Discovery of Putative Dual Inhibitor of Tubulin and EGFR by Phenotypic Approach on LASSBio-1586 Homologs.

Combretastatin A-4 (CA-4, 1) is an antimicrotubule agent used as a prototype for the design of several synthetic analogues with anti-tubulin activity, such as LASSBio-1586 (2). A series of branched and unbranched homologs of the lead-compound 2, and vinyl, ethinyl and benzyl analogues, were designed and synthesized. A comparison between the cytotoxic effect of these homologs and 2 on different human tumor cell lines was performed from a cell viability study using MTT with 48 h and 72 h incubations. In general, the compounds were less potent than CA-4, showing CC50 values ranging from 0.030 µM to 7.53 µM (MTT at 72 h) and 0.096 µM to 8.768 µM (MTT at 48 h). The antimitotic effect of the target compounds was demonstrated by cell cycle analysis through flow cytometry, and the cellular mechanism of cytotoxicity was determined by immunofluorescence. While the benzyl homolog 10 (LASSBio-2070) was shown to be a microtubule stabilizer, the lead-compound 2 (LASSBio-1586) and the methylated homolog 3 (LASSBio-1735) had microtubule destabilizing behavior. Molecular docking studies were performed on tubulin protein to investigate their binding mode on colchicine and taxane domain. Surprisingly, the benzyl homolog 10 was able to modulate EGFR phosphorylate activity in a phenotypic model. These data suggest LASSBio-2070 (10) as a putative dual inhibitor of tubulin and EGFR. Its binding mode with EGFR was determined by molecular docking and may be useful in lead-optimization initiatives.

Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis.

The Sonic Hedgehog signaling (Shh) pathway has been implicated in both proliferation of myoblast cells and terminal differentiation of muscle fibers, and contradictory results of these effects have been described. To clarify the role of Shh during myogenesis, we decided to study the effects of recombinant Shh and the distribution of Gli-1 during in vitro and in situ embryonic chick skeletal muscle differentiation at later stages of development. Gli-1 was found in small aggregates near the nucleus in mononucleated myoblasts and in multinucleated myotubes both in vitro and in situ chick muscle cells. Some Gli-1 aggregates colocalized with gamma-tubulin positive-centrosomes. Gli-1 was also found in striations and at the subsarcolemmal membrane in muscle fibers in situ. Recombinant Shh added to in vitro grown muscle cells induced the nuclear translocation of Gli-1, as well as an increase in the number of myoblasts and in the number of nuclei within myotubes. We suggest that Gli-1 aggregates observed in chick muscle cells near the nuclei of myoblasts and myotubes could be a storage site for the rapid cellular redistribution of Gli-1 upon specific signals during muscle differentiation.

γ-Secretase Inhibition Induces Muscle Hypertrophy in a Notch-Independent Mechanism.

A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ-secretase. Notch-1 is one of the substrates of γ-secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch-1, a number of proteins have been identified to undergo proteolysis by γ-secretase. To date, the specific role of γ-secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ-secretase inhibitor DAPT (N-N[-(3,5-Difluorophenacetyl-l-alanyl)]-S-328 phenylglycine-t-butyl-ester) induces muscle hypertrophy. Knockdown of Notch-1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ-secretase-dependent effects on muscle hypertrophy in chick myogenic cells are Notch-1-independent. We also investigate the effects of γ-secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label-free proteomic approach. Data overview of interaction network obtained from STRING show that after γ-secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ-secretase in skeletal muscle hypertrophy.

Cellular migration, transition and interaction during regeneration of the sponge Hymeniacidon heliophila.

Sponges have a high capacity for regeneration and this process improves biomass production in some species, thus contributing to a solution for the biomass supply problem for biotechnological applications. The aim of this work is to characterize the dynamics of cell behavior during the initial stages of sponge regeneration, using bright-field microscopy, confocal microscopy and SEM. We focused on the first 20 h of regeneration, during which blastema formation and epithelium initialization occur. An innovative sponge organotypic culture of the regenerating internal region is described and investigated by confocal microscopy, cell transplantation and vital staining. Cell-cell interaction and cell density are shown to affect events in morphogenesis such as epithelial/mesenchymal and mesenchymal/epithelial transitions as well as distinct cell movements required for regeneration. Extracellular matrix was organized according to the morphogenetic process observed, with evidence for cell-signaling instructions and remodeling. These data and the method of organotypic culture described here provide support for the development of viable sponge biomass production.