The Induction of Apoptosis in A375 Malignant Melanoma Cells by Sutherlandia frutescens

Sutherlandia frutescens is a medicinal plant indigenous to Southern Africa and is commonly known as the “cancer bush.” This plant has traditionally been used for the treatment of various ailments, although it is best known for its claims of activity against “internal” cancers. Here we report on its effect on melanoma cells. The aim of this study was to investigate whether an extract of S. frutescens could induce apoptosis in the A375 melanoma cell line and to outline the basic mechanism of action. S. frutescens extract induced apoptosis in A375 cells as evidenced by morphological features of apoptosis, phosphatidylserine exposure, nuclear condensation, caspase activation, and the release of cytochrome c from the mitochondria. Studies in the presence of a pan-caspase inhibitor allude to caspase-independent cell death, which appeared to be mediated by the apoptosis inducing factor. Taken together, the results of this study show that S. frutescens extract is effective in inducing apoptosis in malignant melanoma cells and indicates that further in vivo mechanistic studies may be warranted

The apoptosis inducing effects of Sutherlandia spp. extracts on an oesophageal cancer cell line

AIM OF STUDY: Oesophageal cancer is the ninth most common cancer in the world and the second most common cancer among South African men. It also has one of the lowest possibilities of cure, with the 5-year survival rate estimated to be only 10% overall. Sutherlandia frutescens, or the "cancer bush", is a medicinal plant indigenous to southern Africa that is believed to have anti-cancer and anti-proliferative properties. The aim of this study was to investigate the potential apoptosis-inducing effects of two S. frutescens extracts and one Sutherlandia tomentosa extract on the SNO oesophageal cancer cell line. MATERIALS AND METHODS: Cell viability and morphology of SNO cells were evaluated following exposure to the extracts. Apoptotic markers including cytochrome c translocation and phosphatidylserine externalisation were quantified by flow cytometry. The activity of caspases 3 and 7 was evaluated with spectrofluorometry. Apoptosis was evaluated in the presence of the pan-caspase inhibitor, Z-VAD-fmk. The effect of the extracts was compared to non-cancerous peripheral blood mononuclear cells (PBMCs). RESULTS: Time- and dose-response studies were conducted to establish treatment conditions of 2.5 and 5mg/ml of crude plant extracts. Microscopy studies revealed that S. frutescens- and S. tomentosa-treated SNO cells had morphological features characteristic of apoptosis. Annexin V/propidium iodide flow cytometry confirmed that the extracts do, in fact, induce apoptosis in the SNO cells. Caspase inhibition studies seem to indicate that extracts A (S. frutescens (L.) R. Br. subsp. microphylla from Colesberg), B (S. frutescens (L.) R. Br. subsp. microphylla from Platvlei) and C (S. tomentosa Eckl. & Zeyh from Stil Bay) are able to induce caspase-dependent as well as -independent cell death. The S. frutescens and S. tomentosa extracts were found to be more cytotoxic to cancerous SNO cells when compared to the PBMCs. CONCLUSIONS: S. frutescens and S. tomentosa extracts show promise as apoptosis-inducing anti-cancer agents

Anticancer activity of silver(I) cyclohexyldiphenylphosphine complexes toward SNO cancer cells

<p>Silver and its derivatives have been and are currently used as antimicrobial and antibacterial agents. However, their use is rather limited to date as anticancer agents. Thus, this study focuses on the synthesis and characterization of three silver(I) complexes which have the ability to adopt different geometries, and to induce cancer cell death in SNO esophageal cancer cells. Silver thiocyanate was reacted in different ratios with cyclohexyldiphenylphosphine to obtain 1:1 (complex <b>1</b>), 1:2 (complex <b>2</b>), and 1:3 (complex <b>3</b>) molar ratios of silver(I):cyclohexyldiphenylphosphine complexes. These complexes were characterized using conventional spectroscopic techniques, which included ¹H, ¹³C, and ³¹P NMR, FTIR, and microanalysis (%C, H, N, S). In addition, the single-crystal X-ray structures of complexes <b>1</b> and <b>2</b> were determined. Moreover, all three complexes displayed toxic activity toward the malignant SNO cells with IC₅₀ concentrations below 5 μM as determined with an alamarBlue® viability assay. Morphological and flow cytometric analyses were included to identify the possible mode of cancer cell death. These biochemical assays revealed that all three treatments induced apoptosis due to the presence of specific apoptotic markers.</p

A novel 2-methoxyestradiol analogue is responsible for vesicle disruption and lysosome aggregation in breast cancer cells

BACKGROUND : 2-Methoxyestradiol (2ME2) is an endogenous metabolite of 17-β-estradiol with anti-proliferative and anti-angiogenic properties. Due to 2ME2’s rapid metabolism and low oral bioavailability in in vivo settings, 2ME2 analogues have been designed to alleviate these issues. One of these compounds is 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). A previous work alluded to the ability of ESE-16 to induce autophagic cell death. Therefore, we investigated the mode of action of ESE-16 by studying its effects on autophagy, vesicle formation, and lysosomal organisation. SUMMARY : Vesicle formation and autophagy induction were analysed by transmission electron microscopy (TEM), monodansylcadaverine (MDC) staining and Lysotracker staining, while autophagosome turnover was analysed using microtubule-associated protein 1A/1B-light chain 3 (LC3 lipidation) analysis. MDC staining of acidic vesicles revealed an increase both in the number and size of vesicles after ESE-16 exposure. This was confirmed by TEM. Lysotracker staining indicated an increase in the size of lysosomes, as well as changes in their distribution within the cell. However, autophagy was not induced, since LC3 lipidation did not increase after exposure to ESE-16. KEY MESSAGES : This study showed that ESE-16 exposure leads to the aggregation of acidic vesicles, identified as lysosomes, not accompanied by an induction of autophagy. Therefore, ESE-16 disrupts normal endocytic vesicle maturation likely through the inhibition of the microtubule function.Grants awarded to Prof AM Joubert from the Medical Research Council of South Africa (AOS536, AOW110, AK076; AL343), the Cancer Association of South Africa (AOW228, A0V741, AK246), RESCOM, School of Medicine (Faculty of Health Sciences, University of Pretoria), the National Research Foundation of South Africa (N00375, AL239) and the Struwig-Germeshuysen Cancer Research Trust of South Africa (AN074).https://www.karger.com/PHA2019-04-19hj2018Physiolog

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field