Evodiamine induces centrosome amplification with subsequent G2/M cell cycle arrest in primary vascular smooth muscle...

Evodiamine (EVO) is a natural product found in Tetradium ruticarpum. It inhibits vascular smooth muscle cell (VSMC) proliferation, a key mechanism in the pathogenesis of atherosclerosis and restenosis. This study characterizes the mechanism of action behind the antiproliferative activity of evodiamine in platelet derived growth factor (PDGF)-activated VSMC. We confirmed the antiproliferative activity of EVO (0.3 and 1 µmol/L) in cultured primary VSMC by resazurin conversion and bromo-deoxyuridine (BrdU) incorporation assays, respectively, and its ability to arrest VSMC in G2/M by flow cytometric cell cycle analysis. Annexin V- Fluorescein Isothiocyanate (FITC)/propidium iodide (PI) staining and western blot analysis of caspase-3 cleavage detected low levels of apoptosis in response to 3 µmol/L EVO. We demonstrate that EVO (3 µmol/L) induces mitotic catastrophe (MC), as evidenced by characteristic nuclear morphology observed by confocal microscopy and polyploidy detected by flow cytometric DNA content analysis. Mechanistically, we rule out DNA damage as a cause of MC by western blot analysis of phospho-Ser139 histone H2A.X (γH2A.X). Instead, EVO induces centrosome amplification involving polo-like kinase 4 (PLK4) signaling. This is evident in cells co-treated with EVO (3 µmol/L) and the PLK4 inhibitor centrinone B (CENB) at 125 nmol/L by blunted centrosome amplification and cell cycle arrest. The study concludes with a proteomic analysis of purified centrosomes, which identifies candidates involved in this mechanism. In conclusion, evodiamine induces mitotic catastrophe via centrosome amplification in VSMC, positioning it as an antiproliferative agent with a distinct mechanism.