Sumari Marais

In vitro effects of 2-methoxyestradiol on morphology, cell cycle progression, cell death and gene expression changes in the tumorigenic MCF-7 breast epithelial cell line

In the present study, the antiproliferative mechanism of action of 1 microM 2-methoxyestradiol (2ME) was investigated in the MCF-7 cell line. Measurement of intracellular cyclin B and cytochrome c protein levels, reactive oxygen species formation, cell cycle progression and apoptosis induction were conducted by means of flow cytometry. Morphological changes were evaluated using transmission electron microscopy and fluorescent microscopy by employing Hoechst 33342 and acridine orange. Gene expression changes were conducted by means of microarrays. 2ME-treated cells demonstrated an increase in cyclin B protein levels, hydrogen peroxide formation, intracellular levels of cytochrome c, as well as an increase in early and late stages of apoptosis. In addition, morphological data revealed the presence of autophagic processes. Fluorescent microscopy showed an increase in acridine orange staining and electron microscopy revealed an increase in vacuolar formation in 2ME-treated cells. The gene expression of several genes associated with mRNA translation, autophagy-related processes and genes involved in microtubule dynamics were affected. The study contributes to the mechanistic understanding of 2MEs growth inhibition in MCF-7 cells and highlights the possibility of both apoptotic and autophagic processes being activated in response to 2ME treatment in this cell line.

In vitro effects of Sutherlandia frutescens water extracts on cell numbers, morphology, cell cycle progression and cell death in a tumorigenic and a non-tumorigenic epithelial breast cell line

Sutherlandia frutescens is a South African herb traditionally used for internal cancers, diabetes, a variety of inflammatory conditions and recently to improve the overall health in cancer and HIV/AIDS patients. The in vitro effects of S. frutescens extracts were evaluated on cell numbers, morphology, cell cycle progression and cell death. Dose-dependent studies (2-10 mg/ml) revealed a decrease in malignant cell numbers when compared to their controls. S. frutescens extracts (10 mg/ml) decreased cell growth in a statistically significantly manner to 26% and 49% (P<0.001) in human breast adenocarcinoma (MCF-7) and human non-tumorigenic epithelial mammary gland cells (MCF-12A) respectively after 72 h of exposure. Cell density was significantly compromised and hypercondensed chromatin, cytoplasmic shrinking, membrane blebbing and apoptotic bodies were more pronounced in the MCF-7 cell line. Both S. frutescens-treated cell lines exhibited and increased tendency for acridine orange staining, suggesting increased lysosomal and/or autophagy activity. Flow cytometry showed an increase in the sub G(1) apoptotic fraction and an S phase arrest in both the 5 mg/ml and 10 mg/ml S. frutescens-treated cells. S. frutescens induced an increase in apoptosis in both cell lines as detected by Annexin V and propidium iodide flow cytometric measurement. At 10 mg/ml, late stages of apoptosis were more prominent in MCF-7 S. frutescens-treated cells when compared to the MCF-12A cells. Transmission electron microscopy revealed hallmarks of increased vacuolarization and hypercondensed chromatin, suggesting autophagic and apoptotic processes. The preliminary study demonstrates that S. frutescens water extracts exert a differential action mechanism in non-tumorigenic MCF-12A cells when compared to tumorigenic MCF-7 cells, warranting future studies on this multi-purpose medicinal plant in southern Africa.

Commercial Honeybush (Cyclopia spp.) Tea Extract Inhibits Osteoclast Formation and Bone Resorption in RAW264.7 Murine Macrophages—An in vitro Study

Honeybush tea, a sweet tasting caffeine-free tea that is indigenous to South Africa, is rich in bioactive compounds that may have beneficial health effects. Bone remodeling is a physiological process that involves the synthesis of bone matrix by osteoblasts and resorption of bone by osteoclasts. When resorption exceeds formation, bone remodeling can be disrupted resulting in bone diseases such as osteoporosis. Osteoclasts are multinucleated cells derived from hematopoietic precursors of monocytic lineage. These precursors fuse and differentiate into mature osteoclasts in the presence of receptor activator of NF-kB ligand (RANKL), produced by osteoblasts. In this study, the in vitro effects of an aqueous extract of fermented honeybush tea were examined on osteoclast formation and bone resorption in RAW264.7 murine macrophages. We found that commercial honeybush tea extract inhibited osteoclast formation and TRAP activity which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts without cytotoxicity. Furthermore, honeybush tea extract decreased expression of key osteoclast specific genes, matrix metalloproteinase-9 (MMP-9), tartrate resistant acid phosphatase (TRAP) and cathepsin K. This study demonstrates for the first time that honeybush tea may have potential anti-osteoclastogenic effects and therefore should be further explored for its beneficial effects on bone.

Rooibos tea extracts inhibit osteoclast formation and activity through the attenuation of NF-κB activity in RAW264.7 murine macrophages

Rooibos tea is a naturally sweet and aromatic tea that is native to the Western Cape province of South Africa. Rooibos is usually fermented to produce the traditional reddish brown colour and has been found to have numerous health benefits. These include beneficial effects on osteoblasts; however, its effects on osteoclast formation and activity are unknown. Osteoclasts are large, multinucleated cells responsible for bone resorption. Binding of RANKL to its receptor on osteoclast precursors triggers the NF-κB signalling pathway leading to the formation of osteoclasts. Certain bone destructive diseases, such as osteoporosis, are characterised by overactive osteoclasts. The inhibition of osteoclasts may offer a potential mode to prevent these diseases. The polyphenol contents of both fermented and unfermented tea extracts were similar although the radical scavenging activity of fermented rooibos tea was lower. Both tea extracts were not cytotoxic and inhibited osteoclast formation. Fermented rooibos tea extract caused a greater reduction in osteoclast resorption and the associated gene expression when compared with unfermented rooibos tea. Both tea extracts were shown to attenuate NF-κB activity. Fermented rooibos was found to have a more potent inhibitory effect on osteoclasts than unfermented rooibos extract and therefore may have a beneficial effect on bone health.

2-Methoxyestradiol-bis-sulphamate disrupts microtubule network, arrests cell cycle and induces apoptosis in an esophageal carcinoma cell line

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.

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

Volgens die w�reldgesondheidsorganisasie is servikale karsinoom die tweede mees algemene kanker wat in Suid-Afrikaanse vroue voorkom, dus is die toets van nuwe middels om hierdie soort kanker te behandel, van groot belang.