Microtubule-disrupting agents have been studied for decades for their potential anticancer activity and resulted in discovery of an endogenous 17β-estradiol derivative, 2-methoxyestradiol (2ME2). Since 2ME2 possesses low bioavailability, several analogues with improved efficacy was in silico-designed to target tumourigenic cells. This study investigated the influence of an 17β-estradiol analogue, (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) on cell growth, cytotoxicity, metabolism, morphology, cell cycle progression, reactive oxygen species generation and induction of cell death via apoptosis in two adenocarcinoma cell lines (MCF-7 and MDA-MB-231) and the non-tumourigenic epithelial breast cell line
(MCF-12A).
Crystal violet staining and the real-time xCELLigence approach indicated statistically significant antiproliferative activity in an estrogen-independent manner (0.4 μM; 24 h) in all three cell lines. Influence on morphological demonstrated several apoptotic hallmarks including compromised cell density, apoptotic bodies, shrunken cells, hypercondensed chromatin and several cells trapped in metaphase culminating in apoptosis. Cell cycle progression studies revealed apoptosis induction and cells blocked in the G2M phase. Apoptosis induction was verified by means of Annexin V-FITC.EMBS-treated cells demonstrated a reduced mitochondrial membrane potential. Furthermore, autophagy characteristics were observed including vacuoles and autophagosomes. Mitotic indices demonstrated an increase in cells possessing abnormal morphology associated with apoptosis and the number of cells trapped in metaphase culminating in apoptosis. This was confirmed by cell cycle progression studies that revealed apoptosis induction and a G2M block. Apoptosis induction was verified by means of Annexin V-FITC and additional flow cytometry studies indicated EMBS-treated cells demonstrated a reduced mitochondrial membrane potential.
Fluorescent microscopy exhibited increased lysosomal staining suggesting autophagy induction which was verified by conducting flow cytometry employing LC3B conjugated to DyLight 488. Flow cytometry studies also demonstrated that EMBS exposure resulted in statistically significant increased hydrogen peroxide and superoxide production. EMBS exposure resulted in a statistically significant increase in p53 protein expression, decreased Bcl-2 expression and a decrease in pBcl-2(s70) phosphorylation supporting the notion that EMBS utilises crosstalk pathways to induce both autophagy and apoptosis. These results were observed in all three cell lines with caspase 6 and 8 activation being more prominent in the tumourigenic cell lines and cell growth recovering after 24 h exposure in the non-tumourigenic MCF-12A cell line.
Further research will focus on the molecular signal transduction utilized by EMBS and an in-depth analysis of specific anticancer targets identified in vitro and subsequent in vivo investigation. Thus this study contributes to the discovery of targets for cancer therapies that will aid in the design of microtubule disrupting agents. / Thesis(PhD)--University of Pretoria, 2014. / Physiology / PhD / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/40255 |
| Date | January 2014 |
| Creators | Visagie, M.H. (Michelle Helen) |
| Contributors | Joubert, Annie M., Birkholtz, Lyn-Marie |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | © 2013 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. |
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