Vol. 2, 747-751, August 2003
Molecular Cancer Therapeutics
© 2003 American Association for Cancer Research
Naamidine A Intensifies the Phosphotransferase Activity of Extracellular Signal-regulated Kinases Causing A-431 Cells to Arrest in G1
Robyn D. James,
David A. Jones,
William Aalbersberg and
Chris M. Ireland1
Department of Medicinal Chemistry [R. D. J., C. M. I.] and Division of Molecular Pharmacology, Huntsman Cancer Institute [D. A. J.], University of Utah, Salt Lake City, Utah 84112, and Institute of Applied Sciences, The University of the South Pacific, Suva, Fiji Islands [W. A.]
1 To whom requests for reprints should be addressed, at Department of Medicinal Chemistry, University of Utah, 30 South 2000 East, Room 307, Salt Lake City, UT 84112-9453
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Abstract
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The binding of epidermal growth factor to its receptor activates the mitogen-activated protein kinase pathway. This pathway has been identified as a vital link between membrane-bound Ras and nuclear events and, therefore, is a potential target for chemotherapeutic drugs. We reported previously that naamidine A (NA), an alkaloid from the calcareous sponge Leucetta chagosensis, potently inhibited epidermal growth factor-stimulated DNA synthesis. In this current study, we demonstrate that in addition to its antimitogenic effects (complete inhibition of DNA synthesis at 0.78 µM in A-431 cells after 30 h), NA at 1.56 µM caused cells to arrest in the G1 phase of the cell cycle. In vitro kinase, in-gel kinase, and Western blotting experiments demonstrate that extracellular signal-regulated kinase (ERK) 1 and ERK2 are primary molecular targets for NA in A-431 cells. Treatment with NA at concentrations between 0.78 and 3.13 µM produces changes in the phosphorylation states of the ERKs, and strongly induces the phosphotransferase activity of the ERK enzymes. Our data indicate that treatment with NA generates a robust ERK signal. NA is the first small molecule reported to cause this effect on the ERK kinases and consequent G1 cell cycle arrest.
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Introduction
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Mutations in growth factor signaling pathways are consistently linked to a variety of chronic diseases, including cancer (1). The interaction of growth factors with their specific membrane-bound receptors can trigger a cascade of intracellular biochemical signals that may either activate or repress the transcription of certain genes. Tumor cells frequently display a significantly reduced dependence on this type of exogenous growth stimulation.
The MAPK2 pathway is one of the most studied growth factor cascades (2, 3). Binding of EGF to epidermal growth factor receptor may activate the MAPK pathway, which can culminate in cellular responses including transcription, division, adhesion, and death (4–6). Activation of the ERKs, key members in the MAPK cascade, can result in the activation of several transcription factors, triggering proliferation (1). It has been shown that compounds that interfere with EGF binding to its cellular receptor, or alter the EGF signal at some point in the pathway, have exploitable antiproliferative effects on transformed cells that depend on EGF for growth (7, 8).
NA is a dibenzylated 2-aminoimidazole alkaloid from the calcareous sponge Leucetta chagosensis Dendy, 1913 (Clathrinida: Leucettidae). Its chemical structure (Fig. 1) was elucidated by Carmely and Kashman in 1987 (9). We demonstrated previously that NA possesses the ability to inhibit EGF-stimulated DNA synthesis and inhibit the growth of implanted squamous cell tumors in nude mice (10). These studies also suggested that the molecular target of NA was downstream of the receptor, because neither the extracellular nor the tyrosine kinase domain of the receptor appeared to be affected by treatment with NA (10). In the current study, we report that NA is a potent inhibitor of mitogenesis in A-431 cells by completely halting DNA synthesis after 30 h and arresting the cells in the G1 phase of the cell cycle. We hypothesize based on in vitro kinase, in-gel kinase, and Western blotting experiments that this effect results from changes in the phosphorylation state and phosphotransferase activity of the ERKs. These enzymes propagate the activation of several proteins, which can trigger proliferation, differentiation, or cell death. Treatment with NA strongly induces the phosphotransferase activity of the ERK enzymes in A-431 cells indicating that the ERKs are primary molecular targets of NA. NA is the first small molecule reported to cause this effect on the ERK kinases and consequent G1 cell cycle arrest. This type of robust ERK signal generated by transfection of activated raf or MEK has been reported previously to induce and sustain increased levels of p21CIP1 in various cell lines resulting in G1 arrest (11, 12).
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Materials and Methods
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Chemicals.
The L. chagosensis used in the current study was collected at Kandavu, Fiji, in the Usborne Passage off the Great Astrolabe Reef in January 2001. It was collected using SCUBA between 2 and 10 meters below the surface. NA was isolated from the sponge as reported previously (10).
Cell Culture and Growth Inhibition Assays.
The human epidermoid carcinoma cell line A-431 was purchased from American Type Culture Collection (Manassas, VA). The human colon tumor cell line HCT116 was obtained from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). Both cell lines were maintained at 37°C and 5% CO2. The A-431 cell line was cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 1 mM MEM sodium pyruvate, 50 units/ml penicillin, and 50 µg/ml streptomycin. All of the cell culture reagents were purchased from Invitrogen Corporation (Carlsbad, CA). General cytotoxicity was determined by measuring the cellular metabolism of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (Acros Organics, Denver, CO) from its dissolved form to insoluble purple formazan by a method reported previously (13).
Western Blot Analysis.
Treated and untreated A-431 cells were disrupted using lysis buffer [25 mM Tris (pH 7.4), 150 mM NaCl, 1 mM CaCl2, 1% Triton X-100, 53 mM sodium fluoride, 2 mM sodium orthovanadate, and 1 ng each of leupeptin, aprotinin, and phenylmethylsulfonyl fluoride]. Cell lysates (10 µg protein) were loaded onto 4–12% Bis-Tris gradient NuPAGE gels (Invitrogen Corporation) and electrophoresed at 200 V for 35–45 min, and transferred to polyvinylidene fluoride membrane at 300 mA for 1 h. The membranes were immunostained with anti-ERK, anti-phospho-ERK (1:1000; Cell Signaling Technology, Beverly, MA), H-Ras, phospho-Raf-1, phospho-c-Jun, phospho-Elk1, and c-Fos antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-poly(ADP-ribose) polymerase (1:1,000; BD PharMingen, Los Angeles, CA) for 1 h at room temperature. The membranes were then incubated with goat antirabbit IgG or antimouse IgG horseradish peroxidase conjugates (1:10,000; Santa Cruz Biotechnology). Luminol reagent (Santa Cruz Biotechnology) was used for chemiluminescence detection.
[3H]Thymidine Incorporation Mitogenesis Assay.
Aqueous solutions of [methyl-3H] thymidine (1.0 µCi/µl) were purchased from Perkin-Elmer (Boston, MA). Wallac Optiphase "SuperMix" scintillation fluid was purchased from Fisher Chemicals (Loughborough, England). Samples were counted using a Packard liquid scintillation analyzer, model 1900TR (Meriden, CT). A-431 cells were starved 16 h and then treated with 16.5 pM EGF, 3.17 nM TyrAG1478, NA, or DMSO for the indicated number of hours. The final concentration of DMSO did not exceed 1% (v/v). Cells were incubated with 2–4 µCi/ml of [3H]thymidine for 1–4 h. Cells were lysed by aspirating medium and adding 500 µl of 25% (v/v) perchloric acid per well. Wells were washed twice with 10% (v/v) perchloric acid. Macromolecules were solubilized with 500 µl 1.0 M NaCl and neutralized with HCl. After protein quantitation, the solution was added to 10 ml of scintillation fluid and allowed to quench 3 days before counting. Counts were normalized to protein content.
Cell Cycle Analysis by Flow Cytometry.
A-431 cells were starved for 16 h and then treated with 1.55 µM NA or DMSO, and 16.5 pM EGF as necessary. The final concentration of DMSO did not exceed 0.2% (v/v). Cells were trypsinized and resuspended in ice-cold PBS/absolute ethanol (1:2). After 24 h at 4°C, samples were submitted to the University of Utah Flow Cytometry Resource Facility for analysis.
In Vitro MAPK Assay.
All of the chemicals used in the in vitro kinase assays were purchased from Upstate Biotechnology (Lake Placid, NY). The assay was performed according to the manufacturer’s suggested protocol. Briefly, 20 µl of the appropriate enzyme solution (5 ng/µl) and various concentrations of NA or DMSO were incubated at 30°C for 20 m with gentle agitation. The final DMSO concentration did not exceed 2% (v/v). Ten µl of a magnesium/ATP solution and 20 µg MBP were added, and the samples were incubated at 30°C for 30 min with gentle agitation. The reaction was quenched by the addition of SDS sample buffer, and samples were electrophoresed, transferred to a polyvinylidene fluoride membrane, and probed with a monoclonal 
-phospho-MBP antibody purchased from Upstate Biotechnology. Proteins were detected with Luminol reagent (Santa Cruz Biotechnology).
In-Gel MAPK Assay.
A-431 cells were starved for 16 h and treated with NA or DMSO. The final DMSO concentration did not exceed 0.2% (v/v). Cells were then stimulated with 16.5 pM EGF and lysed after 15 min. Gels were prepared by dissolving 0.5 mg/ml MBP in 12.5% polyacrylamide before casting. Samples were electrophoresed through the MBP-impregnated gels, and proteins were renatured and kinase activity reactivated according to a procedure reported previously (14). [
-32P]ATP was purchased from Perkin-Elmer. The gels were dried and exposed to a PhosphorImager overnight and/or Kodak BioMax MR film for 3–5 days.
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Results
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The Effects of NA on EGF-mediated Mitogenesis.
It has been shown that ERK activation is necessary for progression through the G1 phase of the cell cycle (15). Cells will usually progress from G1 to S between 6 and 12 h after the activation of the EGF cascade. We examined the effects of NA on EGF stimulated DNA synthesis by monitoring incorporation of [3H]thymidine. NA decreased EGF-stimulated DNA synthesis in both a dose- and time-dependent manner as shown in Fig. 2. The data revealed little effect after 3 h, which is an expected response. However, after 30 h of treatment, all of the DNA synthesis was completely abrogated in cells treated with even the lowest concentration (0.78 µM) of NA.
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Fig. 2 The effect of NA treatment on [3H]thymidine incorporation by A-431 cells over a time course. All DPM values given were the average of two replicate experiments with the background values deducted. Error bars correlate with ± SD from the mean for each data point. DNA synthesis is totally abrogated after 30 h for every concentration of NA investigated. P < 0.05, significantly different from A-431 cells treated with EGF alone.
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The Effect of NA on the Cell Cycle.
FACS analysis of NA-treated A-431 cells revealed a considerable arrest in the G1 phase of the cell cycle after 18 and 30 h of treatment compared with treatment with DMSO alone (Fig. 3). Data for the 18 h time point are very similar to that shown for the 30 h time point and, therefore, are not shown. Cell populations differed from 38.56% in G0/G1, 48.99% in S, and 12.45% in G2/M in the untreated cells to 72.87% in G0/G1, 20.39% in S, and 6.75% in G2/M in the treated cells after 30 h treatment. As expected based on [3H]thymidine incorporation data, very little effect was seen at the 3-h time point. Additionally, we saw no increase in sub-G0 cellular debris that is typical of cell death. Consistent with this, Western analysis of NA-treated cells showed no evidence of poly(ADP-ribose) polymerase cleavage, an event typical of cells undergoing apoptosis (Ref. 16; data not shown).
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Fig. 3 FACS analysis of NA-treated cells. Cells were treated with DMSO (top) or 1.56 µM NA (bottom) for 30 h. After treatment, cells were harvested and populations determined by FACS. Data presented are from a representative experiment. G1-arrest in treated cells is evident, of which the percentages increased from 38.56% in untreated populations to 72.87% in treated populations.
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The Effect of NA on the MAPK Cascade.
Because treatment with NA induced growth arrest, we next examined the levels of phosphorylated ERK1/2 in cells responding to NA. Surprisingly, EGF-stimulated levels of p-ERK1/2 were increased by NA in a dose-dependent manner (Fig. 4A). Nonphosphorylated ERK protein was included as a control to verify that the p-ERK antibody did not appreciably cross-react with nonphosphorylated ERK protein (data not shown). It appeared that ERK2 (p42) phosphorylation was induced to a greater degree than ERK1 (p44). However, it is important to note that both total and phospho-ERK2 levels detected in untreated cells (Fig. 4, Lane 6) are approximately twice that of ERK1. Therefore, the induction of both enzymes by treatment with NA may be roughly equivalent relative to their cellular protein levels. To establish that the observed response was because of a change in the ratio of phosphorylated to unphosphorylated protein, and not a change in the level of total cellular protein, the membrane was stripped and reprobed with a control ERK antibody, which detected ERK protein regardless of phosphorylation state (Fig. 4B). This antibody revealed that the total cellular level of ERK protein is unaffected by treatment with NA.
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Fig. 4 Effect of NA on ERK phosphorylation. Cells were treated with NA for 6 h. Protein lysates were isolated, and the levels of phosphorylated (A) and total (B) ERK proteins determined by Western analysis. Lanes: (1) 3.13 µM NA and EGF, (2) 1.56 µM NA and EGF, (3) 0.78 µM NA and EGF, (4) 3.13 µM NA only, (5) EGF and vehicle, and (6) vehicle only.
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We also examined protein and phosphorylation levels of H-Ras, phospho-Raf, MEK1/2, phospho-MEK1/2, phospho-Elk-1, phospho-c-Jun, and c-Fos. All were found to be unaffected by treatment with NA (data not shown).
In-Gel MAPK Assay.
Because treatment with NA appeared to enhance ERK1/2 phosphorylation levels, we next decided to examine whether NA also increased ERK1/2 enzymatic activities. To accomplish this, we used an in-gel kinase assay that detects activity in situ after SDS-PAGE (14, 17). The induction of phosphotransferase activity for both the ERK1 and ERK2 enzymes in the dose response experiment can be seen clearly (Fig. 5). Moreover, the induction of ERK2 is approximately twice that of ERK1.
In Vitro Kinase Assay.
Finally, we measured the effect of NA on the phosphotransferase activity of the purified ERKs using an in vitro assay. This assay quantitated the phosphorylation of MBP, a specific substrate for the ERK enzymes. The results of the ERK1 in vitro assay revealed that NA elicited a dose-dependent induction of the phosphotransferase activity of the enzyme (Fig. 6). The ERK1 induction by 50 µM NA is slightly less than that of 25 µM, because the compound begins to precipitate in polar solvents at that concentration. Interestingly, when the assay was performed with the ERK2 enzyme (supplied as the GST-fusion protein), no induction of phosphotransferase activity was observed (Fig. 7). Human ERK1 and ERK2 enzymes are 83% sequence homologous and 95% similar (18), with the main difference being that residues 9 through 27 on the human ERK1 enzyme are missing from the ERK2 enzyme. These missing residues make up the 2 kDa mass difference for the two proteins. Given this similarity, the lack of activity against GST-ERK2 may have been because of interference by the GST tag.
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Fig. 6 In vitro MAPK assay results for ERK1. The recombinant ERK1 enzyme was incubated with various concentrations of NA for 20 min at which time MBP and ATP solutions were added. Protein levels recognized by an -p-MBP antibody were visualized by Luminol.
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Fig. 7 In vitro MAPK assay results for ERK2. The recombinant ERK2 enzyme was incubated with various concentrations of NA for 20 min at which time MBP and ATP solutions were added. Protein levels recognized by an -p-MBP antibody were visualized by Luminol. No significant background in an MBP-only control was observed (data not shown).
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Discussion
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Since the discovery of oncogenic Ras and its role in tumorigenicity, a great deal of attention has been paid to signal transduction research and to developing drugs that target transduction pathways. The MAPK pathway has been identified as a vital link between membrane-bound Ras and nuclear events and, therefore, has great pharmacological potential as the target for chemotherapeutic drugs. Because they overexpress EGF receptors, potential activators of the MAPK pathway, human epidermoid carcinoma A-431 cells are a model platform for the biological testing of NA.
NA was discovered in high-throughput screens as a potential effector of the MAPK pathway. It exhibited potent ability to inhibit EGF-stimulated DNA synthesis and was, therefore, additionally investigated as a potential lead compound for chemotherapeutic agents. Treatment with NA causes A-431 cells to arrest in the G0-G1 phase of the cell cycle and completely abrogates DNA synthesis after 30 h. The in vitro MAPK data, together with the in-gel MAPK data, convincingly demonstrate that ERK1 and ERK2 are molecular targets of NA. Because the phosphotransferase activity of ERK1 in the in vitro assay is induced by treatment with NA, it is possible that it may elicit its effect by direct interaction with the ERK proteins. There is abundant literature illustrating that a robust and sustained ERK signal induces G1 arrest (11, 12, 15, 19). For example, Pumiglia and Decker (15) provide evidence that a sustained increase in MAPK activity, in response to induction of an activated form of the Raf-1 proto-oncogene (
RAF-1:ER), can lead to inhibition of cyclin-dependent kinase activity and growth arrest in G1. It is clear that treatment with NA causes an amplified ERK signal after 6 h. FACS data reveals that NA-treated cells arrest in the G1 phase of the cell cycle.
NA is the first small molecule known to elicit this effect on the ERK kinases and, thus, represents a valuable tool for studying the effects of modulation of the MAPK pathway on cell proliferation.
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Footnotes
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2 The abbreviations used are: MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; ERK, extracellular-regulated kinase; NA, Naamidine A; MEK, mitogen-activated protein/extracellular-regulated kinase kinase; MBP, myelin basic protein; FACS, fluorescence-activated cell sorter; p, phosphorylated; GST, glutathione S-transferase. 
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.
Received 6/10/02;
revised 1/ 2/03;
accepted 3/26/03.
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Abstract
Introduction
3.13 µM is significantly different from cells treated with EGF alone.