Differential anti-cancer signaling exerted by an in silico-designed compound in tumorigenic and non-tumorigenic breast cells

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.PhysiologyPhDunrestricte

In vitro cell signaling events of 2-methoxyestradiol-bis-sulphamate in a breast adenocarcinoma- and a non-tumorigenic breast epithelial cell line

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.Dissertation (MSc)--University of Pretoria, 2011.Physiologyunrestricte

2-Methoxyestradiol-bis-sulphamate refrains from inducing apoptosis and autophagy in a non-tumorigenic breast cell line

BACKGROUND: Anticancer research resulted in the discovery of a promising antimitotic metabolite, 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate, a bis-sulphamoylated analogue exerts antiproliferative- and antimitotic activity. Investigating the anticancer potential of 2-methoxyestradiol-bis-sulphamate requires demonstrating the influence of 2-methoxyestradiol-bis-sulphamate on non-tumorigenic cells. This project focused on the in vitro effects of 2-methoxyestradiol-bis-sulphamate on the non-tumorigenic MCF-12A breast epithelial cell line. METHODS The in vitro influence of 2-methoxyestradiol-bis-sulphamate was investigated on cell cycle progression, possible induction of apoptosis and autophagy and reactive oxygen species generation. Cell cycle progression was done using flow cytometry in conjunction with ethanol fixation and propidium iodide staining. Displaying effects on the mitochondrial membrane potential was achieved utilizing flow cytometry and the MitoCapture TM Mitochondrial apoptosis detection kit. Autophagy detection was done by means of flow cytometry and anti-LC3B conjugated to DyLight 488. Reactive oxygen species generation was conducted employing flow cytometry and 2,7-dichlorofluorescein diacetate and hydroethidine. RESULTS: This study demonstrated that 2-methoxyestradiol-bis-sulphamate did not affect cell cycle progression or reactive oxygen species in a statistically significant manner in the non-tumorigenic MCF-12A cell line. In addition, 2-methoxyestradiol-bis-sulphamate did not statistically significantly induce apoptosis or autophagy. CONCLUSION: Reports indicate that 2-methoxyestradiol-bis-sulphamate induces apoptosis and autophagy in several tumorigenic cell lines. The anticancer ability of 2-methoxyestradiol-bis-sulphamate is due to its antimitotic activity. However, this study demonstrates the promising notion that 2-methoxyestradiol-bis-sulphamate does not affect the non-tumorigenic MCF-12A cells. This project contributes to the embedded scientific knowledge regarding the differential death mechanisms used by 2-methoxyestradiol-bis-sulphamate on tumorigenic and non-tumorigenic cell lines.http://www.cancerci.com/content/12/1/37am2013ay201

In vitro influence of 2-methoxyestradiol-bis-sulphamate on cell numbers, reactive oxygen species production and autophagy induction in a breast adenocarcinoma- and a non tumorigenic breast epithelial cell line

This study indicates that 2-methoxyestradiol-bissulphamate induces both apoptosis and autophagy and contributes to the unraveling of the action mechanism of 2-methoxyestradiol-bis-sulphamate.http://www.satnt.ac.z

2-Methoxyestradiol-bis-sulfamate induces apoptosis and autophagy in a tumorigenic breast epithelial cell line

In anticancer research where the focus is on finding agents that induces cell death while leaving nontumorigenic cells less affected, a novel 2-methoxyestradiol derivative has come forth. 2-Methoxyestradiol-bis-sulfamate (2-MeOE2bisMATE) is a 2-methoxyestradiol derivative produced by bis-sulphamoylation, which possesses increased antiproliferative activity and biological availability. Several questions remain regarding the type of cell death mechanisms and possible induction of autophagy by 2-MeOE2bisMATE. The aim of this in vitro study was to investigate the cell death mechanisms exerted by 2-MeOE2bisMATE in an adenocarcinoma cell line (MCF-7) by analyzing its influence on cell growth, morphology, and possible induction of cell death. Spectrophotometry (crystal violet staining), transmission electron microscopy (TEM), light microscopy (hematoxylin and eosin staining), and fluorescent microscopy (Hoechst 33342, propidium iodide and acridine orange) were employed. Spectrophotometrical studies indicated that 2-MeOE2bisMATE decreased cell numbers to 75% inMCF- 7 cells after 24 h and to 47% after 48 h of exposure. TEM demonstrated membrane blebbing, nuclear fragmentation, and chromatin condensation indicating the hallmarks of apoptosis. Light microscopy revealed the presence of several cells blocked in metaphase, and apoptotic cells were also observed. Fluorescent microscopy demonstrated increased lysosomal staining; suggesting the induction of autophagy. 2-MeOE2bisMATE shows therapeutic potential, as an, anticancer agent, and the investigation of the cell death mechanisms used by 2-MeOE2bisMATE, thus, warrants further investigation.The National Research Foundation, and the Struwig-Germeshuysen Cancer Research Trust of South Africa.http://www.springerlink.com/content/0300-817

In vitro cell signaling events of 2-methoxyestradiol-bis-sulphamate in a breast adenocarcinoma- and a non-tumorigenic breast epithelial cell line

2-Methoxyestradiol-bissulphamate shows therapeutic potential, however, the exact mechanism of action remains elusive. Data obtained from this study contributes to the embedded scientific knowledge regarding the signalling mechanism exerted by 2-methoxyestradiol-bis-sulphamate.This paper was initially delivered at the Annual Congress of the Biological Sciences Division of the South African Academy for Science and Art, ARC-Plant Protection Research Institute, Roodeplaat, Pretoria, South Africa on 01 October 2010.http://www.satnt.ac.zaam201

17-beta-estradiol analog inhibits cell proliferation by induction of apoptosis in breast cell lines

Microtubules are important targets when studying potential anticancer agents since disturbance of these microtubule dynamics results in cell cycle arrest and cell death. 2-Methoxyestradiol is a naturally occurring metabolite that exerts antiproliferative activity and induces apoptosis. Due to limited biological accessibly and rapid metabolic degradation, several analogs were synthesized. This study investigated the antiproliferative influence of an 2-methoxyestradiol analog, (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(sulfamate) (EMBS) on cell proliferation, morphology and apoptosis induction in a estrogen receptor-positive breast adenocarcinoma cells line (MCF-7), estrogen receptor-negative highly metastatic breast cell line (MDAMB- 231) and a non-tumorigenic breast epithelial cell line (MCF-12A). Spectrophotometry results indicated that EMBS exerted differential antiproliferative activity in the three cell lines. Cell growth of the breast adenocarcinoma and highly metastatic breast cell line reached a plateau effect at 0.4 lM after 24 h of exposure. Light microscopy and polarization-optical transmitted light differential interference contrast demonstrated compromised cell density, cells blocked in metaphase and the presence of apoptotic characteristics after EMBS exposure for 24 h in all three cell lines. Transmission electron microscopy and scanning electron microscopy revealed hallmarks of apoptosis namely the presence of apoptotic bodies, shrunken cells and cell debris in EMBS-exposed cells. This investigation demonstrated that EMBS does exert antimitotic activity and induces apoptosis contributing to elucidating the signal transduction of EMBS in tumorigenic and non-tumorigenic breast cell lines. Findings warrant in-depth analysis of specific targets in vitro and subsequent in vivo investigation for anticancer therapy.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-0029hb2014ay201

Sulphamoylated estradiol analogue induces reactive oxygen species generation to exert its antiproliferative activity in breast cancer cell lines

2-Methoxyestradiol (2ME), a 17β-estradiol metabolite, exerts anticancer properties in vitro and in vivo. To address 2ME’s low bioavailability, research led to the in silico design of sulphamoylated 2ME analogues. However, the role of oxidative stress induced in the activity exerted by sulphamoylated compounds remains elusive. In the current study, the influence of 2-Ethyl-17-oxoestra-1,3,5(10)-trien-3-yl sulphamate (ESE-one) on reactive oxygen species (ROS) induction and its effect on cell proliferation, as well as morphology, were assessed in breast tumorigenic cells (MCF-7 and MDA-MB-231). Fluorescent microscopy showed that sulphamoylated estradiol analogues induced hydrogen peroxide and superoxide anion, correlating with decreased cell growth demonstrated by spectrophotometry data. ESE-one exposure resulted in antiproliferation which was repressed by tiron (superoxide inhibitor), trolox (peroxyl inhibitor) and N,N0 -dimethylthiourea (DMTU) (hydrogen peroxide inhibitor). Morphological studies demonstrated that tiron, trolox and DMTU significantly decreased the number of rounded cells and shrunken cells in MCF-7 and MDA-MB-231 cells induced by ESE-one. This in vitro study suggests that ESE-one induces growth inhibition and cell rounding by production of superoxide anion, peroxyl radical and hydrogen peroxide. Identification of these biological changes in cancer cells caused by sulphamoylated compounds hugely contributes towards improvement of anticancer strategies and the ROS-dependent cell death pathways in tumorigenic breast cells.Cancer Association of South Africa, Medical Research Council, Struwig Germeshuysen Trust, School of Medicine Research Committee of the University of Pretoria, South African National Research Foundation and Department of Physiology and the School of Medicine, Faculty of Health Sciences, University of Pretoria.http://www.mdpi.com/journal/moleculespm2020Physiolog

In vitro assessment of a computer‑designed potential anticancer agent in cervical cancer cells

BACKGROUND : Computer-based technology is becoming increasingly essential in biological research where drug discovery programs start with the identification of suitable drug targets. 2-Methoxyestradiol (2ME2) is a 17β-estradiol metabolite that induces apoptosis in various cancer cell lines including cervical cancer, breast cancer and multiple myeloma. Owing to 2ME2’s poor in vivo bioavailability, our laboratory in silico-designed and subsequently synthesized a novel 2ME2 analogue, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol), using receptor- and ligand molecular modeling. In this study, the biological effects of ESE-15-ol (180 nM) and its parent molecule, 2ME2 (1 μM), were assessed on morphology and apoptosis induction in cervical cancer cells. RESULTS : Transmission electron microscopy, scanning electron microscopy and polarization-optical transmitted light differential interference contrast (PlasDIC) images demonstrated morphological hallmarks of apoptosis including apoptotic bodies, shrunken cells, vacuoles, reduced cell density and cell debris. Flow cytometry analysis showed apoptosis induction by means of annexin V-FITC staining. Cell cycle analysis showed that ESE-15-ol exposure resulted in a statistically significant increase in the G2M phase (72%) compared to 2ME2 (19%). Apoptosis induction was more pronounced when cells were exposed to ESE-15-ol compared to 2ME2. Spectrophotometric analysis of caspase 8 activity demonstrated that 2ME2 and ESE-15-ol both induced caspase 8 activation by 2- and 1.7-fold respectively indicating the induction of the apoptosis. However, ESE-15-ol exerted all of the above-mentioned effects at a much lower pharmacological concentration (180 nM) compared to 2ME2 (1 μM physiological concentration). CONCLUSION : Computer-based technology is essential in drug discovery and together with in vitro studies for the evaluation of these in silico-designed compounds, drug development can be improved to be cost effective and time consuming. This study evaluated the anticancer potential of ESE-15-ol, an in silico-designed compound in vitro. Research demonstrated that ESE-15-ol exerts antiproliferative activity accompanied with apoptosis induction at a nanomolar concentration compared to the micromolar range required by 2ME2. This study is the first study to demonstrate the influence of ESE-15-ol on morphology, cell cycle progression and apoptosis induction in HeLa cells. In silico-design by means of receptor- and ligand molecular modeling is thus effective in improving compound bioavailability while preserving apoptotic activity in vitro.The Cancer Association of South Africa, the Struwig Germeshuysen Trust, RESCOM (School of Medicine, Research Committee of the Faculty of Health Sciences, University of Pretoria), the National Research Foundation and the Medical Research Council of South Africa.http://www.biolres.com/am2016Physiolog

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

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.The Cancer Association of South Africa, the Struwig Germeshuysen Trust, RESCOM (Research Council of the University of Pretoria), the South African National Research Foundation and Medical Research Council.http://www.cellandbioscience.comam201