Copper ions are essential for biological function yet are severely detrimental when present in excess. At the molecular level, copper ions catalyze the production of hydroxyl radicals that can irreversibly alter essential bio-molecules. Hence, selective copper chelators that can remove excess copper ions and alleviate oxidative stress will help assuage copper-overload diseases. However, most currently available chelators are non-specific leading to multiple undesirable side-effects. The challenge is to build chelators that can bind to copper ions with high affinity but leave the levels of essential metal ions unaltered. Here we report the design and development of redox-state selective Cu ion chelators that have 10 8 times higher conditional stability constants toward Cu 2+ compared to both Cu + and other biologically relevant metal ions. This unique selectivity allows the specific removal of Cu 2+ ions that would be available only under pathophysiological metal overload and oxidative stress conditions and provides access to effective removal of the aberrant redox-cycling Cu ion pool without affecting the essential non-redox cycling Cu + labile pool. We have shown that the chelators provide distinct protection against copper-induced oxidative stress in vitro and in live cells via selective Cu 2+ ion chelation. Notably, the chelators afford significant reduction in Cu-induced oxidative damage in Atp7a −/− Menkes disease model cells that have endogenously high levels of Cu ions. Finally, in vivo testing of our chelators in a live zebrafish larval model demonstrate their protective properties against copper-induced oxidative stress. Because everything is dissolved in water, the water is present as a huge excess. Generating a little bit more during the reaction is going to make no effective difference to the total concentration of the water in terms of moles of water per dm 3. We have reported that the GFP+ cells exhibit increased dissemination from primary tumor, and when injected via tail vein, generate larger metastasis compared to GFP− cells 37. Therefore, to determine if TM selectively targets GFP+ cells in vivo, we generated cohorts of mice with orthotopic immunocompetent ML1 and immunodeficient LM2 cells stably expressing the SOX2/OCT4-GFP promoter reporter system. Oral TM treatment reduced the percentage of GFP+ cells in the primary tumor, compared to control untreated mice as determined by microscopy imaging of tumor cells (mCherry+) and reporter positive GFP cells (Fig. 4e, Supplementary Fig. 3d). Consistent with microscopy analysis, flow cytometric analysis of primary tumors showed a marked reduction of GFP+ cells (Fig. 4f, Supplementary Fig. 3e). Gating strategy for in vivo LM2 and ML1 models is provided in Supplementary Fig. 4. TM treatment effectively reduced disseminated tumor cells (mCherry+) in early lungs (lungs harvested during primary tumor resection, without overt metastatic outgrowth) as determined by flow cytometry analysis (Fig. 4g). Taken together, these results suggest that copper depletion-mediated reduction in OXPHOS impairs invasion in vitro and targets a specific population of metastatic tumor cells in vivo and that TM targets a subpopulation of metastatic tumor cells that rely on OXPHOS. Erler, J. T. et al. Lysyl oxidase is essential for hypoxia-induced metastasis. Nature 440, 1222–1226 (2006). Venegas, V. & Halberg, M. C. Measurement of mitochondrial DNA copy number. Methods Mol. Biol. 837, 327–335 (2012).

Intra-cellular Cu i

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Some copper supplements are not compatible with oral contraceptives; this needs to be properly discussed with your prescribing GP prior to self-medicating. How to Take Copper Supplements Richmond S, et al. (2013). Copper bracelets and magnetic wrist straps for rheumatoid arthritis – analgesic and anti-inflammatory effects: A randomised double-blind placebo controlled crossover trial. Reading copper deficiency symptoms online, and self-diagnosing based on where you think your copper levels are at, is risky business. It is not recommended to take this route, even if you are only intending to take 10 mg of copper or less. Whether you are looking at the replacement of individual water molecules or an overall reaction producing the final complex ion, a stability constant is simply the equilibrium constant for the reaction you are looking at.

Highly concentrated copper supplements are gaining more and more popularity. These are tinctures that offer safe doses of pure copper in liquid form, administered via a dropper as opposed to mixing into a full glass of water. If you can’t rely on your diet to provide you with the necessary levels of daily copper intake, then a copper supplement is the next best thing. LeBleu, V. S. et al. PGC-1α mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis. Nat. Cell Biol. 16, 1001–1015 (2014). 992–1003. For transwell assays, cells (25,000 cells for bulk LM2 or FACS sorted SOX2/OCT4− GFP− and SOX2/OCT4+GFP+; 40,000 for MDA-MB-468; 60,000 cells for ML1) were plated in triplicates onto the top chamber of 6.5 mm inserts, coated with Matrigel (Corning #356231), in 24-well plates with serum-free DMEM. The cells were treated with TM or vehicle for 48 h before plating the cells for invasion. The bottom chamber contained complete DMEM media with 10% FBS. The cells migrated for 24 h. After migration, the insert was washed twice with fresh PBS and fixed using Kwik-Diff™ Staining kit (ThermoFisher), according to the manufacturer’s protocol. After staining, images were obtained using a computerized Zeiss microscope (Axiovert 200 M) and analyzed using Axiovision 4.6 software (Carl Zeiss Inc.). For assays using AICAR (1.25 mM) and A-769662 (12.5 µM), cells were either pre-treated with a drug or control for 24 h before plating for invasion. For rescue experiments with copper, cells were treated with 0.5 µM CuCl 2 for at least 2 h before plating the cells in the top chamber and continued with CuCl 2 treatment for the remainder of the experiment. For rescue experiments with siAMPK (SCBT) or siControl (SCBT), cells were either treated with TM (0.5 µM) or not (controls) for 24 h prior to transfecting siAMPK (10 nM) or siControl (10 nM) using Lipofectamine3000. Cells (25,000) were plated on Matrigel-coated inserts for invasion assay, 48 h after transfection with siAMPK or siControl in the presence or absence of TM. Inserts were stained after 24 h of plating. LC–MS proteomic analysis

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Warburg, O., Wind, F. & Negelein, E. The metabolism of tumors in the body. J. Gen. Physiol. 8, 519–530 (1927).

Samples were reconstituted in 1 mL of 2% ACN/25 mM ABC. Peptides were fractionated into 48 fractions. An Ultimate 3000 HPLC (Dionex) coupled to an Ultimate 3000 Fraction Collector using a Waters XBridge BEH130 C18 column (3.5 um 4.6 × 250 mm) was operated at 1 mL/min. Buffer A consisted of 100% water, buffer B consisted of 100% acetonitrile, and buffer C consisted of 25 mM ABC. The fractionation gradient operated as follows: 1% B to 5% B in 1 min, 5% B to 35% B in 61 min, 35% B to 60% B in 5 min, 60% B to 70% B in 3 min, 70% B to 1% B in 10 min, with 10% C the entire gradient to maintain pH. The 48 fractions were then concatenated to 12 fractions (i.e., fractions 1, 13, 25, 37 were pooled, followed by fractions 2, 14, 26, 38, etc.) so that every 12th fraction was used to pool. Pooled fractions were vacuum-centrifuged then reconstituted in 1% ACN/0.1% FA for LC–MS/MS. Department of Pulmonary and Critical Care Medicine, Weill Cornell Medicine, New York, NY, 10065, USA Sammons, S., Brady, D., Vahdat, L. & Salama, A. K. Copper suppression as cancer therapy: the rationale for copper chelating agents in. Melanoma Manag. 3, 207–216 (2016). Copper’s role in Alzheimer’s disease is similarly unclear. According to research from 2017, some studies associate Alzheimer’s disease with copper deficiency and recommend increasing copper levels, while others link the disease to overly high levels of copper. This second structure is known as a chelate from a Greek word meaning a crab's claw. You can picture the copper ion as being nipped by the claw of the 1,2-diaminoethane molecule.Lowndes, S. & Harris, A. Copper chelation as an antiangiogenic therapy. Oncol. Res. 14, 529–539 (2004). Mitochondrial content was also determined using MitoTraker deep red (used at 50 nM), according to manufacturer’s protocol, via flow cytometric analysis. Electron microscopy Goodwin, P. J. et al. Effect of metformin vs placebo on and metabolic factors in NCIC CTG MA.32. J. Natl Cancer Inst. 107, djv006 https://doi.org/10.1093/jnci/djv006 (2015).

The enthalpy changes of the two reactions are fairly similar. You might expect this because in each case you are breaking two bonds between copper and oxygen atoms and replacing them by two bonds between copper and nitrogen atoms. Chelates are much more stable than complex ions formed from simple monodentate ligands. The overall stability constants for the two ions are: ionYi, Y. et al. Transcriptional suppression of AMPKalpha1 promotes breast cancer metastasis upon oncogene activation. Proc. Natl Acad. Sci. USA 117, 8013–8021 (2020). Each capsule is a serving of 3 mg, which is slightly higher than most supplements on the market. Daily use can be safely prescribed by a doctor. 3) Good State Liquid Ionic Copper Copper water bottles followed as a portable and convenient way of taking nutrient-dense, copper infused water with you anywhere and everywhere. With enough consumption, one could potentially eliminate the need for any kind of copper supplement, although you should consult with your doctor before attempting to remedy a copper deficiency exclusively through use of a copper water bottle. In addition, drinking from copper is safe with the risk of overdose far less likely relative to using copper supplements. Are Copper Bracelets a Form of Copper Supplement? Even the most gentle of dietary supplements come with their fair share of risks when taken on a regular basis. Ramchandani, D. et al. Nanoparticle delivery of miR-708 mimetic impairs breast cancer metastasis. Mol. Cancer Ther. 18, 579–591 (2019).