Fig. 3

ANGII triggered classical AGTR1 signaling and the transactivation of EGFR in ovarian cancer cells that resulted in cancer dissemination inside the abdominal cavity. a Activation of the classical AGTR1 pathway. Western blotting of p-AKT and p-ERK1/2 with or without ANGII treatment. GAPDH is the loading control. b Losartan suppressed the activation of the AKT/ERK pathway by ANGII. Immunoblots of p-AKT and p-ERK1/2 with ANGII and/or losartan treatment. c ANGII transactivates EGFR in a ligand-dependent manner. Western blotting of MMP2, EGFR, and p-EGFR, with GAPDH as a control. d Losartan suppressed the ANGII-mediated transactivation. Western blotting of p-EGFR, EGFR, p-Gab1, p-Shc, and GAPDH. e Schematic representation of ANGII administration to the animals. f Left: tumors inside the peritoneal cavity were dissected and representative images presented. Right: the number and weight of tumor nodes inside the peritoneal cavity after ANGII (1 μg/kg) treatment (each group n = 6). g Administration of losartan (40 μg/kg) reduced the effect of ANGII (100 nM) on ovarian cancer cell development inside the peritoneal cavity of mice. Upper: tumors removed from the peritoneal cavity with representative images for each group (each group n = 6). Lower: the number of tumor nodes and tumor weights. h AGTR1-CAM strongly enhanced ovarian cancer cell metastasis inside the peritoneal cavity. AGTR1-CAM cell lines were i.p. injected into NOD-SCID mice and were evaluated after 30 days (n = 4). Upper: tumor metastasis into a new organ inside the peritoneal cavity (“1” indicates metastatic tumor inside an ovary; “2” indicates metastatic tumor in the liver; “3” indicates metastatic tumor in the spleen). Lower: the number of tumor nodes and tumor weight in the peritoneal cavity of mice. i The number of metastatic tumors in the mice. j Cell death of MCS was assessed by Annexin V-FITC and PI assay by flow cytometry after treatment with ANGII (100 nM) and/or losartan (10 μM). Necrotic cells in each group were quantified. k Cell necrosis inside MCS was detected by flow cytometry with different combinations of treatment: ANGII (100 nM), losartan (10 μM), CGP42112 (50 nM) and/or ANG(1–7) (100 nM). Necrotic cells in each group were quantified accordingly. All of the data in this figure are presented as means ± SEM from at least three experiments. Significant differences are indicated (* p < 0.05, ** p < 0.01, *** p < 0.001 against control)