hctr1  (Novus Biologicals)

Journal: Journal of Clinical Medicine

Article Title: Targeting Copper in Cancer Imaging and Therapy: A New Theragnostic Agent

doi: 10.3390/jcm12010223

Figure Lengend Snippet: Schematic representation of copper metabolism at the cellular and molecular level ( 64 Cu as copper ions). 64 Cu 2+ ions are bound to plasma proteins which carry them to the external membrane, where they are reduced to Cu 2+ by reductases before their uptake into cells. Reduced copper ions are then transported across the cell membrane by the human copper transporter 1 (hCTR1). In the cell, Cu 2+ ions are closely bound by copper chaperones (cytochrome c oxidase copper chaperone (COX17), copper chaperone for SOD1 (CCS), and antioxidant protein (ATOX1)), which deliver copper ions to the cytosol (via SOD1), mitochondria (via COX) and trans-Golgi network (via copper transporting ATPase A/B). Interestingly, glutathione (GSH) binds excess Cu 2+ to prevent oxidative damage, thus protecting the cell from copper toxicity. Analogous mechanisms are carried out by metallothionein (MT). When intracellular copper is too high, hCTR1 is internalized and destroyed and copper transporting ATPase A/B (ATP7A and ATP7B) transfer from the TGN to the plasma membrane to help in the excretion of copper from the cell (adapted and modified from Michniewicz F. et al.: Copper: An Intracellular Achilles’ Heel Allowing the Targeting of Epigenetics, Kinase Pathways, and Cell Metabolism in Cancer Therapeutics. Chem Med Chem 2021 , 16 , 2315–2329. Copyright Wiley-VCH GmbH. Reproduced with permission).

Article Snippet: A high intracellular concentration of copper is allowed by an elevated expression of hCTR1, and reduced tumor 64 Cu uptake and tumor growth inhibition caused by RNA-mediated hCTR1 knockdown have been shown, suggesting that hCTR1 is a promising novel theragnostic target.

Techniques: Clinical Proteomics, Membrane, Modification

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Dual Action of Acidic Microenvironment on the Enrichment of the Active Metabolite of Disulfiram in Tumor Tissues.

doi: 10.1124/dmd.120.000317

Figure Lengend Snippet: Fig. 5. Intracellular level of Cu2+ after treatment with CuCl2 (A) and intracellular level of CuET (B) after treatment with CuCl2 followed by DTC in hCTR1–MDA-MB-231 cells and vector–MDA-MB-231 cells. Data are expressed as mean 6 S.D. (n = 3). *P , 0.05; **P , 0.01; ***P , 0.001 (two-tailed Student’s t test).

Article Snippet: The hCTR1 lentivirus was custom-made by Cyagen Biosciences (Suzhou, China).

Techniques: Plasmid Preparation, Two Tailed Test

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Dual Action of Acidic Microenvironment on the Enrichment of the Active Metabolite of Disulfiram in Tumor Tissues.

doi: 10.1124/dmd.120.000317

Figure Lengend Snippet: Fig. 6. Intracellular level of CuET after treatments with the mixture of DTC (3 mM) and CuCl2 (3 mM) (A) and CuCl2 (3 mM) followed by DTC (3 mM) (B) in hCTR1–MDA-MB-231 cells and vector–MDA-MB-231 cells. Data are expressed as mean 6 S.D. (n = 3). *P , 0.05; **P , 0.01; ***P , 0.001 (two-tailed Student’s t test).

Article Snippet: The hCTR1 lentivirus was custom-made by Cyagen Biosciences (Suzhou, China).

Techniques: Plasmid Preparation, Two Tailed Test

Journal: International Journal of Nanomedicine

Article Title: Overcoming Radiation Resistance by Iron-Platinum Metal Alloy Nanoparticles in Human Copper Transport 1-Overexpressing Cancer Cells via Mitochondrial Disturbance

doi: 10.2147/ijn.s283147

Figure Lengend Snippet: Figure 1 γ-GCS overexpressed SR3A-13 and SR3A-14 cells exhibited the radioresistant phenomena and enhanced uptake of FePt NPs. (A) Western blotting analysis of γ- GCS and hCtr1 protein levels in SR3A, SR3A-13 and SR3A-14 cells. β-actin was used as a loading control. (B) Representative colony formation photographs of SR3A, SR3A- 13 and SR3A-14 cells irradiated with or without 8 Gy radiation. Noted that number of cells per dish initially plated varied with the dose so that the number of colonies surviving was in the range that could be counted conveniently. (C) Cell survival curves for SR3A series cells exposed to radiation. The surviving fractions of SR3A-13 and SR3A-14 cells were significantly higher than that of the control SR3A cells. (D) Total iron and platinum content determined by ICP-OES (***P < 0.001). Error bars represent ± S.D. (E) Representative TEM images of SR3A, SR3A-13 and SR3A-14 cells treated with FePt NPs. Note that FePt NPs were mainly found in vesicles located in the cytoplasm. Shown in the bottom are high power view of images in red squares shown above.

Article Snippet: The membranes were then incubated with each primary antibody, β-actin (43 kDa, MAB1501, Merck Millipore, Darmstadt, Germany), hCtr1 (25 kDa, NB100-402, Novus Biologicals, LLC, USA), and γ-GCS (73 kDa, sc-166382, Santa Cruz, CA, USA) at 4°C for 16 hours.

Techniques: Western Blot, Control, Irradiation

Journal: International Journal of Nanomedicine

Article Title: Overcoming Radiation Resistance by Iron-Platinum Metal Alloy Nanoparticles in Human Copper Transport 1-Overexpressing Cancer Cells via Mitochondrial Disturbance

doi: 10.2147/ijn.s283147

Figure Lengend Snippet: Figure 4 Elevated hCtr1 expression confers enhanced uptake/transport activity of FePt NPs and induces mitochondria dysfunction. (A) Western blotting analysis of γ-GCSh and hCtr1 protein levels in SR3A-hCtr1-WT cells. β-actin was used as a loading control. (B) The uptake/transport of 1 mg/mL FePt NPs for 24 hrs was significantly increased in SR3A-hCtr1-WT cells as compared with SR3A cells by ICP-OES measurement (***P < 0.001). Error bars represent ± S.D. (C) Representative TEM images of SR3A-hCtr1- WT cells treated with FePt NPs (left). Shown in the right is high-power view of image in red square demonstrating abnormal mitochondria in SR3A-hCtr1-WT cells with increasing membrane density and losing ridges after FePt NPs treatment (red arrowheads). (D) The OCR levels were significantly decreased in SR3A-hCtr1-WT cells treated with FePt NPs, in a time (left)- and concentration (right)-dependent manner.

Article Snippet: The membranes were then incubated with each primary antibody, β-actin (43 kDa, MAB1501, Merck Millipore, Darmstadt, Germany), hCtr1 (25 kDa, NB100-402, Novus Biologicals, LLC, USA), and γ-GCS (73 kDa, sc-166382, Santa Cruz, CA, USA) at 4°C for 16 hours.

Techniques: Expressing, Activity Assay, Western Blot, Control, Membrane, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Overcoming Radiation Resistance by Iron-Platinum Metal Alloy Nanoparticles in Human Copper Transport 1-Overexpressing Cancer Cells via Mitochondrial Disturbance

doi: 10.2147/ijn.s283147

Figure Lengend Snippet: Figure 5 hCtr1 expression significantly enhances FePt NPs-induced radiosensitivity. (A) SR3A-hCtr1-WT cells were treated with 1 mg/mL FePt NPs for 24 hours then irradiated with or without X-rays. Clonogenic assay shows significant decrease of surviving colony numbers in the combination treatment (left). Surviving fractions in these treatments shown (right). (B) ROS was considerably increased after combined treatment of FePt NPs and X-rays in SR3A-hCtr1-WT cells. (C) OCR measured by Seahorse XF24 analyzer was markedly attenuated in SR3A-hCtr1-WT cells treated with FePt NPs and X-rays.

Article Snippet: The membranes were then incubated with each primary antibody, β-actin (43 kDa, MAB1501, Merck Millipore, Darmstadt, Germany), hCtr1 (25 kDa, NB100-402, Novus Biologicals, LLC, USA), and γ-GCS (73 kDa, sc-166382, Santa Cruz, CA, USA) at 4°C for 16 hours.

Techniques: Expressing, Irradiation, Clonogenic Assay

Journal: International Journal of Nanomedicine

Article Title: Overcoming Radiation Resistance by Iron-Platinum Metal Alloy Nanoparticles in Human Copper Transport 1-Overexpressing Cancer Cells via Mitochondrial Disturbance

doi: 10.2147/ijn.s283147

Figure Lengend Snippet: Figure 6 Enhancement of radiation therapy efficacy by utilizing FePt NPs in SR3A-hCtr1-WT-bearing mice under various treatments. (A) Schematic drawing of experimental design for assessing the efficacy of FePt NPs and irradiation (6 Gy) in vivo. (B) Representative H&E and immunostainings of hCtr1 and iron in tumor tissues of SR3A-hCtr1- WT-bearing mice, square was the region magnified 400X in each tumor sections. (C) Tumor growth inhibition of SR3A-hCtr1-WT-subcutaneous xenograft. Growth reduction was seen in the FePt NPs- and X-rays irradiation-treated groups (*p < 0.05; **p < 0.01), but greater reduction was seen in the group of combined treatment with FePt NPs and X-rays (***P<0.01). Error bars represent ± S.D. (D) No significant changes of body weights of the mice among all the treatment groups.

Article Snippet: The membranes were then incubated with each primary antibody, β-actin (43 kDa, MAB1501, Merck Millipore, Darmstadt, Germany), hCtr1 (25 kDa, NB100-402, Novus Biologicals, LLC, USA), and γ-GCS (73 kDa, sc-166382, Santa Cruz, CA, USA) at 4°C for 16 hours.

Techniques: Irradiation, In Vivo, Inhibition

Journal: International Journal of Nanomedicine

Article Title: Overcoming Radiation Resistance by Iron-Platinum Metal Alloy Nanoparticles in Human Copper Transport 1-Overexpressing Cancer Cells via Mitochondrial Disturbance

doi: 10.2147/ijn.s283147

Figure Lengend Snippet: Scheme 1 The schematic illustration of the GSH-overexpressed small-cell lung cancer cells exhibit elevated expression of hCtr1 and are resistant to X-rays irradiation. Radiation resistance can be overcome by enhanced uptake/transport of FePt NPs due to the overexpressed hCtr1 through the mechanisms of ROS outburst and mitochondria dysfunction.

Article Snippet: The membranes were then incubated with each primary antibody, β-actin (43 kDa, MAB1501, Merck Millipore, Darmstadt, Germany), hCtr1 (25 kDa, NB100-402, Novus Biologicals, LLC, USA), and γ-GCS (73 kDa, sc-166382, Santa Cruz, CA, USA) at 4°C for 16 hours.

Techniques: Expressing, Irradiation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Rapid diagnosis of cisplatin-sensitive and resistant cervical squamous cell carcinomas by reverse transcription loop-mediated isothermal amplification

doi:

Figure Lengend Snippet: Primer design for the detection of hCTR1 mRNA by RT-LAMP

Article Snippet: One half was frozen within 30 min after surgery in liquid nitrogen and stored at -80°C until molecular analysis, while the other half was fixed with 10% buffered formalin and embedded in paraffin for routine histopathological examination with H&E staining and IHC examination with monoclonal anti-hCTR1 antibody (Novus, Littleton, USA).

Techniques: Sequencing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Rapid diagnosis of cisplatin-sensitive and resistant cervical squamous cell carcinomas by reverse transcription loop-mediated isothermal amplification

doi:

Figure Lengend Snippet: Sensitivity of LAMP reaction to detect hCTR1. A. The turbidity curve of LAMP technique using a known number of hCTR1 cell copies after 60 min amplification. B. The result of LAMP reaction after 45 min amplification measured by turbidimeter. Lane 1 to 7 was loaded with serially diluted hCTR1 cell copies from one million to one. Lane 8 was loaded as ddH2O as blank control.

Article Snippet: One half was frozen within 30 min after surgery in liquid nitrogen and stored at -80°C until molecular analysis, while the other half was fixed with 10% buffered formalin and embedded in paraffin for routine histopathological examination with H&E staining and IHC examination with monoclonal anti-hCTR1 antibody (Novus, Littleton, USA).

Techniques: Amplification, Control

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Rapid diagnosis of cisplatin-sensitive and resistant cervical squamous cell carcinomas by reverse transcription loop-mediated isothermal amplification

doi:

Figure Lengend Snippet: Representative hCTR1 over-expressed (A) cervical squamous cell carcinoma and low-expressed carcinoma (B) cases analyzed by immunohistochemical staining and RT-LAMP. In RT-LAMP reaction, carcinoma RNA was loaded into lane 1 to 4 as duplicates, while adjacent normal tissue RNA was loaded into lane 5 to 8 as duplicates. *, P<0.05; **, P>0.05.

Article Snippet: One half was frozen within 30 min after surgery in liquid nitrogen and stored at -80°C until molecular analysis, while the other half was fixed with 10% buffered formalin and embedded in paraffin for routine histopathological examination with H&E staining and IHC examination with monoclonal anti-hCTR1 antibody (Novus, Littleton, USA).

Techniques: Immunohistochemical staining, Staining

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Rapid diagnosis of cisplatin-sensitive and resistant cervical squamous cell carcinomas by reverse transcription loop-mediated isothermal amplification

doi:

Figure Lengend Snippet: Primer design for the detection of hCTR1 mRNA by RT-LAMP

Article Snippet: RT- LAMP primers targeting hCTR1 mRNA were designed by Primer ExplorerV4 (Eiken Chemical Co, Tokyo, Japan) as indicated in .

Techniques: Sequencing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Rapid diagnosis of cisplatin-sensitive and resistant cervical squamous cell carcinomas by reverse transcription loop-mediated isothermal amplification

doi:

Figure Lengend Snippet: Sensitivity of LAMP reaction to detect hCTR1. A. The turbidity curve of LAMP technique using a known number of hCTR1 cell copies after 60 min amplification. B. The result of LAMP reaction after 45 min amplification measured by turbidimeter. Lane 1 to 7 was loaded with serially diluted hCTR1 cell copies from one million to one. Lane 8 was loaded as ddH2O as blank control.

Article Snippet: RT- LAMP primers targeting hCTR1 mRNA were designed by Primer ExplorerV4 (Eiken Chemical Co, Tokyo, Japan) as indicated in .

Techniques: Amplification, Control

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Rapid diagnosis of cisplatin-sensitive and resistant cervical squamous cell carcinomas by reverse transcription loop-mediated isothermal amplification

doi:

Figure Lengend Snippet: Representative hCTR1 over-expressed (A) cervical squamous cell carcinoma and low-expressed carcinoma (B) cases analyzed by immunohistochemical staining and RT-LAMP. In RT-LAMP reaction, carcinoma RNA was loaded into lane 1 to 4 as duplicates, while adjacent normal tissue RNA was loaded into lane 5 to 8 as duplicates. *, P<0.05; **, P>0.05.

Article Snippet: RT- LAMP primers targeting hCTR1 mRNA were designed by Primer ExplorerV4 (Eiken Chemical Co, Tokyo, Japan) as indicated in .

Techniques: Immunohistochemical staining, Staining