Unexpected profile of sphingolipid contents in blood and bone marrow plasma collected from patients diagnosed with acute myeloid leukemia
Wątek et al. Lipids in Health and Disease
Unexpected profile of sphingolipid contents in blood and bone marrow plasma collected from patients diagnosed with acute myeloid leukemia
Marzena Wątek 3
Bonita Durnaś 0
Tomasz Wollny 3
Marcin Pasiarski 0 3
Stanisław Góźdź 0 3
Michał Marzec 2
Anna Chabowska 5
Przemysław Wolak 0
Małgorzata Żendzian-Piotrowska 4
Robert Bucki 0 1
0 Faculty of Medicine and Health Sciences of the Jan Kochanowski University in Kielce , Kielce , Poland
1 Department of Microbiological and Nanobiomedical Engineering, Medical University of Bialystok , 15-222 Bialystok , Poland
2 Department of Pathology and Laboratory Medicine, University of Pennsylvania , Philadelphia, PA , USA
3 Department of Hematology, Holy Cross Oncology Center of Kielce , Artwińskiego 3, 25-734 Kielce , Poland
4 Department of Hygiene, Epidemiology and Ergonomics Department Medical University of Bialystok , 15-222 Bialystok , Poland
5 Regional Blood Transfusion Center in Bialystok , 15-950 Bialystok , Poland
Background: Impaired apoptotic pathways in leukemic cells enable them to grow in an uncontrolled way. Moreover, aberrations in the apoptotic pathways are the main factor of leukemic cells drug resistance. Methods: To assess the presence of potential abnormalities that might promote dysfunction of leukemic cells growth, HPLC system was used to determine sphingosine (SFO), sphinganine (SFA), sphingosine-1-phosphate (S1P) and ceramide (CER) concentration in the blood collected from patients diagnose with acute myeloblastic leukemia (AML; n = 49) and compare to values of control (healthily) group (n = 51). Additionally, in AML group concentration of SFO, SFA, S1P and CER was determined in bone marrow plasma and compared to respective values in blood plasma. The concentration of S1P and CER binding protein - plasma gelsolin (GSN) was also assessed in collected samples using immunoblotting assay. Results: We observed that in AML patients the average SFO, SFA and CER concentration in blood plasma was significantly higher (p < 0.001) compare to control group, when blood plasma S1P concentration was significantly lower (p < 0.001). At the same time the CER/S1P ratio in AML patient (44.5 ± 19.4) was about 54% higher compare to control group (20.9 ± 13.1). Interestingly the average concentration of S1P in blood plasma (196 ± 13 pmol/ml) was higher compare to its concentration in plasma collected from bone marrow (154 ± 21 pmol/ml). Conclusions: We hypothesize that changes in profile of sphingolipids concentration and some of their binding protein partners such as GSN in extracellular environment of blood and bone marrow cells in leukemic patients can be targeted to develop new AML treatment method(s).
Sphingolipids; Ceramide; S1P; Acute myeloid leukemia; Hematological malignancies; Plasma gelsolin
Glycosphingolipids (GSLs) are a family of biomolecules
characterized by their pleiotropic effects on cell function
including signalling, regulation of cell proliferation and
apoptosis. Certain family members act as bioactive
lipids, relaying outside-in signals during inflammatory
reactions. The complexity of GSL biological functions is
derived in part from their localization to both intra- and
extracellular compartments. A number of GSLs are
involved in the differentiation of acute myeloid leukaemia
(AML) cells, as indicated by their increased expression
in the bone marrow of patients with AML compared to
healthy subjects [
]. Within the GSL family, sphingosine
(SFO) functions as an endogenous mediator of the
apoptotic signal. When HL-60 cells were exposed to SFO or
its methylated derivative, N,N-dimethylsphingosine,
intranuclear DNA fragmentation and morphological
changes characteristic to apoptosis were observed [
Certain proteins such as gelsolin (GSN) may act as
universal carriers/scavengers of sphingosine-1-phosphate
(S1P), serving to interfere with S1P activity [
interactions may be of importance in settings where the
concentration of both substances is outside of
homeostatic ranges [
]. GSN changes might be of grate
importance since it is a multifunctional
Ca2+/polyphosphoinositide-regulated actin-binding protein [
human blood plasma, the GSN concentration is 150–
300 μg/ml, and muscle is its principal birthplace [
Based on gelsolin’s ability to depolymerize actin
filaments, its function as actin scavenger has been
]. Another discussed role of gelsolin is its
involvement in regulation of inflammatory and
cancerpromoting processes through interactions with different
bioactive lipids including LPA, PAF and LPS, S1P
]. Human blood plasma gelsolin levels decline
markedly in a variety of acute clinical conditions
including major trauma , prolonged hyperoxia [
acute oxidant lung injury [
] malaria [
] and liver injury [
]. Interestingly, the level
of GSN is significantly increased in HIV-1 infected
patients compared to healthy volunteers . The
connection between decreasing gelsolin amount and
tissue injury and the possibility of therapeutic top up
of GSN to stop biological pathways of damaging
cascades are active areas of investigation [
The over-expression of sphingosine kinase-1 in
chemosensitive HL-60 cells results in a significant inhibition
of apoptosis, which is mediated by inhibition of
mitochondrial cytochrome c. The incubation of
chemoresistant cells with cell-permeable ceramide (CER) leads
to the inhibition of sphingosine kinase-1 and supresses
apoptosis. Moreover, F-12509a, a new inhibitor of
sphingosine kinase induces CER accumulation and
reduced S1P, which initiates the same sensitivity to
chemotherapy as chemo-sensitive and chemo-resistant cells;
This effect is inhibited by adding S1P or overexpressing
sphingosine kinase-1 [
]. Inhibition of the sphingosine
kinase-1 activity coupled with an increase in CER
generation has been shown in chemosensitive HL-60 cells. On
the contrary, the high activity of sphingosine kinase-1
but no ceramide generation during anti-cancer
treatment has been shown in chemo-resistant HL-60 cells.
S1P is present in plasma with high-density lipoproteins
and albumin complexes [
]. There has been a
significant effort to assess their involvement in cell
proliferation, differentiation and apoptosis during malignant
development. It is well documented that the increased
intracellular availability of CER induces DNA
degradation and death of human leukemic HL-60 and U937
cell lines [
]. Using traditional in vitro cellular models
(HL-60 cell line), many early studies revealed that
changes in sphingolipid metabolism in leukemic cells
can induce apoptosis and differentiation, which may
provide therapeutic benefit [
]. The increase in
ceramide concentration correlates with the differentiation
of HL-60 cells induced by treatment with vitamin D3
that is associated with increased of neutral SMase
activity in cell extracts and ceramide relesaed via hydrolysis
of sphingomyelin [
]. CER accumulation plays also a
key role in the death of AML-M2 cells induced by
] and blockage of protein kinase C
increases the pro-apoptotic influence that ceramide has on
human leukemia cells [
]. Manipulation of CER
metabolism to increase its production and accumulation
synergistically enhances the apoptotic effect of resveratrol in
HL60 cells. More importantly, gene expression analysis
showed that resveratrol induces apoptosis by
overexpression of genes that generate CER and a decrease in
the expression of sphingosine kinase-1 and genes that
synthesize glukozylceramid [
]. The accumulation of
ceremide during differentiation was also observed in
EL4 thymoma cells, cerebral Purkinji cells, and U937
monoblastic leukemia cells [
]. Furthermore, in
lymphoblastic leukemia cells apoptosis in response to
doxorubicin and vinca alkaloid is promoted by the
accumulation of ceramide [
]. Clinical observations indicate
that anti-leukemic agents, effective in eradicating blasts,
are relatively ineffective in eliminating leukemic
progenitor cells as indicated by a high recurrence rate in cases
of acute myelogenous leukaemia with high index of
complete remission (70%). This interpretation
underscores the natural chemo-resistance of cells that form
the myeloid leukaemia progenitor compartment. In the
last few years, studies have shown that similar cell
perturbations may cause effects such as quick apoptotic
death, differed mitotic death or non-lasting cytostatic
effect. Intracellular signalling is responsible for the specific
response to a given stimuli. These signals are mediated
in part by ceramide (cell death signal mediator) and
diacylglycerol and phosphoinositide-3 phosphates (PI3P)
(survival signal mediator). These observations underline
different possibilities of pharmacological
manipulations favouring the death of cells or their resistance
to anti-cancer agents [
Human blood and bone marrow specimen collection was
performed in the Holy Cross Oncology Center of Kielce.
The experiments were performed according to the
principles outlined in the Declaration of Helsinki and approved
by the Ethical Committee of the Jan Kochanowski
University in Kielce. At the time of patient recruitment,
written consent was obtained from all subjects and all
patients gave their informed consent prior to inclusion in the
study. We collected blood and bone marrow plasma from
patients with AML and blood samples from healthy
subjects. Samples were obtained from patients while
undergoing required diagnostic testing. Control samples were
obtained from healthy volunteers during testing to exclude
hematological diseases. Informed consent was obtained
prior to collection and use of the test material. The samples
of marrow and whole blood were placed into EDTA tubes
(K2 EDTA 5.4 mg) at room temperature, and were
centrifuged at 3200 rpm for 10 min to separate plasma which
was then transferred to fresh plastic tubes and frozen
−70 °C until use. Clinical and laboratory characteristics of
the patient groups are given in Table 1. AML was diagnosed
in patients using standard hematological identification.
Diagnosis of AML was confirmed by phenotypic and
cytological bone marrow testing. Because of the small
sample size we did not analyze sphingolipid
concentrations relative to cytogenetic group or FAB diagnosis.
Evaluation of S1P in blood plasma and CSF samples
The sphingosine-1-phosphate concentration was
measured by the method described in [
]. Briefly, acidified
methanol and an internal standard (30 pmol of C17-S1P,
Avanti Polar Lipids) were added to 250 μl of plasma or
CSF and then the samples were ultrasonicated in
icecold water for 1 min. The lipids were then extracted
with chloroform, 1 M NaCl, and 3 N NaOH. The
alkaline aqueous phase containing S1P was transferred to a
fresh tube. Residual S1P in the chloroform phase was
reextracted twice with a methanol/1 M NaCl (1:1, v/v)
solution and then all aqueous fractions were combined.
The amount of S1P was determined indirectly after
dephosphorylation to sphingosine using alkaline
phosphatase (bovine intestinal mucosa, Fluka, Milwaukee, WA).
To improve the extraction yield of released sphingosine,
chloroform was carefully placed at the bottom of the
reaction tubes. The chloroform fraction containing
dephosphorylated sphingoid base was washed three times
with alkaline water (pH adjusted to 10.0 with
ammonium hydroxide) and then evaporated under a nitrogen
stream. The dried lipid residues were re-dissolved in
ethanol, converted to their o-phthalaldehyde derivatives,
and analyzed using an HPLC system (ProStar, Varian
Inc.) equipped with a fluorescence detector and a C18
reversed-phase column (Varian Inc. OmniSpher 5,
4.6150 mm). The isocratic eluent composition of
acetonitrile (Merck): water (9:1, v/v) and a flow rate of 1 ml/
min were used. The column temperature was
maintained at 33 °C by use of a column oven.
After being thawed, gel sample buffer was added to
plasma or bon marrow samples that were then boiled
and subjected to electrophoresis on 10% polyacrylamide
gels in the presence of SDS. Recombinant human plasma
gelsolin (rhpGSN) was loaded as a standard in each gel
in a concentration range comparable to the gelsolin
concentration in the samples. After electrophoresis, proteins
were transferred to PVDF membranes (Amersham,
Biosciences Little Chalfont, UK), which were blocked by
incubation in 5% (w/v) non-fat dry milk dissolved in
TBS-T (150 mM NaCl, 50 mM TRIS, 0.05% Tween 20,
pH = 7.4). Following transfer, proteins were probed with
a monoclonal anti-human gelsolin antibody (Sigma, St
Louis, MO, USA). Both antibodies were used at 1:10,000
dilution in TBS-T. HRP-conjugated secondary antibodies
were used at 1:20,000 dilution in TBS-T. Immunoblots
were developed with the Fuji Film LAS-300 system using
an ECL Plus HRP-targeted chemiluminescent substrate
(Amersham, Biosciences Little Chalfont, UK). Densitometry
analysis was performed using Image Gauge (version 4.22)
software (Fuji Photo Film Co, USA).
A 1.6 104
Outline of blood sphingolipids
We observed a statistically significant increase in the
concentration of SFO, SFA (p < 0.001) and CER (p < 0.05)
in the blood of patients with AML compared to the
control group (Fig. 1a–d). Conversely, the S1P concentration
in blood plasma from AML patients was lower (p < 0.001)
compared to the control group (Fig. 1c). Based on the
assumption that the ultimate cellular effect of molecules
with opposite functions (CER promotes apoptosis while
S1P promotes proliferation) is based on their relative
abundance we evaluated the ratio of ceramide to SFO and
ceramide to S1P in both control and AML blood plasma
samples (Fig. 1e). The ratio between CER/S1P in the blood
of AML patients was significantly higher compared to the
control group (Fig. 1f, p<0.001). Additionally, we assessed
the correlation between concentrations of CER and S1P in
the blood plasma of the AML patients and control group
(Fig. 2a, b). Interestingly, a weak positive correlation was
been found between the concentration of ceramide and
S1P in the blood of AML group (R = 0.3375). In the
control group this trend was opposite (R = 0.3663) (Fig. 2a
and b, respectively).
Characterization of bone marrow sphingolipids
Although 49 AML patients were included in the study,
only 13 bone marrow samples were collected at volumes
large enough to perform HPLC. Despite these
limitations, comparison of SFA, SFO, S1P and CER blood and
bone marrow plasma concentrations revealed
measurable differences. The concentrations of SFO and SFA
were significantly higher (p < 0.001) in bone marrow
plasma (Fig. 3a and b), while the concentration of S1P
was significantly higher (p < 0.01) in blood plasma
(Fig. 3c). There was no difference between the
concentration of ceramide in bone marrow and blood
plasma collected from AML subjects (Fig. 3d). The
ratio between ceramides and S1P in the bone marrow
was significantly higher (p < 0.001) than in the blood
from AML patients (Fig. 3e). The correlation of the ratio
of the CER/S1P in the blood as compared to the bone
marrow did not reveal any trend (R = 0.0624). The
ceramide/S1P ratio in the bone marrow and blood from
AML patients had a low positive correlation (R = 0.0624,
Fig. 3f ). The correlation values between the concentration
of sphingolipids (SFO, S1P, ceramide) in the blood and
bone marrow of patients with AML was positive
(Fig. 4a–d). No trend was observed when the
correlation of SFA in bone marrow versus SFA in blood
plasma was plotted. Interestingly a strong positive
correlation between S1P (R = 0.6253) and ceramide (R = 0.7089)
concentration in bone marrow and blood plasma
(Figs. 4c, d) was recorded.
Gelsolin concentration in blood and bone marrow plasma
As shown in Fig. 5, in a limited number (n = 13) of bone
marrow samples, the range of measured gelsolin
concentration was 76.5–158.9 μg/ml and was comparable to lower
plasma gelsolin concentration in plasma of ALM patients
(45.3–132.7 μg/ml) when determined using quantitative
immunoblotting. The average gelsolin concentration in
plasma obtained from subjects included in control group
was within the range of 87.9 ± 274.3 μg/ml. Comparing all
samples, the lowest gelsolin concentrations were observed
in blood of patients diagnosed with ALM.
There is increasing evidence to support the hypothesis
that the use of infrared and many chemotherapeutic
agents on leukemic cells results in apoptosis in part
through changes in sphingolipid metabolism. It has been
shown that the accumulation of ceramide produced in
the process of sphingomyelin hydrolysis as well as in the
de novo synthesis play an important role as a mediator
of the leukemic cell death [
]. The CD95(APO-1/Fas)
antigen plays an important role in activating apoptosis
in leukemic T cell acute lymphoblastic leukaemia as well
as mediating cellular DNA fragmentation. Interestingly,
previous studies demonstrate that ceramide plays an
important role in the up regulation of CD95(APO-1/Fas)
]. Additionally, doxorubicin, daunorubicin, and
nucleoside analogues such as cytarabine or fludarabine,
which are used in induction chemotherapy of acute
leukemias, are able to induce apoptosis both through their
incorporation into cellular DNA as well as by
stimulating ceramide generation. Finally, several reports suggest
that the cytostatic drugs AraC, fludarabin, and VCR
activate apoptosis by inducing ceramide production [
In chronic myeloid leukemia S1P enhances the
antiapoptotic protein Mcl-1 (myeloid cell leukemia-1) and
its binding to the S1P receptor type 2 (S1PR2) [
acute myeloid leukemia, S1P induces mitogenic signaling
through activation of NF-kB [
] resulting in inhibition
of apoptosis in U937 and HL-60 cells [
S1P inhibits classical apoptosis in T acute lymphoblastic
leukemia (T-ALL) [
] and SPHK1 level is increased in
B-ALL, contributing to the development of murine
BCR/ABL1 ALL [
]. Results from these studies and
others lead to the conclusion that decreased levels of
ceramide and/or S1P are important regulators for the
resistance of leukemic cells to drug-induced apoptosis
]. This motion is also supported by reported
correlation of SK1 and S1P receptor expression in tumours
with patient survival and tamoxifen resistance in ER+
breast cancer [
]. On the other hand in cancer
associated with inflammation in contrast to oncogenic
SphK1, either overexpression or down-regulation of
SphK2 has been shown to inhibit cell growth and
promote apoptosis in a cell-dependent manner [
The studies shown here demonstrate that dynamic
changes in the extracellular concentration of lipids
belonging to sphingolipid signal transduction have
practical significance for AML patients. They suggest that, as
in tumours derived from epithelial cells, the regulation
of S1P and ceramide-mediated signal transmission may
be a target for the treatment hematopoietic
malignancies. Indirect evidence for this motion is the study
demonstrating that de novo ceramide production induces
spontaneous neutrophil apoptosis through the activation
of the caspase pathway. Studies on the role of ceramide
performed using the Molt-4 human leukaemia cell line
have confirmed that the ceramide molecules synthesized
de novo activate apoptosis in the presence of etoposide
]. Ceramide induces apoptosis through the
mitochondrial pathway, partly through the influence of Bcl-2
proteins. Studies conducted by Bose et al. demonstrate that
daunorubicin induces apoptosis of leukaemia U937 and
P388 by increasing the levels of ceramide [
abillity of FTY720P to induce apoptosis of AML cells in
leukemic mice suggests the ability of extracellular S1P to
affect cellular signalling pathway initiated by the
interaction of S1P with cell surface receptors [
outcome of sphingolipids concentration changes during
ALM development might also be affected by changes of
plasma level of their protein binding regulators such as
plasma gelsolin. Changes in plasma gelsolin can result
from disturbed gelsolin-actin interaction, binding of
gelsolin to cellular mediators, or modulation of gelsolin
synthesis in response to actin release [
] and might
affect the extracellular network of biopolymers [
Our experiments demonstrate significantly increased
plasma levels of sphingolipids in patients with acute
leukaemia, which is associated with decrease of plasma
gelsolin. The concentrations of SFO, SFA and ceramide are
higher in AML patients while the concentration of S1P
is reduced. It is possible that impaired sphingolipid
metabolism plays a role in AML disease progression.
Previously described studies demonstrating changes in
the activity of enzymes involved in sphingolipid
metabolism (glucocerebrosidase, galactocerebrosidase,
sphingomyelinase, acid phosphatase) in leukemic cells support
this hypothesis [
]. These conclusions are further
supported by observations demonstrating differences in
sphingolipid expression between healthy donors and
AML patients (AML patients had higher Lc3 and nLc4
expression than healthy subjects) [
The current dogma is that platelets are the main
source of S1P in the blood. Low platelet levels in AML
patients may partly explain the observation of low
plasma concentrations of S1P, particularly in patients
who experience, during the so called repression
mechanism, the reduction of level of platelets in the blood [
Generated data strongly indicate reduced concentrations
of lipids of the sphingolipid pathway involved in signal
transmission and suggest their participation in the
development of AML. The findings also suggest the
involvement of sphingolipids in AML cell differentiation. It is
known that SFO and ceramide exhibit pro-apoptotic
properties, while S1P promotes cell growth and survival
]. High concentrations of pro-proliferative (S1P) and
low concentrations of pro-apoptotic sphingolipids
(ceramide) might be expected in patients with a disease with
active leukemic cell proliferation. Furthermore, our data
suggest that the measured phospholipid concentration
profile may be lacking some informational molecule,
which initiates cell death. This observation is in line with
previous reports indicating that low levels of ceramide
and elevated sphingolipid pathway enzymes are
associated with acute leukemia cell chemoresistance [
sphingolipids and sphingolipids pathway enzymes may
represent potential therapeutic targets. In various
cancers, SPHK1 mRNA was significantly higher compared
to normal tissue and the bone marrow from AML
patients has much higher SPHK1 gene expression controls
]. Overall, a descriptive nature of presented data and
lack of functional studies that prove the implication of
these findings in leukemogenesis represent a limitation
of our study that in the future should be address with
knockdown experiments, use of inhibitors to manipulate
glycosphingolipids levels in vitro or in mouse models of
blood cancer development.
Based on our results, we conclude that sphingolipids
and their associated pathwyas may be used for either the
identification or forecast for the development of AML.
We also predict that the use of sphingolipids analogs will
be included in the spectrum of AML treatment.
AML: Acute myeloblastic leukemia; CER: Ceramide; FTY720P: phosphoric acid
ester of myriocin (structural analog of sphingosine); HPLC: High performance
liquid chromatography; LPA: Lysophosphatidic acid; LPS: Lipopolysaccharides;
PAF: Platelet activating factor; S1P: Sphingosine-1-phosphate; SFA: Sphinganine;
SFO: Sphingosine; SPHK1: Sphingosine kinase 1; VCR: Vincristine
This work was supported by the National Science Center, Poland under Grant:
UMO-2015/17/B/NZ6/03473 (to RB) and Medical University of Bialystok grant:
N/ST/ZB/16/003/1118 (to MŻP).
Availability of data and materials
All data generated or analysed during this study are included in this
MW, BD, TW, SG and RB contributed to the conception and design of research;
MW, MP, PW, MM, AC, MŻP performed the experiments and collected data;
MW, MŻP and RB analysed the data. MW prepared the figures; RB and MM
drafted the article and revised it critically. All authors participated in article writing.
All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
All authors have reviewed the manuscript and have agreed to its submission.
The authors declare that they have no competing interests.
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