The Angiogenesis Inhibitor SU5416 Has Long-lasting Effects on Vascular Endothelial Growth Factor Receptor Phosphorylation and Function

SU5416 Chemical Synthesis.

SU5416 (3-[2,4-dimethylpyrrol-5-yl methylidenyl]-2-indolinone) was synthesized at the facilities of SUGEN, Inc., as described previously (35) .

Cell Culture.

Unless otherwise indicated, cell culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). A431 human epidermoid carcinoma cells and NCI-HT-29 human colon carcinoma cells were obtained from American Type Culture Collection (ATCC; Rockville, MD) and were propagated using standard tissue culture procedures in the medium suggested by ATCC. HUVECs were obtained from Clonetics (San Diego, CA) and were maintained in EGM (Clonetics) containing 2% FBS. NIH 3T3 cells, engineered to overexpress the murine VEGF receptor Flk-1 (3T3/Flk-1 cells), were obtained from Dr. Axel Ullrich (Max-Planck Institut fur Biochemie, Martinsried, Germany) and were maintained in DMEM supplemented with 10% FBS and 2 mm glutamine. Cells were propagated at 37°C in a humidified atmosphere of 5% CO₂ using standard cell culture techniques.

s.c. Xenograft Model in Athymic Mice.

Female nu/nu mice (8–12 weeks old; 20 g) were obtained from Charles River (Wilmington, MA). Animals were maintained under clean-room conditions in sterile filter-top cages with Aspen Chip bedding housed on HEPA-filtered ventilated racks. Animals received sterile rodent chow and water ad libitum. Animal experiments were conducted as described previously (28) , in accordance to Institutional Animal Care and Use Committee guidelines in the SUGEN Animal Facility, which has been accredited by Association for Assessment and Accreditation of Laboratory Animal Care, International.

Tumor cells to be implanted into athymic mice were harvested at or near confluence by incubation with 0.05% trypsin-EDTA. Cells were pelleted by centrifugation at 450 × g for 5–10 min, and cell pellets were resuspended in sterile PBS to a concentration of 3–5 × 10⁷ cells/ml. Cells (3–5 × 10⁶/animal) were implanted s.c. into the hind flank region of mice on day 0 as described previously (20) . i.p. bolus injections (50 μl) of SU5416 in DMSO or vehicle control (DMSO alone) were started one day after the implantation of cells and continued with the regimens described until termination of the experiment, when tumors in the vehicle-treated control animals grew to an average size of approximately 1000 mm3 . Tumor growth was measured twice weekly using Vernier calipers for the duration of the treatment. Tumor volumes were calculated as the product of length × width × height. Ps were calculated using the two-tailed Student’s t test based on measurements taken on the last day of each experiment.

Endothelial Cell Proliferation Assays.

Endothelial cell proliferation assays were similar to the endothelial cell mitogenesis assays described previously (28) . Briefly, HUVECs were plated in 96-well flat-bottomed tissue culture plates (1.5 × 104 cells/100 μl/well) in starvation medium composed of EBM (Clonetics) containing 0.5% FBS. After an overnight incubation to quiesce the cells, the medium was replaced with starvation medium containing 3-fold serial dilutions of SU5416 (16.7–0.02 μm). Two or 3 h later, the medium containing SU5416 was removed and replaced with starvation medium containing VEGF (20 ng/ml; R&D Systems, Minneapolis, MN), aFGF (1 ng/ml; Boehringer Mannheim, Indianapolis, IN), or no ligand (media control) as indicated. At the indicated times after removal of SU5416-containing medium, the relative number of cells in triplicate wells was determined using the SRB (Sigma Chemical Co., St. Louis, MO) assay (36) .

Surface Expression of KDR.

Surface expression of KDR on HUVECs was assessed using standard flow cytometry procedures and commercially available antibodies. Briefly, serum-starved monolayers of HUVECs were incubated in the presence or absence of SU5416 at the indicated concentration (1 or 5μ m) and for the indicated time (3 or 24 h). After treatment, HUVECs were released from the tissue culture plates by incubation with 5 mm EGTA in Ca²⁺/Mg²⁺-free PBS. Released cells were harvested by centrifugation, washed in PBS containing 1% FBS, and then incubated for 1 h with a 1:100 dilution of a monoclonal antibody directed against an epitope on the extracellular domain of KDR (Sigma Chemical Co.). The cells were then washed with PBS containing 1% FBS to remove free primary antibody before being incubated for 45 min with a 1:100 dilution of PE-conjugated F(ab′)₂ fragment of goat-antimouse IgG as secondary (2°) antibody (Caltag, Burlingame, CA). After a final wash to remove free secondary antibody, cells were analyzed for surface expression of KDR using a Becton Dickinson FACScan.

¹²⁵I-VEGF Binding Assays.

VEGF binding to KDR and Flt-1 on HUVECs was assessed using ¹²⁵I-VEGF. Briefly, confluent monolayers of HUVECs in 12-well tissue culture plates were serum starved overnight in starvation medium (EBM containing 0.5% FBS). The following day, the cells were incubated in the presence or absence of SU5416 at the indicated concentration (1 or 5 μm) and for the indicated time to determine the effect of a short (3 h) or long (24 h) exposure to compound. At the end of the treatment period, the cell monolayers were washed three times at room temperature with binding medium [DMEM containing 25 mm HEPES (pH 7.4) and 0.15% gelatin]. After the third wash, 0.5 ml of binding medium containing 3-fold serial dilutions of ¹²⁵I-VEGF (100 μCi/μg; New England Nuclear, Boston, MA) ranging in concentration from 0.23 to 500 pm was added to duplicate wells to determine total ¹²⁵I-VEGF binding. Separate 0.5-ml aliquots of the same dilutions of ¹²⁵I-VEGF in binding medium containing 100 nm nonradioactive VEGF (R&D Systems) were added to another set of duplicate wells containing HUVECs to determine nonspecific ¹²⁵I-VEGF binding. After a 2-h incubation at room temperature, the binding medium was removed, the cells were washed three times with ice-cold PBS containing 0.1% BSA (Sigma Chemical Co.), and the cells were lysed with the addition of 0.5 ml of 0.1 n NaOH. The amount of radioactivity in the binding buffer that was removed from the cells and that was in the cell lysate was counted in a Packard gamma counter to determine free and bound radioactivity, respectively. Specifically bound counts were determined by subtracting nonspecific from total counts at each free ¹²⁵I-VEGF concentration. Scatchard analyses (37) of the data were used to determine dissociation constants (K_D) for the binding of ¹²⁵I-VEGF to Flt-1 and KDR.

Cellular Flk-1 and ERK 1/2 Phosphorylation Assays.

The effect of SU5416 on VEGF-dependent tyrosine phosphorylation of Flk-1 was determined as described previously (28) . Briefly, twenty confluent monolayers of 3T3/Flk-1 cells or HUVECs in 6-well or 10-cm tissue culture plates, respectively, were quiesced by serum starvation overnight in starvation medium (EBM containing 0.5% FBS). SU5416 was then added to 10 wells to a final concentration of 5μ m; the remaining 10 wells did not receive SU5416. After a short (2 or 3 h as indicated) exposure to SU5416, the medium was removed from five of the SU5416-treated wells, cell monolayer was washed, and fresh starvation medium was added; these samples were referred to as the “wash” SU5416 samples. The other five SU5416-treated samples did not have the compound washed out but, rather, were constantly exposed to SU5416 for the duration of the experiment. Immediately (0 h) or 6, 24, 32, or 48 h after the washout procedure, VEGF was added to a final concentration of 100 ng/ml to one well each of the untreated, the SU5416 wash, and the constant SU5416 wells; no VEGF was added to another untreated well that was subsequently referred to as the unstimulated sample. After a 10-min VEGF stimulation at 37°C, the medium was removed, the cell monolayer washed with ice cold PBS, and the cells scraped into HNTG buffer [20 mm HEPES (pH 7.5), 150 mm NaCl, 0.2% Triton X-100, and 10% glycerol) containing 1 mm NaVO₄, 50 mm NaF, and protease inhibitors. Preparation of cell lysates, separation of cellular proteins (30 μg), and immunoblotting with antiphosphotyrosine antibody (SUGEN reagent) were performed as described previously (28) . Immunoreactive proteins were detected using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were then stripped with elution buffer (Pierce, Rockford, IL), and reprobed with an antibody directed against Flk-1/KDR (Santa Cruz Biotechnology, Santa Cruz, CA) to determine the amount of Flk-1/KDR in each lane.

A similar approach using antiphospho ERK 1/2 and anti-ERK 1/2 antibodies (New England Biolabs, Beverly, MA) was used to determine the effect of SU5416 on VEGF-dependent phosphorylation of ERK 1/2.

Flk-1 Receptor Kinase Assay.

The biochemical Flk-1 receptor tyrosine kinase assay was performed as described previously (28) . Briefly, detergent-solubilized proteins (50 μg) from 3T3/Flk-1 cells were added to each well of a polystyrene ELISA plate that had been precoated with a monoclonal antibody (SUGEN reagent) that recognizes Flk-1. After an overnight incubation at 4°C to allow the Flk-1 to be captured by the adsorbed antibody, the wells were washed three times with deionized water and once with TBST [50 mm Tris (pH 7.0), 150 mm NaCl, and 0.1% Triton X-100] to remove nonabsorbed proteins. Kinase reaction buffer [25 mm Tris, (pH 7.0), 100 mm NaCl, 10 mm MnCl₂, 2% glycerol, 0.5 mm DTT, and 0.1% Triton X-100] was then added to each well. The kinase reaction resulting in the autophosphorylation of Flk-1 on tyrosine residues was initiated with the addition of ATP (10–30μ m final concentration) and was allowed to proceed at room temperature for 60 min. The reaction was then stopped with the addition of EDTA to a final concentration of 20 mm. The wells were washed as above and then probed for 45 min with a biotinylated monoclonal antibody directed against phosphotyrosine (UBI, Lake Placid, NY). The wells were then washed to remove unbound antiphosphotyrosine antibody prior to the addition of avidin-conjugated horseradish peroxidase H (Vector Laboratories, Burlingame, CA) for 30 min. After washing, substrate[ 3,3′,5,5′-tetramethyl benzidine dihydrochloride and H₂O₂ (Turbo-TMB; Pierce)] was added to the wells. After 15 min, reactions were stopped with the addition of H₂SO₄, and the relative amount of colored product in each well was determined based on A_{450 nm} values measured using a 96-well microtiter plate reader. In experiments to determine the inhibitory effect of SU5416, 3-fold serial dilutions of SU5416 were added to the captured Flk-1 enzyme just prior to the addition of ATP to start the autophosphorylation reaction.

[¹⁴C]SU5416 Uptake and Washout Assays.

Confluent monolayers of HUVECs in 24-well tissue culture plates were serum starved overnight in starvation medium (EBM containing 0.5% FBS). To determine the rate of uptake of[ ¹⁴C]SU5416 by the HUVECs, on the following day, the medium was replaced with 1 ml of starvation medium containing 4 μm [¹⁴C]SU5416 (13.56 mCi/mmol; SynPep, Dublin, CA). At the times indicated in Fig. 6⇓ , the medium was harvested from duplicate wells, the cell monolayers were washed twice with PBS, and the cells were lysed with 0.4 ml/well of 0.1 n NaOH. The radioactivity in the starvation medium and cell lysates was determined by scintillation counting (Beckman counter) after the addition of 5 ml of Ready Safe Liquid Scintillation Cocktail (Beckman, Palo Alto, CA). To determine the time course of the washout of [¹⁴C]SU5416 from HUVECs, serum-starved cells in 24-well plates were initially incubated with starvation medium containing 4 μm [¹⁴C]SU5416 for 3 h. Cell monolayers were then washed twice with PBS, and the medium was replaced with fresh starvation medium without SU5416. At the indicated times after washout, medium was removed from duplicate wells, and the amount of cell-associated [¹⁴C]SU5416 was determined as described above. The concentration of cell-associated[ ¹⁴C]SU5416 was calculated using 8μ l/10⁶ HUVECs as the cell volume, which was determined using the equation V= 4 /₃Π r3 for a sphere (where V = volume and r = radius of cell) and an average cell radius of 12.5 μm for HUVECs released from tissue culture plates by trypsinization based on visual inspection of cells in a hemacytometer.

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Fig. 6.

Uptake and washout of [¹⁴C]SU5416 from HUVECs. Serum-starved HUVECs were incubated in starvation medium containing 4 μm [¹⁴C]SU5416. In A, for the uptake experiment, the amount of cell-associated radioactivity was determined at the indicated times after the addition of starvation medium containing 4 μm[ ¹⁴C]SU5416. In B, for the washout experiment, cells were initially incubated with starvation medium containing 4 μm [¹⁴C]SU5416 for 3 h. The cells were then washed, and the medium replaced with fresh starvation medium that did not contain SU5416. The amount of cell-associated radioactivity was then determined in aliquots of cells harvested at the indicated time after removal of the medium containing[ ¹⁴C]SU5416. Prior to the washout, the amount of cell-associated radioactivity was ∼3000 cpm/sample. Background counts in a similar experiment using a structurally related compound that is not sequestered in cells were less than 50 cpm.

Long-lasting Effect of SU5416 on A431 Tumor Growth.

The majority of the animal experiments carried out with SU5416 have used daily administration of the compound because of the rapid clearance of the compound from plasma (28, 29, 30, 31) . However, twice-weekly administration of SU5416 at 50 mg/kg was as efficacious as daily administration of 25 mg/kg SU5416 in inhibiting the growth of A375 human melanoma xenograft tumors (29) . Because A375 xenografts are very sensitive to the antitumor activity of SU5416 (28) , the possibility existed that SU5416 was able to exert antitumor activity with infrequent administration because the A375 xenograft model was particularly sensitive to SU5416. To address this question, subsequent experiments were conducted using A431 human epidermoid carcinoma and NCI-HT-29 human colon carcinoma tumors, which are less sensitive than A375 tumors to the antitumor activity of SU5416 in athymic mice (29) .

As shown in Table 1⇓ , SU5416, administered i.p. daily at 25 mg/kg/day, inhibited A431 tumor growth by 51% (P = 0.0063), which is comparable with previous results with this tumor model (28) . Administration of SU5416 at 25 mg/kg twice weekly did not cause significant inhibition of tumor growth. In contrast, i.p. administration of SU5416 at 50 mg/kg once, two or three times weekly resulted in the statistically significant inhibition of A431 tumor growth comparable with that seen with daily administration of SU5416 at 25 mg/kg/day (Table 1)⇓ . A similar result was obtained with NCI-HT-29 tumors, in that once weekly administration of SU5416 at 50 mg/kg was as efficacious as daily administration of the compound at 25 mg/kg/day (Table 1)⇓ . Thus, the ability of SU5416 to inhibit the growth of tumors with infrequent dosing is evident in all of the three (A375, A431, and NCI-HT-29) tumor models tested, which suggests that the long-lived inhibitory activity of SU5416 would be evident with other tumor models susceptible to inhibition by daily administration of SU5416.

Long-lasting Effect of SU5416 on HUVEC Proliferation in Culture.

The durability of the inhibitory activity of SU5416 in an in vitro assay was directly determined using a HUVEC proliferation assay. In this assay, the ability of a 3-h exposure to SU5416 to provide prolonged inhibition of VEGF-induced proliferation of HUVECs was determined. The results of a representative assay are shown in Fig. 1⇓ .

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Fig. 1.

Duration of SU5416 inhibition of VEGF-dependent survival of HUVECs. Quiescent HUVECs were exposed to serial dilutions of SU5416 in starvation medium. After 3 h, the medium was replaced with fresh starvation medium containing 20 ng/ml VEGF (▪), 1 ng/ml aFGF (•), or no ligand (▴, media control). The relative number of cells was determined based on readout from a SRB assay immediately (0 h; A) and 48 h (B) after removal of the SU5416-containing medium. The relative number of cells was also determined in wells in which the cells had been restimulated with the indicated ligand for 21 h beginning 51 h after removal of the SU5416-containing medium (72 h; C). Data, mean values from triplicate wells in a representative experiment.

At 0 h (immediately after the three-hour exposure to SU5416 and prior to addition of ligand), the SRB signal (indicative of relative cell number) is similar in all of the test wells regardless of SU5416 concentration and in all of the three treatment groups (Fig. 1⇓ A). With increasing time (48-h data are shown in Fig. 1⇓ B), fewer cells were detected in the media control samples, consistent with the inability of HUVECs to propagate or survive under serum-starvation conditions. The decrease in the number of cells with an increasing time of serum starvation was confirmed by a visual inspection of the cultures and did not vary with SU5416 concentration. In the aFGF-stimulated samples, there was an increase in the number of cells at the 48-h time point regardless of SU5416 treatment, which indicated that aFGF is still able to induce HUVEC proliferation in the presence of SU5416. This result is consistent with the demonstration that SU5416 does not block the mitogenic/proliferative response of HUVECs stimulated with aFGF (28) . In contrast, a dose-dependent effect of SU5416 was observed in samples stimulated with VEGF (Fig. 1⇓ C). In these samples, there was no decrease in the number of cells in wells initially exposed to low concentrations (<0.6μ m) of SU5416, which indicated that, in these wells, VEGF induced proliferation of the HUVECs despite the prolonged serum starvation. However, there was a marked decrease in the number of cells in wells exposed to the higher concentrations of SU5416, such that the number of cells in the VEGF-stimulated wells exposed to the two highest concentrations of SU5416 were the same as those in the media control wells. These data indicate that the two highest concentrations of SU5416 completely blocked the proliferative effect of the VEGF, and that the IC₅₀ value for SU5416 in this assay is 1.5–2 μm. This IC₅₀ value is higher than the 40-nm value reported for HUVECs exposed to SU5416 for the duration of the mitogenesis assay (48 h; Ref. 28 ) but is comparable with the IC₅₀ value of ∼1μ m obtained in the mitogenesis assay when HUVECs are exposed to SU5416 during only the first 0.5–5 h of the assay.3

To confirm that an initial 3-h exposure to SU5416 was still having an inhibitory effect more than 48 h after removal of the compound, the cells were given a second stimulation with VEGF or aFGF 51 h after the washout, and the relative number of cells was determined based on the SRB signal 21 h later (72 h after the washout). As shown in Fig. 1⇓ C, at the 72 h time point there was a further decrease in the number of cells in unstimulated (media control) wells and in VEGF-stimulated wells that contained cells initially treated with the higher concentrations of SU5416. Again, SU5416 had no effect on the proliferative response of HUVECs stimulated with aFGF. Thus, even 72 h after a short exposure to SU5416, the effect of VEGF stimulation is specifically inhibited in HUVECs.

Effect of SU5416 on VEGF Binding via Flk-1/KDR.

To investigate the basis for the long-lived in vitro inhibitory activity of SU5416, the effect of SU5416 on the surface expression of KDR and its ability to bind VEGF were investigated. As shown in Fig. 2⇓ , similar levels of KDR are detected on the surface of untreated HUVECs or of HUVECs treated with 1 μm SU5416 for 3 h. Similar results were obtained with HUVECs incubated with a higher concentration of SU5416 (5 μm) and for a longer period of time (24 h; data not shown). Thus, SU5416 treatment of HUVECs does not decrease the surface expression of KDR.

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Fig. 2.

SU5416 does not decrease KDR expression on HUVECs. Serum-starved HUVECs were untreated (left panel) or treated with 1 μm SU5416 (right panel) for 3 h prior to labeling with a primary antibody directed against an extracellular epitope on Flk-1/KDR. Histograms based on FACS analysis indicate relative fluorescence of cells stained with secondary antibody (2° Ab) or both primary and secondary antibodies (KDR). Data are from a representative experiment.

Alternatively, SU5416 could inhibit VEGF-dependent HUVEC proliferation by blocking the binding of VEGF to its receptors through a secondary effect on receptor conformation. To address this question, the binding of ¹²⁵I-VEGF to SU5416-treated and untreated HUVECs was investigated. As previously reported (38) , Scatchard analysis of specific ¹²⁵I-VEGF binding data enabled the separate evaluation of the binding of VEGF to KDR and Flt-1 (a second VEGF receptor expressed on HUVECs). The dissociation constants (K_D) determined for VEGF binding to Flt-1 and KDR were 17 pm and 390 pm, respectively, which are comparable with reported values (38) . As shown in Table 2⇓ , SU5416 treatment did not effect either the affinity (K_D) or the number of functional KDR or Flt-1 binding sites on HUVECs.

Long-lasting Effect of SU5416 on Flk-1/KDR Phosphorylation and Subsequent Signaling.

Because the long-lasting inhibitory activity of SU5416 could not be attributed to alterations in VEGF binding to cellular receptors, the durability of the effect of SU5416 on VEGF-dependent Flk-1/KDR phosphorylation was investigated. As shown in Fig. 3⇓ , a 3-h pretreatment with SU5416, followed by a washout, was sufficient to inhibit VEGF-dependent phosphorylation of Flk-1 in 3T3/Flk-1 cells for 48 h, the longest time period tested. Importantly, the inhibition of Flk-1 phosphorylation observed in the washout sample at 48 h was comparable with that seen in the sample from cells exposed to SU5416 for the full 48 h (compare the third and fourth lanes of the 48-h sample).

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Fig. 3.

Durable inhibition of Flk-1 phosphorylation by SU5416 in 3T3/Flk-1 cells. Serum-starved 3T3/Flk-1 cells were either untreated or treated with 5 μm SU5416 for 3 h. For the wash sample, the SU5416-containing medium was replaced with fresh starvation medium; for the other treated sample, the cells were exposed to SU5416 for the entire experiment. At 0 or 42 h after the washout, cells were either untreated or stimulated with VEGF (100 ng/ml) for 10 min. After stimulation, cell lysates were prepared and subjected to Western blot analysis using antiphosphotyrosine, (α-pTyr) or anti-Flk-1 (α-Flk-1) antibodies. The position of Flk-1 is indicated (arrow). Data are from a representative experiment.

Having demonstrated that SU5416 produced a long-lasting inhibition of VEGF-dependent Flk-1 phosphorylation, we next investigated the ability of SU5416 to confer long-term inhibition of VEGF-dependent signaling in HUVECs. Phosphorylation of ERK 1/2 (p42/p44^{MAP kinase}) was selected as an indicator of VEGF-dependent Flk-1/KDR activation in these studies because it is known to be an early downstream target of Flk-1/KDR activation (39) , because a significant portion of this abundant protein is phosphorylated in response to VEGF stimulation of KDR in HUVECs, and because reliable reagents are available to detect phosphorylated ERK 1/2 separate from total ERK 1/2 in cell extracts. As shown in Fig. 4⇓ , even 32 h after the removal of SU5416 from the medium, VEGF-dependent phosphorylation of ERK 1/2 was inhibited to an extent similar to that observed in HUVECs exposed to SU5416 for the full 32-h period (compare the third and fourth lanes in the 32-h sample). Thus, the long-lived inhibition of the VEGF-dependent mitogenesis/proliferation of HUVECs by SU5416 in vitro is attributable to a long-lived inhibition of the VEGF-dependent signaling via Flk-1/KDR.

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Fig. 4.

Durability of SU5416 inhibition of VEGF-dependent phosphorylation of ERK 1/2 in HUVECs. Serum-starved HUVECs were either untreated or treated with 5 μm SU5416 for 2 h. For the wash sample, the SU5416-containing medium was replaced with fresh starvation medium; for the other treated sample, the cells were exposed to SU5416 for the entire experiment. At 0 or 32 h after the washout, cells were either untreated or stimulated with VEGF (100 ng/ml) for 10 min. After stimulation, cell lysates were subjected to Western blot analysis to detect phosphorylated ERK 1/2 (α-phospho ERK 1/2) or total ERK 1/2 (ERK 1/2). Data are from a representative experiment.

To determine whether the long-lived effect of SU5416 on ligand-dependent phosphorylation of ERK 1/2 was specific for VEGF stimulation, the ability of SU5416 to inhibit ERK 1/2 phosphorylation in response to aFGF was investigated. As shown in Fig. 5⇓ , SU5416 did not inhibit ERK 1/2 phosphorylation in aFGF-stimulated HUVECs (Fig. 5⇓ , right side of each panel) under conditions that VEGF-dependent ERK 1/2 phosphorylation was inhibited (Fig. 5⇓ , left side of each panel). This result, which is consistent with the inability of SU5416 to inhibit aFGF-dependent mitogenesis in HUVECs, demonstrates that the long-lasting inhibition by SU5416 retains its specificity for VEGF-dependent signals. It should be noted that time points beyond 48 h were not investigated because preliminary results indicated that the response of HUVECs to ligand stimulation in serum-starvation medium was more variable at later time points even in the absence of SU5416.

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Fig. 5.

Specificity of SU5416 inhibition of ERK 1/2 phosphorylation in HUVECs. Serum-starved HUVECs were either untreated or treated with 5 μm SU5416 for 2 h. For the wash sample, the SU5416-containing medium was replaced with fresh starvation medium; for the other treated sample the SU5416-containing medium was left on the cells for the entire experiment. At 0 or 32 h after the washout, cells were either untreated or stimulated with VEGF (100 ng/ml) or aFGF (1 ng/ml) for 10 min. After stimulation, cell lysates were subjected to Western blot analysis using antibodies that specifically recognize phosphorylated ERK 1/2 (phospho ERK 1/2) or total ERK 1/2 (ERK1/2). Data are from a representative experiment.

SU5416 Inhibition of Flk-1 Tyrosine Kinase Activity.

Previous biochemical studies have indicated that SU5416 is a potent, competitive (with respect to ATP) inhibitor of the tyrosine kinase activity of Flk-1 (K_i = 0.16μ m for SU5416, K_m = 0.53 μm for ATP; Refs. 28 , 29 ). One possible explanation for the long-lasting inhibitory activity of SU5416 would be that it acts as an irreversible inhibitor of the Flk-1/KDR tyrosine kinase activity. To directly address this possibility, the durability of the inhibitory activity of SU5416 was determined in a biochemical kinase assay. In this assay, SU5416 inhibited Flk-1 kinase activity (IC₅₀ = 0.75 μm when 30μ m ATP was used as substrate) only if SU5416 was present when the reaction was initiated with the addition of ATP. In contrast, little or no inhibitory activity was observed if SU5416 was removed by washing prior to the addition of ATP even after preincubations for as long as 30 min with high (20μ m) concentrations of compound. Thus, SU5416 is not an irreversible inhibitor of Flk-1 kinase activity.

Concentration of SU5416 in HUVECs.

The demonstration that SU5416 is not an irreversible inhibitor of Flk-1 tyrosine kinase activity but is able to inhibit VEGF-stimulated Flk-1 tyrosine phosphorylation for prolonged periods in cells suggested that SU5416 may be sequestered intracellularly. To directly address this possibility, the uptake and washout of SU5416 was determined in HUVECs incubated in starvation medium containing 4 μm[ ¹⁴C]SU5416.

As shown in Fig. 6⇓ A, SU5416 is rapidly taken up into HUVECs, reaching a plateau after 2 h. The cellular concentration of[ ¹⁴C]SU5416 after 3-h incubation in medium containing 4 μm[ ¹⁴C]SU5416 was determined to be approximately 450 μm, which indicated that the compound is concentrated in the cell. The majority of the cellular SU5416 present at the end of the uptake period also rapidly washes out of the cells. As shown in Fig. 6⇓ B, 30 min after replacing the SU5416-containing medium with compound-free medium, the cellular concentration of SU5416 dropped 10-fold compared with the amount present at the end of the 3-h uptake period. However, even after more than 90% of the compound washed out, the cellular SU5416 concentration was ∼25 μm. Importantly, there was no further decrease in the cellular concentration of SU5416, so that even 48 h after removal of compound, the cellular concentration of SU5416 was still ∼25 μm. This concentration exceeds the concentration of SU5416 required to inhibit Flk-1/KDR phosphorylation and VEGF-stimulated HUVEC proliferation. Also of note is the fact that the ¹⁴C recovered from cells at the end of the uptake and washout periods coeluted with SU5416 when subjected to high-performance liquid chromatography analysis,4 which indicated that the cell-associated radioactivity was SU5416. Thus, these data indicate that SU5416 is concentrated in cells, and that the cells maintain an inhibitory concentration of SU5416 for a prolonged period even when the compound is no longer present in the medium.

For comparison, similar studies were performed using[ ¹⁴C]SU6668, a compound structurally related to, but less hydrophobic than, SU5416 (40) . In these experiments, SU6668 was found: to be taken up at a much slower rate than SU5416; to achieve a lower final cellular concentration; and to rapidly and completely wash out of the cells when the compound was removed from the medium (data not shown). Consistent with the rapid and efficient washout of this compound, SU6668 did not have long-lasting activity in any of the functional assays described above.