The association of N-palmitoylethanolamine with the FAAH inhibitor URB597 impairs melanoma growth through a supra-additive action

<p>Abstract</p> <p>Background</p> <p>The incidence of melanoma is considerably increasing worldwide. Frequent failing of classical treatments led to development of novel therapeutic strategies aiming at managing advanced forms of this skin cancer. Additionally, the implication of the endocannabinoid system in malignancy is actively investigated.</p> <p>Methods</p> <p>We investigated the cytotoxicity of endocannabinoids and their hydrolysis inhibitors on the murine B16 melanoma cell line using a MTT test. Enzyme and receptor expression was measured by RT-PCR and enzymatic degradation of endocannabinoids using radiolabeled substrates. Cell death was assessed by Annexin-V/Propidium iodine staining. Tumors were induced in C57BL/6 mice by s.c. flank injection of B16 melanoma cells. Mice were injected i.p. for six days with vehicle or treatment, and tumor size was measured each day and weighted at the end of the treatment. Haematoxylin-Eosin staining and TUNEL assay were performed to quantify necrosis and apoptosis in the tumor and endocannabinoid levels were quantified by HPLC-MS. Tube formation assay and CD31 immunostaining were used to evaluate the antiangiogenic effects of the treatments.</p> <p>Results</p> <p>The <it>N</it>-arachidonoylethanolamine (anandamide, AEA), 2-arachidonoylglycerol and <it>N</it>- palmitoylethanolamine (PEA) reduced viability of B16 cells. The association of PEA with the fatty acid amide hydrolase (FAAH) inhibitor URB597 considerably reduced cell viability consequently to an inhibition of PEA hydrolysis and an increase of PEA levels. The increase of cell death observed with this combination of molecules was confirmed in vivo where only co-treatment with both PEA and URB597 led to decreased melanoma progression. The antiproliferative action of the treatment was associated with an elevation of PEA levels and larger necrotic regions in the tumor.</p> <p>Conclusions</p> <p>This study suggests the interest of targeting the endocannabinoid system in the management of skin cancer and underlines the advantage of associating endocannabinoids with enzymatic hydrolysis inhibitors. This may contribute to the improvement of long-term palliation or cure of melanoma.</p

Oligodeoxyribonucleotide phosphorothioates kill procyclic Trypanosoma brucei brucei: Quantitative determination of their LD50

Phosphorothioates kill the procyclic form of T. brucei brucei by a non antisense but sequence dependent effect. The alamar Blue(TM) method allowed an easy microscale determination of their antiparasitic effect. The LD50 of the sequences tested was in the range of 11-20 mu M. (C) 1997 Elsevier Science Ltd

Heat shock protein 90 transfection reduces ischemia-reperfusion-induced myocardial dysfunction via reciprocal endothelial NO synthase serine 1177 phosphorylation and threonine 495 dephosphorylation

Objectives-The interaction of the heat shock protein 90 (Hsp90) with the endothelial NO synthase ( eNOS) has been shown to account for a sustained production of NO in vitro. Here, we examined whether overexpression of Hsp90 in a pig model of cardiac infarct could preserve the myocardium from the deleterious effects of ischemia-reperfusion. Methods and Results-Percutaneous liposome-based gene transfer was performed by retroinfusion of the anterior interventricular vein before left anterior descending occlusion and reperfusion. We found that recombinant Hsp90 expression in the ischemic region of the heart led to a 33% reduction in infarct size and prevented the increase in postischemic left ventricular end diastolic pressure observed in mock-transfected animals. Regional myocardial function, assessed by subendocardial segment shortening in the infarct region, was increased in Hsp90-transfected animals at baseline and after pacing. All these effects were completely abrogated by administration of the NOS inhibitor N-G-nitro-L-arginine methyl ester. We further documented in vivo and in cultured endothelial cells that the cardioprotective effects of Hsp90 were associated to its capacity to act as an adaptor for both the kinase Akt and the phosphatase calcineurin, thereby promoting eNOS serine 1177 phosphorylation and threonine 495 dephosphorylation, respectively. Conclusions-Hsp90 is a promising target to enhance NO formation in vivo, which may efficiently reduce myocardial reperfusion injury

The type and composition of alginate and hyaluronic-based hydrogels influence the viability of stem cells of the apical papilla

Objective. The goal of the present work was to evaluate in vitro and in vivo the influence ofvarious types and compositions of natural hydrogels on the viability and metabolic activityof SCAPs. Methods. Two alginate, three hyaluronic-based (CorgelTM) hydrogel formulations and Matrigelwere characterized for their mechanical, surface and microstructure properties using rhe-ology, X-ray photoelectron spectroscopy and scanning electron microscopy, respectively. Acharacterized SCAP cell line (RP89 cells) was encapsulated in the different experimentalhydrogel formulations. Cells were cultured in vitro, or implanted in cyclosporine treatedmice. In vitro cell viability was evaluated using a Live/Dead assay and in vitro cellularmetabolic activity was evaluated with a MTS assay. In vivo cell apoptosis was evaluatedby a TUNEL test and RP89 cells were identified by human mitochondria immunostaining.Results. Hydrogel composition influenced their mechanical and surface properties, and theirmicrostructure. In vitro cell viability was above 80% after 2 days but decreased significantlyafter 7 days (60–40%). Viability at day 7 was the highest in Matrigel (70%) and then in Corgel1.5 (60%). Metabolic activity increased over time in all the hydrogels, excepted in alginateSLM. SCAPs survived after 1 week in vivo with low apoptosis (<1%). The highest number ofRP89 cells was found in Corgel 5.5 (140 cells/mm2)

Fibrin hydrogels as Stem Cells of the Apical Papilla (SCAP) Scaffold for regenerative medicine

Evaluation of survival, proliferation and neurodifferentiation of dental stem cells from the apical papilla (SCAP) in fibrin hydrogels. We hypothesized that fibrin composition will influence cell behavior. Methods: Modulus, pore and fiber size were measured. SCAP in vitro viability, proliferation and neural differentiation, as well as in vivo proliferation and angiogenesis were studied. Results: Hydrogel moduli were influenced by fibrin formulation but not hydrogel morphology, SCAP in vitro viability and proliferation. In total 60% of SCAP expressed PanNeurofilament in vitro without induction in Fibrinogen50-Thrombin10. SCAP proliferated when implanted in vivo and stimulated host endothelial cell infiltration. Conclusion: Fibrinogen30-Thrombin10 or Thrombin50 would be more favorable to in vitro SCAP viability and in vivo proliferation, while Fibrinogen50-Thrombin50 would be more adapted to neurodifferentiatio

Alginate-and hyaluronic acid-based hydrogel properties influence dental stem cell viability

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Fitter mitochondria are associated with radioresistance in human head and neck SQD9 cancer cells

The clinical management of head and neck squamous cell carcinoma (HNSCC) commonly involves chemoradiotherapy, but recurrences often occur that are associated with radioresistance. Using human SQD9 laryngeal squamous cell carcinoma cancer cells as a model, we aimed to identify metabolic changes associated with acquired radioresistance. In a top-down approach, matched radiosensitive and radioresistant SQD9 cells were generated and metabolically compared, focusing on glycolysis, oxidative phosphorylation (OXPHOS) and ROS production. The cell cycle, clonogenicity, tumor growth in mice and DNA damage-repair were assessed. Mitochondrial DNA (mtDNA) was sequenced. In a bottom-up approach, matched glycolytic and oxidative SQD9 cells were generated using FACS-sorting, and tested for their radiosensitivity/radioresistance. We found that acquired radioresistance is associated with a shift from a glycolytic to a more oxidative metabolism in SQD9 cells. The opposite was also true, as the most oxidative fraction isolated from SQD9 wild-type cells was also more radioresistant than the most glycolytic fraction. However, neither reduced hexokinase expression nor OXPHOS were directly responsible for the radioresistant phenotype. Radiosensitive and radioresistant cells had similar proliferation rates and were equally efficient for ATP production. They were equally sensitive to redox stress and had similar DNA damage repair, but radioresistant cells had an increased number of mitochondria and a higher mtDNA content. Thus, an oxidative switch is associated with but is not responsible for acquired radioresistance in human SQD9 cells. In radioresistant cells, more and fitter mitochondria could help to preserve mitochondrial functions upon irradiation

Fitter Mitochondria Are Associated With Radioresistance in Human Head and Neck SQD9 Cancer Cells

The clinical management of head and neck squamous cell carcinoma (HNSCC) commonly involves chemoradiotherapy, but recurrences often occur that are associated with radioresistance. Using human SQD9 laryngeal squamous cell carcinoma cancer cells as a model, we aimed to identify metabolic changes associated with acquired radioresistance. In a top-down approach, matched radiosensitive and radioresistant SQD9 cells were generated and metabolically compared, focusing on glycolysis, oxidative phosphorylation (OXPHOS) and ROS production. The cell cycle, clonogenicity, tumor growth in mice and DNA damage-repair were assessed. Mitochondrial DNA (mtDNA) was sequenced. In a bottom-up approach, matched glycolytic and oxidative SQD9 cells were generated using FACS-sorting, and tested for their radiosensitivity/radioresistance. We found that acquired radioresistance is associated with a shift from a glycolytic to a more oxidative metabolism in SQD9 cells. The opposite was also true, as the most oxidative fraction isolated from SQD9 wild-type cells was also more radioresistant than the most glycolytic fraction. However, neither reduced hexokinase expression nor OXPHOS were directly responsible for the radioresistant phenotype. Radiosensitive and radioresistant cells had similar proliferation rates and were equally efficient for ATP production. They were equally sensitive to redox stress and had similar DNA damage repair, but radioresistant cells had an increased number of mitochondria and a higher mtDNA content. Thus, an oxidative switch is associated with but is not responsible for acquired radioresistance in human SQD9 cells. In radioresistant cells, more abundant and fitter mitochondria could help to preserve mitochondrial functions upon irradiation