2-methoxyestradiol induces caspase-independent, mitochondria-centered apoptosis in DS-sarcoma cells.

The anti-cancer potential of the natural estrogen metabolite 2-methoxyestradiol is associated with microtubuli interaction, anti-angiogenetic effects and inhibition of superoxide dismutase leading to apoptosis. The effectors of apoptotic signaling through 2-methoxyestradiol, however, are cell type-dependent. We investigated the effect of 2-methoxyestradiol on several events associated with apoptosis in rat DS-sarcoma cells. Translocation of the pro-apoptotic protein Bax to mitochondria was identified as an initial apoptotic event that was accompanied by a decrease in mitochondrial transmembrane potential and the formation of reactive oxygen species (ROS) followed by mitochondrial release of apoptosis inducing factor and endonuclease G. In addition, 2-methoxyestradiol treatment caused upregulation of death receptor ligands FasL and TNFalpha and induced caspase-8 activation. The pan caspase inhibitor Z-VAD-FMK did not suppress apoptotic cell death, however, indicating that the major pro-apoptotic effect of 2-methoxyestradiol is mediated by a caspase-independent mechanism. Furthermore, ROS do not seem to play a pivotal role in the toxic/apoptotic effect of 2-methoxyestradiol in DS-sarcoma cells because supplementation with various antioxidants provided only limit protection. Colony formation was not affected by antioxidants. Therefore, in DS-sarcoma cells, the breakdown of mitochondrial integrity with the subsequent release of mitochondrial nucleases is the main factor in 2-methoxyestradiol mediated cell death. Copyright 2003 Wiley-Liss, Inc.