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.. Assays fo
Trang 1Hexadecylphosphocholine 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-14C]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 CTtrans-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
A2[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: cmarco@ugr.es
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)
Trang 2hepatic 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-14C]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
M A T E R I A L S A N D M E T H O D S
Materials
[methyl-14C]choline chloride (55 CiÆmol)1), [methyl-14C]
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 mM L-glutamine, 1% nonessential amino acids, and
an antibiotic solution (100 unitsÆml)1 penicillin and
100 lgÆmL)1streptomycin) at pH 7.4 (complete medium)
Cells were seeded on tissue-culture plates (NuncTM) for
adherent cells at densities of 3· 104cellsÆcm)2and incubated
at 37C in a humidified atmosphere of 95% air and 5%
CO2 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· 106 cells per
60-mm dish) HePC was dissolved in phosphate-buffered
saline (NaCl/Pi, 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-14C]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 37C for 6 h in complete medium containing [methyl-14C]choline (60 lM,
55 CiÆmol)1) and supplemented with 50 or 100 lMHePC, 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/Pi 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
NH3 (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 37C either in a medium containing 50 lM HePC or with no supplement as control The medium was then removed and the cells immediately exposed to a medium containing [methyl-14C]choline (60 lM, 55 CiÆmol)1) for 3 min at
37C The incorporation of [14C]choline was stopped by medium aspiration followed by two washes with ice-cold NaCl/Picontaining 580 lMcholine 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/Pi, collected by low-speed centrifugation and suspended in an ice-cold homogenizing buffer containing 0.145M NaCl, 10 mM Tris/HCl, 1 mM EDTA, 10 mMKF, 0.2 mMphenylmethanesulfonyl 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 4C
to yield a particulate fraction and the cytosolic supernatant The membrane pellet was suspended in ice-cold 0.25M sucrose, 10 mM Tris/HCl, 1 mM EDTA, 0.2 mM phenyl-methanesulfonyl fluoride (pH 7.4) The protein concentra-tion of each fracconcentra-tion was then measured The determinaconcentra-tion
of marker enzymes indicated that this centrifugation
Trang 3procedure 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-14C]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-14C]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 lM/500 lM) in the final reaction mixture,
unless stated otherwise, whilst membrane CTactivity was
assayed without liposomes
Diacylglycerol cholinephosphotransferase assay
Diacyl-glycerol cholinephosphotransferase (CPT) was assayed by
measuring the rate of incorporation of CDP-[methyl-14C]
choline into PC, according to the method reported by
Cornell [24], using at least 50 lg of membrane protein
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
theSPSS9.0 program Values of P < 0.05 were considered to
be statistically significant
R E S U L T S
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 lMcaused 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 lM
Thus in subsequent experiments we used concentrations
equal to or lower than 100 lMto 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 lM
concentra-tions for 48 h A concentration of 25 lMdid not change the
growth rate but after 48 h concentrations of 50 and 75 lM
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-14C]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 lMHePC It is interesting to note that the label in betaine, the product of choline oxidation, fell after exposure to 100 lM 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.
Trang 40.64 ± 0.02 nmolÆmg protein)1 in 100 lM HePC-treated
cells) whilst concentrations of 50 or 100 lMHePC 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 CTactivity
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 lM 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-14C]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-14C]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 lM HePC for up to 6 h did not alter choline uptake by HepG2 cells to any significant extent (i.e 51.3 ± 2.6 pmolÆmin)1 per 106 cells in control vs 50.3 ± 1.8 pmolÆmin)1per 106cells 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 lMHePC 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 CPTactivity measured in the particulate fraction were affected by HePC (Table 2) The exposure of HepG2 cells to HePC resulted in a significant inhibition in CTactivity 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 CTin the cell is to calculate the total activity of both forms for each dish (3· 106cells) Thus we determined the effect of 50 lMand 100 lMdoses of HePC upon the contribution of particulate and cytosolic CTto 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-14C]choline
(60 l M , 55 CiÆmol)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 ± 0.21a 2.02 ± 0.13a
Oleate 1 m M 5.46 ± 0.31 4.70 ± 0.11 4.30 ± 0.25 b
Significant differences from controls areaP < 0.002;bP < 0.03.
Fig 3 Effect of HePC on [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-14C]choline (60 l M , 55 Ci Æ 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.
Trang 5total enzyme activity in each dish The results in Fig 4
indicate that HePC caused a dose-dependent increase in
cytosolic CTactivity and that this was accompanied by a
concomitant decrease in membrane-bound CTactivity,
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 CTactivity 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)1Æmg protein)1 in control cells vs
1.22 ± 0.08 nmolÆmin)1Æmg protein)1 in HePC-treated cells, n¼ 3; *P < 0.05)
One possibility we must also bear in mind is that HePC might inhibit CTat the membrane level To test this hypothesis we incubated in vitro the corresponding subcel-lular fraction in the presence of 50 lMand 100 lMHePC 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 CTin the presence
of different amounts of PC/oleate liposomes As can be seen
in Fig 5, the analysis of CTactivity in the absence of liposomes demonstrates the existence in HepG2 cells of an active form of CTin the cytosol, which may correspond to the H-form previously described in this cell line [25] However, CTactivity was about five times higher in the presence of liposomes than in their absence
Cytosolic CTactivity was inhibited by HePC in the presence of activating-lipid levels equal to or lower than
100 lM, 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 lMHePC mark-edly increased soluble CTactivity 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
D I S C U S S I O N
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)1Æmg protein)1 Enzyme activity Control 50 l M HePC treatment
CT(cytosol) 9.57 ± 0.28 10.4 ± 0.97
CT(membranes) 1.82 ± 0.10 1.32 ± 0.09 a
Trang 6have 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 lM 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 lMwe 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 [14C]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 CPTremained
unalterated by HePC whilst CTactivity was significantly
modified It is well documented that CTis inactive in the
cytosolic form but becomes active when bound to
mem-branes, and therefore one possible mode of CTregulation
would be via translocation between membranes and cytosol
[33] After 6 h exposure to 50 lM HePC, CTactivity 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 CTactivity These results demonstrate that HePC affects only the distribution of CTand not its total activity HePC therefore appears to act on the CTtranslocation mechanism It is widely accepted that oleate stimulates PC synthesis in a number of cells and tissues, promoting the translocation of cytosolic CTto 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 CTto 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 vitroto analyse the effect of HePC on CTactivity 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 CTwas 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
A C K N O W L E D G E M E N T S
This work was supported by a grant from DGES (PM97-0179).
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