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The influence of iron and TfR1 on the ascorbate effect in human pancreatic cancer cell lines. To investigate the effect of iron on ascorbate action, the three human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 were either incubated simultaneously for 6 h with ascorbate at different concentrations and 100 µM ferric chloride (FC) (A) or treated with ascorbate after 24 h preincubation with ferric chloride (B). The intracellular ROS levels were determined after treatment by flow cytometry using the DCFH-DA assay. The percentage of DCF-positive cells is shown as a measure of intracellular ROS accumulation. Statistically significant differences between the combination treatment with iron and ascorbate treatment alone are marked by asterisks. Three independent experiments were performed. The intracellular LIP after 24-h incubation with ferric chloride was determined by flow cytometry using the <t>calcein</t> <t>AM</t> assay (C). The relative labile iron pool is shown in relation to the untreated control. Three independent experiments were performed. The basal protein expression of TfR1 in the pancreatic cancer cell lines and the non-malignant pancreatic ductal epithelial cell line HPDE6c7 was determined by western blotting, as well as the influence of ferric chloride on TfR1 expression in the three pancreatic cancer cell lines (D). The western blot results were also analyzed densitometrically. A representative experiment is shown for basal expression. For TfR1 expression after iron treatment, two independent experiments were performed, a representative western blot is shown. The influence of the 24-h ferric chloride treatment as well as ascorbate treatment was additionally determined at the mRNA level by qPCR (E). Error bars represent the mean ± SD, statistical analysis with one-way ANOVA and subsequent Dunnett's multiple comparisons test, confidence interval 95%. *P≤0.05, **P≤0.01, and ***P≤0.001. Asc, ascorbate; DCFH-DA, dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; FC, ferric chloride; LIP, labile iron pool; ROS, reactive oxygen species; TfR1, transferrin receptor 1.
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The influence of iron and TfR1 on the ascorbate effect in human pancreatic cancer cell lines. To investigate the effect of iron on ascorbate action, the three human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 were either incubated simultaneously for 6 h with ascorbate at different concentrations and 100 µM ferric chloride (FC) (A) or treated with ascorbate after 24 h preincubation with ferric chloride (B). The intracellular ROS levels were determined after treatment by flow cytometry using the DCFH-DA assay. The percentage of DCF-positive cells is shown as a measure of intracellular ROS accumulation. Statistically significant differences between the combination treatment with iron and ascorbate treatment alone are marked by asterisks. Three independent experiments were performed. The intracellular LIP after 24-h incubation with ferric chloride was determined by flow cytometry using the <t>calcein</t> <t>AM</t> assay (C). The relative labile iron pool is shown in relation to the untreated control. Three independent experiments were performed. The basal protein expression of TfR1 in the pancreatic cancer cell lines and the non-malignant pancreatic ductal epithelial cell line HPDE6c7 was determined by western blotting, as well as the influence of ferric chloride on TfR1 expression in the three pancreatic cancer cell lines (D). The western blot results were also analyzed densitometrically. A representative experiment is shown for basal expression. For TfR1 expression after iron treatment, two independent experiments were performed, a representative western blot is shown. The influence of the 24-h ferric chloride treatment as well as ascorbate treatment was additionally determined at the mRNA level by qPCR (E). Error bars represent the mean ± SD, statistical analysis with one-way ANOVA and subsequent Dunnett's multiple comparisons test, confidence interval 95%. *P≤0.05, **P≤0.01, and ***P≤0.001. Asc, ascorbate; DCFH-DA, dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; FC, ferric chloride; LIP, labile iron pool; ROS, reactive oxygen species; TfR1, transferrin receptor 1.
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The influence of iron and TfR1 on the ascorbate effect in human pancreatic cancer cell lines. To investigate the effect of iron on ascorbate action, the three human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 were either incubated simultaneously for 6 h with ascorbate at different concentrations and 100 µM ferric chloride (FC) (A) or treated with ascorbate after 24 h preincubation with ferric chloride (B). The intracellular ROS levels were determined after treatment by flow cytometry using the DCFH-DA assay. The percentage of DCF-positive cells is shown as a measure of intracellular ROS accumulation. Statistically significant differences between the combination treatment with iron and ascorbate treatment alone are marked by asterisks. Three independent experiments were performed. The intracellular LIP after 24-h incubation with ferric chloride was determined by flow cytometry using the <t>calcein</t> <t>AM</t> assay (C). The relative labile iron pool is shown in relation to the untreated control. Three independent experiments were performed. The basal protein expression of TfR1 in the pancreatic cancer cell lines and the non-malignant pancreatic ductal epithelial cell line HPDE6c7 was determined by western blotting, as well as the influence of ferric chloride on TfR1 expression in the three pancreatic cancer cell lines (D). The western blot results were also analyzed densitometrically. A representative experiment is shown for basal expression. For TfR1 expression after iron treatment, two independent experiments were performed, a representative western blot is shown. The influence of the 24-h ferric chloride treatment as well as ascorbate treatment was additionally determined at the mRNA level by qPCR (E). Error bars represent the mean ± SD, statistical analysis with one-way ANOVA and subsequent Dunnett's multiple comparisons test, confidence interval 95%. *P≤0.05, **P≤0.01, and ***P≤0.001. Asc, ascorbate; DCFH-DA, dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; FC, ferric chloride; LIP, labile iron pool; ROS, reactive oxygen species; TfR1, transferrin receptor 1.
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The influence of iron and TfR1 on the ascorbate effect in human pancreatic cancer cell lines. To investigate the effect of iron on ascorbate action, the three human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 were either incubated simultaneously for 6 h with ascorbate at different concentrations and 100 µM ferric chloride (FC) (A) or treated with ascorbate after 24 h preincubation with ferric chloride (B). The intracellular ROS levels were determined after treatment by flow cytometry using the DCFH-DA assay. The percentage of DCF-positive cells is shown as a measure of intracellular ROS accumulation. Statistically significant differences between the combination treatment with iron and ascorbate treatment alone are marked by asterisks. Three independent experiments were performed. The intracellular LIP after 24-h incubation with ferric chloride was determined by flow cytometry using the <t>calcein</t> <t>AM</t> assay (C). The relative labile iron pool is shown in relation to the untreated control. Three independent experiments were performed. The basal protein expression of TfR1 in the pancreatic cancer cell lines and the non-malignant pancreatic ductal epithelial cell line HPDE6c7 was determined by western blotting, as well as the influence of ferric chloride on TfR1 expression in the three pancreatic cancer cell lines (D). The western blot results were also analyzed densitometrically. A representative experiment is shown for basal expression. For TfR1 expression after iron treatment, two independent experiments were performed, a representative western blot is shown. The influence of the 24-h ferric chloride treatment as well as ascorbate treatment was additionally determined at the mRNA level by qPCR (E). Error bars represent the mean ± SD, statistical analysis with one-way ANOVA and subsequent Dunnett's multiple comparisons test, confidence interval 95%. *P≤0.05, **P≤0.01, and ***P≤0.001. Asc, ascorbate; DCFH-DA, dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; FC, ferric chloride; LIP, labile iron pool; ROS, reactive oxygen species; TfR1, transferrin receptor 1.
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The influence of iron and TfR1 on the ascorbate effect in human pancreatic cancer cell lines. To investigate the effect of iron on ascorbate action, the three human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 were either incubated simultaneously for 6 h with ascorbate at different concentrations and 100 µM ferric chloride (FC) (A) or treated with ascorbate after 24 h preincubation with ferric chloride (B). The intracellular ROS levels were determined after treatment by flow cytometry using the DCFH-DA assay. The percentage of DCF-positive cells is shown as a measure of intracellular ROS accumulation. Statistically significant differences between the combination treatment with iron and ascorbate treatment alone are marked by asterisks. Three independent experiments were performed. The intracellular LIP after 24-h incubation with ferric chloride was determined by flow cytometry using the <t>calcein</t> <t>AM</t> assay (C). The relative labile iron pool is shown in relation to the untreated control. Three independent experiments were performed. The basal protein expression of TfR1 in the pancreatic cancer cell lines and the non-malignant pancreatic ductal epithelial cell line HPDE6c7 was determined by western blotting, as well as the influence of ferric chloride on TfR1 expression in the three pancreatic cancer cell lines (D). The western blot results were also analyzed densitometrically. A representative experiment is shown for basal expression. For TfR1 expression after iron treatment, two independent experiments were performed, a representative western blot is shown. The influence of the 24-h ferric chloride treatment as well as ascorbate treatment was additionally determined at the mRNA level by qPCR (E). Error bars represent the mean ± SD, statistical analysis with one-way ANOVA and subsequent Dunnett's multiple comparisons test, confidence interval 95%. *P≤0.05, **P≤0.01, and ***P≤0.001. Asc, ascorbate; DCFH-DA, dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; FC, ferric chloride; LIP, labile iron pool; ROS, reactive oxygen species; TfR1, transferrin receptor 1.
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The influence of iron and TfR1 on the ascorbate effect in human pancreatic cancer cell lines. To investigate the effect of iron on ascorbate action, the three human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 were either incubated simultaneously for 6 h with ascorbate at different concentrations and 100 µM ferric chloride (FC) (A) or treated with ascorbate after 24 h preincubation with ferric chloride (B). The intracellular ROS levels were determined after treatment by flow cytometry using the DCFH-DA assay. The percentage of DCF-positive cells is shown as a measure of intracellular ROS accumulation. Statistically significant differences between the combination treatment with iron and ascorbate treatment alone are marked by asterisks. Three independent experiments were performed. The intracellular LIP after 24-h incubation with ferric chloride was determined by flow cytometry using the <t>calcein</t> <t>AM</t> assay (C). The relative labile iron pool is shown in relation to the untreated control. Three independent experiments were performed. The basal protein expression of TfR1 in the pancreatic cancer cell lines and the non-malignant pancreatic ductal epithelial cell line HPDE6c7 was determined by western blotting, as well as the influence of ferric chloride on TfR1 expression in the three pancreatic cancer cell lines (D). The western blot results were also analyzed densitometrically. A representative experiment is shown for basal expression. For TfR1 expression after iron treatment, two independent experiments were performed, a representative western blot is shown. The influence of the 24-h ferric chloride treatment as well as ascorbate treatment was additionally determined at the mRNA level by qPCR (E). Error bars represent the mean ± SD, statistical analysis with one-way ANOVA and subsequent Dunnett's multiple comparisons test, confidence interval 95%. *P≤0.05, **P≤0.01, and ***P≤0.001. Asc, ascorbate; DCFH-DA, dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; FC, ferric chloride; LIP, labile iron pool; ROS, reactive oxygen species; TfR1, transferrin receptor 1.
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The influence of iron and TfR1 on the ascorbate effect in human pancreatic cancer cell lines. To investigate the effect of iron on ascorbate action, the three human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 were either incubated simultaneously for 6 h with ascorbate at different concentrations and 100 µM ferric chloride (FC) (A) or treated with ascorbate after 24 h preincubation with ferric chloride (B). The intracellular ROS levels were determined after treatment by flow cytometry using the DCFH-DA assay. The percentage of DCF-positive cells is shown as a measure of intracellular ROS accumulation. Statistically significant differences between the combination treatment with iron and ascorbate treatment alone are marked by asterisks. Three independent experiments were performed. The intracellular LIP after 24-h incubation with ferric chloride was determined by flow cytometry using the calcein AM assay (C). The relative labile iron pool is shown in relation to the untreated control. Three independent experiments were performed. The basal protein expression of TfR1 in the pancreatic cancer cell lines and the non-malignant pancreatic ductal epithelial cell line HPDE6c7 was determined by western blotting, as well as the influence of ferric chloride on TfR1 expression in the three pancreatic cancer cell lines (D). The western blot results were also analyzed densitometrically. A representative experiment is shown for basal expression. For TfR1 expression after iron treatment, two independent experiments were performed, a representative western blot is shown. The influence of the 24-h ferric chloride treatment as well as ascorbate treatment was additionally determined at the mRNA level by qPCR (E). Error bars represent the mean ± SD, statistical analysis with one-way ANOVA and subsequent Dunnett's multiple comparisons test, confidence interval 95%. *P≤0.05, **P≤0.01, and ***P≤0.001. Asc, ascorbate; DCFH-DA, dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; FC, ferric chloride; LIP, labile iron pool; ROS, reactive oxygen species; TfR1, transferrin receptor 1.

Journal: Oncology Reports

Article Title: Role of iron and TfR1 in the application of high-dose ascorbate against pancreatic cancer

doi: 10.3892/or.2026.9083

Figure Lengend Snippet: The influence of iron and TfR1 on the ascorbate effect in human pancreatic cancer cell lines. To investigate the effect of iron on ascorbate action, the three human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 were either incubated simultaneously for 6 h with ascorbate at different concentrations and 100 µM ferric chloride (FC) (A) or treated with ascorbate after 24 h preincubation with ferric chloride (B). The intracellular ROS levels were determined after treatment by flow cytometry using the DCFH-DA assay. The percentage of DCF-positive cells is shown as a measure of intracellular ROS accumulation. Statistically significant differences between the combination treatment with iron and ascorbate treatment alone are marked by asterisks. Three independent experiments were performed. The intracellular LIP after 24-h incubation with ferric chloride was determined by flow cytometry using the calcein AM assay (C). The relative labile iron pool is shown in relation to the untreated control. Three independent experiments were performed. The basal protein expression of TfR1 in the pancreatic cancer cell lines and the non-malignant pancreatic ductal epithelial cell line HPDE6c7 was determined by western blotting, as well as the influence of ferric chloride on TfR1 expression in the three pancreatic cancer cell lines (D). The western blot results were also analyzed densitometrically. A representative experiment is shown for basal expression. For TfR1 expression after iron treatment, two independent experiments were performed, a representative western blot is shown. The influence of the 24-h ferric chloride treatment as well as ascorbate treatment was additionally determined at the mRNA level by qPCR (E). Error bars represent the mean ± SD, statistical analysis with one-way ANOVA and subsequent Dunnett's multiple comparisons test, confidence interval 95%. *P≤0.05, **P≤0.01, and ***P≤0.001. Asc, ascorbate; DCFH-DA, dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; FC, ferric chloride; LIP, labile iron pool; ROS, reactive oxygen species; TfR1, transferrin receptor 1.

Article Snippet: A total of 24 h after seeding, the cells were harvested by trypsinization, washed twice with PBS, and incubated after addition of PBS with 500 nM calcein AM (cat. no. C1359, MedChemExpress) for 15 min at 37°C in the dark.

Techniques: Incubation, Flow Cytometry, DCFH-DA Assay, Calcein AM Assay, Control, Expressing, Western Blot