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Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, sphingomyelin is not only a structural component of cellular membranes but also serves as the precursor for the potent bioactive lipids ceramide and S1P.3
A ceramide, S1P rheostat has been hypothesized to determine the fate of the cell such that the relative cellular concentrations of ceramide and S1P determine whether a cell proliferates or undergoes apoptosis. Ceramide is produced by the hydrolysis of sphingomyelin in response to stresses, including exposure to chemotherapeutic drugs. Ceramide induces apoptosis in proliferating cells by a mechanism that remains to be elucidated (4
, 5)
. Alternately, ceramide can be additionally hydrolyzed by the action of ceramidase to produce sphingosine. Sphingosine is then rapidly phosphorylated by SK to produce S1P. Ceramidase and SK are activated by a number of growth factors and intracellular oncoproteins, leading to rapid increases in the intracellular levels of S1P and depletion of ceramide levels. This situation promotes cell proliferation and inhibits apoptosis in tumor cells.
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In this study, we demonstrate that SK mRNA is frequently overexpressed in a variety of human solid tumors, additionally supporting the consideration of SK as a new molecular target for cancer chemotherapy. Additionally, we developed an assay for recombinant human SK activity and screened a library of synthetic compounds, resulting in the identification of a panel of inhibitors of this enzyme. These compounds are selective toward SK in comparison with other lipid and protein kinases and are not competitive inhibitors of the ATP-binding site of SK. Furthermore, the compounds are antiproliferative toward a panel of human tumor cell lines. Antiproliferation is concomitant with induction of apoptosis. The compounds inhibit S1P formation in intact cells and maintain activity toward cells that express the drug transport proteins P-glycoprotein or MRP1. A prototypical SK inhibitor was found to inhibit tumor growth in vivo. Overall, we have identified a series of potent, structurally novel inhibitors of SK that have potential utilities as antiproliferative drugs.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Synthesis of Compound V.
Benzofuran-3-one exists in equilibrium between the keto- and enol tautomers and underwent an aldol condensation with 3,4-dihydroxy-benzaldehyde in acetic anhydride to form 3,4-diacetoxyaurone (1). Hydrolysis of 1 in a mild hydrochloric acid solution gave 2-(3,4-dihydroxy-benzylidene)-benzofuran-3-one (compound V). For these reactions, solvents were dried and distilled before use, and reactions requiring anhydrous conditions were conducted under an atmosphere of nitrogen. Purifications were done by column chromatography on silica (Merck, silica gel 60, 230–400 mesh). The identities of 1 and compound V were verified by NMR spectroscopy on a Bruker 200-MHz instrument. Chemical shifts relative to trimethylsilane for 1H- and 13C-NMR spectra follow. Mass spectral data were provided by Mass Consortium (San Diego). For 3,4-diacetoxyaurone (1): 1H NMR(200 MHz, CDCl3) 
2.32–2.35(s,s, 6H, 2CH3), 6.82 (s, 1H, HC =), 7.24–7.36 (m, 3H, Ar-H), 7.67–7.83 (m, 4H, Ar-H); 13C NMR(200 MHz, CDCl3) 20.65, 20.69, 111.0, 113.0, 123.7, 123.9, 124.8, 126.0, 129.9, 130.9, 131.2, 137.1, 142.5, 147.2, 166.2; MS m/z (rel intensity) 339 (MH+, 30), 297 (100), 255 (40)
. For 2-(3,4-dihydroxy-benzylidene)-benzofuran-3-one (compound V): 1H NMR(200 MHz, Acetone-d6) 6.75 (s, 1H, HC =), 6.94–6.98 (d, J = 8 Hz, 1H, Ar-H), 7.31–7.49 (m, 3H, Ar-H), 7.63 (s, 1H, Ar-H), 7.74–7.81 (m, 2H, Ar-H), 8.32 (s, 1H, OH), 8.65 (s,1H, OH); 13C NMR(200 MHz, CDCl3) 114.5, 114.6, 117.4, 119.8, 125.1, 125.6, 126.9, 138.3; MS m/z (rel intensity) 255 (MH+, 100). We have also completed the synthesis of compound IV; however, the S atom appears to favor the enol tautomer in the starting material, and this dramatically reduces the rate of the aldol condensation.
Analysis of SK RNA Expression.
SK mRNA levels were examined using the Cancer-Profiling Array from Clontech, which consists of 241 paired samples of cDNA made from the tumor and adjacent normal tissue from individual patients. The array was hybridized with a 32P-labeled cDNA probe corresponding to a 1-kb fragment of the open reading frame of human SK. The membrane was then washed to remove nonspecific binding, and bound probe was visualized using a phosphorimager. The relative intensities of bound probe were quantified using Pathways 4 software, and the ratios of the relative intensities of tumor samples and the corresponding nontumor samples were determined.
Recombinant Human SK and Cell Lines.
Previous studies have demonstrated that functional human SK can be expressed in Escherichia coli (15)
. The open reading frame of human SK cDNA was subcloned into a pGEX bacterial expression vector (Pharmacia), which results in expression of the enzyme as a fusion protein with GST. The fusion protein was then expressed and purified on a column of immobilized glutathione (Glutathione-Sepharose 4B; Pharmacia) according to the manufacturer’s specifications. T-24 human bladder carcinoma cells and JC mouse mammary adenocarcinoma cells were obtained from American Type Culture Collection. MCF-7 human breast adenocarcinoma cells and NCI/ADR, a P-glycoprotein-overexpressing cell line (16)
, were obtained from the Division of Cancer Treatment of the National Cancer Institute. MCF-7/VP cells that overexpress MRP1, but not P-glycoprotein (17)
, were obtained from Drs. Schneider and Cowan. The cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum and 50 µg/ml gentamicin sulfate.
SK Assay.
A medium-throughput assay suitable for screening for inhibitors of recombinant human SK has been established. The activity assay is based upon the method of Louie et al. (18)
. Briefly, 5 µg of purified GST-SK fusion protein were combined with 12 nM sphingosine, which contained a 100-fold dilution of [3-3 H]sphingosine (20 Ci/mmol; American Radiolabeled Chemicals), 1 mM ATP, 1 mM magnesium chloride, and 200 µl of assay buffer [20 mM Tris HCl (pH 7.4), 20% glycerol, 1 mM ß-mercaptoethanol, 1 mM EDTA, 20 mM zinc chloride, 1 mM sodium orthovanadate, 15 mM sodium fluoride, and 0.5 mM 4-deoxypyridoxine]. Assays were run for 30 min at 25°C with shaking and contained either 1% DMSO or 5 µg/ml test compound, which corresponds to concentrations of 10–25 µM. The reactions were terminated with 50 µl of concentrated ammonium hydroxide, followed by extraction of the assay mixture with chloroform:methanol (2:1). The aqueous portion was transferred to scintillation vials and radioactivity was quantified as a measure of [3 H]]S1P formation using a Beckman LS 3801 Scintillation Counter. The intra-assay coefficient of variation was <10%, whereas interassay variation was 
20%.
ATP Competition Assays.
For the ATP competition assays, SK activity was determined according to the method of Spiegel (19)
. Briefly, 5 µg of purified human GST-SK fusion protein was incubated with 10 µM sphingosine, 5 mM MgCl2, vehicle (DMSO), or 2 µM test inhibitor in assay buffer. Reactions were initiated by the addition of ATP containing 1 µCi of [
-32P]ATP (EasyTides, 3000 Ci/mmol; New England Nuclear) and incubated with shaking for 30 min at 37°C. ATP concentrations were varied from 50 to 1000 µM. Reactions were terminated by the addition of 1 N HCl and then extracted with 200 µl of chloroform:methanol:HCl (100:200:1). The organic layer was loaded onto a G60 silica gel TLC plate (Whatman), and S1P was resolved from ATP using a mobile phase consisting of 1-butanol:methanol:water:acetic acid (8:2:2:1). Plates were exposed to radiography film for 2 h, and S1P bands were scraped and counted for activity.
Cellular S1P Formation Assay.
MDA-MB-231 human breast cancer cells were grown to confluency in 24-well tissue culture plates and deprived of serum overnight. Cells were then pretreated with 1% vehicle (DMSO), 20 µM DMS, or 20 µg/ml of each inhibitor for 5 h. Next, each well was dosed with 1 µM sphingosine containing 250 nCi [3-3 H]sphingosine and incubated for 15 min. Cells were washed three times with PBS and lysed with 50 µl of ammonium hydroxide. PBS (200 µl) was added to each well, and lysate was transferred to Eppendorf tubes followed by the addition of 600 µl of chloroform:methanol (2:1) and vortexing. The organic phase containing [3 H]]sphingosine and the aqueous phase containing [3 H]]S1P were individually transferred to scintillation vials and counted.
Cell Proliferation Assay.
T24, MCF-7, MCF-7/VP, and NCI/ADR cells were plated into 96-well tissue culture plates at 15% confluency. After 24 h, cells were treated with various concentrations of inhibitors. After an additional 48 h, cell survival was assayed using the sulforhodamine B assay (20)
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Apoptosis Assay.
T24 cells, which were shown previously to undergo apoptosis in response to anticancer drugs (21)
, were incubated with vehicle alone (DMSO) or 10 µM SK inhibitor for 24 h and assayed for apoptosis using the fluorescein-labeled TUNEL Cell Death Detection Kit (Roche) according to the manufacturer’s specifications. TUNEL-positive cells were identified by fluorescence microscopy and quantified based upon multiple randomly chosen views for each set of treated wells. The percentage of TUNEL-positive cells in each field was determined by dividing the number of TUNEL-positive cells by the total number of cells viewed in the bright field mode. The results shown were confirmed by triplicate wells in duplicate experiments.
ERK2, PKC, and PI3k Assays.
The kinase activity of recombinant human ERK2 was measured using the mitogen-activated protein kinase indirect ELISA assay kit from Upstate Signaling. The assay measures myelin basic protein phosphorylation in the presence of vehicle (DMSO) or various concentrations of each inhibitor and was performed according to the manufacturer’s specifications. Inhibition of human recombinant purified PKC
(Upstate) was tested using the Creb kinase indirect ELISA assay kit (ImmuneChem, Burnaby, BC, Canada) and performed as per specifications. PI3K immunoprecipitation and activity assays were performed according to the methods of Guan et al. (22
, 23)
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Antitumor Evaluation.
A syngenic mouse tumor model that uses a transformed murine mammary adenocarcinoma cell line (ATCC CRL-2116) and Balb/C mice was recently developed in our laboratory.4
Animal care and procedures were in accordance with guidelines and regulations of the Institutional Animal Care and Use Committee of the Penn State College of Medicine. Animals were housed under 12-h light/dark cycles, with food and water provided ad libitum. BALB/c female mice (Charles River), 6–8 weeks old, were injected s.c. with 1 x 106 JC cells suspended in PBS. After palpable tumor growth, 2–3 weeks after injection, tumor volume was determined (day 1) using calipers measuring the length (L) and width (W) of the tumor. Tumor volume was calculated using the equation: (L x W2)/2. Animals were randomized into four groups (n = 5/group). Treatment was then administered on days 1, 5, 9, and 15 and consisted of i.p. administration of either 100 µl of DMSO (control) or compound V in DMSO at a dose of 75 mg/kg. On day 18, animals were euthanized, and tumors were excised and weighed. Whole body weight and tumor volume measurement were performed each day of treatment. Ps were determined using unpaired t tests using GraphPad InStat (San Diego, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the expression of SK message was generally higher in breast tumors compared with adjacent normal breast epithelium. Increases in expression up to 4-fold were found in 80% of the individuals, whereas decreases in the tumor compared with the normal specimen were rare. There were insufficient numbers of samples to allow stratification of the tumors by stage of disease, but the overall pattern appears to hold for all stages. Similar increases in expression of SK in tumors were observed in all of the tissue types analyzed, and the pooled data are presented in the bottom panel of Fig. 2
. Statistically significant differences were found for most of the tumor types (Ps ranged from 0.09 to 0.00007 for different tissues), indicating that up-regulation of SK may be a common property of tumors.
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3 mg of recombinant protein/liter of culture were typical. The SK assay uses [3 H]sphingosine as the substrate and is based on partitioning of [3 H]S1P into the aqueous phase with [3 H]sphingosine remaining in the organic phase. As a negative control, GST without fused SK was expressed in E. coli and purified. As demonstrated in Fig. 3
, the GST control did not increase the appearance of [3 H] in the aqueous phase. In contrast, recombinant hSK-GST caused increases of at least 3-fold after 30 min of incubation, indicating that all of the increased radioactivity in the aqueous phase derives from SK activity. Inclusion of 2% DMSO, the solvent for the chemical library, did not affect the SK activity, whereas 25 µM DMS totally suppressed SK activity.
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16,000 compounds was screened for inhibitors of human SK. The screening assays were conducted at a fixed concentration of 5 µg/ml compound, which corresponds to concentrations of 10–25 µM. The library was screened using pools of eight compounds/sample followed by deconvolution to identify individual active compounds. The hit rate, defined as at least 80% inhibition of SK activity, was 0.8%. Therefore, sufficient numbers of hits were identified and verified to provide confidence that low molecular weight, nonlipid SK inhibitors could be identified by these methods.
Potency of Novel SK Inhibitors.
Additional analysis of the SK inhibitors detected in the screen described above has revealed several active chemotypes. An active compound from each of four of these chemotypes was selected for additional characterization (Fig. 4)
. Compounds I, II, III, and IV (at 5 µg/ml) inhibited SK activity by 99, 85, 99, and 89%, respectively, and serve as prototypes for nonlipid SK inhibitors. Although the screen provided rapid identification of compounds that inhibit SK activity, additional studies were necessary to determine their potencies. Therefore, the effects of compounds I–IV were determined at multiple concentrations, and IC50s for each compound were calculated. As summarized in Table 1
, the compounds demonstrated IC50s in the sub- to low micromolar range, making them more potent inhibitors of SK than any previously reported compound.
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. The Vmaxs show significant decreases with all of the test compounds versus vehicle alone. In contrast, KMs were not increased such that ATP concentrations up to at least 10 times the KM were unable to overcome inhibition by the compounds. Therefore, these compounds are not competitive inhibitors at the ATP-binding site of SK.
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) and a lipid kinase (PI3K). The results are summarized in Table 1
. As shown, all five compounds demonstrated highest potency toward SK, with most having no inhibition of the other kinases at any concentration tested. Compound I showed some activity toward ERK2 but only at 10-fold higher concentrations than those required to inhibit SK. Compound II was the most selective as no inhibition was seen with any of the kinases tested. Compound III was 10–20-fold less potent toward ERK2 and PI3k than SK. Compound IV and its analogue compound V had weak activity toward ERK2 and moderate activity toward PI3k. None of the compounds inhibited PKC-. Thus, although modest inhibition of certain other kinase was observed, these compounds demonstrate a reasonable degree of selectivity toward SK.
Inhibition of Endogenous SK in Intact Cells.
Although the SK inhibitors demonstrated potency toward the purified enzyme, it was important to determine their ability to inhibit endogenous SK in an intact cell model. A survey of several human tumor cell lines indicated that the breast cancer cell line MDA-MB-231 expresses high levels of SK activity (data not shown). As shown in Fig. 5
, the positive control DMS as well as each of the inhibitors decreased S1P formation. TLC analysis confirmed the decrease in activity was attributable to decreased S1P formation (data not shown). In all cases, activity was decreased by at least 50% when compared with vehicle-treated cells. Compound III was the most potent, with SK activity levels inhibited to <15%. These results demonstrate that the compounds inhibit not only purified but endogenous SK in intact cells.
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. The IC50s (in µM) indicate the concentration of a compound that inhibits tumor cell proliferation by 50%. As the data show, these compounds are antiproliferative at sub- to low micromolar concentrations that correspond quite well with the IC50s for inhibiting SK activity in vitro. An important finding is that these SK inhibitors are effective against tumor cells demonstrating the MRP because of overexpression of either P-glycoprotein or MRP1. As these transporters are often overexpressed in tumor cells, particularly after the patient has been treated with cytotoxic drugs, the SK inhibitors may be effective therapeutic agents for resistant tumors.
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, all of the SK inhibitors induced apoptosis, manifested as a significant increase in TUNEL-positive cells compared with vehicle-treated controls. The known SK inhibitor DMS also induced apoptosis as previously described (26)
. Thus, all of the SK inhibitors inhibit cancer cell proliferation and induce apoptosis consistent with the hypothesized consequence of reducing S1P levels.
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. This bioisosteric replacement was expected to produce a compound with comparable activity and resulted in compound V. Biological evaluations demonstrated that the IC50s for inhibition of SK and tumor cell proliferation by compound V were 2 µM, indicating that it is somewhat less potent than compound IV. i.p. administration of compound V to normal female Swiss-Webster mice did not result in any immediate or delayed toxicity at doses up to at least 75 mg/kg. Repeated injections of compound V into the same mice over a period of 15 days also failed to induce overt toxicity, indicating that this compound was suitable for additional in vivo studies. Antitumor activity was assessed by determining the effects of compound V in a syngenic tumor model that uses the mouse JC mammary adenocarcinoma cells growing s.c. in immunocompetent BALB/c mice. These cells express elevated levels of SK activity relative to nontransformed cells, as well as the MRP attributable to P-glycoprotein activity. As indicated in Fig. 8
, tumor growth in animals treated with compound V was significantly lower (>50% decreased at day 18) than tumor growth in control animals. No significant difference in the body weights of animals in the two groups was observed, indicating the lack of overt toxicity from compound V.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phosphorylation of sphingosine by SK is the only known mechanism for the production of S1P in cells. The Mr 49,000 enzyme was initially isolated from rat kidney and demonstrated KMs 5 and 93 µM for sphingosine and ATP, respectively (44)
. The human isoform was recently cloned and displays similar physical and biochemical characteristics (15)
. Shortly thereafter, a second SK isoform was cloned; however, this species displays much lower activity and different kinetic profiles than the type 1 enzyme (45)
. RNA-encoding SK can be detected in most tissues, with higher levels in lung and spleen (46)
. Interestingly, a number of studies have shown that a variety of proliferative factors, including PKC activators (37)
, FCS and platelet-derived growth factor (47)
, epidermal growth factor (48)
, and tumor necrosis factor (49)
rapidly elevate cellular SK activity. Although the signaling links between mitogenic factors and SK remain unclear, these results have led to consideration of the role of SK in cancer.
Recently, an oncogenic role of SK has been directly demonstrated (7)
. In these studies, transfection of SK into NIH 3T3 fibroblasts was sufficient to promote foci formation and cell growth in soft agar and to allow these cells to form tumors in NOD/SCID mice. Additionally, inhibition of SK by transfection with a dominant-negative SK mutant or by treatment of cells with the nonspecific SK inhibitor DMS blocked transformation mediated by oncogenic H-Ras. Because elevated activation of Ras proteins, either by mutation of ras genes or increased stimulation by upstream activators, frequently occurs in cancer, it is likely that SK plays a significant role in this disease. Another study showed that EDG4, a receptor that specifically binds S1P, is a marker for ovarian cancer cells (50)
. S1P has also been implicated in angiogenesis because it induces motility and mitogenesis in smooth muscle cells and promotes endothelial cell differentiation (51)
. Results presented herein provide the first indications that SK may be commonly overexpressed in a variety of human tumors. Specifically, levels of mRNA encoding SK were found to be 2-fold higher in tumors of the breast, colon, lung, ovary, stomach, uterus, kidney, and rectum compared with normal tissue from the same patient. Overall, these studies suggest that inhibition of SK may be an effective therapy for cancer and other hyperproliferative diseases.
Despite the high level of interest in sphingolipid-derived signaling, there are very few demonstrated inhibitors of the enzymes of this pathway. In particular, the field suffers from a lack of potent and selective inhibitors of SK. Pharmacological studies to date have used sphingosine analogues, especially DMS; however, as indicated above, these lipids are known to inhibit several protein kinases (8, 9, 10) . Therefore, selective and potent inhibitors of SK are required for both basic research and as lead compounds for developing novel anticancer agents. Very recently, a few natural product inhibitors of SK have been isolated (12, 13, 14) . Although these inhibitors are moderately potent, with in vitro Kis ranging from 2 to 58 µM, their selectivity and large-scale production capabilities remain unknown. Clearly, potent inhibitors of SK that can be easily synthesized would be highly desirable for evaluating this enzyme as a therapeutic target for cancer therapy. To this end, we initiated a program to identify and evaluate potent and structurally novel inhibitors of SK.
Because no structural data for SK is currently available to enable a computational docking approach to identifying SK inhibitors, we screened a large library of diverse synthetic compounds using purified recombinant human SK fused to GST. This identified a series of low molecular weight, drug-like molecules that inhibit this enzyme. Although too few and diverse for pharmacophore mapping, several of these hits segregated into four chemotypes, and a representative of each of these chemotypes, i.e. compounds I–IV, was additionally characterized. Each compound was highly potent toward purified SK with IC50s lower than any compounds described in the literature.
An important question to address is the mechanism of inhibition of SK by these compounds. Because most inhibitors of other kinases act by competing with ATP for the highly conserved nucleotide-binding domain, it has been difficult to identify selective kinase inhibitors. A recent study by Pitson et al. (49)
demonstrated that the amino acid sequence of the putative nucleotide-binding domain of human SK-1 [SGDGX(17-21)K] is significantly different from all other known nucleotide-binding sites. In our screen for inhibitors of SK, we used high levels of ATP (10 times the KM) in the hopes of identifying compounds that bind at sites other than the nucleotide-binding site. Importantly, competition experiments demonstrated that excess levels of ATP did not remove the inhibition of recombinant human SK, indicating that these compounds are not competitive inhibitors at the ATP-binding site.
We further explored potential interactions with nucleotide-binding domains by determining the effects of the SK inhibitors on a small panel of protein and lipid kinases. None of the compounds inhibited PKC- at doses up to at least 100 times their IC50 for SK, indicating that they do not act as generic kinase inhibitors. Compound I has some activity toward ERK2, whereas compounds IV and V were reasonably potent inhibitors of PI3K. It is important to note that although these compounds are not totally selective for SK, the purpose of lead optimization is to enhance the potency and selectivity of screening hits. In particular, compound II is the most selective SK inhibitor and so may be the most attractive candidate for additional medicinal chemistry efforts.
Compounds I–IV demonstrated cytotoxicities toward human cancer cell lines that closely parallel their potencies for inhibition of purified human SK. The sulforhodamine B staining assay determines the extent of cell survival upon drug exposure (19) . Concordant with the S1P:ceramide rheostat model for cell survival, we hypothesized that these compounds enter cells and inhibit SK activity, thereby decreasing S1P levels, increasing ceramide levels, and resulting in apoptosis. Our hypothesis was affirmed by detecting elevated levels of apoptosis as determined by TUNEL staining upon exposure of T24 cells to each of the SK inhibitors. Additionally, cells treated with the SK inhibitors demonstrated morphological features of apoptosis that paralleled cytotoxicity. We have demonstrated that each chemotype does indeed decrease S1P production in intact cells, indicating that endogenous SK is sensitive to these compounds. The antiproliferative activities of these compounds toward drug-resistant cell lines suggest that they may be effective agents for cancers that are unresponsive to many current drugs. The issue of multidrug resistance is important because numerous established and experimental therapeutics, including tyrosine kinase inhibitors (52) , provide excellent examples of target-based drug development that resulted in suboptimal clinical activity because the compounds interact with the multidrug transporters P-glycoprotein and/or MRP1. During the process of selecting candidates for additional drug development, priority is often now given to compounds that are not substrates for these transporters.
To test whether these inhibitors could serve as chemotherapy agents, an analogue of compound IV was synthesized and tested for antitumor activity. Compound V was selected for synthesis because an expedient synthetic route was apparent. The methods can be used with a variety of commercially available aldehydes to generate a focused combinatorial library. Compound V was found to have moderate antitumor activity in vivo under conditions that were not toxic to the animals. Administering the compound on a more frequent dosing schedule or by an alternate route may increase this antitumor effect. It should be recognized that compound V is not expected to be the most effective of the SK inhibitors. It was simply used to provide proof-of-principle in these initial studies, and this was successful in providing the first evidence that a SK inhibitor can have antitumor activity in vivo in the absence of toxicity to the animal. We do note the presence of the conjugated cathecol ring in compounds IV and V that may be readily metabolized by cytochrome P450. The acetylated intermediate of compound V (compound 1, Fig. 7
) had similar potency toward purified SK and similar cytotoxicity as compound V (data not shown), indicating that the free hydroxyls are not required for activity. Additional structure-activity analyses of these series of SK inhibitors are in progress.
In conclusion, SK is critically important in the regulation of tumor cell proliferation and apoptosis and so represents a potentially important target for the development of new anticancer drugs. We have now demonstrated that SK expression is significantly elevated in a variety of solid tumors and have identified several low molecular weight compounds that potently inhibit this enzyme in close relationship with their antiproliferative activities. The identification of an SK inhibitor with antitumor activity in vivo additionally substantiates the hypothesis that SK is an attractive target for new therapeutics. Additional development of these compounds through medicinal chemistry efforts and early-stage ADME (Absorption, Distribution, Metabolism and Elimination) profiling will be critical in lead optimization. Analysis of their cellular and in vivo effects will also be crucial for evaluating the clinical potential of this new class of targeted compounds.
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FOOTNOTES |
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1 Supported by NIH Grant R24 CA788243 (to C. D. S.). 
2 To whom requests for reprints should be addressed, at Department of Pharmacology, H078, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033. Phone: (717) 531-1672; Fax: (717) 531-5013; E-mail: cdsmith{at}psu.edu
3 The abbreviations used are: S1P, sphingosine-1-phosphate; DMS, D-erythro-N,N-dimethylsphingosine; NMR, nuclear magnetic resonance; GST, glutathione S-transferase; SK, sphingosine kinase; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; MRP, multidrug resistance phenotype; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; PI3K, phosphatidylinositol 3'-kinase; CREB, cyclic AMP response element-binding protein.
4 B. D. Lee, K. J. French, Y. Zhuang, and C. D. Smith, submitted for publication.
Received 9/10/02. Revised 6/23/03. Accepted 7/ 9/03.
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 I. Ader, L. Brizuela, P. Bouquerel, B. Malavaud, and O. Cuvillier Sphingosine Kinase 1: A New Modulator of Hypoxia Inducible Factor 1{alpha} during Hypoxia in Human Cancer Cells Cancer Res., October 15, 2008; 68(20): 8635 - 8642. [Abstract] [Full Text] [PDF]
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D. Shida, X. Fang, T. Kordula, K. Takabe, S. Lepine, S. E. Alvarez, S. Milstien, and S. Spiegel Cross-talk between LPA1 and Epidermal Growth Factor Receptors Mediates Up-regulation of Sphingosine Kinase 1 to Promote Gastric Cancer Cell Motility and Invasion Cancer Res., August 15, 2008; 68(16): 6569 - 6577. [Abstract] [Full Text] [PDF]
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S. W. Paugh, B. S. Paugh, M. Rahmani, D. Kapitonov, J. A. Almenara, T. Kordula, S. Milstien, J. K. Adams, R. E. Zipkin, S. Grant, et al. A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia Blood, August 15, 2008; 112(4): 1382 - 1391. [Abstract] [Full Text] [PDF]
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D. R. Gude, S. E. Alvarez, S. W. Paugh, P. Mitra, J. Yu, R. Griffiths, S. E. Barbour, S. Milstien, and S. Spiegel Apoptosis induces expression of sphingosine kinase 1 to release sphingosine-1-phosphate as a "come-and-get-me" signal FASEB J, August 1, 2008; 22(8): 2629 - 2638. [Abstract] [Full Text] [PDF]
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S. Bu, B. Kapanadze, T. Hsu, and M. Trojanowska Opposite Effects of Dihydrosphingosine 1-Phosphate and Sphingosine 1-Phosphate on Transforming Growth Factor-{beta}/Smad Signaling Are Mediated through the PTEN/PPM1A-dependent Pathway J. Biol. Chem., July 11, 2008; 283(28): 19593 - 19602. [Abstract] [Full Text] [PDF]
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D. Pchejetski, N. Doumerc, M. Golzio, M. Naymark, J. Teissie, T. Kohama, J. Waxman, B. Malavaud, and O. Cuvillier Chemosensitizing effects of sphingosine kinase-1 inhibition in prostate cancer cell and animal models Mol. Cancer Ther., July 1, 2008; 7(7): 1836 - 1845. [Abstract] [Full Text] [PDF]
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 K. Takabe, S. W. Paugh, S. Milstien, and S. Spiegel "Inside-Out" Signaling of Sphingosine-1-Phosphate: Therapeutic Targets Pharmacol. Rev., June 1, 2008; 60(2): 181 - 195. [Abstract] [Full Text] [PDF]
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C. A. Corcoran, Q. He, S. Ponnusamy, B. Ogretmen, Y. Huang, and M. S. Sheikh Neutral Sphingomyelinase-3 Is a DNA Damage and Nongenotoxic Stress-Regulated Gene That Is Deregulated in Human Malignancies Mol. Cancer Res., May 1, 2008; 6(5): 795 - 807. [Abstract] [Full Text] [PDF]
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 G. Yan, S. Chen, B. You, and J. Sun Activation of sphingosine kinase-1 mediates induction of endothelial cell proliferation and angiogenesis by epoxyeicosatrienoic acids Cardiovasc Res, May 1, 2008; 78(2): 308 - 314. [Abstract] [Full Text] [PDF]
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T. M. Leclercq, P. A. B. Moretti, M. A. Vadas, and S. M. Pitson Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity J. Biol. Chem., April 11, 2008; 283(15): 9606 - 9614. [Abstract] [Full Text] [PDF]
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V. Anelli, C. R. Gault, A. B. Cheng, and L. M. Obeid Sphingosine Kinase 1 Is Up-regulated during Hypoxia in U87MG Glioma Cells: ROLE OF HYPOXIA-INDUCIBLE FACTORS 1 AND 2 J. Biol. Chem., February 8, 2008; 283(6): 3365 - 3375. [Abstract] [Full Text] [PDF]
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 J. Guo, D. Liu, D. Nikolic, D. Zhu, J. M. Pezzuto, and R. B. van Breemen In Vitro Metabolism of Isoliquiritigenin by Human Liver Microsomes Drug Metab. Dispos., February 1, 2008; 36(2): 461 - 468. [Abstract] [Full Text] [PDF]
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 M. Kim, M. Kim, N. Kim, V. D. D'Agati, C. W. Emala Sr, and H. T. Lee Isoflurane mediates protection from renal ischemia-reperfusion injury via sphingosine kinase and sphingosine-1-phosphate-dependent pathways Am J Physiol Renal Physiol, December 1, 2007; 293(6): F1827 - F1835. [Abstract] [Full Text] [PDF]
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H. M. Sankala, N. C. Hait, S. W. Paugh, D. Shida, S. Lepine, L. W. Elmore, P. Dent, S. Milstien, and S. Spiegel Involvement of Sphingosine Kinase 2 in p53-Independent Induction of p21 by the Chemotherapeutic Drug Doxorubicin Cancer Res., November 1, 2007; 67(21): 10466 - 10474. [Abstract] [Full Text] [PDF]
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 K. Itagaki, J. K. Yun, J. A. Hengst, A. Yatani, C. J. Hauser, Z. Spolarics, and E. A. Deitch Sphingosine 1-Phosphate Has Dual Functions in the Regulation of Endothelial Cell Permeability and Ca2+ Metabolism J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 186 - 191. [Abstract] [Full Text] [PDF]
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G. Ding, H. Sonoda, H. Yu, T. Kajimoto, S. K. Goparaju, S. Jahangeer, T. Okada, and S.-i. Nakamura Protein Kinase D-mediated Phosphorylation and Nuclear Export of Sphingosine Kinase 2 J. Biol. Chem., September 14, 2007; 282(37): 27493 - 27502. [Abstract] [Full Text] [PDF]
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D. J. Kusner, C. R. Thompson, N. A. Melrose, S. M. Pitson, L. M. Obeid, and S. S. Iyer The Localization and Activity of Sphingosine Kinase 1 Are Coordinately Regulated with Actin Cytoskeletal Dynamics in Macrophages J. Biol. Chem., August 10, 2007; 282(32): 23147 - 23162. [Abstract] [Full Text] [PDF]
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 A. Gomez-Brouchet, D. Pchejetski, L. Brizuela, V. Garcia, M.-F. Altie, M.-L. Maddelein, M.-B. Delisle, and O. Cuvillier Critical Role for Sphingosine Kinase-1 in Regulating Survival of Neuroblastoma Cells Exposed to Amyloid-beta Peptide Mol. Pharmacol., August 1, 2007; 72(2): 341 - 349. [Abstract] [Full Text] [PDF]
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 F. Doll, J. Pfeilschifter, and A. Huwiler Prolactin upregulates sphingosine kinase-1 expression and activity in the human breast cancer cell line MCF7 and triggers enhanced proliferation and migration Endocr. Relat. Cancer, June 1, 2007; 14(2): 325 - 335. [Abstract] [Full Text] [PDF]
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 I. D. Jung, J. S. Lee, Y. J. Kim, Y.-I. Jeong, C.-M. Lee, T. Baumruker, A. Billlich, Y. Banno, M. G. Lee, S.-C. Ahn, et al. Sphingosine kinase inhibitor suppresses a Th1 polarization via the inhibition of immunostimulatory activity in murine bone marrow-derived dendritic cells Int. Immunol., April 1, 2007; 19(4): 411 - 426. [Abstract] [Full Text] [PDF]
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S. Bonnaud, C. Niaudet, G. Pottier, M.-H. Gaugler, J. Millour, J. Barbet, L. Sabatier, and F. Paris Sphingosine-1-Phosphate Protects Proliferating Endothelial Cells from Ceramide-Induced Apoptosis but not from DNA Damage-Induced Mitotic Death Cancer Res., February 15, 2007; 67(4): 1803 - 1811. [Abstract] [Full Text] [PDF]
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S. Spiegel and S. Milstien Functions of the Multifaceted Family of Sphingosine Kinases and Some Close Relatives J. Biol. Chem., January 26, 2007; 282(4): 2125 - 2129. [Full Text] [PDF]
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J. Radeff-Huang, T. M. Seasholtz, J. W. Chang, J. M. Smith, C. T. Walsh, and J. H. Brown Tumor Necrosis Factor-{alpha}-stimulated Cell Proliferation Is Mediated through Sphingosine Kinase-dependent Akt Activation and Cyclin D Expression J. Biol. Chem., January 12, 2007; 282(2): 863 - 870. [Abstract] [Full Text] [PDF]
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 L. W. Maines, K. J. French, E. B. Wolpert, D. A. Antonetti, and C. D. Smith Pharmacologic Manipulation of Sphingosine Kinase in Retinal Endothelial Cells: Implications for Angiogenic Ocular Diseases Invest. Ophthalmol. Vis. Sci., November 1, 2006; 47(11): 5022 - 5031. [Abstract] [Full Text] [PDF]
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P. Mitra, C. A. Oskeritzian, S. G. Payne, M. A. Beaven, S. Milstien, and S. Spiegel Role of ABCC1 in export of sphingosine-1-phosphate from mast cells PNAS, October 31, 2006; 103(44): 16394 - 16399. [Abstract] [Full Text] [PDF]
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K. J. French, J. J. Upson, S. N. Keller, Y. Zhuang, J. K. Yun, and C. D. Smith Antitumor Activity of Sphingosine Kinase Inhibitors J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 596 - 603. [Abstract] [Full Text] [PDF]
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 M. Yadav, L. Clark, and J. S. Schorey Macrophage's Proinflammatory Response to a Mycobacterial Infection Is Dependent on Sphingosine Kinase-Mediated Activation of Phosphatidylinositol Phospholipase C, Protein Kinase C, ERK1/2, and Phosphatidylinositol 3-Kinase J. Immunol., May 1, 2006; 176(9): 5494 - 5503. [Abstract] [Full Text] [PDF]
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D. Pchejetski, M. Golzio, E. Bonhoure, C. Calvet, N. Doumerc, V. Garcia, C. Mazerolles, P. Rischmann, J. Teissie, B. Malavaud, et al. Sphingosine Kinase-1 as a Chemotherapy Sensor in Prostate Adenocarcinoma Cell and Mouse Models Cancer Res., December 15, 2005; 65(24): 11667 - 11675. [Abstract] [Full Text] [PDF]
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M. Maceyka, H. Sankala, N. C. Hait, H. Le Stunff, H. Liu, R. Toman, C. Collier, M. Zhang, L. S. Satin, A. H. Merrill Jr., et al. SphK1 and SphK2, Sphingosine Kinase Isoenzymes with Opposing Functions in Sphingolipid Metabolism J. Biol. Chem., November 4, 2005; 280(44): 37118 - 37129. [Abstract] [Full Text] [PDF]
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 K. R. Johnson, K. Y. Johnson, H. G. Crellin, B. Ogretmen, A. M. Boylan, R. A. Harley, and L. M. Obeid Immunohistochemical Distribution of Sphingosine Kinase 1 in Normal and Tumor Lung Tissue J. Histochem. Cytochem., September 1, 2005; 53(9): 1159 - 1166. [Abstract] [Full Text] [PDF]
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E. Le Scolan, D. Pchejetski, Y. Banno, N. Denis, P. Mayeux, W. Vainchenker, T. Levade, and F. Moreau-Gachelin Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression Blood, September 1, 2005; 106(5): 1808 - 1816. [Abstract] [Full Text] [PDF]
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N. C. Hait, S. Sarkar, H. Le Stunff, A. Mikami, M. Maceyka, S. Milstien, and S. Spiegel Role of Sphingosine Kinase 2 in Cell Migration toward Epidermal Growth Factor J. Biol. Chem., August 19, 2005; 280(33): 29462 - 29469. [Abstract] [Full Text] [PDF]
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 Y. Kariya, A. Kihara, M. Ikeda, F. Kikuchi, S. Nakamura, S. Hashimoto, C.-H. Choi, Y.-M. Lee, and Y. Igarashi Products by the sphingosine kinase/sphingosine 1-phosphate (S1P) lyase pathway but not S1P stimulate mitogenesis Genes Cells, June 1, 2005; 10(6): 605 - 615. [Abstract] [Full Text] [PDF]
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J. Min, P. P. Van Veldhoven, L. Zhang, M. H. Hanigan, H. Alexander, and S. Alexander Sphingosine-1-Phosphate Lyase Regulates Sensitivity of Human Cells to Select Chemotherapy Drugs in a p38-Dependent Manner Mol. Cancer Res., May 1, 2005; 3(5): 287 - 296. [Abstract] [Full Text] [PDF]
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 S. M. Pitson, P. Xia, T. M. Leclercq, P. A.B. Moretti, J. R. Zebol, H. E. Lynn, B. W. Wattenberg, and M. A. Vadas Phosphorylation-dependent translocation of sphingosine kinase to the plasma membrane drives its oncogenic signalling J. Exp. Med., January 3, 2005; 201(1): 49 - 54. [Abstract] [Full Text] [PDF]
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 J. Min, D. Traynor, A. L. Stegner, L. Zhang, M. H. Hanigan, H. Alexander, and S. Alexander Sphingosine Kinase Regulates the Sensitivity of Dictyostelium discoideum Cells to the Anticancer Drug Cisplatin Eukaryot. Cell, January 1, 2005; 4(1): 178 - 189. [Abstract] [Full Text] [PDF]
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M. Yamanaka, D. Shegogue, H. Pei, S. Bu, A. Bielawska, J. Bielawski, B. Pettus, Y. A. Hannun, L. Obeid, and M. Trojanowska Sphingosine Kinase 1 (SPHK1) Is Induced by Transforming Growth Factor-{beta} and Mediates TIMP-1 Up-regulation J. Biol. Chem., December 24, 2004; 279(52): 53994 - 54001. [Abstract] [Full Text] [PDF]
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J. Min, A. L. Stegner, H. Alexander, and S. Alexander Overexpression of Sphingosine-1-Phosphate Lyase or Inhibition of Sphingosine Kinase in Dictyostelium discoideum Results in a Selective Increase in Sensitivity to Platinum-Based Chemotherapy Drugs Eukaryot. Cell, June 1, 2004; 3(3): 795 - 805. [Abstract] [Full Text] [PDF]
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T. A. Taha, W. Osta, L. Kozhaya, J. Bielawski, K. R. Johnson, W. E. Gillanders, G. S. Dbaibo, Y. A. Hannun, and L. M. Obeid Down-regulation of Sphingosine Kinase-1 by DNA Damage: DEPENDENCE ON PROTEASES AND p53 J. Biol. Chem., May 7, 2004; 279(19): 20546 - 20554. [Abstract] [Full Text] [PDF]
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) or compound V at a dose of 75 mg/kg (
) was administered i.p. on days 1, 5, 9, and 15. Tumor growth is expressed as the volume relative to day 1 for each animal. * indicates Ps <0.05, as determined by unpaired t tests. The insert indicates the body weight of the animals during this experiment.