Spindle checkpoint activatio is required for Combrestatin ST2151 and CA4-phpsphate induced-mitotic catastrophe in primary endothelial and lung tumor-treated cells

Combretastatins are tubulin binding agents, structurally related to colchicines and with a high potential as vascular targeting agents for their capability to induce vascular-mediated tumour necrosis. Molecular mechanisms leading to checkpoint apoptosis have been investigated in endothelial primary cells (HUVEC) and tumor cancer cells (H460) after treatment with ST2151 or combretastatin CA4. We found that both compounds induce depolymerisation and rearrangement of spindle microtubules and an increasingly aberrant organization of metaphase chromosomes in a dose- and time-dependent manner. Both H460 and HUVEC cells were arrested at the pro-metaphase stage at different extent (44,0% vs 17,8% for CA4; 42,6% vs 18,8% for ST2151, respectively) with consequent p53 accumulation and spindle assembly checkpoint activation as evaluated by kinetochore localization of Bub-1 and MAD1 antibodies. Prolonged checkpoint activation led to mitochondrial membrane permeabilization (MMP) alterations, partial activation of caspase-3 and –9, PARP cleavage and DNA fragmentation. On the other hand caspase-2 and -8 were not activated by drug treatment. Interestingly, after 24 h exposure the percentage of cell death was far higher in H460 compared to HUVEC (94,5 % vs 31,5% for CA4; 59,3% vs 20,3% for ST2151). The presence of multinucleated cells, strongly supported the notion that combretastatin, particularly the ST2151 triggers a mitotic catastrophe pathway. Furthermore, we found persistence of cell death induction as evaluated at 24-72 hours from drug washout (in H460 cells persistence of 80-85% cell death). Experiments are in progress, to ascertain the activation of checkpoints at long times from drug removal. Hundred % of cells arrested in metaphase after drugs washout displayed mitotic chechkpoint activation The different pharmacological behaviour of ST2151 respect to CA-4 does not simply correlate with an anti-tubulin mechanism, suggesting that additional molecular targets may be involved, or alternatively, ST molecules might have different binding site or mode of interaction with tubulin.