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

ADIPONECTIN AND CARDIOVASCULAR RISK PREDICTION: STRATIFICATION OF CHEST PAIN PATIENTS BY A CLUSTER ANALYSIS

Cardiovascular disease (CVD) remains the major cause of death and there is the need to a better stratification of CVD patients. By an unbiased statistical approach we sought to identify clusters of patients to better stratify their risk. 202 patients with chest pain (63% males, age 62?12 yr) undergone to CT coronary angiography (CCTA) were prospectively included and classified using K-means cluster analysis of clinical, imaging and bio-humoral data. The most relevant classification resulted in three phenotypes distinguished according to Framingham score and HMW adiponectin plasma levels. Presence and severity of disease as assessed by CCTA were verified trough these phenotypes. By K-means cluster analysis, we identified CVD phenotypes allowing to stratify patients requiring different diagnostic and therapeutic approach

Myocardial interleukin-6 in the setting of left ventricular mechanical assistance: relation with outcome and C-reactive protein

Background: In left ventricular assist device (LVAD) recipients, plasma levels of interleukin (IL)-6 are associated with Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profiles, reflecting postoperative risk. However, it is not clear how the cardiac. Conclusions: Cardiac IL-6 levels do not contribute to improve risk profile of LVAD recipients in relation to clinical inpatient post-implantation. Instead, plasma IL-6 and serum CRP concentrations are more effective in predicting the severity of the clinical course in the early phase of LVAD therapy. level of IL-6, detectable on the tissue samples at the time of implantation, can contribute to predict the post-operative outcome

Multicentre multi-device hybrid imaging study of coronary artery disease: results from the EValuation of INtegrated Cardiac Imaging for the Detection and Characterization of Ischaemic Heart Disease (EVINCI) hybrid imaging population

AIMS: Hybrid imaging provides a non-invasive assessment of coronary anatomy and myocardial perfusion. We sought to evaluate the added clinical value of hybrid imaging in a multi-centre multi-vendor setting. METHODS AND RESULTS: Fourteen centres enrolled 252 patients with stable angina and intermediate (20-90%) pre-test likelihood of coronary artery disease (CAD) who underwent myocardial perfusion scintigraphy (MPS), CT coronary angiography (CTCA), and quantitative coronary angiography (QCA) with fractional flow reserve (FFR). Hybrid MPS/CTCA images were obtained by 3D image fusion. Blinded core-lab analyses were performed for CTCA, MPS, QCA and hybrid datasets. Hemodynamically significant CAD was ruled-in non-invasively in the presence of a matched finding (myocardial perfusion defect co-localized with stenosed coronary artery) and ruled-out with normal findings (both CTCA and MPS normal). Overall prevalence of significant CAD on QCA (>70% stenosis or 30-70% with FFR 640.80) was 37%. Of 1004 pathological myocardial segments on MPS, 246 (25%) were reclassified from their standard coronary distribution to another territory by hybrid imaging. In this respect, in 45/252 (18%) patients, hybrid imaging reassigned an entire perfusion defect to another coronary territory, changing the final diagnosis in 42% of the cases. Hybrid imaging allowed non-invasive CAD rule-out in 41%, and rule-in in 24% of patients, with a negative and positive predictive value of 88% and 87%, respectively. CONCLUSION: In patients at intermediate risk of CAD, hybrid imaging allows non-invasive co-localization of myocardial perfusion defects and subtending coronary arteries, impacting clinical decision-making in almost one every five subjects

New fluorescent probes for the assessment and imaging of total intracellular magnesium

Sebbene il magnesio sia essenziale per la maggior parte dei processi biologici, si conosce ancora poco sulla sua distribuzione e compartimentalizzazione intracellulare, soprattutto a causa dell’inadeguatezza delle tecniche attualmente disponibili. Per questo motivo, particolare interesse ha recentemente suscitato una famiglia di molecole fluorescenti, diaza-18-crown-6 8-idrossichinoline (DCHQ1 e suoi derivati), che mostrano un’alta specificità e affinità per il magnesio (superiore a quella delle sonde commerciali), che consente di mappare il magnesio totale intracellulare. L’approccio sintetico alle molecole DCHQ è stato ottimizzato mediante riscaldamento alle microonde: con questa nuova metodica è stato possibile sintetizzare una famiglia di derivati con caratteristiche di fluorescenza, uptake, ritenzione e localizzazione intracellulare potenziate rispetto alla capostipite DCHQ1. Il derivato acetometossi estere (DCHQ3), idrolizzato dalle esterasi cellulari, ha mostrato un miglior uptake e ritenzione intracellulare; le lunghe catene laterali alchiliche della sonda DCHQ4, invece, hanno conferito a questo derivato maggiore lipofilicità e, di conseguenza, maggiore affinità per le membrane; con l’inserimento di gruppi laterali aromatici, infine, si sono ottenute due sonde (DCHQ5 e DCHQ6) molto fluorescenti e altamente ritenute all’interno delle cellule anche dopo i lavaggi. Il derivato fenilico DCHQ5 si è dimostrato, inoltre, utilizzabile anche per saggi fluorimetrici quantitativi del magnesio totale in campioni cellulari molto piccoli; in più, grazie all’alta ritenzione cellulare, è stato usato per monitorare e quantificare l’efflusso di magnesio attraverso la membrana plasmatica in risposta a stimolazione con cAMP. I risultati presentati in questa tesi mostrano che i DCHQ-derivati potranno rappresentare in futuro uno strumento versatile per lo studio della distribuzione e dell’omeostasi del magnesio cellulare. In particolare la sonda DCHQ5 ha mostrato l’ulteriore peculiarità di essere eccitabile sia nell’UV che nel visibile, e potrebbe essere quindi utilizzata con successo in un’ampia varietà di misure di fluorescenza, fornendo un contributo importante per la comprensione del ruolo di questo importante elemento.Although magnesium is essential for a number of biological processes crucial for cell life, its distribution and intracellular compartimentalization have not been thoroughly elucidated yet, mainly because of the inadequacy of the available techniques to map intracellular magnesium distribution. For this reason, particular interest has been recently raised by a family of fluorescent molecules, diaza-18-crown-6 8-hydroxyquinolines (DCHQ1 and its derivatives), that show a remarkable affinity and specificity for magnesium, higher than all the commercially available probes, thus permitting the detection of the total intracellular magnesium. The synthetic approach to DCHQ has been optimized using microwave heating: with this new procedure a variety of substituted DCHQ derivatives with improved fluorescence, uptake and selective localization with respect to the original reference material (DCHQ1) could be easily generated. Enhanced uptake has been achieved with an acetoxymethyl ester derivative (DCHQ3) that is recognized by the intracellular esterases. Moreover, the insertion of two long hydrophobic side chains (DCHQ4) allowed a better staining of the membranes due to its high affinity to the lipophilic environment. Finally, the introduction of aromatic side groups (DCHQ5 and DCHQ6) enhanced the fluorescence response in cells and also improved intracellular uptake and retention of the probes even after washing. The phenyl-derivative DCHQ5, in particular, can also be used for quantitative fluorimetric assays of total magnesium in very small samples; moreover, thanks to the high cellular retention, DCHQ5 has been used in monitoring and quantifying the total Mg efflux across the membrane in response to cAMP stimulation. Finally, its unique feature of being excitable both in the UV and visible range of wavelength, together with the high fluorescence in response to cation binding, led us to hypothesize that this molecule could act as an effective tool for shedding light on total intracellular magnesium distribution and homeostasis

Folate Receptor Expression and Folate Receptor Targeted Chemotherapy in Cisplatin-sensitive and – resistant Human cancer cell lines Marverti G.a, Pirondi S.a, MarracciniC.a, Frassineti C.a, Helleman J.b, Berns E.M.J.J. b, and Costi, M.P. cThe 4th International Symposium on Folate Receptors and Transporters Cozumel, Mexico October 7 - 11, 2012

Folate Receptor Expression and Folate Receptor Targeted Chemotherapy in Cisplatin-sensitive and – resistant Human cancer cell lines Marverti G.a, Pirondi S.a, MarracciniC.a, Frassineti C.a, Helleman J.b, Berns E.M.J.J. b, and Costi, M.P. c aDept. Biomedical Sciences and cDept. Pharmaceutical Sciences, Via Campi 287-183, University of Modena and Reggio Emilia, 41125, Modena, Italy; Dept. of Medical Oncology, bErasmus University Medical Center - Daniel den Hoed Cancer Center, PO Box 2040, 3000 CA, Rotterdam,The Netherlands. Targeted drug delivery systems promise to expand the therapeutic windows of drugs by increasing delivery to the target tissue as well as the target–non-target tissue ratio. Interest in exploiting the folate receptor (FR) for drug targeting applications has rapidly increased because it is a tumor-associated antigen that is overex- pressed in greater than 90% of human ovarian carcinomas and associated with increased biological aggressive- ness of this tumor [1]. The most significant advantage to emerge from studies of FR-mediated delivery has been the surprisingly low level of toxicity to normal tissues which constitute one of the most significant merits of the FR-targeting strategy. Therefore, FR presents an attractive target for tumor-selective drug delivery. The final goal of this research is to analyze the effects of oligopeptides, designed to inhibit thymidylate syn- thase (TS) activity by interfering with its dimerization, without causing TS over-expression and the develop- ment of cellular drug resistance against the traditional TS-targeted compounds. The over-expression of TS and the others folate cycle enzymes, is one of the major mechanisms of resistance to cisplatin (cDDP), encountered in most of resistant human ovarian cancer cell lines [2], accounting for the more efficient DNA repair and syn- thesis. To this aim, we have chosen two cisplatin-sensitive human ovarian cancer cell lines, 2008 and A2780, and their –resistant counterparts, C13* and A2780/CP cell lines, respectively, in order to display and study possi- ble different responses modulated by cDDP-resistance. At first, the cell lines have been tested for their total levels of FR and for functionally active receptors (FR or also other receptors?). The quantification of functional FR by the microfiltration method [3] and by an alterna- tive method, which deduces FR amount from the folic acid (FA) binding to FR [4] indicate that 2008 and C13* cells present a 3-4 fold higher expression of FR than A2780 and A2780/CP cells. However, time-dependent and concentration-dependent studies revealed that despite their higher expression of FR and the higher [3H]folic acid binding capacity, 2008 and C13* cell lines appeared to saturate earlier than A2780 and A2780/CP cell lines since they accumulated less [3H]folic acid at concentrations higher than 150 nM. Substrate specificity studies of the saturable uptake process, revealed that the 30 nM [3H]Folic acid uptake in A2780 cells was more affected than in the resistant line A2780/CP to the presence of 20μM of unlabeled folic acid (FA) and methotrexate (MTX), respectively (I do not understand this sentence, A2780 is more sensitive to unlabeled folic acid and mtx after labeled FA treatment? How do you measure this sensitivity? Do you mean affected/decreased uptake?). On the contrary, 30 nM [3H]Folic acid uptake was unaffected by unlabeled FA and MTX in both 2008 and C13* cell lines.  To evaluate the presence of ATP-dependent process affecting FA uptake, experiments were also performed at 4°C and compared with those at 37°C. The accumulation of [3H]folic acid was greatly reduced at 4°C in comparison to 37°C both in 2008 and C13* cells, whereas only about 6- and 4-fold reductions were detected in A2780 and A2780/CP cells, respectively . These results are, at least partly, in accordance with the competitive uptake studies, since they suggest that in the resistant A2780/CP cells the accumulation is about 50% energy-dependent, carrier mediated and partly or slightly inhibited by competitive compounds. On the contrary, the sensitive A2780 cells are more sensitive to both temperature and folate analogs. However, the second cell couple (2008 and C13* cells), confirming to accumulate less [3H]folic acid, show an uptake more affected by low temperature and thus mostly energy- dependent, but not significantly affected by competitive compounds. To confirm the observed differences in FR expression and [3H]folic acid uptake, immunoblot analysis of FRα are in progress in a larger panel of human ovarian cancer cells including OAW28, COV504, IGROV1 and TOV112D cell lines. 1. Leamon CP and Reddy JA. Advanced Drug Delivery Review 2004, 56, 1127-41. 2. Scanlon KJ et al. Cancer Commun 1990, 2(10), 339-43. 3. Parker N, et al. Anal Biochem 2005;338: 284–93. 4. Mauritz, R. et al. Cancer Chemother Pharmacol 2008, 62:937–48. Acknowledgement This work has been supported by AIRC-DROC 10474 project to MPC

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