Duramycin-induced calcium release in cancer cells

Introduction: Duramycin through binding with phosphatidylethanolamine (PE) has shown potential to be an effective anti-tumour agent. However its mode of action in relation to tumour cells is not fully understood. Methods: PE expression on the surface of a panel of cancer cell lines was analysed using duramycin and subsequent antibody labelling then analysed by flow cytometry. Cell viability was also assessed via flow cytometry using annexin V and propidium iodide (PI). Calcium ion (Ca²⁺) release by tumour cells in response to duramycin was determined by spectrofluorometry following incubation with Fluo-3, AM. Confocal microscopy was performed on the cancer cell line AsPC-1 to assess real time cell response to duramycin treatment. Results: Duramycin was able to detect cell surface PE expression on all 15 cancer cell lines screened, which was shown to be duramycin concentration dependent. However higher concentrations induced necrotic cell death. Duramycin induced calcium ion (Ca²⁺) release from the cancer cell lines also in a concentration and time dependent manner. Confocal microscopy showed an influx of PI into the cells over time and induced morphological changes. Conclusion: Duramycin induces Ca²⁺ release from cancer cell lines in a time and concentration dependent relationship

Repeated supra-maximal sprint cycling with and without sodium bicarbonate supplementation induces endothelial microparticle release

Under normal homeostatic conditions, the endothelium releases microparticles (MP), which are known to increase under stressful conditions and in disease states. CD105 (endoglin) and CD106 (vascular cell adhesion molecule-1) are expressed on the surface of endothelial cells and increased expression in response to stress may be observed. A randomised-controlled double-blinded study aimed to examine the use of endothelial microparticles as a marker for the state of one’s endothelium, as well as whether maintaining acid-base homeostasis affects the release of these MP. This study tested seven healthy male volunteers, who completed a strenuous cycling protocol, with venous blood analysed for CD105+ and CD106+ MP by flow cytometry at regular intervals. Prior to each trial participants consumed either 0.3 g·kg-1 body mass of sodium bicarbonate (NaHCO3), or 0.045 g·kg-1 body mass of sodium chloride (NaCl). A significant rise in endothelial CD105+MP and CD106+MP (p < 0.05) was observed at 90 minutes post exercise. A significant trend was shown for these MP to return to resting levels 180 minutes post exercise in both groups. No significance was found between experimental groups, suggesting that maintaining acid-base variables closer to basal levels has little effect upon the endothelial stress response for this particular exercise mode. In conclusion, strenuous exercise is accompanied by MP release and the endothelium is able to rapidly recover in healthy individuals, whilst maintaining acid-base homeostasis does not attenuate the MP release from the endothelium after exercise

Implications of a pre-exercise alkalosis-mediated attenuation of HSP72 on its response to a subsequent bout of exercise

The aim of this study was to investigate if a pre-exercise alkalosis-mediated attenuation of HSP72 had any effect on the response of the same stress protein after a subsequent exercise. Seven physically active males [25.0 ± 6.5 years, 182.1 ± 6.0 cm, 74.0 ± 8.3 kg, peak aerobic power (PPO) 316 ± 46 W] performed a repeated sprint exercise (EXB1) following a dose of 0.3 g kg⁻¹ body mass of sodium bicarbonate (BICARB), or a placebo of 0.045 g kg⁻¹ body mass of sodium chloride (PLAC). Participants then completed a 90-min intermittent cycling protocol (EXB2). Monocyte expressed HSP72 was significantly attenuated after EXB1 in BICARB compared to PLAC, however, there was no difference in the HSP72 response to the subsequent EXB2 between conditions. Furthermore there was no difference between conditions for measures of oxidative stress (protein carbonyl and HSP32). These findings confirm the sensitivity of the HSP72 response to exercise-induced changes in acid–base status in vivo, but suggest that the attenuated response has little effect upon subsequent stress in the same day

Chemotherapy treatment of multiple myeloma patients increases circulating levels of endothelial microvesicles

Correspondenc

Procoagulant tumor microvesicles attach to endothelial cells on biochips under microfluidic flow

Tumor patients are at a high risk of venous thromboembolism (VTE), and the mechanism by which this occurs may involve tumor-derived microvesicles (MVs). Previously, it has been shown that tumor MVs become attached to endothelial cells in static conditions. To investigate whether this process occurs under physiologically relevant flow rates, tumor MVs were perfused across a microfluidic device coated with growing human umbilical vein endothelial cells (HUVECs). Cell lines were screened for their ability to form tumor spheroids, and two cell lines, ES-2 and U87, were selected; spheroids formed were transferred to a microfluidic chip, and a second endothelial cell biochip was coated with HUVECs and the two chips were linked. Media flowed through the spheroid chip to the endothelial chip, and procoagulant activity (PCA) of the tumor media was determined by a one-stage prothrombin time assay. Tumor MVs were also quantified by flow cytometry before and after interaction with HUVECs. Confocal images showed that HUVECs acquired fluorescence from MV attachment. Labeled MVs were proportionally lost from MV rich media with time when flowed over HUVECs and were not observed on a control chip. The loss of MV was accompanied by a proportional reduction in PCA. Flow cytometry, confocal microscopy, and live flow imagery captured under pulsatile flow confirmed an association between tumor MVs and HUVECs. Tumor MVs attached to endothelial cells under physiological flow rates, which may be relevant to the VTE pathways in cancer patient

Doxorubicin Enhances Procoagulant Activity of Endothelial Cells after Exposure to Tumour Microparticles on Microfluidic Devices

The majority of cancer patients undergoing chemotherapy have a significantly increased risk of venous thromboembolism via a mechanism not yet fully elucidated but which most probably involves tumour microparticles (MP) combined with damaged/activated endothelium. Tumour cell lines (ES-2 and U87) were cultured as 3D spheroids and transferred to biochips connected through to a second chip precultured with an endothelial cell layer (human umbilical vein endothelial cells [HUVECs]). Media were introduced with and without doxorubicin (DOX) to the spheroids in parallel chips under constant flow conditions. Media samples collected pre- and post-flow through the biochip were analysed for tissue factor microparticles (TFMP) and procoagulant activity (PCA). HUVECs were also harvested and tested for PCA at a constant cell number. TFMP levels in media decreased after passing over HUVECs in both conditions over time and this was accompanied by a reduction in PCA (indicated by a slower coagulation time) of the media. The relationship between PCA and TFMP was correlated (r = −0.85) and consistent across experiments. Harvested HUVECs displayed increased PCA when exposed to tumour spheroid media containing TFMP, which was increased further after the addition of DOX, suggesting that the TFMP in the media had bound to HUVEC cell surfaces. The enhanced PCA of HUVECs associated with the DOX treatment was attributed to a loss of viability of these cells rather than additional MP binding. The data suggest that tumour MP interact with HUVECs through ligand-receptor binding. The model described is a robust and reproducible method to investigate cytotoxic agents on tumour spheroids and subsequent downstream interaction with endothelial cells

Implications of a Pre-Exercise Alkalosis Mediated Attenuation of HSP72 on its Response to a Subsequent Bout of Exercise

The aim of this study was to investigate if a pre-exercise alkalosis mediated attenuation of HSP72 had any effect on the response of the same stress protein after a subsequent exercise. Seven physically active males (25.0 ± 6.5 years, 182.1 ± 6.0 cm, 74.0 ± 8.3 kg, peak aerobic power (PPO) 316 ± 46 W) performed a repeated sprint exercise (EXB1) following a dose of 0.3 g kg-1 body mass of sodium bicarbonate (BICARB), or a placebo of 0.045 g kg-1 body mass of sodium chloride (PLAC). Participants then completed a 90-min intermittent cycling protocol (EXB2). Monocyte expressed HSP72 was significantly attenuated after EXB1 in BICARB compared to PLAC, however there was no difference in the HSP72 response to the subsequent EXB2 between conditions. Furthermore there was no difference between conditions for measures of oxidative stress (protein carbonyl and HSP32). These findings confirm the sensitivity of the HSP72 response to exercise induced changes in acid-base status in vivo, but suggest that the attenuated response has little effect upon subsequent stress in the same day

High-throughput fabrication of hepatic cell clusteroids with enhanced growth and functionality for tissue engineering applications

Culturing of cells as three-dimensional (3D) clusters can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome and expensive than two-dimensional (2D) cultures. Current methods for the preparation of tissue spheroids require complex materials, involve tedious facilities and are generally not scalable. We report a novel inexpensive and up-scalable method for the preparation of large quantities of viable cell clusters (clusteroids) of hepatocytes (Hep-G2). The method has a high throughput potential and is based on an aqueous two-phase system (ATPS) of stable water-in-water (w/w) Pickering emulsions, formed of dextran (DEX) drops and poly ethylene oxide (PEO) continuous phase stabilized with whey protein particles. This system enabled the rapid fabrication of cell clusteroids from Hep-G2 cells. Here, the interfacial tension of the aqueous phase in the emulsion droplets, where the cells partition preferentially, is used to wrap the cells in separate compartments, and then the droplets are shrank by changing the balance of ATPS, thus rapidly driving the cells from larger and loosely packed DEX drops to mostly spherical clusters. Cell-Cell adhesion was strongly promoted within the w/w Pickering emulsion droplets which helped the formation of the 3D clusteroids. These were collected after subsequent dilution of the emulsion with culture media. The collected hepatic clusteroids were incorporated into an alginate hydrogel in media to study their proliferation and the cell function compared with individual cells under the same conditions. Our results confirmed that urea and albumin production, which are both linked to hepatocyte cell function, was strongly reinforced in the clusteroid based tissues compared to the one formulated with individual cells. This methodology could potentially extend the w/w emulsion cell clustering platform in tissue generation and preparation of large quantities of organoids for drug tests and replacement of animal models

Immobilised-enzyme microreactors for the identification and synthesis of conjugated drug metabolites

The study of naturally circulating drug metabolites has been a focus of interest, since these metabolites may have different therapeutic and toxicological effects compared to the parent drug. The synthesis of metabolites outside of the human body is vital in order to conduct studies into the pharmacological activities of drugs and bioactive compounds. Current synthesis methods require significant purification and separation efforts or do not provide sufficient quantities for use in pharmacology experiments. Thus, there is a need for simple methods yielding high conversions whilst bypassing the requirement for a separation. Here we have developed and optimised flow chemistry methods in glass microfluidic reactors utilising surface-immobilised enzymes for sulfonation (SULT1a1) and glucuronidation (UGT1a1). Conversion occurs in flow, the precursor and co-factor are pumped through the device, react with the immobilised enzymes and the product is then simply collected at the outlet with no separation from a complex biological matrix required. Conversion only occurred when both the correct co-factor and enzyme were present within the microfluidic system. Yields of 0.97 ± 0.26 mg were obtained from the conversion of resorufin into resorufin sulfate over 2 h with the SULT1a1 enzyme and 0.47 mg of resorufin glucuronide over 4 h for UGT1a1. This was demonstrated to be significantly more than static test tube reactions at 0.22 mg (SULT1a1) and 0.19 mg (UGT1a1) over 4 h. With scaling out and parallelising, useable quantities of hundreds of micrograms for use in pharmacology studies can be synthesised simply

Advanced biomedical applications based on emerging 3D cell culturing platforms

It is of great value to develop reliable in vitro models for cell biology and toxicology. However, ethical issues and the decreasing number of donors restrict the further use of traditional animal models in various fields, including the emerging fields of tissue engineering and regenerative medicine. The huge gap created by the restrictions in animal models has pushed the development of the increasingly recognized three-dimensional (3D) cell culture, which enables cells to closely simulate authentic cellular behaviour such as close cell-to-cell interactions and can achieve higher functionality. Furthermore, 3D cell culturing is superior to the traditional 2D cell culture, which has obvious limitations and cannot closely mimic the structure and architecture of tissues. In this study, we review several methods used to form 3D multicellular spheroids. The extracellular microenvironment of 3D spheroids plays a role in many aspects of biological sciences, including cell signalling, cell growth, cancer cell generation, and anti-cancer drugs. More recently, they have been explored as basic construction units for tissue and organ engineering. We review this field with a focus on the previous research in different areas using spheroid models, emphasizing aqueous two-phase system (ATPS)-based techniques. Multi-cellular spheroids have great potential in the study of biological systems and can closely mimic the in vivo environment. New technologies to form and analyse spheroids such as the aqueous two-phase system and magnetic levitation are rapidly overcoming the technical limitations of spheroids and expanding their applications in tissue engineering and regenerative medicine