C22:0- and C24:0-dihydroceramides Confer Mixed Cytotoxicity in T-Cell Acute Lymphoblastic Leukemia Cell Lines

Abstract

We previously reported that fenretinide (4-HPR) was cytotoxic to acute lymphoblastic leukemia (ALL) cell lines in vitro in association with increased levels of de novo synthesized dihydroceramides, the immediate precursors of ceramides. However, the cytotoxic potentials of native dihydroceramides have not been defined. Therefore, we determined the cytotoxic effects of increasing dihydroceramide levels via de novo synthesis in T-cell ALL cell lines and whether such cytotoxicity was dependent on an absolute increase in total dihydroceramide mass versus an increase of certain specific dihydroceramides. A novel method employing supplementation of individual fatty acids, sphinganine, and the dihydroceramide desaturase-1 (DES) inhibitor, GT-11, was used to increase de novo dihydroceramide synthesis and absolute levels of specific dihydroceramides and ceramides. Sphingolipidomic analyses of four T-cell ALL cell lines revealed strong positive correlations between cytotoxicity and levels of C22:0-dihydroceramide (ρ = 0.74-0.81, P ≤ 0.04) and C24:0-dihydroceramide (ρ = 0.84-0.90, P ≤ 0.004), but not between total or other individual dihydroceramides, ceramides, or sphingoid bases or phosphorylated derivatives. Selective increase of C22:0- and C24:0-dihydroceramide increased level and flux of autophagy marker, LC3B-II, and increased DNA fragmentation (TUNEL assay) in the absence of an increase of reactive oxygen species; pan-caspase inhibition blocked DNA fragmentation but not cell death. C22:0-fatty acid supplemented to 4-HPR treated cells further increased C22:0-dihydroceramide levels (P ≤ 0.001) and cytotoxicity (P ≤ 0.001). These data demonstrate that increases of specific dihydroceramides are cytotoxic to T-cell ALL cells by a caspase-independent, mixed cell death mechanism associated with increased autophagy and suggest that dihydroceramides may contribute to 4-HPR-induced cytotoxicity. The targeted increase of specific acyl chain dihydroceramides may constitute a novel anticancer approach.

Full text

C22:0- and C24:0-dihydroceramides Confer Mixed
Cytotoxicity in T-Cell Acute Lymphoblastic Leukemia Cell
Lines
Michael W. Holliday Jr.1, Stephen B. Cox2, Min H. Kang1,3, Barry J. Maurer1,4*
1 School of Medicine Cancer Center, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America, 2 Research and Testing
Laboratory, Lubbock, Texas, United States of America, 3 Departments of Cell Biology & Biochemistry and Pharmacology, Texas Tech University Health
Sciences Center, Lubbock, Texas, United States of America, 4 Departments of Cell Biology & Biochemistry, Pediatrics and Medicine, Texas Tech University
Health Sciences Center, Lubbock, Texas, United States of America
Abstract
We previously reported that fenretinide (4-HPR) was cytotoxic to acute lymphoblastic leukemia (ALL) cell lines in vitro
in association with increased levels of de novo synthesized dihydroceramides, the immediate precursors of
ceramides. However, the cytotoxic potentials of native dihydroceramides have not been defined. Therefore, we
determined the cytotoxic effects of increasing dihydroceramide levels via de novo synthesis in T-cell ALL cell lines
and whether such cytotoxicity was dependent on an absolute increase in total dihydroceramide mass versus an
increase of certain specific dihydroceramides. A novel method employing supplementation of individual fatty acids,
sphinganine, and the dihydroceramide desaturase-1 (DES) inhibitor, GT-11, was used to increase de novo
dihydroceramide synthesis and absolute levels of specific dihydroceramides and ceramides. Sphingolipidomic
analyses of four T-cell ALL cell lines revealed strong positive correlations between cytotoxicity and levels of C22:0-
dihydroceramide (ρ = 0.74–0.81, P ≤ 0.04) and C24:0-dihydroceramide (ρ = 0.84–0.90, P ≤ 0.004), but not between
total or other individual dihydroceramides, ceramides, or sphingoid bases or phosphorylated derivatives. Selective
increase of C22:0- and C24:0-dihydroceramide increased level and flux of autophagy marker, LC3B-II, and increased
DNA fragmentation (TUNEL assay) in the absence of an increase of reactive oxygen species; pan-caspase inhibition
blocked DNA fragmentation but not cell death. C22:0-fatty acid supplemented to 4-HPR treated cells further
increased C22:0-dihydroceramide levels (P ≤ 0.001) and cytotoxicity (P ≤ 0.001). These data demonstrate that
increases of specific dihydroceramides are cytotoxic to T-cell ALL cells by a caspase-independent, mixed cell death
mechanism associated with increased autophagy and suggest that dihydroceramides may contribute to 4-HPR-
induced cytotoxicity. The targeted increase of specific acyl chain dihydroceramides may constitute a novel anticancer
approach.
Citation: Holliday Jr. MW, Cox SB, Kang MH, Maurer BJ (2013) C22:0- and C24:0-dihydroceramides Confer Mixed Cytotoxicity in T-Cell Acute
Lymphoblastic Leukemia Cell Lines. PLoS ONE 8(9): e74768. doi:10.1371/journal.pone.0074768
Editor: Leah J Siskind, University of Louisville, United States of America
Received January 27, 2013; Accepted August 6, 2013; Published September 9, 2013
Copyright: © 2013 Holliday et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funded by the National Cancer Institute (Grant # R01 CA100895, cancer.gov) and the Tyler’s Team Leukemia Fund (teamtylerfoundation.com)
to BJM. Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: SBC is employed by Research and Testing Laboratory, LLC. A patent application by the Texas Tech University System (TTUS) is
pending, on which BJM is an Inventor. As TTUS has a revenue-sharing policy for faculty-inventors, should this patent be issued and commercialized, BJM
may share in patent-derived revenues. Patent application: Num. 20120121691 A1, dated May 17, 2012, entitled, " Method for Increasing the Production of
a Specific ACYL-Chain Dihydroceramide(s) for Improving the Effectiveness of Cancer Treatments." Assignee: Texas Tech University System. There are no
further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on
sharing data and materials, as detailed online in the guide for authors.
* E-mail: barry.maurer@ttuhsc.edu
Introduction
The synthetic retinoid N-(4-hydroxyphenyl)retinamide
(fenretinide, 4-HPR) has demonstrated cytotoxic activity in vitro
to cell lines of multiple cancer types, including T-cell acute
lymphoblastic leukemia (ALL) [1–4]. Mechanisms of action of 4-
HPR include increased reactive oxygen species (ROS) levels
in certain cancer cell lines [4–9]. 4-HPR also stimulated the de
novo sphingolipid pathway leading to a time- and dose-
dependent increase of dihydroceramides in multiple model
systems [9–15].
Dihydroceramides are the direct precursors of ceramides in
the mammalian de novo sphingolipid pathway (Figure 1). The
rate-limiting enzyme of the pathway, serine
palmitoyltransferase (SPT), regulates sphinganine synthesis.
The family of dihydroceramide synthases (CerS 1-6) acylate
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sphinganine with a fatty acyl chain to form a dihydroceramide,
with each CerS utilizing a preferred subset of fatty acyl-CoAs
whose acyl chains vary both in carbon length (14- to 30-) and
degree of saturation [16–18]. Carbons 4 and 5 of the
sphinganine backbone of the dihydroceramide are reduced by
dihydroceramide desaturase (DES1) to yield the corresponding
ceramide [19]. We previously reported that 4-HPR increased
the activities of serine palmitoyltransferase and
dihydroceramide synthase in a neuroblastoma cell line
resulting in an increased ‘ceramides’ fraction and that 4-HPR
increased ceramides coincident with cytotoxicity in a dose- and
time-dependent manner in acute lymphoblastic leukemia cell
lines [2,20]. Recent work with more advanced methodologies
has demonstrated that 4-HPR specifically increases
dihydroceramides due to concurrent inhibition of
dihydroceramide desaturase 1 (DES1) [13–15].
Extensive literature supports that intracellular ceramides
have death-signaling properties, but such studies have rarely
distinguished the relative activity of individual ceramide species
[21,22]. In contrast, there is much less data on the bioactive
properties of dihydroceramides, the saturated precursors of
ceramides. Such investigations have relied mainly on the use
of exogenous, synthetic, cell penetrant, very short saturated
acyl chain (C2:0 – C8:0) dihydroceramides [23–27], although
several more recent reports have reported the possible
involvement of native acyl chain dihydroceramides in cell death
processes [28–33]. Given the observed association between
increased dihydroceramides and 4-HPR-induced cytotoxicity,
we hypothesized that the cytotoxic activities of artificial very
short-acyl chain dihydroceramides are not representative of
native acyl chain dihydroceramides, and that the cytotoxic
potential of dihydroceramides is acyl chain length and/or
saturation dependent.
The difficulty in directly assessing the cytotoxic potentials of
native acyl chain dihydroceramides over-induced by
pharmacological agents (i.e. ‘ceramide-stress’) arises from the
technical challenge of exogenously delivering such large
amphipathic sphingolipids into cells. Further, the approach of
increasing native dihydroceramides through overexpression of
the various ceramide synthases is limited by the intracellular
availability of precursor substrate, sphinganine, and the use of
multiple fatty acyl-CoA’s by any given ceramide synthase
family member (Figure 1). Therefore, an aim of the current
study was to develop a biochemical system to mimic
pharmacologically-induced ceramide stress (i.e., selectively
increase the levels of native acyl chain dihydroceramides and
ceramides via de novo synthesis). To achieve this, cells were
exogenously supplemented with a minimally-cytotoxic
concentration of sphinganine to increase the de novo synthesis
of dihydroceramides, and with GT-11, a competitive inhibitor of
DES1, to decrease the conversion of the resulting
dihydroceramides to their corresponding ceramides, thus
broadly mimicking the dihydroceramide-increasing effects of 4-
HPR [34]. α-Cyclodextrin was then employed as a water
soluble carrier to deliver selected fatty acids to sphinganine ±
GT-11 treated cells to increase the synthesis of the
corresponding acyl chain dihydroceramide [35]. We also
sought to distinguish whether dihydroceramide cytotoxicity was
associated with an increase in the total mass of
dihydroceramides, irrespective of acyl chain composition, or
with an increase in levels of specific acyl chain
dihydroceramides. Cell death was characterized by measures
of autophagy, apoptosis, caspase dependency and ROS
levels. We also determined how the levels of dihydroceramides
in 4-HPR-treated cells were affected by supplementation with
specific fatty acids and the effects of such supplementation on
4-HPR-induced cytotoxicity.
Figure 1. Schematic of the de novo ceramide
pathway. Rate-limiting enzyme, serine palmitoyltransferase
(SPT), condenses serine and palmitoyl-CoA to 3-
ketosphinganine, which is subsequently reduced to
sphinganine. Dihydroceramide synthases 1-6 (CerS 1-6), each
utilizing a preferred subset of fatty acid-derived acyl-CoAs, add
a fatty acyl chain (green) to sphinganine to produce
dihydroceramides. Dihydroceramide desaturase (DES1)
converts dihydroceramides to ceramides by introduction of a
4,5-trans double bond into the sphinganine backbone of
dihydroceramide. 4-HPR stimulates both SPT and CerS in
certain cancer cell lines. Both 4-HPR and GT-11, a synthetic
ceramide derivative, inhibit DES1. Asterisks (*) indicate
variable carbon length and saturation.
doi: 10.1371/journal.pone.0074768.g001
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Our results indicate that increased levels of certain, but not
all, native acyl chain dihydroceramides are cytotoxic to T-cell
ALL cell lines. Our results also suggest that supplementation of
dihydroceramide-increasing anticancer agents with specific
fatty acids, whether administered separately or by incorporation
into a formulation vehicle, might result in increased efficacy,
possibly in a cancer or cancer type-specific manner.
Materials and Methods
Materials
Sphinganine ([2S,3R]-2-aminooctadecane-1,3-diol) and
GT-11 (N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-
cyclopropenyl)ethyl]octanamide) were from Avanti Polar Lipids
(Alabaster, AL, USA), and prepared in ethanol at 10 mM and 1
mM, respectively. ABT-737 (4-[4-[[2-(4-
chlorophenyl)phenyl]methyl]piperazine-1-yl]-N-[4-[[(2R)-4-
(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-
nitrophenyl]sulfonylbenzamide) was from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Boc-D-FMK was from
Imgenex (San Diego, CA, USA). Fenretinide, (4-HPR, (2E,4E,
6E,8E)-N-(4-hydroxyphenyl)-3,7-dimethyl-9-(2,6,6-
trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenamide),
graciously provided by the National Cancer Institute (NCI)
Developmental Therapeutics Program (DTP) of the National
Institutes of Health (NIH, Bethesda, MD, USA), was prepared
in ethanol (10 mM). Chloroform (ethanol-stabilized) and other
solvents were obtained from Sigma Aldrich (St. Louis, MO,
USA) or Fisher Scientific (Pittsburg, PA, USA). α-Cyclodextrin
(Acros Organics, Geel, Belgium) was dissolved (15 mM) in
RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA).
Sphingolipid standards were from Avanti Polar Lipids.
Radiolabeled 3H-sphinganine, docosanoic [1-C14] and
tetracosanoic acids [1-C14] (50 mCi/mmol) were from American
Radiolabeled Chemicals (St. Louis, MO, USA). Fatty acids (FA)
were purchased from Sigma Aldrich, and included the
following: tetradecanoic acid, hexadecanoic acid, octadecanoic
acid, (Z)-ocadec-9-enoic acid, icosanoic acid, (Z)-icos-11-enoic
acid, docosanoic acid, (Z)-docos-13-enoic acid, tetracosanoic
acid and (Z)-tetracos-15-enoic acid. Fatty acids were dissolved
in a solution of methanol/chloroform (1:2, v:v) at 10 mM and
stored in PFTE-capped borosilicate vials.
Fatty acid solubilization
Fatty acids were solubilized by modification of Singh and
Kishimoto [35]. Fatty acid stock was added to a glass flask and
dried under nitrogen (~10 PSI). α-Cyclodextrin (15 mM in
RPMI-1640) was added at 27.3 mL/µmol FA. The sealed flask
was then sonicated three times for 5 minutes each using a
Branson 2510 Bath Sonicator (30°C). The fatty acid solution
was then sterilized by filtration (0.22 µm PVDF filter, EMD
Millipore, Billerica, MA, USA) and diluted 3 parts FA to 1 part
RPMI-1640 medium. The efficiency of fatty acid solubilization
and fatty acid cellular uptake was demonstrated using 14C-
C22:0- and 14C-C24:0-fatty acid tracers, thin layer
chromatography separation, and liquid scintillation counting
(data not shown); fatty acid solutions were prepared to ≥15 µM,
and used at a final concentration of 5 µM in whole cell culture
medium.
Cell culture
T-cell ALL cell lines, CCRF-CEM and p53 gene mutated
MOLT-4, were from American Type Culture Collection
(Manassas, VA, USA) and grown at 5% O2/5% CO2 and 20%
O2/5% CO2, respectively. The T-cell ALL cell lines COG-
LL-317h and COG-LL-332h were obtained from the TTUHSC
Cancer Center Cell Repository and grown at 5% O2/5% CO2.
Cell line identities were verified by the Children’s Oncology
Group Cell Culture and Xenograft Core using the AmpF/STR
Identifiler system (Applied Biosystems, Carlsbad, CA, USA),
and mycoplasma testing was performed. Cell lines were
maintained in RPMI-1640 medium supplemented with 10%
fetal bovine serum (FBS, Invitrogen, USA) in humidified 37°C
incubators. For all experiments, cells were seeded at 2.5x105
cells/mL in RPMI-1640 supplemented with 15% FBS, with a
final FBS content of 10% after addition of fatty acids and/or
drugs in serum free medium. Control treatments consisted of
fatty acid and drug vehicles.
Cytotoxicity assay
Cytotoxicity was measured using DIMSCAN (Bioimaging
Solutions, Inc., San Diego, CA, USA), a semi-automated,
fluorescence-based, digital imaging microscopy method with a
4-log dynamic range of detection [36]. Briefly, digital image
thresholding of cells treated with a fluorophore (fluorescein
diacetate) and quencher (2′4′5′6′-tetrabromofluorescein)
enabled capture and quantification of live cell fluorescence via
a CCD camera. For cytotoxicity assays, cells were seeded
(2.5x104 cells/well) into a 96-well flat bottom plate (BD-Falcon)
in 100 µL of whole medium one hour prior to addition of the
various treatment reagents added in 50 µL and assayed at +48
hours of treatment exposure. Cytotoxicity of each treatment
condition was measured in two or more separate experiments
except where otherwise indicated.
LC/MS/MS analysis of sphingolipids
For sphingolipid analysis, cells were seeded (7.5x106 cells)
one hour before treatment. After six hours of treatment, cells
were washed with ice-cold phosphate-buffered saline (PBS)
and stored at -80°C for sphingolipid analysis. Each treatment
was prepared in triplicate and repeated a minimum of two
times. Sphingolipids were separated using an Agilent 1200
HPLC and determined by ESI/MS/MS performed on a Applied
Biosystems SCIEX 4000 QTRAP Hybrid Triple Quadrupole/
Linear Ion Trap mass spectrometer, operating in a multiple-
reaction monitoring, positive ionization mode as described
previously with modifications [37]. Specifically, 50 µL of a
solution (1 pM) of internal sphingolipid standards (including
C17-sphingosine, C17-sphinganine, C17-sphingosine-1-
phosphate, and C17-ceramide) was added to each cell pellet.
Cells were extracted twice with ethyl acetate/isopropyl alcohol/
water (60:28:12; v:v). Sample was divided for quantitative and
lipid phosphate analyses. For LC/MS/MS, sample was re-
dissolved in mobile phase A (ammonium formate [1 mM] and
formic acid [0.2%] in methanol). Samples were injected (10 µL)
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and separated on a Spectra C8SR, 150 x 3.0 mm, 3 µm
particle size column (Peeke Scientific, Redwood City, CA)
using gradient-elution with mobile phase A and B (ammonium
formate [2 mM] and formic acid [0.2%] in water). Data
acquisition, peak integration and analyte quantitation were
performed using ABI/SCIEX Analyst 1.4.2 Software.
Sphingolipid data were normalized to lipid phosphate as
previously described [38]. Briefly, lipids were extracted using
the method of Bligh and Dyer [39]. Disposable borosilicate
tubes (Kimble Chase, Vineland, NJ, USA) were used so that
acid washing was not necessary. Sample organic phase was
isolated and a known volume was separated to a new tube and
dried at 80°C. Phosphate standards and dried samples were
then heated with ashing buffer (water:10 N H2SO4:70% HClO4
[40:9:1]) at 160°C overnight. Samples were subsequently
incubated with ammonium molybdate and ascorbic acid, and
absorbance (820 nM) was measured using a SpectraMax M2e
(Molecular Devices, Silicon Valley, CA, USA).
Immunoblotting
Treated cells were washed with PBS and lysed on ice with
RIPA Buffer (Thermo, Fisher) supplemented with protease
inhibitors (EMD Millipore) and 2% Triton-X100 (to ensure
complete LC3B-II solubilization). Cellular membranes were
disrupted by sonication. Protein content was measured using
the BCA assay as directed (Thermo, Fisher). Proteins were
separated using Bis-Tris (Invitrogen) gels. Proteins were
transferred to PVDF membranes (Invitrogen) using a Semi-Dry
Western Blot method (Thermo, Fisher). Blots were blocked in
5% milk/TBST and the lower molecular weight regions were
probed with the either anti-LC3B (Cell Signaling [CS], Beverly,
MA) or anti-caspase 3 (CS) antibodies. Higher molecular
weight regions of each blot were probed with anti-β-actin
(Santa Cruz Biotechnology [SBT]), which served as loading
control. The secondary antibodies used were HRP-anti-rabbit
(CS) and HRP-anti-mouse (SBT). LC3B-transfected, HEK-293
cell lysate was included as a positive control (Novus
Biologicals, Littleton, CO). Blots were developed using ECL
Substrate (Invitrogen) and visualized using a VersaDoc MP
5000 (Bio-Rad, Hercules, CA, USA) equipped with a 50 mm,
f1.4 fixed focal length lens. Data were analyzed using Quantity
One Software (Bio-Rad).
Assay of DNA fragmentation by flow cytometry
Treated cells were stained using the Apo-Direct kit (a TUNEL
(Terminal deoxynucleotide transferase dUTP Nick End
Labeling) assay variant)(BD Pharmingen, San Diego, CA,
USA) as directed. Cells (including compensation controls) were
analyzed on an LSR II Custom Flow Cytometer System (BD
Biosciences, San Jose, CA, USA). Doublet discrimination and
data analysis was performed using FlowJo (v10, Tree Star,
Inc., Ashland, OR, USA) software for Macintosh.
Detection of reactive oxygen species (ROS) and
mitochondrial depolarization by flow cytometry
For ROS assay, treated cells were stained as previously
described with 2’, 7’-dichlorofluorescein diacetate (125 µM;
Sigma Aldrich) [4]. 4-HPR (10 µM) and H2O2 (120 mM) were
employed as positive controls. Each treatment was assayed in
triplicate in two independent experiments. Mitochondrial
depolarization was assayed using the MitoProbe JC-1 kit
(Invitrogen), and analyzed on an LSR II Custom Flow
Cytometer System in single experiments. Data were analyzed
using FacsDiva (v6.0) software.
Real-time PCR
Total RNA was extracted with the RNeasy Mini-Kit (Qiagen,
Valencia, CA, USA), with QIAshredder columns employed for
improved cell lysis efficiency. RNA quality and quantity was
measured by spectrophotometry using a NanoDrop 1000
(Thermo, Fisher). First-strand cDNA synthesis was performed
using the High Capacity cDNA Reverse Transcription Kit
(Applied Biosystems). TaqMan Gene Expression Master Mix
and Primer/Probe sets (Applied Biosystems) were used as
recommended with 100 ng of cDNA per reaction (20 µL), and
real-time quantitative PCR was performed on an Applied
Biosystems 7900HT Fast Real-Time PCR System employing
the standard method. The following TaqMan primer/probe sets
(Applied Biosystems) were used: GAPDH, Hs03929097_g1;
HPRT1, Hs02800695_m1; CerS1, Hs00242151_m1; CerS2,
Hs00371958_g1; CerS3, Hs00698859_m1; CerS4,
Hs00226114_m1; CerS5, Hs00908757_m1; CerS6,
Hs00826756_m1. Real-time PCR samples were assayed in
triplicate and data were analyzed using the 2(-ΔΔCT) method [40].
Data were normalized to GAPDH and calibrated to the CerS1
mRNA of CCRF-CEM cells.
Statistical Analysis
T-tests (two-tailed) were used to compare sphingolipid levels
and cytotoxicity (quantified as the surviving fraction) of
treatment groups with those of control groups. Analysis of
variance was used to determine whether addition of fatty acids
significantly affected the cytotoxicity of sphinganine ± GT-11.
Spearman’s non-parametric rank correlation was used to
quantify relationships between cytotoxicity and sphingolipid
analyte levels. SigmaPlot 11 and Microsoft Excel 2011 were
employed for statistical testing. Correlation analyses were
performed in the R Statistical Environment (2.15.0).
Results
Dihydroceramides were cytotoxic to T-cell ALL cell
lines
Incorporation of exogenous sphinganine into de novo
synthesized intracellular dihydroceramides/ceramides was
demonstrated utilizing 3H-sphinganine (not shown). Treatment
of CCRF-CEM cells with sphinganine resulted in a 2.9-fold
increase (P ≤ 0.001) in total dihydroceramides (Figure 2A), a
1.5-fold increase (P ≤ 0.001) of total ceramides and significant
increases (P ≤ 0.05) in each of the individual ceramides
assayed (Figure 2C). Addition of DES1-inhibitor, GT-11, to
sphinganine (i.e., sphinganine + GT-11) resulted in a 9.6-fold
increase (P ≤ 0.001) of total dihydroceramides, including
significant increases (P ≤ 0.05) in all individual
dihydroceramides except C20:1-dihydroceramide (Figure 2B).
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Sphinganine + GT-11 also resulted in a decrease of both total
(P ≤ 0.001) and individual ceramides (P ≤ 0.02) relative to
sphinganine-alone.
Sphinganine-alone (1–4 µM) and GT-11-alone (0.5 µM) were
minimally cytotoxic (Figure 2D). However, the combination of
sphinganine + GT-11 increased cytotoxicity (P ≤ 0.001) in a
sphinganine concentration-dependent manner in each of the
cell lines tested. Thus, the combination of sphinganine and
GT-11 increased dihydroceramide levels in CCRF-CEM cells,
and increased cytotoxicity in all four T-cell ALL cell lines.
Addition of specific fatty acids increased levels of
dihydroceramides and ceramides
To increase levels of specific acyl chain dihydroceramides
and ceramides, CCRF-CEM cells were treated with
sphinganine ± GT-11 and supplemented with individual fatty
acids (FA) (C14:0-, C16:0-, C18:0-, C18:1-, C20:0-, C20:1-,
C22:0-, C22:1-, C24:0- or C24:1-FA). Fatty acids were
solubilized with α-cyclodextrin. Incorporation of solubilized fatty
acid into de novo ceramides was demonstrated using 14C-
tetracosanoic acid (C24:0-FA) (not shown). Of the fatty acids
tested, C14:0-, C16:0, C18:0, C20:0-, C22:0-, C22:1-, C24:0-
and C24:1-FA supplemented to sphinganine significantly
increased levels (P ≤ 0.05) of the corresponding acyl chain
dihydroceramides relative to sphinganine with no fatty acid
Figure 2. Effects of sphinganine and GT-11 on dihydroceramides, ceramides and cytotoxicity. A-C) CCRF-CEM cells were
treated with sphinganine (4 µM, S) ± GT-11 (0.5 µM, G) for six hours and sphingolipids analyzed. A) Total dihydroceramides
(DHCer) and ceramides (Cer) were normalized to drug/fatty acid vehicle-treated cells (control) and plotted as fold change (Y-axis).
Error bar, propagated standard deviation. Individual dihydroceramides (B) and ceramides (C) were normalized to control and plotted
as fold change (Z-axis). Dihydroceramides and ceramides are identified by acyl chain (x:y), where x is the number of carbons and y
is the number of double bonds in the acyl chain (X-axis). Significant (A, P ≤ 0.001; B and C, P ≤ 0.05) fold changes between
treatment and vehicle control levels are indicated by asterisks (*). D) Cytotoxicity of sphinganine and GT-11. Indicated cell lines
were treated with sphinganine (0-4 µM) and/or GT-11 (0.5 µM). The cytotoxicity of GT-11-alone is represented by the sphinganine
(0 µM) data point. Cytotoxicity assayed by DIMSCAN cytotoxicity assay at +48 hours. Data were normalized to vehicle-treated
control and plotted as Survival Fraction (Y-axis). Error bar, SEM. Sphinganine + GT-11 resulted in significantly increased (P ≤
0.001) cytotoxicity relative to sphinganine-only for all concentrations and across all cell lines.
doi: 10.1371/journal.pone.0074768.g002
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(Figure 3A). C14:0-, C16:0-, C20:0-, C22:0-, C22:1-, C24:0-
and C24:1-FA supplemented to sphinganine + GT-11
significantly increased levels (P ≤ 0.05) of the corresponding
acyl chain dihydroceramides relative to sphinganine + GT-11
with no fatty acid (Figure 3B). C14:0-, C16:0-, C18:0-, C20:0-,
C20:1-, C22:0-, C22:1-, C24:0- and C24:1-FA supplemented to
sphinganine significantly increased levels (P ≤ 0.05) of the
corresponding acyl chain ceramides relative to sphinganine-
alone in CCRF-CEM cells (Figure S1). Certain fatty acids
increased the corresponding ceramide when given in
combination with sphinganine + GT-11, likely due to incomplete
DES1 inhibition in the presence of high dihydroceramide
substrate levels, but not to the extent observed when the fatty
acid was combined with sphinganine-alone. Certain fatty acids
also resulted in concurrent increases of longer and/or
desaturated acyl chain dihydroceramides and ceramides due to
intracellular modification of the fatty acids by elongases and
desaturases prior to acylation of sphinganine [41]. As an
example, when C22:0-FA was added to sphinganine + GT-11,
in addition to the observed 29-fold increase (P ≤ 0.001) of
C22:0-dihydroceramide, there was a 3-fold (P ≤ 0.01) and 19-
fold (P ≤ 0.001) increase of C22:1- and C24:0-
dihydroceramides, respectively, compared to sphinganine +
GT-11 without fatty acid.
Addition of specific fatty acids increased cytotoxicity
Certain, but not all, fatty acids increased (P ≤ 0.001) the
cytotoxicity of sphinganine ± GT-11 (Figure 3C) in a
sphinganine concentration-dependent manner. In general,
longer chain fatty acids increased cytotoxicity to a greater
degree than did shorter chain fatty acids.
Levels of C22:0- and C24:0- dihydroceramides
positively correlated with cytotoxicity
Since the intracellular metabolism of certain fatty acids
resulted in an increase of multiple dihydroceramides and/or
ceramides, to identify individual level-dependent cytotoxic
relationships, quantitative sphingolipid levels and cytotoxicity
data were analyzed using the Spearman’s non-parametric rank
correlation. Correlations were evaluated using sphingolipids
assayed at +6 hours of treatment and cytotoxicity assayed at
+48 hrs, as serial assay demonstrated that minimal further
changes in sphingolipid profiles occurred at later time points
(not shown). Additionally, sphingolipid data used for correlation
analyses were taken at a fixed exogenous sphinganine
concentration (1 µM) corresponding to a modest cell kill fraction
to minimize possible confounding secondary changes in
sphingolipid profiles resulting from death in large fractions of
the cells. Of all sphingolipid analytes, only levels of C22:0- (ρ =
0.74, P ≤ 0.001) and C24:0- (ρ = 0.84, P ≤ 0.001)
dihydroceramides demonstrated strong positive correlations
with cytotoxicity (for scatter plots, see Figure S2). No other
consistent positive or negative correlations were observed
between cytotoxicity and other sphingolipid analytes (total or
individual dihydroceramides, total or individual ceramides,
sphingoid bases, including sphinganine, or phosphorylated
sphingoid bases). The absolute amounts of the
dihydroceramides which correlated with cytotoxicity, and the
amounts of their corresponding ceramides, are shown in Table
1. C22:1-dihydroceramide levels increased to a larger absolute
amount than C22:0-dihydroceramide, but did not correlate with
cytotoxicity; therefore, this species served as a negative control
to exclude effects based on increase of total dihydroceramide
mass irrespective of acyl chain.
Characterization of dihydroceramide-induced cell death
mechanisms
Because 4-HPR increases reactive oxygen species (ROS)
levels in association with cytotoxicity in certain cancer cell
lines, ROS levels were measured in treated CCRF-CEM and
MOLT-4 cells to determine if an increase of dihydroceramides
was contributory to ROS increase. Compared to treatment with
4-HPR, an increase in ROS was not observed with sphinganine
and/or GT-11 treatments, with or without C22:0-FA, at doses
that increased dihydroceramides and conferred cytotoxicity
(Figure 4A). Mitochondrial depolarization proceeded
concurrently with, but did not precede, cell death in cells
treated with sphinganine/GT-11/C22:0-FA (not shown). To
assess markers of apoptosis, DNA fragmentation was assayed
by terminal deoxynucleotidyltransferase duty nick end labeling
(TUNEL) and caspase cleavage by immunoblotting. Cells
treated with C22:1-FA plus sphinganine and GT-11 exhibited
minimal TUNEL positivity at +24 hrs (Figure 4B and Figure S3).
In contrast, treatment with C22:0-FA plus sphinganine ± GT-11
increased TUNEL positivity relative to controls (Figure 4C).
Increased procaspase-3 cleavage was increased by C22:0-FA
plus sphinganine ± GT-11 (combinations that increased
cytotoxicity), but procaspase-3 cleavage was not increased by
Table 1. Absolute sphingolipid levels.
Treatment
Species Basal S S + FA S + G S + G + FA
Fatty
Acid
C22:0-
DHCer
0.13 ±
0.03
0.93 ±
0.09 45 ± 4 2.0 ± 0.2 57 ± 4 C22:0-FA
C22:0-Cer 0.37 ±
0.04 0.6 ± 0.1 7.7 ± 0.4 0.37 ±
0.04 4.6 ± 0.3 C22:0-FA
C22:1-
DHCer
0.18 ±
0.01 1.2 ± 0.1 53 ± 5 2.3 ± 0.2 73 ± 7 C22:1-FA
C22:1-Cer 0.05 ±
0.01
0.16 ±
0.03 2.5 ± 0.4 0.07 ±
0.01
1.05 ±
0.07 C22:1-FA
C24:0-
DHCer
0.16 ±
0.05
0.61 ±
0.04 78 ± 6 1.4 ± 2 99 ± 10 C24:0-FA
C24:0-Cer 0.37 ±
0.05
0.40 ±
0.06 4.4 ± 0.8 0.28 ±
0.04 2.6 ± 0.4 C24:0-FA
Effects of specific fatty acids on absolute levels of corresponding dihydroceramides
and ceramides in CCRF-CEM cells treated with sphinganine ± GT-11. Absolute
levels (pmol sphingolipid/nmol lipid phosphate) are shown (± SEM) of intracellular
sphingolipids from untreated cells (Basal), or cells treated with sphinganine (S, 1
µM) or sphinganine and GT-11 (G, 0.5 µM) with and without supplemented with
fatty acid (FA). The specific fatty acids used to supplement cells are indicated in
the fifth column.
doi: 10.1371/journal.pone.0074768.t001
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Figure 3. Effects of specific fatty acids on sphinganine ± GT-11-induced dihydroceramides and cytotoxicity. A, B) Effects
on dihydroceramides. CCRF-CEM cells were treated with (A) sphinganine (1 µM), or (B) sphinganine (1 µM) + GT-11 (0.5 µM), and
supplemented with the indicated fatty acids (5 µM) for six hours, followed by sphingolipid assay. To evaluate the effects resulting
from addition of each fatty acid, data for (A) and (B) were normalized either to cells that received sphinganine-only with no fatty acid
supplementation (A), or to sphinganine + GT-11 without fatty acid (B), and plotted as fold change (Z-axis). Fatty acids are identified
by x:y, where x is the number of carbons and y is the number of double bonds in the fatty acid chain (Y-axis). Significant (P ≤ 0.05)
differences from sphinganine-only are indicated by asterisks (*). C) Effects on cytotoxicity. CCRF-CEM cells were treated with
sphinganine-only (0-4 µM), or sphinganine (0-4 µM) + GT-11 (0.5 µM), supplemented with the indicated fatty acids (5 µM).
Cytotoxicity assayed by DIMSCAN cytotoxicity assay at +48 hours. Data were normalized to control and plotted as survival fraction
(Y-axis). Error bar, SEM. Data, grouped by sphinganine dose, were analyzed by one-way ANOVA.
doi: 10.1371/journal.pone.0074768.g003
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C22:1-FA plus sphinganine ± GT-11 (combinations that
minimally effected cytotoxicity)(Figure 5A). To determine
whether cytotoxicity was dependent on caspase-mediated DNA
fragmentation, the effect of a pan-caspase inhibitor, Boc-D-
FMK, on C22:0-dihydroceramide-mediated cell death was
assessed. The addition of Boc-D-FMK to ABT-737, a known
inducer of apoptosis in ALL cell lines [4], abrogated both
ABT-737-induced TUNEL positivity and reduction in the G1 cell
population (Figure 4C), as well as reduced ABT-737-induced
cytotoxicity (Figure 4D). In contrast, while Boc-D-FMK
abrogated C22:0-FA plus sphinganine + GT-11 induced
TUNEL positivity (Figure 4C), it did not decrease C22:0-FA
plus sphinganine + GT-11 induced cytotoxicity, suggesting that
under conditions of caspase inhibition cell death proceeded via
a non-apoptotic mechanism (Figure 4D). Interestingly, the
proportion of cells treated with C22:0-FA plus sphinganine +
GT-11 that were in G1 significantly reduced relative to controls,
while the proportion of cells in G2 remained similar or
increased.
Lipidation of LC3B-I to LC3B-II, a marker of increased
autophagic vacuolization, increased in cells treated with C22:0-
FA plus sphinganine ± GT-11 to a much greater extent than
with C22:1-FA (Figure 5A). To determine whether LC3B-II level
elevation was due to increased LC3B-I lipidation (flux) or to
decreased LC3B-II degradation, cells were pretreated with
protease inhibitors of LC3B-II degradation, E64d and pepstatin-
A (each at 10 µg/mL), prior to exposure to C22:0-FA or C22:1-
FA plus sphinganine + GT-11. E64d and pepstatin-A
pretreatment resulted in a marked increase of LC3B-II in cells
compared to controls for both C22:0-FA and C22:1-FA,
suggesting an increase of autophagic flux (Figure 5B).
Interestingly, addition of 3-methyladenine (0.1-10 mM), a
putative autophagy inhibitor, neither increased nor decreased
the cytotoxicity of C22:0-FA plus sphinganine ± GT-11 in
CCRF-CEM cells (not shown), suggesting that autophagy was
not directly linked to the cell death mechanism.
C22:0- and C24:0-dihydroceramides positively
correlated with cytotoxicity in other ALL cell lines
Given the results in CCRF-CEM cells, the effects of C18:0-,
C22:0- and C22:1-FA supplemented to sphinganine ± GT-11
were tested in three additional T-cell ALL cell lines (MOLT-4,
COG-LL-317h and COG-LL-332h). C22:1-FA was also tested
because, while minimally affecting the cytotoxicity of
sphinganine + GT-11 in CCRF-CEM cells, it resulted in
absolute levels of C22:1-dihydroceramide similar to those of
C22:0-dihydroceramide after treatment with C22:0-FA, thus
effectively serving as a negative control for a possible non-
specific (acyl chain-independent) mass effect of increased
dihydroceramides on cytotoxicity. C22:0-FA added to
sphinganine + GT-11 increased C22:0-dihydroceramide in
MOLT-4, COG-LL-317h, and COG-LL-332h cells by 29-fold (P
≤ 0.001), 28-fold (P ≤ 0.001), and 224-fold (P ≤ 0.001)
respectively (Figure 6A), relative to sphinganine + GT-11 with
no fatty acid. C22:1-FA added to sphinganine + GT-11
increased C22:1-dihydroceramide in MOLT-4, COG-LL-317h
and COG-LL-332h cells by 11-fold (P ≤ 0.001), 17-fold (P ≤
0.001), and 35-fold (P ≤ 0.001), respectively, relative to
Figure 4. Mechanisms of C22:0-dihydroceramide induced
cell death. A) Reactive oxygen species levels in sphinganine
and/or GT-11 treated CCRF-CEM cells supplemented with or
without C22:0-fatty acid. CCRF-CEM cells were treated with
sphinganine (1 µM) and/or GT-11 (0.5 µM), both with and
without C22:0-FA (5 µM). 4-HPR (10 µM) and H2O2 (120 mM,
not shown) were employed as positive controls. Cells were
stained with 2’, 7’-dichlorofluorescein diacetate and
fluorescence analyzed after 6 hours by flow cytometry. Data
were normalized to control. Error bars, SEM. B & C) Effect of
pan-caspase inhibition on TUNEL positivity. CCRF-CEM cells
were pre-treated with pan-caspase inhibitor, Boc-D-FMK (80
µM), or DMSO (final concentration = 0.33%, Boc-D-FMK
vehicle control), for one hour prior to treatment with ABT-737 (1
µM, positive control), or C22:0-FA plus sphinganine (1 or 2 µM,
S) + GT-11 (0.5 µM, G). C22:1-FA plus sphinganine (1 µM) +
GT-11 (0.5 µM, G) and Boc-D-FMK alone included as controls.
Cells analyzed by TUNEL assay at +24 hrs. Shown are
histograms representative of three separate experiments.
Histograms are of indicated treatments analyzed by PI counter-
stain of TUNEL samples. D) Effect of pan-caspase inhibition on
cytotoxicity. CCRF-CEM cells were pre-treated with pan-
caspase inhibitor, Boc-D-FMK (80 µM, Boc), for one hour prior
to treatment with ABT-737 (1 µM), C22:0-FA plus sphinganine
(2 µM, S) + GT-11 (0.5 µM, G). Cytotoxicity assessed at +12
and +24 hrs by DIMSCAN cytotoxicity assay and represented
as Survival Fraction. Asterisks (*) represent significant (P ≤
0.05) effects of Boc treatment.
doi: 10.1371/journal.pone.0074768.g004
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sphinganine + GT-11 with no fatty acid. Presumptive
intracellular elongation of C22:0-FA to form C24:0-FA, and of
C22:1-FA to form C24:1-FA, was observed as indicated by
concurrent elevations of C24:0- and C24:1-dihydroceramides,
respectively. C18:0-FA affected dihydroceramide and ceramide
levels to a much lesser extent. C22:0-FA addition consistently
increased (P ≤ 0.001) cytotoxicity of sphinganine-alone and of
sphinganine + GT-11 (Figure 6B), in all three cell lines, in a
sphinganine concentration-dependent manner. As observed in
CCRF-CEM cells, C18:0- and C22:1-FA minimally affected
cytotoxicity. Spearman’s non-parametric rank correlation
analyses of the cytotoxicity data and sphingolipid levels of each
cell line revealed significant, strong positive correlations
between cytotoxicity and the absolute levels of C22:0- and
C24:0-dihydroceramides in MOLT-4 [(C22:0-DHCer, ρ = 0.81,
P ≤ 0.01), (C24:0-DHCer, ρ = 0.86, P ≤ 0.01)], COG-LL-317h
[(C22:0-DHCer, ρ = 0.79, P ≤ 0.02), (C24:0-DHCer, ρ = 0.88, P
Figure 5. Caspase cleavage and LC3B-I/II turnover. A)
CCRF-CEM cells treated with drug/fatty acid vehicle (C),
sphinganine (1 µM, S), or sphinganine (1 µM) + GT-11 (0.5 µM)
(SG), were supplemented with the indicated fatty acids (5 µM).
After 12 hours, total proteins were extracted and procaspase-3
(35 kb), activated caspase-3 (17/19 kb), and LCB3-I/II (14/16
kb), were detected by immunoblotting. β-Actin served as
loading control. Treatment with pan-Bcl-2 inhibitor, ABT-737 (1
µM, A), and LC3B-transfected, HEK-293 cell lysate, were used
as positive controls. C22:1-fatty acid was used as a negative
control for C22:0-fatty acid. Lanes rearranged to ease
interpretation. Data representative of three separate
experiments are shown. B) Assessment of LC3B-II flux. CCRF-
CEM cells were pretreated with or without protease inhibitors
(Pepstatin-A and E64d) and treated as described. After 12
hours, total proteins were extracted and LC3B-I/II analyzed by
immunoblotting. Data representative of three separate
experiments are shown, except for C22:1-fatty acid.
doi: 10.1371/journal.pone.0074768.g005
≤ 0.004)], and COG-LL-332h [(C22:0-DHCer, ρ = 0.77, P ≤
0.04), (C24:0-DHCer, ρ = 0.90, P ≤ 0.002)]. No other consistent
strong positive or negative correlations were observed.
C22:0-fatty acid increased 4-HPR cytotoxicity
Because 4-HPR increased dihydroceramides in susceptible
cell lines, but a cause and effect relationship between
dihydroceramide increase and fenretinide-induced cytotoxicity
remained unclear, the effects of fatty acid supplementation on
4-HPR-induced cytotoxicity and dihydroceramide levels were
determined. T-cell ALL cell lines were exposed to 4-HPR ±
C18:0 or C22:0-FA. Similar to the effects of specific fatty acids
on sphinganine + GT-11, the addition of C22:0-FA, but not
C18:0-FA, increased (P ≤ 0.001) 4-HPR-induced cytotoxicity in
all four ALL cell lines (Figure 7A). C24:0-fatty acid increased
the cytotoxicity of low 4-HPR concentrations in all cell lines (P ≤
0.001); C22:1-fatty acid minimally to moderately increased 4-
HPR cytotoxicity in a cell line-specific manner (Figure S4). The
effects of fatty acid co-treatment on sphingolipid levels in
fenretinide-treated cells were analyzed in the COG-LL-317h
and COG-LL-332h cell lines. C22:0-FA addition increased
C22:0-dihydroceramide levels in COG-LL-317h and COG-
LL-332h cells, 10-fold (P ≤ 0.001) and 6-fold (P ≤ 0.001),
respectively, over cells treated with 4-HPR-alone (Figure 7B).
C18:0-FA increased (P ≤ 0.01) C18:0-dihydroceramide levels
in COG-LL-317h cells, but not in COG-LL-332h cells (Figure
7B). However, this increase of C18:0-dihydroceramide in COG-
LL-317h cells occurred in the absence of a corresponding
increase in cytotoxicity (Figure 7A) suggesting that the
observed effects of C22:0-dihydroceramide levels on
fenretinide-induced cytotoxicity was acyl chain-specific.
Discussion
We previously demonstrated that 4-HPR induced cytotoxicity
in T-cell ALL cell lines was associated with increased levels of
de novo synthesized long and very long acyl chain (i.e., native)
dihydroceramides (2). However, a causal relationship between
the increase of dihydroceramides and 4-HPR-induced
cytotoxicity was not clear. The aim of the current study was to
elucidate the cytotoxic potentials of native acyl chain
dihydroceramides produced via de novo synthesis in a
controlled manner and determine by correlation analysis if such
cytotoxicity was due to increases in total dihydroceramide
mass or to the increase of discrete dihydroceramide species.
Therefore, we supplemented cells with minimally to non-
cytotoxic amounts of normal sphinganine ± GT-11, a DES1
inhibitor, ± individual fatty acids (as acyl chain precursors). We
then determined how the manipulation of specific
dihydroceramides (via supplementation of individual fatty acids)
affected fenretinide cytotoxicity. While this model lacked the
specific serine palmitoyltransferase (SPT) and dihydroceramide
synthase (CerS) stimulating properties of 4-HPR, it did allow for
the manipulation and assessment of multiple individual
dihydroceramides in isolation from other cytotoxic effects of
fenretinide, such as an increase in reactive oxygen species
(ROS).
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Results using tracer radiolabeling and tandem mass
spectroscopy demonstrated that exogenous sphinganine
supplemented in non-cytotoxic amounts was incorporated into
cellular sphingolipids and successfully increased levels of
dihydroceramides and ceramides (Figure 2) with a sphingolipid
profile that was similar but distinctive to each cell line (not
shown). The addition of minimally-toxic amounts of GT-11 to
sphinganine further increased most dihydroceramides at the
expense of the corresponding ceramides (Figure 2 and Table
1). It was further determined using radiolabeling and tandem
mass spectroscopy that, within the limitations of intracellular
metabolism (i.e., shortening, elongation, and desaturation of
fatty acids, and of possible cell line-specific activity of the
dihydroceramide synthases), supplementation of nontoxic
amounts of individual fatty acids successfully increased the
level of the corresponding dihydroceramide in most cases
(Figures 3 and 6, Table 1). Thus, the intracellular levels of
individual native acyl chain dihydroceramides derived from de
novo synthesis could be manipulated and their cytotoxic
potentials assessed. It should be emphasized that the levels of
dihydroceramides attained in this model represent levels likely
not found in cancer cells in the absence of strong
pharmacological stimulation of de novo synthesis, and not
experienced in normal cells, even in dihydroceramide
desaturase null mice [42]. Further, no inferences should be
made on the cytotoxic potential of dihydroceramides and
ceramides in other cellular pools, such as those derived from
activation of various sphingomyelinases.
Figure 6. Effects of specific fatty acids on sphinganine + GT-11-induced dihydroceramide accumulation and
cytotoxicity. A) Effects of fatty acids on dihydroceramide levels. MOLT-4, COG-LL-317h, and COG-LL-332h, cell lines were
treated with sphinganine (1 µM) + GT-11 (0.5 µM) supplemented with the indicated fatty acid (5 µM) for six hours and sphingolipids
analyzed. Data are normalized to cells that received sphinganine + GT-11 without fatty acid supplementation and dihydroceramide
levels plotted as fold change (Z-axis). Significant (P ≤ 0.05) fold change differences are indicated by asterisks (*). B) Effects of fatty
acids on cytotoxicity. Cell lines were treated with sphinganine (0-4 µM) ± GT-11 (0.5 µM) and supplemented with C18:0-, C22:0- or
C22:1-fatty acids (5 µM). Cytotoxicity assessed at +48 hours by DIMSCAN cytotoxicity assay. Data were normalized to controls and
plotted as Survival Fraction (Y-axis). Error bar, SEM.
doi: 10.1371/journal.pone.0074768.g006
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Correlation analysis was employed to obtain insight into the
cytotoxic properties of individual dihydroceramides. Results
showed that only the absolute levels of both C22:0- and C24:0-
dihydroceramides strongly correlated with cytotoxicity in the
CCRF-CEM, MOLT-4, COG-LL-317h, and COG-LL-332h T-cell
ALL cell lines. No other consistent relationships were observed
between cytotoxicity and total or other individual
dihydroceramides, total or individual ceramides, or levels of the
other sphingolipid species (sphinganine, sphinganine-1-P,
sphingosine, sphingosine-1-P). Further, supplementation with
C22:1-FA increased absolute levels of its corresponding
dihydroceramide to a greater extent than did C22:0-FA without
increasing cytotoxicity (Figure S5), evidencing that cytotoxicity
did not correlate with total dihydroceramide mass, but rather
with an increase of specific dihydroceramides. A limitation was
that a targeted increase of dihydroceramide could not be
achieved in all cases (e.g., C18:1-dihydroceramide, Figure 3)
and, therefore, the cytotoxic potential of some
dihydroceramides could not be assessed; further, it cannot be
excluded that the cytotoxic potential of a given
dihydroceramide is cancer cell line-, or cancer type-,
dependent.
Results also demonstrated that the cytotoxic potentials of
dihydroceramides were dependent on both acyl chain length
and saturation status as, although both C22:0- and C22:1-
dihydroceramides were increased to similar absolute levels,
only levels of C22:0-dihydroceramide positively correlated with
cytotoxicity (Figure S2). Significantly, supplementing 4-HPR-
Figure 7. Effects of specific fatty acids on 4-HPR-induced dihydroceramide levels and cytotoxicity. A) Effects on
dihydroceramide levels. COG-LL-317h and COG-LL-332h cells were treated with 4-HPR (1 µM) with or without C18:0- or C22:0-
fatty acids (5 µM) for six hours and sphingolipids analyzed. Data were normalized to cells that received 4-HPR without fatty acid
supplementation and plotted as fold change (Z-axis). Significant (P ≤ 0.001) differences from 4-HPR without fatty acid indicated by
asterisks (*). B) Effects on cytotoxicity. CCRF-CEM, MOLT-4, COG-LL-317h, and COG-LL-332h cell lines were treated with 4-HPR
(0-9 µM) ± C18:0- or C22:0-fatty acids (5 µM) and cytotoxicity assessed at +48 hours by DIMSCAN cytotoxicity assay. Data were
normalized to controls and represented as Survival Fraction (Y-axis). Error bar, SEM. Significant (P ≤ 0.001) differences in
cytotoxicity from 4-HPR without fatty acid are indicated by asterisks (*).
doi: 10.1371/journal.pone.0074768.g007
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treated cells with nontoxic amounts of C22:0-FA and C24:0-FA
increased 4-HPR-induced cytotoxicity (Figure 7 and Figure S4)
that, in the case of C22:0-FA supplementation, was in
association with increased C22:0- and C24:0-dihydroceramide
levels (the direct effect of C24:0-FA supplementation on
sphingolipid levels was not assayed). In contrast, C18:0-FA
minimally increased 4-HPR-induced cytotoxicity despite
increasing C18:0-dihydroceramide, and C22:1-FA minimally
increased 4-HPR cytotoxicity despite its ability to selectively
increase C22:1- and C24:1-dihydroceramides in sphinganine +
GT-11 treated cells, suggesting that the increase of certain
dihydroceramides, but not all dihydroceramides, may contribute
to 4-HPR cytotoxicity. As the targeted increase of C22:0-
dihydroceramide did not increase ROS in association with
increased cytotoxicity, these results also likely exclude a role
for ROS as a downstream mediator of dihydroceramide-driven
cytotoxicity. These results support previous reports that 4-HPR-
induced increase of ROS in susceptible cell lines occurs
through a process that is mechanistically distinct from the
elevation of dihydroceramides (although 4-HPR does not
increase ROS in association with cytotoxicity in all cell lines)
[8,43].
We had previously observed both apoptotic and non-
apoptotic cell death in neuroblastoma cells treated with 4-HPR,
a p53-independent agent [9]. In CCRF-CEM cells, we observed
that the targeted increase of C22:0-dihydroceramide induced
cytotoxicity associated with an increase in flux and levels of
autophagy marker LC3B-II and caspase-dependent apoptosis
(as evidenced by TUNEL-positive DNA cleavage). Interestingly,
preventing DNA cleavage by pan-caspase inhibition did not
significantly reduce cytotoxicity, indicating the presence of a
concurrent, caspase-independent, non-apoptotic death
mechanism (Figure 4). 3-methyladenine, a putative autophagy
inhibitor, did not affect the cytotoxicity of C22:0-FA plus
sphinganine ± GT-11 in CEM cells. Further, increased LC3B-II
flux was observed in cells treated with C22:1-fatty acid,
sphinganine and GT-11, suggesting that autophagy was a
concurrent process that did not subserve either a strong pro-
death or pro-life function in this context. The role of autophagy
in dihydroceramide-associated cell death is being further
investigated via the knockdown of autophagy-initiating proteins,
Beclin-1 and ATG7 (work in progress).
Of note, it has recently been suggested that sphinganine
level increase resulting from hydrolysis of dihydroceramides by
alkaline ceramidase 2 (ACER2) is the mechanism of 4-HPR-
induced cytotoxicity in tumor cells [44]. However, although the
dihydroceramide profiles of cells treatment with sphinganine +
GT-11 did not perfectly mimic those induced by 4-HPR in the
same cell line likely due to the stimulatory effects of 4-HPR on
specific CerS family members (not shown), we observed that
intracellular sphinganine levels did not correlate with
cytotoxicity, and that exogenous C22:0-FA increased C22:0-
dihydroceramide levels and 4-HPR cytotoxicity in the absence
of a further increase of sphinganine. Together, these findings
suggest that specific dihydroceramides can mediate
cytotoxicity independently of sphinganine.
It is significant that the results indicate that dihydroceramide
cytotoxicity is both acyl chain length- and saturation-dependent
as the individual ceramide synthase family members (CerS
1-6) each have specific fatty acyl-CoA substrate preferences. If
dihydroceramides do contribute to 4-HPR cytotoxicity, we
speculate that the CerS expression/activity profile of a given
tumor might be a biomarker partially predictive of its response
to 4-HPR. Interestingly, CerS2 was the most highly expressed
(mRNA) CerS in the T-cell ALL cell lines tested (Figure S6),
and the acyl-CoA preferences of CerS2 include C22:0- and
C24:0-acyl chains [45]. We are currently determining how
CerS2 knock-down affects the cytotoxicity of sphinganine +
GT-11 ± C22:0- and C24:0-FA in these cell lines (work in
progress).
In summary, the present study reports a novel method for
manipulating levels of individual dihydroceramides in whole
cells and evidence that dihydroceramides produced through de
novo synthesis can confer level-dependent cytotoxicity to
cancer cells in a manner dependent upon the length and
saturation status of their acyl chain; specifically, C22:0- and
C24:0-dihydroceramides exhibited level-dependent cytotoxicity
independent of ceramide levels in four T-cell ALL cell lines.
Additionally, as co-treatment with nontoxic amounts of C22:0-
FA enhanced 4-HPR cytotoxicity in these cell lines in
association with an increase of C22:0-dihydroceramide, the
data suggest that certain dihydroceramides may contribute to
the overall mechanism of 4-HPR cytotoxicity. This latter
observation also suggests the possibility that addition of
specific fatty acids, perhaps in a tumor or tumor type-specific
manner, either through dietary or intravenous supplementation
or via incorporation into drug delivery vehicles, might be
employed to improve the clinical efficacy of dihydroceramide-
increasing anticancer agents.
Based on these findings, we are currently evaluating the
effects of an oral, organized lipid complex formulation of 4-HPR
enriched with C22:0-FA acid on intra-tumor dihydroceramide
levels and cytotoxicity in human pediatric T-cell ALL xenograft
models [46,47].
Supporting Information
Figure S1. Effects of fatty acids on ceramide levels. CCRF-
CEM cells were treated with (left) sphinganine (1 µM) or (right)
sphinganine (1 µM) + GT-11 (0.5 µM) and supplemented with
the indicated fatty acids (5 µM) for six hours with subsequent
sphingolipid analysis. To evaluate the effects resulting from
addition of each fatty acid, data for (A) & (B) were normalized
either 1) to cells that received sphinganine-only with no fatty
acid supplementation (A) or, 2) to sphinganine + GT-11 without
fatty acid (B), and plotted as fold change (Z axis) ceramide.
Fatty acids are identified by x:y, where x is the number of
carbons and y is the number of double bonds in the fatty acid
chain (Y axis). Significant (P ≤ 0.05) differences are indicated
by asterisks (*).
(PDF)
Figure S2. Relationships between cytotoxicity and C22:0-,
C22:1- and C24:0-dihydroceramides. Absolute levels (X-axis)
of C22:0-DHCer (left), C22:1-DHCer (middle), and C24:0-
DHCer (right) were plotted against the Killed Fraction (Y-axis)
C22:0-/C24:0-Dihydroceramides Confer Cytotoxicity
PLOS ONE | www.plosone.org 12 September 2013 | Volume 8 | Issue 9 | e74768

of the respective treatment as measured using DIMSCAN.
CCRF-CEM cells were treated with sphinganine (1 µM) ±
GT-11 (0.5 µM) with and without fatty acid supplementation
(C14:0-, C16:0-, C18:0-, C18:1-, C20:0-, C20:1-, C22:0-,
C22:1-, C24:0- and C24:1-fatty acids (5 µM)). Open circles
indicate treatment with sphinganine ± FA; closed circles
indicate treatment with sphinganine + GT-11 ± FA. X and Y
error bars are SEM. Spearman correlation coefficients (ρ) are
shown.
(PDF)
Figure S3. TUNEL positivity in sphinganine and/or GT-11
treated CCRF-CEM cells supplemented with C22:0-FA or
C22:1-FA. Cells were treated as indicated with sphinganine (1
µM, S), GT-11 (0.5 µM, G) and C22:0-FA or C22:1-FA. After
+24 hours, cells were fixed and subsequently analyzed by
TUNEL assay. C22:1-fatty acid served as a negative control for
C22:0-fatty acid. Shown are singlet histograms representative
of three independent experiments. Increased TUNEL positivity
is observed versus similar treatment in Figure 4C. This is due
to the presence of DMSO, vehicle of Boc-D-FMK, which may
interfere with cyclodextrin inclusion complexes and reduce the
effective fatty acid concentration.
(PDF)
Figure S4. Effects of specific fatty acids on 4-HPR-
induced cytotoxicity. CCRF-CEM, MOLT-4, COG-LL-317h,
and COG-LL-332h cell lines were treated with 4-HPR (0-9 µM)
± C18:0-, C22:0-, C22:1-, or C24:0-fatty acids (5 µM) and
cytotoxicity assessed at +48 hours by DIMSCAN cytotoxicity
assay. Data were normalized to controls and represented as
Survival Fraction (Y-axis). Error bar, SEM. Significant (P ≤
0.001) differences in cytotoxicity from 4-HPR without fatty acid
are indicated by asterisks (*).
(PDF)
Figure S5. Total dihydroceramide levels. CCRF-CEM cells
were treated drug vehicles (C), GT-11 (G) (0.5 µM),
sphinganine (S) (1 µM), or sphinganine + GT-11. The indicated
fatty acids (5 µM) were supplemented as indicated, and “No
FA” indicates treatment with fatty acid vehicle. Cells were
treated for six hours, followed by sphingolipid assay. Plotted
are absolute total dihydroceramide levels (Z axis). Fatty acids
are identified by x:y, where x is the number of carbons and y is
the number of double bonds in the fatty acid chain.
(PDF)
Figure S6. CerS mRNA levels in T-cell ALL cell lines. Two-
step RT-PCR was performed using mRNA extracted from
untreated CCRF-CEM, MOLT-4, COG-LL-317h and COG-
LL-332h cell lines. Data were normalized to GAPDH and
calibrated to the CerS1 mRNA of CCRF-CEM cells (Y axis).
Data normalized to HPRT1 instead of GAPDH were similar.
Error bar, SEM. CerS3 mRNA was minimally detectable.
(PDF)
Acknowledgements
The authors thank Daniel M. Hardy, C. Patrick Reynolds, Ina L.
Urbatsch and Harry M. Weitlauf for their guidance and critical
appraisal of this manuscript. Certain intellectual property
related to the present report may be retained by the Texas
Tech University System.
Author Contributions
Conceived and designed the experiments: MWH MHK BJM.
Performed the experiments: MWH. Analyzed the data: MWH
SBC MHK BJM. Contributed reagents/materials/analysis tools:
MHK BJM. Wrote the manuscript: MWH SBC MHK BJM.
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