Sphingosine kinase activity confers resistance to apoptosis by fumonisin B1 in human embryonic kidney (HEK-293) cells

Chemico-Biological Interactions 151 (2004) 33–42

Sphingosine kinase activity confers resistance to apoptosis byfumonisin B1 in human embryonic kidney (HEK-293) cells

Neelesh Sharma, Quanren He, Raghubir P. Sharma∗

Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7389, USA

Received 23 September 2004; received in revised form 22 October 2004; accepted 23 October 2004

Abstract

Fumonisin B1 induces cytotoxicity in sensitive cells by inhibiting ceramide synthase due to its structural similarity to thelong-chain backbones of sphingolipids. The resulting accumulation of sphingoid bases has been established as a mechanism forfumonisin B1 cytotoxicity. We found that despite the accumulation of sphinganine, human embryonic kidney (HEK-293) cellsare resistant to fumonisin B1 toxicity; 25�M fumonisin B1 exposure for 48 h did not increase apoptosis in these cells, whileit did so in sensitive porcine kidney epithelial (LLC-PK1) cells. In this study,dl-threo-dihydrosphingosine, the sphingosinekinase inhibitor (SKI), considerably increased the sensitivity of HEK-293 cells to fumonisin B1. Treatment of these cells with25�M fumonisin B1 and 2.5�M SKI increased apoptosis. Sphingoid bases, sphinganine or sphingosine, added to cell culturesinduced apoptosis by themselves and their effects were potentiated by SKI or fumonisin B1. Addition of physiological amountso atH erties.T in renderingt©

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f sphingosine-1-phosphate prevented the toxic effects induced by SKI inhibition and fumonisin B1. Results indicated thEK-293 cells are resistant to fumonisin B1 due to rapid formation of sphingosine-1-phosphate that imparts survival propaken together, these findings suggest that sphingoid base metabolism by sphingosine kinase may be a critical eventhe HEK-293 cells relatively resistant to fumonisin B1-induced apoptosis.

2004 Elsevier Ireland Ltd. All rights reserved.

eywords:Fumonisin B1; Sphingosine-1-phosphate; Human embryonic kidney cells; Sphingosine kinase

. Introduction

Fumonisins are structurally related toxic and car-inogenic mycotoxins produced byFusarium verticil-ioides, a common fungal contaminant of maize. Fu-

∗ Corresponding author. Tel.: +1 706 542 2788;ax: +1 706 542 3015.

E-mail address:[email protected] (R.P. Sharma).

monisin B1 is the most abundant fumonisin and occnaturally in contaminated foods and feeds[1]. Fumon-isin B1 toxicity is specific to tissue and also dependspecies and gender of the treated animal. Fumonis1causes equine leukoencephalomalacia and porcinmonary edema[2,3]. In vivo studies demonstrated thfumonisin B1-induced apoptosis in kidney and liverrodents[4–6]. It was hepatocarcinogenic in male BIX rats [7] and a nephrocarcinogen in male F34

009-2797/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2004.10.003

34 N. Sharma et al. / Chemico-Biological Interactions 151 (2004) 33–42

rats, whereas it was hepatocarcinogenic in only femaleB6C3F1 mice[8,9]. An association between humanesophageal cancer and fumonisin B1 in areas highlycontaminated with these mycotoxins was reported[10].

Fumonisins are structurally related to sphingoidbases and cause inhibition of ceramide synthase(sphinganine- or sphingosine-N-acyltransferase) lead-ing to accumulation of corresponding free sphingoidbases, sphingoid base metabolites, and depletion ofmore complex sphingolipids[11,12]. Physiologicalconsequences possible after ceramide synthase inhi-bition include apoptosis caused by accumulation offree sphingoid bases (sphinganine and sphingosine), in-creased proliferation caused by increased sphingosine-1-phosphate, or/and decreased ceramide and alteredlipid raft function due to disruption of complex sph-ingolipids synthesis/transport[12].

It has been shown that fumonisin B1-induced apop-tosis, necrosis, and inhibition of proliferation in pigrenal epithelial (LLC-PK1) cells [13], human coloniccells HT29[14] and human keratinocytes[15] dependon sphingoid base accumulation. However, other stud-ies demonstrated that neoplastic African green mon-key kidney cells (COS-7)[16] and primary hepatocytes[17] were resistant to the toxic effects of fumonisin B1despite sphinganine accumulation.

Ceramide and sphingosine are interconvertable sph-ingolipid messengers; intensive investigations in pastdecade have confirmed the role of ceramide and sph-ingosine in inducing apoptosis. However, there is con-s osineiB edi-a pto-s ateswHf e ors ouldi on-v

d sig-n eink andh einB inf ter-i ased

on the observation that Bcl-2 can protect p53−/− cellsfrom fumonisin B1-induced apoptosis[26].

Levels of free sphingoid bases decrease con-sistently in fumonisin B1-treated cells between 48and 72 h indicating the induction of sphingoid basemetabolism [19]. Catabolism of sphingoid basesrequires two independent and sequential steps. Thefirst step involves phosphorylation of sphingoid basesto their 1-phosphate derivatives and is mediated bysphingosine kinase[27]. The second step involvescleavage of the resulting product by sphingosine-1-phosphate lyase into ethanolamine phosphate andcorresponding aldehyde[17]. Due to relatively lowlevels of sphingosine-1-phosphate in cells, it is evidentthat phosphorylation of sphingosine by sphingosinekinase is the rate-limiting step in this process.

Sphingosine kinase in human tissues is widelyexpressed with highest levels in adult lung, spleen,kidney, heart, and brain[28]. Activation of sphingosinekinase and formation of sphingosine-1-phosphate arelinked to cell growth and survival[29–31]. Diverse ex-ternal stimuli, particularly growth and survival factorsstimulate sphingosine kinase and intracellularly gen-erated sphingosine-1-phosphate has been implicatedin their mitogenic and anti-apoptotic effects[32,33].

In our preliminary experiments, we found that hu-man embryonic kidney (HEK-293) cells were resis-tant to cytotoxic effects of up to 50�M fumonisin B1whereas porcine kidney (LLC-PK1) cells show signifi-cant toxic response with 10�M fumonisin B1 [13,19].W sis-tc sinet nasea 1-p steda ofs phin-gD e ki-n

2

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iderable debate whether the ceramide or sphings the primary inducer of apoptosis[18]. In fumonisin

1 sensitive cells, the same final pathway may mte apoptosis by which sphingoid bases affect apois; accumulation of sphingoid bases directly correlith their cytotoxicity[19]. In HL-60 cells[20,21]andep3B hepatoma cells[22], fumonisin B1 did not af-

ect the apoptosis directly induced by sphingosinphinganine indicating that these sphingoid bases cndependently produce apoptosis without getting certed to ceramide.

Recent data suggest that sphingoid bases coulal mitochondrial apoptosis by inhibiting the protinase Akt, responsible for Bad phosphorylationence leading to inhibition of anti-apoptotic protcl-2 by Bad[23–25]. Events leading to apoptosis

umonisin B1-treated cells have not been characzed, although a role for Bax has been suggested b

e hypothesized that the HEK-293 cells are reant to apoptotic effects of fumonisin B1 due to rapidonversion of accumulated sphinganine or sphingoo their respective phosphates by sphingosine kind the antiapoptotic and proliferative role of thehosphate derivatives. In the present study, we tebove hypothesis by employing specific inhibitorphingosine kinase, the enzyme that converts soid bases to their respective phosphates.dl-threo-ihydrosphingosine was employed as sphingosinase inhibitor (SKI).

. Materials and methods

.1. Materials

Fumonisin B1 (purity > 95%) was obtained frorogramme on Mycotoxin and Experimental Carc

N. Sharma et al. / Chemico-Biological Interactions 151 (2004) 33–42 35

genesis (Tygerberg, South Africa). Human EmbryonicKidney cells (HEK-293) were obtained from Ameri-can type culture collection (CRL-1573, ATCC, Man-assas, VA). Dulbecco’s Modified Eagle’s Medium waspurchased from Gibco (Carlsbad, CA). Sphingolipidsanddl-threo-dihydrosphingosine were procured fromBiomol (Plymouth Meeting, PA). Annexin V and pro-pidium iodide were purchased from Molecular Probes(Eugene, OR). All other reagents were obtained fromSigma (St. Louis, MO) and were of tissue culturegrade.

2.2. Preparations of fumonisin B1 andsphingolipid stock solutions

Fumonisin B1 was dissolved in phosphate-bufferedsaline (PBS) at 1 mM, and then diluted into growthmedium and added to cultures to achieve proper fi-nal concentrations. Relatively high concentration offumonisin B1 was used, as the HEK-293 cells are notresponsive to it up to 50�M concentration. Control cul-tures were treated with similar dilutions of the vehiclebut without fumonisin B1. Free sphinganine, free sph-ingosine, anddl-threo-dihydrosphingosine were firstdissolved in ethanol to a concentration of 50 mM andthen were prepared in 1.0 ml of 1.5 mM fatty acid–freebovine serum albumin (BSA) to get a concentration of1 mM, as previously described[19]. An equal volumeof absolute ethanol was added into BSA and processedas above as a BSA vehicle control.

2

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2.4. Hoechst 33258 and propidium iodide staining

Apoptotic changes in the nuclear chromatin of cellswere evaluated by staining with the DNA binding flu-orochrome Hoechst 33258 (bis-benzimide). Cell deathwas also analysed using propidium iodide (PI), a mem-brane impermeable dye, which binds to DNA by in-tercalating between the bases with little or no se-quence preference. At the end of treatment, super-natant was removed and 50�l of 30% glycerol/PBSsolution of Hoechst 33258 (8�g/ml) or PI (1�g/ml)was added. The plates were read at 346 nm/460 nmor 535 nm/617 nm (excitation/emission) for Hoechst33258 or PI fluorescence, respectively, using a Spec-tramax Gemini fluorescent plate reader (Molecular De-vices, Irvine, CA).

2.5. Mitochondrial function assay

3[4,5-Dimethyl thiazolyl-2]2,5-diphenyl tetra-zolium bromide (MTT) was employed to observe cellviability. Cells were seeded at 3× 104 cells/well in96-well plates and treated with the indicated concen-tration of treatments in total volume of 200�l. Cellswere incubated with addition of 20�l MTT (5 mg/ml)4 h before the end of treatment. At the end of treatment,120�l of media containing MTT was taken out fromeach well and 100�l of 0.02N HCl–isopropanol(warm) was added to dissolve formazan crystals.The absorbance of each well was measured usinga ent,I

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.3. Cell culture and treatment

HEK-293 cells were grown in Dulbecco’s Modifiagle’s Medium containing 100 U/ml penicillin a0% non-heat-inactivated fetal bovine serum (Atlaiologics, Atlanta, GA) in 5% CO2 at 37◦C. Cells wererown in 75 cm2 culture flasks and sub cultured wh

he cells reached 70–80% confluence. For all exents, cells were seeded in the CD 293 chemically

ned media (Gibco, Carlsbad, CA), which was devf any active biolipids, containing 0.5% fetal boverum, 2 mM glutamine and 5�g/ml insulin (Sigmat. Louis, MO). Cells were seeded 18 h before trent with fumonisin B1, sphingosine, sphinganineKI in their second or third passages. Concentraf each chemical and time point for the treatments wptimized in preliminary trials.

scanning spectrophotometer (Bio-Tek InstrumNC, Winooski, VT, USA).

.6. Determination of intracellular free sphingoidases

At the end of 48 h treatment with fumonisin B1, theedium was aspirated, and cells were washedith ice-cold PBS and then scraped into 1 mlold PBS. An aliquot (0.1 ml) of cell suspensionBS was transferred to another tube, spun at 200×gnd 4◦C for 5 min. The cells were lysed and stot −85◦C until analysis of total protein by Bradfoeagent (Bio-Rad Laboratories, Hercules, CA) usi6-well plate according to the manufacturer’s protoree sphingoid bases were extracted from the remer of cells by using the modified method descrireviously[34]. The relative amounts of free sphing


nine and sphingosine in base-treated cell extracts weredetermined by high performance liquid chromatogra-phy (HPLC), as described earlier. Sphingoid bases werequantified based on the recovery of a C20 sphinganineinternal standard. The limit of detection for C20 was26.8 fmol/assay (equivalent to 1 fmol/�g protein).

2.7. Statistical analysis of data

Each experiment was repeated three times withreproducible results consistently. The results are ex-pressed as mean± standard error of combined resultsfrom three independent experiments with each per-formed in triplicates. Differences among treatmentswere analyzed statistically by one-way analysis of vari-ance (ANOVA) followed by Duncan’s multiple rangetest. The level ofp≤ 0.05 was considered significantfor all comparisons.

3. Results

3.1. Fumonisin B1 and SKI decreased the viabilityof HEK-293 cells when used together

HEK-293 cells are resistant to cytotoxic effects offumonisin B1 considering that even 50�M fumonisinB1 for 72 h could not increase significant LDH releasefrom these cells (data not shown). Fumonisin B1 at25�M and SKI at 2.5�M alone had no effect on via-b if-i herw(

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Fig. 1. HEK-293 cells were treated with 25�M fumonisin B1 and2.5�M SKI for 72 h. Cell viability was determined by MTT assay.Fumonisin B1 and SKI alone were not toxic to HEK-293 cells buttogether they decreased cell viability significantly. Similar findingswere noted in three independent experiments. The results expressedas mean± S.E. are combined results from three experiments witheach experiment performed in triplicates. Asterisk (*) indicates sig-nificantly different value atp< 0.05 from the control.

Necrotic cell death was confirmed by nuclear stain-ing with membrane impermeant dye propidium iodide,which is taken up only by necrotic cells as their cellmembrane is damaged. To confirm apoptosis, cells

Fig. 2. HEK-293 cells are resistant to apoptotic effects of fumonisinB1. Exposure to 25�M fumonisin B1 alone for 48 h did not induceapoptosis in HEK-293 cells whereas, treatment with SKI and fu-monisin B1 together for 48 h induced apoptosis. Induction of apop-tosis measured by Hoechst 33258 fluorescence at 346 nm/460 nm.Mean± S.E.,n= 9; asterisk (*) indicates significantly different valueat p< 0.05 from the fumonisin B1 only treated group; (¶) indicatessignificantly different value atp< 0.05 from the control group (nottreated with either fumonisin B1 or SKI).

ility of HEK-293 cells. Cell viability decreased signcantly in HEK-293 cell culture, when treated togetith 25�M fumonisin B1 and 2.5�M SKI for 72 h

Fig. 1), determined by MTT assay.

.2. Induction of apoptosis in HEK-293 cellsreated with fumonisin B1 and SKI

Exposure to 25�M fumonisin B1 alone for 48 h didot induce apoptosis in HEK-293 cells, however, trent with 25�M fumonisin B1 and 2.5�M SKI to-ether for 48 h resulted in increased apoptosis, mured by the fluorescence of Hoechst 33258 (Fig. 2).

Cell death in HEK-293 culture was a combinatf apoptosis and necrosis. Cells treated with fumon1 and SKI together had∼20% apoptotic cells an8% necrotic cells, while in control wells less th% cells were undergoing either apoptosis or necr


Fig. 3. (a) HEK-293 cells were treated with 25�M fumonisin B1 and 2.5�M SKI for 48 h. Annexin V and Hoechst 33258 staining were used todetect apoptosis. Cells treated with fumonisin B1 and SKI together had significantly greater number of cells stained with Annexin V and Hoechst33258 compared to control, fumonisin B1 and SKI alone. (b) Overlay of few propidium iodide stained necrotic cells, and Hoechst 33258 andAnnexin V staine for apoptotic cells, was used to differentiate between necrosis and apoptosis. Similar findings were noted in three independentexperiments.

were stained with Annexin V and Hoechst 33258 dye.Early apoptotic cells stain with Annexin V and Hoechst33258, which can bind to exposed phosphotidyl ser-ine and fragmented nuclear chromatin, respectively,but exclude PI as their cell membrane is still intact.Cells treated with fumonisin B1 and SKI together hadsignificantly greater number of cells stained with An-nexin V and Hoechst 33258 compared to control andSKI alone (Fig. 3a). Visual inspection under Olym-pus IX71 inverted fluorescence microscope (OlympusAmerica, Melville, NY) confirmed the presence of in-creased number of cells exhibiting bright green stain-ing of Annexin V on cell surface and blue staining ofHoechst 33258 on nucleus indicating apoptotic cells.

Overlay of Annexin V, Hoechst 33258 and PI stain-ing in fumonisin B1 and SKI treated cells showed pre-dominance of apoptotoic cells stained with only An-nexin V and Hoechst 33258 and presence of somenecrotic cells which were stained by all three dyes (PI,Annexin V and Hoechst 33258) (Fig. 3b).

3.3. Increased accumulation of intracellularsphingoid bases in cells treated with fumonisin B1

and SKI

HEK-293 cells were treated with 25�M fumonisinB1 and 2.5�M SKI for 48 h. Treatment of cells to-gether with SKI and fumonisin B1 caused higher ele-vation of intracellular level of sphinganine compared tofumonisin B1 treatment alone. Fumonisin B1 alone didnot increase intracellular level of sphingosine, whereasexposure of cells to fumonisin B1 and SKI togethercaused significantly higher accumulation of sphingo-sine (Fig. 4).

3.4. Fumonisin B1 and SKI-induced apoptosis wasfurther increased by sphingoid bases

Sphingoid bases are known to induce apoptosis. Wetreated HEK-293 cells with 5�M sphinganine or 5�Msphingosine alone or in the presence of 25�M fumon-


Fig. 4. HEK-293 cells were treated with 25�M fumonisin B1 and 2.5�M SKI for 48 h. Treatment of cells together with SKI and fumonisin B1

caused higher elevation of intracellular level of sphinganine compared to fumonisin B1 treatment alone. Fumonisin B1 alone did not increaseintracellular level of sphingosine, whereas exposure of cells to fumonisin B1 and SKI together caused significantly higher accumulation ofsphingosine. Mean± S.E.,n= 9; asterisk (*) indicates significantly different value atp< 0.05 from the fumonisin B1 only treated group; (¶)indicates significantly different value atp< 0.05 from the control group.

isin B1 and 2.5�M SKI for 48 h. Apoptosis was mea-sured by recording the fluorescence of Hoechst 33258at 346 nm/460 nm. Sphingosine, when added to cellcultures induced apoptosis by itself. Fumonisin B1 andsphingosine treated cells had more apoptotic cells com-pared to fumonisin B1 treatment alone. Sphingosineincreased the apoptosis induced by the combined treat-ment of fumonisin B1 and SKI (Fig. 5). Sphinganinetreatment followed the similar trend and had the syn-ergistic effects with SKI and fumonisin B1 to induceapoptosis except that at 5�M concentration sphinga-nine itself could not induce apoptosis (Fig. 6).

3.5. Sphingosine-1-phosphate prevented theapoptosis induced by fumonisin B1 and SKI

HEK-293 cells were treated with 25�M fumon-isin B1, 2.5�M SKI and 1, 5 and 10 nM concen-trations of sphingosine-1-phosphate for 48 h (Fig. 7).Sphingosine-1-phosphate significantly reduced theapoptosis induced by fumonisin B1 and SKI. Apop-tosis was measured by Hoechst 33258 fluorescenceat 346 nm/460 nm. A similar trend was shown whensphinganine-1-phosphate (1–100 nM) was used in-stead of sphingosine-1-phosphate, however, the protec-tion was not statistically significant (data not shown).Direct addition of either sphingosine-1-phosphateor sphinganine-1-phosphate in concentrations greaterthan 100 nM was cytotoxic in this cell line.

Fig. 5. HEK-293 cells were treated with 25�M fumonisin B1,2.5�M SKI and 5�M sphingosine (So) for 48 h. Sphingosine whenadded to cell cultures induced apoptosis by itself and its effect was po-tentiated by sphingosine kinase inhibitor and fumonisin B1. Apopto-sis was measured by Hoechst 33258 fluorescence at 346 nm/460 nm.Mean± S.E., n= 9; (¶) indicates significantly different value atp< 0.05 from the fumonisin B1-treated group; (#) indicates signif-icantly different value atp< 0.05 from the group treated with sph-ingosine only; asterisk (*) indicates significantly different value atp< 0.05 from the group treated with fumonisin B1 and SKI together.

4. Discussion

We found that HEK-293 cells are resistant to fu-monisin B1-induced apoptosis. Results in the currentstudy showed that inhibition of sphingosine kinase en-zyme that converts sphingoid bases to their respective1-phosphates makes HEK-293 cells sensitive to fumon-


Fig. 6. HEK-293 cells were treated with 25�M fumonisin B1,2.5�M SKI and 5�M sphinganine (Sa) for 48 h. Sphinganine alsohad synergistic effect with SKI and fumonisin B1 in making HEK-293 cells more sensitive to fumonisin B1 apoptosis. Apoptosiswas measured by Hoechst 33258 fluorescence at 346 nm/460 nm.Mean± S.E., n= 9; (¶) indicates significantly different value atp< 0.05 from the fumonisin B1 treated group; asterisk (*) indicatessignificantly different value atp< 0.05 from the group treated withfumonisin B1 and SKI together.

isin B1-induced apoptosis. In HEK-293 cells, the rapidformation of sphingoid base-1-phosphate is the puta-tive mechanism of protection from fumonisin B1 tox-icity. In most cell lines, fumonisin B1 blocks de novoceramide biosynthesis and causes accumulation of freesphingoid bases but only selected cell lines are sensitiveto toxic effects of fumonisin B1, while others are resis-

Fig. 7. HEK-293 cells were treated with 25�M fumonisin B1,2.5�M SKI and 1, 5 and 10 nM sphingosine-1-phosphate (S1P) for48 h. Sphingosine-1-phosphate prevented the apoptosis induced byfumonisin B1 and SKI together. Apoptosis was measured by Hoechst33258 fluorescence at 346 nm/460 nm. Mean± S.E.,n= 9; (¶) indi-cates significantly different value atp< 0.05 from the fumonisin B1-treated group; asterisk (*) indicates significantly different value atp< 0.05 from the group treated with fumonisin B1 and SKI together.

tant[16,17]. Accumulation of sphingoid bases has beenestablished as a mechanism for fumonisin B1-inducedapoptosis in various types of cells.

For fumonisin B1, the IC50 for inhibition of ce-ramide synthase is 0.1�M in primary hepatocytes anddoses as low as 1�M caused almost complete inhibi-tion of this enzyme leading to accumulation of sphin-goid bases as quickly as in 3 h[11]. Elevation of sph-inganine level by almost 20-fold in 24 h treatment withfumonisin B1 is a common finding with most of thecell lines. Sphingoid bases are toxic to cells[18] andare responsible for fumonisin B1 cytotoxicity [13]. In-hibition of fumonisin B1 cytotoxicity by myriocin (in-hibits serine palmitoyltransferase activity) correlatedwell with this conclusion[14]. Since level of sphin-gosine depends on the rate of turnover of the complexsphingolipids it either does not change or increases lit-tle after fumonisin B1 treatment[11].

It has been a question as why increased levels ofsphinganine do not cause toxicity in some cells. Eventhough sphinganine levels rise significantly withinhours with as low as 1�M fumonisin B1 treatment insensitive and insensitive cells, high doses of fumonisinB1 and long incubation periods (>24 h) are requiredto produce toxicity even in sensitive cells. Levels offree sphingoid bases decreased consistently in fumon-isin B1-treated cells between 48 and 72 h, which in-dicated temporal sphingoid base metabolism[19]. InJ774A.1 cells, exogenous sphingoid bases initially ac-cumulated and then were rapidly metabolized to their1 -t emmo inei asec

ndsu ands ands tiona ands toa torsl se,l vels,w eo s re-p ere

-phosphate derivatives; fumonisin B1 caused inhibiion of acylation of sphingoid bases and diverted thore towards phosphorylation[19]. About one-thirdf the ethanolamine within phoshatidylethanolam

n J774A.1 cells was derived from long-chain batabolism when fumonisin B1 was added[35].

The role of sphingoid bases in cell death depepon the balance between levels of ceramidephingosine both of which mediate apoptosis,phingosine-1-phosphate that promotes proliferand survival. For instance, increases in ceramidephingosine levels by TNF� and Fas ligand leadpoptosis of T lymphocytes, whereas survival fac

ike protein kinase C stimulate sphingosine kinaeading to increased sphingosine-1-phosphate lehich suppress apoptosis[36]. Recently, the balancf sphingosine-1-phosphate and sphingosine waorted to have a role in activation of mast cells, wh


high intracellular concentration of sphingosine inhib-ited cytokine production by preventing activation ofextracellular signal-regulated kinase (ERK) pathway,while high intracellular concentration of sphingosine-1-phosphate activated ERK to stimulate cytokine pro-duction [37]. Study by Olivera et al.[38] empha-sizes the importance of sphingosine kinase in deter-mining cell fate as expression of sphingosine kinasein HEK-293 cells markedly increased production ofsphingosine-1-phosphate, which induced proliferation.

The threo enantiomers of sphingosine and dihy-drosphingosine were not phosphorylated by sphingo-sine kinase and were found to be potent competitive in-hibitors of platelet sphingosine kinase activity in vitro[39]. Substrate and inhibition studies showed that themixture ofdl-threo-dihydrosphingosine was more po-tent and highly specific inhibitor of platelet sphingosinekinase[39]. dl-threo-Dihydrosphingosine inhibits theproduction of sphingosine-1-phosphate by competitiveinhibition of sphingosine kinase and is useful to investi-gate the function of sphingosine-1-phosphate in signaltransduction processes.

In the current study, SKI increased sensitivity ofHEK-293 cells to fumonisin B1 toxicity as treatmentof these cells with fumonisin B1 and SKI together in-creased apoptosis, whereas fumonisin B1 alone or in-hibitor alone were not cytotoxic. Inhibition of sphingo-sine kinase bydl-threo-dihydrosphingosine preventedconversion of sphingoid bases to their 1-phosphatederivatives and resulted in higher accumulation ofsH tiono sso oxice hin-g cellc ectsw andf

tosisc dedte ine-1 igherc on-i en-t tedt nase

and degradation by sphingosine-1-phosphate lyase andphosphohydrolase. Recently, two different kinds of en-zymes with sphingosine-1-phosphate phosphohydro-lase (SPP) activity, SPP-1[40] and SPP-2[41] havebeen characterized in HEK-293 cells. These enzymesare known to degrade exogenously added sphingosine-1-phosphate in cell cultures to sphingosine and sub-sequently produce apoptosis[40]. It is possible thatin our study, higher concentrations of sphingosine-1-phosphate increased the apoptosis by getting convertedto sphingosine by sphingosine-1-phosphate phospho-hydrolase. It would be desirable to look at the effectof higher concentrations of sphingosine-1-phosphateafter inhibiting the sphingosine-1-phosphate phospho-hydrolase enzyme but the lack of specific inhibitor ofthis enzyme restricts such possibility.

In summary, our results indicate that HEK-293 cellsare resistant to apoptotic effects of fumonisin B1 due tohigher activity of sphingosine kinase, which enhancescell survival by forming sphingosine-1-phosphate. Ac-tivity of sphingosine kinase enzyme may have substan-tial bearing on different responses of fumonisin B1 inother cell types. It is also possible that fumonisin B1 orSKI sensitized cells to each other by some other mech-anisms. However, our findings are applicable to onlyHEK-293 cells and further investigations are requiredto determine whether or not this is a general mecha-nism for resistance to fumonisin B1 in other cells invitro and tissues in vivo.

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phingosine and sphinganine in fumonisin B1-treatedEK-293 cells. Increased apoptosis with co-incubaf SKI and fumonisin B1 can be explained by the lof protective role of sphingosine-1-phosphate and tffects of increased levels of sphingoid bases. Spoid bases (sphinganine or sphingosine) added toultures induced apoptosis by itself and their effere potentiated by sphingosine kinase inhibitor

umonisin B1, supporting our hypothesis.Sphingosine-1-phosphate prevented the apop

aused by sphingosine kinase inhibition, when ado cells treated with fumonisin B1 and SKI. How-ver, only very small concentrations of sphingos-phosphate were able to prevent apoptosis and honcentrations further increased the toxicity of fumsin B1 and SKI (data not shown). Intracellular concration of sphingosine-1-phosphate is tightly regulahrough its synthesis catalyzed by sphingosine ki

cknowledgements

Studies reported in this paper were supported iny USPHS grant ES09403 from the National Instif Environmental Health Sciences and the Centecademic Excellence in Toxicology at the Universf Georgia.

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Sphingosine kinase activity confers resistance to apoptosis by fumonisin B1 in human embryonic kidney (HEK-293) cells