ORIGINAL ARTICLE

Sphingolipid Content in the Human Uterus and Pair-MatchedUterine Leiomyomas Remains Constant

Knapp Paweł • Adrian Chabowski •

Jan Gorski

Received: 3 August 2012 / Accepted: 6 November 2012 / Published online: 14 December 2012

� AOCS 2012

Abstract In the present work we sought to investigate the

content of sphingolipids (sphingosine, sphinganine, sphin-

gosine-1-phosphate and ceramide) in human fibroids and

pair-matched healthy uterus tissue. We demonstrated that,

in uterine leiomyomas, the contents of sphingosine, sphin-

ganine, sphingosine-1-phosphate and ceramide remains

quite constant. However, a trend towards elevation of cer-

amide and simultaneous reduction of sphingosine-1 phos-

phate levels was also noticed. Additionally, in uterine

leiomyomas we found relevant activation of both PTEN and

MAPK(ERK1/2) signaling pathways with only a minor

change in AKT activity and relatively absent HIF-1a/

AMPK activation. In conclusion, rather modest changes in

sphingolipids are correlated with the activation of PTEN

and MAPK(ERK1/2) signaling proteins in human uterine

leiomyomas.

Keywords PTEN � MAPK(ERK1/2) � AKT � HIF-1a �Ceramide � Sphingolipids � Uterine leiomyomas

Abbreviations

S1P Sphingosine-1-phosphate

CER Ceramide

SPH Sphingosine

SPA Sphinganine

PTEN Phosphatase and tensin homolog

MAPK(ERK1/2) Mitogen activated protein kinase

AKT V-akt murine thymoma viral oncogene

homolog 1

HIF-1a Hypoxia inducible factor alpha

Introduction

Uterine leiomyomas (fibroids) are the most common

benign tumors in women. They start to be clinically

apparent in up to 25 % of all women and up to 30–40 %

women over 40 years old. However, the majority may still

remain undiagnosed mostly because fibroids are highly

asymptomatic. Importantly, hysterectomy is the only

effective medical treatment for this condition as the path-

ogenesis of uterine fibroids and their biology are still

poorly understood. For some, neoplastic transformation

may likely occur, with apparent changes in signaling

pathways [1] and altered sphingolipid metabolism [2]. It

appears that, at least in some cases of leiomyomas, the key

intracellular signaling pathways regulating cell prolifera-

tion and cell death are also disrupted. The important issue

regarding leiomyomas may not be the etiology per se but

rather why some fibroids grow faster (with increased risk

for neoplastic transformation) whereas others remain dor-

mant (and benign) for long time. Obviously, growth of

tumors depends on the balance between cell proliferation

and cell death, processes mediated by activation or deac-

tivation of different signaling pathways such as PI3

K/PKB/AKT, MAPK(ERK1/2) or AMPK [3]. Much less is

known regarding sphingolipid-based signaling pathways

and just a few studies related changes in sphingolipid

content with PI3 K/PKB/AKT or MAPK(ERK1/2) signal-

ing. It seems likely, that the major player on the crossroads

K. Paweł

Department of Gynecology and Gynecological Oncology,

Medical University of Bialystok, 24a Skłodowskiej Str.,

15-269 Białystok, Poland

A. Chabowski (&) � J. Gorski

Department of Physiology, Medical University of Bialystok,

2C Mickiewicza Str., 15-222 Białystok, Poland

e-mail: adrian@umwb.edu.pl

123

Lipids (2013) 48:245–250

DOI 10.1007/s11745-012-3746-2

of sphingolipid metabolism is ceramide (CER). Its pro-

duction is proceed by two key pathways: sphingomyelin

degradation evoked by increased activity of sphingomye-

linase, and by condensation of palmito-CoA with serine in

de novo synthesis. The rate limiting enzyme of the latter

pathway is serine palmitoyltransferase and as a result

3-ketosphinganine is formed, which is rapidly converted to

sphinganine (SPA). Next steps are acylation of sphinganine

to dihydroceramide by ceramide synthase and conversion

of the latter to CER by the dihydroceramide desaturase.

The main pathway of CER degradation is deacylation to

sphingosine (SPH) by ceramidase. Several lines of evi-

dence indicated that increased levels of CER and SPH,

SPA may influence tumor-suppressor processes such as

apoptosis, growth arrest and differentiation [4–6]. Sphin-

gosine may either be reacylated to CER or converted to

sphingosine-1-phosphate (S1P) by sphingosine kinase. In

opposite to CER, SPA (and SPH), S1P is regarded as a

tumor-promoting compound involved in cell proliferation,

transformation, inflammation and angiogenesis [4–6]. So

far, there are evidence for CER induced apoptosis of cancer

cells via induction of caspase-related enzymes [7]. Studies

in vitro, confirmed that, pharmacological induction of CER

accumulation causes apoptosis and based on this finding

CER is considered as major anti-tumor compound [5]. CER

acts also as a negative regulator of the PI3 K/AKT pathway

as this was shown not only in healthy [6], but also in neo-

plastic cells [8]. Interestingly, another bioactive sphingo-

lipid, namely S1P, likely favors the opposite effects

enhancing cellular cell proliferation and survival rather than

induction of apoptosis [4]. Likely, since the effects of CER

are inhibitory to PI3 K/AKT signaling, S1P, most probably,

activates this pathway [9]. Nonetheless, sphingolipids are a

complex and diverse group of lipids that has gained much

attention as intracellular signaling molecules involved in

cell differentiation, cell cycle arrest and apoptosis.

Considering the involvement of different signaling

pathways (such as PI3 K/AKT/MAPK(ERK1/2) and/or

sphingolipid-mediated) on cell growth and survival we

examined the content of selected sphingolipids in healthy

human myometrium and uterine leiomyomas as well

as the expression of total and phosphorylated AKT,

MAPK(ERK1/2), PTEN and AMPK proteins.

Materials and Methods

Patients and Tissue Collection

The present study conforms with the principles outlined in

the Declaration of Helsinki and was approved by the Eth-

ical Committee for Human Studies of the Medical Uni-

versity of Bialystok. All patients gave their informed

consent prior to their inclusion in the study. Between

January 2010 and December 2011 the group of women

were investigated in the Department of Gynecology and

Gynecological Oncology, Medical University of Bialystok,

Poland. Diagnosis of fibroids were first confirmed by two

independent ultrasonographers in out-patient clinic. Nor-

mal myometrial specimens and fibroids were obtained from

45 patients, aged 38–51 years old (average age 42.3 years)

undergoing hysterectomy with no hormonal or other

medications history treatment for at least 6 months before

surgery. The average body mass index of the patients was

22.5 kg/m2. All patients included in the study declared

having a regular menstrual period (27–31 days). There

were no histories of internal disease (hypertension, diabe-

tes, or other endocrine disease) and blood biochemical

examinations abnormalities in these women. Surgical

procedures were routinely performed in the follicular phase

of the menstrual cycle.

The sizes of the dissected fibroids ranged from 10 up to

50 mm in diameter. Uterine leiomyomas included for the

study were 35–40 mm in diameter and situated within the

myometrial wall of the uterus. The specimens included for

the study were obtained just under the capsule of the tumor

in each case. Healthy myometrial tissues were obtained

from surrounding normal myometrium situated more than

20 mm away from the fibroid capsule and were used as

paired controls. All specimens were examined by pathol-

ogist to exclude adenomyosis or any malignant changes

and/or inflammatory infiltration. The tissue samples used

for this study were confirmed as histologically ordinary

fibroids. All specimens after dissection were immediately

frozen in liquid nitrogen and were stored at -80 �C until

use.

Ceramide Content

Prior to sample homogenization, an internal standard

(C17-sphingosine, Avanti Polar Lipids, UK) was included

and then the samples were homogenized. Subsequently,

lipids were extracted as described recently [2]. CER

present in the obtained chloroform phase (50 ll) was

hydrolyzed with 1 M KOH in 90 % methanol (90 �C for

60 min). The content of free SPH, liberated from Cer was

then analyzed by means of an HPLC instrument (ProStar,

Varian, Inc., Palo Alto, CA) equipped with a fluorescence

detector and a C18 reversed-phase column (Varian, Inc.,

OmniSpher 5, 4.6 9 150 mm). The calibration curve was

prepared using N-palmitoylsphingosine (Avanti Polar

Lipids, UK) as a standard. The chloroform extract used

for the analysis of CER level contained also small

amounts of free sphingoid bases. Therefore, the content of

CER was corrected for the level of free SPH determined

in the same sample.

246 Lipids (2013) 48:245–250

123

Sphingosine, Sphinganine and Sphingosine-1-

Phosphate Content

The CER derivatives were measured as described previously

[2, 10]. Prior to samples homogenization and ultrasonica-

tion, internal standards were included (C17-sphingosine

and C17-S1P, Avanti Polar Lipids, Alabaster, AL). The

sphingoid bases were converted to their o-phthalaldehyde

derivatives and analyzed on a HPLC system as indicated

above.

Western Blot Analyses

Routine western blotting procedures were used to detect

proteins as described previously [11]. Briefly, the

expression of selected proteins (total and phosphorylated

forms of AKT, MAPK(ERK1/2), PTEN, and AMPK, Cell

Signaling Technology, US, and HIF-1a, b-actin, Abcam,

EU) was determined in homogenates from healthy myo-

metrium (n = 45), and fibroids (n = 45). The tissue was

homogenized in a lysis RIPA buffer (HEPES 20 mmol/L,

EDTA 2 mmol/L, EGTA 2 mmol/L, Triton 1 %, PMSF

5 lmol/L, Na3VO4 50 lmol/L) containing a protease

inhibitor cocktail (Sigma, St. Louis, USA). After centri-

fugation at 10,000g for 10 min at 4 �C, protein concen-

tration was determined by use of the BCA protein assay

kit (Pierce). The total proteins (90 lg) were denatured

and separated using 10 % SDS–polyacrylamide gel elec-

trophoresis with subsequent transfer to a nitrocellulose

membrane (Bio-Rad, CA, USA). Equal protein concen-

trations were loaded in each lane as also confirmed by

Ponceau staining the blot membrane. Membranes were

blocked at room temperature for 2 h in Tris-buffered

saline/Tween 20 (TBST) (20 mmol/L Tris, pH 7.6;

137 mmol/L NaCl; 0.1 % Tween 20) containing 10 %

nonfat dry milk. After that, the membranes were immu-

noblotted with adequate selected primary (as described

above) and secondary antibodies (Santa Cruz Biotech-

nology, HRP conjugated). Signals obtained by western

blotting were quantified by densitometry (Biorad, Poland).

The relative expression (optical density arbitrary units) of

each protein (total and phosphorylated) was calculated as

a respective ratio and HIF-1a was related to b-actin

expression.

Statistical Analysis

Data are presented as means ± SE. Statistical significance

was assessed using the Student T test for paired samples,

and/or two-way ANOVA followed by Newman–Keuls

post hoc test. Differences were considered significant at

p \ 0.05.

Results

Sphingolipid Content

The content of both SPH and SPA was relatively similar in

the human uterus and uterine leiomyomas (Fig. 1a,b). In

contrast to SPH and SPA levels, a trend towards a decrease

in S1P (-26 %, p = 0.091, Fig. 1c) with concomitant, but

not significant, increase in CER (?11 %, p [ 0.5, Fig. 1d)

in uterine leiomyomas as compared to normal uterus was

observed. This trend was further enhanced by difference in

the ratio of S1P to CER between the tissues examined (data

not shown).

Signaling Pathways Protein Expression

The expression of both total and phosphorylated MAP-

K(ERK1/2) proteins was significantly higher in leiomyo-

mas compared with patient-matched myometrium, which

resulted in an increased ERK1/2 vs p ERK1/2 ratio

(?42 %, p \ 0.05, Fig. 2). Similarly, although to a lesser

degree, PTEN and phospho-PTEN (Ser380) expression was

greater in leiomyomas and the respective PTEN/pPTEN

ratio was increased (?35 %, p \ 0.05, Fig. 2). Concomi-

tantly, a trend toward an increase in total and phosphory-

lated AKT (Ser473) in fibroids was observed (?7 %,

p [ 0.05, Fig. 2) and no significant changes were noticed

for either HIF-1a (?6 %, p [ 0.05, Fig. 2) or total or

phosphorylated AMPK (Thr172) (-4 %, p [ 0.05, Fig. 2).

Rang Spearman Correlations

We found only a positive correlation between the content

of CER and activation of either MAPK(ERK1/2) and

PTEN/pPTEN kinases in the group of uterine leiomyomas.

Accordingly no other significant relations were observed

between sphingolipids and activation of signaling proteins

in this group (Table 1).

Discussion

The present study demonstrated that in uterine leiomyomas

the content of basic sphingolipids remains quite constant.

However, a trend towards an increase in CER content with

a concomitant, but not significant decrease in S1P level

was observed. These changes were accompanied by a

significant increase in phosphorylation of PTEN and

ERK1/2 proteins, but not AKT or AMPK kinases. Aware

of the influence of estrogen and estrogen-related genes that

are thought to play a key role in the growth of uterine

leiomyomas [12, 13], we included exclusively patients in

the proliferative phase of their menstruation cycle. The

Lipids (2013) 48:245–250 247

123

results were pair-matched since the samples were from the

same patient. Importantly, we eliminated also small

(\30 mm) and large ([50 mm) fibroids from our study,

since it has been shown that more extensive leiomyomas

show higher levels of AKT compared with small ones [14].

It was also shown that the content of PTEN protein may be

size and cycle-depended [15]. Furthermore, modest size of

the samples was chosen to prevent the influence of hypoxia

in the core of large fibroids, since ischemia is a well-known

factor activating AMPK kinase, which modulates substrate

metabolism of the cells [16]. Accordingly, we observed no

changes in HIF-1a expression between healthy myome-

trium and fibroid tissues, although, we need to stress that

the content of HIF-1a in both was relatively low, as also

reported by others [17]. Based on the aforementioned

observations, we can speculate that the observed changes in

signaling pathways were related to the growth potential of

the fibroid cells and were not affected by hypoxia. We can

also suggest that the observed activation of PTEN protein

with subsequent modest activation of AKT, at least in part,

0,0

0,1

0,1

0,2

0,2

Sphingosine

nmol

x g

-1 w

et w

eigh

t

B

0,5

0,7

0,9

Sphinganine

nmol

x g

-1 w

et w

eigh

t

A

Sphingosine-1-Phosphate

0,0

0,1

0,1

0,2

0,2

nmol

x g

-1 w

et w

eigh

t

D

15,0

16,0

17,0

18,0

19,0

20,0

Ceramide

nmol

x g

-1 w

et w

eigh

t

C

Fig. 1 Sphingolipid content

(sphingosine, sphinganine,

ceramide, sphingosine-1-

phosphate) in healthy and pair-

matched leiomyomas (n = 35),

as determined by HPLC

(nM/g wt)

Fig. 2 Representative western blots of the examined signaling

proteins MAPK(ERK1/2) PTEN, AKT and AMPK (optical density

units, ODU) presented as the total vs the phosphorylated isoform of

the respective protein. Healthy myometrium protein expression of

respective signaling kinases activation was set at 100 %

248 Lipids (2013) 48:245–250

123

was a consequence of increased CER levels in fibroids. It is

well recognized that an increased intracellular level of CER

induces the activity of phosphatase PPA2, which subse-

quently dephosphorylates AKT proteins. Probably, CER

accumulation could also recruit PTEN to the plasma

membrane (i.e., lipid rafts) with resultant AKT deactivation

[18]. It is tempting to speculate that the observed trend

towards an increase in CER content induced inhibition of

the AKT pathway that may decrease the cell proliferation

potential and promote apoptosis of leiomyoma cells.

However, this effect is speculative, since others have

reported either no difference in PTEN activation, between

healthy smooth muscles and leiomyosarcomas or atypical

myomas and/or leiomyomas [19]. In our study, the activity

of AKT was rather modest, due to either PTEN activation

and/or CER accumulation, but others have also reported

higher levels of phosphorylated Akt (Ser 473), however,

exclusively in leiomyomas from the growing period of

tumors [15].

Sphingolipid metabolism in benign tumors is poorly

investigated, as only very few studies presents data

regarding the CER/S1P axis [20–22]. So far, the existence

of this interplay has been shown in an animal model of rat

uterine leiomyoma cells (ELT3). In these cells SphK1/S1P

axis controlled cell survival and proliferation [23] and

SphK1 activation required former activation of MAP-

K(ERK1/2). Correspondingly, we and others [24] observed

an increase in the level of phosphorylated MAPK(ERK1/2)

in the leiomyoma compared to the matched myometrium.

Several studies had shown that MAPK(ERK1/2) activation

plays a prominent role in the regulation of cell cycle pro-

gression and cellular proliferation and constitutively acti-

vated MAPK(ERK1/2) (phospho-p44/42 MAPK(ERK1/2))

is highly expressed in leiomyomas [25].

Interestingly, our previous study showed that the content

of SPA, SPH, CER (and dihydroceramide) as well as S1P

in human endometrial cancers is markedly augmented

comparing to the healthy tissue. The accumulation of SPA

and CER was accompanied by a concomitant increased

activation of serine palmitoyltransferase which is the first

rate-limiting enzyme involved in the de novo CER syn-

thesis pathway [2]. In the present study we may speculate

that in fibroids, augmented CER formation was accompa-

nied by an increased rate of its degradation by ceramidase,

since we observed a relatively similar level of SPH

(Fig. 1). This observation is in line with the studies

showing upregulation of acid ceramidase in several benign

human tumors [26, 27]. However, in our study fibroids

were characterized by rather modest increase in the content

of CER, likely due to the medium size of examined uterine

leiomyomas. Based on our present (fibroids) and previous

study (endometrial cancer) we may also speculated that

with the neoplastic progression towards more aggressive

neoplasms (fibroids vs endometrial cancer) there is a

marked increase in sphingolipid content (reported here and

in [2]).

In conclusion we report quite stable content of sphin-

golipids in human uterine leiomyomas when compared to

healthy myometrium or endometrial cancer [2]. This lack

of significant changes was accompanied by modest acti-

vation of either PTEN and MAPK(ERK1/2) kinases, but

not AMPK.

Acknowledgments The study was supported by the Medical Uni-

versity of Białystok (#3-18788, 3-18710, 3-18577).

Conflict of interest The authors declare no conflict of interest.

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Rang Spearman

correlations (r)

pERK/

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* Significance set at p \ 0.05

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