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Experimental Therapeutics |
Departments of Internal Medicine [G. W. K.] and Microbiology/Immunology [G. W. K., S. H.], Virginia Commonwealth University, McGuire Veterans Affairs Medical Center, Richmond, Virginia 23249, and SUGEN, Incorporated, South San Francisco, California 94080 [D. K., C. L., S. V., L. S., G. M., K. E. L.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Kit is normally expressed in hemopoietic cells, melanocytes, other neural crest derivatives, and germ cells, as well as a variety of solid tumors (1 , 3) . Coexpression of both the Kit receptor and its ligand SCF has been reported in myeloid leukemia (4) , neuroblastoma (5) , breast tumors (6) , colon tumors (7) , gynecological tumors (8) , testicular germ cell tumors (9) , and at least 70% of SCLC cell lines and tumor specimens (10, 11, 12, 13) . These observations suggest the possibility of an autocrine growth loop involving the SCF/Kit system in these malignancies. In addition, mutations that cause constitutive activation of Kit have been reported in gastrointestinal stromal tumors, seminomas, and systemic mastocytosis associated with other hematological disorders such as acute myelogenous leukemia (14, 15, 16) . The fact that Kit activation is likely to contribute to the development of the aforementioned tumors has led to efforts to block Kit-mediated mitogenic signaling.
One possible approach to interfering with Kit-mediated signaling would be to block the binding of SCF to Kit. Cohen et al. (5) showed that the functional blockade of Kit with a MoAb induced a reduction in growth of neuroblastoma cell lines. Similar results were obtained using breast tumor cell lines coexpressing SCF and Kit (6) . Ricotti et al. (17) also reported that treatment of sarcoma cell lines with neutralizing Kit MoAb resulted in a significant increase in apoptosis and also demonstrated that treatment with c-kit antisense oligonucleotides resulted in growth inhibition. Introduction of mutations within the kinase domain that eliminate enzymatic activity, resulting in a dominant-negative receptor, is another way of abolishing signal transduction by the Kit receptor. Interference with a preexisting autocrine loop by transfection of the H209 SCLC cell line, which coexpresses high levels of Kit and SCF, with a kinase-defective c-kit gene construct resulted in a loss of growth factor independence (18) .
Another attractive approach is to develop small molecule inhibitors that block the tyrosine kinase activity of the receptor. Screening studies have identified several classes of compounds as tyrosine kinase inhibitors (19, 20, 21) . In addition to being potential antitumor agents, these compounds have proven to be useful research tools for understanding the physiological functions of specific tyrosine kinases. Certain tyrosine kinase inhibitors that block SCF-dependent proliferation have been reported. AG1295 and AG1296, quinoxalines that compete for ATP binding, have been shown to inhibit SCF-mediated Kit activation as well as SCF-mediated growth of SCLC cells (22) . STI571, a 2-phenylaminopryimidine derivative originally designed as an ATP competitive inhibitor of Bcr-Abl (23) , is also an effective Kit inhibitor and blocked SCF-mediated growth of SCLC (24) . Substituted indolinones have been identified as a versatile scaffold for the development of protein kinase inhibitors (25) . By altering the substituents on the pyrrole and oxindole rings, it is possible to tune the potency and selectivity of the compounds for inhibition of various kinases. These compounds have proven to be especially useful for inhibition of kinases of the PDGFr superfamily (25 , 26) that includes the VEGF receptors and Kit. Activity against both the wild type and mutationally activated Kit has been demonstrated for some members of this biochemical family (27) . In this manuscript, we have examined the ability of six related indolinone-based compounds to inhibit Kit kinase and to block Kit-mediated responses and growth of SCLC cells. We have also attempted to correlate activity in both biochemical and cellular assays with unique structural features of these related compounds.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and MTT Cellular Growth Assays.
The SCLC cell lines used that express c-kit were H526, H510, H432, H209, and WBA, whereas H146 cells do not express the receptor (12)
. SCLC cells were grown in RPMI 1640 (Bio-Whitaker, Walkersville, MD) medium that was supplemented with 10% (volume for volume) heat-inactivated fetal bovine serum (Life Technologies, Inc.), 2 mM L-glutamine, and 50 units/ml penicillin-50 units/ml streptomycin. Non-neoplastic MRC-5 pulmonary fibroblasts (ATCC CCL-171; Manassas, VA) were maintained in Eagle‘s minimum essential medium (Bio-Whitaker) supplemented with 10% heat-inactivated fetal bovine serum. CHO/GyrB-Kit cells (a stable clone of CHO-K1 cells expressing a GyrB-Kit chimeric protein) were grown in Ham‘s F12 medium containing 10% fetal bovine serum and 1 mg/ml G418 (Life Technologies, Inc.). All of the cells were maintained routinely in a humidified chamber at 37°C and 5% carbon dioxide in air. When grown in the absence of serum, 0.1% BSA (Sigma Chemical Co.) was added to the medium. Where indicated, serum-free medium was supplemented with saturating concentrations of recombinant SCF (100 ng/ml) or IGF-1 (20 ng/ml). Cells were treated with SCF and IGF-1 after preincubation in serum-free medium overnight. Cellular growth was assessed using the MTT (Sigma Chemical Co.) colorimetric dye reduction method. This assay is dependent on the ability of mitochondrial dehydrogenases within viable cells to reduce the MTT dye to a blue formazan product. The amount of dye reduced is directly correlated to the number of viable SCLC cells, as determined by other assays (28)
. Duplicate plates containing eight replicate wells/each assay condition were seeded at a density of 1 x 104 cells in 0.1 ml of medium, and data were expressed as the change in absorbance at 540 nM over 72 h, relative to initial values obtained 3 h after plating.
GyrB/Kit Kinase Assays.
Biochemical Kit autophosphorylation kinase assays were performed with immunoprecipitated GyrB/Kit. A confluent dish of CHO/GyrB-Kit cells were lysed in 2.5 ml of ice-cold radioimmunoprecipitation assay buffer containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1.4 µM E64, 10 µM bestatin, 1 µM leupeptin, 0.3 µM aprotinin, and 1 µM pepstatin A; Sigma Chemical Co.) and 20 mM EDTA and then diluted with an equal volume of 20 mM HEPES, 150 mM NaCl, 0.5% Triton X-100, and 10% glycerol. After centrifugation, the cleared lysate was incubated with protein A beads (175 µl; Pierce Chemical, Rockford, IL) and anti-Kit rabbit polyclonal antibodies (150 µl; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at 4°C. Washed beads were incubated with the indicated concentration of test compounds in kinase buffer (25 mM HEPES, 100 mM NaCl, 5 mM MgCl2, and 5 mM MnCl2) containing 5 µM ATP for 30 min at room temperature. The kinase reaction was stopped by the addition of 2x reducing SDS-PAGE sample buffer and boiling. After fractionation by SDS-PAGE and transfer to nitrocellulose, the amount of autophosphorylation was detected with antiphosphotyrosine antibodies (biotin-PY99; Santa Cruz Biotechnology) and enhanced chemiluminescence (Amersham, Arlington Heights, IL) reagents. The amount of Kit protein loaded in each lane was visualized in stripped blots with anti-Kit goat polyclonal antibodies (Santa Cruz Biotechnology).
Cellular Kit kinase assays were performed with CHO/GyrB-Kit cells in 6-well plates. Cells were serum-starved in DMEM containing 0.1% BSA overnight and then incubated with the test compound for 2 h. Coumermycin (1 µM; final concentration) was added to the cells to initiate kinase dimerization. After 30 min of incubation, the cells were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors, 20 mM EDTA, and 1 mM orthovanadate and then diluted in an equal volume of 20 mM HEPES, 150 mM NaCl, 0.5% Triton X-100, and 10% glycerol, containing protease inhibitors and orthovanadate. After centrifugation, Kit was immunoprecipitated from the cleared lysate, subjected to SDS-PAGE, transferred to nitrocellulose, and probed for phosphotyrosine and Kit protein, as described above.
Biochemical Kinase Assays.
Biochemical kinase assays to evaluate the potency of compounds for inhibition of PDGFr, EGFr, and FGFr were performed as described previously (29
, 30)
. In brief, all of the kinase assays were performed at twice the Km for ATP to make the IC50 values comparable. The cytoplasmic domains of EGFr, FGFr1, or IGF-1r or the kinase domain of Zap70 were expressed in SF9 cells as a glutathione S-transferase fusion protein and purified on glutathione agarose columns. The purified protein was added to microtiter plate wells coated with poly-GluTyr (4:1), together with the test compound and the appropriate kinase buffer. The amount of substrate phosphorylation was then measured with antibodies to phosphotyrosine. Lysates of cells overexpressing PDGFrß or GyrB/Kit were transferred to wells coated with antibodies to the COOH termini to immunopurify the kinase. The test compound and appropriate kinase buffer were added to the wells, and the amount or autophosphorylation was measured with antibodies to phosphotyrosine.
IP of Kit from SCLC.
H526 cells were grown until confluence and then serum-starved overnight in serum-free medium. The cells were both pretreated with DMSO vehicle or various concentrations of inhibitors for 30 min and then either left unstimulated or stimulated with 100 ng/ml SCF for 5 min. They were then washed twice with cold PBS and lysed in a Dounce homogenizer with a tight-fitting pestle, using ice-cold lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP40, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.2 mM Na3VO4, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 10 µg/ml leupeptin. Protein concentrations were determined by the BCA assay (Pierce). The lysate, containing 1.0–1.5 mg of protein, was transferred to a microfuge tube and centrifuged for 10 min at 10,000 x g. The supernatant was transferred to a clean microfuge tube, followed by IP with 10 µg of anti-Kit MoAb (K45; NeoMarkers, Fremont, CA) for 2 h at 4°C. For control experiments, 10 µg of nonimmune mouse IgG was used for IP. Immune complexes were harvested by the addition of protein A + G agarose beads for an additional 1-h incubation followed by centrifugation. Beads were then washed four times with lysis buffer and then once in cold PBS. The pellet was aspirated to dryness and boiled for 5 min in an equal volume of 2 x SDS sample loading buffer and centrifuged to obtain the supernatant, which was loaded onto a 10% SDS polyacrylamide gel. Western blotting was carried out using standard procedures. Staining was accomplished using an anti-phosphotyrosine MoAb (PY-20; Transduction Laboratories, Lexington, KY) and the anti-Kit 3D6 antibody (Boehringer-Mannheim, Indianapolis, IN). Visualization was accomplished using the enhanced chemiluminescence system.
Molecular Modeling.
A homology model for the catalytic domain of Kit was generated using the Modeler program (Molecular Simulations, Inc., San Diego, CA). The "open form" of FGFr1 cocrystallized with SU6668 was used as reference, and the sequence alignment was based on that of Hanks and Quinn (31)
with slight modifications. Because the sequence homology between FGFr1 and Kit is high (44%), the Kit model and FGFr1 crystal structures have very similar overall folding. The final Kit model was obtained after simple energy minimization. SU6597 was docked into the Kit model based on the crystal structure of SU6668 in FGFr1 (29)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The relative potencies of these compounds for inhibition of receptor tyrosine kinases in the PDGFr superfamily were compared in screening biochemical assays and are shown in Table 1
. Although they demonstrate varying potencies toward the different family members as well as unrelated kinases, they tend to be selective inhibitors of Kit kinase (Table 1)
. None of the compounds inhibited EGFr kinase, and most demonstrated poor potency for inhibition of IGF-1r and Zap70 kinases. Of these compounds, only SU5416 and SU6668 were tested for inhibition of Abl kinase. SU5416 did not inhibit Abl kinase up to a concentration of 20 µM (the highest concentration tested), whereas SU6668 demonstrated an IC50 value of 12.2 ± 1.3 µM. These observations confirm and extend previously reported activities for 3-substituted indolinones as kinase inhibitors (25
, 26)
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. As expected, novobiocin was unable to activate the Kit kinase fusion protein.
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illustrates a comparison of the biochemical and cellular potencies of these compounds for inhibition of Kit kinase autophosphorylation. Fig. 1C
demonstrates that comparable amounts of protein were present in each lane and provides an internal control. SU5416 demonstrated nM biochemical potency for inhibition of Kit kinase. It was also effective at inhibiting Kit kinase in cells, where its apparent IC50 value was less than 10-fold greater than that observed in the biochemical assay. The 5-Cl analogue of SU5416, SU5614, demonstrated slightly greater potency in both the biochemical and cellular assays than those observed for SU5416. SU6668 exhibited better potency in the biochemical Kit kinase assay than SU5416, with its 5-Cl analogue, SU6597, being even more potent. However, SU6668 and SU6597 displayed a significant discrepancy between their biochemical and cellular potencies, with their biochemical potencies more than 100-fold better than their cellular potencies. SU6663 demonstrated slightly better potency for inhibition of Kit kinase in the biochemical assay than was observed for SU5416, but it did not appear to be as potent as SU6668. Although its biochemical potency was slightly better than that for SU5416, its cellular potency was much weaker. There was also a large discrepancy between its biochemical and cellular potencies, as observed with SU6668 and SU6597. The discrepancy between biochemical and cellular potencies was even greater for SU6561 than for SU6663, because its IC50 value in the biochemical assay was lower. The relative activity of SU6597 and SU6561, the two most active compounds in the biochemical kinase assay, can be more accurately estimated from the data shown in Fig. 1D
, in which lower compound concentrations were used.
Kit Kinase Homology Model.
To try to understand the differences in biochemical potency observed for the various compounds, a homology model for Kit kinase complexed with SU6597 was developed (Fig. 2)
. This model was based on the crystal structure of FGFr1 with SU6668 (29)
. As observed with other indolinone-based kinase inhibitors, the nitrogen and oxygen of the oxindole core form hydrogen bonds with the peptide backbone of Glu671 and Cys673. The carboxylate substituent can interact with the peptide amide of Asp677 and the side chain of Asn680. The ability of the carboxylate moiety to form additional contacts suggests a rationale for the observation that the acidic compounds tend to be more potent than SU5416. However, the homology model cannot explain why addition of the 5-Cl to the oxindole of SU6668 or SU6663 increases the potency of the compound for inhibition of Kit kinase.
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Although addition of the carboxylate increased biochemical potency, it decreased cellular potency. The discrepancy between biochemical and cellular potency becomes even more apparent with SU6561 and SU6597, which contain both the carboxylate and the 5-Cl substituents and, therefore, are very potent biochemical Kit kinase inhibitors but work less well in cellular assays. Such weaker apparent potencies in cellular assays may result from such factors as membrane permeability, protein binding, and higher physiological ATP concentrations than those used in biochemical kinase assays.
Inhibition of SCF-induced Kit Autophosphorylation.
To determine whether these tyrosine kinase inhibitors block SCF-induced Kit activation in tumor cells, we used H526 SCLC cells, which express high levels of Kit and low levels of endogenous SCF and can be stimulated by the addition of SCF in serum-free medium. These cells were incubated in serum-free medium overnight, followed by 30 min of incubation with vehicle or various concentrations of the compounds. They were then stimulated with 100 ng/ml SCF for 5 min and lysed for Kit IP. Phosphorylated Kit was detected on Western blots with an antiphosphotyrosine antibody. Representative Western blots for three compounds are illustrated in Fig. 3
. Stimulation of H526 cells with SCF resulted in phosphorylation of a Mr 145,000–160,000 protein consistent with autophosphorylation of Kit. To confirm the identity of this band as Kit and to assure that equal amounts of Kit were immunoprecipitated, the Western blot was stripped and restained with an anti-Kit MoAb. The percentage change in Kit autophosphorylation was calculated as the ratio of the density of the tyrosine-phosphorylated Kit band divided by the density of the Kit band detected by Western blotting between samples analyzed on the same gel. Table 2
shows the concentration of the compounds necessary to produce a 50% inhibition of SCF-induced Kit autophosphorylation in H526 cells.
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Interestingly, all of the compounds blocked the SCF-stimulated cell growth in a dose-dependent fashion (Fig. 4)
. At a concentration of 5 µM, growth of H526 stimulated by the addition of SCF was completely inhibited by SU5416 and almost completely blocked by SU6597, whereas this effect was observed at 10 µM for SU6663 and SU6668. Increasing the concentration of SU5416 to 10 µM not only eliminated basal autocrine growth, presumably mediated by endogenously produced SCF, but also lead to cell death, whereas up to 20 µM SU6597, SU6663, and SU6668 was needed to have a similar effect. Although SU5614 significantly inhibited growth stimulated by SCF at 1 µM, this compound precipitated out of solution in tissue culture media at concentrations of 2 µM or greater, which accounts for the flat dose-response at higher concentrations. At 10 µM SU5416 and 20 µM for SU6597 and SU6663, growth inhibition was observed in the presence of IGF-1. This inhibition could be attributable to complete inhibition of background endogenous SCF-Kit autocrine growth and/or partial inhibition of IGF-1r signaling, because these concentrations approach or exceed the biochemical IC50 for the IGF-1r (Table 1)
. However, SU5416 concentrations up to 40 µM had no effect on IGF-1-mediated Akt activation (data not shown). It is also interesting to note that the potency for inhibition of IGF-1-mediated growth does not correlate especially well with the ability to inhibit IGF-1r in the biochemical assay (Table 1)
. Although these observations make it less likely that the drugs are directly affecting IGF-1r signaling, an effect on other endogenous growth factor receptors and regulatory kinases cannot be excluded. Table 2
lists the potencies of the five soluble compounds as inhibitors of SCF-stimulated growth in H526 cells. The order of potency from most potent to least potent is SU5416, SU6597, SU6663, SU6668, and SU6561.
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illustrates that the treatment of serum-stimulated H526 cells with the compounds resulted in a dose-dependent growth inhibition. SU5416 and SU6597 inhibited H526 growth by more than 50% at concentrations between 10–20 µM, whereas SU6668, SU6561, and SU6663 required higher doses (concentrations between 20–30 µM) to achieve a similar inhibitory pattern. SU5614 was again found to be insoluble at concentrations greater than 1 µM and, therefore, results at higher concentrations cannot be interpreted. Calculated IC50 values for the soluble compounds are shown in Table 2
, demonstrating that the order of potency of these compounds for inhibition of serum-stimulated growth is similar to that for SCF-stimulated growth. SU5416 appears to be the most potent inhibitor followed by SU6597.
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and 5
demonstrate that SU5416 and SU6597 appeared to be consistently more potent inhibitors of cell growth than the other compounds. Therefore, we chose SU5416 and SU6597 for further evaluation of their growth inhibitory potential in serum-containing medium. Six SCLC cell lines, derived from both untreated and chemotherapy-treated patients and having both classic and variant features (33)
, were incubated in serum-containing medium for 72 h in the presence of increasing concentrations of drug, and cell growth was measured by MTT assay. All of the cell lines exhibited a dose-dependent growth inhibition, as illustrated in Fig. 6
. At a concentration of 20 µM, SU5416 induced an approximately 80% inhibition of H526, H146, and WBA growth and resulted in significant cell death of H209, H510, and H187 cells (Fig. 6A)
. A higher dose of SU6597 was required to induce growth inhibition of all of the six cell lines (Fig. 6B)
. At a concentration 30 µM, SU6597 inhibited more than 80% of H209 and H510 growth, whereas it had a little effect on H146 growth and no effect on WBA growth. The growth of all of the cell lines was completely inhibited at 40 µM SU6597. Interestingly, H146, which is the only one of these cell lines that does not express Kit, also displayed a moderate degree of growth inhibition in the presence of SU5416 and significant cell death in 40 µM SU6597. This effect could be explained by nonspecific inhibition of cell growth at high drug concentrations or inhibition of additional tyrosine kinases other than Kit that are required for SCLC cell growth.
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, MTT growth assays revealed that growth was moderately slowed by SU5416 and SU6597, especially at 30–40 µM concentrations. Cytotoxicity was absent, however, as evidenced by the cells remaining tightly adherent to the culture dish without morphological evidence of apoptosis or necrosis (data not shown). These data illustrate a clear differential cytotoxic effect of these compounds for SCLC cells relative to pulmonary fibroblasts.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have investigated the effect of six related tyrosine kinase inhibitors on SCF-stimulated Kit tyrosine phosphorylation and SCF-induced growth stimulation in SCLC cells. The data demonstrate that they were able to block SCF-stimulated Kit autophosphorylation in a dose-dependent manner and selectively inhibit SCF-stimulated growth. IC50 values for inhibition of SCF-induced Kit phosphorylation in H526 SCLC cells were in reasonable agreement with the potency observed in the CHO cell assay. The IC50 values for inhibition of SCF-stimulated growth of H526 SCLC cells were 10–200-fold higher than those observed for inhibition of Kit phosphorylation. Despite this differential, inhibition of SCF-mediated growth in serum-free medium appeared to be relatively specific, because little or no inhibition of IGF-1-mediated growth occurred at the IC50 values for SCF-mediated growth. The differences between the IC50 values for Kit phosphorylation and SCF-stimulated growth could be explained by several possible factors. They could be an indication that 50% inhibition of receptor autophosphorylation may not be sufficient to suppress growth, because it is not known how many functional receptors or phosphorylated tyrosines are needed to elicit a proliferative response. It may be a very small percentage of the total receptor capacity and, therefore, enough compound to inhibit autophosphorylation by 90% or more might be required to produce effects on cell growth. In addition, our results could be attributable to modifying factors in long-term culture, including nonspecific binding to media components, tissue culture plasticware, or cellular metabolism that lessens the effect of these compounds. Another possible explanation is that inhibition of another kinase in addition to Kit is required to inhibit cell growth, and this unidentified kinase requires a higher dose of these compounds for effective inhibition.
To determine whether these compounds could inhibit SCLC proliferation in an environment containing a complex mix of growth factors representative of in vivo conditions, we tested their effects on H526 growth in medium containing 10% fetal bovine serum. The IC50 values for serum-stimulated growth were somewhat higher than those observed for SCF-stimulated growth in serum-free medium. The higher IC50 values could be attributable to the larger amount of proteins and lipids in 10% fetal bovine serum that could potentially bind the compounds. In addition to the low concentration of SCF present in serum, several other unidentified growth factors in serum could stimulate SCLC growth through other tyrosine kinase receptors and/or other signaling pathways involving tyrosine kinases. Therefore, an additional explanation for the less potent inhibitory effect of these compounds on serum-stimulated growth may be that higher concentrations may be necessary to inhibit these alternative tyrosine kinase-containing pathways. On the basis of their biochemical inhibitory profile (Table 1)
, it is possible that some inhibition of IGF-1r or FGFr signaling, for instance, may be necessary for optimal inhibition of serum-stimulated growth.
The two most potent inhibitors, based on inhibition of Kit phosphorylation and SCF-mediated growth, SU5416 and SU6597, were selected for study using six representative cell lines grown in serum-containing medium. We found that the compounds exhibited dose-dependent growth inhibition in all of the cell lines. Clearly, the response of SCLC cells in serum may not be entirely attributed to inhibition of Kit, because SU5416 and SU6597 also inhibited growth of the H146 cell line, which does not express Kit. However, the growth inhibition appeared to be relatively selective for SCLC, because only moderate growth suppression of MRC-5 cells by high concentrations of SU5416 and SU6597 was observed. Cytotoxicity was not observed for either compound in the MRC-5 assay. This is in marked contrast to the SCLC assays where 30–40 µM concentrations of either drug induced extensive cytotoxicity. This cytotoxic effect is also in striking contrast to the results seen with the quinoxaline tyrosine kinase inhibitor AG1296 (22)
and the 2-phenylaminopyrimidine compound STI571 (24)
, which inhibited growth without significant cytotoxicity in serum-containing medium. The broader spectrum of activity of SU5416 and SU6597 (Table 1)
could account for the increase in cytotoxicity relative to these other inhibitors. In addition to the induction of selective cytotoxicity, these compounds have additional properties that theoretically could make them excellent agents for use in the treatment of SCLC. Both compounds exhibit activity against receptors that are involved in the induction of tumor angiogenesis (Table 1)
. It has already been demonstrated that in vivo tumor growth can be significantly inhibited by SU5416 based solely on its ability to inhibit angiogenesis, because the drug had no significant effect on the in vitro growth of the tumor cell lines tested (30)
. In SCLC, therefore, the in vivo potency could be even greater, based on the ability of the drugs to inhibit kinases required for both growth and tumor angiogenesis. In addition, it would be expected that the development of drug resistance would be hampered by the fact that these agents block multiple kinases involved in regulating tumor growth that lie on two distinct biological pathways intrinsic to both the tumor and nonmalignant endothelial cells.
In summary, we have shown that indolinone tyrosine kinase inhibitors can be potent Kit kinase inhibitors and can block SCF-mediated Kit activation and SCF- and serum-stimulated growth of SCLC cells. To develop these compounds as therapeutic drugs for SCLC, their efficacy in animal models would have to be determined. However, based on our results, it is reasonable to conclude these Kit inhibitors or their derivatives may have a role not only in the treatment of SCLC but also in the treatment of other cancers and human diseases where Kit activation occurs.
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FOOTNOTES |
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1 Supported in part by a Merit Review Award from the Department of Veterans Affairs. 
2 To whom requests for reprints should be addressed, at Richmond Veterans Affairs Medical Center (111K), 1201 Broad Rock Boulevard, Richmond, VA 23249. Phone: (804) 675-5446; Fax: (804) 675-5447; E-mail: gkrystal{at}hsc.vcu.edu
3 The abbreviations used are: SCF, stem cell factor; SCLC, small cell lung cancer; MoAb, monoclonal antibody; IGF-1, insulin-like growth factor-1; PDGFr, platelet-derived growth factor receptor; EGFr, epidermal growth factor receptor; FGFr, fibroblast growth factor receptor; IP, immunoprecipitation; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide; GyrB, B subunit of bacterial DNA gyrase.
Received 6/15/00. Accepted 3/13/01.
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N. C. Wolff, D. E. Randle, M. J. Egorin, J. D. Minna, and R. L. Ilaria Jr. Imatinib Mesylate Efficiently Achieves Therapeutic Intratumor Concentrations in Vivo but Has Limited Activity in a Xenograft Model of Small Cell Lung Cancer Clin. Cancer Res., May 15, 2004; 10(10): 3528 - 3534. [Abstract] [Full Text] [PDF]
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