Andre Stander

Docking, Synthesis, and in vitro Evaluation of Antimitotic Estrone Analogs

In the present study, Autodock 4.0 was employed to discover potential carbonic anhydrase IX inhibitors that are able to interfere with microtubule dynamics by binding to the Colchicine binding site of tubulin. Modifications at position 2′ of estrone were made to include moieties that are known to improve the antimitotic activity of estradiol analogs. 2‐ethyl‐3‐O‐sulphamoyl‐estra‐1,3,5(10),15‐tetraen‐3‐ol‐17‐one estronem (C9) and 2‐ethyl‐3‐O‐sulphamoyl‐estra‐1,3,5(10)16‐tetraene (C12) were synthesized and tested in vitro. Growth studies were conducted utilizing spectrophotometrical analysis with crystal violet as DNA stain. Compounds C9 and C12 were cytotoxic in MCF‐7 and MDA‐MB‐231 tumorigenic and metastatic breast cancer cells, SNO non‐keratinizing squamous epithelium cancer cells and HeLa cells after 48 h exposure. Compounds C9 inhibited cell proliferation to 50% of the vehicle‐treated controls from 110 to 160 nm and C12 at concentrations ranging from 180 to 220 nm. Confocal microscopy revealed abnormal spindle morphology in mitotic cells. Cell cycle analysis showed an increase in the number of cells in the G2/M fraction after 24 h and an increase in the number of cell in the sub‐G1 fraction after 48 h, indicating that the compounds are antimitotic and able to induce apoptosis.

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.

Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells

Cancer is the second leading cause of death in South Africa. The critical role that microtubules play in cell division makes them an ideal target for the development of chemotherapeutic drugs that prevent the hyperproliferation of cancer cells. The new in silico-designed estradiol analogue 2-ethyl-3-O-sulfamoylestra-1,3,5(10)16-tetraene (ESE-16) was investigated in terms of its in vitro antiproliferative effects on the esophageal carcinoma SNO cell line at a concentration of 0.18 μM and an exposure time of 24 h. Polarization-optical differential interference contrast and triple fluorescent staining (propidium iodide, Hoechst 33342 and acridine orange) revealed a decrease in cell density, metaphase arrest, and the occurrence of apoptotic bodies in the ESE-16-treated cells when compared to relevant controls. Treated cells also showed an increase in the presence of acidic vacuoles and lysosomes, suggesting the occurrence of autophagic processes. Cell death via autophagy was confirmed using the Cyto-ID autophagy detection kit and the aggresome detection assay. Results showed an increase in autophagic vacuole and aggresome formation in ESE-16 treated cells, confirming the induction of cell death via autophagy. Cell cycle progression demonstrated an increase in the sub-G1 fraction (indicative of the presence of apoptosis). In addition, a reduction in mitochondrial membrane potential was also observed, which suggests the involvement of apoptotic cell death induced by ESE-16 via the intrinsic apoptotic pathway. In this study, it was demonstrated that ESE-16 induces cell death via both autophagy and apoptosis in esophageal carcinoma cells. This study paves the way for future investigation into the role of ESE-16 in ex vivo and in vivo studies as a possible anticancer agent.

Differential signaling involved in Sutherlandia frutescens-induced cell death in MCF-7 and MCF-12A cells

ETHNOPHARMACOLOGICAL RELEVANCE The scientific study of natural products traditionally used in anticancer preparations has yielded several therapeutically relevant compounds. One of these traditional preparations with potentially beneficial properties is aqueous extracts of Sutherlandia frutescens, a shrub indigenous to the Western Cape region of South Africa. The aims of this study were to evaluate in vitro efficacy of these preparations on the MCF-7 breast adenocarcinoma and MCF-12A non-tumorigenic cell lines in terms of cell proliferation, cell morphology and possible induction of cell death. MATERIALS AND METHODS Crystal violet staining was used to evaluate cell proliferation, light-and fluorescence microscopy were used to investigate both intracellular and extracellular morphological features of apoptosis and autophagy (e.g. membrane blebbing, condensed chromatin and intracellular lysosomes), while flow cytometry quantified cell cycle changes and induction of apoptosis through analysis of the flip-flop translocation of phosphatidylserine. RESULTS Crystal violet staining showed a time- and dose specific response to aqueous Sutherlandia frutescens extracts, revealing exposure to 1mg/ml aqueous extract for 48h to be ideal for comparing the differential effects of Sutherlandia frutescens in the MCF-7 and MCF-12A cell lines. Microscopy showed distinct morphological changes with hallmarks of apoptosis being observed in both cell lines. Flow cytometry revealed a decrease in actively cycling cells in both cell lines, and a 4.36% increase in phosphatidylserine translocation in the MCF-7 cell line, indicative of apoptosis induction, while fluorescence microscopy showed evidence of the induction of autophagy. CONCLUSIONS Analyses revealed the carcinogenic MCF-7 cell line to be more susceptible to the cytostatic and cytotoxic effects of aqueous extracts of Sutherlandia frutescens when compared to the non-tumorigenic MCF-12A cell line, thus warranting further research into the exact cellular mechanisms involved and the possible synergistic activities of Sutherlandia frutescens ingredients.

Characterization of Carbonic Anhydrase Isozyme Specific Inhibition by Sulfamated 2-Ethylestra Compounds

This work was supported by NIH Grant GM25154 and by grants from the Medical Research Council of South Africa (AG374, AK076), the Cancer Association of South Africa (AK246), the Struwig-Germeshuysen Cancer Research Trust of South Africa (AJ038) and RESCOM University of Pretoria (A0R984).

In silico design and bioevaluation of selective benzotriazepine BRD4 inhibitors with potent antiosteoclastogenic activity

The bromodomain (BRD) and extra‐terminal domain (BET) protein family bind to acetylated histones on lysine residues and act as epigenetic readers. Recently, the role of this protein family in bone loss has been gaining attention. Earlier studies have reported that benzotriazepine (Bzt) derivatives could be effective inhibitors of BET proteins. In this study, using in silico tools we designed three Bzt analogs (W49, W51, and W52). By docking, molecular simulations, and chemiluminescent Alpha Screen binding assay, we show that the studied analogs were selective at inhibiting BRD4 when compared to BRD2. Furthermore, we tested the effectiveness of these analogs on osteoclast formation and function. Among the examined analogs, Bzt‐W49 and Bzt‐W52 were found to be the most potent inhibitors of osteoclastogenesis without cytotoxicity in murine RAW264.7 osteoclast progenitors. Both the compounds also inhibited osteoclast formation without affecting cell viability in human CD14+ monocytes. Moreover, owing to attenuated osteoclastogenesis, actin ring formation and bone resorptive function of osteoclasts were severely perturbed. In conclusion, these results suggest that the novel BRD4‐selective Bzt analogs designed in this study could be explored further for developing therapeutics against bone loss diseases characterized by excessive osteoclast activity.

An endometrial histomorphometric study of CD56 + natural killer cells in women with unexplained infertility

51 SAJOG • September 2017, Vol. 23, No. 2 Background. The number of peripheral blood and endometrial natural killer cells varies greatly during implantation and the first trimester of pregnancy and is thought to play a role in the maintenance of a healthy pregnancy. However, the role of endometrial CD56+ natural killer (NK) cells as an immunological mechanism in unexplained infertility is yet unknown. Objectives. The study aimed to enumerate the concentrations of CD56+ NK cells in endometrial samples, and to statistically compare these numbers between fertile and infertile women. Methods. A histomorphometric analysis was conducted using haematoxylin and eosin staining and an immunohistochemical approach was used for quantifying cell numbers. Results. Fifty samples were collected in equal parts between a study group of infertile female subjects (mean (standard deviation) age 35 (4), range 26 42 years) and a control group of multiparous fertile individuals (mean (SD) age 43.4 (6.3), range 30 55). The mean number of CD56+ NK cells present at different depths for both the study and control groups did not differ significantly. Age and group (study or control) were not significantly related to the mean number of CD56+ NK cells. However, for the late secretory phase the mean number of CD56+ NK cells was significantly higher than for the early phase. Conclusion. Our findings could not identify a statistically significant correlation between the number of CD56+ NK cells and infertility.

In vitro evaluering van 'n nuwe antimitotiese estradiol analoog en dichloor asynsuur in menslike borsadenokarsinoom en normale borsepiteelselle : referaatopsomming

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.

In vitro evaluation of a novel antimitotic estradiol analog and dichloroacetic acid on breast adenocarcinoma and breast non-tumorigenic cells

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.

In vitro evaluering van ’n nuwe antimitotiese estradiol analoog en dichloor asynsuur in menslike borsadenokarsinoom en normale borsepiteelselle

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.