Journal: International Journal of Molecular Medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple-negative breast cancer metastasis via the inhibition of CXCR4 expression

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: The HER3 pathway is associated with the DMFS of patients with TNBC. (A) c-Met, EGFR, IGF1R, IR and HER3 protein phosphorylation levels were measured using Luminex ® 200. The experiments were performed in duplicate and repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with 4T1 cells. (B) The expression levels of p-HER3, HER3, p-Akt, Akt, p-mTOR, mTOR, p-ERK and ERK were detected using western blot analysis. The expression levels of β-actin were used as internal controls. Quantification of signals is presented as fold of changes relative to phosphorylated proteins vs. total proteins. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with 4T1 cells. (C) Kaplan-Meier Plotter analysis indicated that DMFS was associated with HER3 expression (low expression group, n=212; high expression group, n=212) among 424 TNBC cases (log-rank, P=0.0035). DMFS, distant metastasis-free survival; TNBC, triple-negative breast cancer; HER3, human epidermal growth factor receptor 3; EGFR, epidermal growth factor receptor; IGF1R, insulin-like growth factor 1 receptor; IR, insulin receptor; mTOR, mammalian target of rapamycin; p-phosphorylated.

Article Snippet: The mouse TNBC cell line, luciferase-expressing 4T1 (cat. no. JCRB1447), was obtained from the Japanese Collection of Research Bioresources (JCRB).

Techniques: Phospho-proteomics, Luminex, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple-negative breast cancer metastasis via the inhibition of CXCR4 expression

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: Knockdown of HER3 decreases the migration, invasion and metastasis of metastatic TNBC cells by inhibiting Akt and mTOR. (A-C) The 4T1-L8 cells were transfected with HER siRNA (50 nM) or control siRNA. (A) The expression levels of HER3, p-Akt, Akt, p-mTOR and mTOR were detected using western blot analysis. The expression levels of β-actin were used as internal controls. Quantification of signals is presented as fold of changes relative to phosphorylated protein vs. total proteins or total proteins vs. β-actin. The experiments were repeated four times. Data are presented as the mean ± SD. * P<0.05, compared with control siRNA. (B) The number of cells stained with trypan blue counted on days 1, 2 and 3. The experiments were performed in triplicate and repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with control siRNA. (C) Cell migration and invasion analysis was performed using the cell culture insert. The cells passing through the cell culture insert were counted. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with control siRNA. Representative images of cells transfected with control siRNA and HER3 siRNA are shown. Scale bars, 100 µ m. (D) The 4T1-L8 cells transfected with HER siRNA (50 nM) or control siRNA were injected into BALB/c mice via the tail vain. Metastasis in the lungs was monitored using IVIS. After 8 days, the mice were sacrificed and the number of metastatic nodules in the lungs were counted. The results are expressed as the mean ± SD. * P<0.05, compared with control siRNA. TNBC, triple-negative breast cancer; HER3, human epidermal growth factor receptor 3; mTOR, mammalian target of rapamycin; p-phosphorylated.

Article Snippet: The mouse TNBC cell line, luciferase-expressing 4T1 (cat. no. JCRB1447), was obtained from the Japanese Collection of Research Bioresources (JCRB).

Techniques: Knockdown, Migration, Transfection, Control, Expressing, Western Blot, Staining, Cell Culture, Injection

Journal: International Journal of Molecular Medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple-negative breast cancer metastasis via the inhibition of CXCR4 expression

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: The HER3/Akt/mTOR pathway promotes the migration and invasion of metastatic triple-negative breast cancer cells by increasing CXCR4 expression. (A) The expression levels of CCR2, CCR7 and CXCR4 were detected using western blot analysis. The expression levels of β-actin were used as internal controls. Quantification of signals is presented as fold of changes relative to total proteins vs. β-actin. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with 4T1 cells. (B) Cell migration and invasion analysis was performed using the cell culture insert. The cells passing through the cell culture insert were counted. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with SDF-1(-). Representative images of SDF-1-untreated 4T1 or 4T1-L8 and SDF-1-treated 4T1 or 4T1-L8 cells are shown. Scale bars, 100 µ m. (C and D) 4T1-L8 cells were transfected with HER siRNA (50 nM) or control siRNA (Stealth™ RNAi Negative Control). (C) The expression levels of CXCR4 were detected using western blotting. The expression levels of β-actin were used as internal controls. Quantification of signals is presented as fold change or relative levels of CXCR4 vs. β-actin. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with control siRNA. (D) Cell migration and invasion analysis was performed using the cell culture insert. The cells passing through the cell culture insert were counted. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with control siRNA. Representative images of control siRNA and HER3 siRNA are shown. Scale bars, 100 µ m. HER3, human epidermal growth factor receptor 3; mTOR, mammalian target of rapamycin; CCR, chemokine receptor; CXCR4, C-X-C chemokine receptor type 4; SDF-1, stromal derived factor-1.

Article Snippet: The mouse TNBC cell line, luciferase-expressing 4T1 (cat. no. JCRB1447), was obtained from the Japanese Collection of Research Bioresources (JCRB).

Techniques: Migration, Expressing, Western Blot, Cell Culture, Transfection, Control, Negative Control, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple-negative breast cancer metastasis via the inhibition of CXCR4 expression

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: Everolimus decreases the expression of CXCR4 via mTOR inhibition. (A and B) 4T1-L8 cells were treated with everolimus (1, 5 and 10 µ M). (A) The expression levels of p-mTOR, mTOR and CXCR4 were detected using western blot analysis. The expression levels of β-actin were used as internal controls. Quantification of signals is presented as the fold change or relative levels of phosphorylated protein vs. total protein or total protein vs. β-actin. The experiments were repeated three times. Data are presented as the mean ± SD. * P<0.05, compared with the control. (B) The number of cells stained with trypan blue counted on days 1, 2 and 3. The experiments were performed in triplicate and repeated three times. Data are presented as the mean ± SD. mTOR, mammalian target of rapamycin; CCR, chemokine receptor; CXCR4, C-X-C chemokine receptor type 4; p-phosphorylated.

Article Snippet: The mouse TNBC cell line, luciferase-expressing 4T1 (cat. no. JCRB1447), was obtained from the Japanese Collection of Research Bioresources (JCRB).

Techniques: Expressing, Inhibition, Western Blot, Control, Staining

Journal: International Journal of Molecular Medicine

Article Title: HER3/Akt/mTOR pathway is a key therapeutic target for the reduction of triple-negative breast cancer metastasis via the inhibition of CXCR4 expression

doi: 10.3892/ijmm.2023.5283

Figure Lengend Snippet: Everolimus inhibits the metastasis of triple-negative breast cancer cells in vivo . The 4T1-L8 cells were injected via the tail vein into BALB/c mice. The mice were then randomly divided into three groups, namely, the control, 5 mg/kg everolimus, and 10 mg/kg everolimus groups. Metastasis in the lungs was monitored using an in vivo imaging system. After 8 days, the mice were sacrificed, and the number of metastatic nodules in the lungs was counted. The results are expressed as the mean ± SD. * P<0.05, compared with the control.

Article Snippet: The mouse TNBC cell line, luciferase-expressing 4T1 (cat. no. JCRB1447), was obtained from the Japanese Collection of Research Bioresources (JCRB).

Techniques: In Vivo, Injection, Control, In Vivo Imaging

Journal: Journal of Extracellular Vesicles

Article Title: Baohuoside I chemosensitises breast cancer to paclitaxel by suppressing extracellular vesicle/CXCL1 signal released from apoptotic cells

doi: 10.1002/jev2.12493

Figure Lengend Snippet: EV‐Apo promotes the invasion and chemoresistance of breast cancer cells co‐cultured with macrophages. (a) A schematic diagram of EV‐Apo and EV‐alive isolation procedures. (b) Representative TEM images of EV‐Apo and EV‐alive as well as their size distribution analysis by a Flow NanoAnalyzer. (c) Expression of exosome markers detected by Western blotting. n = 3. (d) Comparison of the secretion quantity difference between EV‐Apo and EV‐alive through nanoparticle tracking analysis (NTA). n = 3. (e) A schematic diagram of the co‐culture system. 4T1 cells and Raw264.7 cells were co‐cultured using the transwell co‐culture system. (f) Cell migration and invasion assays of 4T1 cells cultured alone or co‐cultured with Raw264.7 cells. The cells were treated as indicated for 24 h. n = 3. (g) Apoptosis rates of 4T1 cells detected by Annexin V‐FITC/PI staining and flow cytometry. 4T1 cells were cultured alone or co‐cultured with Raw264.7 cells and treated as indicated for 48 h. n = 3. ** p < 0.01.

Article Snippet: TNBC 4T1 cells and Raw264.7 macrophages were purchased from KeyGen Biotech (Nanjing, China).

Techniques: Cell Culture, Isolation, Expressing, Western Blot, Comparison, Co-Culture Assay, Migration, Staining, Flow Cytometry

Journal: Journal of Extracellular Vesicles

Article Title: Baohuoside I chemosensitises breast cancer to paclitaxel by suppressing extracellular vesicle/CXCL1 signal released from apoptotic cells

doi: 10.1002/jev2.12493

Figure Lengend Snippet: BHS synergizes with paclitaxel to suppress the invasion and chemoresistance of breast cancer cells co‐cultured with macrophages. (a) Cytotoxicity of BHS in 4T1 cells and Raw264.7 cells detected by the CCK8 assay. n = 3. (b) Cell viability of 4T1 cells in the co‐culture system after treatments with the combination of 1.25−5 µM BHS and 50 nM PTX determined with the CCK8 assay. n = 3. (c–e) BHS synergized with paclitaxel to suppress the colony formation, invasion, and apoptosis chemoresistance of the co‐cultured breast cancer cells. 4T1 cells were cultured alone or co‐cultured with Raw264.7 cells and treated as indicated for 24 or 48 h. n = 3. (f) A schematic diagram of the EV‐Apo BHS isolation procedure. (g) A representative TEM image of EV‐Apo BHS . (h) Size distribution of EV‐Apo BHS detected by a Flow NanoAnalyzer. (i) An immunoblot analysis of EV‐Apo BHS detecting EV‐positive and negative biomarkers. n = 3. * p < 0.05, ** p < 0.01.

Article Snippet: TNBC 4T1 cells and Raw264.7 macrophages were purchased from KeyGen Biotech (Nanjing, China).

Techniques: Cell Culture, CCK-8 Assay, Co-Culture Assay, Isolation, Western Blot

Journal: Journal of Extracellular Vesicles

Article Title: Baohuoside I chemosensitises breast cancer to paclitaxel by suppressing extracellular vesicle/CXCL1 signal released from apoptotic cells

doi: 10.1002/jev2.12493

Figure Lengend Snippet: BHS chemosensitises breast cancer cells via modulating paclitaxel‐induced EV‐Apo signalling. (a, b) Proliferation (a) and colony formation (b) efficacies of 4T1 cells when treated as indicated for 48 h in the presence or absence of a Raw264.7 macrophage co‐culture. EVs were used at a concentration of 100 µg/mL. n = 3. (c) Migration and invasion efficacies of 4T1 cells when treated as indicated for 24 h. n = 3. (d–e) Apoptosis rates of 4T1 cells when treated as indicated for 48 h detected by Annexin V‐FITC/PI staining and flow cytometry. n = 3. (f) Expression levels of EMT‐related proteins in 4T1 cells when treated as indicated for 48 h. The concentration of paclitaxel was 50 nM. n = 3. * p < 0.05, ** p < 0.01.

Article Snippet: TNBC 4T1 cells and Raw264.7 macrophages were purchased from KeyGen Biotech (Nanjing, China).

Techniques: Co-Culture Assay, Concentration Assay, Migration, Staining, Flow Cytometry, Expressing

Journal: Journal of Extracellular Vesicles

Article Title: Baohuoside I chemosensitises breast cancer to paclitaxel by suppressing extracellular vesicle/CXCL1 signal released from apoptotic cells

doi: 10.1002/jev2.12493

Figure Lengend Snippet: EV‐Apo is crucial for BHS to synergize with paclitaxel to suppress breast cancer chemoresistance and invasion. (a) Alix knockdown (left) significantly decreased the EV concentration (right) in the culture medium of 4T1 cells. The successful generation of 4T1/shAlix cells was validated by Western blotting. n = 3. (b–e) Alix knockdown significantly synergized with paclitaxel to inhibit the proliferation (b), colony formation (c–d), and apoptosis resistance (e) of breast cancer cells in the macrophage co‐culture system. 4T1 cells and 4T1/shAlix cells were pre‐treated with 5 µµ BHS, 50 nM paclitaxel or their combination for 48 h. n = 3. (f–g) Both Alix knockdown and BHS significantly synergized with paclitaxel to inhibit the migration and invasion of co‐cultured breast cancer cells. 4T1 cells and 4T1/shAlix cells were pre‐treated with BHS, paclitaxel or their combination for 24 h. n = 3. (h) The expression levels of EMT‐related proteins in 4T1 cells and 4T1/shAlix cells when the cells were pre‐treated as indicated for 48 h. n = 3. * p < 0.05, ** p < 0.01.

Article Snippet: TNBC 4T1 cells and Raw264.7 macrophages were purchased from KeyGen Biotech (Nanjing, China).

Techniques: Knockdown, Concentration Assay, Western Blot, Co-Culture Assay, Migration, Cell Culture, Expressing

Journal: Journal of Extracellular Vesicles

Article Title: Baohuoside I chemosensitises breast cancer to paclitaxel by suppressing extracellular vesicle/CXCL1 signal released from apoptotic cells

doi: 10.1002/jev2.12493

Figure Lengend Snippet: BHS inhibits EV‐Apo signalling by suppressing ILV biogenesis via CXCL1. (a) The size distribution and concentrations of different EVs isolated from the culture medium of 4T1 cells after treatments as indicated for 48 h. PTX, 50 nM; BHS, 5 µM; CXCL1‐NA, 5 µg/mL; rCXCL1, 30 ng/mL. n = 3. (b) Western blotting assay of the expression levels of EV markers in 4T1 cells after treatments as indicated for 48 h. n = 3. (c) Immunofluorescence analysis of CD81 protein expression level in 4T1‐Luc cells when treated as indicated for 48 h. n = 3. (d) TEM analysis of ILVs in MVBs of 4T1 cells when treated with 50 nM PTX, 5 µM BHS or their combination for 48 h. n = 3. (e) Biotin‐tagged BHS was synthesized chemically and its successful synthesis was confirmed by the mass spectrum (left) and 1H NMR spectrum (right). (f) A total ion current chromatogram of the pull‐down samples obtained by LC‐MS/MS. (g) FLOT1 and FLOT2 were selected by intersecting BHS‐interacted proteins with EV biogenesis regulatory proteins. * p < 0.05, ** p < 0.01.

Article Snippet: TNBC 4T1 cells and Raw264.7 macrophages were purchased from KeyGen Biotech (Nanjing, China).

Techniques: Isolation, Western Blot, Expressing, Immunofluorescence, Synthesized, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Extracellular Vesicles

Article Title: Baohuoside I chemosensitises breast cancer to paclitaxel by suppressing extracellular vesicle/CXCL1 signal released from apoptotic cells

doi: 10.1002/jev2.12493

Figure Lengend Snippet: BHS decreases ILV biogenesis by directly inhibiting the FLOT2/RAB31 interaction. (a) A CETSA analysis of the thermal stability of FLOT2 in 4T1 cells. n = 3. (b) A Co‐IP analysis of the interaction between 1.25 and 5 µM BHS and FLOT2 in 4T1 cells. n = 3. (c) Immunofluorescence analysis and ultra‐high‐resolution microscopy observation of FLOT2 and CD63 expression levels in 4T cells when treated with 5 µM BHS‐Biotin for 48 h. n = 3. Yellow arrows indicate high distribution regions and colocalization regions of BHS‐Biotin and FLOT2, whereas CD63 exhibits low expression in these regions. White arrows indicate low BHS‐Biotin distribution regions, whereas CD63 exhibits high expression in these regions. (d) Western blotting analysis of FLOT2 expression levels in 4T cells when treated with 1.25−5 µµ BHS for 48 h. n = 3. (e) Computer‐aided virtual docking between BHS and FLOT2 (left) and subsequent virtual alanine scanning (right) were conducted to predict the potential binding between BHS and the 104 LQTL 107 motif of the FLOT2 protein. (f) A Co‐IP analysis of the interaction between 1.25 and 5 µM BHS‐Biotin and Flag‐tagged FLOT2 L104A in 4T1/FLOT2 L104A mutation cells. n = 3. (g) A Co‐IP analysis of the interaction activity between RAB31 and FLOT2 in 4T1 cells when treated as indicated for 48 h. n = 3. (h) Successful generation of 4T1/FLOT2‐OE cells validated by Western blotting. n = 3. (i) An immunofluorescence analysis of CD63 and FLOT2 protein expression levels in 4T1 and 4T1/FLOT2‐OE cells. n = 3. (j) A TEM analysis of ILVs per MVBs of 4T1 cells and 4T1/FLOT2‐OE cells when treated with 5 µM BHS for 48 h. n = 3. * p < 0.05, ** p < 0.01.

Article Snippet: TNBC 4T1 cells and Raw264.7 macrophages were purchased from KeyGen Biotech (Nanjing, China).

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Microscopy, Expressing, Western Blot, Binding Assay, Mutagenesis, Activity Assay