2-Methoxy-estradiol (2-ME2), a natural metabolite of 17β-estradiol, was shown to inhibit angiogenesis and the proliferation of many human cancer cells both in vivo and in vitro, but not in normal cells. 2-ME was also shown to induce apoptosis in several tumor cell types through a variety of molecular mechanisms. However, the precise mechanism of action of 2-ME2 remains unknown. In the present study, we examined the role of 2-ME2 in the regulation of apoptosis in human Jurkat T-lymphocytes. Specifically, we investigated the regulation of topoisomerase II (topo II) activity and expression by 2-ME2 as well as the involvement of Bcl-2 in 2-ME2 induced apoptosis of Jurkat cells. We found that 2-ME2 inhibited replication and induced apoptosis of Jurkat cells in a dose and time-dependent manner. Cell cycle protein expression analysis demonstrated that inhibition of cell replication by 2-ME2 was due to the reduction of cyclins D3 and E and the tumor suppressor protein p21Cip1/Waf1, and the induction of p16INK4A. 2-ME2- induced apoptosis was associated with a reduction in expression and phosphorylation (hence inactivation) of Bcl-2, induction of Bak protein expression, activation of caspases 3 and 9 and proteolytic cleavage of PARP. In order to further examine the role of Bcl-2 in 2-ME2 induced Jurkat cell apoptosis and replication, Jurkat cell lines were infected with recombinant retrovectors carrying the puromycin resistance gene with or without human Bcl-2 cDNA and selected with puromycin to generate Jurkat-Puro and Jurkat Bcl-2 cells. 2-ME2 inhibited the replication and induced apoptosis of Jurkat-Puro cells whereas overexpression of Bcl-2 blocked 2-ME2 induced apoptosis of Jurkat cells. In addition, topoisomerase-II activity assays demonstrated that 2-ME2-induced the activation of topo-II in Jurkat and Jurkat-Puro cells but not in Jurkat-Bcl-2 cells. The increase in topo-II activity induced by 2-ME2 correlated with increased phosphorylated topo-II expression in Jurkat and Jurkat-Puro cells but not in Jurkat-Bcl-2 cells. It seems that the increased topo-II activity is at least partly due to the increased levels of phosphorylated topo II. These results taken together suggest that activation of topo-II by 2-ME2 is an important component of the mechanism that leads to apoptosis.
Ackowledgements: Supported by Cyprus Research Promotion Foundation, grant number 0603/25 and General Secretariat of Research and Technology, Ministry of Development in the framework of a Bilateral Agreement between Greece and Cyprus to AC and EK.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA