Anti-proliferation effect of 3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one (BJ-601) on human vascular endothelial cells: G0/G1 p21-associated cell cycle arrest.
The aim of this study was to examine the anti-proliferation effect of 3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one (BJ-601) on human vascular endothelial cells and its possible molecular mechanism underlying. Our data showed that BJ-601 at a range of concentrations (0-40 microM) dose- and time-dependently decreased cell number in cultured human dermal microvascular endothelial cells (HDMVECs), but not human fibroblasts. The BJ-601-induced growth inhibition in HDMVECs was reversible. [3H]thymidine incorporation demonstrated that BJ-601 arrested the HDMVECs at the G0/G1 phase of the cell cycle. Western blot analysis revealed that BJ-601 (0-40 microM) dose-dependently increased the levels of the protein p21, but not of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase 2 (CDK2), and CDK4 in HDMVECs. Immunoprecipitation showed that the formation of the CDK2-p21 complex, but not CDK2-p27, CDK4-p21 and CDK4-p27 complexes, was increased in the BJ-601-treated HDMVECs. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in a dose-dependent manner in the BJ-601-treated HDMVECs. Pretreatment of HDMVECs with a p21 antisense oligonucleotide, which blocked the expression of p21 protein, reversed the BJ-601-induced inhibition of [3H]thymidine incorporation into HDMVECs. Moreover, cotreatment of the endothelial cells with protein kinase C (PKC) inhibitor, staurosporine, prevented the BJ-601-induced decrease of [3H]thymidine incorporation into HDMVECs. Administration of BJ-601 dose-dependently inhibited capillary-like tube formation of HDMVECs in Matrigel. In conclusion, these data suggest that BJ-601 inhibits HDMVECs proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally causes retardation of the cell cycle at the G0/G1 phase.
Yu
Chung-Hsun
CH
Graduate Institute Cellular and Molecular Biology, Taipei Medical University, Taipei, Taiwan, ROC.
Wu
Jender
J
Su
Yi-Fan
YF
Ho
Pei-Yin
PY
Liang
Yu-Chih
YC
Sheu
Ming-Thau
MT
Lee
Wen-Sen
WS
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D042846:CDC2-CDC28 Kinases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D016193:G1 Phase / Q000187:drug effects*; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism; D016192:Resting Phase, Cell Cycle / Q000187:drug effects*; D013843:Thiazines / Q000494:pharmacology*
C487059:3-amino-2-imino-3,4-dihydro-2H-1,3-benzothiazin-4-one; D020533:Angiogenesis Inhibitors; D013843:Thiazines; D011493:Protein Kinase C; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2