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

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

Copper(I) and Copper(II) Binding to R1 and R3 Fragments of Tau Protein

Tau (τ) is a 441-mer peptide present in significant amounts in neurons, where it contributes to the stabilization of microtubules. Insoluble amyloid aggregates of tau are associated with over 20 neurological disorders known as tauopathies, among which is Parkinson’s [1]. In neurons, tau binds tubulin through its microtubule binding domain which comprises four repeats (R1-R4) characterized by the presence of histidine residues. These regions are potential binding sites for metal ions [2]. The elucidation of the binding capacities toward metal ions, especially those redox active such as copper(II), may shed light on the biomolecular processes that underlie the progression of tauopathies [3]. In this contribution we examine the thermodynamic stability of copper(I) and copper(II) adducts with two peptide fragments which are encompassed in the R1 and R3 repeats of tau, namely Ac- 257VKSKIGSTENLKHQGGG273-NH2 (R1τ, HL1 in its neutral form) and Ac-323GSLGNIHHKPGGG335- NH2 (R3τ, L3 in its neutral form). Copper(II) binding to R1τ (HL1) at pH 7.4 is dominated by the formation of [Cu(HL1)]2+, where (L1)- is tridentate. The copper(II) equatorial coordination positions are occupied by the imidazole ring of His269, two amido nitrogens, and a water molecule. As for the R3τ (L3) peptide, at pH 7.4 the two most abundant species are [CuL3]2+ and [Cu(L3H-1)]+ (in a ratio of ca. 1:4, Figure 1, left). While copper(II) coordination mode in [Cu(L3H-1)]+ is similar to that in [Cu(HL1)]2+, that of [CuL3]2+ is different and possibly most interesting. Spectroscopic data suggest that in [CuL3]2+ two imidazole donors and one amido nitrogen are equatorially coordinated to copper(II), plus a water molecule (Figure 1, right). The presence of this tandem HisHis fragment makes this peptide interesting in view of the stabilization of copper(I). Indeed, spectroscopic competition titration using a metallochromic indicator clearly showed that copper(I) binds significantly to R3τ at neutral pH but not to R1τ. The catalytic activity in reactions of oxidation of catecholes and the NMR features of these complexes will be discussed in terms of the speciation of the thermodynamic stability of these complexes with copper in both oxidation states. The authors acknowledge MIUR for financial support through the project "Metal ions, dopamine, and oxidative stress in Parkinson's disease” (PRIN 2015T778JW). References [1] M. Goedert, D. S. Eisenberg, R. A. Crowther, Annu. Rev. Neurosci. 40 (2017) 189–210. J.A. White "Biological inorganic chemistry" (1973) Oxford University Press, Oxford. [2] M. G. Savelieff, S. Lee, Y. Liu, M. H. Lim, ACS Chem. Biol. 8 (2013) 856–865. [3] A. Soragni, B. Zambelli, M. D. Mukrasch, J. Biernat, S. Jeganathan, C. Griesinger, S. Ciurli, E. Mandelkow, M. Zweckstetter, Biochemistry 47 (2008) 10841–10851

Copper binding to R1 and R3 fragments of Tau protein

Tau is a 441-mer peptide present in significant amounts in neurons, where it contributes to the stabilization of microtubules. Insoluble amyloid aggregates of tau are associated with over 20 neurological disorders known as tauopathies, among which is Parkinson.[1] In neurons, tau binds tubulin through its microtubule binding domain which comprises four repeats (R1- R4) characterized by the presence of histidine residues. These regions are potential binding sites for metal ions.[2] The elucidation of the binding capacities toward metal ions, especially those redox active such as copper(II), may shed light on the biomolecular processes that underlie the progression of tauopathies.[3] In this contribution we examine the stability and the structural models of Cu(II) adducts with two peptide fragments which are encompassed in the R1 and R3 repeats of tau (Fig. 1). Copper(II) binding to R1 (HL) starts at pH 4. The relevant species at pH 7.4 is [CuLH]2+, where R1 is tridentate: the copper(II) equatorial coordination positions are occupied by the imidazole ring, two amidic nitrogen atoms, and a water molecule. As for the R3 peptide, at pH 7.4 the two most abundant species are [CuL]2+ and [CuLH-1]+ (in a ratio of ca. 1:2). In the case of [CuL]2+, the two imidazole groups of R3 and one deprotonated amidic nitrogen atom are bound to the equatorial plane. In [CuLH-1]+, a further amidic nitrogen bounds the metal ion in the equatorial plane, most likely pushing one imidazole group to the axial position. The redox and NMR behavior of these complexes will be discussed in terms of their speciation. Having the Cu(II)-tau peptides binding constants in our hands, we decided to investigate the copper(I) adducts with the R1 and R3 tau fragments via spectrophotometric competition titrations with the metallochromic ligand ferrozine (Fz).[4] The titration of a Cu(I) solution with ferrozine causes the formation of the chromophoric complex [CuI(Fz)2]3- which presents two characteristic absorption bands at 470 nm and 600 nm. By back titrating this solution with the R1 and R3 fragments, we observed a decrease in the absorbance values (Fig. 2). The addition of R3 to [CuI(Fz)2]3- causes a significative change in the absorbance, which decreases of almost 0.40 units. As for the R1 peptide, a decrease of only 0.20 units is observed and can be fully accounted by dilution effects. Data treatment using HypSpect program yields a log β value of 5.9(1) for the Cu(I)-R3 complex. Data treatment for the back titration of [CuI(Fz)2]3- with R1 confirms the absence of significant interactions of Cu(I) with R1. NMR data suggest that the binding of Cu(I) to R3 occurs at the tandem HH site, as it occurs for Cu(II). The authors acknowledge MIUR for financial support through the project "Metal ions, dopamine, and oxidative stress in Parkinson's disease” (PRIN 2015T778JW). References: [1] M. Goedert, D. S. Eisenberg, R. A. Crowther, Annu. Rev. Neurosci. 2017, 40, 189-210. [2] M. G. Savelieff, S. Lee, Y. Liu, M. H. Lim, ACS Chem. Biol., 2013, 8, 856-865. [3] A. Soragni, B. Zambelli, M. D. Mukrasch, J. Biernat, S. Jeganathan, C. Griesinger, S. Ciurli, E. Mandelkow, M. Zweckstetter, Biochemistry, 2008, 47, 10841-51. [4] B. Alies, B. Badei, P. Faller, C. Hureau, Chem. Eur. J., 2012, 18, 1161-1167

Cryopreservation affects platelet macromolecular composition over time after thawing and differently impacts on cancer cells behavior in vitro

Cryopreservation affects platelets’ function, questioning their use for cancer patients. We aimed to investigate the biochemical events that occur over time after thawing to optimize transfusion timing and evaluate the effect of platelet supernatants on tumor cell behavior in vitro. We compared fresh (Fresh-PLT) with Cryopreserved platelets (Cryo-PLT) at 1 h, 3 h and 6 h after thawing. MCF-7 and HL-60 cells were cultured with Fresh- or 1 h Cryo-PLT supernatants to investigate cell proliferation, migration, and PLT-cell adhesion. We noticed a significant impairment of hemostatic activity accompanied by a post-thaw decrease of CD42b+ , which identifies the CD62P−-population. FTIR spectroscopy revealed a decrease in the total protein content together with changes in their conformational structure, which identified two sub-groups: 1) Fresh and 1 h Cryo-PLT; 2) 3 h and 6 h cryo-PLT. Extracellular vesicle shedding and phosphatidylserine externalization (PS) increased after thawing. Cryo-PLT supernatants inhibited cell proliferation, impaired MCF-7 cell migration, and reduced ability to adhere to tumor cells. Within the first 3 hours after thawing, irreversible alterations of biomolecular structure occur in Cryo-PLT. Nevertheless, Cryo-PLT should be considered safe for the transfusion of cancer patients because of their insufficient capability to promote cancer cell proliferation, adhesion, or migration. What is the context?Transfusion of Fresh platelets (Fresh-PLT) with prophylaxis purposes is common in onco-hematological patients.Cryopreservation is an alternative storage method that allows to extend platelet component shelf life and build supplies usable in case of emergency.It is well established that cryopreservation affects platelet function questioning their use in onco-hematological patients.It is still unknown how platelet impairment, induced by cryopreservation, occurs over time after thawing, nor how the by-products of PLT deterioration may impact on cancer cell behavior. Transfusion of Fresh platelets (Fresh-PLT) with prophylaxis purposes is common in onco-hematological patients. Cryopreservation is an alternative storage method that allows to extend platelet component shelf life and build supplies usable in case of emergency. It is well established that cryopreservation affects platelet function questioning their use in onco-hematological patients. It is still unknown how platelet impairment, induced by cryopreservation, occurs over time after thawing, nor how the by-products of PLT deterioration may impact on cancer cell behavior. What is new?In this study, we deeply characterized the functional and morphological changes induced by cryopreservation on platelets by comparing Fresh-PLT with Cryo-PLT at 1 h, 3 h and 6 h after thawing. Afterwards, we evaluated the effect of PLT supernatants on cancer cell behavior in vitro.The data presented show that within 3 hours after thawing Cryo-PLT undergo to irreversible macromolecular changes accompanied by increase of peroxidation processes and protein misfolding.After thawing the clot formation is reduced but still supported at all-time points measured, combined with unchanged phosphatidylserine expression and extracellular vesicles release over time.Cryo-PLT supernatants do not sustain proliferation and migration of cancer cells. In this study, we deeply characterized the functional and morphological changes induced by cryopreservation on platelets by comparing Fresh-PLT with Cryo-PLT at 1 h, 3 h and 6 h after thawing. Afterwards, we evaluated the effect of PLT supernatants on cancer cell behavior in vitro. The data presented show that within 3 hours after thawing Cryo-PLT undergo to irreversible macromolecular changes accompanied by increase of peroxidation processes and protein misfolding. After thawing the clot formation is reduced but still supported at all-time points measured, combined with unchanged phosphatidylserine expression and extracellular vesicles release over time. Cryo-PLT supernatants do not sustain proliferation and migration of cancer cells. WHAT is the impact?Cryo-PLT may be considered a precious back-up product to be used during periods of Fresh-PLT shortage to prevent bleeding in non-hemorrhagic patients.It is desirable to make it logistically feasible to transfuse cryopreserved platelets within 1 hour of thawing to maintain the platelets in their best performing condition. Cryo-PLT may be considered a precious back-up product to be used during periods of Fresh-PLT shortage to prevent bleeding in non-hemorrhagic patients. It is desirable to make it logistically feasible to transfuse cryopreserved platelets within 1 hour of thawing to maintain the platelets in their best performing condition.</p