A chrysin derivative inhibits cell transformation
Chrysin (5,7 dihydroxyflavone) a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF induced neoplastic transformation of JB6 P+ cells compared to chrysin. It attenuated cell cycle progression of EGF stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of Rb phosphorylation at both Ser795 and Ser807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage dependent and independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and Rb phosphorylation at Ser795 and Ser807/811. IP kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF stimulated JB6 P+ cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP independent http:/www.jbc.org/cgi/doi/10.1074/jbc.M113.464669 The latest version is at JBC Papers in Press. Published on July 25, 2013 as Manuscript M113.464669 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on August 16, 2013 http:/www.jbc.org/ Downloaded from A chrysin derivative inhibits cell transformation 2 manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP noncompetitive Cdk inhibitor with anti tumor effects, which acts by binding inside the Cdk2 allosteric pocket. The present study provides new insights for creating a general pharmacophore model to design and develop novel ATP noncompetitive agents with chemopreventive or chemotherapeutic potency. Carcinogenesis involves at least three distinct stages including initiation, promotion and progression. Tumor promotion requires chronic exposure to tumor promoters, such as 12 O tetradecanoylphorbol 13 acetate (TPA) (1,2) and epidermal growth factor (EGF) (3,4) that complete the conversion of a cell to the neoplastic state. In contrast to initiation, the tumor promotion stage is prolonged and also reversible if tumor promoter treatment is ceased (5) making it a potential target for prevention strategies. One of the molecular mechanisms underlying EGF triggered neoplastic cell transformation is EGF induced aberrant cell cycle progression, which is one of the hallmarks of tumor formation and progression. The cyclin dependent kinase (Cdk) family of serine/threonine kinases was identified as a key regulator of cell cycle progression. At least 9 Cdks (Cdk1 9) have been identified and Cdk1 through 7 are involved in cell cycle control. Cell cycle progression is driven by the sequential and periodic activation of various Cdk cyclin complexes (6) Mouse embryonic fibroblasts (MEFs) lacking Cdk4 are resistant to transformation by oncogenes (7) When co transfected with activated H Ras, Cdk4 displayed oncogenic potential by provoking foci formation in primary rat embryonic fibroblasts (8) and generating malignant human epidermal tumorigenesis (9) MEFs expressing disrupted Cdk2 exhibit reduced susceptibility to transformation by oncogenes (10,11) Activation of Cdk2 is required for foci formation induced by cyclin E and SV40 small T antigen co expression in human fibroblasts (12) Thus, Cdk2 and Cdk4 are closely associated with oncogenic transformation. Chemoprevention is acknowledged as an important and practical strategy for the management of cancer. Accumulating research evidence suggests that many dietary phytochemicals have anticancer activity, low toxicity and few adverse side effects making them safe for human use. These natural or dietary compounds could be a source of prototype molecules from which to synthetically develop new chemotherapeutic agents with potent anticancer properties. Chrysin (5,7 dihydroxyflavone) a natural flavonoid widely distributed in many plant extracts, and honey and propolis, reportedly has chemopreventive properties such as anti proliferative and pro apoptosis activities against various cancers (13,14) However, the anticancer activity of chrysin observed in in vivo studies has been disappointing (15,16) To address this issue, several chrysin derivatives have been synthesized in recent years (17 19) suggesting the feasibility of improving the biological activities of chrysin as an antitumor agent that is more potent, with lower toxicity and minimal side effects by modifying its structure. The majority of protein kinase inhibitors are ATP competitive (type I) agents, which typically bind to the ATP pocket that is highly conserved across most of the kinases of the human genome. The lack of selectivity is an issue with type I inhibitors, which can lead to so called "off target" effects (20) The relatively poor selectivity of type I inhibitors can be addressed by type II inhibitors, which bind not only the ATP pocket but, in addition, interact with a site adjacent to the pocket. Type III inhibitors bind to regions that are remote from the ATP pocket. These regions are typically not highly conserved across all the kinases, providing for better selectivity (21) Type IV inhibitors target protein kinases distal to the substrate binding pocket and type V are bi substrate and bivalent inhibitors (22) Types II IV are allosteric (noncompetitive) inhibitors with distinct allosteric binding characteristics. To date, only a small number of noncompetitive inhibitors have been identified (21,23) Most were identified serendipitously and were later determined to be ATP noncompetitive agents through examination of X ray co structures (24) Although comparatively few agents remain in by guest on August 16, 2013 http:/www.jbc.org/ Downloaded from A chrysin derivative inhibits cell transformation 3 development, in particular phytochemicals, chemical strategies for converting known type I inhibitors into corresponding type II inhibitors with different kinase selectivity profiles and exceptionally potent cellular activity have been reported (24) This raises the possibility that natural phytochemicals could serve as core scaffolds that can be further designed and developed to obtain inhibitors with the desired spectrum of inhibitory activities. Because of the important role of Cdks in carcinogenesis, these kinases have long been considered ideal targets for anticancer agents. As a result, many Cdk inhibitors have been developed, some of which have progressed to clinical trials. However, none are currently approved for clinical use because the numerous potential drug leads are ATP competitive type I compounds, leading to a lack of target selectivity. An ever increasing demand exists for the development of ATP noncompetitive Cdk inhibitors, and especially those from natural and dietary sources. Indeed, progress has been made in identifying Cdk inhibitors that act through novel mechanisms. A novel structural pocket present on Cdk2, which is conserved on Cdks 1, 4, and 6, has been identified. Small molecules, identified by a high throughput in silico screening of this pocket, exhibit cytostatic effects and act by decreasing the function of Cdks in cells by binding to this site (25) Recently, an allosteric ligand binding site, away from the ATP site, in Cdk2 was also discovered. Binding of two ANS molecules is accompanied by substantial structural changes in Cdk2, resulting in a C helix conformation that is incompatible for cyclin A association (26) A phytochemical Cdk inhibitor described as an ATP noncompetitive inhibitor has also been reported. However, a mechanism of action that is distinct from that of ATP competitive inhibitors remains undisclosed (27) Here, we report that a modified chrysin derivative, compound 69407, inhibits EGF induced anchorage independent growth of JB6 P+ cells and suppresses anchorage dependent and independent growth of A431 human epidermoid carcinoma cells. It also exhibited tumor suppression effects in an A431 mouse xenograft model. Compound 69407 was shown to be an ATP noncompetitive inhibitor of Cdks. The present study investigated a possible mechanism by which ATP did not compete with compound 69407 for binding to Cdk2. Our results provide new information for creating a general pharmacophore model through which the design and development of new ATP noncompetitive agents (based on parental phytochemicals) with potent activity that target therapeutically relevant kinases with minimal or no side effects. EXPERIMENTAL PROCEDURES Reagents Compound 69407 (97% purity) was purchased from InterBioScreen (Moscow, Russia) Chrysin 97% purity) and other chemical reagents, including Tris, NaCl, and SDS, for molecular biology and buffer preparation were purchased from Sigma Aldrich (St. Louis, MO) R roscovitine was from Selleck Chemicals (Houston, TX) Restriction enzymes and a DNA ligation kit (v.2.0) were purchased from TAKARA Bio, Inc. (Shiga, Japan) CNBr Sepharose 4B beads were obtained from Amersham Pharmacia Biotech (Piscataway, NJ) and the protein assay kit was from Bio Rad Laboratories (Hercules, CA) Active Cdk2/cyclin E was purchased from Millipore (Dundee, UK) Active Cdk4/cyclin D1, active Cdk6/cyclin D1, active Cdk7/cyclin H1/MNAT1, recombinant human Rb protein (773 928aa; substrate for Cdk4/cyclin D1 and Cdk6/cyclin D1) and MBP protein (Myelin Basic Protein; substrate for Cdk7/cyclin H1/MNAT1) were purchased from SignalChem (Richmond, BC) Recombinant human histone H1 (substrate for Cdk2/cyclin E) was purchased from New England Biolabs (Ipswich, MA) Antibodies for immuno