recombinant hepcidin  (Labeo Technologies Inc)

Journal: Nature metabolism

Article Title: Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells.

doi: 10.1038/s42255-021-00406-7

Figure Lengend Snippet: qPCR analysis for hepcidin ( Hamp ) after treatment with FG4592 (100 μM) or vehicle for 16 hours in (A) HCT116 cells (N=3 biologically independent cell replicates) and (B) enteroids generated from mice with inducible, colon epithelial deletion of APC and p53 and activation of KRAS (N=3 biologically independent samples). (C) qPCR analysis of Hamp in the colon of mice with embryonic, intestinal epithelial-specific overexpression of HIF-2α (HIF-2α OE ) compared to wild-type mice (HIF-2α WT ) (HIF-2α WT N=6 and HIF-2α OE N=10 biologically independent samples from independent animals). (D) qPCR analysis of Hamp in the colon of colon epithelial-specific HIF-2α WT and HIF-2α OE mice that are also deficient for APC for 30 days HIF-2α WT N=3 and HIF-2α OE N=3 biologically independent samples from independent animals). (E) qPCR analysis of Hamp in HCT116 cells treated for 24 hours with conditioned media (CM) from RAW 264.7 macrophages that had been treated with 10 ng/mL LPS for 16 hours (N=3 biologically independent cell replicates). (F) Relative luciferase activity of the human hepcidin promoter in HCT116 cells treated with vehicle (Veh), live bacteria (Live), or heat-killed bacteria (HK), or (G) vehicle (Veh) or bacteria-derived metabolites (N=3 independent cell replicates). (H) Methylation status of the human hepcidin promoter in human colorectal cancer tissue (Normal N=37 and Tumor N=313). (I) HCT116 cells treated with vehicle (Veh) or 5AZA (10 μM) for 72 hours and then treated with vehicle (Veh) and/or FG4592 (100 μM) for 16 hours and analyzed via Western blot analysis for DNA methyltransferase 1 (DNMT1) and (J) qPCR analysis for HAMP expression (N=3 biologically independent cell replicates). (K) HCT116 cells pretreated with or without FG4592 (100 μM) for 16 hours and then administered a panel of epigenetic modifiers for 24 hours. qPCR was used to measure the expression of HAMP and the HIF-2α target, ANKRD37 (N=3 biologically independent cell replicates for each treatment). (L) Patient-derived enteroids were either pretreated with or without FG4592 (100 μM) for 16 hours and were then administered a panel of epigenetic modifiers for 24 hours. qPCR was used to measure the expression of HAMP and the HIF-2α target, ANKRD37 (N=3 biologically independent samples for each treatment). Data represent the mean ± SEM. Significance was determined by 2-tailed, unpaired t test (A-E, H) or 1-way ANOVA with Tukey’s post hoc (F, J, K-L). **** P < 0.0001.

Article Snippet: The following concentrations were used for cell treatments: 250 ng/ml doxycycline, 100 μM FG4592 (Selleckchem), 1 mg/ml human recombinant hepcidin (Bachem), 200 μM DFO (Sigma-Aldrich), 10 μM 5AZA (Cayman, Ann Arbor, MI), 10 ng/mL LPS (Sigma-Aldrich), 100 μM Adenosine (A4036–5G, Sigma) 100 μM Cytosine (C3056–1G, Sigma), 100 μM Thymidine (T1895–1G, Sigma), 100 μM Guanosine (G6264–1G, Sigma).

Techniques: Generated, Activation Assay, Over Expression, Luciferase, Activity Assay, Derivative Assay, Methylation, Western Blot, Expressing

Journal: Nature metabolism

Article Title: Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells.

doi: 10.1038/s42255-021-00406-7

Figure Lengend Snippet: (A) qPCR analysis for the ferroportin transcript in late passages of cells that were made stable for a doxycycline (dox) inducible ferroportin GFP overexpression construct, following 16 hour treatment with dox (250 ng/mL) (N=3 biologically independent cell replicates for each cell line and treatment, HEK293 p=.0001, IEC6 p=0.0021, HT29 p<0.0001, SW480 p=0.0103) (B) Western blot analysis for GFP and ferroportin (FPN) in stable, normal ferroportin GFP overexpressing cell lines following 16 hour treatment with dox (250 ng/mL). (C and D) Cell growth MTT assay in HEK293 ferroportin GFP (C) (N=3 biologically independent cell replicates for each cell line, treatment, and time point, p<0.0001) and (D) IEC6 ferroportin GFP cells (N=3 biologically independent cell replicates for each cell line, treatment, and time point, p=0.0008) treated with vehicle, dox (250 ng/mL), or dox (250 ng/mL) and recombinant hepcidin (1 μg/mL) (E-H) Representative crystal violet staining images (E) and quantification (F) of HEK293 ferroportin GFP (N=3 biologically independent cell replicates for each cell line and treatment, p=0.0005) and IEC6 ferroportin GFP cells, images (G) and quantitation (H) (N=3 biologically independent cell replicates for each cell line and treatment, p=0.0025) ten days following treatment with vehicle, dox (250 ng/mL), or dox (250 ng/mL) and recombinant hepcidin (1 μg/mL) Data represent the mean ± SEM. Significance was determined by 2- tailed, unpaired t test (A) or by 1-way ANOVA with Tukey’s post hoc (C,D,F,H). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 comparing within each cell line or across treatment groups.

Article Snippet: The following concentrations were used for cell treatments: 250 ng/ml doxycycline, 100 μM FG4592 (Selleckchem), 1 mg/ml human recombinant hepcidin (Bachem), 200 μM DFO (Sigma-Aldrich), 10 μM 5AZA (Cayman, Ann Arbor, MI), 10 ng/mL LPS (Sigma-Aldrich), 100 μM Adenosine (A4036–5G, Sigma) 100 μM Cytosine (C3056–1G, Sigma), 100 μM Thymidine (T1895–1G, Sigma), 100 μM Guanosine (G6264–1G, Sigma).

Techniques: Over Expression, Construct, Western Blot, MTT Assay, Recombinant, Staining, Quantitation Assay

Journal: Nature metabolism

Article Title: Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells.

doi: 10.1038/s42255-021-00406-7

Figure Lengend Snippet: (A-D) Patient-derived CRC tumor enteroids were treated with DFO for 7-days at the indicated concentrations. (A-B) Bright field images (A) and quantification of enteroid size from bright field images (B) (N=3 biologically independent replicates for each treatment, 10 μM p=0.0082 and 100 μM p=0.0015). (C) Immunohistochemistry Ki67 staining (D) and quantification of Ki67+ nuclei (D) (N=3 biologically independent replicates for each treatment, 10 μM p<0.0001 and 100 μM p<0.0001). (E) Pyrimidine and purine metabolite abundance in patient-derived enteroids after 4-days of DFO treatment (10 μM) measured via metabolomics (N=3 biologically independent replicates for each treatment, Thymidine p=0.0024, Thymine p<0.0001, CMP p=0.0015, CDP p=0.0250, dAMP p=0.0274, ATP p=0.0164, Adenine p=0.0243, GMP p=0.0014, IMP p=0.0058, XMP p=0.0059, L-Glutamine p=0.0095, Carbamoyl phosphate p=0.0274, UMP p=0.0022, UDP p=0.0175, Uridine p=0.0019, Uracil p=0.0138, and AICAR p=0.0160). (F G) Schematics of where DFO-mediated iron chelation impinges on pyrimidine and purine synthesis; blue is down regulated, red is upregulated, and black is undetected or unchanged. (H) Nucleotide abundance in tumors from sporadic CRC, colon epithelial hepcidin or ferroportin knockout model deficient or wild-type for colon epithelial hepcidin or ferroportin (Hamp +/+ , Hamp ΔCE , Fpn +/+ , and Fpn ΔCE ) (Hamp fl/fl N=7, Hamp ΔCE N=8, Fpn fl/fl N=5, and Fpn ΔCE N=5 biologically independent samples, [Hamp] Thymidine p=0.0317, Deoxycytidine p<0.0001, Cytosine p<0.0001, Cytidine 0.0001, pUracil 0..0001, Adenosine p=0.0189, Xantine p=0.0015, Hypoxanthine p==.0010, Guanine p=0.0410, Inosine p<0.0001, and Orotate p=0.0053; [Fpn] Thymine p<0.0001, Deoxycytidine p<0.0001, Cytosine p= 0.0067, Cytidine p<0.0001, Deoxyuridine p=0.0301, Adenosine p<0.0001, Adenine p<0.0001, Guanine p<0.0001, Uridine 5-monophosphate p=0.0075, Uridine p<0.0001, Uracil p<0.0001, Guanosine p<0.0001, Inosine p=.0001, Orotate P<0.0001.). Data represent the mean ± SEM. Significance was determined by 2-tailed, unpaired t test (E and H) or by 1-way ANOVA with Tukey’s post hoc (B and D). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001 comparing treatment groups. # P < 0.05 is specific for metabolomics changes in tumors from the ferroportin (Fpn) knockout model.

Article Snippet: The following concentrations were used for cell treatments: 250 ng/ml doxycycline, 100 μM FG4592 (Selleckchem), 1 mg/ml human recombinant hepcidin (Bachem), 200 μM DFO (Sigma-Aldrich), 10 μM 5AZA (Cayman, Ann Arbor, MI), 10 ng/mL LPS (Sigma-Aldrich), 100 μM Adenosine (A4036–5G, Sigma) 100 μM Cytosine (C3056–1G, Sigma), 100 μM Thymidine (T1895–1G, Sigma), 100 μM Guanosine (G6264–1G, Sigma).

Techniques: Derivative Assay, Immunohistochemistry, Staining, Knock-Out

Journal: Nature metabolism

Article Title: Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells.

doi: 10.1038/s42255-021-00406-7

Figure Lengend Snippet: (A) Seahorse analysis of mitochondrial metabolism in HCT116 and (B) SW480 cells 24 hours after DFO administration (10μM) (N=4 biologically independent cell replicates for each treatment condition). (C) NAD+/NADH ratio in HCT116 (N=6 biologically independent cell replicates, p=0.0218) and (D) SW480 (Vehicle N=3 and DFO N=4 biologically independent cell replicates, p=0.0222) treated with DFO (10μM). (E) Aspartate levels in HCT116 cells (N=5 biologically independent cell replicates, p<0.0001) and (F) SW480 cells (N=5 biologically independent cell replicates, p<0.0001) treated with DFO (10μM). (G) Aspartate abundance in patient-derived enteroids treated with DFO (100μM for 72 hours) (N=5 biologically independent replicates, p=0.0019) and (H) sporadic tumors from wild-type or colon-specific hepcidin-deficient mice (Hamp) (N=5 biologically independent samples from independent animals, p=0.0014), and (I) sporadic tumors from wild-type or colon-specific ferroportin-deficient mice (Fpn) (N=5 biologically independent samples from independent animals, p=0.0006). (J) Cell growth in HCT116 cells (N=8 biologically independent cell replicates, p<0.0001) and (K) SW480 cells (N=8 biologically independent cell replicates, p<0.0001) following DFO treatment (10μM) with and without aspartate supplementation (100μM). (L and M) Western blot analysis of mitochondrial enzymes following 24-hours of DFO (10μM) treatment in HCT116 cells (L) and SW480 cells (M). Data represent the mean ± SEM. Significance was determined by 2-tailed, unpaired t test (C-I) or one-way ANOVA followed by Tukey’s post hoc (J and K). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 compared to vehicle or between genotypes.

Article Snippet: The following concentrations were used for cell treatments: 250 ng/ml doxycycline, 100 μM FG4592 (Selleckchem), 1 mg/ml human recombinant hepcidin (Bachem), 200 μM DFO (Sigma-Aldrich), 10 μM 5AZA (Cayman, Ann Arbor, MI), 10 ng/mL LPS (Sigma-Aldrich), 100 μM Adenosine (A4036–5G, Sigma) 100 μM Cytosine (C3056–1G, Sigma), 100 μM Thymidine (T1895–1G, Sigma), 100 μM Guanosine (G6264–1G, Sigma).

Techniques: Derivative Assay, Western Blot