T.V. Mqoco

Flow cytometric analysis of platelets type 2 diabetes mellitus reveals ‘angry’ platelets

BackgroundThe function of platelets have extended way beyond the horizon of haemostasis and thrombosis, and are recognised as active participants in vascular inflammation, as well as in prothrombotic complications of cardiovascular diseases. We describe and compare platelet function in type II diabetes (with and without cardiovascular manifestation) and healthy individuals using scanning electron microscopy and flow cytometry.MethodsThirty subjects were recruited per group and informed consent was obtained from all participants. Diabetic patients were recruited from the diabetic clinic of the Steve Biko Academic Hospital (South Africa). Blood samples were drawn from all participants so that platelet specific antigens were analyzed in citrated whole blood. The platelet parameters used in the study were platelet identifiers (CD41 and CD42) and markers of platelet activation (CD62 and CD63).ResultsResults show that, compared to healthy individuals, both diabetic groups showed a significant difference in both platelet identifiers (CD41-PE, CD42b-PE) as well as markers indicating platelet activation (CD62P-PE and CD63-PE).InterpretationThe flow cytometric data shows that the platelet surface receptors and platelet activation are statistically elevated. This is suggestive of enhanced platelet activation and it appears as if platelets are displaying ‘angry’ behaviour. The lysosomal granules may play a significant role in diabetes with cardiovascular complications. These results were confirmed by ultrastructural analysis.

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.

Sulphamoylated 2-Methoxyestradiol Analogues Induce Apoptosis in Adenocarcinoma Cell Lines

2-Methoxyestradiol (2ME2) is a naturally occurring estradiol metabolite which possesses antiproliferative, antiangiogenic and antitumor properties. However, due to its limited biological accessibility, synthetic analogues have been synthesized and tested in attempt to develop drugs with improved oral bioavailability and efficacy. The aim of this study was to evaluate the antiproliferative effects of three novel in silico-designed sulphamoylated 2ME2 analogues on the HeLa cervical adenocarcinoma cell line and estrogen receptor-negative breast adenocarcinoma MDA-MB-231 cells. A dose-dependent study (0.1–25 μM) was conducted with an exposure time of 24 hours. Results obtained from crystal violet staining indicated that 0.5 μM of all 3 compounds reduced the number of cells to 50%. Lactate dehydrogenase assay was used to assess cytotoxicity, while the mitotracker mitochondrial assay and caspase-6 and -8 activity assays were used to investigate the possible occurrence of apoptosis. Tubulin polymerization assays were conducted to evaluate the influence of these sulphamoylated 2ME2 analogues on tubulin dynamics. Double immunofluorescence microscopy using labeled antibodies specific to tyrosinate and detyrosinated tubulin was conducted to assess the effect of the 2ME2 analogues on tubulin dynamics. An insignificant increase in the level of lactate dehydrogenase release was observed in the compounds-treated cells. These sulphamoylated compounds caused a reduction in mitochondrial membrane potential, cytochrome c release and caspase 3 activation indicating apoptosis induction by means of the intrinsic pathway in HeLa and MDA-MB-231 cells. Microtubule depolymerization was observed after exposure to these three sulphamoylated analogues.

In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17‐β‐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‐β‐estradiol analogue, was tested on the breast adenocarcinoma MCF‐7 cell line. The in vitro influence of 24 h exposure to 0.18 μM 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. Copyright

Sulphamoylated estradiol analogue induces antiproliferative activity and apoptosis in breast cell lines

Research into potential anticancer agents has shown that 2-methoxyestradiol exerts antiproliferative activity in vitro and in vivo in an estrogen receptor-independent manner. Due to its limited biological accessibility and rapid metabolic degradation, several new analogues have been developed in recent years. This study investigated the in vitro effects of a novel in silicodesigned compound (C16) in an estrogen receptor-positive breast adenocarcinoma epithelial cell line (MCF-7), an estrogen receptor-negative breast adenocarcinoma epithelial cell line (MDA-MB-231) and a nontumorigenic breast cell line (MCF-12A). Light microscopy revealed decreased cell density, cells blocked in metaphase and the presence of apoptotic characteristics in all three cell lines after exposure to C16 for 24 h. Polarizationoptical transmitted light differential interference contrast revealed the presence of several rounded cells and decreased cell density. The xCELLigence real-time label-independent approach revealed that C16 exerted antiproliferative activity. Significant inhibition of cell growth was demonstrated after 24 h of exposure to 0.2 μM C16 in all three cell lines. However, the non-tumorigenic MCF-12A cell line recovered extremely well after 48 h when compared to the tumorigenic cell lines. This indicates that C16 acts as an antiproliferative agent, possesses antimitotic activity and induces apoptosis in vitro. These features warrant further investigation.

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.