A 2-methoxyestradiol bis-sulphamoylated derivative induces apoptosis in breast cell lines

Michelle Helen Visagie; Lyn-Marie Birkholtz; Anna Margaretha Joubert

doi:10.1186/s13578-015-0010-5

A 2-methoxyestradiol bis-sulphamoylated derivative induces apoptosis in breast cell lines

Cell Biosci. 2015 Apr 22:5:19. doi: 10.1186/s13578-015-0010-5. eCollection 2015.

Authors

Michelle Helen Visagie¹, Lyn-Marie Birkholtz², Anna Margaretha Joubert¹

Affiliations

¹ Department of Physiology, University of Pretoria, Private Bag X 323, Arcadia, 0007 South Africa.
² Department of Biochemistry, Centre for Sustainable Malaria Control, University of Pretoria, Private Bag X20, Hatfield, Pretoria 0028 South Africa.

Abstract

Introduction: 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.

Conclusion: 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.

Keywords: Apoptosis; Bcl-2; Caspase; EMBS; p53; xCELLigence.