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CELLULAR AND MOLECULAR

Stimulation of Extracellular Signal-Regulated Kinase Pathway by Suramin with Concomitant Activation of DNA Synthesis in Cultured Cells

Department of Molecular Cell Signaling, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan

Received for publication August 24, 2003
Accepted October 14, 2003.

	Abstract

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Suramin is a well known antitrypanosomal drug and a novel experimental agent for the treatment of several cancers, yet the molecular mechanisms through which suramin exerts its effects on cell functions are not completely clear. In this study, we investigated the potential of suramin to activate the mitogen-activated protein kinase cascade in cultured Chinese hamster ovary (CHO) cells. The treatment of CHO cells with suramin increased the enzyme activity of extracellular signal-regulated kinases (ERK1/2) approximately 10-fold dose and time dependently. The EC₅₀ value was approximately 2.4 µM. This activation is inhibited by PD98059 and wortmannin/LY294002, indicating a crucial role for mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K), respectively. Suramin-mediated stimulation of PI3K was confirmed by the observation that suramin stimulates the phosphorylation of protein kinase B (Akt) in a wortmannin-sensitive manner. Furthermore, cAMP response element-binding protein, a transcription factor, was also activated by suramin in a MEK-dependent manner. The suramin-induced phosphorylation of cGMP-dependent protein kinase was also suggested by a solid-phase kinase assay. The suramin effects on CHO cells were shown to have a concomitant increase in DNA synthesis, which was attenuated by PD98059. Similar activation of ERK1/2 activity by suramin was observed in other cell lines such as Chinese hamster lung or PC12 cells, but not in RBL2H3, ECV304, and OVK18 cells, indicating a cell-type specific mechanism for suramin. These results indicate that suramin induces mitogenic activity in several cell lines through the pathway from PI3K to MEK and ERK.

Mitogen-activated protein kinases (MAP kinases), a family of serine/threonine kinases regulating diverse cellular activities, are divided into three classes: extracellular signal-regulated kinases (ERKs), Jun amino-terminal kinases (JNKs), and p38 MAP kinases. JNKs and p38 MAP kinases mediate signals in response to cytokines and environmental stress, whereas ERK subtypes are classically recognized as key transducers in the signaling cascade mediating cell proliferation in response to growth factors (Davis, 1995). Two major isoforms of ERK, p44 (ERK1), and p42 (ERK2) have been identified in mammalian systems. A major pathway involved in ERK1 and ERK2 (ERK1/2) stimulation in a variety of cell types requires the sequential activation of Ras, Raf, and MAP kinase kinase (MEK) (Davis, 1993).

In this article, as a simple model for a suramin-responsive cell signaling study, we examined the effect of suramin on the ERK cascade in cultured Chinese hamster ovary (CHO) cells commonly used in recombinant technology, with the expectation of the suppressive effect of suramin in ERK signaling that may explain the antiangiogenesis activity. To our surprise, suramin was found to significantly stimulate the ERK cascade with a concomitant increase in DNA synthesis.

	Materials and Methods

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Materials. Suramin was purchased from Wako Pure Chemicals (Osaka, Japan). An anti-ERK1/2 antibody was purchased from Zymed Laboratories (South San Francisco, CA). An anti-phosphotyrosine antibody (4G10) was from Seikagaku Co. (Tokyo, Japan). An anti-phospho-ERK1/2 antibody that recognizes dual phosphorylated, active ERK1/2 was purchased from Promega (Madison, WI). Antibodies recognizing dual phosphorylated p38 and JNK were also from Promega. Antibodies against MEK and phospho-MEK, Akt, and phospho-Akt (Ser473), and cAMP response element-binding protein (CREB) and phospho-CREB (Ser133) were from Cell Signaling Technology Inc. (Beverly, MA). The MEK-1 inhibitor PD98059, protein kinase C (PKC) inhibitor GF109203X, and Ras inhibitor FTI-277 were obtained from Calbiochem (San Diego, CA). CHO cells, okadaic acid-resistant CHO cells (CHO/OAR2–3 and CHO/OAR6–6), and human ovarian carcinoma (OVK18) cells were purchased from the Cell Resource Center of the Biomedical Research Institute of Development, Aging and Cancer (Tohoku University, Sendai, Japan). Chinese hamster lung cells were from the JCRB Cell Bank (National Institute of Health Sciences, Tokyo, Japan). Syrian hamster smooth muscle cells (DDT₁MF-2) and human prostate cancer cells (PC-3) were from American Type Culture Collection (Manassas, VA). Human transformed endothelium cells from umbilical cord (ECV304) were provided from Dr. K. Takahashi (Tokyo Medical University). PhosphoSpots cellulose strips were purchased from Jerini Bio Tools (Berlin, Germany). An ERK1/2 enzyme assay kit was purchased from Amersham Biosciences Inc. (Piscataway, NJ).

Cell Culture, Stimulation, and Extraction. CHO cells were maintained in F-12 medium supplemented with 10% fetal calf serum and 100 µg/ml kanamycin sulfate at 37°C in a humidified incubator containing 5% CO₂. Other cultured cells were maintained in various media according to the protocols from the providers. CHO cells grown to 70 to 90% confluence in six-well plates were serum-starved for approximately 10 h in F-12 medium and then stimulated with suramin in the presence or absence of various drugs. The stimulation was stopped by washing three times with 5 ml of ice-cold phosphate-buffered saline containing 1 mM EDTA and 1 mM Na₃VO_4. Cells were scraped into 400 µl of lysis buffer [20 mM Tris-HCl (pH 7.4), 0.5% Nonidet P-40, 50 mM -glycerophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM Na₃VO₄, 0.1 mM Na₂MbO₄, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml of each leupeptin, pepstatin A, antipain, and chymostatin]. Cellular extracts were sonicated and centrifuged at 20,000g for 10 min, and the supernatant was saved as the soluble extract. The extract was stored at –80°C until ERK1/2 activity assays.

Western Blotting. For Western blot analyses, the washed cells were solubilized with sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and separated by 10% SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA). The membranes were blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline/Tween buffer (20 mM Tris-Cl, 500 mM NaCl, and 0.1% Tween 20) and then probed with the appropriate primary and horseradish peroxidase-conjugated secondary antibodies. Bound antibodies were visualized using ECL reagent (Amersham Biosciences Inc.). In some cases, the same blot was subsequently stripped at room temperature for 30 min in a restore Western blot stripping buffer (Pierce Chemical, Rockford, IL) and reprobed with anti-nonphospho protein antibodies to determine the amount of indicated proteins loaded in the gel. When necessary, band intensity was quantified with a scanner and analyzed by comparing the value obtained for phosphorylated protein with the value obtained for nonphosphorylated protein using Atto Densitograph, version 2 (Atto Corp., Tokyo, Japan).

ERK1/2 Assays. The phosphorylation and activation of ERK1/2 in CHO cells were detected by Western blotting using an anti-phospho-ERK1/2 antibody as described above. The kinase activity was also determined with an ERK1/2 enzyme assay system (Amersham Biosciences Inc.) that measures the incorporation of [-³²P]ATP into a synthetic peptide (KRELVEPLTPAGEAPNQALLR) as a specific substrate for ERK1/2. Briefly, the cytosolic extracts (15 µl) were incubated at 30°C for 20 min with the substrate buffer (10 µl, containing HEPES, sodium orthovanadate, 0.05% sodium azide, and the synthetic peptide, pH 7.4), the assay buffer (containing HEPES, ATP and magnesium chloride, pH 7.4) and 1 µCi of [-³²P]ATP. Reactions were terminated by the addition of 10 µl of stop reagent containing orthophosphoric acid, and spotted onto P81 phosphocellulose paper discs, which were then washed twice with 75 mM orthophosphoric acid, twice with water, air-dried, and counted using liquid scintillation counting. The activated ERK activity was expressed as the percentage of basal activity or the amount of ³²P incorporated per minute per milligram of protein.

Specificity of Kinase Activation. The kinase specificity of suramin-induced activation was examined using a filter-based technique (PhosphoSpots-assay). The PhosphoSpots test strip containing covalently bound substrate peptides to indicate various kinases was phosphorylated by incubating with 480 µg of suramin-stimulated or basal CHO cell extract in 1.2 ml of kinase buffer containing 50 mM MOPS (pH 7.2), 150 mM NaCl, 30 mM MgCl₂, 4 mM dithiothreitol, 12.5 mM 2-mercaptoethanol, 2 mM EGTA, 1 mM Na₃VO₄, 100 µM okadaic acid, 100 µM ATP, and 1 µCi of [-³²P]ATP at 22°C for 30 min. The reaction was stopped by washing the filter extensively according to the manufacturer's instructions. The phosphorylation was quantified using a bio-image analyzer (Fuji BAS 2000; Fuji Photofilm, Tokyo, Japan).

DNA Synthesis. CHO cells were plated onto 24-well culture plates and grown to confluence. The cells were growth-arrested in serum-free F-12 medium for 40 h and thereafter stimulated by suramin for the following 24 h. Four hours before the end of the stimulation period, cells were pulse-labeled with [³H]thymidine (1 µCi/ml). The experiments were terminated by washing the cells with cold phosphate-buffered saline, precipitation of the acid-insoluble materials with 10% trichloroacetic acid, and extraction of the DNA with 0.5 ml of 0.1 N NaOH/0.1% SDS. Aliquots of 0.4 ml were added to 5 ml of scintillant. [³H]Thymidine incorporation into the cellular DNA was determined by liquid scintillation spectrometry using a -scintillation counter.

	Results

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Activation of ERK by Suramin. Incubation of CHO cells with suramin stimulated ERK1/2 activity in a concentration- and time-dependent manner as shown in Fig. 1. The ERK activity was determined using ERK1/2-specific peptide as a substrate. Maximal activation of ERK1/2 (approximately 10-fold) was observed after 5 to 10 min and then quickly declined (Fig. 1A), although the activation was sustained with continued exposure of CHO cells to suramin for several hours. The activation by suramin was concentration-dependent, with an EC₅₀ value of approximately 2.4 µM (Fig. 1B). It should be noted that a high dose of suramin (>300 µM) suppressed ERK activation from its toxic effect (Fig. 1B). As shown in Fig. 2, A and B, suramin stimulated concentration- and time-dependent increases in 44-kDa (ERK1) and 42-kDa (ERK2) phosphorylation in CHO cells as demonstrated by Western blotting using an antibody to the dual phosphorylated forms of ERK1/2. Similarly, suramin was demonstrated to activate ERK1/2 activity of CHO cells when cultured in normal conditions, i.e., in the presence of 10% fetal calf serum (data not shown). In contrast, no phosphorylated forms of JNK and p38 MAP kinase were detected in the same blots using antibodies to the dual phosphorylated forms of JNK and p38 MAP kinase, indicating no apparent activation of JNK and p38 by suramin (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 1. Suramin activates ERK1/2 in a time- and concentration-dependent manner. A, time course of suramin-mediated ERK1/2 activity in CHO cells. Quiescent cultures of semi-confluent CHO cells were stimulated with suramin (10 µM) for the indicated time. B, concentration-response curve for suramin-mediated ERK1/2 activation. Quiescent cultures of CHO cells were stimulated with the indicated concentrations of suramin for 10 min. The ERK1/2 activity of cell lysates was determined using the ERK1/2 enzyme assay system (Amersham Biosciences Inc.) as described under Materials and Methods. Each point is the mean of three independent experiments.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Suramin stimulates ERK1/2 phosphorylation in a time- and concentration-dependent manner. A and B, CHO cells were treated with suramin as described in Fig. 1. The cell lysates were collected for Western blotting using an antibody specific for dual phosphorylated ERK1/2 (top, P-ERK1/2) or an antibody against total ERK (bottom, ERK1/2). C, suramin-induced phosphorylation of ERK1/2 (P-ERK1/2) was significantly inhibited by the MEK inhibitor PD98059 or PI3K inhibitor wortmannin. CHO cells were pretreated with PD98059 (5–10 µM) or wortmannin (3–30 nM) and then 10 µM suramin was added to the culture medium for 10 min.

Suramin-Induced ERK Activation Is Dependent on MEK and PI3K. ERK1/2 activation by suramin was inhibited by MEK inhibitor PD98059 (Figs. 2C and 3A). A similar strong inhibition of suramin-stimulated ERK1/2 activity was observed with another MEK inhibitor, U0126 (data not shown). These data indicate that suramin did not directly cause ERK1/2 activation (phosphorylation) but mediated ERK1/2 activation via a sequential MEK/ERK pathway. In fact, the time- and dose-dependent phosphorylation of MEK by suramin was demonstrated (Fig. 4, A and B) and the promoted phosphorylation was significantly inhibited in the presence of MEK inhibitor PD98059 (Fig. 4C), indicating MEK activation by suramin. The activation of ERK1/2 was also dependent on PI3K activation because it was dose dependently inhibited by PI3K inhibitors wortmannin or LY294002 (Figs. 2C; and 3, B and C).

View larger version (21K): [in this window] [in a new window]   Fig. 3. MEK and PI3K inhibitors block suramin-induced ERK1/2 activation. Semiconfluent quiescent cultures of CHO cells were pretreated with indicated concentrations of MEK inhibitor PD98059 (A), PI3K inhibitor wortmannin (B), or LY294002 (C) for 20–30 min before treatment with 10 µM suramin. After 10 min, cell lysates were collected for ERK1/2 assay as described under Materials and Methods. The ERK1/2 activity was expressed as the percentage of suramin-stimulated ERK activity.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Suramin-induced phosphorylation of MEK was blocked by PD98059 or wortmannin. A and B, CHO cells were treated with suramin as described in Fig. 3. The cell lysates were collected for Western blotting using an antibody specific for dual phosphorylated MEK1/2 (top, P-MEK) or an antibody against MEK1/2 (bottom, MEK). C, suramin-induced phosphorylation of MEK1/2 was significantly inhibited by the MEK inhibitor PD98059 (5–10 µM) or PI3K inhibitor wortmannin (10–30 nM). +, denotes 10 µM suramin.

Suramin Stimulates Akt Phosphorylation. The effect of suramin on the phosphorylation pattern of Akt (protein kinase B) in CHO cells was investigated because Akt phosphorylation is considered to reflect the activation of PI3K (Andjelkovic et al., 1996). Time-course experiments indicate a rapid phosphorylation of Akt within 5 min of stimulation with suramin followed by a rapid decrease after 30 min (Fig. 5B). Akt phosphorylation by suramin occurs in a concentration-dependent manner, with a maximum effect with 30 µM (Fig. 5A). PI3K is likely to be involved in the suramin-induced phosphorylation of Akt because it is blocked with PI3K inhibitors wortmannin (Fig. 5C) or LY294002 (data not shown). Significant inhibition of suramin-induced phosphorylation of Akt by PD98059 was not detected under the experimental conditions (Fig. 5C). Because it is shown that the Ras Raf MEK ERK1/2 and the PI3K Akt routes are stimulated via activation of insulin receptors (White, 1997), the extent of phosphorylation of Akt and ERK1/2 by suramin and insulin was examined in CHO cells (Fig. 5D). Suramin preferentially phosphorylated ERK1/2 and insulin preferentially phosphorylated Akt, suggesting Akt phosphorylation is not necessarily required for suramin-induced ERK phosphorylation.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Suramin stimulates Akt phosphorylation in a time- and concentration-dependent manner. A and B, CHO cells were treated with suramin as described in Fig. 3. The cell lysates were collected for Western blotting using an antibody specific for phosphorylated Akt (top, P-Akt) or an antibody against Akt (bottom, Akt). C, suramin-induced phosphorylation of Akt was significantly inhibited by the PI3K inhibitor wortmannin (10–50 nM) but was not inhibited by MEK inhibitor PD98059 (5–10 µM). +, denotes 10 µM suramin. D, phosphorylation of Akt and ERK1/2 by suramin or insulin in CHO cells. Quiescent cultures of CHO cells were stimulated with 10 µM suramin or 20 nM insulin for 10 min and the cell lysates were analyzed by Western blotting using antibody specific for phosphorylated Akt (top, P-Akt), phosphorylated ERK1/2 (middle, P-ERK), or nonphosphorylated ERK1/2 (bottom, ERK).

Suramin Stimulates CREB Phosphorylation. Phosphorylation of CREB, one of the transcriptional factors, is thought to modify transcriptional activity. To establish whether suramin was capable of signaling to CREB through ERK1/2, phosphorylated CREB were determined with an antibody that recognizes the Ser133 phosphorylation site on CREB. As shown in Fig. 6, suramin (10 µM) stimulated the time-dependent phosphorylation of CREB. This stimulation was significantly inhibited in the presence of the MEK inhibitor PD98059. The extent of CREB phosphorylation by suramin was determined by densitometric scanning and found that suramin increased CREB phosphorylation about 3-fold, and more than one-half of the phosphorylation was attenuated by PD98059 (Fig. 7).

View larger version (48K): [in this window] [in a new window]   Fig. 6. Suramin-induced CREB phosphorylation in CHO cells. A, time course of CREB phosphorylation by suramin in CHO cells. Quiescent cultures of CHO cells were stimulated with 10 µM suramin for the indicated time. The cell lysates were collected for Western blotting using an antibody specific for phosphorylated CREB (top, P-CREB) or an antibody against CREB (bottom, CREB). B, effect of MEK inhibitor PD98059 on the suramin-induced phosphorylation of CREB. CHO cells were pretreated with PD98059 (5–10 µM) as shown in Fig. 2 and then 10 µM suramin was added to the culture medium for 10 min.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Quantification of time course of suramin-induced CREB phosphorylation and the inhibitory effect of PD98058 on CREB phosphorylation. The Western blots of CREB phosphorylation from two to three independent experiments performed as described in Fig. 6 were quantified by densitometry as described under Materials and Methods. Data were normalized by the protein amount based on nonphospho-CREB immunoreactivity and expressed by setting the basal response 1-fold.

PKC Is Not Involved in Suramin-Induced ERK Activation. The possible involvement of PKC in suramin-induced ERK1/2 activation was examined. Phorbol 12-myristate 13-acetate (PMA) treatment is known to cause acute activation of PKC, but depletes the level of PKC after prolonged treatment (Newton, 1995). Treatment of CHO cells with 100 nM PMA strongly activated ERK1/2 in CHO cells within 10 min (Fig. 8A), demonstrating that ERK1/2 can be activated via a PKC-dependent pathway in these cells. However, ERK1/2 activation by suramin was not significantly affected by PMA treatment for 16 h, which completely depleted the PMA-induced activation of ERK1/2 (Fig. 8A). Furthermore, pretreatment of CHO cells with GF109203X, an inhibitor of conventional PKC, before suramin treatment showed only 30% blocking of the suramin activation of ERK1/2 (Fig. 8B). No effect of the cAMP-dependent protein kinase inhibitor H89 was observed in the suramin-induced ERK1/2 activation (Fig. 8B).

View larger version (26K): [in this window] [in a new window]   Fig. 8. Lack of involvement of PKC in suramin-mediated ERK1/2 activation. A, quiescent semi-confluent CHO cells were pretreated with or without 100 nM PMA for 16 h. The CHO cells were then treated by 100 nM PMA for 10 min and ERK1/2 activity was determined. Similarly, the treated CHO cells were also stimulated by suramin (10 µM, for 5 min). B, inhibitor of PKC (GF109203X) blocks PMA-induced ERK1/2 activation but does not significantly block ERK1/2 activation by suramin. The cAMP-dependent protein kinase inhibitor H89 is ineffective in suramin-induced ERK1/2 activation. CHO cells were pretreated for 30 min with 1 µM GF109203X or 5 µM H89 before the addition of 10 µM suramin.

Effect of Ras Inhibitor on Suramin-Induced ERK Activation. To determine whether the ERK activation by suramin in CHO cells is mediated primarily by the Ras/MEK/ERK signaling pathway, the effect of the farnesyl-transferase inhibitor FTI-277, a Ras inhibitor (Lerner et al., 1995), on suramin-induced ERK phosphorylation was examined. As shown in Fig. 9A, no inhibition of suramin-induced ERK phosphorylation was observed by treatment with FTI-277, whereas insulin-induced ERK phosphorylation (White and Kahn, 1994) was significantly inhibited by FTI-277 under similar conditions (Fig. 9B).

View larger version (32K): [in this window] [in a new window]   Fig. 9. Suramin-induced ERK phosphorylation was not blocked by FTI-277. A, confluent quiescent cultures of CHO cells were pretreated with the indicated concentrations of FTI-277, a Ras inhibitor, for 16 h and then 100 µM suramin was added to the medium for 10 min. The cell lysates were collected for Western blotting using an antibody specific for dual phosphorylated ERK1/2 (top) or an antibody against ERK1/2 (bottom). The quantification of the Western blot by densitometric scan shows no inhibition by FTI-277. Data were normalized by the protein amount based on nonphospho-ERK1/2 immunoreactivity. (B) FTI-277-pretreated CHO cells as described above were also stimulated by 200 nM insulin for 10 min and the cell lysates were analyzed by Western blotting using an antibody specific for dual phosphorylated ERK1/2 (top) or an antibody against ERK1/2 (bottom).

Effect of Tyrosine Kinase Inhibitors on Suramin-Induced ERK Activation. The involvement of tyrosine kinase in suramin-dependent ERK activation was examined by the preincubation of CHO cells with various tyrosine kinase inhibitors. Suramin-induced ERK activation was not significantly attenuated in the presence of protein tyrosine kinase inhibitors, such as genistein (up to 100 µM), PP1 (a Src-family selective inhibitor, up to 10 µM), and AG1478 (an epidermal growth factor receptor inhibitor, up to 100 nM). It was also found that neither epidermal growth factor alone (25 ng/ml) nor in combination with suramin (10 µM) modified ERK activity in CHO cells (data not shown). These data indicate that no direct involvement of protein tyrosine kinases in suramin-induced ERK activation is likely in CHO cells.

Protein Kinases Modulated by Suramin in CHO Cells. To characterize further the kinase activity stimulated by suramin in CHO cells, in vitro solid-phase phosphorylation of a set of recognition sequence peptides for 15 well characterized protein kinases was performed (Fig. 10). The extent of phosphorylation of various synthetic substrates by extracts from CHO cells before and after treatment with suramin was compared (indicated as "Ratio" in the figure). It was found that the phosphorylation of two synthetic peptides for ERK1/2 (Fig. 10, no.10 and 12) was stimulated approximately 2–3-fold. It should be noted that the phosphorylation of a peptide substrate for cGMP-dependent protein kinase (Fig. 10, no. 2) was also significantly stimulated.

View larger version (30K): [in this window] [in a new window]   Fig. 10. Specificity of protein kinases responsive to suramin. Quiescent CHO cells were stimulated with 10 µM suramin for 5 min. PhosphoSpots test strip (Jerini Bio Tools) containing covalently bound substrate peptides for indicated kinases was phosphorylated by incubating [-32P]ATP with cell extract from basal (control) or suramin-stimulated (suramin) cells as described under Materials and Methods. The extent of phosphorylation was determined by a bio-image analyzer. The ratio of phosphorylation (suramin-treated/control) was shown below each peptide spot. The protein kinases and the corresponding substrate peptides are, cAMP-dependent protein kinase (1), cGMP-dependent protein kinase (2), protein kinase C (3 and 4), Ca2+/calmodulin-dependent protein kinase II (5 and 6), casein kinase II (7), cdc2-kinase (8), p34cdc2-kinase (9), ERK1/2 (10–12), casein kinase I (13), S6 kinase (14), myosin light chain kinase (15), insulin receptor tyrosine kinase (16), csk tyrosine kinase (17), Raf-1 kinase (18), abl tyrosine kinase (19), and p60 c-src tyrosine kinase (20).

Suramin Stimulates DNA Synthesis. To determine the mitogenic effect of suramin-induced DNA synthesis, the ability of suramin to stimulate [³H]thymidine uptake was tested in CHO cells. As shown in Fig. 11, suramin significantly stimulated [³H]thymidine incorporation in a dose-dependent manner. The suramin-stimulated DNA synthesis was inhibited in the presence of 10 µM PD98059 (Fig. 11, a closed circle) or 100 nM wortmannin (Fig. 11, a triangle), indicating that the suramin-stimulated DNA synthesis is MEK- and PI3K-dependent. The involvement of PKC in the DNA synthesis is unlikely because 1 µM GF109203X, a PKC inhibitor, showed no significant effect on the suramin-stimulated DNA synthesis (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 11. Stimulation of DNA synthesis by suramin. DNA synthesis was measured by [3H]thymidine incorporation into CHO cells cultured in the presence of indicated concentrations of suramin as described under Materials and Methods. Data are mean of four wells from three separate experiments. **, p < 0.01 versus unstimulated. A closed circle and a triangle were [3H]thymidine incorporation in the presence of 10 µM PD98059 and 100 nM wortmannin, respectively. **, p < 0.01 versus incorporation with 10 µM suramin.

Effect of Suramin on ATP-Stimulated ERK1/2 Activity. ERK1/2 activity of CHO cells is activated by the addition of ATP or UTP via P2 purinergic receptors (Dickenson et al., 1998). Because suramin is also known as a P2 purinergic receptor antagonist, ERK1/2 activation by ATP (or UTP) in CHO cells was examined in the presence of suramin. As shown in Fig. 12, suramin showed almost additive activation of ERK1/2 in the presence of ATP or UTP and was unable to modulate ATP- or UTP-stimulated ERK1/2 activity. Other P2 receptor antagonists, XAMR0721 and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, also failed to attenuate ATP-stimulated ERK1/2 activity. These results suggest that the suramin-, XAMR0721- or pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid-sensitive P2 receptors are not involved in ATP (or UTP)-induced ERK1/2 activation under the present experimental conditions.

View larger version (34K): [in this window] [in a new window]   Fig. 12. Effect of suramin on ATP- or UTP-induced activation of ERK1/2. Quiescent semi-confluent CHO cells were stimulated with 10 µM ATP or UTP for 5 min in the presence or absence of suramin (10 µM) or other P2 purinergic receptor antagonists (100 µM). ERK1/2 activity of treated cells was determined as described under Materials and Methods. PPADS, pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid.

ERK Activation by Suramin in Various Cell Lines. To examine whether suramin-induced ERK1/2 activation is restricted to CHO cells, various cultured cell lines were tested for their sensitivity to suramin. As shown in Fig. 13, ERK1/2 in four cell lines derived from Chinese hamster tissues, including okadaic acid-resistant CHO cell lines OAR2-3 and OAR6-6 (Tohda et al., 1997), was significantly activated by suramin. Human cell lines OVK18 (human ovarian carcinoma) and transformed human endothelium cells ECV304 (Takahashi et al., 1990), both exert high endogenous ERK1/2 activity, showed no significant activation of ERK1/2 by suramin. RBL2H3 (rat mast cell line) also showed no response to suramin. Some activation of ERK1/2 by suramin was observed in other cell lines originating from smooth muscle cells (DDT₁MF-2) and chromaffin cells (PC12). Human prostate carcinoma cells (PC-3) showed a significant increase in ERK1/2 activity by suramin under experimental conditions.

View larger version (28K): [in this window] [in a new window]   Fig. 13. Comparison of suramin-induced ERK1/2 activity among various cell lines. Quiescent semiconfluent cultured cells were activated with 10 µM suramin for 5 to 10 min. ERK1/2 activity of cell lysates prepared from each cell before and after suramin activation was determined as described under Materials and Methods. ERK1/2 activity was expressed as the percentage of total ERK activity for CHO cells (the total ERK activity = basal ERK activity + suramin-activated ERK activity, ERK activity was normalized with the protein amount). Specific activities of ERK1/2 of basal and suramin-activated CHO cells were estimated to be 80 and 900 pmol/min/mg protein, respectively.CHO/OAR2-3 and CHO/OAR6-6, okadaic acid-resistant CHO cells; CHL, Chinese hamster lung; OVK18, human ovarian carcinoma cells; RBL2H3, rat mast cell line established from peripheral blood with basophilic leukemia; ECV304, human transformed endothelium cells from umbilical cord; DDT1MF-2, smooth muscle cell line from Syrian hamster vas deferens; PC12, rat pheochromocytoma; PC-3, human prostatic carcinoma cells.

	Discussion

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Suramin has attracted attention as an anticancer drug able to disrupt growth factor stimulatory pathways, i.e., antiproliferative activity against cancer cells. However, several studies have demonstrated that suramin can induce the proliferation of some epithelial cancer cell lines (Cardinali et al., 1992; Foekens et al., 1992; Shin et al., 1997). Moreover, it is reported that suramin does not decrease but increases the tyrosine phosphorylation of a series of proteins in several cell lines (Sartor et al., 1992). A modest stimulation in MAP kinase pathways by suramin in glioma cell lines or PC12 cells was reported (Kuratsu et al., 1995; Gill et al., 1996). However, the suramin-dependent cellular signaling mechanism inducing such cellular proliferation or MAP kinase activation was not well documented in those studies.

Our study was initiated from the observation that suramin did not inhibit but enhanced the proliferative activity of ATP, a P2 purinergic receptor agonist, in CHO cells, with a concomitant increase in MAP kinase activity (Nakata, 1998). This was against expectations because suramin is a well known P2 receptor antagonist. Surprisingly, very few systematic studies on the suramin-induced activation of cellular MAP kinases have been conducted, although suramin has been studied extensively over the past 10 years as an anticancer agent. It is therefore important to elucidate the molecular mechanism of suramin underlying such cellular responses.

In this study, the ability of suramin to stimulate ERK cascades was examined in cultured cells to determine whether this activity could be correlated with a proliferative outcome such as DNA synthesis. The results show that suramin enhances ERK activity rapidly even at nontoxic concentrations in CHO cells, and although the activation quickly declined, it was sustained for several hours with continued exposure of CHO cells to suramin. Suramin was also found to stimulate the phosphorylation of Akt and CREB in addition to MEK. The signaling mechanism inducing ERK activation by suramin was studied using key inhibitors for key signaling proteins.

PD98059, an MEK inhibitor, was able to block suramin-dependent ERK1/2 activation in CHO cells in addition to CREB phosphorylation, suggesting the pathway of suramin MEK ERK in CHO cells. Wortmannin or LY294002, PI3K inhibitors, also inhibited suramin-induced MEK and ERK activation in addition to Akt phosphorylation. Thus, PI3K was likely to be present upstream of the MEK/ERK signaling mechanism. Evidence for the involvement of the PI3K Akt pathway in suramin-induced ERK activation was not obtained, because the phosphorylation of Akt was not parallel with the phosphorylation of ERK in CHO cells (Fig. 5D).

CREB is a stimulus-induced transcription factor able to promote the expression of target genes in response to different stimuli such as peptide hormones, synaptic activation, and growth factors. All these stimuli activate kinase cascades, culminating in the phosphorylation of CREB at Ser133, which is required for the onset of CREB-mediated responses (Shaywitz and Greenberg, 1999). In our study, suramin was effective in promoting CREB phosphorylation at Ser 133. Because this rapid suramin-induced phospho-CREB formation was inhibited by the MEK inhibitor PD98059, suramin may functionally control gene expression via the MEK/ERK signaling pathway. In fact, the DNA synthesis determined by the incorporation of [³H]thymidine into cellular DNA was significantly increased by suramin in a PD98059-sensitive manner; thereby the MEK ERK CREB cascade is likely to be involved in mitogenic signaling by suramin in CHO cells.

Ras participation upstream of the suramin-induced ERK pathway was not confirmed in this study because FTI-277, a Ras inhibitor, showed no apparent inhibitory effect on suramin-induced ERK1/2 activation, although insulin-induced ERK1/2 activation was significantly inhibited by FTI-277 in a parallel experiment. Insulin-induced ERK activation was reportedly mediated through the Ras/Raf/MEK pathway (White and Kahn, 1994). More detailed studies are necessary to confirm the participation of the Ras/Raf pathway in the suramin-ERK pathway.

It is shown that PKC is not involved in suramin-induced ERK1/2 activation (Fig. 8). Because ERK was activated by PMA within 10 min in this experiment, and because PMA has been demonstrated to rapidly activate PKC (Newton, 1995), suramin may mediate the phosphorylation of ERK1/2 via PKC activation in this system. However, chronic 16-h treatment by PMA depletes the conventional and novel isozymes of PKC (Newton, 1995), and this treatment had no significant effect on suramin-induced ERK activation in this study. In addition, an inhibitor of conventional PKCs GF109203X showed no significant blocking of the suramin activation of ERK. Although PMA depletes the conventional and novel isozymes of PKC, it does not deplete the atypical isozymes PKC- and PKC- (Newton, 1995). In fact, it has been reported that suramin preferentially activated PKC- among PKC isozymes (Gschwendt et al., 1998). Therefore, we cannot rule out these PMA-insensitive, atypical PKC isozymes as mediators of suramin-induced ERK1/2 activation.

To determine protein kinases modulated by suramin in CHO cells, the phosphorylation of various protein kinase peptide substrates was performed in vitro using peptide libraries on cellulose paper (Tegge and Frank, 1998). As shown in the autoradiogram (Fig. 10), a significant increase in the phosphorylation of peptide substrates was observed only in the peptides for ERK and cGMP-dependent protein kinase. The physiological relevance of the increase in cGMP-dependent protein kinase phosphorylation remains to be elucidated.

ATP or UTP activates ERK via P2 purinergic receptors (Dickenson et al., 1998; Tai et al., 2001). In the latter literature, the treatment of granulosa-luteal cells either with suramin, staurosporin (a PKC inhibitor), or PD98059 significantly attenuated the ATP-induced activation of ERK. In contrast, the ATP- or UTP-mediated activation of ERK was not inhibited by suramin or suramin derivatives in the present study. Although the precise reason why suramin does not attenuate ATP/UTP-mediated ERK activation in CHO cells is unknown, it is apparent that suramin, a P2 receptor antagonist, does not influence ERK activity in CHO cells via the interaction with P2 receptor systems.

It is important to examine whether the suramin-induced activation of ERK1/2 is restricted to CHO cells. A variety of cell lines were tested, including those originating from Chinese hamster tissues or from ovary cells (Fig. 13). Human prostate carcinoma cells were also examined because suramin has been assessed as a therapeutic agent for prostate cancer. When the suramin-induced ERK activation was compared, significant activation was seen in every Chinese hamster cell line, and some activation was also observed in PC12 and PC-3 cells. It is apparent that CHO cells showed the greatest sensitivity to suramin among the cultured cell lines. It is also suggested that cells exerting low specific ERK activity in basal conditions respond well to suramin. In contrast, ERK of OVK18, ECV304, and RBL2H3 cells that showed high activity in basal conditions was not significantly stimulated by suramin. These results suggest that Chinese hamster cells express specific suramin-response factors that can transfer the suramin signal to the ERK cascade.

As it is generally assumed that suramin, a large molecule with a strong anionic nature, is not capable of readily penetrating cell membranes over a short period, suramin may interact with cell surface proteins such as receptors or G proteins where signals can readily be transmitted inside the cells. In fact, immobilized suramin was shown to exert a growth stimulatory effect in several cell lines (Lokshin et al., 1999). However, the possibility that suramin exerts its effect after entering the cells cannot be completely ruled out because it was incorporated into Chinese hamster fibrosarcoma cells after exposure for 24 h (Bojanowski et al., 1992).

The major finding of this study was that suramin-mediated DNA synthesis involves marked and transient ERK1/2 activation. Furthermore, these data describe a mechanism for ERK1/2 activation resulting from PI3K. Similar ERK activation in a PI3K-dependent manner was reported in response to integrin or insulin-like growth factor (King et al., 1997; Moelling et al., 2002). In addition, it is of interest to note that PMA, a PKC activator, induces cell growth arrest in myeloid cell lines (Das et al., 2000) but stimulates fibroblast proliferation (Hussaini et al., 2000). As observed with suramin, PMA properties are linked to ERK activation. Although further studies are necessary to describe more precisely the mechanisms linking suramin to PI3K and MEK, a model of suramin-mediated signaling consistent with the data presented in this report is shown (Fig. 14). It is interesting to hypothesize a putative suramin response factor leading to the activation of PI3K. The pathway bifurcates with the phosphorylation of two PI3K substrates, Akt and MEK, directly or indirectly. This study also strongly indicates that the effects of suramin on various cell lines are more complex than the simple inhibition of heparin- and nonheparin-binding growth factors suggested previously (Cardinali et al., 1992). Future studies will be directed toward the identification of the signaling molecules responsible for suramin on the cell surface.

View larger version (18K): [in this window] [in a new window]   Fig. 14. Possible signaling pathways for suramin signal transduction. Suramin binds to a putative cell surface protein, resulting in the activation of PI3K. PI3K activation followed by the activation of MEK/ERK cascade activates DNA synthesis in the nucleus.

	Footnotes

 
This work was supported in part by grants for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. It was also supported by the CREST program of the Japan Science and Technology Agency.

DOI: 10.1124/jpet.103.058230.

ABBREVIATIONS: MAP kinase, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated protein kinase 1 and 2; JNK, c-Jun NH₂-terminal kinase; MEK, mitogen-activated protein kinase kinase; CHO, Chinese hamster ovary; Akt, protein kinase B; CREB, cAMP response element-binding protein; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PI3K, phosphatidylinositol 3-kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PD98059, 2'-amino-3'-methoxyflavone; GF109203X, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide; FTI-277, farnesyl transferase inhibitor 277; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene; H89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide; XAMR0721, 8-(3,5-dinitrophenylene carbonylimino)-1,3,5-naphthalenetrisulfonic acid.

Address correspondence to: Dr. Hiroyasu Nakata, Department of Molecular Cell Signaling, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. E-mail: nakata{at}tmin.ac.jp

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