Title page for ETD etd-07112011-123356


Document Type Master's Dissertation
Author Visagie, Michelle Helen
Email michellevisagie@yahoo.com
URN etd-07112011-123356
Document Title In vitro cell signaling events of 2-methoxyestradiol-bis-sulphamate in a breast adenocarcinoma- and a non-tumorigenic breast epithelial cell line
Degree MSc
Department Physiology
Supervisor
Advisor Name Title
Prof A M Joubert Supervisor
Keywords
  • 2-MeOE2bisMATE
  • autophagy
  • MCF-12A
  • apoptosis
  • MCF-7
Date 2011-04-08
Availability unrestricted
Abstract
2-Methoxyestradiol, an endogenous metabolite of 17-β-estradiol exerts antipropliferative, antiangiogenic and antitumor effects in vitro and in vivo and is currently in clinical trials phase II for various types of cancers including breast cancer. Due to low oral bioavailability and rapid metabolic degradation, several analogues have been developed in recent years. 2-Methoxyestradiol-bis-sulphamate (2-MeOE2bisMATE), a novel bissulphamoylated derivative of 2-methoxyestradiol exerts in vitro antipropliferative effects. Although 2-MeOE2bisMATE holds therapeutic potential as an anticancer agent, several questions remain regarding the signal transduction and exact mechanism of action used by 2-MeOE2bisMATE.

In vitro effects of 2-MeOE2bisMATE were investigated in a breast adenocarcinoma cell line (MCF-7) and a non-tumorigenic epithelial breast cell line (MCF-12A) by analysing its influence on cell growth, cytotoxicity, morphology, cell cycle progression, mitochondrial membrane potential, reactive oxygen species production and induction of apoptosis and autophagy.

Spectrophotometrical studies indicated that 2-MeOE2bisMATE decreased cell numbers to 47% in MCF-7 cells and to 79% in MCF-12A cells after 48h of exposure. Haematoxylin and eosin staining revealed several 2-MeOE2bisMATE-treated cells with the presence of apoptotic bodies. Transmission electron microscopy demonstrated membrane blebbing, nuclear fragmentation and chromatin condensation indicating the occurrence of apoptosis. Increased lysosomal staining was revealed by fluorescent microscopy using propidium iodide, Hoechst 33342 and acridine orange; suggesting cell death via autophagy. Data obtained employing flow cytometry using rabbit polyclonal anti-LC3B conjugated to DyLight 488 verified the induction of autophagy in 2-MeOE2bisMATE-treated cells. In addition, cell cycle progression revealed an apoptotic sub-G1 peak, confirming the induction of apoptosis by 2-MeOE2bisMATE. Reactive oxygen species generation increased when cells were exposed to 2-MeOE2bisMATE. Annexin V-FITC and the investigation of a possible reduction in the mitochondrial membrane potential verified induction of apoptosis by 2-MeOE2bisMATE. All of the above-mentioned results were observed more prominently in the tumorigenic MCF-7 cell line when compared to the non-tumorigenic MCF-12A cell line.

Data obtained from this in vitro study contributes to the embedded scientific knowledge regarding the signaling transduction mechanism exerted by 2-MeOE2bisMATE.

2010, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.

Please cite as follows:

Visagie, MH 2010, In vitro cell signaling events of 2-methoxyestradiol-bis-sulphamate in a breast adenocarcinoma- and a non-tumorigenic breast epithelial cell line, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-07112011-123356/ >

E11/309/gm

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