title: Inhibitory effects and underlying mechanism of 7-hydroxyflavone phosphate ester in HeLa cells.
abstract: Chrysin and its phosphate ester have previously been shown to inhibit cell proliferation and induce apoptosis in Hela cells; however, the underlying mechanism remains to be characterized. In the present study, we therefore synthesized diethyl flavon-7-yl phosphate (FP, C(19)H(19)O(6)P) by a simplified Atheron-Todd reaction, and explored its anti-tumor characteristics and mechanisms. Cell proliferation, cell cycle progression and apoptosis were measured by MTS, flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling techniques, respectively in human cervical cancer HeLa cells treated with 7-hydroxyflavone (HF) and FP. p21, proliferating cell nuclear antigen (PCNA) and cAMP levels in Hela cells were analyzed by western blot and radioimmunoassay. Both HF and FP inhibited proliferation and induced apoptosis in HeLa cells via induction of PCNA/p21 expression, cleaved caspase-3/poly (ADP-ribose) polymerase (PARP)-1, elevation of cAMP levels, and cell cycle arrest with accumulation of cells in the G0/G1 fraction. The effects of FP were more potent than those of HF. The interactions of FP with Ca(2+)-calmodulin (CaM) and Ca(2+)-CaM-phosphodiesterase (PDE)1 were explored by electrospray ionization-mass spectrometry and fluorescence spectra. FP, but not HF, formed non-covalent complexes with Ca(2+)-CaM-PDE1, indicating that FP is an inhibitor of PDE1, and resulting in elevated cellular cAMP levels. It is possible that the elevated cAMP levels inhibit growth and induce apoptosis in Hela cells through induction of p21 and cleaved caspase-3/PARP-1 expression, and causing down-regulation of PCNA and cell cycle arrest with accumulation of cells in the G0/G1 and G2/M fractions. In conclusion, FP was shown to be a Ca(2+)-CaM-PDE inhibitor, which might account for its underlying anti-cancer mechanism in HeLa cells. These observations clearly demonstrate the special roles of phosphorylated flavonoids in biological processes, and suggest that FP might represent a potential new drug for the therapy of human cervical carcinoma.
authors.affiliation: MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People's Republic of China.
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.forename: Jiang
authors.initials: J
authors.lastname: Du
authors.forename: Liguo
authors.initials: L
authors.lastname: Liu
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Chen
authors.forename: Fang
authors.initials: F
authors.lastname: Yang
authors.forename: Qi
authors.initials: Q
authors.lastname: Jin
mesh_terms: D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002147:Calmodulin / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D047309:Flavones / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D010755:Organophosphates / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism
chemical_list: D016588:Anticarcinogenic Agents; D002147:Calmodulin; D050759:Cyclin-Dependent Kinase Inhibitor p21; D047309:Flavones; D005419:Flavonoids; D010755:Organophosphates; D010710:Phosphates; D010726:Phosphodiesterase Inhibitors; D018809:Proliferating Cell Nuclear Antigen; C574792:diethyl flavon-7-yl phosphate; D000242:Cyclic AMP; D011065:Poly(ADP-ribose) Polymerases; D010727:Phosphoric Diester Hydrolases; D053148:Caspase 3; C429845:7-hydroxyflavone