PMC

Gray bars, vehicle treatment; black bars, norepinephrine (A) or epinephrine (B) treatment. Results represent the mean ± SEM. *P < 0.05.

(A) SKOV3ip1 or EG cells were plated and treated with either norepinephrine (NE) or epinephrine (Epi) and then subjected to Western blot analysis for FAK or pFAK^Y397. (B) SKOV3ip1 cells kept in suspension were treated with norepinephrine, followed by Western blot for FAK and pFAK^Y397. (C) SKOV3ip1 cells were treated with norepinephrine, then subjected to immunofluorescence analysis for pFAK^Y397 and actin. Quantification of pFAK^Y397 staining is shown in the graph. Scale bars: 10 μm. (D) Effect of 10 μM norepinephrine with or without FAK silencing with siRNA on anoikis. Results represent the mean ± SEM. *P ≤ 0.01.

Effects of control siRNA-DOPC or FAK siRNA-DOPC on stress-induced in vivo (C) SKOV3ip1 or (D) HeyA8 tumor growth. (E) Effect of stress on tumor cell apoptosis in the SKOV3ip1 model. (F) Effect of propranolol or FAK siRNA-DOPC on tumor cell apoptosis in ascites as a measure of anoikis using the 2774 model. Results represent the mean ± SEM; n = 10 mice per group. *P < 0.01.

(C) Effect of 10 μM norepinephrine with or without propranolol or ADRB1- or ADRB2-targeted (β1 or β2) siRNA in SKOV3ip1 cells on anoikis. (D) Effect of β1 and β2 targeted siRNA on pFAK^Y397 and FAK in SKOV3ip1 cells. In A, B, and D, the immunoblot is shown at the top, and quantification of band intensity relative to total FAK intensity is shown below. For all panels, results represent the mean ± SEM of triplicate experiments. *P < 0.01.

(A) SKOV3ip1 cells stimulated with 10 μM norepinephrine were treated with either the Src inhibitor PP2 or its inactive counterpart PP3 followed by immunoblotting for FAK and pFAK^Y397. In addition, the effect of Src silencing with siRNA was examined on norepinephrine-mediated FAK activation. (B) SYF-null cells transfected with either empty vector (EV) or Src (WT Src) were stimulated with 10 μM norepinephrine, followed by immunoblotting for FAK and pFAK^Y397. (C) SKOV3ip1 cells treated with 10 μM norepinephrine were subjected to immunoprecipitation for Src (left) or FAK (right), followed by immunoblotting for FAK and Src. (D) Cells stimulated with 10 μM norepinephrine were treated with thapsigargin followed by Western blot for pFAK^Y397 and FAK (left). Cells stimulated with 10 μM norepinephrine were treated with EGTA followed by immunoprecipitation for FAK, then immunoblotting for FAK and Src (right). (E) SKOV3ip1 cells stimulated with 10 μM norepinephrine were treated with cytochalasin D (Cyt D), followed by Western blot for pFAK^Y397 and FAK. In A, D, and E, the immunoblot is shown at the top, and quantification of band intensity relative to total FAK intensity is shown below.

(A) Representative images of human ovarian tumors with low or high immunohistochemical staining for FAK and pFAK^Y397. Original magnification, ×200. (B) Kaplan-Meier curves of disease-specific mortality for patients with epithelial ovarian carcinoma based on FAK or pFAK^Y397 expression. The log-rank test (2-sided) was used to compare differences between groups. Percentage of ovarian cancers with high FAK or pFAK^Y397 expression based on CESD scores of at least 16 (C) or tumoral norepinephrine (NE) levels (greater versus less than median value of 0.84 pg/mg) (D).