Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Article Snippet: The human breast cancer cell lines SKBR3, BT474, MDA-MB-453 (American Type Culture Collection) and JIMT-1 (Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were cultured in DMEM, MEM or RPMI-1640 (all Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

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: Influence of high-dose ascorbate alone or in combination with ferric iron on the viability of human pancreatic cancer cell lines. The cell lines BxPC-3, MIA PaCa-2 and PANC-1 were either treated for 24 h with the indicated ascorbate concentrations alone (A), in the form of coincubation simultaneously with ascorbate and 100 µM FC (B), or incubated with 100 µM FC for 24 h as a preincubation before ascorbate treatment (C). Triton™ X-100 at 0.1% (v/v) served as positive control. Cell viability was measured after treatment by MUH assay. The results are presented as a percentage of fluorescence intensity relative to the untreated control. Three independent experiments were performed in duplicates. 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; FC, ferric chloride; MUH, 4-methylumbelliferyl heptanoate.

Article Snippet: The human pancreatic carcinoma cell lines BxPC-3, MIA PaCa-2, and PANC-1 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

Techniques: Incubation, Positive Control, Fluorescence, Control

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: Investigation of the effect of high-dose ascorbate treatment on different apoptosis markers in human pancreatic cancer cell lines. A possible induction of apoptosis in the human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 was investigated by testing caspase-3 cleavage by flow cytometry (A) and western blotting (B). Cells were treated for 6 h with the indicated ascorbate concentrations. 20 µM STS served as positive control. Three independent experiments were performed. Morphological nuclear changes were detected by fluorescence microscopy. The white scale bars represent 25 µm. (C). Cells were treated with the indicated ascorbate concentrations for 6 h and then fixed. 10 µM STS served as positive control. The nuclei were stained with DAPI (blue), the cytoskeleton with phalloidin (red). A representative experiment is shown. Cell cycle analysis was performed by flow cytometry (D). Cells were treated with the indicated ascorbate concentrations for 24 h, fixed, stained with PI, and subsequently detected. Treatment with 1 µM STS for 20 h served as positive control. Three independent experiments were performed. 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 and ***P≤0.001. Asc, ascorbate; DAPI, diamidino-2-phenylindole; PI, propidium iodide; STS, staurosporine.

Article Snippet: The human pancreatic carcinoma cell lines BxPC-3, MIA PaCa-2, and PANC-1 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

Techniques: Flow Cytometry, Western Blot, Positive Control, Fluorescence, Microscopy, Staining, Cell Cycle Assay

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: Investigation of the effect of high-dose ascorbate treatment on different ferroptosis markers in human pancreatic cancer cell lines. The human pancreatic cancer cell lines BxPC-3, MIA PaCa-2, and PANC-1 were treated with or without the ferroptosis inhibitor DFO for 24 h, after which cell viability was measured using the MUH assay (A). Triton™ X-100 at 0.1% (v/v) served as positive control. The results are presented as a percentage of the fluorescence intensity of the untreated control. Three independent experiments were performed in duplicates. As further signs of ferroptosis, protein expression of TfR1, GPX4, and LC3B-II was detected by western blotting (B) and quantified densitometrically (C). Cells were treated with the indicated ascorbate concentrations for 6 h, after which a western blot was performed. 5 µM RSL3 or 60 µM CQ together with 500 nM RAPA were used as positive controls. Signal intensity was analyzed densitometrically and normalized to GAPDH. One representative experiment out of two is shown. Error bars represent the mean ± SD, statistical analysis with one-way ANOVA and subsequent Dunnett's multiple comparisons test, confidence interval 95%. **P≤0.01 and ***P≤0.001. Asc, ascorbate; CQ, chloroquine; DFO, deferoxamine mesylate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GPX4, glutathione peroxidase 4; MUH, 4-methylumbelliferyl heptanoate; RAPA, rapamycin; RSL3, RAS-selective lethal 3; TfR1, transferrin receptor 1.

Article Snippet: The human pancreatic carcinoma cell lines BxPC-3, MIA PaCa-2, and PANC-1 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

Techniques: Positive Control, Fluorescence, Control, Expressing, Western Blot

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: The human pancreatic carcinoma cell lines BxPC-3, MIA PaCa-2, and PANC-1 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

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