Teresa Martínez

Aplidin induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation.

Aplidin® is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin® alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin® generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin® activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin®-induced JNK activation and cytotoxicity. Our results show that Aplidin® induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.

JNK activation is critical for Aplidin-induced apoptosis.

Aplidin™ is an antitumor drug that induces apoptosis and activates EGFR, Src, JNK and p38MAPK. Here, we show that Aplidin™ induces c-JUN, JUN B, JUN D, c-FOS, FRA-1 and FOS B genes of the activator-protein (AP)-1 family, and also p65/RELA, a major component of nuclear factor-kappa B (NF-κB). Concordantly, Aplidin™ increases AP-1 and NF-κB activity. c-FOS induction depends on EGFR, Src and JNK/p38MAPK. In contrast, induction of c-JUN does not require EGFR activity and p65/RELA induction is only partially dependent on these kinases. We used several genetically deficient cells to identify the critical target of Aplidin™. Mouse embryo fibroblasts (MEFs) deficient for src, yes and fyn, and those lacking all p38MAPK isoforms displayed normal Aplidin™ sensitivity (IC50=12 nM). In contrast, MEFs lacking jnk1 and jnk2, which do not express any JNK isoform, were much less sensitive (IC50>500 nM). Furthermore, cells lacking c-jun or expressing a c-Jun protein in which JNK targets Ser63/73 were mutated (c-JunAA) showed intermediate sensitivity (IC50=60 nM). Additionally, Aplidin™ has higher cytotoxic activity against proliferating than quiescent cells, which is reflected in higher JNK activation. We conclude that phosphorylation by JNK of c-Jun and additional substrate(s) is crucial for Aplidin™ activity.

Plitidepsin Has a Dual Effect Inhibiting Cell Cycle and Inducing Apoptosis via Rac1/c-Jun NH2-Terminal Kinase Activation in Human Melanoma Cells

Melanoma is the most aggressive skin cancer and a serious health problem worldwide because of its increasing incidence and the lack of satisfactory chemotherapy for late stages of the disease. The marine depsipeptide Aplidin (plitidepsin) is an antitumoral agent under phase II clinical development against several neoplasias, including melanoma. We report that plitidepsin has a dual effect on the human SK-MEL-28 and UACC-257 melanoma cell lines; at low concentrations (≤45 nM), it inhibits the cell cycle by inducing G1 and G2/M arrest, whereas at higher concentrations it induces apoptosis as assessed by poly-(ADP-ribose) polymerase cleavage and the appearance of a hypodiploid peak in flow cytometry analyses. Plitidepsin activates Rac1 GTPase and c-Jun NH2-terminal kinase (JNK). In addition, it induces AKT and p38 mitogen-activated protein kinase (MAPK) phosphorylation. By using inhibitors, we found that JNK and p38 MAPK activation depends on Rac1 but not on phosphatidylinositol 3-kinase (PI3K), whereas AKT activation is independent of Rac1 but requires PI3K activity. Plitidepsin cytotoxicity diminishes by Rac1 inhibition or by the blockage of JNK and p38 MAPK using 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), but not by PI3K inhibition using wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). It is remarkable that plitidepsin and dacarbazine, the alkylating agent most active for treating metastatic melanoma, show a synergistic antiproliferative effect that was paralleled at the level of JNK activation. These results indicate that Rac1/JNK activation is critical for cell cycle arrest and apoptosis induction by plitidepsin in melanoma cells. They also support the combined use of plitidepsin and dacarbazine in in vivo studies.

Behaviour in solution of humic acids from lignite in the presence of calcium ions

Abstract Humic acids (HA) extracted from Spanish lignite, were studied to discover their Ca-complexing capacity and their coagulation threshold. It was found that the concentration of Ca + + ions necessary for the complete flocculation increased with the increase of pH and ionic strength (μ) and it was linearly dependent on the concentration of HA. The calcium-complexing capacity of HA increased with the increase of pH and registered lower values as μ was increased. The i.r. spectra of the Ca-humic acid complexes show that the metal is complexed mainly by carboxylic groups from the HA.

Conservación del germoplasma de especies leñosas con técnicas de cultivo in vitro y alamacenamiento en frío

El mantenimiento in vitro de tejidos vegetales constituye un procedimiento esencial para la conservacion y el intercambio de recursos geneticos. El cultivo in vitro se define como un proceso en el que las celulas, tejidos, o organos vegetales son cultivadas en condiciones asepticas en un ambiente controlado. Esta metodologia ofrece la posibilidad de almacenar un gran numero de muestras en un espacio reducido, ademas de permitir la conservacion de aquellas especies con semillas de poca viabilidad, cultivos de propagacion clonal en masa, cultivos altamente heterocigoticos o cultivos que requieren ser propagados vegetativamente para conservar su integridad genetica. Los cultivos establecidos in vitro se pueden conservar a corto plazo (desde 1 semana a dos meses) y a medio plazo manipulando las condiciones de crecimiento. La conservacion a largo plazo de cultivos in vitro se puede lograr usando condiciones de almacenamiento en nitrogeno liquido o crioconservacion. El objetivo de este trabajo ha sido describir las principales metodologias para conservacion in vitro a corto y medio plazo de germoplasma de una serie de especies lenosas lo que ha permitido al Departamento de Fisiologia Vegetal del Instituto de Investigaciones Agrobiologicas de Galicia (CSIC) el establecimiento de un banco de germoplasma de especies lenosas.