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

The role of oxidative stress in the effects mediated by a garlic constituent (diallyl trisulfide) in cancer cell lines.

https://drive.google.com/file/d/1ZfJmcecqhTfX83sfE9Bbl6Com5BaSGiV/view?usp=sharinghttps://drive.google.com/drive/folders/1EyJ2qye7hyxflwGFto6JFfDZlvUGs-WD?usp=sharinghttps://drive.google.com/drive/folders/1c98-HLU80ylK-o44vS0dR3uBtKEcWQO7?usp=sharin

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

Comparison of liposomal encapsulated and non-formulated fulvic acid on proliferation, oxidative stress and cell death in cancer cell lines

National Research Foundationhttps://drive.google.com/file/d/1e2m2Y5LoP9EYUziKsd0w8qCJW8nQdvOr/view?usp=sharinghttps://drive.google.com/drive/folders/1CEIJlI-XNUtxMQ6_4CAtIqNxoBb90IH3?usp=sharinghttps://drive.google.com/drive/folders/1ge_ZUDz07kS3X6dw4FLdlPkQ_YMd7xyl?usp=sharin

In vitro effects of papaverine on cell migration and vascular endothelial growth factor in cancer cell lines

Papaverine (PPV) is a benzylisoquinoline alkaloid isolated from Papaver somniferum that exerts antiproliferative activity. However, several questions remain regarding the biochemical pathways affected by PPV in tumourigenic cells. In this study, the influence of PPV on cell migration (light microscopy), expression of vascular endothelial growth factor (VEGF) B, VEGF R1, VEGF R2, and phosphorylated focal adhesion kinase (pFAK) were investigated using spectrophotometry in MDA-MB-231-, A549- and DU145 cell lines. The migration assay revealed that, after 48 h, PPV (100 µM) reduced cell migration to 81%, 91%, and 71% in MDA-MB-231-, A549-, and DU145 cells, respectively. VEGF B expression was reduced to 0.79-, 0.71-, and 0.73-fold after 48 h of exposure to PPV in MDA-MB-231-, A549- and DU145 cells, while PPV exposure of 48 h increased VEGF R1 expression in MDA-MB-231- and DU145 cells to 1.38 and 1.46. A fold decrease in VEGF R1 expression was observed in A549 cells to 0.90 after exposure to 150 µM. No statistically significant effects were observed on VEGF R2- and FAK expression after exposure to PPV. This study contributes to the understanding of the effects of a phytomedicinal alkaloid compound in cancer cells and may provide novel approaches to the application of non-addictive alkaloids.The Cancer Association of South Africa, Medical Research Council, the Struwig Germeshuysen Trust, School of Medicine Research Committee of the University of Pretoria and the South African National Research Foundation.https://www.mdpi.com/journal/ijerphPhysiolog

Molecular farming of pembrolizumab and nivolumab

DATA AVAILABILITY STATEMENT : Data is contained within the article.Immune checkpoint inhibitors (ICIs) are a class of immunotherapy agents capable of alleviating the immunosuppressive effects exerted by tumorigenic cells. The programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint is one of the most ubiquitous checkpoints utilized by tumorigenic cells for immune evasion by inducing apoptosis and inhibiting the proliferation and cytokine production of T lymphocytes. Currently, the most frequently used ICIs targeting the PD-1/PD-L1 checkpoint include monoclonal antibodies (mAbs) pembrolizumab and nivolumab that bind to PD-1 on T lymphocytes and inhibit interaction with PD-L1 on tumorigenic cells. However, pembrolizumab and nivolumab are costly, and thus their accessibility is limited in low- and middle-income countries (LMICs). Therefore, it is essential to develop novel biomanufacturing platforms capable of reducing the cost of these two therapies. Molecular farming is one such platform utilizing plants for mAb production, and it has been demonstrated to be a rapid, low-cost, and scalable platform that can be potentially implemented in LMICs to diminish the exorbitant prices, ultimately leading to a significant reduction in cancer-related mortalities within these countries.The Cancer Association of South Africa, the Medical Research Council, the Struwig Germeshuysen Trust School of Medicine Research Committee of the University of Pretoria and the South African National Research Foundation.https://www.mdpi.com/journal/ijmsPhysiologySDG-03:Good heatlh and well-bein