Table 1.
Up-regulated pathways in recurrent stage II+IIIA triple negative breast cancer.
Fig 1.
Effect of brain-derived neurotrophic factor (BDNF) on the MDA-MB-231 cell line.
MDA-MB-231 cells (1 x 104/well)were seeded and transferred into a 12-well plates containing low serum medium, followed by treatment with BDNF (0-, 12,5-, 25-, and 50 ng/mL)for 1 day, 2 days and 3 days. Then the cells were trypsinized and subjected to a trypan blue dye exclusion assay as described in Methods. (Two way ANOVA, p>0.05, n = 6).
Fig 2.
The effects of brain-derived neurotrophic factor (BDNF) on autoregulation, migration and migration related proteins signaling in MDA-MB-231 cells.
MDA-MB-231 (4 X105 / well) cells cultured with low serum medium were treated with BDNF (0-, 12,5-, 25-, and 50 ng/mL) for 24 h or 6 h, and analyzed with Western blot (A) or real-time PCR (B), respectively. TkrB FL and TrkB T1 indicated full length and T1 domain of TrkB, respectively. Cells at a density of 2×104 cells were seeded into a 3.5 cm Petri dish containing an insert for migration assay (C) as described in Methods. Protein expressions of Rac, CDC42, and Rho (D) were quantified (E) and gene expression of COX2, MMP2, MMP9, and MMP13 were analyzed with Western blot (F,G) and real-time PCR (H). Data were expressed as mean + SEM. *, p< 0.05 compared to vehicle group by one way ANOVA, n = 4–5).
Fig 3.
The effects of brain-derived neurotrophic factor (BDNF) on autoregulation and the expression of angiogenesis- related signaling proteins in HUVEC cells.
HUVEC cells (4 X105 / well) cultured with low serum medium were treated with BDNF (0-, and 50 ng/mL) for 24 h or 6 h, and analyzed with Western blot (A) or real-time PCR (B), respectively. Protein expressions of Rac, CDC42, and Rho (C) and metastasis-related proteins such as COX2, MMP2, MMP9, and MMP13 (D) were analyzed with Western blot. The angiogenic factors such as VEGFA, BEGFR2, and eNOS were analyzed with Western blot (E) and real-time PCR (F), respectively. Data were expressed as mean + SEM. *, p< 0.05 compared to vehicle group by Mann-Whitney U test, n = 4).
Fig 4.
Effects of different inhibitors on brain-derived neurotrophic factor (BDNF) induced migratory activity in the MDA-MB-231 and HUVEC cell lines.
MDA-MB-231 cells (2×104) were seeded into a 3.5 cm Petri dish containing an insert for migration assay (A). Inhibitors of ERK, PI3K, and TrkB were pretreated1h before administration of BDNF (50 ng/mL) for 24 h and migratory area (mm2) were measured (B) (repeatedly measured one way ANOVA, n = 9)as described in Methods. After with or without pretreatment of inhibitors of ERK, PI3K, and TrkB, BDNF (50 ng/mL)-induced eNOS and COX2 expression in MDA-MB-231 cells (C, D) and HUVEC line (E, F). Data were expressed as mean ± SEM. *, p< 0.05 compared to vehicle group by Mann-Whitney U test, n = 5–9).
Fig 5.
The effects of brain-derived neurotrophic factor (BDNF) and TrkB on the migratory activity of the MDA-MB-231 line.
The cell morphology is presented (A) and the protein expression of BDNF and TrkB (B) were validated in the parent, the △BDNF cell line and the △TrkB cell line. MDA-MB-231 parent cells or the △BDNF and the △TrkB cell lines (2×104) were seeded into a 3.5 cm Petri dish containing an insert for migration assay (C). The migratory area was quantified (D). Data were expressed as mean ± SEM. *, p< 0.05 compared to vehicle group; #, p< 0.01 compared to positive control group by Repeatedly measured one way ANOVA, n = 5).
Fig 6.
Ingenuity pathway analysis (IPA) of gene expression levels in MDA-MB-231 cells.
MDA-MB-231 cells (4 X105 / well) cultured with low serum medium were treated with brain-derived neurotrophic factor (BDNF) (50 μg/mL), followed by RNA extraction and analysis of gene expression by oligonucleotide microarray assay. Then, the possible BDNF-related pathway was analyzed using Ingenuity pathway analysis (IPA). The metalloprotease network (A) and the calmodulin (B) network were modulated by BDNF treatment.
Fig 7.
Correlation with their prognosis of brain-derived neurotrophic factor (BDNF) expression levels using a tissue array of tumor slices from patients.
The protein expression levels of BDNF in a tissue array were measured after immunohistochemical staining. The degree of positiveness for protein expression was measured in a semi-quantified manner and is expressed as (0), <10%, (1), 11–25%, (2), 26–50%, (3) >50% of the tumor cells examined. Disease-free survival (DFS) and Overall survival (OS) were defined in Methods. The Kaplan–Meier method was used to estimate the cumulative incidence of DFS and OS and Log-rank (Mantel-Cox) Test were used for comparisons.
Fig 8.
Correlation with their prognosis of TrkB expression levels using a tissue array of tumor slices from patients.
The protein expression levels of TrkB in a tissue array were measured after immunohistochemical staining (A). The degree of positiveness for protein expression was measured in a semi-quantified manner and is expressed as (0), <10%, (1), 11–25%, (2), 26–50%, (3) >50% of the tumor cells examined. Disease-free survival (DFS, B) and Overall survival (OS, C) were defined in Methods. The Kaplan–Meier method was used to estimate the cumulative incidence of DFS and OS and Log-rank (Mantel-Cox) Test were used for comparisons.