Abstract
Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3',4'-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G(1) phase. As shown by Western blot, 7,3',4'-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G(1) phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3',4'-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3beta/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3',4'-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3',4'-THIF provide a molecular basis for the possible development of new chemoprotective agents.
Highlights
The complex process of carcinogenesis is believed to be comprised of three stages that include initiation, promotion, and progression
The D-type cyclins are initially activated in response to mitogenic signals and preferentially bind to and activate CDK4 during the early G1 stage of the cell cycle [6]
The association of CDK2 with cyclin E and with cyclin A is believed to coordinate events as cells progress from G1 through S phase [7]. phosphorylated Rb (pRb) is initially phosphorylated on multiple sites by CDK4 and phosphorylated by cyclin E-bound CDK2
Summary
Chemicals—Eagle’s minimum essential medium (MEM), basal medium Eagle, gentamicin, and L-glutamine were purchased from Invitrogen. Western Blot Analysis—Cells were cultured in 10-cm dishes (4 ϫ 105 cells/dish) for 24 h and starved in 0.1% FBS-MEM for an additional 36 h. Of 200 mM ATP, and 1 g/l Rb-C fusion protein were added, and the reaction mixture was incubated at 30 °C for an additional 30 min. Direct Pulldown Assays—The recombinant cyclin D1-CDK4 or cyclin E-CDK2 complex or PI3K (2 g) was incubated with 7,3Ј,4Ј-THIF-conjugated Sepharose 4B (or Sepharose 4B as a negative control) beads (100 l, 50% slurry) in immunoprecipitation reaction buffer (50 mM Tris-HCl, (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM dithiothreitol, 0.01% Nonidet P-40, 0.02 mM phenylmethylsulfonyl fluoride) containing 2 g/ml bovine serum albumin and 1ϫ protease inhibitor mixture at 4 °C with gentle rocking overnight. A probability value of p Ͻ 0.05 was used as the criterion for statistical significance
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