Zampanolida, un novedoso agente estabilizador de microtubulos quew se une covalentemente al sitio de paclitaxel

Trabajo presentado en el XI Congreso de la Sociedad de Biofísica de España: "XXV años y más allá", celebrado en Murcia (España) , del 1 al 4 de junio de 201

βIII-tubulin overexpression in cancer: Causes, consequences, and potential therapies

Class III β-tubulin (βIII-tubulin) is frequently overexpressed in human tumors and is associated with resistance to microtubule-targeting agents, tumor aggressiveness, and poor patient outcome. Understanding the mechanisms regulating βIII-tubulin expression and the varied functions βIII-tubulin may have in different cancers is vital to assess the prognostic value of this protein and to develop strategies to enhance therapeutic benefits in βIII-tubulin overexpressing tumors. Here we gather all the available evidence regarding the clinical implications of βIII-tubulin overexpression in cancer, describe factors that regulate βIII-tubulin expression, and discuss current understanding of the mechanisms underlying βIII-tubulin-mediated resistance to microtubule-targeting agents and tumor aggressiveness. Finally, we provide an overview of emerging therapeutic strategies to target tumors that overexpress βIII-tubulin

Pten regulates spindle pole movement through Dlg1-mediated recruitment of Eg5 to centrosomes

Phosphatase and tensin homologue (Pten) suppresses neoplastic growth by negatively regulating PI(3)K signalling through its phosphatase activity. To gain insight into the actions of non-catalytic Pten domains in normal physiological processes and tumorigenesis, we engineered mice lacking the PDZ-binding domain (PDZ-BD). Here, we show that the PDZ-BD regulates centrosome movement and that its heterozygous or homozygous deletion promotes aneuploidy and tumour formation. We found that Pten is recruited to pre-mitotic centrosomes in a Plk1-dependent fashion to create a docking site for protein complexes containing the PDZ-domain-containing protein Dlg1 (also known as Sap97) and Eg5 (also known as Kif11), a kinesin essential for centrosome movement and bipolar spindle formation. Docking of Dlg1-Eg5 complexes to Pten depended on Eg5 phosphorylation by the Nek9-Nek6 mitotic kinase cascade and Cdk1. PDZ-BD deletion or Dlg1 ablation impaired loading of Eg5 onto centrosomes and spindle pole motility, yielding asymmetrical spindles that are prone to chromosome missegregation. Collectively, these data demonstrate that Pten, through the Dlg1-binding ability of its PDZ-BD, accumulates phosphorylated Eg5 at duplicated centrosomes to establish symmetrical bipolar spindles that properly segregate chromosomes, and suggest that this function contributes to tumour suppression

Peloruside- and laulimalide-resistant human ovarian carcinoma cells have βI-tubulin mutations and altered expression of βII- and βIII-tubulin isotypes

Peloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on tubulin remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide. Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on β-tubulin nor was there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the βI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a β-tubulin–binding site for peloruside A and laulimalide. To determine whether the different resistance phenotypes of the cells were attributable to any other tubulin alterations, the β-tubulin isotype composition of the cells was examined. Increased expression of βII- and βIII-tubulin was observed in L4 cells only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two drugs, peloruside A and laulimalide, that have a similar mode of action

Zampanolide, a potent new microtubule stabilizing agent, covalently reacts with the taxane luminal site in both tubulin alpha-beta-heterodimers and microtubules

13 páginas, 3 figuras, 1 tabla -- PAGS nros. 686-698Zampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to β-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,β-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates β-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and suggest that microtubule nucleation induction by MSAs may proceed through an allosteric mechanismThis work was supported in part by grants BIO2010-16351 and CTQ2009-08536 from Ministerio de Economia y Competitividad to J.F.D. and J.J.B., respectively, and grant S2010/BMD-2457 BIPEDD2 from Comunidad Autónoma de Madrid to J.F.D., the Cancer Society of New Zealand, and the Wellington Medical Research Foundation (J.M.). The CNIC is supported by the Ministerio de Economía y Competitividad and the Fundación Pro CNICPeer reviewe