Department of Neurophysiology, University of Cologne, Robert-Koch-Strasse 39, D-50931 Cologne, Germany
* Author for correspondence (e-mail: mw{at}physiologie.uni-koeln.de )
Accepted 21 May 2002
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Summary |
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Key words: Electric field, Multicellular tumor spheroid, ATP release, Anion channel, Intracellular calcium
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Introduction |
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In the present study we report on the underlying mechanisms of change in
intracellular Ca2+ and the subsequent growth stimulation of
multicellular tumor spheroids, which are well established model systems for
avascular micrometastases (Sutherland,
1988; Acker et al.,
1987
; Mueller-Klieser et al.,
1986
) with significant endogenous drug resistance
(Wartenberg et al., 2000
;
Wartenberg et al., 1998
;
Olive and Durand, 1994
;
Olive et al., 1997
). Changes
in intracellular Ca2+ following EMF treatment of cells have been
reported previously and have been attributed mainly to transmembrane
Ca2+ influx through voltage-gated Ca2+ channels, and/or
activation of stretch-activated cation channels which, upon opening, permit
the influx of cations including Ca2+
(Cho et al., 1999
;
Ihrig et al., 1997
;
Onuma and Hui, 1988
). As a
further mechanism of intracellular Ca2+ elevation in response to
EMF fields the stimulation of cell membrane receptors with subsequent
activation of phospholipase C, generation of inositol triphosphate and
Ca2+ release from intracellular stores has been discussed
(Eichwald and Kaiser, 1995
;
Eichwald and Kaiser, 1993
). In
previous studies, we have demonstrated that DC electrical fields transiently
elevate intracellular Ca2+ in multicellular prostate tumor
spheroids by a mechanism involving an elevation of intracellular ROS
(Wartenberg et al., 1997
).
This elevation of intracellular Ca2+ was mediated by
Ca2+ release from intracellular stores and resulted in growth
stimulation of multicellular tumor spheroids. The data of the present study
indicate that electrical field treatment of multicellular tumor spheroids
results in the release of intracellular ATP via anion channels to the
extracellular compartment, which then may activate purinergic receptors and
elicit a transient Ca2+ response. ATP release has been reported to
occur in a variety of preparations after mechanical stretch
(Mitchell, 2001
;
Mitchell et al., 1998
;
Cotrina et al., 1998
;
Sabirov et al., 2001
;
Verderio and Matteoli, 2001
;
Ostrom et al., 2001
) and, in
prostate cancer cells grown in monolayer culture, it has been demonstrated by
us to elicit a Ca2+ wave propagating radially from the site of
mechanical perturbation (Sauer et al.,
2000
). It is demonstrated that a comparable mechanism may be
involved in the elevation of intracellular Ca2+ and growth
stimulation following treatment of multicellular tumor spheroids with DC
electrical fields.
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Materials and Methods |
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Culture technique of multicellular tumor spheroids
The human androgen-insensitive prostate cancer cell line DU-145 was used
for the experiments. Monolayers were cultivated in 25 cm2 tissue
culture flasks (Greiner, Solingen, Germany) in 5% CO2-humidified
air at 37°C. The monolayer cultures were trypsinized and replated once a
week. Cell culture medium was Ham's F-10 medium (Invitrogen, Fernwald,
Germany) supplemented with 10% fetal calf serum (Invitrogen), 100 IU/ml
penicillin, and 100 µg/ml streptomycin (Invitrogen). Spheroids were grown
from single cells seeded in siliconized 250 ml spinner flasks (Integra
Biosciences, Fernwald, Germany) at a cell density of 1x105
cells/ml. The spinner flask medium (175 ml) was stirred at 25 rpm, using a
stirrer system (Cell Spin, Integra Biosciences) and partly changed every day.
For the experiments, tumor spheroids with a diameter of 100±50 µm
were used.
Electrical field treatment and confocal laser scanning
microscopy
Electrical field pulses were applied to multicellular tumor spheroids under
the optical control (fluorescence/transmission mode) of an inverted confocal
laser scanning microscope (LSM 410; Zeiss, Jena, Germany) using a 25x
oil immersion objective, numerical aperture 0.8 (Zeiss, Neofluar). Processing
of images (512x512 pixels, 8 bit) was carried out using the
Time-software facilities of the confocal setup. Full-frame images were
acquired and stored automatically at 2 second time intervals to a 16-megabyte
video memory of the confocal setup. Each series of images was scaled between
pixel intensity 0 (background fluorescence) and 255 (maximum fluorescence in
that series). The minimum, maximum, mean, standard deviation and integrated
sum of the pixel values in a region of interest (selected using an overlay
mask) were written to a data file and routinely exported for further analysis
to the commercially available Sigma Plot (Jandel Scientific, Erkrath, Germany)
graphic software.
For electropulse experiments, multicellular tumor spheroids were suspended
in a low-ionic content
N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid
(Hepes) (5 mM, pH 7.2)-buffered `pulsing medium' that contained 255 mM
sucrose, 1 mM CaCl2 and 1 mM MgCl2, and had a
conductivity of 500 µScm-1. They were then placed in an
incubation chamber between stainless steel electrodes with an electrode area
of 0.4 cm2 and an electrode distance of 0.2 cm. The electrodes were
connected to a custom-made voltage generator, which gave square electric
pulses. Voltage pulses of a field strength of 750 Vm-1 (1.5 V at
the electrodes) and a duration of 60 seconds were applied to multicellular
tumor spheroids. The total current in the chamber was 1.5 mA. The magnetic
flux density in the proximity of the tumor spheroids was calculated to 0.079
µT, which is below the average laboratory noise level for low-frequency
EMFs (9.5 µT) (Cameron et al.,
1993
). In control experiments without tumor spheroids we ensured
that no water hydrolysis, ROS production, or pH and temperature shifts
occurred in the pulsing chamber during the duration of the experiments.
Ca2+ imaging during electrical field exposure
[Ca2+]i was monitored using the fluorescent dye
fluo-3,AM (Molecular Probes, Eugene, OR). Multicellular tumor spheroids were
loaded for 45 minutes in F-10 cell culture medium with 10 µM fluo-3,AM
dissolved in dimethyl sulfoxide (final concentration 0.1%) and PluronicTM
(Molecular Probes; final concentration <0.025%). After loading, the tumor
spheroids were rinsed in `pulsing buffer' and placed in the experimental
chamber, and then electropulses were applied. For fluorescence excitation, the
488 nm line of an argon ion laser of the confocal setup was used. Emission was
recorded by the use of a 515 nm longpass filter. Because fluo-3 does not
permit use of ratio measurements to determine absolute free Ca2+
levels, data are presented in arbitrary units as percentage of fluorescence
variation (F/F0) with respect to the resting level
F0.
Antibody staining
The polyclonal rabbit c-fos (AB-2) antibody was obtained from Calbiochem
(Bad Soden, Germany) and was used in a dilution of 1:100. Multicellular tumor
spheroids were fixed in ice-cold methanol-acetone for 60 minutes at -20°C,
washed with phosphate-buffered saline (PBS) plus 0.1% Triton X-100 and blocked
against unspecific binding in 10% nonfat milk powder for 60 minutes.
Incubation with the primary antibody was performed for 60 minutes. As
secondary antibody, a Cy3-labelled goat anti-rabbit IgG (H+L) antibody
(Jackson ImmunoResearch Laboratories, West Grove, PA) (concentration 1.2 mg
ml-1) was used at a dilution of 1:100.
Bioluminescence experiments
ATP release from multicellular tumor spheroids was determined using a
luciferin-luciferase assay (Sigma, Deisenhofen, Germany) in a
chemiluminescence apparatus (Bioluminiscence Analyzer XP2000, SKAN AG, Basel,
Switzerland) under dim light. For data sampling the output of the
photomultiplier tube of the setup was connected to a multimeter (Voltcraft
M-3610D, Conrad electronics, Hirschau, Germany) and a Tandon 286/N personal
computer (Tandon, Moorpark, CA). An aliquot of approximately 50 tumor
spheroids was washed three times in `pulsing buffer' and treated with DC
electrical fields. Subsequently, 100 µl of the supernatant was removed and
pressure injected via a light-tight access into a 3 ml glass cuvette
containing the luciferase cocktail consisting of 50 µl of the ATP assay mix
and 1.5 ml ATP assay mix dilution buffer (Sigma). In control experiments the
background chemiluminescence signal from supernatants of tumor spheroids that
were not treated with electrical fields was analyzed and set to 100%. For the
experiments with anion channel inhibitors, cells were preincubated for 20
minutes in `pulsing buffer' that was supplemented with the respective
inhibitor. We have previously assured that none of the applied anion channel
inhibitors interfered in the applied concentrations with the activity of
luciferase enzyme activity (Sauer et al.,
2000).
Statistical analysis
Data are given as mean values±standard deviation, with n
denoting the number of experiments. Student's t-test for unpaired
data was applied as appropriate. A value of P<0.05 was considered
significant.
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Results |
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|
Involvement of purinergic receptor stimulation in the
electrical-field-induced a Ca2+ response
The Ca2+ response elicited by DC electrical field treatment of
multicellular tumor spheroids may arise from the stimulation of purinergic
receptors. Various subtypes of purinergic receptors have been demonstrated to
be present in DU-145 tumor spheroids
(Janssens and Boeynaems, 2001;
Sauer et al., 2001
). To
investigate this issue tumor spheroids were repetitively treated with 10 µM
ATP and subsequently with a single electrical field pulse (750 Vm-1
for 60 seconds). Addition of ATP to the incubation medium resulted in a
transient elevation of [Ca2+]i
(Fig. 2A). Repetitive treatment
of tumor spheroids with ATP resulted in a decline of the amplitude of the
Ca2+ response. Subsequent treatment with a DC electrical field
pulse failed to raise [Ca2+]i, presumably owing to an
interference of the signal transduction of purinergic receptors with the
signalling cascade elicited by the electrical field pulse
(Fig. 2A; n=3). To
yield further information on the involvement of purinergic receptors in the
electrical-field-evoked [Ca2+]i response, tumor
spheroids were pretreated for 60 minutes with the purinergic receptor
antagonist suramin (300 µM; n=4;
Fig. 2B) or for 30 minutes with
2 U/ml apyrase (n=3; Fig.
2C), which scavenges extracellular ATP. Under both experimental
conditions the [Ca2+]i transient in response to
electrical field treatment was totally abolished, indicating that stimulation
of purinergic receptors as well as ATP in the extracellular medium is a
prerequisiste for electrical-field-induced Ca2+ signaling.
|
ATP release upon treatment of multicellular tumor spheroids with DC
electrical fields
In a recent study we have demonstrated that DU-145 cancer cells grown in
monolayer culture release ATP via anion channels upon mechanical stimulation
(Sauer et al., 2000). Since
similar signal transduction cascades may prevail in Ca2+ signaling
upon electrical field treatment, ATP release from tumor spheroids was
investigated by the use of luciferase-based bioluminescence
(Fig. 3). It was demonstrated
that within 1 minute after electrical field treatment of multicellular tumor
spheroids with a single electrical field pulse, ATP bioluminescence increased
to 424±72% (n=9) compared with the untreated control (set to
100%). Pretreatment of tumor spheroids for 20 minutes with the anion channel
inhibitors tamoxifen (50 µM; n=4), niflumic acid (200 µM;
n=4), and NPPB (50 µM; n=4) significantly reduced
electrical-field-evoked bioluminescence to 121±45%, 145±64%, and
150±22%, respectively, indicating that ATP may be released to the
supernatant via anion channels.
|
Inhibition of the electrical-field-evoked Ca2+ response by
anion channel inhibitors
We assumed that the elevation of [Ca2+]i following
treatment of tumor spheroids with a DC electrical field pulse may be caused by
ATP release through anion channels and subsequent stimulation of purinergic
receptors. To verify this assumption [Ca2+]i changes
upon electrical field treatment were recorded in the presence of anion channel
inhibitors. As shown in Fig. 4,
preincubation of tumor spheroids for 60 minutes with either tamoxifen (50
µM; n=10), niflumic acid (200 µM; n=9) or NPPB (50
µM; n=7) totally abolished the Ca2+ response,
indicating that ATP released via anion channels elicits the Ca2+
response during electrical field treatment. Comparable results were achieved
with the anion channel inhibitor DIDS (data not shown), which has also been
shown to antagonize purinergic receptor stimulation
(Zhang et al., 2000b).
|
Inhibition of DC electrical-field-induced c-Fos induction and growth
stimulation of multicellular tumor spheroids by suramin and anion channel
antagonists
Treatment of multicellular prostate tumor spheroids with DC electrical
fields accelerates tumor growth by a mechanism involving a transient elevation
of [Ca2+]i (Sauer et
al., 1997; Wartenberg et al.,
1997
). Consequently it should be expected that inhibition of the
electrical-field-induced Ca2+ response blunts the stimulation of
tumor growth. To investigate this issue tumor spheroids were treated with a
single electrical field pulse, and tumor growth was investigated 6 days after
electrical field treatment (Fig.
5A). Furthermore, the protein levels of the growth-associated
immediate early reponse gene c-fos were evaluated 1 hour after
electrical field treatment (Fig.
6). To exclude that the applied anion channel inhibitors exerted
toxic side effects, tumor spheroids were pretreated for 30 minutes with
niflumic acid (200 µM), tamoxifen (50 µM) or NPPB (50 µM), and
subsequently 10 µM ATP was added, which has been demonstrated to stimulate
tumor spheroid growth (Sauer et al.,
2001
). After a further 30 minutes the medium was completely
exchanged, and tumor spheroid growth was evaluated after 24 hours
(Fig. 5B). Electrical field
treatment of tumor spheroids accelerated tumor growth (n=6), which
resulted in a 38±11-fold (V/V0) increase in tumor spheroid
volume in the electrical-field-treated sample compared with the untreated
control, which exerted a 12±4-fold volume (V/V0) increase
during the 6 days of experimental observation
(Fig. 5A). When 10 µM ATP
were exogenously added to tumor spheroids a significant growth stimulation was
observed that was unchanged in the presence of anion channel inhibitors
(Fig. 5B; n=3),
excluding that the applied anion channel blockers exerted toxic effects on the
tumor spheroids. Incubation of tumor spheroids for 1 hour with anion channel
inhibitors alone did not impair tumor spheroid growth (data not shown).
Furthermore, a pronounced increase in the expression of c-Fos protein
(n=3) with a maximum effect after 1 hour was obvious
(Fig. 6A,B). The growth
stimulation observed upon electrical field treatment could be efficiently
inhibited when tumor spheroids were preincubated for 60 minutes with the anion
channel inhibitor niflumic acid (n=3), as well as with the purinergic
receptor antagonist suramin (Fig.
5A; n=3). Additionally, the elevation of c-Fos protein
was totally abolished in the presence of suramin, as well as after
preincubation with the anion channel inhibitors niflumic acid, tamoxifen and
NPPB (Fig. 6A,B; n=3).
Hence it is concluded that the increased c-Fos expression and growth
stimulation of multicellular tumor spheroids by DC electrical field treatment
requires the stimulation of purinergic receptors by ATP released to the
extracellular space via anion channels.
|
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Discussion |
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Extracellular pathways for the propagation of Ca2+ waves have
been reported for several preparations including basophil leukemic cells
(Osipchuk and Cahalan, 1992),
hepatocytes (Schlosser et al.,
1996
), ciliary epithelial cells
(Homolya et al., 2000
) and
osteoblastic cell lines (Jorgensen et al.,
1997
). It was demonstrated that these extracellular pathways of
Ca2+ wave propagation were based on activation of purinergic
receptors of the P2Y class that activate phospholipase C, resulting in the
generation of Ins(1,4,5)P3 and intracellular
Ca2+ release from Ins(1,4,5)P3-sensitive
Ca2+ stores. It was shown that mechanical stimulation of cells
resulted in release of ATP stored inside the cell to the extracellular space,
which subsequently stimulated purinergic receptors in an autocrine and
paracrine manner and elicited intracellular Ca2+ responses. Since
it was recently demonstrated by our group that comparable mechanisms of ATP
release were existent in prostate cancer cells of the DU-145 cell line
(Sauer et al., 2000
), we
tested whether electrical DC field treatment increased ATP in the
extracellular medium. Indeed we found that within approximately 1 minute after
electrical field treatment substantial amounts of ATP could be detected in the
supernatant of electrical-field-treated tumor spheroids. It has been recently
demonstrated by us that under hypotonic conditions DU-145 prostate cancer
cells grown in monolayer culture release approximately 1.6 pmol ATP per
105 cells, which is sufficient to raise an intracellular
(Ca2+]i response
(Sauer et al., 2000
). In the
present study the role of released ATP in the electrical-field-induced
Ca2+ response was investigated by preincubation of tumor spheroids
with the purinergic receptor antagonist suramin (1 hour) and the ATP scavenger
apyrase (30 minutes). Both experimental conditions totally abolished the
ATP-induced [Ca2+]i transient, which indicates that ATP
in the extracellular medium and purinergic receptor stimulation are
prerequisites for electrical-field-induced Ca2+ signaling.
To investigate the release mechanisms for intracellular ATP, tumor
spheroids were preincubated with antagonists of anion channels that
significantly inhibited ATP release. ATP release through anion channels has
been described for a variety of preparations
(Mitchell, 2001;
Mitchell et al., 1998
;
Cotrina et al., 1998
;
Sabirov et al., 2001
) and
their relationship to the cystic fibrosis transmembrane conductance regulator
(CFTR) has been critically discussed
(Schwiebert, 2001
). Recently
it has been shown that CFTR may not by itself release ATP but may regulate the
activity of a separate anion channel
(Braunstein et al., 2001
).
Anion channels with less selectivity for chloride versus other halides or
larger anions are prime candidates for putative ATP channels. These include
the outwardly rectifying Cl- channel (ORCC) as well as plasma
membrane forms of the voltage-dependent anion channel (VDAC). Recently, an
ATP-conducting anion channel that was activated under hyperpolarizing
conditions was characterized in Xenopus oocytes. During
hyperpolarizing pulses the permeability of this channel was more than 4000
times higher for ATP than that for Cl-
(Bodas et al., 2000
).
Furthermore, it has been demonstrated that the multidrug resistance
transporter P-glycoprotein-associated Cl- channel, which belongs to
the ATP-binding cassette (ABC) transporter superfamily, may regulate ATP
release channels (Roman et al.,
2001). We have previously shown that multicellular tumor spheroids
of different origin, including spheroids of the DU-145 cell line, express
intrinsic P-glycoprotein with the development of quiescent cell areas in the
depth of the tissue. The size class of tumor spheroids (diameter 100±50
µm) used in the present study express low, but detectable, levels of
intrinsic P-glycoprotein that could serve as a mediator for ATP release.
The physiological function of ATP release to the extracellular medium is
still a matter of debate. It has been argued that, in ciliary epithelial cells
of the eye, released ATP may modulate aquous humor flow by autocrine and
paracrine mechanisms within the two cell layers of this epithelium
(Mitchell et al., 1998). In
liver cells (Wang et al.,
1996
; Feranchak et al.,
2000
) and bilary epithelial cells
(Roman et al., 1999
), recovery
from swelling is mediated by an autocrine pathway involving conductive release
of ATP. In endometrial (Chan et al.,
1997
), intestinal (Merlin et
al., 1994
), and epidymal epithelial cells
(Chan et al., 1995
), regulation
of Cl- secretion is mediated by extracellular ATP. Recently, it has
been demonstrated that ATP released constitutively from Madin-Darby canine
kidney (MDCK), COS-7 and HEK-293 cells modulates phosphatidylinositol
signaling and turnover as well as cAMP production. It was assumed that
autocrine and paracrine ATP signaling occurs constantly in the extracellular
milieu and may establish a `set point' for multiple signal transduction
pathways or signaling molecules (Insel et
al., 2001
; Ostrom et al.,
2000
). In the tumor spheroid model used in the present study it is
demonstrated that electrical field treatment with a single electrical field
pulse increased c-Fos expression and accelerated tumor growth. c-Fos
activation in response to EMF fields has been previously reported in HeLa
cells that were transiently transfected with plasmids containing upstream
regulating regions of c-fos (Rao
and Henderson, 1996
). The activation of c-fos was shown
to be sensitive to the presence of extracellular Ca2+
(Karabakhtsian et al., 1994
).
In the present study, inhibiting ATP release by pretreatment with anion
channel blockers and antagonizing activation of purinergic receptors by
suramin significantly inhibited c-Fos elevation and growth stimulation after
electrical field treatment of multicellular tumor spheroids. This clearly
indicates that the observed effect of the electrical field on the acceleration
of tumor growth was caused by ATP release and purinergic receptor
stimulation.
The mitogenic effect of ATP has been shown for several preparations
including prostate cancer cells
(Wartenberg et al., 1999;
Sauer et al., 2001
), smooth
muscle cells (Wang et al.,
1992
; Erlinge,
1998
), ovarian tumor cells
(Popper and Batra, 1993
),
renal proximal tubule cells (Paller et
al., 1998
), and mesangial cells
(Schulze-Lohoff et al., 1992
).
By contrast, EMF fields have been repeatedly reported to accelerate tumor cell
growth in vitro and, although contradictory data exist
(Gurney and van Wijngaarden,
1999
; van Wijngaarden et al.,
2001
; Jahn, 2000
),
epidemiological studies have suggested that longterm exposure to EMF fields
increase the incidence of several cancers
(Robinson et al., 1999
;
Loomis et al., 1998
;
Bianchi et al., 2000
;
Villeneuve et al., 2000
;
Caplan et al., 2000
). The
signal transduction pathways underlying the growth-stimulating and
tumor-promoting effects of EMF fields are far from being sufficiently
investigated. Hence, the data of the present study performed with the complex
3D neoplastic tissue of multicellular tumor spheroids may have extended impact
on our understanding of how electrical fields promote either benign or
neoplastic prostate tumor growth.
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