Monoterpene Glycoside ESK246 from Pittosporum Targets LAT3 Amino Acid Transport and Prostate Cancer Cell Growth

Isolationand Identification of ESK242 and ESK246

ESK246(trivial name venuloside A) was isolated as an optically active clearoil ([α]_D +39, c 0.1, MeOH) witha molecular formula C₂₃H₃₆O₇, asestablished from HRESIMS measurements. The ¹H NMR spectrumof ESK 246 in C₆D₆ was well resolved and showed 21 resonances composed of 2 sp²-hybridized methines, 6 sp³-hybridized methines,3 diastereotopic methylene pairs, and 7 sp³-hybridizedmethyls (SI Table S1). The ¹³C NMR spectrum showed two carbonyls (δ_C 168.9, 165.8),three other quaternary carbons (δ_C 157.7, 133.6 and79.4), two olefinic resonances (δ_C 121.4 and 116.4),four oxymethines (δ_C 95.9, 73.8, 70.4, 70.3, and70.0), one other methine (δ_C 44.4), three methylenes(δ_C 31.3, 26.96, and 24.0), and seven methyls (δ_C 27.02, 23.9, 23.53, 23.52, 20.7, 20.2, and 16.6). Interpretationof 2D NMR data allowed for the identification of four partial structuresdepicted in Figure 2B, namely an acetyl substituent(fragment A), a 3-methylbut-2-enoyl group (fragment B), a sugarmoiety (fragment C), and a ten-carbon aglycone unit (fragment D).Characteristic chemical shifts for the methyl (δ_H 1.87, 3H, s, δ_C 20.7) and carbonyl resonances (δ_C 168.9, C-1″) were used to assign fragment A as theacetyl substituent. Fragment B had two methyl vinyl resonances (δ_H 2.14, d, J = 1.0 Hz, δ_C 27.02 and 1.45, d, J = 1.0 Hz; δ_C 20.2), coupled to each other and to an olefin proton (δ_H 5.77, δ_C 116.4), in addition to a quaternarycarbon (δ_C 157.7) and an ester carbonyl resonance(δ_C 165.8) consistent with a senecioyl (3-methylbut-2-enoyl)moiety. Partial structure C contained an anomeric proton, four otheroxymethines and a doublet methyl resonance, consistent with the presenceof a six-deoxy sugar moiety. The ¹H NMR chemical shiftsand coupling constants for H-1′ (δ_H 4.47,d, J = 7.8 Hz), H-2′ (δ_H 5.66,dd, J = 10.3, 7.8 Hz), H-3′ (5.16, dd, J = 10.4, 3.2 Hz), H-4′ (3.72, br d, J = 3.2 Hz), H-5′ (3.10, br q, J = 6.3 Hz),and H-6′-Me (1.17, d, J = 6.3 Hz) protons,together with the ROESY data showed that the glycosyl moiety was β-fucopyranoside.The downfield shift of H-2′ and H-3′ resonances suggestedthat the C-2 and C-3 hydroxyl groups on the sugar moiety were esterified.Chemical shifts assigned to the acetyl and senecioyl substituentswere positioned on C-2′ and C-3′ respectively followingcrucial ¹H–¹³C HMBC correlations fromthe H-2′ glycosidic resonance to C-1″ carbonyl on theacetyl group, as well as the glycosidic H-3′ to C-1‴carbonyl on the senecioyl group (Figure 2B).The sugar residue in ESK246 was therefore concluded to be 2′-acetyl-3′-senecioyl-β-fucopyranoside.Signals for the aglycone fragment D, C₁₀H₁₇ assuggested by the molecular formula, were composed of an olefinic multiplet,three diastereotopic methylene pairs, a single methine and three methylresonances, consistent with the presence of an α-terpineol moiety.Crucial HMBC correlation from the anomeric H-1′ to the quaternaryC-8 carbon (δ_C 79.4) (Figure 2B) connected substructure D to the sugar unit to complete the planarstructure of the glycoside.

Figure 2

Identification of compound structure. (A) Structuresof two newnatural products ESK242 and ESK246. (B) Crucial NMR correlations usedto establish the planar structure of ESK246. Bold lines indicate ¹H–¹H COSY correlations that were used toidentify the 1,1-dimethyl vinyl spin system (fragment B), fucopyranosidespin system (fragment C) and the terpineol spin system (fragment D).Red arrows depict the crucial ¹H–¹³CHMBC correlations used to identify the position of the aglycone substituentand the esterification sites of the fucopyranoside residue. (C) Syntheticroute to ESK246. Reagents used: (a) α-terpineol, Ag₂CO₃, CH₂Cl₂, rt, 48 h. (b) i. NaOMe,MeOH, rt, 3 h. ii. 3,3-Dimethylacryloyl chloride, py, CH₂Cl₂, 0 °C, 18 h. iii. Ac₂O, py,CH₂Cl₂, 0 °C, 3 h. In addition to the synthesisof diastereomers of α-terpineol shown, an identical syntheticroute was used with the (4S)-α-terpineol startingmaterial.

The absolute configuration ofthe natural product was determinedvia comparison of chiro-optical and NMR spectral data with that ofthe synthetically prepared analogues. Hydrolysis of ESK246 in 5% HClin MeOH afforded a mixture of methyl-3-O-senecioyl-β-fucopyranoside, methyl-3-O-senecioyl-α-fucopyranoside and methyl-4-O-senecioyl-α-fucopyranoside (see Supporting Information). Comparison of the optical rotation values of the ESK246 hydrolysis-sourcedα- and β-anomers of methyl-3-O-senecioyl-fucopyranosidewith that of the synthetic α- and β-anomers of methyl-3-O-senecioyl-d-fucopyranoside establishedthe absolute configuration of fucose residue as d (see Supporting Information). The d-configurationof the fucose sugar is typical of plant-sourced glycosides, whereas l has been reported from bacteria- and plant-sourced polysaccharides,as well as glycolipids and glycoproteins of animal origin.²⁶ Resolving the absolute configuration of theα-terpineol proved to be a challenge as the acid hydrolysiswork-up and isolation did not yield any terpineol product. The absoluteconfiguration of the natural product was therefore resolved with totalsynthesis of the racemic and (4S)-α-terpineoldiastereomers of ESK246. Starting from d-fucose and the readilyavailable (4rac)- and (4S)-α-terpineols,a four-step synthesis was achieved (Figure 2C and Supporting Information text). Koenigs–Knorrglycosylation of bromo-fucosyl donor with α-terpineol affordedthe fully protected α-terpineol fucoside, which was subsequentlydeprotected under basic conditions (NaOMe/MeOH) to give the α-terpineolfucoside. Utilizing the inherent difference in reactivity of the fucosehydroxyl groups,²⁷ selective acylationat the 3′O position of α-terpineol fucosidewith 3,3-dimethylacryloyl chloride was achieved.²⁸ Introduction of the acetate moiety at the 2′O position was carried out under basic conditions usingacetic anhydride to give the final product.

In the ¹H NMR spectrum of the C-4 epimeric mixure ofESK246, the greatest signal dispersion between the two isomers wasobserved with the H-9 and H-10 resonances (SIFigure S2). These two sets of resonances were therefore usedas a point of differentiation in the determination of the absoluteconfiguration of ESK246. In an ¹H NMR-based titration experimentdepicted in SI Figure S3, addition of thenaturally occurring ESK246 to the C-4 epimeric mixture of α-terpineol-8-O-β-d-(2′-acetyl, 3′-senecioyl)fucopyranoside resulted in the enhancement of the resonances associatedwith the (4R)-α-terpineol epimer, thereforesecuring the stereochemistry of the natural product as (4R)-α-terpineol-8-O-β-d-(2′-acetyl,3′-senecioyl) fucopyranoside.

ESK242 (trivial name venulosideB) was isolated as an opticallyactive clear oil ([α]_D +20, c 0.1,MeOH) with a molecular formula C₂₃H₃₆O₇, as established from HRESIMS measurements. The compound was isomericwith ESK246 and showed an identical number of ¹H and ¹³C resonances in the NMR spectra. The most significant differencesbetween the NMR spectra of the two constitutional isomers were centeredon the two methyl vinyl resonances H-4‴ (δ_H 2.05, m; δ_C 16.0) and H-5‴ (δ_H 1.94, m; δ_C 20.8) as well as an olefinicmethine resonance at H-3‴ (δ_H 5.79, m; δ_C 139.3). In contrast to ESK246, which had a senecioyl groupat C-3, ESK242 showed two methyl vinyl resonances each coupled toan olefin proton, consistent with a 1,2-dimethyl vinyl group. A ROESYexperiment (τ_mix = 400 ms) assigned the geometryof the double bond to be cis, based on the observationof a correlation between H-3‴ and H-5‴ methyl resonances,consistent with the presence of an angeloyl group. Crucial ¹H–¹³C HMBC correlations from the glycosidic H-3′to C-1‴ placed the angeloyl group on C-3′ of the sugarmoiety. Interpretation of 1D and 2D NMR data (SI Table S1) established the structure of ESK242 as α-terpineol-2′-acetate-3′-angeloyl-β-fucopyranoside.Based on the structural homology with ESK246 and the close correspondenceof the NMR chemical shifts (SI Table S1) the absolute configuration of ESK242 is proposed to be (4R)-α-terpineol-8-O-β-d-(2′-acetyl, 3′-angeloyl) fucopyranoside.

Effects ofESK242 and ESK246 on Leucine Uptake in Prostate CancerCell Lines

To examine the inhibitory effects of ESK242 andESK246, we next investigated the impact of these new compounds onLNCaP and PC-3 prostate cancer cell lines. We have previously shownthat LAT3 is the dominant leucine transporter in LNCaP cells, whileLAT1 plays a more important role in PC-3 cells (SI Figure S4A).¹⁶ In the presenceof compounds ESK242 and ESK246, leucine uptake was decreased in adose-dependent manner in LNCaP cells (Figure 3A). The IC₅₀ of ESK242 is 29.6 ± 1.2 μM, whilethe IC₅₀ of ESK246 is 8.12 ± 1.2 μM. Both compoundshad an IC₅₀ more than 2 orders of magnitude lower thanthe universal LAT inhibitor, BCH, whose IC₅₀ is 4060 ±1.1 μM (Figure 3A). In contrast, neithercompound substantially inhibited leucine uptake at the same concentrationin PC-3 cells, in which LAT1 is more abundantly expressed (SI Figure S4B).

Figure 3

(A) Inhibition of LAT3-mediated [³H]-l-leucineuptake in LNCaP cells. The IC₅₀ of BCH, ESK242 and ESK246was calculated to be 4060 ± 1.1 μM, 29.6 ± 1.2 μM,and 8.12 ± 1.2 μM, respectively. (B–E) [³H]-l-leucine uptake assay in the presence or absence of50 μM ESK242 or ESK246 in oocytes expressing LAT1/4F2hc (B),LAT2/4F2hc (C), LAT3 (D), or LAT4 (E). Data show mean ± SEM (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001.

Characterization of ESK242 and ESK246 by Amino Acid Uptake Assayin Oocytes

The differential effects on leucine uptake inLNCaP and PC-3 cells suggested these compounds selectively inhibitindividual LAT family members. To test the specificity of the purifiedcompounds, we expressed LAT1/4F2hc, LAT2/4F2hc, LAT3 or LAT4 in Xenopus laevis oocytes and [³H]-l-leucineuptake assays were performed. After 4 days, transporters were expressedon the plasma membrane of oocytes, as shown by an increase in [³H]-l-leucine uptake compared to uninjected oocytes(SI Figure S5A). To demonstrate expressionof the LAT3 transporter on the surface of oocytes, we performed asurface biotinylation assay, which confirmed LAT3 expression in injectedoocytes only (SI Figure S5B). The optimalconcentration of leucine, and duration of leucine uptake, was determinedby dose response and time course experiments, showing 1 mM l-leucine for 30 min to be optimal (SI FiguresS5C and D). After treatment with 50 μM ESK242 or ESK246for 30 min, LAT1/4F2hc transport activity was significantly inhibitedby ESK242 (34.2%; Figure 3B). LAT3-mediatedleucine transport was significantly inhibited by both ESK242 (47.9%)and ESK246 (47.3%; Figure 3D). Neither compoundsignificantly inhibited leucine uptake mediated by LAT2/4F2hc (Figure 3C) or LAT4 (Figure 3E). Theseresults suggest that ESK246 is a preferential LAT3 inhibitor and ESK242inhibits both LAT1 and LAT3. We next determined the IC₅₀ of both compounds for LAT3 in oocytes. The IC₅₀ of ESK246was calculated at 146.7 ± 2.4 μM, which is lower than theIC₅₀ of ESK242 at 281.8 ± 1.3 μM (Figure 4A), suggesting that ESK246 has a higher affinityfor LAT3 than ESK242.

Figure 4

ESK242 and ESK246 inhibit LAT3 via a mixed mode of inhibition.(A) Increasing doses of ESK242 and ESK246 inhibit [³H]-l-leucine transport in oocytes expressing LAT3 with IC₅₀ values of 281.8 ± 1.3 μM and 146.7 ± 2.4 μM,respectively. (B–C) [³H]-l-leucine doseresponse in the absence or presence of 50 μM or 500 μMESK242 (B) or ESK246 (C). Data are fitted to the Michaelis–Mentenequation and normalized to the maximal rate of transport of [³H]-l-leucine alone. An Eadie–Hofstee transformationwas performed (inset, B–C). Data show mean ± SEM (n = 5).

In order to investigatethe nature of inhibition of LAT3 by ESK242and ESK246, leucine dose responses were performed in the absence orpresence of 50 μM and 500 μM of each inhibitor. As theconcentration of inhibitor increases, the K_m of leucine for LAT3 (6.3 ± 0.5 mM) increases slightly in thepresence of 50 μM ESK242 (7.5 ± 0.5 mM) but decreases to2.7 ± 0.5 mM in the presence of 500 μM ESK242 (Figure 4B). In the presence of 50 μM and 500 μMESK246, the K_m of leucine for LAT3 decreasesto 4.7 ± 0.6 mM and 2.1 ± 0.3 mM, respectively (Figure 4C) suggesting a competitive mechanism of actionfor these compounds. The maximal rate of uptake also decreases asthe inhibitor concentration increases, which would not be expectedfor a purely competitive inhibitor suggesting that these compoundsmay be acting in a mixed competitive/noncompetitive manner. An Eadie–Hofsteetransformation of the data (Figure 4B and C,inset) also demonstrates that both ESK242 and ESK246 are acting asmixed inhibitors.

Effects of ESK242 and ESK246 on mTORC1 Signalingand Cell Growthin Prostate Cancer Cell Lines

Having established that ESK242and ESK246 inhibit leucine uptake in LNCaP cells with an IC₅₀ between 8–30 μM (Figure 3A),we next assessed the downstream effects of leucine deprivation onsignaling and cell growth. Leucine uptake mediated by LAT3 has beenshown to regulate mTORC1 activation in prostate cancer cells.¹⁶ Both ESK242 and ESK246 suppressed phosphorylation(activation) of the mTORC1 target protein p70S6K (Figure 5A), consistent with a mechanism consequent to inhibitionof LAT3-mediated leucine transport in LNCaP cells. We next examinedcell viability in the presence of compounds using an MTT assay inprostate cancer cells. Both ESK242 and ESK246 led to lower cell viabilityin LNCaP cells after 3 days treatment (Figure 5B). Only compound ESK242, but not ESK246, significantly inhibitedcell viability in PC-3 cells, (SI Figure S6A), which may be due to ESK242 inhibition of both LAT1 and LAT3. Todetermine whether decreased cell viability is due to activation ofapoptosis or suppression of proliferation, we first examined apoptosisusing flow cytometry to detect “flipped” Annexin-V proteinlevels in the plasma membrane. Cells exposed to UV showed increasedAnnexin-V flipping indicative of apoptosis (Figure 5C). Neither ESK242 nor ESK246 induced apoptosis, consistentwith previous studies examining BCH (Figure 5C).¹⁶ Therefore, it is likely that theeffects on viability are mediated through inhibition of cell cycleand proliferation. To test this, we next examined BrdU (bromodeoxyuridine,a thymidine analogue) incorporation in DNA using flow cytometry andshowed the BrdU incorporation rate was decreased after BCH, ESK242,or ESK246 treatment (Figure 5D,E). CompoundESK246 showed the highest level of inhibition of BrdU incorporation(4.9%; 17.2% of control), which was significantly lower than eitherBCH (14.7%; 51.6% of control) or ESK242 treatment (23.5%; 82.7% ofcontrol; Figure 5E). Our previous studies haveshown that reduced extracellular amino acid levels inhibit expression of the cell cycleregulator CDK1 and the ubiquitination enzyme UBE2C in prostate cancerand melanoma.^17,29 Therefore, we examined expressionof CDK1 and UBE2C in the presence of BCH, ESK242, and ESK246, showingexpression of both proteins was down regulated by ESK246 (SI Figure S6B).

Figure 5

Effects of ESK242 and ESK246 in LNCaPcells. (A) RepresentativeWestern blots (from n = 3) of p70S6K phosphorylationafter BCH (10 mM), ESK242 (50 μM), and ESK246 (50 μM)inhibition. GAPDH was used as the loading control. (B) MTT cell viabilityassay (n = 3) in LNCaP cells incubated with BCH (10mM), ESK242 (50 μM) and ESK246 (50 μM). Two-way ANOVAtest was performed, **P < 0.01, ***P < 0.001. (C) Analysis of apoptosis (n = 3) inLNCaP cells using Annexin-V and PI staining after inhibition withBCH (10 mM), ESK242 (50 μM), and ESK246 (50 μM). (D,E)Analysis of cell proliferation using BrdU incorporation in LNCaP cellsinhibited with BCH (10 mM), ESK242 (50 μM) and ESK246 (50 μM).Representative flow cytometry analysis (D) and quantification (E)from 3 separate experiments are shown. One-way ANOVA test was performed,*P < 0.05, **P < 0.01, ***P < 0.001.

Conclusions

We have utilized a function-based drugdiscovery platform that enabled high-throughput screening of LAT3-specificnatural products from the Nature Bank fraction library. Using thisplatform, we identified two novel monoterpene glycosides, ESK242,a dual LAT1 and LAT3 inhibitor, and ESK246, which is more selectivefor inhibition of LAT3 in both oocytes and LNCaP cells. Notably, ESK246,inhibits LAT-mediated leucine transport in LNCaP cells with a ∼500-foldlower IC₅₀ compared to the leucine analogue BCH. We anticipatethat derivatives of these inhibitors could be used as prostate cancertherapeutics as well as to investigate the biological function ofLAT1 and LAT3 during development and in disease.

General Chemical Experimental Procedures

UV spectrawere recorded on a Jasco V650 UV/vis spectrophotometer. NMR spectrawere recorded at 30 °C on either a Varian 500 or 600 MHz UnityINOVA spectrometer. The latter spectrometer was equipped with a tripleresonance cold probe. The ¹H and ¹³C NMR chemicalshifts were referenced to the solvent peaks for benzene-d₆ at δ_H 7.20 and δ_C 128.0,respectively, and for DMSO-d₆ at δ_H 2.50 and δ_C 39.43, respectively. HRESIMSwere recorded on a Bruker Daltronics Apex III 4.7e Fourier-transformmass spectrometer. Silica gel chromatography was performed using Mercksilica gel 60 (0.015–0.040 mm). A Waters 600 pump equippedwith a Waters 996 PDA detector and a Waters 717 autosampler were usedfor HPLC. A Phenomenex Onyx monolythic column [100 × 10 mm] wasused for semipreparative HPLC separations. All solvents used for chromatography,UV, and MS were Lab-Scan HPLC grade (RCI Lab-Scan, Bangkok, Thailand),and the H₂O was Millipore Milli-Q PF filtered. All startingmaterials and reagents for the synthesis were obtained from commercialsuppliers, Sigma-Aldrich, Carbosynth, Alfa Aesar, Merck Millipore,and used without further purification. High-performance liquid chromatography(HPLC) grade solvents were obtained from Labscan, and purified usinga PureSolv MD 5 solvent purification system from Innovative Technology.Reactions were performed in flame-dried glassware under positive N₂ pressure, with magnetic stirring. Rubber septa and syringeswere used for liquid transfers. Thin layer chromatography (TLC) wasperformed on 0.25 mm Merck silica gel 60 F254 plates and plates visualizedby staining with Seebach’s stain; phosphomolybdic acid/cerium(IV)sulfate 2.5:1 in H₂O/H₂SO₄ 94:6 (mL).

Plant Material

Pittosporum venulosum (F.Muell) (Nature Bank code 11711.8) was collected in July 1995from State Forest 144, Mt. Windsor Tableland, Queensland, Australia.The plant was identified by P.I. Forster and S.J. Figg. A voucherspecimen (PIF17245) has been lodged with the Queensland Herbarium.

Isolation of ESK246 and ESK242

Pittosporumvenulosum (10 g) was dried, ground, and sequentially extractedin hexane (250 mL), CH₂Cl₂ (250 mL) and MeOH(2 × 250 mL). All extracts were combined and reduced under pressureto yield a dark green oil (1.62 g). The combined organic extract wasthen subjected to a solvent–solvent partition with the compoundsof interest concentrated in the hexane fraction. The hexane fraction(370 mg) was subjected to flash silica oxide chromatography (10 cm× 4 cm) eluting with a gradient from 100% hexane to 100% ethylacetate (EtOAc). Fraction eluting with 8:2 (hexane/EtOAc) yieldedESK242 (149 mg, 1.49% dry weight), while the fraction eluting with6:4 (hexane/EtOAc) yielded ESK246 (58 mg, 0.58% dry weight). The purityof ESK242 and ESK246 was confirmed by ¹H NMR and ¹³C NMR (SI Figures S7–S10) to be>95% (SI Table S2).

Cell Lines

Human prostate cancercell lines LNCaP-FGCand PC-3 were purchased from ATCC (Rockville, MD). LNCaP cells havebeen passaged directly from original low-passage stocks (2009), andwe confirmed PC-3 cell identity by STR profiling in 2010 (Cellbank).Cells were cultured in RPMI 1640 medium (Life Technologies) containing10% (v/v) fetal bovine serum (FBS), penicillin–streptomycinsolution (Sigma-Aldrich), and 1 mM sodium pyruvate (Life Technologies).Cells were maintained at 37 °C in a fully humidified atmospherecontaining 5% CO₂.

Leucine Uptake Assay

The [³H]-l-leucine uptake was performed asdetailed previously.¹⁶ Briefly, cells werecultured in 6-well platesin RPMI media. After collecting and counting, cells (3 × 10⁴/well) were incubated with 0.3 μCi [³H]-l-leucine (200 nM; PerkinElmer) in leucine-free RPMI media (LifeTechnologies) with 10% (v/v) dialyzed FBS for 15 min at 37 °C.For high-throughput screening of Nature Bank fractions, LNCaP cells(10⁴/well) were incubated with 0.3 μCi [³H]-l-leucine (200 nM) in HBSS with 10% (v/v) dialyzed FBSand 50 mM l-glutamine. Cells were directly added into 96-wellplates containing preloaded fractions from Nature Bank. DMSO 0.5%(v/v) was used as the negative control and 10 mM BCH was used as thepositive control. Cells were collected, transferred to filter paperusing a 96-well plate harvester (Wallac PerkinElmer), dried, exposedto scintillation fluid, and counts measured using a liquid scintillationcounter (PerkinElmer).

Cell Viability Assay

Cells in exponentialgrowth phasewere harvested and seeded (1 × 10⁴/well) in a flat-bottomed96-well plate. The cells were incubated overnight in RPMI media, priorto culture with or without each inhibitor. MTT solution (10 μL;3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide; Millipore)was added to each well for 4 h, prior to addition of 100 μLof isopropanol/HCl solution and mixed thoroughly. The plates wereimmediately read at 570 nm/630 nm in a PolarStar plate reader (BMG).Results were plotted as percentages of the absorbance observed incontrol wells (vehicle/DMSO).

Oocyte Uptake Assay

Human LAT1, LAT2, LAT3, LAT4, and4F2hc cDNAs were subcloned into the oocyte transcription vector pOTV,respectively. The resulting transporter cDNAs were linearized with SpeI. Complementary RNA (cRNA) was transcribed with T7 RNApolymerase and capped with 5′-7-methylguanosine using the mMessagemMachine kit (Ambion). All cRNAs were purified using a NucAway SpinColumn (Ambion).

Stage V oocytes were harvested from Xenopus laevis as described previously,³⁰ and all surgical procedures followed a protocol approvedunder the Australian Code of Practice for the Care and Use of Animalsfor Scientific Purposes. Oocytes were injected with 23 nL of cRNAmix containing 1:1 LAT1/4F2hc, LAT2/4F2hc, LAT3, or LAT4 cRNA, respectively(4.6 ng of cRNA in total), and incubated in standard frog Ringer’ssolution (ND96:96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mMCaCl₂, 5 mM HEPES, pH 7.5) supplemented with 50 μg/mLgentamycin, 2.5 mM sodium pyruvate, and 0.5 mM theophylline at 16–18°C. Four days after injection, 5 oocytes per group were incubatedin 500 μL of uptake solution (standard frog Ringer’ssolution, ND96) containing 500 nM [³H]-l-leucinein a final concentration of 1 mM l-leucine for 30 min atRT. The LAT inhibitor BCH was used at 10 mM and ESK242 and ESK246were used at various concentrations, as indicated in the text. Uptake wasterminated by three rapid washes in ice-cold ND96 followed by lysisin 50 mM NaOH and 50% SDS. [³H]-l-leucine uptakewas measured by scintillation counting using a Trilux β-counter(PerkinElmer Life Science).

Biotinylation Assay

Surface proteinson Xenopuslaevis oocytes were isolated using the Cell Surface ProteinIsolation Kit (Thermo Scientific Pierce). The oocytes were incubatedfor 30 min at 4 °C on an orbital shaker with Sulfo-NHS-SS-biotin.After three washes with PBS, the oocytes were sonicated in 500 μLof Lysis buffer containing protease inhibitors. The lysate was incubatedon ice for 30 min and spun for 2 min at 10 000g. The labeled proteins were isolated from the supernatant using NeutrAvidinagarose and eluted with SDS-PAGE sample buffer containing 50 mM DTT.The eluted proteins were then analyzed by Western blotting as detailedbelow.

BrdU Incorporation Assay

Cells were seeded at a densityof 2 × 10⁵ in 6-well plates and allowed to adhereovernight. After serum starvation, cells were incubated with eitherDMSO, 10 mM BCH, 50 μM ESK242, or 50 μM ESK246 for 22h. At the end of the treatment, BrdU (150 μg/mL) was added toculture media and incubated for another 2 h, followed by detachmentusing Tryple (Life Technologies). Cells were fixed and stained usingthe Becton Dickinson APC BrdU flow cytometry kit (BD). The BrdU antibodywas diluted 1 in 50. Nuclei were counterstained by 7-AAD (7-aminoactinomycin).The cells were analyzed on a Canto II flow cytometer (BD) withpostanalysis performed using FlowJo software (Tree Star Inc.).

ApoptosisAssay

Cells were seeded at a density of 2× 10⁵ in 6-well plates, allowed to adhere overnight,before incubation with DMSO, 10 mM BCH, 50 μM ESK242, or 50μM ESK246 for 48 h, respectively. Positive control group cellswere irradiated in a UV Stratalinker 2400 (Stratagene) with a 400 000μJ dosage and incubated in fresh media for 16 h. Cells weredetached using Tryple and resuspended in 1 mL of binding buffer (HEPES-bufferedPBS supplemented with 2.5 mM calcium chloride) containing antiannexinV-APC (BD) and incubated for 15 min in the dark at RT. PI solution(20 μg/mL) was added, and the cells were analyzed on a CantoII flow cytometer (BD) with postanalysis performed using FlowJo software.

Western Blots

Cells were seeded at a density of 2 ×10⁵ in 6-well plates, allowed to adhere overnight, beforeincubation with DMSO, 10 mM BCH, 50 μM ESK242, or 50 μMESK246 for 6 h or 3 d. Cells were lysed by the addition of lysis buffer(200 μL) with protease inhibitor Cocktail III (BioprocessingBiochemical) and 1 mM Na₃VO₄ (Sigma). Equalprotein (micro-BCA method; Pierce, IL) was loaded on 4–12%gradient gels (Life Technologies), electrophoresed, and transferredto PVDF membrane. The membrane was blocked with 2.5% (w/v) BSA inPBS-Tween20, and incubated with the primary and secondary antibodies.The secondary HRP-labeled antibodies were detected using enhancedchemiluminescence reagents (Pierce) on a Kodak Imager (Kodak). Antibodiesused in this study were against LAT1 (Cosmo Bio), LAT3 (a kind giftfrom Kunimasa Yan, Kyorin University, Tokyo, Japan), α-tubulin(Santa Cruz), p-p70S6K, p70S6K, (Cell Signaling), UBE2C (Boston Biochem),CDK1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam).Horseradish peroxidase-conjugated donkey antimouse IgG, donkey antirabbitIgG, and goat antimouse IgM were used as secondary antibodies (Millipore).

Statistical Analysis

Data are expressed as mean ±SEM. Experiments were performed in triplicate, except where notedin the Figure Legend. All data were analyzed using a one-way ANOVAtest, apart from MTT assays that used a two-way ANOVA test in GraphPadPrism v6 (GraphPad Software, Inc.).