title: A 2-methoxyestradiol bis-sulphamoylated derivative induces apoptosis in breast cell lines.
abstract: BACKGROUND
  Research involving antimitotic compounds identified 2-methoxyestradiol (2ME2), as a promising anticancer endogenous metabolite. Owing to its low bioavailability, several in silico-designed 2ME2 analogues were synthesized. Structure-activity relationship studies indicated that an already existing 17-β-estradiol analogue, namely (8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) to exert potential in vitro anticancer activity.
  
  
  METHODS
  This study investigated the in vitro apoptotic influence of EMBS in an estrogen receptor-positive breast adenocarcinoma epithelial cell line (MCF-7); an estrogen receptor-negative breast epithelial cell line (MDA-MB-231) and a non-tumorigenic breast cell line (MCF-12A). Cell cycle progression, a phosphatidylserine flip, caspase 6-, 7- and 8 enzyme activity levels, Bcl-2 phosphorylation status at serine 70 and Bcl-2- and p53 protein levels were investigated to identify a possible action mechanism for apoptotic induction.
  
  
  RESULTS
  The xCELLigence real-time label-independent approach revealed that EMBS exerted antiproliferative activity in all three cell lines after 24 h of exposure. A G2M block was observed and apoptosis induction was verified by means of flow cytometry using propidium iodide and Annexin V-FITC respectively. EMBS-treated cells demonstrated a reduced mitochondrial membrane potential. EMBS exposure resulted in a statistically significant increase in p53 protein expression, decreased Bcl-2 protein expression and a decrease in pBcl-2(s70) phosphorylation status in all three cell lines. Results support the notion that EMBS induces apoptosis in all three cell lines.
  
  
  CONCLUSIONS
  This study includes investigation into the apoptotic hallmarks exerted by EMBS after exposure of three cell lines namely MCF-7-, MDA-MDA-231- and MCF-12A cells. Increased caspase 6-, caspase 7- and caspase 8 activities, upregulation of p53 protein expression and a decrease in phosphorylation status of Bcl-2 at serine 70 in tumorigenic and non-tumorigenic lines were demonstrated.
authors.affiliation: Department of Physiology, University of Pretoria, Private Bag X 323, Arcadia, 0007 South Africa.
authors.forename: Michelle Helen
authors.initials: MH
authors.lastname: Visagie
authors.affiliation: Department of Biochemistry, Centre for Sustainable Malaria Control, University of Pretoria, Private Bag X20, Hatfield, Pretoria 0028 South Africa.
authors.forename: Lyn-Marie
authors.initials: LM
authors.lastname: Birkholtz
authors.affiliation: Department of Physiology, University of Pretoria, Private Bag X 323, Arcadia, 0007 South Africa.
authors.forename: Anna Margaretha
authors.initials: AM
authors.lastname: Joubert
keywords: Apoptosis; Bcl-2; Caspase; EMBS; p53; xCELLigence