Mol Cancer Ther. 2005;4:595-602
© 2005 American Association for Cancer Research
Novel hydroxyl naphthoquinones with potent Cdc25 antagonizing and growth inhibitory properties
Vincent P. Peyregne1,2,
Siddhartha Kar1,
Seung W. Ham1,
Meifang Wang1,
Ziqiu Wang1 and
Brian I. Carr1
1 Liver Cancer Center, Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania and 2 Department of Surgery and Liver Transplantation, Hospital Edouard Herriot, Lyon, France
Requests for reprints: Brian I. Carr, Liver Cancer Center, Starzl Transplantation Institute, University of Pittsburgh School of Medicine, E1552 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: 412-624-6684; Fax: 412-624-6666. E-mail: carrbi{at}upmc.edu
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Abstract
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Cdc25 phosphatases are important in cell cycle control and activate cyclin-dependent kinases (Cdk). Efforts are currently under way to synthesize specific small-molecule Cdc25 inhibitors that might have anticancer properties. NSC 95397, a protein tyrosine phosphatase antagonist from the National Cancer Institute library, was reported to be a potent Cdc25 inhibitor. We have synthesized two hydroxyl derivatives of NSC 95397, monohydroxyl-NSC 95397 and dihydroxyl-NSC 95397, which both have enhanced activity for inhibiting Cdc25s. The new analogues, especially dihydroxyl-NSC 95397, potently inhibited the growth of human hepatoma and breast cancer cells in vitro. They influenced two signaling pathways. The dual phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was induced, likely due to inhibition of the ERK phosphatase activity in Hep 3B cell lysate but not the dual specificity ERK phosphatase MKP-1. They also inhibited Cdc25 enzymatic activities and induced tyrosine phosphorylation of the Cdc25 target Cdks. Addition of hydroxyl groups to the naphthoquinone ring thus enhanced the potency of NSC 95397. These two new compounds may be useful probes for the biological functions of Cdc25s and have the potential for disrupting the cell cycle of growing tumor cells.
Key Words: Dual specificity phosphatase • small-molecule inhibitor • liver cancer • growth inhibition
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Introduction
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The timing of cell cycle progression is controlled by several cyclin-dependent kinases (Cdk) and their cyclin partners (1). The cyclin-Cdk complexes are kept in an inactive state by phosphorylation on conserved tyrosine and threonine residues (1, 2). Members of the Cdc25 family of phosphatases remove phosphates from the tyrosine and threonine residues within the Cdk catalytic subunit, specifically Thr14 and Tyr15 in both Cdk1 and Cdk2, and thus activate the cyclin-Cdk complexes. The three human Cdc25 homologues, Cdc25A, Cdc25B, and Cdc25C, have been reported to control different phases of the cell cycle. Cdc25A is involved in the G1-S transition and S-phase progression (3), but also probably acts during entry into mitosis (4). Cdc25B acts at the onset of mitosis (5) and Cdc25C catalyses mitotic progression (6). Overexpression of proto-oncogenes Cdc25A and Cdc25B has been reported in several cancers and seems to be associated with a poor prognosis (7). Cdc25s are important in control of cell cycle checkpoints in which they are targeted for inactivation and destruction to arrest the cell cycle and maintain genomic integrity. In G2-M phase, Cdc25B is phosphorylated by p38 (5) and Cdc25C by CHK1/CHK2 (8) with consequent inactivation by 14-3-3 proteins. Genotoxic stress can also induce a G1-S arrest through Cdc25A inhibition. In response to DNA damage, Cdc25A levels rapidly decrease by polyubiquitylation and proteasome-mediated degradation (9).
Because of these functions, Cdc25s represent important targets for the design of cell cycle inhibitors. Among the numerous published molecules (7), only few were found to possess potency against Cdc25s. To date, the most potent Cdc25 inhibitor was NSC 95397, with IC50 values against Cdc25A, Cdc25B, and Cdc25C of 22 ± 5.9, 125 ± 6, and 56.9 ± 17.7 nmol/L, respectively (10). We designed two hydroxyl-derivatives of NSC 95397, namely monohydroxyl-NSC 95397 (M-NSC) and dihydroxyl-NSC 95397 (D-NSC), to increase the hydrophilic properties of the molecule and potentially to enhance its cellular effects because structural similarity to phospholipids has been reported to enhance cytotoxicity on the cell membrane (11, 12).
We have examined the effects of M-NSC and D-NSC, both in cell and cell-free systems, and compared them to their parent NSC 95397 and the well-studied naphthoquinone derivative, Cpd 5. Our results provided evidence that addition of hydroxyl groups to the naphthoquinone ring enhanced the potency of NSC 95397.
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Materials and Methods
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Synthesis of Cpd 5, NSC 95397, M-NSC, D-NSC, and Biotinylated Cpd 5
Synthesis of Cpd 5 has been previously reported (13). NSC 95397 and D-NSC were prepared by the addition of 2-mercaptoethanol to a methanol solution of 2,3-dibromo-1,4-naphthoquinone and 5,8-dihydroxy-1,4-naphthoquinone, respectively. M-NSC was prepared by the addition of triethyl amine to an ether solution of 5-hydroxy-1,4-naphthoquinone and 2-mercaptoethanol to give the intermediate monothioether and then another addition reaction to dithioether analog. All compounds were recrystallized from hexane and ethyl acetate solution. NSC 95397 was initially the kind gift of Dr. J. Lazo (University of Pittsburgh, Pittsburgh, PA; ref. 10). Biotinylated Cpd 5 was synthesized by addition of 2-mercaptoethanol to the known biotin containing 2-bromo-3-methyl-1,4-naphthoquinone (14).
Cell Culture and Growth Inhibition
Human hepatoma Hep 3B, Hep G2, and PLC/PRL/5 and breast cancer cell lines were maintained in Eagle's MEM supplemented with 10% fetal bovine serum. Compounds with or without MEK inhibitors (PD98059, U0126; Calbiochem, San Diego, CA) or thiol-antioxidant glutathione were added in the medium for the indicated times. Cell number was estimated by a DNA fluorometric assay using the fluorochrome Hoechst 33258.
Flow Cytometric Analysis
Hep 3B cells were synchronized in G1-S or G2-M phase by a 24-hour treatment with 1 mmol/L hydroxyurea or 400 nmol/L nocodazole (Sigma Chemical, Co., St. Louis, MO), respectively. Then, cells were washed with cold PBS before immediate treatment with compounds at the IC50 concentration. Cells were harvested at different time points and stained with a solution containing 50 µg/mL propidium iodide and 250 µg/mL RNase A. Flow cytometry analysis was conducted with a Becton Dickinson FACS Star (Becton Dickinson, Franklin Lakes, NJ). A final concentration of 0.025% DMSO was used for all compounds and as a negative control. For positive controls, 24-hour hydroxyurea-treated and nocodazole-treated cells were used.
DNA Synthesis
To determine in vitro DNA synthesis, 5 µCi [3H]thymidine (ICN, Costa Mesa, CA) was added for 3 hours to the cdc25 wt/mut gene–transfected Hep 3B cells after 24 hours of compound treatment. Cells were harvested, DNA was precipitated with 50% trichloroacetic acid, and [3H]thymidine incorporated in the DNA was measured using a liquid scintillation counter.
Phospho–Extracellular Signal-Regulated Kinase Dephosphorylation Assay
We used the previously published method (14). Tyrosine and threonine phosphorylated extracellular signal-regulated kinase 2 (ERK2; NEB, Waltham, MA) was incubated for 1 hour at 25°C in the presence or absence of 10 µmol/L compounds, with either the dual-specificity ERK phosphatase MKP-1 or Hep 3B cell lysate proteins. A phosphatase inhibitor cocktail (Sigma) was used as an inhibitor control. ERK2 was then resolved on a Western blot and probed with phospho-ERK(Thr202, Tyr204) [p-ERK(Thr202, Tyr204); Santa Cruz Biotechnologies, Inc., Santa Cruz, CA].
Cdc25A, Cdc25B, and Cdc25C Activity Assay
Full-length Cdc25A, catalytic site of Cdc25B, and catalytic site of Cdc25C were kind gifts from Dr. J. Rudolph (Duke University, Durham, NC; refs. 15–18). The phosphatase activity was measured as previously reported (14) using the artificial substrate O-methyl fluorescein phosphate (Molecular Probes, Inc., Eugene, OR). The compounds were solubilized in DMSO at a final concentration of 7% DMSO. Reactions were initiated by addition of 1 µg Cdc25A, Cdc25B, or Cdc25C phosphatase. Fluorescence emission was measured with a multiwell plate reader.
Competitive Binding of the Four Compounds and Biotinylated Cpd 5 to Glutathione S-Transferase–Cdc25A, Cdc25B, and Cdc25C
Binding to Cdc25 was assayed as described before (14). Briefly, glutathione S-transferase (GST)-Cdc25A, GST-Cdc25B, and GST-Cdc25C were incubated with 1 µmol/L biotinylated Cpd 5 and 10 µmol/L each of the four compounds to give a compound to biotinylated Cpd 5 ratio of 10. Biotinylated Cpd 5 was immunoprecipitated with antibiotin antibody and Cdc25 bound to biotinylated Cpd 5 in the immunoprecipitate was determined on a Western blot probed with anti-Cdc25A, anti-Cdc25B, and anti-Cdc25C antibodies. MKP-1 protein, whose activity was not inhibited by the compounds, served as a negative control.
Western Blots and Immunoprecipitation
Western blotting and immunoprecipitation were done as previously described (19). Hybond polyvinylidene difluoride membranes and horseradish peroxidase–conjugated secondary antibody were from Amersham Biosciences (Piscataway, NJ). Antibodies were purchased from Santa Cruz Biotechnology (ERK2, Cdc25A, Cdc25B, Cdc25C, Cdk1, Cdk2, Cdk4, phospho-tyrosine, phospho-tyrosine 15 Cdk1), Cell Signaling Technology [Beverly, MA; p-ERK(Thr202, Tyr204)], Zymed Laboratories (South San Francisco, CA; phospho-serine, phospho-threonine), and Sigma (ß-actin).
cdc25A Gene Transfection
The mammalian expression plasmids encoding the full-length wild-type Cdc25A (cdc25A wt) or a catalytically dead mutant cdc25A (C430S, cdc25A mut) were generously provided by Dr. T. Roberts (Dana-Farber Cancer Institute, Boston, MA; ref. 20). Transfections were done by the LipofectAMINE method following the manufacturer's instructions (Invitrogen, Carlsbad, CA). Briefly, Hep 3B cells were plated in 12-well plates and transfected with 0.8 g/well plasmid DNA in Opti-MEM transfection medium using the LipofectAMINE 2000 reagent. Five hours after transfection, the medium was replaced with complete growth medium and the cells were allowed to recover overnight before compound treatment for 24 hours. DNA synthesis was assessed as reported above.
Statistical Analysis
The significance of the differences between treatments was determined by the Student's t test.
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Results
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Growth Inhibition by Four Compounds
We examined our two new analogues and compared their actions, together with the previously described Cpd 5 and NSC 95397, on cell growth inhibition. We found that Cpd 5, NSC 95397, M-NSC, and D-NSC all had potent growth inhibitory activity against Hep 3B, Hep G2, and PLC/PRL/5 hepatoma cells and MCF7 breast cancer cells in culture, with IC50 between 1.2 and 12.9 µmol/L (Fig. 1). A significant difference in potency was noted for D-NSC, when compared with Cpd 5, NSC 95397, and M-NSC (Student's t test, P < 0.001).
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Figure 1. A, chemical structures of K vitamin analogues and median growth inhibitory concentrations in asynchronously growing Hep 3B human hepatoma cells and MCF7 human breast cancer cells. D-NSC was significantly more potent than the three other compounds (Student's t test, P < 0.001). B, growth inhibition in asynchronous Hep 3B cells by the actions of Cpd 5, NSC 95397, M-NSC, and D-NSC at IC50 dose versus DMSO control.
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Growth Inhibition Was Mediated Partly through p-ERK Induction
Because our previously studied vitamin K derivative, Cpd 5, was shown to strongly induce p-ERK, which was required for its growth inhibitory actions (19, 21), we tested the new analogues for their effects on cellular p-ERK levels. All compounds induced a strong and rapid increase in p-ERK amounts in Hep 3B cells, which was sustained over a prolonged period of time (Fig. 2A). NSC 95397 and its derivatives were more potent than Cpd 5 in inducing p-ERK at any given time point (Fig. 2B). Glutathione, as well as the MEK inhibitors PD98059 and U0126, antagonized both their growth inhibitory actions and the p-ERK induction by all compounds (Fig. 2C and D). However, the MEK inhibitors did not fully reverse compound-mediated growth inhibition, especially by D-NSC, although the activation of p-ERK was similar for NSC, M-NSC, and D-NSC. The MEK inhibitors completely antagonized p-ERK induction.
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Figure 2. The K vitamin analogues mediated their growth inhibitory effect through induction of p-ERK. A, time course in Hep 3B cells treated with Cpd 5, NSC 95397, M-NSC, and D-NSC at 10 µmol/L. B, p-ERK was induced in a dose-dependent manner after a 30-min treatment. C, antagonism of compound-induced growth inhibition by glutathione (GSH) and MEK inhibitors (PD98059, U0126). D, inhibition of compound-induced p-ERK expression by glutathione and MEK inhibitors. Hep 3B cells were asynchronous in every experiment described above. In (C) and (D), Cpd 5, NSC 95397, M-NSC, and D-NSC were used at 20 µmol/L, PD98059 at 10 µmol/L, and U0126 at 5 µmol/L. The same membranes were stripped and blotted with ERK2 antibody for the loading control.
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Inhibition of a p-ERK Phosphatase in Compound-Treated Cell Lysates
The sustained induction of p-ERK led us to hypothesize that a phosphatase for active phosphorylated ERK kinase might be inhibited by the actions of the K vitamin analogues. This idea was supported by the ability of our compounds (10 µmol/L), as well as a phosphatase inhibitor cocktail (control), to antagonize the dephosphorylation of p-ERK by lysates from nontreated cells in a cell-free system. However, the p-ERK1/2 phosphatase activity of the dual-specificity ERK phosphatase MKP-1 was not antagonized by any of our compounds at the same concentrations (Fig. 3).
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Figure 3. Inhibition by the four compounds of p-ERK phosphatase activity of MKP-1 and of Hep 3B cell lysate proteins. Tyrosine and threonine phosphorylated ERK2 (p-ERK) was incubated for 60 min at 25°C in a dephosphorylation assay with either MKP-1 or Hep 3B cell lysates. The Hep 3B lysates were cleared of endogenous ERK2 by immunoprecipitation with anti-ERK2 antibody. The dephosphorylation assays were done in the presence or absence of 10 µmol/L each of the four compounds. ERK2 was then resolved by a Western blot and probed with p-ERK(Thr202, Tyr204) antibody. The Western blot was probed with ERK2 antibody for the loading control. A phosphatase inhibitor cocktail (PPI) was used to inhibit the phosphatases as a control.
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Binding of the Four Compounds to Cdc25A, Cdc25B, and Cdc25C
We considered that the phosphatases in Hep 3B lysates might be dual-specificity phosphatases because the compounds rapidly induced tyrosine phosphorylation (Fig. 4A) and, to a lesser extent, serine and threonine phosphorylation (Fig. 4B and C), as previously shown for Cpd 5 (22). Cpd 5 has been shown to inhibit the specific Cdc25 family of dual-specificity phosphatases (14, 23). The ability of the four compounds to bind to GST-tagged Cdc25 isozymes was studied in a cell-free system by assessing their actions as competitive antagonists in the presence of biotinylated Cpd 5, which has previously been reported to bind to the catalytic site of Cdc25B (14). As shown in Fig. 5, NSC 95397 and its hydroxyl derivatives were able to compete with the binding of biotinylated Cpd 5 with the Cdc25s. However, MKP-1, whose p-ERK dephosphorylating activity was not inhibited by the compounds, was not able to compete with biotinylated Cpd 5 for binding to the Cdc25s. The inhibition of a phosphatase action by compound-treated cell lysates and the ability of the compounds to compete with binding to Cdc25A, Cdc25B, and Cdc25C led us to hypothesize that Cdc25 activity might be antagonized by these four K vitamin analogues.
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Figure 5. Competitive binding of the four compounds to GST-Cdc25A, GST-Cdc25B, and GST-Cdc25C. GSTCdc25A (100 mU), GST-Cdc25B (100 IU), and GST-Cdc25C (50 mU) were incubated in a 10 µL reaction volume for 18 h at 4°C, with 1 µmol/L biotinylated Cpd 5 (bt-Cdp 5) and 10 µmol/L of each of the four compounds (Cpd 5, NSC 95397, M-NSC, or D-NSC) or MKP-1 protein (control). Biotinylated Cpd 5 was immunoprecipitated with antibiotin antibody and Cdc25 bound to biotinylated Cpd 5 in the immunoprecipitate was determined on a Western blot probed with anti-Cdc25A, anti-Cdc25B, and anti-Cdc25C antibodies.
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Antagonism of Cdc25A, Cdc25B, and Cdc25C Activity in a Cell-free System
We examined the activity of Cdc25A, Cdc25B, and Cdc25C using the artificial substrate OMPF. In vitro inhibition of Cdc25A, Cdc25B, and Cdc25C activity was found with each of the compounds (Fig. 6). An increase in potency was associated with addition of a bis-thioethanol moiety to the structure (NSC 95397 compared with Cpd 5 for Cdc25B and Cdc25C but not for Cdc25A). With further addition of hydroxyl groups to the naphthoquinone ring, M-NSC and D-NSC were found to be more potent than the parent compound NSC 95397 (for Cdc25A but not for Cdc25B or Cdc25C).
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Figure 6. In vitro inhibition of Cdc25A, Cdc25B, and Cdc25C by the four compounds. Phosphatase assays were done by incubation of full-length Cdc25A (100 mU), catalytic site of Cdc25B (100 IU), or catalytic site of Cdc25C (50 mU) phosphatase (1 µg) for 1 h with Cpd 5, NSC 95397, M-NSC, and D-NSC at increasing concentrations. Enzymatic activity was assessed using the artificial substrate O-methyl fluorescein phosphate. Fluorescence emission from the product was measured over a 10- to 60-min reaction period at ambient temperature with a multiwell plate reader.
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Effects of Cdc25A Gene Transfection on Growth Inhibition by the Compounds
Because we had found that the Cdc25s were targets for the K vitamin analogues, we further examined their action on whole cells after modulation of the target. Growth inhibition of Hep 3B cells by the analogues was examined after transfection of with either a cdc25A wild type (cdc25A wt) or a catalytically inactive cdc25A mutant (cdc25A mut) gene. Inhibition of DNA synthesis after 24 hours of analogue treatment in the Hep 3B cells transfected with the cdc25A mut was found to be reversed when cdc25A wt was transfected (Fig. 7). The differences were statistically significant for all the compounds.
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Figure 7. Cdc25A overexpression reversed the growth inhibitory effect induced by the four compounds. Hep 3B cells were transfected with either a full-length wild-type cdc25A (cdc25A wt) or a catalytically dead mutant cdc25A (cdc25A mut). After overnight recovery, cells were compound-treated at IC50 concentrations for 24 h. [3H]thymidine (5 µCi) was added in the fresh medium and maintained there for a 3-h culture period. Hep 3B cells were harvested, DNA was precipitated, and [3H]thymidine incorporated in the DNA was measured using a liquid scintillation counter.
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Effects of Analogues on Cell Cycle Progression
Cdc25A, Cdc25B, and Cdc25C have a prominent role in control of G1-S and G2-M phases of the cell cycle, respectively (3, 5, 6, 24). Therefore, we predicted that the cell cycle might be affected by our analogues. To selectively assess G1-S or G2-M arrest, cells were treated with low concentrations of compound in the IC50 dose range to study them up to 24 hours. An analysis of cell cycle alterations at the same concentration for all compounds could not be done because of the growth inhibitory potency of DNSC after 6 hours of treatment. After synchronization in either G1-S or G2-M phase of the cell cycle by hydroxyurea or nocodazole, respectively, and subsequent release from the block, a cell cycle arrest in both phases was found with all the analogues but with differential potency (Fig. 8).
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Figure 8. G1-S and G2-M cell cycle arrest after treatment with compounds at IC50 concentration. Hep 3B cells were synchronized in G1-S phase by 1 mmol/L hydroxyurea (A) or in G2-M phase by 400 nmol/L nocodazole (B), then released from the block to be immediately treated with Cpd 5, NSC 95397, M-NSC, and D-NSC at IC50 concentration versus DMSO control, and proportion of cell cycle phases was assessed by flow cytometry after propidium iodide staining.
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Antagonism of Cdc25A, Cdc25B, and Cdc25C Activity in Hep 3B Cells
Because our compounds arrested cells in both G1 and G2 phases of the cell cycle and they were found to inhibit Cdc25 phosphatases in cell-free assays, we determined the tyrosine phosphorylation status of the Cdc25 substrate Cdks (Cdk1, Cdk2, and Cdk4) in Hep 3B cells. The Cdk activities had been shown to be inhibited when they remained tyrosine phosphorylated due to inhibition of Cdc25 activities (3–6). Hep 3B cells were synchronized at the G1-S or the G2-M phases with 1 mmol/L hydroxyurea or 400 nmol/L nocodazole treatment for 24 hours, respectively. The blocks were then released by washing and changing the growth medium. The compounds were added to the culture medium at their respective IC50 concentrations. After 6 hours (G1-S) or 4 hours (G2-M) treatment, the cells were harvested. Cdk2 and Cdk4 (from the G1-S block-released cells) and Cdk1 (from the G2-M block-released cells) were immunoprecipitated and Western blotted. The Western blots were probed with anti-pY and Cdk2, Cdk4, or Cdk1 antibodies. We found that the tyrosine phosphorylation of Cdk1, Cdk2, and Cdk4 were all induced after treatment with our compounds, especially D-NSC (Fig. 9).
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Figure 9. Tyrosine phosphorylation of the Cdc25 substrate Cdks due to compound action. Hep 3B cells were either synchronized at the G1-S or G2-M boundaries, released from hydroxyurea (1 mmol/L) or nocodazole (400 nmol/L) block, and immediately treated with compounds (Cpd 5, NSC 95397, M-NSC, and D-NSC at their IC50 concentrations) versus DMSO control. Six hours after release from the G1-S blocks, the cell lysates were immunoprecipitated with anti-Cdk2 or anti-Cdk4 antibodies, then followed by Western blot using antiphosphotyrosine antibody. The membranes were stripped and probed using anti-Cdk2 and anti-Cdk4 antibodies as loading control. Hep 3B cells were also treated at IC50 concentration of the compounds for 4 h after releasing them from G2-M block and harvested. Cell lysates were immunoprecipitated with anti-Cdk1 antibody and then followed by Western blot using antiphosphotyrosine-Cdk1 (pY-Cdk1) antibody. The same membrane was stripped and probed using anti-Cdk1 antibody as loading control.
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Discussion
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The Cdc25 dual-specificity phosphatases have important roles in control of cell cycle promotion (3, 5, 6, 24) and because there is involvement of Cdc25A and Cdc25B in neoplastic transformation (7), several groups have attempted to develop small-molecule inhibitors as potential targets for anticancer therapy (7, 10, 25–28). Recently, NSC 95397 was reported as the most potent inhibitor of Cdc25A, Cdc25B, and Cdc25C to date, with a mean growth inhibitory concentration of <1 µmol/L against the entire National Cancer Institute 60-tumor-cell panel (10). However, the hydrophobicity of NSC 95397 may have physiochemical effects on the cell membrane, which might, in turn, limit its biological activity (11). We, therefore, designed and synthesized hydroxyl derivatives of NSC 95397 to increase its hydrophilic properties. The addition of polar groups to the naphthoquinone ring resulted in more potent inhibition of both cell growth and Cdc25s compared with parent compound NSC 95397 (Figs. 1 and 6). The relationship between hydroxyl groups on the naphthoquinone ring and potency of cellular actions was supported by several observations. First, DNSC was found to be the most potent growth-inhibitory compound (Fig. 1). Second, NSC 95397 and its derivatives all mediated growth inhibition, at least in part through p-ERK induction, as was previously found for Cpd 5 (21). The new compounds were also more potent than Cpd 5 both in p-ERK induction and in mediating growth inhibition (Fig. 2). Third, the two new analogues were more potent inhibitors of Cdc25 cell-free system. However, differential inhibition was found between the three Cdc25 isoforms. M-NSC and D-NSC were clearly more potent in inhibiting the activity of Cdc25A compared with Cdc25B or Cdc25C. However, they were not significantly different than NSC 95397 in inhibiting the activities of Cdc25B or Cdc25C. These differences might be possibly due to the differences in structure of the Cdc25 isoforms. All four compounds had their action mediated at least partly through ERK phosphorylation because MEK inhibitors could antagonize their growth inhibitory and p-ERK–inducing effects (Fig. 2C and D). However, the MEK inhibitors were not able to completely antagonize growth inhibition, especially by D-NSC, although the p-ERK induction was completely abolished. This probably suggests that growth inhibition might be mediated by signaling pathways other than ERK. A direct effect on Cdc25s and cell cycle was also likely involved.
In Hep 3B cells treated with our compounds, the tyrosine phosphorylation of Cdk1, Cdk2, and Cdk4 were induced especially by D-NSC. This might suggest a direct inhibition of the Cdc25 phosphatases by the compounds.
It has been previously shown that ERK activation can lead to either cell proliferation if transient or cell cycle arrest is sustained (29, 30). Two related mechanisms were previously reported to explain Cpd5-mediated actions. First, that it could cause prolonged induction of p-ERK with subsequent prolonged phosphorylation of its physiologic substrate, the transcription factor Elk-1 (30, 31). Second, Cpd 5 caused inhibition of transcriptional activation of cAMP-responsive element binding protein (CREB), which also involved the ERK pathway (21). Positive transcriptional regulation of cyclin D by the ERK pathway has been well-characterized during the G1-S transition (32–34). Sustained ERK induction might also affect Cdc25A activity. A cross-talk between Cdc25A and Raf/MEK/ERK pathways is supported by a growing body of evidence. It has been reported that Cdc25A is a phosphatase for both epidermal growth factor receptor (19), which transmits activating signals to the Raf/MEK/ERK through Ras (35), as well as for Raf-1, with a consequent significant decrease in Raf-1 kinase activity (20). Furthermore, overexpression of Cdc25A in whole cells caused dephosphorylation of ERK (31). These reports support the hypothesis that Cdc25A regulates ERK phosphorylation status. On the other hand, Raf-1 has been reported to be a kinase for Cdc25A, with a 3- to 4-fold increase in Cdc25A phosphatase activity toward both the artificial substrate pNPP and physiologic substrate GST-cyclin A/Cdk2 (36).
In intact cells, two factors at least could play a role in the differential inhibition of Cdc25 isozymes. They are the ability of each compound to accumulate in the cell as well as their specificity toward the Cdc25s. Drug delivery into the cell may be influenced by the addition of hydroxyl groups, resulting in an increase in hydrophilic properties. Simple diffusion might not be the only mechanism for intracellular accumulation of these K vitamin analogues as we previously reported (37), because K vitamin can accumulate in cells against a concentration gradient. As for the specificity of the different compounds toward their target Cdc25s, the study of enzyme structure and inhibitor binding and interactions, using the previously published crystal structures of the Cdc25A and Cdc25B catalytic domains (38, 39) and molecular modeling, may provide further insights.
It was interesting to find that the p-ERK dephosphorylating activity of the ERK phosphatase MKP-1 was not inhibited by these compounds, although they inhibited the phosphatase activity of Hep 3B lysate proteins. MKP-1 also did not compete for binding to Cdc25s. Therefore, the phosphatase(s), other than MKP-1, in the Hep 3B cell lysate might be involved in the pERK dephosphorylation. Addition of hydroxyl groups to the naphthoquinone ring thus enhanced the growth inhibitory potency of NSC 95397. These two new compounds may represent valuable tools for probing signaling pathways involving Cdc25s.
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Acknowledgments
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We thank Drs. J. Rudolph and T. Roberts for providing Cdc25 phosphatases and wild-type and mutant Cdc25A expression plasmids, respectively.
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Footnotes
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Grant support: NIH grant CA 82723 (B.I. Carr) and scholarships from Association de Recherche contre le Cancer and Hospices Civils de Lyon (V.P. Peyregne).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: S.W. Ham is on sabbatical leave from Department of Chemistry, Chung-Ang University, Seoul, South Korea.
Received 10/ 6/04;
revised 1/14/05;
accepted 2/ 9/05.
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Abstract
Introduction
