In vitro effects of an in silico-modelled 17β-estradiol derivative in combination with dichloroacetic acid on MCF-7 and MCF-12A

OBJECTIVES : To investigate anti-proliferative properties of a novel in silico-modelled 17β-oestradiol derivative (C9), in combination with dichloroacetic acid (DCA), on MCF-7 and MCF-12A cells. MATERIALS AND METHODS : xCELLigence system was employed to determine optimal seeding number for cells, and crystal violet assay was used to assess cell number and to determine IC50 value (24 h) for combination treatment. Light and fluorescent microscopy techniques were used to morphologically detect types of cell death. Flow cytometry was used to analyse cell cycle and apoptosis. RESULTS : Optimal seeding number for 96-well plates was determined to be 5000–10 000 cells ⁄ well for both MCF-7 and MCF-12A cells. IC50 for MCF-7 cells of the combination treatment after 24 h was 130 nM of C9 in conjunction with 7.5 mM of DCA (P < 0.05). In contrast, the same concentration inhibited cell population growth by only 29.3% for MCF- 12As after 24-h treatment (P < 0.05). Morphological studies revealed lower cell density of both types of combination-treated cells. Flow cytometric analyses demonstrated increase in sub-G1 phase in combination- treated MCF-7 cells. CONCLUSIONS : These results demonstrate that the novel 17β-oestradiol derivative C9, in combination with DCA is a potent anti-proliferation treatment, with properties of selectivity towards tumourigenic cells. Thus, this warrants further studies as a potential combination chemotherapeutic agent for further cancer cell lines.This research was supported by grants from the Medical Research Council of South Africa (AL343, AS536), the Cancer Association of South Africa (AS201), the National Research Foundation (NRF) (AL239), the RESCOM of University of Pretoria and the Struwig-Germeshuysen Cancer Research Trust of South Africa (AN074).http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1365-2184

2-Methoxyestradiol-3,17,0,0-bis-sulfamate (2MEBM) induces two types of cell death in a cervical adenocarcinoma cell line

2-MEBM induced apoptosis and autophagy in HeLa cells and also disrupted the cellular microtubule network. This contributes to the understanding of the in vitro mechanism of action of 2MEBM as a potential anticancer drug.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.zaam2014ay201

Cytotoxic activity of pentachlorophenol and its active metabolites in SH-SY5Y neuroblastoma cells

As knowledge regarding mechanisms of pentachlorophenol (PCP) toxicity in neuronal cell lines is limited, the aim of the study was to evaluate the effects of PCP and its active metabolites, tetrachloro-1,4-benzoquinone (TCBQ) and tetrachlorohydroquinone (TCHQ) in human neuroblastoma SH-SY5Y cells. All compounds induced cytotoxic effects in time- and dose-dependent manners, and resulted in differential modes of cell death. Reduced mitochondrial membrane potential (ΔᴪM) and oxidative damage lead to apoptosis and necrosis following TCBQ and PCP exposure, respectively. Time-dependent investigations revealed transient ΔᴪM recovery in TCHQ exposed cells, and redox stress. Sufficient ΔᴪM recovery allowed apoptosis completion in TCHQ exposed cells, whereas overwhelming metabolic and oxidative stress saw a conversion from apoptotic to necrotic-like cell death. The onset of mitochondrial dysfunction preceded that of redox damage for all compounds, indicating that oxidative damage is secondary to ΔᴪM insult. Cytotoxic events were further linked to cell cycle. S phase and G2/M blocks were observed after 12 h exposure to TCBQ and TCHQ, respectively, while a G1 block occurred after 24 h exposure to PCP. This study provides new insight regarding time-dependant toxic effects of PCP and its metabolites in human neuronal cells.The National Research Foundation, South Africa and Research Committee of the School of Medicine, Faculty of Health Sciences, University of Pretoria.http://www.elsevier.com/locate/tiv2020-08-01hj2020PharmacologyPhysiolog

In vitro effects of a new and novel antimitotic compound in human breast adenocarcinoma metastatic epithelial cells

This study contributes to the understanding of molecular mechanisms and cell signaling events associated with in vitro anticancer responses of a new and novel antimitotic compound, 2-etiel-3-sulfamaat- 1,3,5(10)16-tetraeen (C19).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

Short communication : effects of a 17-beta estradiol analogue on gene expression and morphology in a breast epithelial adenocarcinoma cell line : a potential antiproliferative agent

2-Methoxyestradiol is a 17-beta estradiol derivative with antitumor activity. Due to low bioavailability, a new estrogen analogue was in silico-designed. The latter, a sulphamoylated analogue of 2-methoxyestradiol, was evaluated for its potential antiproliferative effect by means of expression microarray analysis and transmission electron microscopy in the highly metastatic breast adenocarcinoma (MDA-MB-231) cell line. Data indicate changes in gene expression pertaining to induction of apoptosis and autophagy as types of cell death, arrest of the cell cycle, and impairment of the cytoskeleton and induction of reactive oxygen species. Transmission electron microscopy confirmed morphological characteristics of apoptosis and autophagy. Altered cell cycle- and cytoskeletal-related gene expression profiles demonstrated that the estradiol analogue acts as an antimitotic agent in this highly metastatic breast cell line. Data also showed that the newly designed estrogen analogue exerts an antiproliferative effect in this cancer cell line culminating in both apoptosis and autophagy as type of cell death paving the way for further investigations into its potential as anticancer agent.Cancer Association of South Africa, the Struwig Germeshuysen Trust, RESCOM (Research Council of the University of Pretoria), National Research Foundation and The Medical Research Council.http://www.biomedres.infohb201

Glu2.53(90) of the GnRH receptor is part of the conserved G protein-coupled receptor structure and does not form a salt-bridge with Lys3.32(121)

Please read abstract in the article.The South African National Research Foundation and an award from the Harry Oppenheimer Trust.http://www.elsevier.com/locate/mcehj2020Physiolog

Signaling pathways of ESE-16, an antimitotic and anticarbonic anhydrase estradiol analog, in breast cancer cells

The aim of this study was to characterize the in vitro action of 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) on non-tumorigenic MCF-12A, tumorigenic MCF-7 and metastatic MDA-MB-231 breast cancer cells. ESE-16 is able to inhibit the activity of a carbonic anhydrase II and a mimic of carbonic anhydrase IX in the nanomolar range. Gene and protein expression studies using various techniques including gene and antibody microarrays and various flow cytometry assays yielded valuable information about the mechanism of action of ESE-16. The JNK pathway was identified as an important pathway mediating the effects of ESE-16 while the p38 stress-induced pathway is more important in MDA-MB-231 cells exposed to ESE-16. Lysosomal rupture and iron metabolism was identified as important mediators of mitochondrial membrane depolarization. Abrogation of Bcl-2 phosphorylation status as a result of ESE-16 also plays a role in inducing mitochondrial membrane depolarization. The study provides a basis for future research projects to develop the newly synthesized compound into a clinically usable anticancer agent either alone or in combination with other agents. Keywords: Antimitotic, anticarbonic anhydrase IX, apoptosis, autophagy, cell cycle arrest, Bcl-2, JNK, p38, mitochondrial membrane depolarization, flow cytometry, gene expression and protein microarray, anticancer.This research was supported by grants from the Medical Research Council of South Africa (AG374, AK076), the Cancer Association of South Africa (AK246), National Research Foundation (NRF) (AL239), the Research Committee of the Faculty of Health Sciences (RESCOM) of University of Pretoria and the Struwig-Germeshuysen Cancer Research Trust of South Africa (AJ038, AN074).http://www.plosone.orgam2013ay201

In vitro evaluation of ESE-15-ol, an Estradiol analogue with nanomolar antimitotic and carbonic anhydrase inhibitory activity

Antimitotic compounds are still one of the most widely used chemotherapeutic anticancer drugs in the clinic today. Given their effectiveness against cancer it is beneficial to continue enhancing these drugs. One way is to improve the bioavailability and efficacy by synthesizing derivatives that reversibly bind to carbonic anhydrase II (CAII) in red blood cells followed by a slow release into the blood circulation system. In the present study we describe the in vitro biological activity of a reduced derivative of 2-ethyl-3-O-sulphamoyl-estradiol (2EE), 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol). ESE-15-ol is capable of inhibiting carbonic anhydrase activity in the nanomolar range and is selective towards a mimic of carbonic anhydrase IX when compared to the CAII isoform. Docking studies using Autodock Vina suggest that the dehydration of the D-ring plays a role towards the selectivity of ESE-15-ol to CAIX and that the binding mode of ESE-15-ol is substantially different when compared to 2EE. ESE-15-ol is able to reduce cell growth to 50% after 48 h at 50–75 nM in MCF- 7, MDA-MB-231, and MCF-12A cells. The compound is the least potent against the non-tumorigenic MCF-12A cells. In vitro mechanistic studies demonstrate that the newly synthesized compound induces mitochondrial membrane depolarization, abrogates the phosphorylation status of Bcl-2 and affects gene expression of genes associated with cell death and mitosis.Grants from the Medical Research Council of South Africa (AG374, AK076), the Cancer Association of South Africa (AK246), National Research Foundation (NRF) (AL239), the Research Committee of the Faculty of Health Sciences (RESCOM) of University of Pretoria, and the Struwig-Germeshuysen Cancer Research Trust of South Africa (AJ038, AN074).http://www.plosone.orgam2013ay201

The in vitro effects of compound-X on growth, morphology, the induction of autophagy and apoptosis, as well as cell cycle progression in a cervical adenocarcinoma cell line

It can be concluded that compound-X induced both autophagy and apoptosis as a means of cell death in HeLa cells.This abstract was initially presented at the annual Biological Sciences Symposium, presented under the protection of the Suid-Afrikaanse Akademie vir Wetenskap en Kuns. The symposium was held at the University of Johannesburg on 01 October 2011.http://www.satnt.ac.z

In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17-beta-estradiol analogue in breast adenocarcinoma cells

2-Methoxyestradiol, a natural metabolite of estradiol, exerts antiproliferative and antitumour properties in vitro and in vivo. Because of its low oral bioavailability, several promising analogues of 2-methoxyestradiol have been developed. In this study, the in vitro influence of the compound, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19), a non-commercially available 17-b-estradiol analogue, was tested on the breast adenocarcinoma MCF-7 cell line. The in vitro influence of 24 h exposure to 0.18 mM of C19 on MCF-7 cells was evaluated on cell morphology, cell cycle progression and possible induction of apoptosis and autophagy. Polarization-optical transmitted light differential interference contrast and fluorescence microscopy revealed the presence of cells blocked in metaphase, occurrence of apoptotic bodies and compromised cell density in C19-treated cells. Hallmarks of autophagy, namely an increase in the number of acidic vacuoles and lysosomes, were also observed in C19-treated samples. An increase in the number of cells present in the sub-G1 fraction, as well as a reduction in mitochondrial membrane potential was observed. No significant alterations in caspase 8 activity were observed. A twofold increase in aggresome formation was observed in C19-treated cells. C19 induced both apoptosis and autophagy in MCF-7 cells.The Medical Research Council of South Africa (AK076; AL343), the Cancer Association of South Africa (AK246), RESCOM, School of Medicine (Faculty of Health Sciences, University of Pretoria), the National Research Foundation of South Africa (AL239) and the Struwig-Germeshuysen Cancer Research Trust of South Africa (AN074). The Bioinformatics and Computational Biology Unit (University of Pretoria) contributed to the in silico-design of the compound.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1099-0844hb2013ay201