Nitric Oxide Suppression Triggers Apoptosis Through the FKHRL1 (FOXO3A)/ROCK Kinase Pathway in Human Breast Carcinoma Cells

Background: The winged helix/forkhead transcriptional factor FKHRL1 (FOXO3a) triggers apoptosis, but its mode of action is not well understood. ROCK kinase is an effector molecule in human breast carcinoma cell apoptosis, but its relation to FKHRL1 is unknown. Because the human breast carcinoma T47D cell line releases a great amount of nitric oxide (NO), I investigated signaling of FKHRL1/ROCK [corrected] kinase during NO suppression.

Methods: Expression of phosphorylated FKHRL1 in T47D cells was analyzed using Western blotting. Apoptosis was evaluated by flow cytometry. Transfection of FKHRL1-HA wild-type and mutant FKHRL1-HA T32A constructs were performed by lipofectamine plus reagent. Measurement of NO generation was performed by Griess reaction.

Results: Nitric oxide suppression promotes FKHRL1 thr-32-enhanced phosphorylation, which was significantly (P < 0.005) sensitive to Y-27632, a specific inhibitor of the ROCK kinase, but not to capase-3 inhibitor or wortnannin, a specific inhibitor of phosphoinositol-3-OH kinase (PI3K). Nitric oxide suppression by N-(G)-monomethyl-L-arginine, an inhibitor of NO synthase, causes a significant (P < 0.001) increase in the apoptosis of T47D cells. However, a significant decrease (P < 0.01) in NO generation and a significant (P < 0.01) increase in apoptosis were observed when FKHRL1-HA wild-type cells were transfected, which caused increased FKHRL1 thr-32 phosphorylation.

Conclusions: This novel unknown phenomenon of breast carcinoma cell apoptosis was triggered by NO suppression, which promotes FKHRL1 thr-32-enhanced phosphorylation and initiates signaling of FKHRL1 to ROCK kinase as an effector molecule. This apoptotic signalling process is caspase-3 as well as PI3K/Akt independent.