ACTIN DISRUPTION INHIBITS BOMBESIN STIMULATION OF FOCAL ADHESION KINASE (PP125(FAK)) IN PROSTATE CARCINOMA

Jasplakinolide is a member of a new class of antitumor agents targetin g the actin cytoskeleton with activity against prostate cancer. Focal adhesion kinase (FAK) is an actin-associated mediator of mitogenic pep tides. We hypothesized that the neuropeptide bombesin would activate F AK in prostate carcinoma, and that disruption of the actin network wou ld block FAK activation and inhibit cell growth. Methods: PC-3 human p rostate carcinoma cells were exposed to 50-200 nM jasplakinolide (Jas) or cytochalasin E (CyE) in cytotoxicity experiments. FAR phosphorylat ion was measured in cells stimulated with 0.01-10 nM bombesin; separat e cells were pretreated 6 hr with 50-500 nM Jas or CyE. Cell lysates a nd anti-FAK immuno-precipitates were subjected to SDS-PAGE, Western bl otting, and detection with anti-actin or anti-phosphotyrosine. Depolym erized G-actin was separated from total actin by ultracentrifugation. Cytoskeletal changes were confirmed by fluorescence microscopy. Result s: Jas (GI(50) = 47 +/- 7 nM) and CyE (GI(50) = 61 +/- 20 nM) potently inhibited PC-3 growth (P < 0.01 vs control). Bombesin rapidly stimula ted tyrosine phosphorylation of FAK in a dose dependent manner. FAK ph osphorylation was inhibited to near-basal levels (50% of bombesin stim ulated) by 500 nM Jas (63%) and 500 nM CyE (61%). Conclusions: Bombesi n stimulated FAK in prostate carcinoma cells. Jasplakinolide, which in duced over-polymerization of actin, and CyE, which depolymerizes actin , both inhibited bombesin-stimulated phosphorylation of FAK and inhibi ted PC-3 cell growth. Actin-disrupting agents block FAK signal transdu ction, which may be critical to their antitumor activity in prostate c arcinoma. (C) 1996 Academic Press, Inc.