title: 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.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Physiology and Toxicology of Reproduction, Jageillonian University, Krakow, Poland. ewa.gregoraszczuk@uj.edu.pl
authors.forename: E
authors.initials: E
authors.lastname: Gregoraszczuk
authors.forename: A
authors.initials: A
authors.lastname: Ptak
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D053181:Caspase 8 / Q000378:metabolism*; D053453:Caspase 9 / Q000378:metabolism*; D045744:Cell Line, Tumor; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D065633:Cytochrome P-450 CYP1B1; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004967:Estrogens / Q000494:pharmacology; D002393:Estrogens, Catechol; D005260:Female; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism
chemical_list: D004967:Estrogens; D002393:Estrogens, Catechol; D004958:Estradiol; C014036:4-hydroxyestradiol; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9