Showing papers by "Faustino Mollinedo published in 2007"

Expression and regulation of the metalloproteinase ADAM-8 during human neutrophil pathophysiological activation and its catalytic activity on L-selectin shedding.

Abstract: A disintegrin and metalloproteinase domain (ADAM) proteins are a family of transmembrane glycoproteins with heterogeneous expression profiles and proteolytic, cell-adhesion, -fusion, and -signaling properties. One of its members, ADAM-8, is expressed by several cell types including neurons, osteoclasts, and leukocytes and, although it has been implicated in osteoclastogenesis and neurodegenerative processes, little is known about its role in immune cells. In this study, we show that ADAM-8 is constitutively present both on the cell surface and in intracellular granules of human neutrophils. Upon in vitro neutrophil activation, ADAM-8 was mobilized from the granules to the plasma membrane, where it was released through a metalloproteinase-dependent shedding mechanism. Adhesion of resting neutrophils to human endothelial cells also led to up-regulation of ADAM-8 surface expression. Neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis expressed higher amounts of ADAM-8 than neutrophils isolated from peripheral blood and the concentration of soluble ADAM-8 in synovial fluid directly correlated with the degree of joint inflammation. Remarkably, the presence of ADAM-8 both on the cell surface and in suspension increased the ectodomain shedding of membrane-bound L-selectin in mammalian cells. All these data support a potential relevant role for ADAM-8 in the function of neutrophils during inflammatory response.

Endoplasmic reticulum stress in the proapoptotic action of edelfosine in solid tumor cells.

Abstract: The endoplasmic reticulum (ER) has been posited as a potential anticancer target. The synthetic antitumor alkyl-lysophospholipid analogue edelfosine accumulates in the ER of solid tumor cells. This ER accumulation of the drug leads to the inhibition of phosphatidylcholine and protein synthesis, G(2)-M arrest, depletion of ER-stored Ca(2+), Bax up-regulation and activation, transcriptional factor growth arrest and DNA damage-inducible gene 153 up-regulation, caspase-4 and caspase-8 activation, and eventually to apoptosis. Edelfosine prompted ER stress apoptotic signaling, but not the survival unfolded protein response. Edelfosine also induced persistent c-Jun NH(2)-terminal kinase (JNK) activation. Gene transfer-mediated overexpression of apoptosis signal-regulating kinase 1, which plays a crucial role in ER stress, enhanced edelfosine-induced JNK activation and apoptosis. Inhibition of JNK, caspase-4, or caspase-8 activation diminished edelfosine-induced apoptosis. Edelfosine treatment led to the generation of the p20 caspase-8 cleavage fragment of BAP31, directing proapoptotic signals between the ER and the mitochondria. bax(-/-)bak(-/-) double-knockout cells fail to undergo edelfosine-induced ER-stored Ca(2+) release and apoptosis. Wild-type and bax(-/-)bak(-/-) cells showed similar patterns of phosphatidylcholine and protein synthesis inhibition, despite their differences in drug sensitivity. Thus, edelfosine-induced apoptosis is dependent on Bax/Bak-mediated ER-stored Ca(2+) release, but phosphatidylcholine and protein synthesis inhibition is not critical. Transfection-enforced expression of Bcl-X(L), which localizes specifically in mitochondria, prevented apoptosis without inhibiting ER-stored Ca(2+) release. These data reveal that edelfosine induces an ER stress response in solid tumor cells, providing novel insights into the edelfosine-mediated antitumor activity. Our data also indicate that mitochondria are indispensable for this edelfosine-induced cell death initiated by ER stress.

Antitumour ether lipids: proapoptotic agents with multiple therapeutic indications

Abstract: Synthetic ether-linked analogues of phosphatidylcholine, collectively referred to as antitumour ether lipids or synthetic antitumour lipids (ATLs), are a versatile class of structurally-related and pharmacologically active alkylphospholipids with manifold biomedical applications that are able to kill tumour cells and protozoan parasites. This cytotoxic action of ATLs relies on their ability to affect signalling processes, inducing apoptosis in the target cell. ATLs comprise two subfamilies of compounds, namely: i) the so-called alkyl-lysophospholipid analogues or alkyl ether phospholipids, exemplified by edelfosine, which shows a selective antitumour action through a unique mechanism of action by intracellular activation of Fas/CD95 death receptor and its co-clustering with membrane rafts; and ii) the alkylphosphocholines (APCs), exemplified by miltefosine, which has become the first orally-effective treatment for visceral leishmaniasis. New clinically promising APC members include perifosine and erucylph...

Mitochondrial-derived ROS in edelfosine-induced apoptosis in yeasts and tumor cells.

Abstract: Aim: To investigate whether a similar process mediates cytotoxicity of 1- O -octadecyl-2- O -methyl- rac -glycero-3-phosphocholine (ET-18-OCH 3 , edelfosine) in both yeasts and human tumor cells. Methods: A modified version of a previously described assay for the intracellular conversion of nitro blue tetrazolium to formazan by superoxide anion was used to measure the generation of reactive oxygen species (ROS). Apoptotic yeast cells were detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. DNA fragmentation and the generation of ROS were measured by cytofluorimetric analysis in Jurkat cells. Results: Edelfosine induced apoptosis in Saccharomyces cerevisiae , as assessed by TUNEL assay. Meanwhile, edelfosine induced a time- and concentration-dependent generation of ROS in yeasts. Rotenone, an inhibitor of the mitochondrial electron transport chain, prevented ROS generation and apoptosis in response to edelfosine in S cerevisiae . α-Tocopherol abrogated the edelfosine-induced generation of intracellular ROS and apoptosis. Edelfosine also induced an increase of ROS in human leukemic cells that preceded apoptosis. The overexpression of Bcl-2 by gene transfer abrogated both ROS generation and apoptosis induced by edelfosine in leukemic cells. Changes in the relative mitochondrial membrane potential were detected in both yeasts and Jurkat cells. Conclusion: These results indicate that edelfosine induces apoptosis in yeasts in addition to human tumor cells, and this apoptotic process involves mitochondria, likely through mitochondrial-derived ROS. These data also suggest that yeasts can be used as a suitable cell model in elucidating the antitumor mechanism of action of edelfosine.

Synthesis of (+)-Thiersindole C

Abstract: The indole diterpene alkaloid (+)-thiersindole C has been synthesised from ENT-halimic acid. Firstly was elaborated the bi-cyclic system, secondly a Fischer indolization was used to obtain the north side chain and finally elongation of the south side chain was achieved with an isoprene unit. The synthesis of (+)-thiersindole C has corroborated the absolute configuration for the natural product (-)-thiersindole C. The synthesized (+)-thiersindole C showed antitumor activity against a number of human tumor cell lines with an IC 50 in the range of 10 -5 M.