Magnesium Modulates Doxorubicin Activity through Drug Lysosomal

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Magnesium modulates doxorubicin activity through drug lysosomal sequestration and trafficking Valentina Trapani, Francesca Luongo, Daniela Arduini, and Federica I Wolf Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00478 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on March 3, 2016

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Chemical Research in Toxicology

Magnesium modulates doxorubicin activity through drug lysosomal sequestration and trafficking

Valentina Trapani,* Francesca Luongo, Daniela Arduini and Federica I. Wolf Istituto di Patologia Generale, Facoltà di Medicina e Chirurgia “A. Gemelli”, Università Cattolica del Sacro Cuore, Rome

*Tel: 0039 06 30154914. E-mail: [email protected] Keywords: Breast cancer, chemotherapy, cytoskeleton, cytotoxicity, TRPM7.

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Abstract

Magnesium is directly involved in the control of cell growth and survival, but its role in cancer biology and therapy is multifaceted; in particular, it is highly controversial whether magnesium levels can affect therapy outcome. Here we investigated whether magnesium availability can modulate cellular responses to the widely used chemotherapeutic doxorubicin. We used an in vitro model consisting of mammary epithelial HC11 cells, and found that high magnesium availability correlated with diminished sensitivity both in cells chronically adapted to high magnesium concentrations, and in acutely magnesium-supplemented cells. Such decrease in sensitivity resulted from reduced intracellular doxorubicin accumulation, in the face of a similar drug uptake rate. We show that high magnesium conditions caused a decrease in intracellular drug retention by altering drug lysosomal sequestration and trafficking. In our model magnesium supplementation correspondingly modulated expression of the TRPM7 channel, which is known to control cytoskeletal organization and dynamics and may be involved in the proposed mechanism. Our findings suggest that magnesium supplementation in hypomagnesemic cancer patients may hinder response to therapy.



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Introduction Magnesium (Mg) has a crucial function in countless biological processes, primarily at three levels: 1) as cofactor for ATP in multiple enzymatic reactions, 2) as a stabilizer for membranes, nucleic acids and protein complexes, 3) as a signaling molecule.1 As such, it is no surprise that it plays a direct role in the control of cell survival and growth. However, in the context of cancer biology and therapy, the role of magnesium is highly debated.2 Experimental reports have demonstrated that magnesium deficiency can promote many steps of carcinogenesis and tumor development,3 while clinical studies have highlighted that chemotherapeutic drugs often induce hypomagnesemia, which may be important for response to therapy.2 To our knowledge, only one study has so far investigated directly whether magnesium status can affect cell sensitivity to anticancer drugs.4 Recent research findings suggested that alterations of magnesium homeostasis may be involved in the development of resistance to the widely used chemotherapeutic doxorubicin (DXR), via modulation of the transient receptor potential melastatin TRPM7 channel.5 TRPM7 is a critical determinant of intracellular magnesium homeostasis, and peculiarly contains both a transmembrane cation channel domain and a cytosolic functional serine/threonine protein kinase domain.6 In this report we sought to investigate whether magnesium availability can modulate cellular responses to DXR. We used an in vitro cellular model that has been extensively characterized in our laboratory. Mammary epithelial HC11 cells were adapted to different Mg concentrations and two lines were derived that stably acquired the ability to grow in culture media containing ≤ 0.05 mM Mg (Low-Mg cells) or ≥ 30 mM Mg (High-Mg cells).7 We have already characterized HC11 cells and their clones in terms of proliferative behavior, and magnesium content and homeostasis.7-9 Here, we set about to assess whether these cell lines display different sensitivity to DXR, and sought to identify the underlying mechanism(s).

Experimental Procedures


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Materials. All cell culture reagents were from Euroclone (Pero, Italy). All chemicals were from Sigma-Aldrich (Milan, Italy). Fluorescent probes and reagents for microscopy were from Invitrogen (Life Technologies Italia, Monza, Italy). Microscopy dishes were from Ibidi (Martinsried, Germany). Doxorubicin was obtained through the courtesy of Nerviano Medical Sciences (Milan, Italy). Cell culture. Mammary epithelial HC11 cells and breast carcinoma MCF7 cells were grown in MEM medium supplemented with 10% FBS, penicillin, streptomycin, and L-glutamine. Control MEM medium contained 0.8 mM MgSO4. High-Mg and Low-Mg HC11 cells were obtained from HC11 cells by stepwise chronic adaptation to 30 and 0.05 mM MgSO4, respectively.7 High-Mg cells were grown in the same medium as HC11 cells, but supplemented with 30 mM MgSO4. Low-Mg cells were cultured in Mg-free MEM medium, supplemented as for parental cells, but with Mg2+-free serum obtained by dialysis, as described previously.7 DXR was added in the medium normally used to grow each cell line. Where indicated, control HC11 or MCF7 cells were grown in culture medium supplemented with 10 mM MgSO4 for 24 h before drug addition, and kept in the same medium during drug treatment. MTT assay. Cells were seeded in 24-well plates at a density of 40,000 cells/well and allowed to adhere for 24 h before drug treatment. DXR (concentration range: 0.1 to 50 µM) was added to culture medium in triplicates. After a 24-h exposure, culture medium was replaced with serum-free medium containing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 1 mg/ml) and cells were incubated for 90 min at 37°C. Finally formazan crystals were dissolved in acidified isopropanol (0.04 N HCl in isopropanol) and absorbance was read at λ= 565 nm. Dose-response data were analyzed by Prism 4 software (non-linear regression, sigmoidal curve, variable slope). Confocal microscopy. For experiments with fixed cells (Figures 1B, 4B, S1B and S3B), cells were plated on sterilized coverslips and exposed to 10 µM DXR for 2 h. After treatment, cells were fixed in 4% paraformaldehyde and mounted on slides with Prolong Antifade reagent. For live experiments (Figures 2, 3, 4C, 4D, S2 and S4), cells were plated on 35-mm microscopy dishes and maintained in culture medium for at least 24h. Live experiments were carried out in a buffer (pH 7.4) containing



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120 mM NaCl, 20 mM HEPES, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM CaCl2, 10 mM glucose, and MgSO4 at the same concentration normally present in the culture medium of each cell line (0.05, 0.8 and 30 mM for Low-Mg, control and High-Mg cells, respectively; 10 mM for acutely Mgsupplemented cells). Intracellular DXR uptake was followed for 5 min after addition of 50 µM DXR to optimize visualization of the drug. Intracellular distribution of DXR, lysosomes and mitochondria was studied in cells treated with 1 µM DXR for 24 h and loaded simultaneously with LysoTracker Green DND-26 (100 nM) and MitoTracker Deep Red 633 (100 nM). Imaging of all samples was performed at a confocal laser scanning system (TCS-SP2, Leica Microsystems GmbH) using the following settings: λex= 488 nm, λem= 505–530 nm for LysoTracker; λex=488 nm, λem=585–615 nm for DXR; and λex= 633 nm, λem= 663–695 nm for MitoTracker. Images for quantification of DXR fluorescence were acquired with identical parameters, i.e. laser output, photomultiplier gain and offset. Mean fluorescence intensity in the regions of interests was measured by Leica Confocal Software. At least 50 cells from five different fields were analyzed for quantification of DXR accumulation. Drug uptake was evaluated as the mean increase in intracellular fluorescence of 10 cells in the same field, and expressed as (F-F0)/F0, where F0 indicates the fluorescence at the time of drug addition. Western blot. Cell were lysed in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.05% Na-deoxycholate, 0.1% SDS) supplemented with protease inhibitors (10 µg/ml leupeptin, 20 µg/ml aprotinin,1 mM PMSF, 1 mM NaVO4, 100 mM NaF). Protein concentration was determined using the Bradford protein assay (Bio-Rad). Cell extracts (50 µg/lane) were resolved by 8% SDSPAGE, transferred to PVDF membranes, and probed with rabbit monoclonal anti-TRPM7 (Abcam), and rabbit polyclonal anti-actin (Sigma Aldrich) antibodies. HRP-conjugated secondary antibodies (GE Healthcare) were detected by use of the ECL Prime Western Blotting Detection Reagent (GE Healthcare) and the ChemiDoc XRS system (Bio-rad). Densitometric analysis was performed using the ImageJ software (NIH, http://imagej.nih.gov/ij/). Statistics. All experiments were repeated independently at least three times. Data are presented as mean ± SE. Statistical significance was assessed using non parametric Mann-Whitney test, or


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Kruskal-Wallis test followed by Dunn’s multiple comparison post-hoc test, for comparing two or more groups, respectively. Statistical significance was set as following: *P

Magnesium Modulates Doxorubicin Activity through Drug Lysosomal

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