Study of Magnesium Homeostasis and Intracellular Compartmentalization in Human Cells by Fluorescent Chemosensors and Synchrotron X-Ray Fluorescence

In this study, we investigated the analytical capabilities of DCHQ5, a new fluorescent chemosensor, belonging to the family of diaza-crown-hydroxyquinolines, for the quantitative assessment of total intracellular magnesium content, and its biological applications. We performed a comparative study of DCHQ5 and DCHQ1, the latter being the mother probe of the series, which showed preliminary encouraging results comparable to atomic absorption spectroscopy. We demonstrated that DCHQ5 is able to accurately quantify the total amount of Mg in a very “small” cellular population, by using a simple spectrofluorimetric assay. Furthermore, DCHQ5 demonstrated to be a versatile tool for different applications: its higher intracellular retentions allow to perform cytofluorimetric assays and two-photon confocal microscopy on whole and viable cells; its photochemical characteristic make it excitable in both UV and visible spectra, and the presence of different lifetimes allow to perform fluorescence life time imaging of intracellular Mg. DCHQ5 was also exploited for studying the involvement of magnesium in the commitment of human adipose-derived mesenchymal stem cells (hASCs) with a mixture of hyaluronic, butyric and retinoic acids (HBR). We found that in normal magnesium availability, hASCs precommitment is associated by an increase of total magnesium content during time and by a block in the G2/M phase of the cell cycle. Moreover, our results demonstrated that magnesium deprivation triggers multilineage enrichments of HBR-induced preconditioning of hASCs. The second part of this research was aimed at comparing single cells elemental analysis performed with synchrotron-based fluorescence and cell population analysis carry out by DCHQ5. We exploited innovative techniques of x-ray fluorescence microscopy by using a multimodal approach in order to achieve within the cells the spatial distribution of the concentration of magnesium and fundamental light elements for life. The combination of classical and innovative analytical techniques can shed new light in the comprehension of magnesium homeostasis

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

Magnesium homeostasis goes awry in chemoresistance -TRPM6, TRPM7 and MagT1 in colon carcinoma LoVo cells

Chemoresistance is one of the most significant factors impeding the progress of cancer therapy (1). It is known that neoplastic cells accumulate magnesium and frequently upregulate one of its transporters, i.e.TRPM7 (2). We have investigated magnesium homeostasis in a model of chemoresistance i.e. colon carcinoma LoVo cells sensitive (LoVo-S) or resistant to doxorubicin (LoVo-R). We observed that LoVo-R have higher amount of total intracellular magnesium than LoVo-S. We studied the expression of some magnesium transporter (TRPM6, TRPM7 and MagT1) by Real Time PCR and Western Blot and found that TRPM6 and 7 are overexpressed in LoVo-S, while MagT1 is upregulated in LoVo-R. In LoVo-S, silencing TRPM7 retards cell growth and shifts the phenotype to one more similar to resistant cells. On the other hand, calpeptin, a calpain inhibitor, upregulates TRPM7, stimulated proliferation and enhances the sensitivity to doxorubicin of LoVo-R. Silencing MagT1 in LoVo-R markedly inhibited cell growth without affecting the response to doxorubicin. We conclude that alterations of magnesium homeostasis play a role in drug resistance

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

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

Implementation of an iterative approach to optimize synchrotron X-ray fluorescence quantification of light elements in single cell

A new approach for elemental quantification and error estimation in single cells, named Single Cell Elemental Quantification Iterative Approach (SCEQIA), has been performed merging two analytical approaches conceptually different. The first one is a multimodal approach designed to identify and quantify the concentration of light elements in biological samples and to calculate errors following error propagation law. The second one is an iterative algorithm conceived to quantify heavy elements in terms of mass fraction in mineral samples and to evaluate errors using an iterative Monte Carlo-based procedure. The fluorescence data and scanning transmission data, provided by synchrotron X-ray microscopic techniques, together with the volumetric data from Atomic Force Microscopy (AFM) of two colon carcinoma cells have been analyzed following SCEQIA approach. The mass fraction and the molar concentration of the elements C, N, O, Na, and Mg have been mapped. The obtained results have been compared with those previously provided with the original multimodal approach above mentioned. The outputs of the two approaches are comparable in terms of elemental quantification (mass fraction and concentration) for both the analyzed samples. On the contrary, the two approaches differ in error estimation: the newly developed SCEQIA approach results less influenced by the uncertainties of AFM measurements

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

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