Actin disruption inhibits bombesin stimulation of focal adhesion kinase (pp125FAK) in prostate carcinoma.
Jasplakinolide is a member of a new class of antitumor agents targeting the actin cytoskeleton with activity against prostate cancer. Focal adhesion kinase (FAK) is an actin-associated mediator of mitogenic peptides. We hypothesized that the neuropeptide bombesin would activate FAR in prostate carcinoma, and that disruption of the actin network would block FAK activation and inhibit cell growth.


METHODS
PC-3 human prostate carcinoma cells were exposed to 50-200 nM jasplakinolide (Jas) or cytochalasin E (CyE) in cytotoxicity experiments. FAK phosphorylation was measured in cells stimulated with 0.01-10 nM bombesin; separate cells were pretreated 6 hr with 50-500 nM Jas or CyE. Cell lysates and anti-FAK immunoprecipitates were subjected to SDS-PAGE, Western blotting, and detection with anti-actin or anti-phosphotyrosine. Depolymerized G-actin was separated from total actin by ultracentrifugation. Cytoskeletal changes were confirmed by fluorescence microscopy.


RESULTS
Jas (GI50 = 47 +/- 7 nM) and CyE (GI50 61 +/- 20 nM) potently inhibited PC-3 growth (P < 0.01 vs control). Bombesin rapidly stimulated tyrosine phosphorylation of FAK in a dose dependent manner. FAK phosphorylation was inhibited to near-basal levels (50% of bombesin stimulated) by 500 nM Jas (63%) and 500 nM CyE (61%).


CONCLUSIONS
Bombesin stimulated FAK in prostate carcinoma cells. Jasplakinolide, which induced over-polymerization of actin, and CyE, which depolymerizes actin, both inhibited bombesin-stimulated phosphorylation of FAK and inhibited PC-3 cell growth. Actin-disrupting agents block FAK signal transduction, which may be critical to their antitumor activity in prostate carcinoma.
Duncan
M D
MD
Surgical Service, Veterans Affairs Medical Center, Georgetown University, Washington, D.C. 20422, USA.
Harmon
J W
JW
Duncan
L K
LK
D000199:Actins / Q000187:drug effects / Q000502:physiology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001839:Bombesin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015815:Cell Adhesion Molecules / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D003572:Cytochalasins / Q000494:pharmacology; D047630:Depsipeptides; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007700:Kinetics; D008297:Male; D010456:Peptides, Cyclic / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011471:Prostatic Neoplasms / Q000201:enzymology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000199:Actins; D000970:Antineoplastic Agents; D015815:Cell Adhesion Molecules; D003572:Cytochalasins; D047630:Depsipeptides; D004791:Enzyme Inhibitors; D010456:Peptides, Cyclic; C057531:jasplakinolide; D019000:Phosphotyrosine; C007215:cytochalasin E; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D001839:Bombesin