Fig. 1 (A) Scheme highlighting mechanisms that can increase intracellular Cu ion levels. Exposure to external agents and mis-folded peptides can cause increased levels of reactive oxygen species (ROS) leading to oxidative stress. Increased oxidative stress leads to protein oxidation releasing Cu ions from proteins and also reduces levels of glutathione (GSH) bound Cu +. Mutations in Cu ion transporters lead to elevated intracellular Cu ion levels. (B) Cu +/Cu 2+ catalyzed Fenton reaction produces reactive hydroxyl radicals, HO˙. Cu 2+ is reduced by cellular reductants like superoxide (O 2˙ −) and hydroascorbate (AscH −) to complete the catalytic cycle. (C) Proposed cell-permeable Cu 2+ chelators that alleviate oxidative stress via selective Cu 2+ chelation.

LM2 and ML1 cells were treated with vehicle (water) or TM (0.5 µM). 2 × 10 6 or 4 × 10 6 cells were digested overnight with 50% HNO 3 + 0.01% digitonin at 65 °C. Digested cells were used to measure copper content with Pinnacle 900z GFAAS machine (Perkin Elmer) against copper standards. Invasion Assays However many examples you take, you always find that a chelate (a complex ion involving multidentate ligands) is more stable than ions with only unidentate ligands. This is known as the chelate effect. While there has been significant progress in understanding the contribution of metabolic reprogramming to tumor growth, the role of metabolism in facilitating metastasis is poorly understood 26. Indeed, the SOX2/OCT4+ cells with high intracellular copper exhibited elevated OXPHOS compared to SOX2/OCT4− cells, a finding consistent with the notion that tumors are metabolically heterogeneous, and that cancer stem cells with high metastatic and tumorigenic potential are more reliant upon OXPHOS than the bulk tumors 58, 59. Indeed, copper enriched SOX2/OCT4+ cells were markedly sensitive to copper depletion, as inactivation of Complex IV significantly reduced OXPHOS and increased glycolysis resulting in impaired invasion and metastatic phenotypes. Together, these findings provide the most direct evidence linking copper-induced metabolic reprograming and metastasis in TNBC. Copper is an essential cofactor for a host of metalloenzymes/proteins that contribute to carcinogenesis 10, 11, 60, 61, so it is conceivable that copper depletion is likely to elicit context-dependent effects based on tumor type and organ-specific microenvironments. Indeed, copper chelation in the RIP-Tag model of pancreatic cancer, expressing the SV40T oncogene under control of the rat insulin promoter, and in human endometrial cancer cells resulted in Complex IV inactivation and HIF-1α stabilization 56, 62 or inhibition of SOD1 63. Similarly, copper depletion was able to overcome resistance to BRAF and MEK1/2 inhibitors 17, and regulated autophagy in lung adenocarcinoma 18. Canto, C. & Auwerx, J. AMP-activated protein kinase and its downstream transcriptional pathways. Cell. Mol. Life Sci. 67, 3407–3423 (2010). To directly establish a link between TM-mediated copper depletion and metastasis in TNBC, we deployed the SOX2/OCT4 promoter reporter lineage tracing system, which we recently reported marks a discrete subpopulation of cancer cells in the primary tumor with increased invasive and metastatic potential 37. Notably, the SOX2/OCT4+ reporter positive metastatic cells exhibited elevated basal intracellular copper levels and exhibited marked sensitivity to TM compared to the reporter negative cells. Global proteomic and metabolomic profiling identified TM-mediated inactivation of copper-dependent mitochondrial Complex IV as the primary metabolic defect in the SOX2/OCT4+ population. TM-mediated destabilization of Complex IV was associated with a significant reduction in the oxidation of cytochrome c that plays a key role in mitochondrial bioenergetics. The mitochondria in TM treated cells showed perturbed (unfolded or improperly folded) cristae morphology, consistent with the observations that cristae shape affects the overall performance of electron transport chain, energy production and metabolic adaptations, making them “bioenergetic membrane of the mitochondrion” 40.Tsang, T. et al. Copper is an essential regulator of the autophagic kinases ULK1/2 to drive lung adenocarcinoma. Nat. Cell Biol. 22, 412–424 (2020). 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). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe96 Analyzer. Pre-treatments with TM were performed in 10 cm plates. For both MDA-MB-231-LM2 and EO771.ML1 20,000 cells were plated per well of the 96-well XFe plate 24 h prior to the assay. LM2 and ML1 cells were then analyzed using Seahorse XFe96 Analyzers according to the manufacturer’s protocol. Seahorse Wave Desktop Software (v2.6.1) was used for measurements. Oligomycin (oligo) and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) concentrations were optimized and adjusted for each cell line (1 µM oligo and 1 µM FCCP was used for LM2, and 2 µM oligo and 0.5 µM FCCP was used for ML1 experiments). Basal respiration, ATP-linked respiration, proton leak, maximal respiration, spare capacity, and non-mitochondrial respiration were then calculated from the OCR Mitostress test profile. Differential glycolytic and mitochondrial ATP production rate was measured in a seahorse assay, using an ATP rate assay kit, according to the manufacturer’s protocol. At the end of each assay, total cellular DNA content was quantified, and acquired values were used for OCR and ECAR values normalization. Briefly, media was aspirated, and cells were stained overnight with 0.5% methylene blue. The next day, methylene blue was replaced with 4% acetic acid in 40% methanol, and after 10 min absorbance was measured at 668 nm. Measurement of glucose consumption and lactate secretion

You will often find these values quoted as log K 1 or whatever. All this does is tidy the numbers up so that you can see the patterns more easily. Form: Copper supplements are available in a variety of forms, including capsules, tablets, and liquids. Some people may prefer one form over another, depending on their personal preferences and needs. Groleau, J. et al. Essential role of copper-zinc superoxide dismutase for ischemia-induced neovascularization via modulation of bone marrow-derived endothelial progenitor cells. Arterioscler. Thromb. Vasc. Biol. 30, 2173–2181 (2010).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 The recommended daily intake for individuals above the age of 19 is around 2 mg per day. A copper supplement is a convenient way of hitting the mark every time, and, since there is such a thing as ‘too much’ copper, a dose controlled supplement is arguably the safest way to manage one’s copper levels.

Menkes syndrome can also cause a copper deficiency. If you have Menkes syndrome, you can absorb copper from the food you eat. But your body doesn’t release it into your bloodstream properly. Erler, J. T. et al. Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche. Cancer Cell 15, 35–44 (2009). Pan, Q. et al. Copper deficiency induced by tetrathiomolybdate suppresses tumor growth and angiogenesis. Cancer Res. 62, 4854–4859 (2002).Copper bracelets have their place in health and wellness, and a potentially important one at that. However, they work via micro-absorption through the skin of the wrist, which means that if any copper is making it into the blood stream it’s only in trace amounts.