Luis García-Fernández

Aplidin ™ induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase C δ

Aplidin™, a new antitumoural drug presently in phase II clinical trials, has shown both in vitro and in vivo activity against human cancer cells. Aplidin™ effectively inhibits cell viability by triggering a canonical apoptotic program resulting in alterations in cell morphology, caspase activation, and chromatin fragmentation. Pro-apoptotic concentrations of Aplidin™ induce early oxidative stress, which results in a rapid and persistent activation of both JNK and p38 MAPK and a biphasic activation of ERK. Inhibition of JNK and p38 MAPK blocks the apoptotic program induced by Aplidin™, demonstrating its central role in the integration of the cellular stress induced by the drug. JNK and p38 MAPK activation results in downstream cytochrome c release and activation of caspases -9 and -3 and PARP cleavage, demonstrating the mediation of the mitochondrial apoptotic pathway in this process. We also demonstrate that protein kinase C delta (PKC-δ) mediates the cytotoxic effect of Aplidin™ and that it is concomitantly processed and activated late in the apoptotic process by a caspase mediated mechanism. Remarkably, cells deficient in PKC-δ show enhanced survival upon drug treatment as compared to its wild type counterpart. PKC-δ thus appears as an important component necessary for full caspase cascade activation and execution of apoptosis, which most probably initiates a positive feedback loop further amplifying the apoptotic process.

Antibacterial Strategies from the Sea: Polymer-Bound Cl-Catechols for Prevention of Biofilm Formation

Inspired by the amino acid 2-chloro-4,5-dihydroxyphenylalanine (Cl-DOPA), present in the composition of the proteinaceous glue of the sandcastle worm Phragmatopoma californica, a simple strategy is presented to confer antifouling properties to polymer surfaces using (but not releasing) a bioinspired biocide. Cl-Dopamine is used to functionalize polymer materials and hydrogel films easily, to prevent biofilm formation on them.

Dexamethasone Induces Lipocalin‐Type Prostaglandin D Synthase Gene Expression in Mouse Neuronal Cells

Lipocalin‐type prostaglandin (PG) D synthase (L‐PGDS) is responsible for the production of PGD2, the main PG in the CNS. PGD2 is an endogenous sleep inducer, and it is involved in the control of odor and pain responses and body temperature. In addition, PGD synthase transports lipophilic molecules in the subarachnoid space and CSF. By northern and western assays we show that the synthetic glucocorticoid dexamethasone, an inhibitor of PG production in most tissues, induces L‐PGDS mRNA and protein in a dose‐ and time‐dependent fashion in mouse neuronal GT1‐7 cells. Accordingly, dexamethasone increases cellular L‐PGDS enzymatic activity. Dexamethasone induced L‐PGDS gene transcription in run‐on assays and activated the mouse L‐PGDS gene promoter in transiently transfected cells. It is interesting that the tumor promoter 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA), which induces the synthesis of PGs in many tissues, inhibited the increase in L‐PGDS expression induced by dexamethasone. In contrast, neither dexamethasone nor TPA affected the expression of cyclooxygenases‐1 and ‐2. Our data demonstrate that dexamethasone induces L‐PGDS gene transcription in neuronal cells.

Identification and localization of a nucleolin homologue in onion nucleoli

A protein homologous to nucleolin, a major nucleolar protein with multifunctional features involved in pre-rRNA synthesis and early processing, has been identified and localized in situ in onion root meristematic cells by different techniques, which have included the use of an antibody raised against hamster nucleolin. The protein was identified on Western blots of nucleolar proteins as a 64-kDa band, by means of the anti-nucleolin antibody, bismuth staining, and the silver staining-nucleolar organizer (Ag-NOR) method. The experiments also suggested that nucleolin could be a target of these two cytochemical stainings. Although the 64-kDa band corresponds to a major nucleolar protein, it is a minor one among total nuclear proteins. The same techniques were used in situ at the ultrastructural level, and the immunogold detection of the nucleolin homologue was quantitatively evaluated. The protein accumulates in the transition area from nucleolar fibrillar centers to the dense fibrillar component, which is considered to be the structural result of ribosomal gene transcription. Out of this transition area, the dense fibrillar component may be divided into two regions, proximal and distal with respect to fibrillar centers, which show, respectively, the significant and unsignificant presence of nucleolin; we interpret this fact as the expression of the topological arrangement of pre-rRNA processing. Fibrillar centers themselves showed a weak but significant labeling with the anti-nucleolin antibody. However, bismuth staining was absent from the interior of fibrillar centers, indicating that the nucleolin in them is not phosphorylated. Ag-NOR staining uniformly covered fibrillar centers and the dense fibrillar component (at least in its proximal region), but it did not stain condensed chromatin inclusions in heterogeneous fibrillar centers, showing that the binding of nucleolin to chromatin is associated with its decondensation. This work provides additional evidence of the high phylogenetic conservation of molecular motifs which take part in ribosome biogenesis.

Nanostructured medical sutures with antibacterial properties

Bacterial repellence in suture materials is a desirable property that can potentially improve the healing process by preventing infection. We describe a method for generating nanostructures at the surface of commercial sutures of different composition, and their potential for preventing biofilm formation. We show how bacteria attachment is altered in the presence of nanosized topographies and identify optimum designs for preventing it without compromising biocompatibility and applicability in terms of nanostructure robustness or tissue friction. These studies open new possibilities for flexible and cost-effective realization of topography-based antibacterial coatings for absorbable biomedical textiles.

Regulation of tau RNA maturation by thyroid hormone is mediated by the neural RNA-binding protein musashi-1.

The tau gene encodes a microtubule-associated protein expressed by neuronal and glial cells. Abnormal deposits of Tau protein are characteristic of several neurodegenerative disorders. Additionally, mutations affecting tau pre-mRNA alternative splicing of exon 10 are associated with frontotemporal dementia and Parkinsonism linked to chromosome 17. In rodents, this process is developmentally regulated by thyroid hormone (T3) causing the predominance of exon 10-containing transcripts. Here we demonstrate that musashi-1 (msi-1) gene is induced by T3 during rat brain development and in N2a cells. T3 increases msi-1 mRNA level in an actinomycin D-sensitive, cycloheximide-resistant fashion without affecting its half-life, which suggests a transcriptional effect. Both ectopic Msi-1 expression and T3 treatment increased the proportion of exon 10-containing tau transcripts. Furthermore, antisense msi-1 expression inhibited T3 action. Our results show that msi-1 mediates the posttranscriptional regulation of tau expression by T3.

PM060184, a new tubulin binding agent with potent antitumor activity including P-glycoprotein over-expressing tumors.

PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganization and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.

Comparison of in vitro and in vivo biological effects of trabectedin, lurbinectedin (PM01183) and Zalypsis® (PM00104).

This study: (i) investigated the in vitro cytotoxicity and mode of action of lurbinectedin (PM01183) and Zalypsis® (PM00104) compared with trabectedin in cell lines deficient in specific mechanisms of repair, (ii) evaluated their in vivo antitumor activity against a series of murine tumors and human xenografts. The antiproliferative activity, the DNA damage and the cell cycle perturbations induced by the three compounds on tumor lines were very similar. Nucleotide Excision Repair (NER) deficient cells were approximately fourfold more resistant to trabectedin, lurbinectedin and Zalypsis®. Cells deficient in non‐homologous end joining (NHEJ), MRN complex and translesion synthesis (TLS) were slightly more sensitive to the three compounds (approximately fivefold) while cells deficient in homologous recombination (HR) were markedly more sensitive (150–200‐fold). All three compounds showed a good antitumor activity in several in vivo models. Lurbinectedin and trabectedin had a similar pattern of antitumor activity in murine tumors and in xenografts, whereas Zalypsis® appeared to have a distinct spectrum of activity. The fact that no relationship whatsoever was found between the in vitro cytotoxic potency and the in vivo antitumor activity, suggests that in addition to direct cytotoxic mechanisms other host‐mediated effects are involved in the in vivo pharmacological effects.

Chitosan-gelatin biopolymers as carrier substrata for limbal epithelial stem cells

The aim of this work was to evaluate semi-synthetic biopolymers based on chitosan (CH) and gelatin (G) as potential in vitro carrier substrata for human limbal epithelial cells (hLECs). To that end, human corneal epithelial cells (HCE) were cultured onto different CH–G membranes. None of the polymers were cytotoxic and cell proliferation was higher when CH was functionalized with G. Expression levels of corneal epithelial markers (K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) were better maintained in HCE cells grown on CH–G 20:80 membranes than other proportions. Consequently, CH–G 20:80 was chosen for the subsequent expansion of hLECs. Cells derived from limbal explants were successfully expanded on CH–G 20:80 membranes using a culture medium lacking components of non-human animal origin. The expression levels found for corneal (K3 and K12) and limbal epithelial stem cells (K15) specific markers were similar to or higher than those found in limbal cells grown onto the control substratum. Our results demonstrate that CH–G 20:80 membranes are suitable for the expansion and maintenance of stem cells derived from the limbal niche. These results strongly support the use of polymers as alternative substrata for the transplantation of cultivated limbal cells onto the ocular surface.

New Interfacial Microtubule Inhibitors of Marine Origin, PM050489/PM060184, with Potent Antitumor Activity and a Distinct Mechanism

We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αβ-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg(2+)-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αβ-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to β-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating β-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.