Angiotensin-(1-7) counteracts the transforming effects triggered by angiotensin II in breast cancer cells

Figure 1. Ang-(1-7) prevents AngII-induced AKT and ERK1/2 phosphorylation in the non-tumorigenic mammary cell line NMuMG Left panels show representative Western blots; right panels show quantification by densitometry. Effect of AngII (10-7M) and/or Ang-(1-7) (10-7M) on AKT (A) and ERK1/2 (B) phosphorylation for the indicated time. WB analyses were performed using phospho-specific antibodies against ERK1/2 and AKT. The same samples were blotted with antibodies to account for total kinase expression (lower panel for each kinase). N of 3 independent experiments; Values shown in bar represent mean± SEM, **P<0.01, ***P<0.001 vs untreated cells, ### P<0.001 vs AngII.

Figure 2. AngII induces ERK1/2 and AKT activation through AT1 receptor while Ang-(1-7) acts through the Mas receptor Western Blot analyses from the non-tumorigenic mammary cell line NMuMG were performed for p-AKT and AKT (A-B) and p-ERK1/2 and ERK1/2 (C-D). Cells were pre-incubated for 5 min with Irbesartan (10-6 M), PD123319 (10-6 M), A779 (10-6 M), or D-Pro (10-6 M), and then stimulated with AngII (10-7M) (A-C) or (B-D) Ang-(1-7) (10-7M) for the indicated time. Blots show representative Western blots. N of 3 independent experiments; values shown in bar represent mean±SEM quantified by densitometry and relative to control-untreated cells. *P<0.05, **P<0.01, ***P<0.001 vs untreated control cells; # P<0.05, ##P<0.01, ### P<0.001 vs AngII or §§P<0.01, §§§P<0.001 vs Ang-(1-7) treated cells.

Figure 3. Ang-(1-7) abolish AngII-induced EMT in the non-tumorigenic mammary cell line NMuMG (A) Cells were treated with AngII (10-7M) and/or Ang-(1-7) (10-7M) for 3 days. The mRNA levels of E-cadherin, fibronectin, N-cadherin and ɑ-SMA were determined by qRT-PCR. mRNA levels have been normalized to GAPDH and relative to control. Bars indicate means ± SEM, n ≥ 3, ***P<0.001 vs control, ###P<0.001 vs AngII, §P<0.05, §§P<0.01 vs Ang-(1-7) treated cells. (B) Upper Panel: influence of AngII (10-7M) and/or Ang-(1-7) (10-7M) on E-cadherin and fibronectin expression and subcellular localization after 3 days of treatments. NMuMG cells were immunostained for E-Cadherin (red) or fibronectin (green). Images are shown at ×400 magnification. Lower Panel: Bright fields of morphological changes induced by AngII (10-7M) on NMuMG treated cells during EMT process.

Figure 4. AngII induces EMT and through AKT activation NMuMG cells were treated for 3 days with AngII (10-7M) in the presence or absence of an AKT inhibitor A6730 (1uM). The mRNA levels of E-Cadherin, Fibronectin, N-cadherin and ɑ-SMA were determined by qRT-PCR. mRNA levels have been normalized to GPDH and relative to control. Bars indicate means ± SEM, n ≥ 3, *P<0.05, **P<0.01, ***P<0.001 vs untreated cells.

Figure 5. Ang-(1-7) blockes AngII induced tumor cell migration (A) Wound healing assay on MDA-MB231 or LM3 cells. Wounds were registered by phase contrast microscopy immediately after scratching (T0) and after 16 h in serum-free medium (T16) treated with AngII (10-7M) and/or Ang-(1-7) (10-7M). Bars show results from 3 independent experiments performed and expressed as fold increase of wound closure at time 16 hrs (T16) compared to control (untreated cells). Representative pictures of wounds at T0 and T16, magnification, 100x. Bars indicate means ± SEM, ***P <0.001 vs control, ###P<0.001 vs AngII. (B) Transwell cell migration assayed with MDA-MB231 cells in serum-free media across 8 mm-pore filters coated with type I collagen. The cells that migrated to the lower portion of the chamber, were fixed and stained with cristal violet, and five fields per well were counted. Bars indicate number of migrated cells, means ± SEM, n ≥ 3 ***P <0.001 vs control or ###P<0.001 vs AngII. (C) AngII (10-7M) and Ang-(1-7) (10-7M) had no effect on cell proliferation measured over 24 h by cell counting and MTS assay in MDA-MB231 cells.

Figure 6. Ang-(1-7) abolishes AngII-induced breast cancer cell invasion, MMP-9 activity and VEGF and MMP-9 expression (A) Transwell cell invasion assayed of MDA-MB231 cell in serum-free media across 8 mm-pore filters coated with matrigel. Cells that migrated to the lower portion of the chamber were fixed and stained with cristal violet, and five fields per well were counted. Bars indicate number of invaded cells, means ± SEM, n ≥ 3, **P <0.01 vs control or #P<0.05 vs AngII. (B) VEGF and MMP-9 mRNA expression levels were determined by qRT-PCR on MDA-MB231 cells treated with AngII (10-7M) and/or Ang-(1-7) (10-7M) for 24 h. mRNA levels have been normalized to GAPDH and relative to control. Bars indicate means ± SEM, n ≥ 3 ***P <0.001 vs control or ###P <0.001 vs AngII. (C) Gelatin-based zymography analysis of MMP-xs9 activity in conditioned medium of MDA-MB231 cells treated as in B. Image shown is one representative of 3 independent experiments (Upper panel). For quantification (ImageJ software), results were normalized to the quantity of proteins in cell lysate and expressed relative to control (lower panel). Bars indicate means ± SEM, n ≥ 3, ***P <0.001 vs control or ##P <0.01 vs AngII. (D)…