Báo cáo khoa học: Hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis and the proliferation of HepG2 cells pot

Hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis
and the proliferation of HepG2 cells
Jose
´
M. Jime
´
nez-Lo
´
pez, Marı
´
a P. Carrasco, Josefa L. Segovia and Carmen Marco
Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Granada, Spain
Hexadecylphosphocholine (HePC) is a synthetic lipid rep-
resentative of a new group of antiproliferative agents,
alkylphosphocholines (APC), which are promising candi-
dates in anticancer therapy. Thus we have studied the action
of HePC on the human hepatoblastoma cell line HepG2,
which is frequently used as a model for studies into hepatic
lipid metabolism. Non-toxic, micromolar concentrations of
HePC exerted an antiproliferative effect on this hepatoma
cell line. The incorporation into phosphatidylcholine (PC) of
the exogenous precursor [methyl-
14
C]choline was substan-
tially reduced by HePC. This effect was not due to any
alteration in choline uptake by the cells, the degradation rate
of PC or the release of PC into the culture medium. As
an accumulation of soluble choline derivatives points to
CTP:phosphocholine cytidylyltransferase (CT) as the target
of HePC activity we examined its effects on the different
enzymes involved in the biosynthesis of PC via CDP–cho-

line. Treatment with HePC altered neither the activity of
choline kinase (CK) nor that of diacylglycerol cholinephos-
photransferase (CPT), but it did inhibit CT activity in
HepG2 cells. In vitro HePC also inhibited the activity of
cytosolic but not membrane-bound CT. Taken together our
results suggest that HePC interferes specifically with the
biosynthesis of PC in HepG2 cells by depressing CT trans-
location to the membrane, which may well impair their
proliferation.
Keywords: alkylphosphocholines; CTP:phosphocholine
cytidylyltransferase; phosphatidylcholine metabolism;
human hepatoblastoma cells; proliferation.
Hexadecylphosphocholine(HePC)isanewmembrane-active
antineoplastic compound belonging to the alkylphospho-
cholines (APC) group, which exert antitumoral activity
against a broad spectrum of established tumour cell lines [1].
It is currently used for the topical palliative treatment of
cutaneous metastases of mammary carcinomas [2]. There is
growing interest in the biological activity of these lipid
analogues as they do not interact with DNA but selectively
inhibit the growth of transformed cells and could well
complement existing DNA-directed anticancer chemo-
therapies.
APC administered either orally or intravenously accu-
mulate in different organs, including the liver [3]. Although
at present the systemic application of HePC to cancer
patients is limited because of the significant gastrointestinal
intolerance it often entails, alternative formulations of APC
treatment towards a variety of tumours are currently being
developed [4,5].

A wide variety of cytotoxic mechanisms have been
attributed to this class of antineoplastic compounds [6].
Their direct incorporation into the cell membrane, which
seems to be the primary site of their activity, suggests that
the molecular mechanism behind their effect involves some
membrane-dependent function. There is considerable evi-
dence to suggest that HePC could interfere with an early
step in lipid-signal transduction events, which might
therefore be responsible for its capacity to inhibit growth
[7].
Previous reports have indicated the ability of HePC to
interfere with phospholipid metabolism. However, a clear
mechanism of action has not been established yet. An
important mechanism for HePC-induced biological effects
may be the inhibition of phospholipase C [8], phospholipase
A
2
[9] or the activation of phospholipase D [10,11] which
may be achieved by protein kinase C (PKC) dependent or
independent mechanism, depending of the cell line investi-
gated [11]. The toxicity of HePC may also be related to a
disruption of calcium homeostasis [12]. Other authors have
shown that exposure of cells to HePC leads to a reduction in
the biosynthesis of phosphatidylcholine (PC) in MDCK
[13], HaCaT [14] and HL60 cells [15] by inhibiting the rate-
limiting enzyme CTP:phosphocholine cytidylyltransferase
(CT). In neuronal axons, on the other hand, HePC does not
seem to act in the same way but rather blocks choline
uptake by the cell [16].
In the light of these findings, our previous experience with

hepatic cells and research into the action of different
xenobiotics on phospholipid metabolism [17,18] has promp-
ted us to investigate the effect of HePC on the tumoral
Correspondence to C. Marco, Department of Biochemistry and
Molecular Biology, Faculty of Sciences, University of Granada,
Av. Fuentenueva s/n, Granada 18071, Spain.
Fax: + 34 958249945, Tel.: + 34 958243086,
E-mail:
Abbreviations: APC, alkylphosphocholines; CK, choline kinase; CP,
choline phosphate; CT, CTP:phosphocholine cytidylyltransferase;
CPT, diacylglycerol cholinephosphotransferase; EMEM, Eagle’s
minimum essential medium; HePC, hexadecylphosphocholine;
LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; SM, sphingomyelin.
Enzymes: choline kinase (CK) (ATP:choline phosphotransferase,
EC 2.7.1.32); CTP:phosphocholine cytidylyltransferase (CT)
(EC 2.7.7.15); diacylglycerol cholinephosphotransferase (CPT)
(CDP–choline:1,2-diacylglycerol cholinephosphotransferase,
EC 2.7.8.2)
(Received 15 May 2002, revised 18 July 2002, accepted 2 August 2002)
Eur. J. Biochem. 269, 4649–4655 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03169.x
hepatic cell line HepG2. We studied its cytotoxic and
cytostatic activity against HepG2 cells and examined its
influence on de novo PC biosynthesis by using the precursor
[methyl-
14
C]choline as radioactive marker. We measured
the effects of HePC on choline incorporation into soluble
intermediates in the CDP–choline pathway as well as PC,
the final product of this biosynthetic pathway. We also

analysed the effect of HePC on the enzyme activities
involved in the de novo biosynthesis of PC.
MATERIALS AND METHODS
Materials
[methyl-
14
C]choline chloride (55 CiÆmol
)1
), [methyl-
14
C]
choline phosphate (56 CiÆmol
)1
) and CDP-[methyl-
14
C]cho-
line (54 CiÆmol
)1
) were supplied by Amersham Pharmacia
Biotech (Freiburg, Germany). Fetal bovine serum was from
Roche Diagnostics (Barcelona, Spain). HePC, Eagle’s
minimum essential medium (EMEM) and thin-layer
chromatography (TLC) plates were from Sigma-Aldrich
(Madrid, Spain). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) was from Molecular Probes
(Leiden, The Netherlands). All other reagents were of
analytical grade.
Cell culture
The human hepatoma cell line HepG2 was obtained from
the European Collection of Animal Cell Cultures (Salisbury,

Wiltshire, UK). Cells were routinely grown in EMEM
supplemented with 10% v/v heat-inactivated fetal bovine
serum, 2 m
ML
-glutamine, 1% nonessential amino acids, and
an antibiotic solution (100 unitsÆml
)1
penicillin and
100 lgÆmL
)1
streptomycin) at pH 7.4 (complete medium).
Cells were seeded on tissue-culture plates (Nunc
TM
)for
adherent cells at densities of 3 · 10
4
cellsÆcm
)2
and incubated
at 37 °C in a humidified atmosphere of 95% air and 5%
CO
2
. The medium was replaced every 2 days and the cells
were subcultured by trypsinization before confluence.
The cells were used in experiments after 6 or 7 days
culture, by which time dense monolayers at about 70%
confluence had formed (approximately 3 · 10
6
cells per
60-mm dish). HePC was dissolved in phosphate-buffered

saline (NaCl/P
i
, pH 7.4) shortly before being added to the
culture medium to the required final concentration.
APC are readily bound to serum proteins such as
albumin or lipoproteins, thus lowering their uptake rate
into the cells and decreasing their biological activity.
Therefore, all experiments were carried out with cells
growing in medium supplemented with serum.
Assays for cell viability and proliferation
The cytotoxic effect of HePC on HepG2 cells was
determined by the MTT test, based upon the conversion
by viable cells of a tetrazolium salt into a blue formazan
product [19]. The toxicity of HePC against HepG2 cells was
also determined by the trypan blue exclusion assay and by
measuring the release of lactate dehydrogenase (LDH)
activity from the cytosol of damaged cells into the extra-
cellular medium. Cell proliferation was determined by
haemocytometer counting.
[Methyl-
14
C]choline incorporation
Cells were subcultured in 12-well culture plates and grown
in EMEM/10% fetal bovine serum as indicated above. Cells
in log-phase growth were incubated at 37 °Cfor6hin
complete medium containing [methyl-
14
C]choline (60 l
M
,

55 CiÆmol
)1
) and supplemented with 50 or 100 l
M
HePC,
whilst untreated cells were used as control. The medium was
then withdrawn and the cells were washed twice and
harvested by scraping with a rubber policeman into ice-cold
NaCl/P
i
. Lipids were extracted from the cells following the
procedure of Bligh and Dyer [20]. The chloroform layer was
dried by evaporation with a stream of nitrogen whilst the
water-methanol phase was collected and used to determine
water-soluble metabolites. We analysed the incorporation
of radioactive choline into PC and sphingomyelin (SM), and
also into soluble intermediaries of the CDP–choline path-
way. The different phospholipids were separated on silica-
gel 60 G TLC plates using a mixture of chloroform/
methanol/acetic-acid/water (60 : 50 : 1 : 4, v/v) as solvent.
The soluble metabolites from choline were separated by
TLC developed in methanol/0.6% NaCl/25% aqueous
NH
3
(50 : 50 : 5, v/v). The spots were made visible by
exposure to iodine vapour and ultraviolet light, and
assigned by means of standards. Radiometric measurements
of lipid spots were made by liquid scintillation using a
Beckman 6000-TA counter (Madrid, Spain). The values
were normalized to the quantity of cell protein determined

by Bradford’s method [21] with BSA as standard.
Choline uptake assay
Cells were preincubated for 5 min, 1.5 h or 6 h at 37 °C
either in a medium containing 50 l
M
HePC or with no
supplement as control. The medium was then removed and
the cells immediately exposed to a medium containing
[methyl-
14
C]choline (60 l
M
,55CiÆmol
)1
)for3minat
37 °C. The incorporation of [
14
C]choline was stopped by
medium aspiration followed by two washes with ice-cold
NaCl/P
i
containing 580 l
M
choline. The lipids were extrac-
ted directly from the attached cells according to Bligh and
Dyer [20]. After lipid extraction the aqueous and organic
phases were separated and the radioactivity of each phase
was measured. Incorporation was determined as the total
amount of radiolabel taken up by the cells.
Enzyme activities in the Kennedy pathway

Proliferating cells from 90-mm dishes were harvested by
scraping into ice-cold NaCl/P
i
, collected by low-speed
centrifugation and suspended in an ice-cold homogenizing
buffer containing 0.145
M
NaCl, 10 m
M
Tris/HCl, 1 m
M
EDTA, 10 m
M
KF, 0.2 m
M
phenylmethanesulfonyl fluoride
(pH 7.4), by using a volume equal to four times the volume
of the cellular pellet. Cells in suspension were sonicated for
2 s with a microprobe in an ice bath, and the homogenate
was centrifuged immediately at 105 000 g for 30 min at 4 °C
to yield a particulate fraction and the cytosolic supernatant.
The membrane pellet was suspended in ice-cold 0.25
M
sucrose, 10 m
M
Tris/HCl, 1 m
M
EDTA, 0.2 m
M
phenyl-

methanesulfonyl fluoride (pH 7.4). The protein concentra-
tion of each fraction was then measured. The determination
of marker enzymes indicated that this centrifugation
4650 J. M. Jime
´
nez-Lo
´
pez et al.(Eur. J. Biochem. 269) Ó FEBS 2002
procedure resulted in less than 8% contamination of the
microsomes and mitochondria in the cytosolic fraction.
Choline kinase assay. Choline kinase (CK) activity was
assayed by measuring the rate of [methyl-
14
C]choline
incorporation into choline phosphate (CP) according to
the method described by Pelech et al. [22], using approxi-
mately 50 lg of cytosolic protein.
CTP:phosphocholine cytidylyltransferase assay. CTP:
phosphocholine cytidylyltransferase (CT) activity was
determined in both the cytosolic and the particulate fraction
by measuring the formation of radiolabelled CDP–choline
from [methyl-
14
C]choline phosphate, as reported in
Weinhold and Feldman [23], using approximately 25 lg
of cytosolic or membrane protein. Enzyme activity in the
cytosolic fraction was measured in the presence of PC/oleate
liposomes (500 l
M
/500 l

M
) in the final reaction mixture,
unless stated otherwise, whilst membrane CT activity was
assayed without liposomes.
Diacylglycerol cholinephosphotransferase assay. Diacyl-
glycerol cholinephosphotransferase (CPT) was assayed by
measuring the rate of incorporation of CDP-[methyl-
14
C]
choline into PC, according to the method reported by
Cornell [24], using at least 50 lgofmembraneprotein.
Data analysis
Data are expressed as means ± SEM, as indicated in the
figure and table legends. Statistical comparisons were made
by variance analysis followed by the Bonferroni test using
the
SPSS
9.0 program. Values of P < 0.05 were considered to
be statistically significant.
RESULTS
Effect of HePC on cell toxicity and cell proliferation
We observed the toxic effect of 6 hours’ exposure to HePC in
proliferating HepG2 cells by measuring the leakage of LDH
into the extracellular medium and quantifying formazan
production from MTT (Fig. 1). HePC concentrations of less
than 100 l
M
caused no LDH to be released into the medium
but at higher doses there was a concomitant increase in LDH
activity. These results were confirmed by the formazan

produced from MTT, which also indicated that the cells were
affected by HePC at concentrations of more than 100 l
M
.
Thus in subsequent experiments we used concentrations
equal to or lower than 100 l
M
to avoid cell lysis.
The antiproliferative effect of HePC has already been
demonstrated in different tumour cells and cell lines [1] but
data are still unavailable for tumoral hepatic cells. To
examine the possible action of HePC on HepG2 prolifer-
ation we treated the cells with 25, 50, or 75 l
M
concentra-
tions for 48 h. A concentration of 25 l
M
did not change the
growth rate but after 48 h concentrations of 50 and 75 l
M
had reduced cell numbers significantly (Fig. 2). This anti-
proliferative action could not be put down to lysis as the
decrease in the number of viable cells was not accompanied
by a significant increase in nonviable cells, as measured by
the trypan blue exclusion assay and LDH release into the
medium.
Inhibition of PC biosynthesis by HePC
We examined the effect of HePC on the biosynthesis of PC
by using choline as exogenous precursor. Cells were incuba-
ted for 6 h with [methyl-

14
C]choline either in the presence or
absence of HePC. HePC inhibited the incorporation of
choline into both PC and SM in a dose-dependent manner
(Fig. 3). As far as the soluble intermediates in the CDP–
choline pathway are concerned, HePC caused a significant
increase in the label of CP and a decrease in that of CDP–
choline compared to controls (Fig. 3). These effects were
markedly enhanced at a concentration of 100 l
M
HePC. It is
interesting to note that the label in betaine, the product of
choline oxidation, fell after exposure to 100 l
M
HePC
(i.e. 0.77 ± 0.04 nmolÆmg protein
)1
in control cells vs.
Fig. 1. Cytotoxic effect of HePC on HepG2 cells. Cells growing in log-
phase were incubated for 6 h with different concentrations of HePC.
The release of LDH into the medium is given as absorbance at 340 nm
(A) and formazan production from MTT as absorbance measured at
570 nm with background subtraction at 630 nm (B). Significant dif-
ference from control is *P < 0.002.
Fig. 2. Effect of HePC on the proliferation of HepG2 cells. Cells
growing in log-phase were incubated with different concentrations of
HePC. Total cell numbers were determined by counting in a haemo-
cytometer. Results are expressed as means ± SEM for four inde-
pendent samples in duplicate. Significant differences from controls are
*P <0.01;**P < 0.001.

Ó FEBS 2002 Hexadecylphosphocholine and HepG2 cells (Eur. J. Biochem. 269) 4651
0.64 ± 0.02 nmolÆmg protein
)1
in 100 l
M
HePC-treated
cells) whilst concentrations of 50 or 100 l
M
HePC failed to
produce any alteration in the radioactivity associated to the
choline pool (0.34 ± 0.03 nmolÆmg protein
)1
in control
cells).
It has been widely demonstrated that oleate activates the
PC synthesis in HepG2 cells increasing the CT activity.
Thence, in another set of experiments we assayed the
combined effect of HePC and oleate. As it can be observed
in Table 1, exposure of HepG2 cells to oleate in the presence
or absence of HePC, significantly increases the choline
incorporation into PC. As oleate drastically reduces the
inhibitory effect of the APC on the PC formation, the HePC
50 l
M
does not significantly alter the synthesis of this
phospholipid in the presence of oleate.
These results suggest that in HepG2 cells, HePC produces
an alteration in PC biosynthesis via CDP–choline, although
a modification in the degradation rate of newly synthesized
PC could also contribute to this effect. To test this latter

possibility we prelabelled the cells with [methyl-
14
C]choline
for 24 h. The radioactive medium was removed and the
labelled cells were incubated for an additional period of 6 h
either in the presence or absence of HePC. The results
indicate that the APC did not affect the rate of intracellular
PC degradation as the radioactive label of PC was similar in
both control and HePC-treated cells and neither was the
secretion of labelled PC altered by exposure to HePC
(results not shown).
Effect of HePC on choline uptake by HepG2 cells
To determine its possible effect on choline uptake the cells
were exposed to HePC for different periods of time, pulsed
with [methyl-
14
C]choline for 3 min and the radioactivity
associated to the cells was measured. We chose this short
time period to avoid intracellular choline metabolism.
Exposure to 50 l
M
HePCforupto6hdidnotalter
choline uptake by HepG2 cells to any significant extent. (i.e.
51.3 ± 2.6 pmolÆmin
)1
per 10
6
cells in control vs.
50.3 ± 1.8 pmolÆmin
)1

per 10
6
cells in HePC-treated cells).
Effect of HePC on enzymes of the CDP–choline pathway
According to the observations made above, and bearing in
mind that the incorporation of choline into PC might be
inhibited by HePC interfering with the biosynthesis of PC,
we went on to analyse the enzyme activities involved in this
synthetic pathway. Cells were exposed to 50 l
M
HePC for
6 h and the cytosolic and particulate fractions were obtained
as described in the Materials and methods section. Neither
CK assayed in the cytosol nor CPT activity measured in the
particulate fraction were affected by HePC (Table 2). The
exposure of HepG2 cells to HePC resulted in a significant
inhibition in CT activity in the particulate fraction without
affecting the activity of cytosolic CT.
A good way of arriving at the real amount of soluble and
membrane-bound CT in the cell is to calculate the total
activity of both forms for each dish (3 · 10
6
cells). Thus we
determined the effect of 50 l
M
and 100 l
M
doses of HePC
upon the contribution of particulate and cytosolic CT to the
Table 1. Reversion by oleate of effect caused by HePC on phosphati-

dylcholine biosynthesis. Proliferating cells were incubated for 6 h in
EMEM/10% fetal bovine serum containing [methyl-
14
C]choline
(60 l
M
,55CiÆmol
)1
) and different concentrations of HePC, in the
presence or absence of oleate 1 m
M
BSA 1%. The incorporation of
[
14
C]choline into PtdCho was determined, as described in the Materials
and methods section. Results are expressed as means ± SEM for four
independent samples.
nmol PCÆmg protein
)1
None 50 l
M
HePC 100 l
M
HePC
Without oleate 3.91 ± 0.11 2.54 ± 0.21
a
2.02 ± 0.13
a
Oleate 1 m
M

5.46 ± 0.31 4.70 ± 0.11 4.30 ± 0.25
b
Significant differences from controls are
a
P < 0.002;
b
P < 0.03.
Fig.3. EffectofHePCon[methyl-
14
C]choline
incorporation into lipids in HepG2 cells. Pro-
liferating cells were incubated for 6 h in
EMEM/10% fetal bovine serum containing
[methyl-
14
C]choline (60 l
M
,55CiÆ mol
)1
)
and different concentrations of HePC. The
incorporation of [
14
C]choline into cellular
lipids and soluble intermediates of the CDP–
choline pathway was determined, as described
in the Materials and methods section. Results
are expressed as means ± SEM for four
independent samples in duplicate. Significant
differences from controls are *P < 0.02;

**P < 0.005.
4652 J. M. Jime
´
nez-Lo
´
pez et al.(Eur. J. Biochem. 269) Ó FEBS 2002
total enzyme activity in each dish. The results in Fig. 4
indicate that HePC caused a dose-dependent increase in
cytosolic CT activity and that this was accompanied by a
concomitant decrease in membrane-bound CT activity,
suggesting that it acts on PC biosynthesis by modulating CT
translocation between the membrane and the cytosol. It
must be considered that the subcellular fractionation
procedure could produce an alteration on the intracellular
distribution of this enzyme. Thence, in order to corroborate
the results above, we carried out the analysis of this enzyme
activity in a homogenate from control and HePC-treated
cells, demonstrating again that the total CT activity was
unaltered by APC whilst the measure in the absence of
liposomes, i.e. membrane-bound enzyme activity showed
again a significant diminution in treated cells (i.e.
1.57 ± 0.06 nmolÆmin
)1
Æmg protein
)1
in control cells vs.
1.22 ± 0.08 nmolÆmin
)1
Æmg protein
)1

in HePC-treated
cells, n ¼ 3; *P < 0.05).
One possibility we must also bear in mind is that HePC
might inhibit CT at the membrane level. To test this
hypothesis we incubated in vitro the corresponding subcel-
lular fraction in the presence of 50 l
M
and 100 l
M
HePC
and analysed the activity of the enzymes in the CDP–choline
pathway. We found that HePC did not in fact affect CK or
CPT, neither did it alter particulate CT activity (results not
shown). Nevertheless, as other authors have reported that
the effects of HePC can be modulated by the quantity of
lipid activator [15], we checked cytosolic CT in the presence
of different amounts of PC/oleate liposomes. As can be seen
in Fig. 5, the analysis of CT activity in the absence of
liposomes demonstrates the existence in HepG2 cells of an
active form of CT in the cytosol, which may correspond to
the H-form previously described in this cell line [25].
However, CT activity was about five times higher in the
presence of liposomes than in their absence.
Cytosolic CT activity was inhibited by HePC in the
presence of activating-lipid levels equal to or lower than
100 l
M
, although the degree of inhibition diminished
concomitantly with an increase in the quantity of liposomes
in the assay mixture (Fig. 5). Surprisingly, in the absence of

PC/oleate liposomes, the addition of 100 l
M
HePC mark-
edly increased soluble CT activity. HePC is a nonbilayer-
forming lipid and so in the assay mixture it must be as
micelles [10], which could act as a system for reconstituting
inactive soluble CT, thus increasing the enzyme activity.
DISCUSSION
It has recently been shown both in vitro and in vivo that
HePC exerts an antineoplastic effect [1,26], especially in the
topical treatment of skin metastases of human mammary
carcinoma [2,5], but to our knowledge no specific studies
Fig. 5. Influence of HePC in vitro on cytosolic CTP:phosphocholine
cytidylyltransferase activity in HepG2 cells. Cells growing in log-phase
were harvested, sonicated briefly, and the cytosolic fraction obtained
by centrifugation. Cytidylyltransferase activity was assayed in the
absence or presence of 100 l
M
HePC and different concentrations of
PC/oleate liposomes (molar ratio 1 : 1). Results are expressed as
means ± SEM for three independent samples in duplicate. Significant
differences from controls are *P <0.05;**P < 0.01.
Fig. 4. Influence of HePC treatment on CTP:phosphocholine
cytidylyltransferase activity in HepG2 cells. Cells growing in log-phase
were incubated for 6 h with 50 l
M
or 100 l
M
HePC, or no additive
(controls). Cytidylyltransferase (CT) activity was assayed both in the

cytosolic supernatant and the particulate pellet. Data are expressed as
percentages of distribution of cytosolic or membrane-bound CT
activities per dish (3 · 10
6
cells). Results are expressed as means ± SEM
for three independent samples in duplicate. Significant differences from
controls are *P <0.04;**P < 0.001.
Table 2. Influence of HePC treatment on enzyme activities in the CDP–
choline pathway. Cells growing in log-phase were incubated for 6 h
with 50 l
M
HePC whilst untreated cells were used as control. The
different enzyme activities in the cytosol and particulate fractions
[choline kinase (CK); CTP:phosphocholine cytidylyltransferase (CT)
and diacylglycerol cholinephosphotransferase (CPT)] were determined
as described in the Materials and methods section. Results are
expressed as means ± SEM for three independent samples in dupli-
cate. Significant difference from controls is *P <0.03.
nmolÆmin
)1
Æmg protein
)1
Enzyme activity Control 50 l
M
HePC treatment
CK 6.22 ± 0.10 5.63 ± 0.23
CT (cytosol) 9.57 ± 0.28 10.4 ± 0.97
CT (membranes) 1.82 ± 0.10 1.32 ± 0.09
a
CPT 1.49 ± 0.07 1.51 ± 0.04

Ó FEBS 2002 Hexadecylphosphocholine and HepG2 cells (Eur. J. Biochem. 269) 4653
have been made into its effects upon hepatic cell lines. Thus
we have examined the action of HePC, as a representative of
the APC group, on the hepatoma cell line HepG2. Firstly,
we evaluated the dose- and time-dependent cytotoxic and
cytostatic activity of HePC against HepG2 cells, proving
that concentrations of more than 100 l
M
for 6 h produce
alterations in plasma membrane permeability and thence a
rapid and unspecific detergent-like lytic effect. Nevertheless,
at concentrations of less than 100 l
M
we found a reduction
in viable cell numbers with no significant signs of toxicity.
The quantities of HePC required to produce this antipro-
liferative action agree with those encountered in MDCK
[13], HeLa [27] and other neoplastic cell lines [15], indicating
that HepG2 cells are moderately sensitive to the toxic and
cytostatic activity of HePC concentrations in the lmolar
range.
Little is known about the biochemical mechanisms by
which HePC, and probably also other lipid analogues,
mediate their antiproliferative activity. These compounds
are directly absorbed into both plasma and intracellular
membranes, where they accumulate [28]. It might be
assumed therefore that they interfere with membrane lipid
composition and metabolic processes, although the effects
of HePC on cell proliferation and metabolism may well
differ depending upon the cell type or line [29] and/or the

uptake rate into the cell [30]. Nevertheless, one consistent
finding is that HePC causes a reduction in the biosynthesis
of PC. In a similar way, our results demonstrate that in the
tumoral hepatic cell line HepG2, HePC hinders the
incorporation of radiolabelled choline into PC, the end-
product of the CDP–choline pathway. This reduction in the
incorporation of choline was not due to any alteration in its
uptake by the cells, a finding which agrees with that of
Geilen et al. [31] working with MDCK cells, but it does go
against observations made by other researchers working
with neuronal cells [16] and KB and Raji cells [32], in which
the authors showed that APC did in fact interfere with the
uptake of choline into the cell, leading to a decrease in PC
biosynthesis. In the present work we exposed the cells to
HePC between 5 min and 6 h and did not observe any
change in the uptake of choline, thus demonstrating that in
this cell line at least the mechanism via which HePC acts is
unrelated to the availability of intracellular choline.
Our results also indicate that the lower radioactivity in
PC from exogenous choline compared to control cells was
not due to any effect that HePC might have had upon the
degradation of cellular PC or its secretion into the medium.
These data contrast with those obtained with other cell lines
such as Raji and KB cells, in which a pronounced decrease
in the incorporation of [
14
C]choline into PC was paralleled
by an increase in the degradation of this phospholipid [32].
It is worth emphasizing that the reduction we observed in
the incorporation of exogenous choline into PC was

accompanied by an accumulation of radiolabel in CP and
a reduction in the radioactivity associated to CDP–choline,
suggesting that some of the enzymes involved in the
biosynthesis of PC must be the main target of HePC.
Cytosolic CK and membrane-bound CPT remained
unalterated by HePC whilst CT activity was significantly
modified. It is well documented that CT is inactive in the
cytosolic form but becomes active when bound to mem-
branes, and therefore one possible mode of CT regulation
would be via translocation between membranes and cytosol
[33]. After 6 h exposure to 50 l
M
HePC, CT activity
diminished by 20% in the particulate fraction of the cells
and increased in the cytosolic fraction, as can be seen when
the values are expressed as percentages of total CT activity.
These results demonstrate that HePC affects only the
distribution of CT and not its total activity. HePC therefore
appears to act on the CT translocation mechanism. It is
widely accepted that oleate stimulates PC synthesis in a
number of cells and tissues, promoting the translocation of
cytosolic CT to the membrane [34]. This prompted us to
investigate the combined effect of oleate and HePC on PC
synthesis. The results obtained demonstrate that the effect
of HePC is clearly buffered by oleate suggesting that this
fatty acid and HePC act in an opposite way. As oleate
favours the translocation of CT to membrane our results
corroborate the interference of HePC on this regulatory
process. Thence, HePC could act on CT activity either by
promoting the release of the enzyme from the membrane to

the cytosol or by hindering the insertion of the cytosolic
form into the membrane.
To explore these possibilities we carried out experiments
in vitro to analyse the effect of HePC on CT activity in the
cytosol and in the particulate fraction isolated from HepG2
cells. With regard to cytosolic CT, we looked into the effect
of HePC in the presence of different amounts of PC/oleate
vesicles as lipid activators. In the presence of low-levels of
activating lipid, the soluble activity was significantly
reduced, suggesting that HePC interferes with the translo-
cation of the soluble form of the enzyme to the lipid vesicles.
This inhibitory effect was not observed in the presence of
high concentrations of PC/oleate liposomes as HePC acts as
a competitive inhibitor of the lipid activator [15]. Moreover,
and in accordance with our hypothesis, membrane-bound
CT was not altered in vitro, demonstrating that HePC does
not promote the release of the enzyme from the membrane
to the cytosol and also that it did not affect the activity in the
membranes.
Our results demonstrate that HePC exerts a specific effect
on the biosynthesis of PC in HepG2 cells at the level of CT
activity. It interferes with the translocation process of CT
from the cytosol to the membrane and finally leads to an
inhibition of the biosynthesis of PC, which could in turn be
responsible for the antiproliferative effect exerted upon the
HepG2 cell line, all of which confirms the potential of this
lipid analogue as an antineoplastic agent.
ACKNOWLEDGEMENTS
This work was supported by a grant from DGES (PM97-0179).
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