Amlodipine, a Ca2+ channel blocker, suppresses phosphorylation of epidermal growth factor receptor in human epidermoid carcinoma A431 cells.
OBJECTIVE
Amlodipine, a dihydropyridine Ca(2+) channel blocker, inhibits the proliferation of human epidermoid carcinoma A431 cells in vitro and in vivo. This study examined the underlying mechanism of this antiproliferative effect in relation to epidermal growth factor receptor (EGFR) signaling.


METHODS
The tyrosine phosphorylated active state of EGFR in A431 cells incubated with the test agents was evaluated by western blot with anti-phosphotyrosine antibody. EGFR phosphorylation levels in A431 xenograft tumors were assessed by immunostaining of matrigel plug sections and western blotting for phosphoEGFR in A431 xenograft tumor homogenates.


RESULTS
In vitro treatment of exponentially growing A431 cells with amlodipine decreased the tyrosine phosphorylation states of EGFR. Amlodipine also suppressed the EGF-stimulated phosphorylation of EGFR and a membrane scaffolding protein, caveolin-1, in serum-starved A431 cells. Amlodipine attenuated the EGF-stimulated phosphorylation of EGFR coimmunoprecipitated with caveolin-1 without affecting the EGFR/caveolin-1 interaction. Crosslinking experiments showed that amlodipine also suppressed the EGF-stimulated phosphorylation of EGFR predimers. Addition of cholesterol abolished these inhibitory effects of amlodipine plus its inhibition of cell growth. Furthermore, treatment of mice with amlodipine (10mg/kg/dayx7days, i.p.) decreased the levels of phosphorylated EGFR in A431 xenograft tumors.


CONCLUSIONS
The results indicated that amlodipine inhibits tyrosine phosphorylation of EGFR in vitro and in vivo, possibly via modulating cholesterol-rich, caveolin-1-containing membrane microdomains.
Yoshida
Junko
J
Department of Pharmacology, School of Medicine, Kanazawa Medical University, Daigaku 1-1, Uchinada, Ishikawa 920-0293, Japan. yayuyo@kanazawa-med.ac.jp
Ishibashi
Takaharu
T
Yang
Mei
M
Nishio
Matomo
M
D017311:Amlodipine / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D015153:Blotting, Western; D002121:Calcium Channel Blockers / Q000494:pharmacology* / Q000627:therapeutic use; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051242:Caveolin 1 / Q000378:metabolism; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D010766:Phosphorylation; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D002121:Calcium Channel Blockers; D051242:Caveolin 1; D017311:Amlodipine; D002784:Cholesterol; C512478:EGFR protein, human; D066246:ErbB Receptors