Abstract C214: Disorazol Z: A highly cytotoxic natural compound with antitumor properties.

Abstract

Abstract In search of novel antitumor agents natural macrocyclic polyketides called Disorazols have been isolated from myxobacterium Sorangium cellulosum[1,2,3]. Here we show that Disorazol Z specifically produced by strain So ce427, possesses outstanding cytotoxicity with single digit to even subnanomolar EC50 values in a highly diverse panel of more than 60 different tumor cell lines. Interestingly, the maximum efficacy by which Disorazol Z inhibited cell growth increased with the incubation time, e.g. in the endometrium carcinoma cell line Hec1A reaching a maximum efficacy of about 25% after 24 hours, but about 75% efficacy after 72 hours at a saturating concentration of 100 nM, being approximately 100–200 fold above the respective EC50 values. Based on this observation, it was speculated that the mechanism of action of Disorazol Z is dependent on progressing through cell cycle. Indeed, this was supported by showing that in the cell line RKOp27Kip inducible expression of the cell cycle inhibitor p27Kip led to a complete loss of Disorazol Z cytotoxicity up to a concentration of 100 nM. In contrast, without cell cycle arrest Disorazol Z demonstrated high cytotoxic activity with an EC50 value of 0.54 nM in the same cell line. Furthermore, cell cycle analysis revealed that Disorazol Z arrested KB/HeLa cells in the G2/M phase of the cell cycle with an IC50 value of 0.8 nM. As expected this Disorazol Z-induced G2/M arrest induced apoptotic events, as demonstrated by Caspase 3/7 activation in HCT-116 cells with an EC50 value of 0.25 nM. In agreement with the above described behaviour Disorazol Z could be identified as tubulin binding agent by inhibiting in vitro tubulin polymerisation with a mean IC50 value of 3.3 μM as well as inducing multipolar spindle formation at low nanomolar concentrations in U-2 OS cells. The high divergence of the in vitro tubulin activity compared to the cellular efficacy may point to additional mechanisms of action for Disorazol Z. This working hypothesis may be supported by the fact that synergistic cytotoxic action of Disorazol Z and the tubulin binding agent Colchicin could be observed with combination indices down to about 0.5. Neither Taxol nor Vinblastine showed such synergism, both tubulin binders behaved nearly additive in combination with Disorazol Z. Currently, experiments are under way to identify the tubulin binding site for Disorazol Z. Additional ongoing studies focus on the evaluation of the mechanisms of action of this novel highly potent agent with antitumor properties. Further, we aim at evaluating the utility of Disorazol Z as cytotoxic component in a drug-targeting approach utilizing GPCR ligands as the targeting moieties for the treatment of GPCR overexpressing cancers.