The role of mitotic spindle alterations in the induction of apoptosis

Microtubules, together with actin and intermediate filaments are the major components of the cytoskeleton of eukaryotic cells and play a critical role in many cellular processes, including cell division, cell motility, intracellular trafficking, and cell shape maintenance. The principal Microtubule Organizing Center (MTOC) in mammalian cells is the centrosome that is composed of a pair of centrioles surrounded by an osmiophilic matrix termed pericentriolar material (PCM). As the microtubule network is highly involved in cell proliferation, it appeared to be a preferential target for cancer therapy. Microtubule Damaging Agents (MDA) represent an important class of anticancer drugs. Although some of this agents promote microtubules stabilization and other induce microtubules disruption, all MDA share the common property of suppressing microtubules dynamics, and thereby microtubules function, leading to the disruption of the mitotic spindle and the blockade of cell cycle progression at the transition from metaphase to anaphase that leads to apoptosis or mitotic catastrophe commitment. Mitotic catastrophe has been described as cell death that occurs from metaphase of mitosis in response to agents that cause mitotic-spindle damage. Recently we focused our attention on the antineoplastic spindle poison agent combretastatin-A4 (CA-4). CA-4 is an MDA isolated from the South African tree Combretum Caffrum that is not recognized by the multi drug resistance pump, a cellular pump which rapidly ejects foreing molecules including many anticancer drugs. MDAs also promote several effects on the centrosome including abnormal centrioles structure, centrosome fragmentation and inappropriate centrosome duplication. CA-4 is able to arrest cells at mitosis in a dose depending manner accompanied by an increasing disorganization of chromosomal arrangement, led to formation of particular structure named 'star-like'. This structures contain pericentrosomal matrix components like γ-tubulin, pericentrin and ninein. Confocal and immunofluorescence microscopy revealed that 'star like' structure are spherical structure formed by residual fragments of microtubules connected to kinetochore. At the centre of this structure are present some component of the pericentriolar matrix like γ-tubulin, pericentrin and ninein. Treatment with CA-4, which produced high frequency of 'star-like' mitosis, was accompanied by mitotic catastrophe commitment characterized by activation of caspases-3/-9 and DNA fragmentation. The mechanisms that lead from microtubules depolymerization to cell death are still not completely clear. Recently it has been demonstrated that Bim, a proapoptotic Bcl-2 protein, is associated to the dynein motor complex and sequestered by microtubules. MDA, through the alteration of microtubules structure, might lead to the release of Bim from cytoskeleton and to its translocation to the mitochondria. Moreover, recent data show that further mechanisms might be at the base of Bim-induced apoptosis such as regulation of Bim transcriptional levels. In a similar way, p53 another well known pro-apoptotic molecule, is associated with cellular microtubules and is transported to the nucleus by dynein. Based on these data, we have investigated whether upon tubulin depolymerization, Bim and p53 could be involved, and eventually in which manner, in the first steps of apoptosis. CA-4 treatment induce the release of Bim from microtubules and its increase at transcriptional level. In addition, Bim silencing strongly reduced apoptotic commitment. It has recently reported that p53 can also induce apoptosis independently of new protein synthesis however the functional importance of p53 in MDA-induced apoptosis of human cancer cells remain poorly understood. Concerning the role of p53 in CA-4-induced apoptosis, we found that treatment determined its release from cytoskeleton and re-localization to mitochondria. Data obtained from this study showed that Combretastatin-treated H460 cells are killed via a mitotic catastrophe mechanism strictly dependent on the achievement of mitotic block, caspases activation and Bim and p53 translocation to mitochondria.