Seminars Archive

Study of the role of Magnesium and of its transporters in drug induced apoptosis: in depth study on intracellular magnesium content in single and large cells population

Abstract
Study of the role of Magnesium and of its transporters in drug induced apoptosis: in depth study on intracellular magnesium content in single and large cells population. This research was a part of a broader framework regarding cellular magnesium homeostasis. Magnesium is a critical cofactor of many enzymatic reactions important for physiological functions, such as nucleic acid metabolism, protein synthesis and energy production. For its widely accepted role in cell proliferation, it has been proposed to have a role in carcinogenesis and tumour progression1. Growing evidences point to a possible role for this cation in the apoptotic process but the available literature is still scarce and contradictory. Nevertheless, in both intrinsic and extrinsic pathways of apoptosis there are variations in cytosolic magnesium, and magnesium appears to be required for the release of cytochrome c from isolated mitochondria2. In this regard, the presence of a Mg selective influx channel in mitochondria (MRS2) suffrages these hypothesis. Indeed, mitochondria plays a pivotal role in programmed cell death and they have emerged to be the major Mg store in the cell3. Given the central role of these organelles in the apoptotic cascade, their ion channels are emerging as promising targets for cancer treatment4. Our aim was to highlight the involvement of magnesium and of its mitochondria transporter in the apoptotic process. We successfully generated an appropriate transgenic cellular system overexpressing Mrs2 channel. Then, we investigated whether Mrs2 could influence the sensitivity to apoptotic treatment in human embryonic kidney cells by transiently overexpressing this channel. The cells were exposed to doxorubicin, as apoptotic insult, and cell cycle analysis and caspase activity assay were performed. As expected doxorubicin treatment induced programmed cell death in control cells evidenced by cell cycle perturbation and a massive caspases activation; conversely, the same treatment did not trigger programmed cell death in cells overexpressing the Mrs2 channel. Furthermore, it has been reported that the expression of Mrs2 channel is associated with resistance to drug-induced apoptosis in cancer cells, which is one of the main mechanisms of MDR (multi drug resistance)6. Therefore, it is important to investigate cellular Mg transport and concentration changes in cancer cells to better understand intracellular signalling and the pathogenesis of tumour progression. With the aim to verify whether magnesium intracellular content and compartmentalization could be used as a signature to distinguish MDR tumour cells from their drug-sensitive counterparts, we performed measurements on LoVo human colon cancer cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin. We used a standard-less approach providing a complete characterization of whole single-cells by combining XRFM, atomic force microscopy (AFM) and scanning transmission X-ray microscopy (STXM). This method allows the quantification of the intracellular spatial distribution and total concentration of fundamental life elements and of light metals such as magnesium in whole dehydrated cells. The comparison of LoVo-S and LoVo-R revealed different patterns in the maps of Mg concentration with higher values within the nucleus in LoVo-R and in the perinuclear region in LoVo-S cells. This feature was not evident for the other elements, suggesting that Mg compartmentalization could be a significant trait of the drug-resistant cells. Next, with the aim to compare single cells analysis with large cell population we exploited the features of a new fluorescent dye DCHQ5, able to quantify the total amount of intracellular Mg in a large populations of cells by traditional fluorimetric technique. Results showed that the average intracellular Mg concentration found in a sample of 27 single cells by XRFMAFM analysis is of the same order of magnitude of that found in a population of 10^5 cells assessed by DCHQ5-assisted fluorimetric assay. Collectively, these findings suggest that magnesium is a critical parameter in apoptosis and in the developing of the MDR phenotype of cancer cells. References [1] Vincenzi et al. Early Magnesium Reduction in Advanced Colorectal Cancer Patients Treated with Cetuximab Plus Irinotecan as Predictive Factor of Efficacy and Outcome, Clin Cancer Res;14(13),2008; [2] Wolf et al., Cell (patho)physiology of magnesium , Clin. Sci 2008; [3] Leanza L. et al Mitochondrial ion channels as oncological targets, Oncogene, 1-13, 2014; [4] Kolisek et al. Mrs2p is an essential component of the major electrophoretic Mg influx system in mitochondria EMBO J. 1236-1244, 20033) 4) [5] Kubota T. et al, Mitochondria are intracellular magnesium stores: investigation by simultaneous fluorescent imagings in PC12 cells. Biochim Biophys Acta. 2005, 1744(1):19- [6] Chen et al. Human mitochondrial Mrs2 protein promotes multidrug resistance in gastric cancer cells by regulating p27, cyclin D1 expression and cytochrome C release, Cancer Bio Ther8:7,1-8,2009.