The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration assays; the neutral red uptake (NRU), resazurin reduction (RES) and sulforhodamine B (SRB) assays, in comparison to the MTT assay. Interference between the MTT assay and three glycolysis inhibitors, 2-deoxyglucose, 3-bromopyruvate and lonidamine, was investigated. Data indicate that the NRU, RES and SRB assays showed the smallest variability across the linear range, while the largest variation was observed for the MTT assay. This implies that these assays would more accurately detect small changes in cell number than the MTT assay. The SRB assay provided the most reproducible results as indicated by the coefficient of determination after a limited number of experiments. The SRB assay also produced the lowest variance in the derived 50% inhibitory concentration (IC50), while IC50 concentrations of 3-bromopyruvate could not be detected using either the MTT or RES assays after 24 hours incubation. Interference in the MTT assay was observed for all three tested glycolysis inhibitors in a cell-free environment. No interferences were observed for the NRU, SRB or RES assays. This study demonstrated that the MTT assay was not the best assay in a number of parameters that must be considered when a cell enumeration assay is selected: the MTT assay was less accurate in detecting changes in cell number as indicated by the variation observed in the linear range, had the highest variation when the IC50 concentrations of the glycolysis inhibitors were determined, and interference between the MTT assay and all the glycolysis inhibitors tested were observed. The SRB assay performed best overall considering all of the parameters, suggesting that it is the most suitable assay for use in preclinical screening of novel therapeutic compounds with oxido-reductive potential.

Background: The estrogen metabolite 2-methoxyestradiol (2ME2) and a number of synthesised derivatives have been shown to bind to microtubules thereby arresting cancer cells in mitosis which leads to apoptosis. In interphase cells, microtubules play an important role in the delivery of proteins to subcellular locations including the focal adhesions. In fact, focal adhesion dynamics and cell migration are in part regulated by microtubules. We hypothesised that novel 2ME2 derivatives can alter cell migration by influencing microtubule dynamics in interphase cells. In this report we describe 2ME2 derivatives that display anti-migratory capabilities in a metastatic breast cancer cell line through their effects on the microtubule network resulting in altered focal adhesion signalling and RhoA activity. Methods: Cell migration was assayed using wound healing assays. To eliminate mitosis blockage and cell rounding as a confounding factor cell migration was also assessed in interphase blocked cells. Fluorescence confocal microscopy was used to visualise microtubule dynamics and actin cytoskeleton organisation while western blot analysis was performed to analyse focal adhesion signalling and RhoA activation. Results: 2ME2 derivatives, ESE-one and ESE-15-one, inhibited cell migration in cycling cells as expected but equally diminished migration in cells blocked in interphase. While no significant effects were observed on the actin cytoskeleton, focal adhesion kinase activity was increased while RhoA GTPase activity was inhibited after exposure to either compound. Microtubule stability was increased as evidenced by the increased length and number of detyrosinated microtubules while at the same time clear disorganisation of the normal radial microtubule organisation was observed including multiple foci. Conclusions: ESE-15-one and ESE-one are potent migration inhibitors of metastatic breast cancer cells. This ability is coupled to alterations in focal adhesion signalling but more importantly is associated with severe disorganisation of microtubule dynamics and polarity. Therefore, these compounds may offer potential as anti-metastatic therapies.

Cancer is a complex disease since it is adaptive in such a way that it can promote proliferation and invasion by means of an overactive cell cycle and in turn cellular division which is targeted by antimitotic drugs that are highly validated chemotherapy agents. However, antimitotic drug cytotoxicity to non-tumorigenic cells and multiple cancer resistance developed in response to drugs such as taxanes and vinca alkaloids are obstacles faced in both the clinical and basic research field to date. In this review, the classes of antimitotic compounds, their mechanisms of action and cancer cell resistance to chemotherapy and other limitations of current antimitotic compounds are highlighted, as well as the potential of novel 17-β estradiol analogs as cancer treatment.

Erythrocytes play an important role in oxygen and carbon dioxide transport. Although erythrocytes possess no nucleus or mitochondria, they fulfil several metabolic activities namely, the Embden-Meyerhof pathway, as well as the hexose monophosphate shunt. Metabolic processes within the erythrocyte contribute to the morphology/shape of the cell and important constituents are being kept in an active, reduced form. Erythrocytes undergo a form of suicidal cell death called eryptosis. Eryptosis results from a wide variety of contributors including hyperosmolarity, oxidative stress, and exposure to xenobiotics. Eryptosis occurs before the erythrocyte has had a chance to be naturally removed from the circulation after its 120-day lifespan and is characterised by the presence of membrane blebbing, cell shrinkage, and phosphatidylserine exposure that correspond to nucleated cell apoptotic characteristics. After eryptosis is triggered there is an increase in cytosolic calcium (Ca²⁺) ion levels. This increase causes activation of Ca²⁺-sensitive potassium (K⁺) channels which leads to a decrease in intracellular potassium chloride (KCl) and shrinkage of the erythrocyte. Ceramide, produced by sphingomyelinase from the cell membrane’s sphingomyelin, contributes to the occurrence of eryptosis. Eryptosis ensures healthy erythrocyte quantity in circulation whereas excessive eryptosis may set an environment for the clinical presence of pathophysiological conditions including anaemia.

Tumourigenic tissue uses modified metabolic signalling pathways in order to support hyperproliferation and survival. Cancer-associated aerobic glycolysis resulting in lactic acid production was described nearly 100 years ago. Furthermore, increased reactive oxygen species (ROS) and lactate quantities increase metabolic, survival and proliferation signalling, resulting in increased tumourigenesis. In order to maintain redox balance, the cell possesses innate antioxidant defence systems such as superoxide dismutase, catalase and glutathione. Several stimuli including cells deprived of nutrients or failure of antioxidant systems result in oxidative stress and cell death induction. Among the cell death machinery is autophagy, a compensatory mechanism whereby energy is produced from damaged and/or redundant organelles and proteins, which prevents the accumulation of waste products, thereby maintaining homeostasis. Furthermore, autophagy is maintained by several pathways including phosphoinositol 3 kinases, the mitogen-activated protein kinase family, hypoxia-inducible factor, avian myelocytomatosis viral oncogene homolog and protein kinase receptor-like endoplasmic reticulum kinase. The persistent potential of cancer metabolism, redox regulation and the crosstalk with autophagy in scientific investigation pertains to its ability to uncover essential aspects of tumourigenic transformation. This may result in clinical translational possibilities to exploit tumourigenic oxidative status and autophagy to advance our capabilities to diagnose, monitor and treat cancer.

Platelets are conventionally defined as playing a vital role in homeostasis and thrombosis. This role has over the years transformed as knowledge regarding platelets has expanded to include inflammation, cancer progression, and metastasis. Upon platelet activation and subsequent aggregation, platelets release a host of various factors, including numerous pro-inflammatory factors. These pro-inflammatory factors are recruiters and activators of leukocytes, aiding in platelets’ immune regulating function and inflammatory function. These various platelet functions are interrelated; activation of the inflammatory function results in thrombosis and, moreover, in various disease conditions, can result in worsened or chronic pathogenesis, including cancer. The role and contribution of platelets in a multitude of pathophysiological events during hemostasis, thrombosis, inflammation, cancer progression, and metastasis is an important focus for ongoing research. Platelet activation as discussed here is present in all platelet functionalities and can result in a multitude of factors and signaling pathways being activated. The cross-talk between inflammation, cancer, and platelets is therefore an ideal target for research and treatment strategies through antiplatelet therapy. Despite the knowledge implicating platelets in these mentioned processes, there is, nevertheless, limited literature available on the involvement and impact of platelets in many diseases, including myeloproliferative neoplasms. The extensive role platelets play in the processes discussed here is irrefutable, yet we do not fully understand the complete interrelation and extent of these processes.