Copper Transporter 1 Expression Research Articles

Inhibition of CTR1 expression improves hypoxia/reoxygenation-induced myoblast injury by blocking cuproptosis

Skeletal muscle ischemia-reperfusion injury (IRI) is a common severe disease with a complex pathological process. This study found that copper chloride (CuCl2) inhibited cell viability in a concentration dependent manner, increased intracellular copper levels and downregulated copper transporter 1 (CTR1) expression. CTR1 upregulation promoted copper uptake by myoblasts and then enhanced cuproptosis, leading to a significant increase in the levels of dihydrolipoamide S-acetyltransferase (DLAT) oligomers, while a significant decrease in the levels of lipoylated (Lip)-dihydrolipoamide S-succinyltransferase (DLST) and Lip-DLAT, ultimately inhibiting cell viability and inducing cell injury. Inducing cuproptosis with elesclomol plus CuCl2 (ES + Cu) further confirmed that “ES + Cu” treatment significantly reduced the contents of adenosine triphosphate (ATP) and glutathione (GSH), decreased the activities of mitochondrial complex I and III, and increased the contents of lactate (LA), malondialdehyde (MDA), creatine kinase (CK) and lactate dehydrogenase (LDH); when tetrathiomolybdate (TTM) was added to inhibit cuproptosis, myoblast injury was recovered significantly. Meanwhile, hypoxia/reoxygenation (H/R) induced CTR1 expression, increased the levels of intracellular copper, DLAT oligomers, LA, MDA, CK and LDH, reduced the levels of Lip-DLST, Lip-DLAT, ATP and GSH, and weakened the activities of mitochondrial complex I and III; after knocking down CTR1 expression, the levels of intracellular copper and the activation of cuproptosis pathway were decreased, and cell viability, injury and inflammation levels were significantly improved. Therefore, cuproptosis can promote myoblast injury, while H/R enhances copper uptake by inducing CTR1 expression, thereby enhancing cuproptosis and inducing cell injury, indicating that cuproptosis is a new mechanism of H/R-induced myoblast injury.

Chronic Corticosterone Exposure Suppresses Copper Transport through GR-Mediated Intestinal CTR1 Pathway in Mice.

Numerous studies have discovered that chronic stress induces metabolic disorders by affecting iron and zinc metabolism, but the relationship between chronic stress and copper metabolism remains unclear. Here, we explore the influence of chronic corticosterone (CORT) exposure on copper metabolism and its regulatory mechanism in mice. Mice were treated with 100 μg/mL CORT in drinking water for a 4-week trial. We found that CORT treatment resulted in a significant decrease in plasma copper level, plasma ceruloplasmin activity, plasma and liver Cu/Zn-SOD activity, hepatic copper content, and liver metallothionein content in mice. CORT treatment led to the reduction in duodenal expression of copper transporter 1 (CTR1), duodenal cytochrome b (DCYTB), and ATPase copper-transporting alpha (ATP7A) at the mRNA and protein level in mice. CORT treatment activated nuclear glucocorticoid receptor (GR) and down-regulated CRT1 expression in Caco-2 cells, whereas these phenotypes were reversible by an antagonist of GR, RU486. Chromatin immunoprecipitation analysis revealed that GR bound to the Ctr1 promoter in Caco-2 cells. Transient transfection assays in Caco-2 cells demonstrated that the Ctr1 promoter was responsive to the CORT-activated glucocorticoid receptor, whereas mutation/deletion of the glucocorticoid receptor element (GRE) markedly impaired activation of the Ctr1 promoter. In addition, CORT-induced downregulation of Ctr1 promoter activity was markedly attenuated in Caco-2 cells when RU486 was added. These findings present a novel molecular target for CORT that down-regulates intestinal CTR1 expression via GR-mediated trans-repression in mice.

Glucose restriction induces ROS-AMPK-mediated CTR1 expression and increases cisplatin efficiency in NSCLC

Cisplatin is one of the principal platinum-based chemotherapeutic agents for many types of cancer, including non-small-cell lung cancer (NSCLC). Copper transporter 1 (CTR1) plays a significant role in increasing cellular cisplatin uptake and sensitivity. The current study found that glucose restriction upregulated AMPK (AMP-activated protein kinase) through reactive oxygen species (ROS) to induce CTR1 expression in NSCLC cells. Direct upregulation of ROS levels also activated AMPK expression. The changes in CTR1 expression were consistent with glucose concentrations and AMPK expression. Feeding a low-carbohydrate ketogenic diet (a glucose restriction diet) to a severe combined immune deficiency (SCID) mouse xenograft model significantly enhanced the efficacy of cisplatin. The tumor size was significantly smaller in the group treated with cisplatin plus the low-carbohydrate ketogenic diet than in the group treated with cisplatin alone. Survival was longer in mice treated with the low-carbohydrate ketogenic diet than in the controls. Mice fed the low-carbohydrate ketogenic diet showed increased expression of CTR1 and AMPK in tumor tissues. These results suggest a novel mechanism whereby glucose restriction induces ROS-AMPK-mediated CTR1 expression in NSCLC, indicating glucose restriction as an effective adjuvant NSCLC therapy.

MO438: Excessive Mitochondrial Copper Load Driven by Upregulated Copper Transporter 1 Contributes to Mitochondrial Dysfunction and Renal Fibrosis

Abstract BACKGROUND AND AIMS Copper is an essential trace element for eukaryotes and a vital cofactor for various enzymes for a multiplicity of functions. Our previous study indicated that intracellular copper overload plays an important role in renal fibrosis. However, the underlying mechanism invoked by copper overload in renal fibrosis remains largely unknown. In this study, we found copper ions in cells were mainly accumulated in mitochondria, which damage the structure and function of mitochondria. Furthermore, copper transporter 1 (CTR1), the major transporter for the copper influx, was significantly increased in fibrotic kidneys. The activity of cytochrome C oxidase (COX), a copper coenzyme mediating the final step in the electron transport chain, was decreased. Therefore, we proposed that CTR1 might be involved in mitochondrial copper overload and renal fibrosis. METHOD The expression of CTR1 was examined in ischemia-reperfusion injury (IRI 28d) mice model. The regulatory mechanisms of CTR1 in vitro were investigated by treating renal tubule epithelium cell line (NRK-52E) with TGF-β1 or copper ions with or without copper chelator tetrathiomolybdate (TM) and in vivo, we use CTR1 transgenic mice subjected to IRI operation. ICP-MS, mitoSOX Red, electron microscopy, realtime-PCR and western blot analysis were applied in the current study. RESULTS We found that stimulated by TGF-β1, COX activity was declined. Mitochondria and cytosol, especially mitochondria, accumulated a large amount of copper in fibrotic kidney tissues. Furthermore, we found Copper transporter 1 (CTR1) expression was increased in fibrotic kidneys from chronic kidney disease patients, experimental fibrosis model mice and in vitro. More importantly, compared with WT mice, CTR1+/- mice subjected to an ischemia-reperfusion injury (IRI) had reduced mitochondrial copper level and ameliorated mitochondrial function and renal fibrosis, as evidenced by improving mitochondrial structure, inhibiting mtROS production and reducing expression of α-smooth muscle actin (α-SMA), collagen I and fibronectin. In addition, after treatment with the copper chelator tetrathiomolybdate (TM), mitochondrial function and kidney fibrosis were improved. CONCLUSION Collectively, our study showed that copper overload in mitochondria could damage the mitochondrial function and lead to renal fibrosis and CTR1 is involved in the mitochondrial copper overload. Copper overload inhibits the activity of COX and impairs mitochondria, subsequently leading to renal fibrosis.

Intestinal uptake of Cu by processes that do not involve copper transporter 1 (CTR1), as determined with Caco2 cell monolayers

Dietary copper absorption across the apical brush border of intestinal cells involves several uptake systems, yet in mammals, only one gene product (CTR1) has as yet been identified as specific for copper uptake across the cell plasma membrane of intestinal and other cells. Some studies have suggested that the important iron uptake system, divalent metal transporter 1 (DMT1), is also involved, and yet another, uncharacterized chloride-dependent system has been implicated. CTR1 takes up Cu(I), and it is generally accepted that dietary copper is first reduced by cell surface reductases before entering the enterocyte. Uptake of Cu(I) by CTR1 is inhibited by Ag(I), while Fe(III) and Cu(II) compete for reduction by the reductases in the brush border. Using Caco2 cell monolayers with tight junctions grown in bicameral chambers, we determined that, whether given as Cu(I) or Cu(II), only about one third of copper uptake was inhibited by an excess of Ag(I), implying that CTR1 accounts for only a portion of intestinal copper uptake. Rates of Cu uptake were measured by following accumulation of radioactive Cu over 60 min in cells and the basal medium (equivalent to blood fluid) after application of 67Cu-labeled Cu(II)-nitrilotriacetate (5 uM) to the apical surface, and were calculated in terms of % dose/h/mg cell protein. Our finding implied that CTR1 does not account for most of the uptake carried out by the enterocytes. Measuring uptake of Cu(II) in the presence of an excess of Fe(III) – to prevent reduction of Cu(II), failed to inhibit copper uptake, implying that not all copper needs to be reduced prior to uptake. To further investigate the chloride-dependent process, reported by Zimnicka et al.[1]) the effects of substituting sulfate for chloride in the culture medium was examined. Chloride-dependent uptake was measured by the fall in uptake rate when sulfate was substituted for chloride, and the effects of substituting with other halides was also examined. We found that substitution of sulfate for chloride reduced rates of copper uptake about 30%, and that the effects of Ag(I) and sulfate substitution were additive. Substituting bromide for chloride made no difference, while iodide substitution actually doubled uptake rates. Thus, a halide dependent system may also be prominent in mediating intestinal copper absorption. Effects of altering the nutritional iron status of the Caco2 cell monolayer was also examined. Pretreatment with excess iron (3 days with 5 uM ferric ammonium citrate) reduced expression of CTR1 and DMT1 mRNAs (determined by qPCR relative to 18S rRNA) but had little effect on rates of copper uptake. However, 48 h depletion of iron with desferrioxamine increased rates of copper uptake. As expected, this also increased expression of mRNA for DMT1. However, the iron deficiency also enhanced expression of CTR1. We conclude that at least in this intestinal model, CTR1 and a halide-dependent uptake process each account for about one third of Cu uptake; that CTR1 expression is enhanced by iron deficiency – which would explain increased uptake of copper in that condition; and that a Cu(II) uptake system may also be present. [1]Zimnicka et al. (2011) Am J Physiol Cell Physiol 300: C588–C599.

Inhibition of Copper Transporter-1 by Ammonium Tetrathiocarbolybdate in the Treatment of Pancreatic Cancer

To examine copper transporter 1 (CTR1) expression in pancreatic carcinoma cells, orthotopic xenograft pancreatic tumor model and clinical samples, and verify the effect of copper chelating agent ammonium tetrathiomolybdate (TM) regulate the expression of CTR1 in pancreatic carcinoma cells and the inhibition of pancreatic carcinoma. The expressions of copper transporter CTR1 and antioxidant protein 1 (ATOX1) in 22 clinical pancreatic ductal carcinoma and paracancer tissues 0.5-1 cm away from the tumor were measured by immunohistochemistry (IHC). PANC-1 cells were used to construct 5 orthotopic xenograft pancreatic tumor of nude mice models. Pancreatic cancer tissues and corresponding normal pancreatic tissues were collected, and the expressions of CTR1 and ATOX1 were detected by IHC and compared with clinical tissues. The proliferation of pancreatic carcinoma cells PANC-1 treated with 10, 30, 50, 100 μmol/L TM for 24 h, 48 h, 72 h was measured by CCK8 assay. The migration abilities of PANC-1 cells treated with 50 μmol/L TM for 24 h, 48 h were detected by scratch test. The expressions of CTR1, vascular endothelial growth factor (VEGF) and CyclinD1 proteins in PANC-1 cells treated with 10, 30, 50, 100 μmol/L TM for 48 h were measured by Western blot. Then the subcutaneous tumor-bearing model of nude mice were established with PANC-1 cells, and the growth of tumor was observed after oral administration of 0.3 mg/d and 1.0 mg/d of TM, respectively. The immunohistochemical results indicated that 19 of the 22 clinical pancreatic ductal cancer tissues of carcinoma patients had high expression of CTR1, and the same high expression of CTR1 was found in the orthotopic transplanted tumor tissues of PANC-1 nude mice. The proliferation inhibition of PANC-1 cells increased with the concentration of TM increased and the treatment time prolonged. The expressions of intracellular CTR1, VEGF and CyclinD1 all decreased with the concentration of TM increased. The cell migration ability decreased after the PANC-1 cells treated with TM. The tumor growth of PANC-1 tumor-bearing nude mice was inhibited after different doses of TM were delivered. The reduction in tumor volume and weight was more pronounced in the high-dose TM group (P<0.05). The expression of CTR1 is abnormally elevated in pancreatic carcinoma, and treatment with copper chelating agent for this target may help to inhibit pancreatic carcinoma.

Preclinical PET imaging study of lung cancer with 64CuCl2.

Human copper transporter 1 (CTR1) has been proven to be overexpressed in many types of cancer cells, and copper (II)-64 chloride (64CuCl2) has been used as an effective tracer for positron emission tomography (PET) imaging in tumor-bearing animal models. Thus, this study aimed to investigate the potential application of 64CuCl2 in PET imaging of lung cancer through targeting CTR1. The expression of CTR1 in a series of lung cancer cell lines was identified by quantitative real-time polymerase chain reaction (Q-PCR), western blot, enzyme-linked immunosorbnent assay (ELISA), and immunofluorescent staining. Then in vitro cell uptake assay of 64CuCl2 was investigated in human lung cancer cell lines with different levels of CTR1 expression. Small animal PET imaging and quantitative analysis were performed in human lung cancer tumor-bearing mice after intravenous injection of 64CuCl2, respectively. The CTR1 expression in multiple human lung cancer cells was identified and confirmed, and H1299 cell lines with high CTR1 expression, H460 with moderate CTR1, and H1703 with low CTR1 were selected for further experiments. In vitro cellular uptake assay displayed that the 64CuCl2 uptake by these three kinds of cells was positively correlated with their CTR1 expressed levels. The blocking experiments testified the specificity of 64CuCl2 to target CTR1. Moreover, small animal PET imaging and quantitative results showed that 64CuCl2 accumulation in H1299, H460, and H1703 tumor-bearing mice were consistent with CTR1 levels and cell uptake experiments. The expression of CTR1 in human lung cancer xenograft model could be successfully visualized by 64CuCl2 PET examination. With the expected growth of PET/CT examination to be an essential strategy in clinical lung cancer management, 64CuCl2 has the potential to be a promising PET imaging agent of lung cancer.

Non‐CTR1 mechanisms of intestinal copper absorption determined with the Caco2 cell culture model

Much is still unknown about how dietary copper is absorbed by the small intestine. The current view is that Cu(II) is reduced to Cu(I) by reductases in the brush border of enterocytes, and then taken up by copper transporter 1 (CTR1), the only as yet identified copper uptake transporter in mammals. Uptake of Cu(I) by CTR1 is inhibited by Ag(I) and can thus be used to assess the extent to which CTR1 contributes to copper uptake. Using Caco2 cell monolayers with tight junctions grown in bicameral chambers, we determined that, whether given as Cu(I) or Cu(II), only about one third of copper uptake was inhibited by an excess of Ag(I). So CTR1 is not the only copper uptake system in enterocytes. Our goals were thus to document the contributions of CTR1 and a potential chloride‐dependent process (reported by Zimnicka et al. [1]) to copper absorption, and to explore whether cellular iron status or the ability to reduce Cu(II) impacts these processes. Caco2 cell monolayers were grown on collagen‐coated filters in Transwell plates until trans‐epithelial electrical resistance reached or exceeded 250 Ohms. Rates of Cu uptake were measured by following accumulation of radioactive Cu over 60 min in cells and the basal medium (equivalent to blood fluid) after application of 67Cu‐labeled Cu(II)‐nitrilotriacetate (5 uM) to the apical surface, and were calculated in terms of % dose/h/mg cell protein. Ascorbate (1 mM) was added to produce Cu(I). Control uptake medium contained all chloride salts. The contribution of CTR1 to uptake was measured by the fall in uptake rate due to 50–100 uM Ag(I). Cu(II) reduction was inhibited by an excess of Fe(III). Chloride dependent uptake was determined by the fall in uptake rate when sulfate was substituted for chloride. Effects of substituting other halide ions for chloride were also recorded. Cellular Fe status was varied by preincubating monolayers for 3 days with 5 uM ferric ammonium citrate, or depleting them with desferrioxamine. Levels of mRNA for CTR1, DMT1, and reductases (STEAPs 2 and 3) were determined by qPCR, relative to 18S rRNA. We found that excess Ag(I) decreased rates of Cu(I) and Cu(II) uptake about 30%. The same was true when sulfate was substituted for chloride. Silver and sulfate effects were additive. Substituting bromide for chloride made no difference, while iodide substitution doubled uptake rates. Fe pretreatment reduced expression of CTR1 and DMT1 mRNAs but had little effect on rates of copper uptake. Fe deficiency greatly enhanced expression not only of DMT1 but also of CTR1 mRNA, and increased rates of copper uptake as well. Inhibition of Cu/Fe reductases with excess Fe(III) did not reduce rates of Cu(II) uptake. These results indicate that at least in this intestinal model, CTR1 and a halide‐dependent uptake process each account for about one third of Cu uptake; that CTR1 expression is enhanced by iron deficiency – which would explain increased uptake of copper in that condition; and that copper can be absorbed not only in the Cu(I) state but also as Cu(II).

Iron and copper transport activities of the mammalian metal‐ion transporters DMT1 and CTR1

Divalent metal‐ion transporter‐1 (DMT1) and the high‐affinity copper transporter‐1 (CTR1) play essential roles in the intestinal absorption of iron and copper respectively. Both transporters are potential therapeutic targets in disorders of metal metabolism. Disagreement remains over whether the metal‐ion specificities of the two transporters overlap. For example, others have concluded that DMT1 transports Cu+ and CTR1 transports Fe2+ based on the results of shRNA knockdown of gene expression in Caco‐2 cells [Espinoza A et al (2012) Biol Trace Elem Res 146, 281]. Since these observations contradicted our published data regarding the substrate profile of DMT1 in vitro [Illing AC et al (2012) J Biol Chem 287, 30485] and in vivo [Shawki A et al (2015) Am J Physiol Gastrointest Physiol 309, G635] or of CTR1 in vitro [Lee J et al (2002) J Biol Chem 277, 4380], we have re‐examined metal‐ion transporter substrate profiles. We tested the hypothesis that DMT1 can transport copper and that CTR1 can transport iron by using radiotracer assays and the voltage clamp in RNA‐injected oocytes expressing human DMT1 or CTR1. Expression of CTR1 in oocytes stimulated up to 20‐fold the uptake of 2 μM 64Cu+ (pH 5.5) but did not stimulate the uptake of 64Cu2+ or 55Fe2+. Expression of DMT1 in oocytes stimulated 80‐fold the uptake of 2 μM 55Fe2+ (pH 6.0) but transport of 64Cu+ or 64Cu2+ did not differ from that in control oocytes. We found that Cu+ inhibited DMT1‐mediated 55Fe2+ uptake in a competitive fashion (Ki ≈ 58 μM). In DMT1‐expressing oocytes, Cu+ evoked inward currents; however, such currents were also observed in control oocytes and their I/V relationships did not resemble those for DMT1 substrates Fe2+ and Cd2+. Addition of Fe2+ or Cd2+ abolished the presteady‐state (transient) currents observed in the absence of metal ion whereas addition of Cu+ or Cu2+ did not. We found, however, that Cu2+ accelerated the decay of the transient currents. We conclude that (1) whereas Cu+ and Cu2+ are capable of interacting with DMT1, neither are transported substrates; and (2) CTR1 mediates transport of Cu+ but not of Cu2+ or Fe2+. We therefore find no evidence of overlapping metal‐ion specificities in DMT1 and CTR1.Support or Funding InformationPHS grants R01‐DK080047, R01‐DK107309, R01‐DK074192, and R25‐HL115473; and the American Physiological Society

EGCG regulates CTR1 expression through its pro-oxidative property in non-small-cell lung cancer cells.

Copper transporter 1 (CTR1)plays an important role in increasing cisplatin intake. Our previous studies showed that CTR1 expression was upregulated by (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, therefore enhanced cisplatin sensitivity in ovary cancer and non-small-cell lung cancer (NSCLC) cells. In the current study in the non-small-cell lung cancer cells, we uncovered a potential mechanism of EGCG-induced CTR1 through its pro-oxidative property. We found that EGCG increased reactive oxygen species (ROS) generation, while in the presence of ROS scavenger N-acetyl-cysteine (NAC), ROS production was eliminated. Changes of CTR1 expression were consistent with the ROS level. Simultaneously, EGCG downregulated ERK1/2 while upregulated lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) through ROS to induce CTR1 expression. Besides, in a nude mouse xenografts model, EGCG treatment raised ROS level, expression of CTR1 and NEAT1 in tumor tissue. Also, ERK1/2 and p-ERK1/2 were suppressed as well. Taken together, these results suggested a novel mechanism that EGCG mediated ROS to regulate CTR1 expression through the ERK1/2/NEAT1 signaling pathway, which provided more possibilities for EGCG as a natural agent in adjuvant therapy of lung cancer.

Abstract 3224: Copper homeostasis: A new player in anti-tumor immune response

Abstract Immunotherapy has shown great potential for treating aggressive cancers and it is becoming the fourth and newest pillar of cancer therapy complementing surgery, cytotoxic therapy, and radiotherapy. In particular, immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown extraordinary clinical efficacy in several types of cancer. This is because tumor cells express molecules, such as the Programmed Death Ligand 1 (PD-L1), to prevent immune cells activity (immune-evasion). The immune checkpoint protein Programmed Death receptor 1 (PD-1) expressed by lymphocytes instructs T-cells not to attack any tumor cell expressing PD-L1. Several therapies anti PD-1/PD-L1 have been approved by FDA, but concerns have been raised about their long term efficacy and safety. Therefore, there is a need for better understanding of the biology and the mechanisms regulating PD-1/PD-L1 axis, to develop different approaches to target this pathway. Drugs modulating the transcriptional and post-transcriptional regulation of PD-L1 could represent new therapeutic strategies for increasing the efficacy and reducing side effects of the current anti PD-L1 antibodies. Copper transporter 1 (CTR-1) and copper levels are elevated in tumors and the use of copper targeting agents is currently under intense investigations. It has been also reported that copper plays a major role in the immune-system, but its activity is unclear. In this study we demonstrated that copper plays a key role in the expression of PD-L1 in cancer cells. Tissue microarrays from neuroblastoma (NB) and glioblastoma patients showed a significant correlation between CTR-1 and PD-L1 expression (p=0.00014 and p=0.012 respectively). In vitro experiments showed that downregulation of CTR-1 caused a decrease of intracellular copper which in turn led to a downregulation of PD-L1 expression in cancer cells. On the other hand, addition of copper into the media clearly induced PD-L1 upregulation. RNA-seq analysis revealed specific pathways and candidate genes associated with tumor copper homeostasis and PD-L1 expression. Consistently, Dextran-Catechin (DC) and TEPA, drugs reducing copper, were able to downregulate PD-L1 expression in tumors. In vivo studies showed that copper lowering drugs prolonged mice survival, and ex vivo immunohistochemistry staining confirmed the downregulation of CTR1 and PD-L1 expression. In addition, 24h and 48h of DC treatments showed an increase of tumor-infiltrating CD4+ and CD8+ lymphocytes and activated Natural Killer cells (NK) cells in NB immune-competent mouse model. In conclusion, there is a strong association between PD-L1 expression and intracellular copper levels. Copper dysregulating agents reduce PD-L1 in vitro and in vivo, highlighting the possibility to enhance tumor immune surveillance by targeting intracellular copper levels. This study shows the potential utility of copper targeting drugs to improve anti-cancer immunotherapies. Citation Format: Florida Voli, Luigi Lerra, Kathleen Kimpton, Federica Saletta, Sylvie Shen, Giuseppe Cirillo, Maria Kavallaris, Orazio Vittorio. Copper homeostasis: A new player in anti-tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3224.

Copper transporter 1 (CTR1) expression by mouse testicular germ cells, but not Sertoli cells, is essential for functional spermatogenesis.

An imbalance in copper (Cu) tissue homeostasis has a degenerative effect on spermatogenesis and male fertility. The high-affinity Cu transporter 1 (CTR1; SLC31A1) is the major protein responsible for Cu acquisition in eukaryotes and is highly expressed in mouse testes. Studies on yeast and Drosophila have demonstrated the conserved essential function of Cu and CTR1 for meiosis and fertility, implying that CTR1 may play an essential function in mammalian spermatogenesis. In mice, spermatogenesis takes place within the seminiferous epithelium, where tight junctions between somatic Sertoli cells (SCs) create a specialized microenvironment for the development of meiotic germ cells (GCs) by tightly regulating the free transport of metabolites and ions to reach these cells. Here, it is demonstrated that within the seminiferous epithelium, CTR1 is expressed on the membrane of primary pachytene spermatocytes and SCs. To examine the physiological significance of CTR1 in spermatogenesis, mice with a GC-specific (Ctr1ΔGC) and SC-specific (Ctr1ΔSC) disruption of the Ctr1 gene were generated. The testis of Ctr1ΔGC mice exhibits a severe progressive loss of GCs starting at postnatal day (PND) 28 leading to testis hypoplasia by adulthood. No spermatogenic recovery was observed in Ctr1ΔGC testis beyond PND 41, despite the presence of FOXO-1 expressing undifferentiated spermatogonial cells. However, Ctr1ΔSC mice displayed functional spermatogenesis and were fertile, even though testicular Cu levels and Cu-dependent cellular activities were significantly reduced. These results reveal, for the first time, the importance of CTR1 expression by GCs for maintaining functional spermatogenesis.

Research progress of copper transporter 1 in platinum-based chemotherapy

Platinum drugs are widely used in the treatment of various solid tumors, but their resistance to platinum is the most significant obstacle to successful treatment. Copper transporter 1 (CTR1) is the specific transporter for copper, and it mainly locates at the plasma membrane and plays a role in pumping copper into the cell. CTR1 is also the major platinum influx transporter and plays a key role in platinum resistance. The expression, polymorphism, and degradation of CTR1 affect platinum resistance in tumors. Therefore, CTR1 may be a potential predictive biomarker of platinum resistance and a therapeutic target for overcoming platinum resistance.

EFFECTS OF CURCUMIN AND NANOCURCUMIN ON CISPLATIN-INDUCED NEPHROTOXICITY IN RAT: COPPER TRANSPORTER 1 AND ORGANIC CATION TRANSPORTER 2 AS DRUG TRANSPORTERS

Objective: This study aimed to investigate the efficacy of curcumin (CMN) and nanocurcumin (NC) at preventing cisplatin (CDPP)-inducednephrotoxicity.Methods: Two membrane transporters, copper transporter 1 (CTR1) and organic cation transporter 2 (OCT2), have been identified involved in activeaccumulation of CDPP into renal tubular cells. We analyzed OCT2 transcription levels in rat kidney tissue and determined whether renoprotectivemechanism of CMN involves CTR1. Rats were randomly divided into five groups: (1) Control, (2) CDPP (7 mg/kg as single dose (i.p.), (3) CDPP+CMN(7 mg/kg CDPP as a single dose, i.p.+100 mg/kg/day of CMN), (4) CDPP+50 mg NC (7 mg/kg CDPP as single dose, i.p.+50 mg/kg/day NC), and(5) CDPP+100 mg NC (7 mg/kg CDPP as single dose, i.p.+100 mg/kg/day NC). Quantitative reverse transcription-polymerase chain reaction wasperformed to calculate relative expression of CTR1 and OCT2 genes in rat kidney.Results: Expression of CTR1 was unassociated with administration of CMN or NC, indicating CTR1 is uninvolved in renoprotective mechanism of CMN.The administration of 100 mg/kg/day NC increased expression of OCT2; this increase was higher compared with normal expression levels. This maybe due to another regulatory mechanism from the CMN itself.Conclusion: NC has a better renoprotective effect compared with curcumin, suggested by the increased OCT2 expression on its administration inCDPP-treated rats.

Curcumin enhances cisplatin sensitivity of human NSCLC cell lines through influencing Cu-Sp1-CTR1 regulatory loop

BackgroundCurcumin is a naturally occurring polyphenol which has been demonstrated to possess diverse biological activities. We previously reported that curcumin is a biologically active copper chelator with antitumor activity. Copper transporter 1 (CTR1) on the plasma membrane of eukaryotic cells mediates both copper as well as anticancer drug cisplatin uptake. PurposeThis study aims to investigate whether curcumin enhances cisplatin sensitivity of human non-small cell lung cancer (NSCLC) through influencing Cu-Sp1-CTR1 regulatory loop. MethodsThe combination effect of curcumin and cisplatin on cell proliferation and apoptosis was determined in vitro and in vivo. Platinum level in A549 cells and tumor tissue was measured by atomic absorption spectrometry (AAS). The binding ability of specificity protein 1 (Sp1) to CTR1 and Sp1 promoters was detected by ChIP assay and luciferase reporter assay system. ResultsHere we show that combined curcumin and cisplatin treatment markedly inhibited A549 cells proliferation and induced its apoptosis. Using a mouse model of A549 xenograft, we demonstrated that curcumin inhibits copper influx and increases uptake of platinum ion in tumor. Curcumin treatment enhances the binding of Sp1 to CTR1 and Sp1 promoters, thus induces CTR1 expression and chemosensitization to cisplatin treatment. This process is regulated by the Cu-Sp1-CTR1 regulatory loop. Moreover, the enhancement mediated by curcumin on cisplatin therapeutic efficacy in cultured human NSCLC cell lines (A549, H460, H1299) was dependent on CTR1. ConclusionsOur results demonstrated copper chelator curcumin enhances the benefits of platinum-containing chemotherapeutic agents and CTR1 could be a promising therapeutic target for non-small cell lung cancer treatment.

Pilot Study of 64CuCl₂ for PET Imaging of Inflammation.

Copper(II) ion (Cu2+) is the essential element for numerous pathophysiological processes in vivo. Copper transporter 1 (CTR1) is mainly responsible for maintaining Cu2+ accumulation in cells, which has been found to be over-expressed in inflammatory tissues. Therefore, we explored the potential application of 64CuCl2 for PET imaging of inflammation through targeting CTR1. The animal models of H2O2 induced muscle inflammation and lipopolysaccaharide induced lung inflammation were successfully established, then imaged by small animal PET (PET/CT) post-injection of 64CuCl2, and PET images were quantitatively analyzed. H&E and immunohistochemical (IHC) staining and western blot experiments were performed for evaluating CTR1 levels in the inflammatory and control tissues. Both inflammatory muscle and lungs can be clearly imaged by PET. PET image quantitative analysis revealed that the inflammatory muscle and lungs showed significantly higher 64Cu accumulation than the controls, respectively (p < 0.05). Furthermore, IHC staining and western blot analysis demonstrated that compared with the controls, CTR1 expression was increased in both the inflammatory muscle and lungs, which was consistent with the levels of 64Cu2+ accumulation in these tissues. 64CuCl2 can be used as a novel, simple, and highly promising PET tracer for CTR1 targeted imaging of inflammation.

NEAT1 acts as an inducer of cancer stem cell-like phenotypes in NSCLC by inhibiting EGCG-upregulated CTR1.

Long non-coding RNAs (lncRNAs) play significant roles in the pathogenesis of various cancers, including lung cancer. In this study, we aimed to investigate the biological function of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in cancer stem cells (CSCs). CSCs have been suggested as the main cause of tumor metastasis, tumor recurrence, and chemotherapy resistance. The copper transporter 1 (CTR1) has been the focus of many recent studies because of its correlation with cisplatin (CDDP) resistance. So far, the mechanism of how NEAT1 regulates CSCs in NSCLC remains unknown. In the current study, lung cancer stem cells were enriched from the parental NSCLC cells. We observed that NEAT1 was up-regulated while copper transporter 1 (CTR1) was down-regulated in the enriched NSCLC cancer stem cells. Knockdown of NEAT1 was able to decrease the CSC-like properties in NSCLC cells, while over-expression of NEAT1 could contribute to the stemness respectively. Meanwhile, appropriate doses of EGCG restrained the stemness triggered by over-expressing NEAT1 via inducing CTR1 expression. Wnt signal pathway and epithelial-to-mesenchymal transition (EMT) process were involved in NEAT1-induced CSCs in NSCLC. These findings may suggest a novel role of NEAT1 for NSCLC treatment.

In vivo [64Cu]CuCl2 PET imaging reveals activity of Dextran-Catechin on tumor copper homeostasis.

Given the strong clinical evidence that copper levels are significantly elevated in a wide spectrum of tumors, copper homeostasis is considered as an emerging target for anticancer drug design. Monitoring copper levels in vivo is therefore of paramount importance when assessing the efficacy of copper-targeting drugs. Herein, we investigated the activity of the copper-targeting compound Dextran-Catechin by developing a [64Cu]CuCl2 PET imaging protocol to monitor its effect on copper homeostasis in tumors.Methods: Protein expression of copper transporter 1 (CTR1) in tissue microarrays representing 90 neuroblastoma patient tumors was assessed by immunohistochemistry. Western blotting analysis was used to study the effect of Dextran-Catechin on the expression of CTR1 in neuroblastoma cell lines and in tumors. A preclinical human neuroblastoma xenograft model was used to study anticancer activity of Dextran-Catechin in vivo and its effect on tumor copper homeostasis. PET imaging with [64Cu]CuCl2 was performed in such preclinical neuroblastoma model to monitor alteration of copper levels in tumors during treatment.Results: CTR1 protein was found to be highly expressed in patient neuroblastoma tumors by immunohistochemistry. Treatment of neuroblastoma cell lines with Dextran-Catechin resulted in decreased levels of glutathione and in downregulation of CTR1 expression, which caused a significant decrease of intracellular copper. No changes in CTR1 expression was observed in normal human astrocytes after Dextran-Catechin treatment. In vivo studies and PET imaging analysis using the neuroblastoma preclinical model revealed elevated [64Cu]CuCl2 retention in the tumor mass. Following treatment with Dextran-Catechin, there was a significant reduction in radioactive uptake, as well as reduced tumor growth. Ex vivo analysis of tumors collected from Dextran-Catechin treated mice confirmed the reduced levels of CTR1. Interestingly, copper levels in blood were not affected by treatment, demonstrating potential tumor specificity of Dextran-Catechin activity.Conclusion: Dextran-Catechin mediates its activity by lowering CTR1 and intracellular copper levels in tumors. This finding further reveals a potential therapeutic strategy for targeting copper-dependent cancers and presents a novel PET imaging method to assess patient response to copper-targeting anticancer treatments.

Correlation of Expression Levels of Copper Transporter 1 and Thymidylate Synthase with Treatment Outcomes in Patients with Advanced Non-small Cell Lung Cancer Treated with S-1/Carboplatin Doublet Chemotherapy

Background:Copper transporter 1 (CTR1) is a critical determinant of the uptake and cytotoxic effect of the platinum drugs carboplatin and cisplatin. Thymidylate synthase (TS) is an enzyme involved in DNA synthesis and is associated with resistance of tumor cells to 5-fluorouracil. We investigated the correlation between CTR1 and TS expression levels and treatment outcomes in patients with advanced non-small-cell lung cancer (NSCLC) treated with S-1/carboplatin doublet chemotherapy.Methods:Twenty-nine patients were enrolled in this study. Tumor expression of CTR1 and TS was measured immunohistochemically and analyzed for correlation with tumor response, progression-free survival (PFS), and overall survival (OS).Results:Tumor response was significantly better in patients with CTR1High tumors than in patients with CTR1Low tumors (64% vs. 18%, P = 0.02). Patients with TSLow tumors had a significantly longer OS (median 21.2 vs. 8.5 months, P = 0.02), but not PFS, than patients with TSHigh tumors. When CTR1 and TS co-expression was analyzed, patients with either CTR1High or TSLow tumors showed a significantly better tumor response (50% vs. 0%, P = 0.01), longer PFS (median 4.2 vs. 2.1 months, P = 0.03), and longer OS (median 21.2 vs. 8.5 months, P = 0.01) than patients with both CTR1Low and TSHigh tumors.Conclusions:Our study suggests that combined CTR1/TS expression status has the potential to be an important predictor of good treatment outcomes in patients with advanced NSCLC treated with S-1/carboplatin doublet chemotherapy.

Prognostic value of copper transporter 1 expression in patients with clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) features a Von Hippel-Lindau mutation, associated with a hypoxia-inducible factor (HIF) imbalance. Copper transporter 1 (CTR1) may also promote tumor progression through the modulation of the HIF pathway by copper. Therefore, the present study explored the prognostic effect of tumor CTR1 expression in patients with ccRCC. A total of 293 patients with ccRCC that underwent nephrectomy were retrospectively enrolled. CTR1 expression was assessed by immunohistochemistry, and its association with clinicopathological features and prognosis were evaluated. The present data indicated that high tumor CTR1 expression was independently associated with poor overall survival (OS) [hazard ratio, 2.291; 95% confidence interval (CI), 1.389–3.777; P<0.001] and disease-free survival (DFS) (hazard ratio, 2.210; 95% CI, 1.299–3.759; P=0.003) rates in patients with ccRCC. Furthermore, CTR1 expression was significantly higher for Mayo Clinic stage, size, grade and necrosis score risk groups, and could be incorporated into several existing prognostic models to improve performance. Nomograms incorporating tumor CTR1 expression with other parameters performed well in the 5- and 8-year OS and DFS rate predictions of patients (concordance index 0.805 and 0.787, respectively). In conclusion, the present study demonstrated that CTR1 expression is a potential independent biomarker for poor prognosis for the recurrence and survival prediction of patients with ccRCC following nephrectomy.