Role of Magnesium and its mitochondrial transporter MRS2 in the modulation of drug-induced apoptosis leading to multidrug resistance phenotype

Magnesium is an essential element for many biological processes crucial for cell life and proliferation. Growing evidences point out a role for this cation in the apoptotic process and in developing multi drug resistance (MDR) phenotype. The first part of this study aimed to highlight the involvement of the mitochondrial magnesium channel MRS2 in modulating drug-induced apoptosis. We generated an appropriate transgenic cellular system to regulate expression of MRS2 protein. The cells were then exposed to two different apoptotic agents commonly used in chemotherapy. The obtained results showed that cells overexpressing MRS2 channel are less responsiveness to pharmacological insults, looking more resistant to the induced apoptosis. Moreover, in normal condition, MRS2 overexpression induces higher magnesium uptake into isolated mitochondria respect to control cells correlating with an increment of total intracellular magnesium concentration. In the second part of this research we investigated whether magnesium intracellular content and compartmentalization could be used as a signature to discriminate MDR tumour cells from their sensitive counterparts. As MDR model we choose colon carcinoma cell line sensitive and resistant to doxorubicin. We exploited a standard-less approach providing a complete characterization of whole single-cells by combining X-Ray Fluorescence Microscopy , Atomic Force Microscopy and Scanning Transmission X-ray Microscopy. This method allows the quantification of the intracellular spatial distribution and total concentration of magnesium in whole dehydrated cells. The measurements, carried out in 27 single cells, revealed a different magnesium pattern for both concentration and distribution of the element in the two cellular strains. These results were then confirmed by quantifying the total amount of intracellular magnesium in a large populations of cells by using DCHQ5 probe and traditional fluorimetric technique

Monitoring magnesium efflux cyclic AMP-induced in HL60 cells by using a new hydroxyquinoline fluorescent chemosensor

Cellular homeostasis of magnesium is still unclear. Several studies documented the occurrence of fluxes of magnesium across the plasmamembrane within minutes from the application of metabolic or hormonal stimuli. These fluxes, however, result in limited variation of free Mg2+ intracellular concentration and large changes in total Mg content. It has been reported that a stimulation with cyclic AMP caused a movement of total magnesium within 10 min after treatment in cardiomyocytes. In this study we tested this hypothesis in HL60 leukemic cells, not excitable but highly proliferating cell model. We evaluated Mg flux by DCHQ5, the phenyl-derivative of hydroxyquinoline fluorescent probe family. We observed a drastic decrease of intracellular total magnesium in the first 3 min. We also verified that at least 10% of the total intracellular amount of magnesium moved in the supernatant of stimulated cells

Intracellular magnesium content changes during mitochondria-mediated apoptosis: in depth study of early events on mitochondrial membrane potential

A recent study showed the antitumor activity of a new indole-derivative – MM-67 – inducing mitochondria-mediated apoptosis and a decrease of intracellular magnesium (Mg) concentration in HT29 colon cancer cells. Aim of this work was to assess cellular Mg levels throughout MM-67-induced apoptosis from the early to the final stage of the process and to evaluate the correlation with mitochondrial membrane potential (ΔΨm) variations. All analysis were performed by flow cytometry: ΔΨm was assessed by using mitochondrial potential sensitive dye DiOC6, while free and total intracellular cation concentrations were assessed by using the commercial probe MagFluo4-AM (Kd=4.7 mM), and the new synthesized DCHQ5 (Kd=8.3 mM), respectively. Our results evidenced that the MM67 induced apoptosis is characterized by a direct correlation between ΔΨ and free intracellular Mg content variations

Imbalance of Mg Homeostasis as a Potential Biomarker in Colon Cancer

Background: Increasing evidences support a correlation between magnesium (Mg) homeostasis and colorectal cancer (CRC). Nevertheless, the role of Mg and its transporters as diagnostic markers in CRC is still a matter of debate. In this study we combined X-ray Fluorescence Microscopy and databases information to investigate the possible correlation between Mg imbalance and CRC. Methods: CRC tissue samples and their non-tumoural counterpart from four patients were collected and analysed for total Mg level and distribution by X-Ray Fluorescence Microscopy. We also reviewed the scientific literature and the main tissue expression databases to collect data on Mg transporters expression in CRC. Results: We found a significantly higher content of total Mg in CRC samples when compared to non-tumoural tissues. Mg distribution was also impaired in CRC. Conversely, we evidenced an uncertain correlation between Mg transporters expression and colon malignancies. Discussion: Although further studies are necessary to determine the correlation between different cancer types and stages, this is the first report proposing the measurement of Mg tissue localisation as a marker in CRC. This study represents thus a proof-of-concept that paves the way for the design of a larger prospective investigation of Mg in CRC

new perspective in the assessment of total intracellular magnesium

Magnesium (Mg) is essential for biological processes, but its cellular homeostasis has not been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular Mg distribution. Recently, particular interest has been raised by a new family of fluorescent probes, diaza-18-crown-hydroxyquinoline (DCHQ), that shows remarkably high affinity and specificity for Mg, thus permitting the detection of the total intracellular Mg. The data obtained by fluori- metric and cytofluorimetric assays performed with DCHQ5 are in good agreement with atomic absorption spectroscopy, confirming that DCHQ5 probe allows both qualitative and quantitative determination of total intracellular Mg

Proof of concept: hypoxanthine from stored red blood cells induces neutrophil activation

BACKGROUND: Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated.MATERIALS AND METHODS: Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO2. Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex assay.RESULTS: In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-alpha- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation.DISCUSSION: Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes

Monitoring magnesium efflux cyclic AMP-induced in HL60 cells by using a new hydroxyquinoline fluorescent chemosensor

Cellular homeostasis of magnesium is still unclear. Several studies documented the occurrence of fluxes of magnesium across the plasmamembrane within minutes from the application of metabolic or hormonal stimuli. These fluxes, however, result in limited variation of free Mg2+ intracellular concentration and large changes in total Mg content. It has been reported that a stimulation with cyclic AMP caused a movement of total magnesium within 10 min after treatment in cardiomyocytes. In this study we tested this hypothesis in HL60 leukemic cells, not excitable but highly proliferating cell model. We evaluated Mg flux by DCHQ5, the phenyl-derivative of hydroxyquinoline fluorescent probe family. We observed a drastic decrease of intracellular total magnesium in the first 3 min. We also verified that at least 10% of the total intracellular amount of magnesium moved in the supernatant of stimulated cells