Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

<p>Abstract</p> <p>Background</p> <p><it>N</it>-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model.</p> <p>Methods</p> <p>CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling.</p> <p>Results</p> <p>No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells.</p> <p>Conclusions</p> <p>In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.</p

Healthy ageing and depletion of intracellular glutathione influences T cell membrane thioredoxin-1 levels and cytokine secretion

Background: During ageing an altered redox balance has been observed in both intracellular and extracellular compartments, primarily due to glutathione depletion and metabolic stress. Maintaining redox homeostasis is important for controlling proliferation and apoptosis in response to specific stimuli for a variety of cells. For T cells, the ability to generate specific response to antigen is dependent on the oxidation state of cell surface and cytoplasmic protein-thiols. Intracellular thiols are maintained in their reduced state by a network of redox regulating peptides, proteins and enzymes such as glutathione, thioredoxins and thioredoxin reductase. Here we have investigated whether any relationship exists between age and secreted or cell surface thioredoxin-1, intracellular glutathione concentration and T cell surface thioredoxin 1 (Trx-1) and how this is related to interleukin (IL)-2 production.Results: Healthy older adults have reduced lymphocyte surface expression and lower circulating plasma Trx-1 concentrations. Using buthionine sulfoximine to deplete intracellular glutathione in Jurkat T cells we show that cell surface Trx-1 is lowered, secretion of Trx-1 is decreased and the response to the lectin phytohaemagglutinin measured as IL-2 production is also affected. These effects are recapitulated by another glutathione depleting agent, diethylmaleate.Conclusion: Together these data suggest that a relationship exists between the intracellular redox compartment and Trx-1 proteins. Loss of lymphocyte surface Trx-1 may be a useful biomarker of healthy ageing. © 2013 Carilho Torrao et al.; licensee Chemistry Central Ltd

CD133 is a marker for long-term repopulating murine epidermal stem cells

Maintenance, repair, and renewal of the epidermis are thought to depend on a pool of dedicated epidermal stem cells (EpiSCs). Like for many somatic tissues, isolation of a nearly pure population of stem cells is a primary goal in cutaneous biology. We use

CD133 is a marker for long-term repopulating murine epidermal stem cells

Maintenance, repair, and renewal of the epidermis are thought to depend on a pool of dedicated epidermal stem cells (EpiSCs). Like for many somatic tissues, isolation of a nearly pure population of stem cells is a primary goal in cutaneous biology. We use

Lysosomal ceramide mediates gemcitabine-induced death of glioma cells

Acid sphingomyelinase-induced ceramide release has been shown by many studies to induce apoptosis in response to various stimuli. However, the mechanisms of acid sphingomyelinase/ceramide-mediated death signaling following treatment with chemotherapeutic drugs have not been fully elucidated thus far. The present study demonstrates that treatment of glioma cells with clinically achievable doses of gemcitabine results in acid sphingomyelinase activation, lysosomal accumulation of ceramide, cathepsin D activation, Bax insertion into the mitochondria, and cell death. Pharmacological inhibition or genetic deficiency of acid sphingomyelinase prevented these events while overexpression of the enzyme sensitized cells to gemcitabine. Likewise, inhibitors of lysosomal functions also prevent gemcitabine-induced cell death. Our data indicate a critical role of the acid sphingomyelinase/ceramide system for gemcitabine-induced signaling and suggest that lysosomal ceramide accumulation mediates cell death induced by a chemotherapeutic drug