Expression Of CTR1 Research Articles

Ethylene biosynthesis and signal transduction are enhanced during accelerated ripening of postharvest tomato treated with exogenous methyl jasmonate

Phytohormone methyl jasmonate (MeJA) plays an important role in fruit ripening. This research aimed to investigate the regulation of MeJA in ethylene biosynthesis and signal transduction during postharvest tomatoes ripening. After mature green cherry tomatoes were infiltrated with MeJA (0.5 mM) or deionized water (control) respectively, fruit colour and firmness, ethylene production, the activities and expressions of ACS and ACO, as well as the expressions of major genes involved in ethylene signal transduction were periodically monitored. Results showed that a significant acceleration in ripening in MeJA-treated fruit was observed, along with the accelerated changes of ethylene production, the enhanced activities and expression levels of ACS and ACO. The expression levels of ETR3, ETR4, ETR6, ETR7, EIN2, EIL2, EIL3, EIL4 and ERF1 were up-regulated (from 1.26- to 6.50-fold) by MeJA during tomatoes ripening. The expression of CTR1, however, was down-regulated (from 0.23- to 0.54-fold). Moreover, the expression of EIL1 was positively regulated at the early ripening stage whereas negatively regulated at the late ripening stage (11.76-/0.39-fold), whereas the expressions of CTR3 and CTR4 showed the opposite expression patterns (0.15-/7.78-fold, 0.48-/2.00-fold). Results suggested the up-regulation of the genes associated with ethylene biosynthesis, as well as the up-regulated transcriptional levels of the most genes involved in ethylene signal transduction, leading to the increase of ethylene production, response and action, would be one of the major mechanisms of MeJA in accelerating postharvest tomatoes ripening.

Mammalian copper homeostasis requires retromer-dependent recycling of the high-affinity copper transporter 1.

ABSTRACTThe concentration of essential micronutrients, such as copper (used here to describe both Cu+ and Cu2+), within the cell is tightly regulated to avoid their adverse deficiency and toxicity effects. Retromer-mediated sorting and recycling of nutrient transporters within the endo-lysosomal network is an essential process in regulating nutrient balance. Cellular copper homeostasis is regulated primarily by two transporters: the copper influx transporter copper transporter 1 (CTR1; also known as SLC31A1), which controls the uptake of copper, and the copper-extruding ATPase ATP7A, a recognised retromer cargo. Here, we show that in response to fluctuating extracellular copper, retromer controls the delivery of CTR1 to the cell surface. Following copper exposure, CTR1 is endocytosed to prevent excessive copper uptake. We reveal that internalised CTR1 localises on retromer-positive endosomes and, in response to decreased extracellular copper, retromer controls the recycling of CTR1 back to the cell surface to maintain copper homeostasis. In addition to copper, CTR1 plays a central role in the trafficking of platinum. The efficacy of platinum-based cancer drugs has been correlated with CTR1 expression. Consistent with this, we demonstrate that retromer-deficient cells show reduced sensitivity to the platinum-based drug cisplatin.

P0735ELAVATED INTRACELLULAR COPPER CONTRIBUTES A UNIQUE ROLE TO KIDNEY FIBROSIS BY LYSYL OXIDASE MEDIATED MATRIX CROSSLINGKING

Abstract Background and Aims Copper is an essential trace element required for many biological processes. Some studies have demonstrated that copper accumulating was related to liver fibrosis and lung fibrosis, but the underlying mechanism is not very clear. Copper is the essential unit of lysyl oxidase (LOXs), which are the key enzymes of crosslinking of extracellular matrix. Method Sprague-Dawley rats were divided into the sham group, unilateral ureteral obstruction (UUO) operated group and UUO treated with copper chelating agents tetrathiomolybdate (TM). Rat kidney fibroblast cells (NRK-49F) were used in vitro. The concentration of copper, the LOXs activity and the degree of cross-linking of extracellular collagen were detected in vivo and vitro. Results (1) The copper concentration in serum, urine and kidney of rats increased significantly at 7 days after UUO surgery; After treatment of TGF-β1, the intracellular copper concentration was increased significantly in cells; The concentration of copper in patients` serum is on the rise with the progression of chronic kidney disease (CKD). (2) The expression of CTR1 was upregulated in the kidneys of UUO rats; The level of CTR1 was increased significantly by TGF-β1 in vitro; (3) Blockage of Smad2/3 suppresses TGF-β1-induced expression of CTR1; (4) Downregulation of CTR1 significantly inhibited the intracellular copper concentration; (5) The activity of LOXs was increased significantly after TGF-β1 treatment; (6) Downregulation of CTR1 significantly inhibited the activity of LOXs and the cross-linking of extracellular collagen induced by TGF-β1 in vitro; (7) The concentration of copper, the degree of collagen cross-linking and the deposition of collagen were decreased in the kidney tissue of UUO rats after treatment with TM. The concentration of intracellular copper, the activity of LOXs and the degree of collagen cross-linking were attenuated with treatment of TM in vitro. Conclusion We firstly found that the intracellular copper accumulating was closely to renal fibrosis. The underlying mechanism was related with the increasing expression of CTR1 and activity of LOXs. Treatment with TM ameliorated the renal fibrosis. This study presented a novel treatment target for renal fibrosis.

Altered Cisplatin Pharmacokinetics during Nonalcoholic Steatohepatitis Contributes to Reduced Nephrotoxicity

Cisplatin is an alkylating antineoplastic agent that is indicated for the treatment of solid malignancies. Cisplatin is preferentially eliminated from systemic circulation via tubular secretion, whereby it exhibits dose‐limiting nephrotoxicity. Interindividual variability in xenobiotic transporter expression is a known contributor to differential cisplatin toxicity and efficacy, and may be the result of genetic, environmental, and pathological contributions. In this study, we aimed to determine if nonalcoholic steatohepatitis (NASH) alters cisplatin pharmacokinetics and if this change elicits differential nephrotoxicity. Sprague Dawley rats fed a control or methionine and choline deficient (MCD) diet to model NASH were given a single bolus dose of cisplatin and sacrificed after 72 h. The MCD diet resulted in a NASH hepatic phenotype that remained unchanged following cisplatin exposure. Drug‐naïve NASH rats also displayed no evidence of differential renal pathology relative to control rats. However, renal necrosis and inflammation were reduced in NASH by 40 and 63% following cisplatin treatment, respectively, relative to healthy controls. Furthermore, kidney weights of cisplatin‐treated control rats were increased by 31%, compared to an 18% increase in NASH. Plasma cisplatin clearance was reduced from 6.78 (control) to 4.04 mL/min in NASH, and cisplatin plasma AUC was significantly increased by 44% in NASH, relative to control. Cumulative urinary elimination of cisplatin was decreased from 73 to 34% of total dose and renal clearance was reduced from 4.64 to 1.49 mL/min in NASH, compared to control. Subsequently, renal intracellular accumulation of cisplatin after 6 h was reduced by 34% in NASH, relative to control. Supporting these findings, expression of proximal tubule cisplatin uptake transporters, Ctr1 and Ctr2, were reduced by 24 and 64%, respectively compared to healthy control rats, whereas expression of Oct1, Oct2, and Oct3 were unchanged. Interestingly, expression of cisplatin efflux transporters Mate1 and Atp7a were reduced by 52 and 31%, respectively, in NASH compared to control. Taken together, these data suggest that NASH alters renal uptake and efflux transporter expression, thereby attenuating cisplatin uptake and clearance in the kidney with a corresponding reduction in renal cell exposure and nephrotoxicity during NASH. As such, this study demonstrates that NASH can influence pharmacokinetics of drugs cleared by renal elimination, which may contribute to adverse drug reactions.Support or Funding InformationThis work is supported by a NIH grant [R01 ES028668] and the Southwest Environmental Health Sciences Center [P30 ES006694]. JLJ is supported by an institutional training grant [T32 ES007091].

Germination and gene expression as affected by aminocyclopropane-1-carboxylic acid in deteriorated soybean seed

ABSTRACTSeed aging leads to reduced germination and vigor, especially in long-term storage, which can significantly reduce crop yields. Hence, identifying genetic mechanisms and providing physiological approaches to alleviate the deterioration process are needed. The current study was conducted in a series of laboratory experiments with soybean (Glycine max (L.) Merr.) in 2015. Accelerated aging durations (0 d, 6 d, and 10 d), priming solutions [dry seed, water, and 50 µM aminocyclopropane-1-carboxylic acid (ACC)] and priming periods (6 h and 12 h) were considered experimental factors. Total RNA was extracted from whole dry and imbibed seeds using hexadecyltrimethylammonium method. Genes and associated accessions studied were: LIG4 (XM_003546387.3), CTR1 (BW653506.1), GAI1 (BW656807.1), NCED1 (AK244131.1), and NCED5 (AK245513.1). With increasing severity of seed deterioration, the expression of LIG4, CTR1, GAI1, and NCED1 significantly increased, whereas a decreasing trend was observed for NCED5 expression. ACC had an inhibitory effect on germination index in aged soybean seed, which was associated with expression level of LIG4. Priming with ACC considerably reduced the inhibitory effect of CTR1, GAI1, and NCED1 in 6-day aged seeds, resulting in a positive effect on germination. It seems that positive or negative effects of ethylene priming on soybean germination depend on severity of seed deterioration.

Correlation of CTR1, ERCC-1, and HSP70 expressions with cisplastin response in cervical cancer stage IIB

Objectives: This study aimed to determine the resistance of cisplatin (chemotherapy drugs) at three point areas including expression of CTR1, ERCC1, and HSP70 in cervical cancer patients.Materials and Methods: This research used a cross sectional approach. The population in this study were patients with stage IIB of cervical carcinoma. The study sample were patients with stage IIB of cervical carcinoma according to the FIGO classification, patients received cisplatin chemotherapy treatment (50 mg/m2 4 times), had good kidney and liver function. The independent variables in this study were the expression of CTR1, ERCC1, and HSP70 and the dependent variable of this study was the response to cisplatin therapy in cervical cancer. Primary data was obtained through MRI examination after the administration of cisplatin 50 mg/m2 every week. The parameters of this study included the expression of CTR1, ERCC1 and HSP70. Data obtained were analyzed using Wilcoxon rank test, Spearman test and categorical regression with a significance level of p <0.05.Results: The treatment of cisplatin therapy in cervical cancer patients had no significant correlation between the expression of CTR1 and ERCC1, but in the expression of HSP70 there was a significant negative correlation which means that the higher the expression of HSP70, the worse the response of therapy.Conclusion: This study showed that HSP70 expression can be used as an indicator in treatment of cisplatin therapy in cancer patients through MRI examination.

The expression of copper transporters associated with the ototoxicity induced by platinum-based chemotherapeutic agents

BackgroundAntitumor agents based on platinum have gained a well-established place in the treatment of several forms of cancer. Their efficiency is hampered by serious toxic effects against healthy tissues as well. Ototoxicity is a serious side effect leading to hearing impairment and represents an important issue affecting the patients’ quality of life. The currently used platinum chemotherapeutics exert different toxicity towards cochlear cells. The aim of our study was to answer some questions regarding the differential uptake and cellular pharmacodynamics of Cisplatin (CDDP), Carboplatin (CBDCA) and Oxaliplatin (L-OHP) in the HEI-OC1 cochlear cell line. MethodsWe studied the expression of copper transporters CTR1, ATP7A and ATP7B which are presumably involved in the uptake, cellular transport and efflux of platinum compounds by immunofluorescence microscopy and flow-cytometry. The cellular uptake of the compounds was evaluated through the determination of intracellular platinum concentration by atomic absorption spectroscopy. The effects of the treatment of HEI-OC1 cells with platinum compounds were also evaluated: cytotoxicity with the Cell Titer Blue viability test, formation of reactive oxygen species with 2′,7’ –dichlorofluorescein diacetate, genotoxicity with the comet assay and apoptosis with the cleaved PARP ELISA test. ResultsCTR1, ATP7A and ATP7B were all expressed by HEI-OC1 cells. The treatment with the platinum compounds led to a modulation of their expression, manifested in a differential platinum uptake. Treatment with Cisplatin led to the highest intracellular concentration of platinum compared to Oxaliplatin and Carboplatin at the same dose. Treatment with CuSO4 reduced platinum uptake of all the compounds, significantly in the case of Cisplatin and Carboplatin. CDDP was the most cytotoxic against HEI-OC1 cells, with an IC50 = 65.79 μM, compared to 611.7 μM for L-OHP and 882.9 μM for CBDCA, at the same molar concentration. The production of ROS was the most intense after CDDP, followed by L-OHP and CBDCA. In the comet assay, at the 100 μM concentration, L-OHP and CBDCA induced DNA adducts while CDDP induced adducts as well as DNA strand breaks. CBDCA and L-OHP lead to a significant increase of cleaved PARP at 24h (p < 0.001), suggesting an important apoptotic process induced by these compounds at the used concentrations. ConclusionsThe results obtained in the current study suggest that the modulation of copper transporters locally may represent a new strategy against platinum drugs ototoxicity.

A Preliminary Study of NER and MMR Pathways Involved in Chemotherapy Response in Bladder Transitional Cell Carcinoma: Impact on progression-free survival.

One of the main genotoxic drugs used in bladder cancer chemotherapy is cisplatin. While it is applied in most types of cancers, resistance to cisplatin is wildly common. In order to overcome drug resistance, it is necessary to determine a predictive marker. This study was conducted to provide basic data for selecting and designing a gene profile for further cohort and RCT studies in the future to improve response to treatment in bladder cancer. The expression levels of ERCC1, MLH1, MSH2, and CTR1 mRNA were determined in the tumor tissue using real-time q-PCR. Progression-free survival (PFS) was analyzed in term of the level of genes expression. The results revealed that the level of ERCC1 mRNA expression was higher in the recurrence (R) group compared to the no recurrence (NR) group. Moreover, the PFS time was increased in the patients with an ERCC1 expression level of below 1.57. The level of MLH1 and MSH2 mRNA expression was lower in the R group compared to the NR group; therefore, PFS time was increased in the patients with MLH1 and MSH2 gene expression levels above the cutoff point. While the level of CTR1 mRNA expression was higher in the R group versus the NR group, the PFS time was longer in the patients with CTR1 expression levels of below 1.265 compared to the patients with high levels of CTR1 expression. It can be concluded that the level of ERCC1, MLH1, MSH2, and CTR1 mRNA expression may be associated with PFS time as possible therapeutic targets for decreasing cisplatin resistance.

Promotion of cadmium uptake and cadmium-induced toxicity by the copper transporter CTR1 in HepG2 and ZFL cells

Cadmium (Cd2+) is considered a human carcinogen as it causes oxidative stress and alters DNA repair responses. However, how Cd2+ is taken up by cells remains unclear. We hypothesized that Cd2+ could be transported into cells via a membrane copper (Cu) transporter, CTR1. CTR1 expression was not affected by Cd2+ exposure at the mRNA or protein level. Stable cell lines overexpressing either hCTR1, in the human liver cell line HepG2, or zCTR1, in the zebrafish liver cell line ZFL, were created to study their responses to Cd2+ insult. It was found that both HepG2 and ZFL cells overexpressing CTR1 had higher Cd2+ uptake and thus became sensitive to Cd2+. In contrast, hCTR1 knockdown in HepG2 cells led to a reduced uptake of Cd2+, making the cells relatively resistant to Cd2+. Localization studies revealed that hCTR1 had a clustered pattern after Cd2+ exposure, possibly in an attempt to reduce both Cd2+ uptake and Cd2+-induced toxicity. These in vitro results indicate that CTR1 can transport Cd2+ into the cell, resulting in Cd2+ toxicity.

Different Sources of Copper Effect on Intestinal Epithelial Cell: Toxicity, Oxidative Stress, and Metabolism.

Copper (Cu) is widely used in the swine industry to improve the growth performance of pigs. However, high doses of copper will induce cell damage and toxicity. The aim of this study was to evaluate toxicity, bioavailability, and effects on metabolic processes of varying copper sources using porcine intestinal epithelial cells (IPEC-J2) as a model. The IPEC-J2 were treated with two doses (30 and 120 μM) of CuSO4, Cu Glycine (Cu-Gly), and Cu proteinate (Cu-Pro) for 10 h, respectively. Cell damage and cellular copper metabolism were measured by the changes in cell viability, copper uptake, oxidative stress biomarkers, and gene/protein expression levels. The results showed that cell viability and ratio of reduced and oxidized glutathione (GSH/GSSG) decreased significantly in all treatment groups; intracellular copper content increased significantly in all treatment groups; total superoxide dismutase (SOD) activity increased significantly in the 120 μM exposed groups; SOD1 protein expression levels were significantly upregulated in 30 μM Cu-Pro, 120 μM Cu-Gly, and 120 μM Cu-Pro treatment groups; intracellular reactive oxygen species (ROS) generation and malondialdehyde (MDA) content increased significantly in 30 μM treatment groups and 120 μM CuSO4 treatment group. CTR1 and ATP7A gene expression were significantly downregulated in the 120 μM exposed groups. While upregulation of ATOX1 expression was observed in the presence of 120 μM Cu-Gly and Cu-Pro. ASCT2 gene expression was significantly upregulated after 120 μM Cu-Glycine and CuSO4 exposure, and PepT1 gene expression was significantly upregulated after Cu-Pro exposure. In addition, CTR1 protein expression level decreased after 120 μM CuSO4 and Cu-Gly exposure. PepT1 protein expression level was only upregulated after 120 μM Cu-Pro exposure. These findings indicated that extra copper supplementation can induce intestinal epithelial cell injury, and different forms of copper may have differing effects on cell metabolism.

The Impact of Integrin-Mediated Matrix Adhesion on Cisplatin Resistance of W1 Ovarian Cancer Cells.

Background: Tumor cell binding to the microenvironment is regarded as the onset of therapeutic resistance, referred to as cell adhesion mediated drug resistance (CAM-DR). Here we elucidate whether CAM-DR occurs in ovarian cancer cells and contributes to still-existing cisplatin resistance. Methods: Cultivation of W1 and cisplatin-resistant W1CR human ovarian cancer cells on collagen-type I (COL1) was followed by whole genome arrays, MTT assays focusing cisplatin cytotoxicity, and AAS detection of intracellular platinum levels. Expression of cisplatin transporters Ctr1 and MRP2 was analyzed. Mechanistic insight was provided by lentiviral β1-integrin (ITGB1) knockdown, or inhibition of integrin-linked kinase (ILK). Results: EC50 values of cisplatin cytotoxicity increased twofold when W1 and W1CR cells were cultivated on COL1, associated with significantly diminished intracellular platinum levels. Transporter deregulation could not be detected at mRNA levels but appears partially responsible at protein levels. The ITGB1 knockdown confirms that CAM-DR follows a COL1/ITGB1 signaling axis in W1 cells; thus, a blockade of ILK re-sensitized W1 cells on COL1 for cisplatin. In contrast, CAM-DR adds to cisplatin resistance in W1CR cells independent of ITGB1. Conclusions: CAM-DR appears relevant for ovarian cancer cells, adding to existing genetic resistance and thus emerges as a target for sensitization strategies.

Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

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.

Phosphorylation of STAT1 serine 727 enhances platinum resistance in uterine serous carcinoma.

Uterine serous carcinoma (USC) is a highly aggressive histological subtype of endometrial cancers harboring highly metastatic and chemoresistant features. Our previous study showed that STAT1 is highly expressed in USC and acts as a key molecule that is positively correlated with tumor progression, but it remains unclear whether STAT1 is relevant to the malicious chemorefractory nature of USC. In the present study, we investigated the regulatory role of STAT1 toward platinum-cytotoxicity in USC. STAT1 suppression sensitized USC cells to increase cisplatin-mediated apoptosis (p < 0.001). Furthermore, phosphorylation of STAT1 was prominently observed on serine-727 (pSTAT1-Ser727), but not on tyrosine-701, in the nucleus of USC cells treated with cisplatin. Mechanistically, the inhibition of pSTAT1-Ser727 by dominant-negative plasmid elevated cisplatin-mediated apoptosis by increasing intracellular accumulation of cisplatin through upregulation of CTR1 expression. TBB has an inhibitory effect on casein kinase 2 (CK2), which phosphorylate STAT1 at serine residues. Sequential treatment with TBB and cisplatin on USC cells greatly reduced nuclear pSTAT1-Ser727, enhanced intracellular accumulation of cisplatin, and subsequently increased apoptosis. Tumor load was significantly reduced by combination therapy of TBB and cisplatin in in vivo xenograft models (p < 0.001). Our results collectively suggest that pSTAT1-Ser727 may play a key role in platinum resistance as well as tumor progression in USC. Thus, targeting the STAT1 pathway via CK2 inhibitor can be a novel method for attenuating the chemorefractory nature of USC.

Oleandrin sensitizes human osteosarcoma cells to cisplatin by preventing degradation of the copper transporter 1.

A major problem in osteosarcoma treatment is cisplatin resistance. We have reported the anti-osteosarcoma effect of oleandrin; however, whether oleandrin sensitizes osteosarcoma to cisplatin is unknown. We investigated the chemosensitization of oleandrin and potential mechanisms in osteosarcoma cells U-2OS, SaOS-2, and MG-63. The median-effect analysis demonstrated that cisplatin+oleandrin exerted synergistic (U-2OS and MG-63) or additive effects (SaOS-2), which were consistent with the changes of the intracellular accumulation of platinum (Pt) and Pt-DNA adducts. Immunohistochemistry staining showed that the expression level of the mature form CTR1, the major influx transporter of cisplatin, was low in osteosarcoma tissue. However, oleandrin with or without cisplatin significantly increased the expression and membrane localization of the mature CTR1. Furthermore, CTR1 knockdown reversed the synergistic effect and decreased cisplatin uptake. The mRNA microarray analysis suggested that oleandrin downregulated the expression of proteasome-related genes, which was verified by the proteasome activity assay. Besides, the proteasome inhibitor MG132 upregulated the expression of the mature CTR1 in U-2OS and MG-63 cells. Overall, we conclude that oleandrin sensitizes osteosarcoma cells to cisplatin in synergistic or additive manners. The synergy results from the enhanced cisplatin uptake via oleandrin-mediated inhibition of proteasome activity and subsequent blockage of the mature CTR1 degradation.

Life-time exposure to waterborne copper II: Patterns of tissue accumulation and gene expression of the metal-transport proteins ctr1 and atp7b in the killifish Poecilia vivipara

The involvement of transporting proteins on copper (Cu) bioaccumulation was evaluated in the killifish Poecilia vivipara chronically exposed to environmentally relevant concentrations of waterborne Cu. Fish (<24 h-old) were maintained under control condition or exposed to different waterborne Cu concentrations (5, 9 and 20 μg/L) for 28 and 345 days in saltwater. Following exposure periods, Cu accumulation and the expression of genes encoding for the high affinity Cu-transporter (ctr1) and the P-type Cu-ATPase (atp7b) were evaluated. Whole-body metal accumulation and gene expression were evaluated in fish exposed to 28 days. Similarly, in fish exposed to 345 days, liver, gills and gut were also evaluated. No fish survival was observed after exposure to 20 μg/L for 345 days. Whole-body Cu accumulation was significantly higher in fish exposed to 20 μg/L Cu for 28 days and in fish exposed to 9 μg/L for 345 days in comparison to control animals. Similarly, tissue Cu accumulation was significantly higher in fish exposed to 9 μg/L for 345 days in comparison to control animal. However, no significant accumulation was observed in fish muscle. Following exposure for 28 days, whole-body ctr1 expression was slightly induced in fish exposed to 9 μg/L. In turn, no significant change in ctr1 expression was observed following exposure to Cu for 345 days. Differently, whole-body atp7b expression was markedly up-regulated in the whole-body of fish exposed Cu for 28 days and in tissues of fish exposed to Cu for 345 days. These findings indicate the expression of atp7b is more responsive to Cu accumulation in P. vivipara than ctr1 expression and, therefore, more suitable to be used as a biomarker of exposure to this metal. Also, we argue that the expression of atp7b is sustained at elevated levels for as much time as fish are maintained in Cu contaminated water.

Nitric oxide increases biofilm formation in Saccharomyces cerevisiae by activating the transcriptional factor Mac1p and thereby regulating the transmembrane protein Ctr1

BackgroundBiofilms with immobilized cells encased in extracellular polymeric substance are beneficial for industrial fermentation. Their formation is regulated by various factors, including nitric oxide (NO), which is recognized as a quorum-sensing and signal molecule. The mechanisms by which NO regulates bacterial biofilms have been studied extensively and deeply, but were rarely studied in fungi. In this study, we observed the effects of low concentrations of NO on biofilm formation in Saccharomyces cerevisiae. Transcriptional and proteomic analyses were applied to study the mechanism of this regulation.ResultsAdding low concentrations of NO donors (SNP and NOC-18) enhanced biofilm formation of S. cerevisiae in immobilized carriers and plastics. Transcriptional and proteomic analyses revealed that expression levels of genes regulated by the transcription factor Mac1p was upregulated in biofilm cells under NO treatment. MAC1 promoted yeast biofilm formation which was independent of flocculation gene FLO11. Increased copper and iron contents, both of which were controlled by Mac1p in the NO-treated and MAC1-overexpressing cells, were not responsible for the increased biofilm formation. CTR1, one out of six genes regulated by MAC1, plays an important role in biofilm formation. Moreover, MAC1 and CTR1 contributed to the cells’ resistance to ethanol by enhanced biofilm formation.ConclusionsThese findings suggest that a mechanism for NO-mediated biofilm formation, which involves the regulation of CTR1 expression levels by activating its transcription factor Mac1p, leads to enhanced biofilm formation. The role of CTR1 protein in yeast biofilm formation may be due to the hydrophobic residues in its N-terminal extracellular domain, and further research is needed. This work offers a possible explanation for yeast biofilm formation regulated by NO and provides approaches controlling biofilm formation in industrial immobilized fermentation by manipulating expression of genes involved in biofilm formation.

Copper accumulation in the brain causes the elevation of oxidative stress and less anxious behavior in Ts1Cje mice, a model of Down syndrome

Elevated oxidative stress (OS) is widely accepted to be involved in the pathogenesis of Down syndrome (DS). However, the mechanisms underlying the elevation of OS in DS are poorly understood. Biometals, in particular copper and iron, play roles in OS. We therefore focused on biometals in the brain with DS. In this study, we analyzed the profile of elements, including biometals, in the brain of Ts1Cje mice, a widely used genetic model of DS. An inductively coupled plasma-mass spectrometry (ICP-MS)-based comparative metallomic/elementomic analysis of Ts1Cje mouse brain revealed a higher level of copper in the hippocampus and cerebral cortex, but not in the striatum, in comparison to wild-type littermates. The expression of the copper transporter CTR1, which is involved in the transport of copper into cells, was decreased in the ependymal cells of Ts1Cje mice, suggesting a decrease in the CTR1-mediated transport of copper into the ependymal cells, which excrete copper into the cerebrospinal fluid. To evaluate the pathological significance of the accumulation of copper in the brain of Ts1Cje mice, we examined the effects of a diet with a low copper content (LoCD) on the elevated lipid peroxidation, the accumulation of hyperphosphorylated tau, and some behavioral anomalies. Reducing the copper concentration in the brain by an LoCD restored the enhanced lipid peroxidation and phosphorylation of tau in the brain and reduced anxiety-like behavior, but not hyperactivity or impaired spatial leaning, in Ts1Cje mice. The findings highlight the reduction of accumulation of copper in the brain may be a novel therapeutic strategy for DS.

Glutaryl Polyamidoamine Dendrimer for Overcoming Cisplatin-Resistance of Breast Cancer Cells

Cisplatin has limited clinical applications due to drug resistance. PAMAM dendrimer was chosen as a vehicle to counteract cisplatin-resistance and its mechanism was assessed. Generation 5 Polyamidoamine dendrimer (G5) was modified by glutaric anhydride (GA) and then conjugated with cisplatin. The cisplatin release of G5-GA-cisplatin was evaluated at pH 5.5 and pH 7.4. The cytotoxicity of G5-GA-cisplatin and free cisplatin was compared in cisplatin-resistant breast cancer cell line MCF-7R. The intracellular platinum content of MCF-7R was determined using ICP-MS. The expression of Ctr1 and ATP7B of MCF-7R cells was also evaluated. An average of 75 amino groups present in the G5 PAMAM surface were converted into glutaric acid (G5-GA75) and platinum loading was 350±21 μg per 1 mg of G5-GA75. G5-Ac75-cisplatin complex exhibited controlled release of cisplatin at different pH over a period of 96 h. After 96 h incubation with G5-Ac75-cisplatin, cell viability was 27.47±2.53%, 12.18±0.65% and 11.62±0.84% using platinum concentration of 1 μg/ml, 3 μg/ml and 5 μg/ml, respectively. Meanwhile, 46.33±5.06% cells survived even in the high platinum concentration of 5 μg/ml after 96 h incubation with free cisplatin. G5-GA75 led to 3-6 times higher cisplatin accumulation than free cisplatin in MCF-7R cells, because MCF-7R cells exhibited lower Ctr1 expression and higher ATP7B expression than MCF-7 cells. The G5-GA75-cisplatin complex displayed greater anticancer activity than free cisplatin in the cisplatin-resistant breast cancer cell line MCF-7R. The low levels of Ctr1 and high levels of ATP7B in MCF-7R caused G5-GA75 to allow the accumulation of cisplatin, which in turn increased the cytotoxicity. Results indicated that glutaryl G5 PAMAM may be a potential carrier for cisplatin targeting in breast cancer.

Effects of lead and lead-melatonin exposure on protein and gene expression of metal transporters, proteins and the copper/zinc ratio in rats.

Human lead (Pb) exposure induces many adverse health effects, including some related to lead accumulation in organs. Although lead bio-distribution in the body has been described, the molecular mechanism underlying distribution and excretion is not well understood. The transport of essential and toxic metals is principally mediated by proteins. How lead affects the expression of metal transporter proteins in the principal metal excretory organs, i.e., the liver and kidney, is unknown. Considering that co-administration of melatonin and lead reduces the toxic effects of lead and lead levels in the blood in vivo, we examined how lead and co-administration of lead and melatonin affect the gene and protein expression of metal transporter proteins (ZIP8, ZIP14, CTR1 and DMT1) in these organs. Rats were exposed intraperitoneally to lead or lead-melatonin. Our results show that Pb exposure induces changes in the protein and gene expression of ZIP8, ZIP14 and CTR1. Alterations in the copper/zinc ratio found in the blood, liver and kidney were likely related to these changes. With DMT1 expression (gene and protein), a positive correlation was found with lead levels in the kidney. Co-administration of melatonin and lead reduced lead-induced DMT1 expression through an unknown mechanism. This effect of melatonin relates to reduced lead levels in the blood and kidney. The metal transport protein function and our results suggest that DMT1 likely contributes to lead accumulation in organs. These data further elucidate the effects of lead on Cu and Zn and the molecular mechanism underlying lead bio-distribution in animals.