Involvement of caspase-9 but not caspase-8 in the anti-apoptotic effects of estradiol and 4-OH-Estradiol in MCF-7 human breast cancer cells.

OBJECTIVES: Evidence is accumulating that certain estradiol metabolites may play a more important role in enhancing breast cancer risk than their parent substance - 17 β-estradiol (E2). Of special interest are the metabolites 2-hydroxyestradiol (2-OH-E2), which can show anticarcinogenic effect, while that of 4-hydroxyestradiol (4-OH-E2) may be rather procarcinogenic. We suggest that local activation of cytochrome P450 enzymes - CYP1A1 and/or CYP1B1 - by E2 could generate active metabolites that affect the apoptosis and thereby promote mammary carcinogenesis. Over the last several years, there has been accumulating evidence that, apart from the receptor-mediated (extrinsic) pathway, also the mitochondrial (intrinsic) pathway plays a role in E2-induced apoptosis. In the present study, we have compared the effect of these metabolites and their parent substance E2 on caspase-8 and caspase-9 activity as well as on the end step of apoptosis DNA fragmentation.
METHODS: MCF-7 human breast cancer cells (ATCC) were routinely cultured in DMEM supplemented with 10 % heat-inactivated FBS. Forty-eight hours before experiments, the medium was removed and replaced by DMEM without phenol red supplemented with 5 % heat-inactivated fetal bovine serum. For determination of caspase-8 and caspase-9 activities, MCF-7 cells were seeded in 48-well culture plates at a density of 15 x 104 cells/well and incubated with 1 nM E2 and its metabolites for 24 h. DNA fragmentation, caspase-8 and caspase-9 activities were determined in cell lysates by ELISAs. The CYP1A1 and CYP1B1 protein expression was evaluated by Western blotting.
RESULTS: E2 had no effect on CYP1A1 protein levels. However an increase in CYP1B1 protein expression was observed within 48 hrs of exposure. None of the compounds tested changed caspase-8 activity as compared to the controls. Statistically significant decrease in caspase-9 activity and DNA fragmentation was observed in the presence of E2 and 4-OH-E2, but no significant effect was found for the metabolite 2-OH-E2.
CONCLUSIONS: It was found that local activation of cytochrome P450 enzyme CYP1B1 by E2 may change the local metabolic activation pathway into 4-OH-E2 as well as the activation of caspase-9 (a part of the intrinsic mitochondrial apoptotic pathway) in the antiapoptotic effect of E2 and 4-OH-E2.