxicity on a pigmented melanoma cell line. The present study confirms the high susceptibility of melanoma cell lines to this drug and shows concentrations producing a 50% decrease in cell content (IC50 values) inversely proportional to the melanin content of each cell line. At cytotoxic concentrations, Hcol A1 induces membrane-damaging effects within 6 h, cytoplasmic vacuolization within 24 h, and non-apoptotic cell death within 48 h. Using a new high-resolution magic-angle spinning nuclear magnetic resonance method, we have shown for the first time that this hederasaponin specifically interacts with melanin. The dissociation constant (2.7 mM) is comparable to those observed with drugs known to interact with melanin. Taking into consideration that the IC50 values were inversely proportional to the melanin in each cell line, our data suggest that, in addition to the delayed membrane injury induced by this drug, the ability of Hcol A1 to bind melanin could contribute to the higher toxicity of Hcol A1 in pigmented melanoma cells

Protein metabolism in the small intestine during cancer cachexia and chemotherapy in mice

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CPOP: An open source C++ cell POPulation modeler for radiation biology applications

International audiencePurpose: Multicellular tumor spheroids are realistic in-vitro systems used in radiation biology research to studythe effect of anticancer drugs or to evaluate the resistance of cancer cells under specific conditions. Whencombining the modeling of spheroids together with the simulation of radiation using Monte Carlo methods, onecould estimate cell and DNA damage to be compared with experimental data. We developed a Cell Population(CPOP) modeler combined to Geant4 simulations in order to tackle how energy depositions are allocated to cells,especially when enhancing radiation outcomes using high-Z nanoparticles. CPOP manages to model large threedimensionalcell populations with independent deformable cells described with their nucleus, cytoplasm andmembranes together with force law systems to manage cell–cell interactions.Methods: CPOP is an opensource platform written in C++. It is divided into two main libraries: a “Modeler”library, for cell geometry modeling using meshes, and a Multi Agent System (MAS) library, simulating all agent(cell) interactions among the population. CPOP is fully interfaced with the Geant4 Monte Carlo toolkit and is ableto directly launch Geant4 simulations after compilation.We modeled a full and realistic 3D cell population from SK-MEL28 melanoma cell population cultured experimentally.The spheroid diameter of 550 ± 40 μm corresponds to a population of approximately 1000 cells havinga diameter of 17.2 ± 2.5 μm and a nucleus diameter of 11.2 ± 2.0 μm. We decided to reproduce cell irradiationsperformed with a X-RAD 320 Biological Irradiator (Precision XRay Inc., North Branford, CT).Results: We simulated the energy spectrum of secondary particles generated in the vicinity of the spheroid andplotted the different energy spectra recovered internally to the spheroid. We evaluated also the impact of AGuIX(Gadolinium) nanoparticles modeled into the spheroid with their corresponding secondary energy spectra.Conclusions: We succeeded into modeling cell populations and combined them with Geant4 simulations. The nextstep will be to integrate DNA geometrical models into cell nuclei and to use the Geant4-DNA physics andradiolysis modeling capabilities in order to evaluate early strand breaks induced on DNA

Simulation de la dose biologique produite par des protons de 65 MeV (faisceau clinique) et des ions carbone

International audienceIntroduction. Pour optimiser les traitements en hadronthérapie, il est primordial de prendre en compte la dose absorbée physique ainsi que l’impact biologique des rayonnements sur les structures irradiées. Pour cela est estimée l’efficacité biologique relative (EBR) qui permet d’évaluer la réponse des cellules à un rayonnement étudié. Ici nous nous intéressons à un faisceau clinique de protons de 65 MeV délivré par la ligne MediCyc du centre Antoine Lacassagne à Nice et un rayonnement d’ions carbones délivrés par le Heavy-Ion Medical Accelerator de Chiba (Japon). Pour prédire cette efficacité biologique relative, il existe différents modèles biophysiques intégrés par des codes Monte Carlo dont certains sont déjà utilisés dans les logiciels de planification de traitement [1]. Parmi ces modèles, le modèle microcinétique (MKM) [2] et le modèle NanOx [1] sont testés dans cette étude. Nous proposons d’utiliser la plateforme Monte Carlo GATE à laquelle sont combinés ces modèles pour prédire la dose biologique.References 1. Monini, Caterina, Étienne Testa, and Michael Beuve. NanOx Prediction of cell survival probabilities for three cell lines. Acta Physica Polonica B 48.10 (2017).2. Y. Kase et al., Microdosimetric calculation of relative biological effectiveness for design of therapeutic proton beams. Journal of Radiation Research, 54. 485–493 (2013)