p her3  (Cell Signaling Technology Inc)

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 downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

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 downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control

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 downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

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: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

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

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 downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control

Journal: European Journal of Medical Research

Article Title: Cigarette smoke extract promotes metastasis and oxaliplatin resistance in colon adenocarcinoma through GDF15/ERBB2/AKT pathway

doi: 10.1186/s40001-025-03233-8

Figure Lengend Snippet: GDF15 influenced AKT signaling and increased GSH levels contributing to L -OHP resistance. A Enrichment analysis of DEGs from transcriptome sequencing in HT29-CSE-P30 and HT29-CSE-P30-sh-GDF15 cells. B Effect of GDF15 on intracellular glutathione (GSH) levels in COAD cells. C Correlation analysis between GDF15 and SLC7A11 expression in colon cancer tissues from the GSE39582 data set, assessed using Pearson’s correlation coefficient. D OS of COAD patients stratified by high vs. low SLC7A11 expression in the GSE39582 cohort, analyzed by Kaplan–Meier curves. E Interaction between GDF15 and ERBB2 proteins in HT29 cells, evaluated by co-immunoprecipitation (Co-IP). F , G WB analysis and quantification of ERBB2, p-ERBB2, AKT, p-AKT, and SLC7A11 protein levels under GDF15 modulation. * P < 0.05, ** P < 0.01, *** P < 0.001. L-OHP oxaliplatin; DEGs differentially expressed genes; OS overall survival; WB western blotting

Article Snippet: The membranes underwent a 2-h blocking with 5% non-fat milk in TBST (TBS containing 0.1% Tween-20), followed by an overnight incubation at 4 °C with primary antibodies: anti-GDF15 (27455-1-AP, 1:1000; Proteintech, Wuhan, China), anti- E -cadherin (20874-1-AP, 1:1000; Proteintech, Wuhan, China), anti- N -cadherin (22018-1-AP, 1:1000; Proteintech, Wuhan, China), anti-ERBB2 (18299-1-AP, 1:1000; Proteintech, Wuhan, China), anti- p -ERBB2 (#2243, 1:1000; CST, Boston, USA), anti-AKT (#9272, 1:1000; CST, Boston, USA), anti- p -AKT (#4060, 1:1000; CST, Boston, USA), anti-SLC7A11 (DF12509, 1:1000; Affinity, JiangSu, China) and anti- β -Tublin (GB15140-100, 1:1000; Servicebio, Wuhan, China).

Techniques: Sequencing, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot

Journal: European Journal of Medical Research

Article Title: Cigarette smoke extract promotes metastasis and oxaliplatin resistance in colon adenocarcinoma through GDF15/ERBB2/AKT pathway

doi: 10.1186/s40001-025-03233-8

Figure Lengend Snippet: CSE promoted metastasis and L -OHP resistance through GDF15/ERBB2/AKT pathway in vitro. A Long-term CSE-exposed colon cancer cells were treated with the highly selective AKT allosteric inhibitor MK-2206, AKT-specific agonist SC79, and knocked down ERBB2 or GDF15. B Levels of the GDF15, E -cadherin, N -cadherin, ERBB2, P-ERBB2, AKT, P-AKT and SLC7A11 proteins were determined using WB assay and quantified using gray scale analysis . C Wound healing assay demonstrated migration capacity (0/24 h imaging). D Transwell invasion/migration assay with crystal violet staining. E CCK-8 assay to detect the cell viability in HT29, HCT116 cells after treatment with L -OHP for 48 h. F Flow cytometry analysis of apoptosis inducted by L -OHP (HT29:10 µM; HCT116: 5 µM) for 48 h. G Levels of GSH were evaluated in HT29, HCT116 cells. * P < 0.05, ** P < 0.01, *** P < 0.001. CSE cigarette smoke extract; L-OHP oxaliplatin

Article Snippet: The membranes underwent a 2-h blocking with 5% non-fat milk in TBST (TBS containing 0.1% Tween-20), followed by an overnight incubation at 4 °C with primary antibodies: anti-GDF15 (27455-1-AP, 1:1000; Proteintech, Wuhan, China), anti- E -cadherin (20874-1-AP, 1:1000; Proteintech, Wuhan, China), anti- N -cadherin (22018-1-AP, 1:1000; Proteintech, Wuhan, China), anti-ERBB2 (18299-1-AP, 1:1000; Proteintech, Wuhan, China), anti- p -ERBB2 (#2243, 1:1000; CST, Boston, USA), anti-AKT (#9272, 1:1000; CST, Boston, USA), anti- p -AKT (#4060, 1:1000; CST, Boston, USA), anti-SLC7A11 (DF12509, 1:1000; Affinity, JiangSu, China) and anti- β -Tublin (GB15140-100, 1:1000; Servicebio, Wuhan, China).

Techniques: In Vitro, Wound Healing Assay, Migration, Imaging, Staining, CCK-8 Assay, Flow Cytometry

Journal: European Journal of Medical Research

Article Title: Cigarette smoke extract promotes metastasis and oxaliplatin resistance in colon adenocarcinoma through GDF15/ERBB2/AKT pathway

doi: 10.1186/s40001-025-03233-8

Figure Lengend Snippet: Overall mode diagram of CSE promoting the metastasis and L -OHP resistance in COAD. CSE significantly promoted the expression of GDF15 in COAD. GDF15 can promote the phosphorylation of ERBB2 and AKT. Phosphorylated AKT drives colon cancer metastasis by promoting EMT, while simultaneously upregulating SLC7A11 expression to increase intracellular GSH levels, thereby inducing chemoresistance. CSE cigarette smoke extract; L-OHP oxaliplatin; COAD colon adenocarcinoma; EMT epithelial–mesenchymal transition; GSH glutathione

Article Snippet: The membranes underwent a 2-h blocking with 5% non-fat milk in TBST (TBS containing 0.1% Tween-20), followed by an overnight incubation at 4 °C with primary antibodies: anti-GDF15 (27455-1-AP, 1:1000; Proteintech, Wuhan, China), anti- E -cadherin (20874-1-AP, 1:1000; Proteintech, Wuhan, China), anti- N -cadherin (22018-1-AP, 1:1000; Proteintech, Wuhan, China), anti-ERBB2 (18299-1-AP, 1:1000; Proteintech, Wuhan, China), anti- p -ERBB2 (#2243, 1:1000; CST, Boston, USA), anti-AKT (#9272, 1:1000; CST, Boston, USA), anti- p -AKT (#4060, 1:1000; CST, Boston, USA), anti-SLC7A11 (DF12509, 1:1000; Affinity, JiangSu, China) and anti- β -Tublin (GB15140-100, 1:1000; Servicebio, Wuhan, China).

Techniques: Expressing, Phospho-proteomics