Anti-angiogenic action of 5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10).
Previously, we demonstrated that 5,5-diphenyl-2-thiohydantoin (DPTH) exerts an anti-proliferation effect on subcultured human umbilical vein endothelial cells (HUVEC). In the present study, we show that 2(naphthalen-2-ylmethylsulfanyl)-5,5-diphenyl-1,5-dihydro-imidazol-4-one (DPTH-N10), a derivative compound of DPTH, exerts a 5 times stronger inhibition of [3H]thymidine incorporation into HUVEC as compared with DPTH and at very low concentrations (0-20 microM) inhibited DNA synthesis and decreased cell number in cultured HUVEC in a concentration- and time-dependent manner, but not in human fibroblasts. [3H]thymidine incorporation analysis demonstrated that treatment of HUVEC with DPTH-N10 arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 in HUVEC increased after DPTH-N10 treatment. In contrast, the protein levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after DPTH-N10 treatment. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not the CDK2-p27, CDK4-p21, and CDK4-p27 complex, was increased in the DPTH-N10-treated HUVEC. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in the DPTH-N10-treated HUVEC. Pretreatment of HUVEC with a p21, but not p27, antisense oligonucleotide reversed the DPTH-N10-induced inhibition of [3H]thymidine incorporation into HUVEC. Taken together, these data suggest that DPTH-N10 inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally interrupts the cell cycle. Capillary-like tube formation, aortic ring culture, and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of DPTH-N10.
Liu
Yuan
Y
Graduate Institute of Medical Sciences, School of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan.
Wu
Jender
J
Ho
Pei-Yin
PY
Chen
Li-Ching
LC
Chen
Chiung-Tong
CT
Liang
Yu-Chih
YC
Cheng
Cheng-Kuo
CK
Lee
Wen-Sen
WS
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000187:drug effects; D015153:Blotting, Western; D002478:Cells, Cultured; D002642:Chick Embryo; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D016193:G1 Phase / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D010672:Phenytoin / Q000031:analogs & derivatives* / Q000494:pharmacology; D016192:Resting Phase, Cell Cycle / Q000187:drug effects; D013867:Thiohydantoins / Q000494:pharmacology*
D020533:Angiogenesis Inhibitors; D013867:Thiohydantoins; C073336:diphenylthiohydantoin; D010672:Phenytoin