 |
INTRODUCTION |
The treatment of cancer is limited by either intrinsic or acquired
expression of multidrug resistance transporters, including Pgp.1 The Pgp-mediated MDR
phenotype is generally characterized by a decreased cellular drug
accumulation owing to an enhanced drug efflux and is caused by the
overexpression of the drug transporter (1-3). Most in vitro
studies have been performed using cell cultures selected for drug
resistance by repetitive treatment with anti-cancer agents or using
recurrent tumors from patients after chemotherapy (3). However, there
is increasing evidence that Pgp-mediated MDR is likewise developing in
the multicellular context of tumor tissues, without previous treatment
with anti-cancer agents (4-8). This intrinsic Pgp-mediated MDR may
represent a so far underestimated common in vivo situation,
which may even worsen the efficacy of chemotherapeutic regimen.
Especially, renal and prostate cancers are known as intrinsic
drug-resistant cancers, which already have a relatively resistant
phenotype to anti-cancer agents even without drug selection (9). This
may explain the low response of hormone-insensitive prostate tumors
toward cytostatic agents (9, 10).
In most strategies developed to reverse the MDR phenotype,
chemical-reversing agents are applied, which have in common the ability
to reverse the MDR phenotype through the binding to the MDR
transporters (11). A more efficient strategy to circumvent MDR
would be to down-regulate the expression of genes coding for the
transporters. This, however, requires knowledge of the molecular mechanisms and signal transduction pathways that are involved in the
regulation of MDR-related genes and elaboration of sophisticated experimental approaches that efficiently down-regulate the drug transporters. Recently, it has been pointed out that the expression of
Pgp may be redox-regulated, because down-regulation of Pgp and reversal
of the MDR phenotype was achieved by treatment of tumor cells with
tumor necrosis factor
(12), which is known to utilize low levels of
ROS in its signal transduction pathway (13). On the other hand, it has
been reported that high levels of ROS resulting in severe cellular
oxidative stress increased the expression of the MRP-1 (14)
and MDR-1b (15) genes.
In previous studies we have reported on an intrinsic Pgp MDR, which
develops in quiescent cell areas of large, non-necrotic multicellular
prostate tumor spheroids derived from the androgen-independent cell
line DU-145 (7, 8). We furthermore demonstrated that the MDR phenotype
could be reversed and that Pgp could be down-regulated upon incubation
of spheroids with chemical agents that raised intracellular ROS and
stimulated cell cycle activity (16). The current study presents
evidence that the development of intrinsic Pgp expression is a general
phenomenon occurring in multicellular tumor spheroids of different
origin, i.e. melanoma, hepatoma, and glioma tumor spheroids.
It is hypothesized that endogenously generated ROS are regulating the
expression of intrinsic Pgp in multicellular tumor spheroids, because
small, exponentially growing spheroids, which robustly generate ROS,
display reduced Pgp levels, whereas up-regulation of Pgp in large,
non-necrotic tumor spheroids coincides with decreased ROS levels. It is
shown that an elevation of intracellular ROS is down-regulating Pgp
expression via the activation of receptor tyrosine kinase signaling
pathways, which results in the phosphorylation of MAPK members and
mitogenic stimulation. The modulation of the intracellular redox state
by BSO or other ROS-generating agents is apparently suitable in
vitro to circumvent the MDR phenotype and may be useful in
vivo to promote the efficacy of chemotherapeutic regimen in
anti-cancer treatment.
 |
EXPERIMENTAL PROCEDURES |
Culture Technique of Multicellular Tumor Spheroids--
The
human prostate cancer cell line DU-145 and the melanoma cell line IGR
were grown in 5% CO2/humidified air at 37 °C with Ham's F-10 medium (Life Technologies, Inc., Gaithersburg, MD). The
glioma cell line Gli36 and the hepatoma cell line Hepa1 were grown in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.). The
cell culture media were supplemented with 10% fetal calf serum (Roche
Molecular Biochemicals, Mannheim, Germany), 2 mM glutamine,
0.1 mM
-mercaptoethanol, 2 mM minimal
essential amino acids, 100 IU/ml penicillin, and 100 µg/ml
streptomycin (ICN Flow, Meckenheim, Germany). Spheroids were grown from
single cells. Cell monolayers were enzymatically dissociated with 0.2% trypsin, 0.05% EDTA (ICN Flow) and seeded in siliconized 250-ml spinner flasks (Integra Biosciences, Fernwald, Germany) with 250 ml of
complete medium and agitated at 20 rpm using a Cell Spin stirrer system
(Integra Biosciences). Cell culture medium was partially (125 ml)
changed every day.
Incubation with BSO, Glutamine-reduced Medium, and
Ebselen--
For incubation with BSO and glutamine-reduced medium,
small multicellular tumor spheroids (diameter 130 ± 50 µm, day
5) were transferred to bacteriological tissue culture plates (diameter 10 cm) (Becton Dickinson, Meylan, France) filled with 10 ml of F10 cell
culture medium. They were subsequently treated for 7 days with 50 µM BSO (Sigma, Deisenhofen, Germany), which was added to
the cell culture medium. For incubation in glutamine-reduced medium,
F10 cell culture medium was supplemented with 10% fetal calf serum,
0.1 mM
-mercaptoethanol, 2 mM minimal
essential amino acids, 100 IU/ml penicillin, and 100 µg/ml
streptomycin, whereas glutamine was omitted as an additive. Tumor
spheroids were cultivated for 7 days in the glutamine-reduced medium.
The cell culture medium was changed every 24 h. Ebselen
(2-phenyl-1,2-benzisoselenazol-3(2H)-one) (Sigma) was dissolved in
Me2SO and was used at a final concentration of 1 µM.
Incubation with EGF and Inhibitors of the Tyrosine Kinase
Signaling Pathway--
EGF was purchased from Calbiochem (San Diego,
CA). Large, non-necrotic tumor spheroids (diameter 300 ± 50 µm,
day 14) were removed from spinner flasks and were incubated for 24 h in 6-cm bacteriological Petri dishes with EGF in a concentration of
2.5, 5, 20, and 50 ng/ml. Subsequently, they were fixed and Pgp
expression was investigated by immunohistochemistry. Inhibitors of the
receptor tyrosine kinase pathway were purchased from Calbiochem. Tumor spheroids were incubated with the EGF receptor kinase inhibitor tyrphostin AG 1478 (10 µM), the
farnesyltransferase inhibitor (E,E)-(2-oxo-2-{[(3,7,11-trimethyl-2,6,10-dodecatrienyl)oxy]amino}ethyl)phosphonic acid, (2,2-dimethyl-1-oxo-propoxyl)methyl ester (FPT inhibitor III) (1 µM), the c-Raf inhibitor
N-[5-(3-dimethylaminobenzamido)-2-methylphenyl]-4-hydroxybenzamide (ZM 336372) (1 µM), the PKC inhibitors
bisindolylmaleimide I (BIM-1) (1 µM) and
bisindolylmaleimide IX (Ro-31-8220) (0.5 µM), and the MAPK kinase inhibitor 2'amino-3'-methoxyflavone (PD98059) (20 µM) for 24 h. Subsequently, the tumor spheroids were
fixed and anti-Pgp immunohistochemistry was performed.
Incubation with H2O2 and Lethal Cell
Staining--
Incubation with H2O2 was
performed with 6-day-old tumor spheroids. Spheroids were incubated for
24 h in 6-cm bacteriological Petri dishes containing 10 ml of F10
cell culture medium supplemented with either 1, 200, 500, or 750 µM H2O2. After the incubation time tumor, spheroids were either fixed for immunohistochemistry or the
incubation medium was exchanged for F10 medium supplemented with 1 µM SYTOX green (Molecular Probes; Eugene, OR), which
intensively stains the nuclei of cells with compromised cell membranes.
Following 1 h of incubation, the percentage of labeled lethal
cells was assessed using the 488-nm band of the argon laser of the
confocal setup and a long-pass LP 515-nm filter set.
Doxorubicin Fluorescence Recording and Confocal Laser Scanning
Microscopy--
The fluorescent cytostatic anti-cancer agent
doxorubicin (Sigma Chemical Co.) (excitation at 543 nm, emission,
long-pass filter LP 570 nm) was used at a concentration of 10 µM unless otherwise indicated. Multicellular tumor
spheroids were loaded with doxorubicin at 37 °C for 90 min. After
recording of doxorubicin fluorescence by confocal laser scanning
microscopy, spheroids were transferred to doxorubicin-free cell culture
medium and incubated at 37 °C for 30 min. Following this incubation
doxorubicin retention in multicellular spheroids was evaluated.
Fluorescence recordings were performed by means of a confocal laser
scanning setup (LSM 410, Carl Zeiss, Jena, Germany), connected to an
inverted microscope (Axiovert 135, Carl Zeiss). Fluorophores were
excited using a 0.5-milliwatt helium-neon laser (single excitation at
543 nm). A 25×, numerical aperture 0.8, oil immersion-corrected objective (Carl Zeiss) was applied. Doxorubicin fluorescence was measured in 3600-µm2 areas in a depth up to 80 µm from
the spheroid periphery.
Immunohistochemical Techniques and Quantitative
Immunohistochemistry--
Antibody staining was performed on whole
mount multicellular spheroids. The monoclonal anti-p27Kip1
and p21WAF-1 antibodies were obtained from PharMingen
(Hamburg, Germany) and were used in a concentration of 2.5 µg/ml. The
monoclonal antibodies directed against the NADPH oxidase subunits
p47phox and p67phox (Dianova Hamburg, Germany) were
used in a concentration of 5 µg/ml. The polyclonal anti-mdr (Ab-1)
antibody (Oncogene Research Products, Cambridge, UK) was used in a
concentration of 5 µg/ml. The anti-active MAPK polyclonal antibody
directed against ERK1,2 (dilution 1:20), the anti-active JNK (dilution
1:20), and the anti-active p38 MAPK (dilution 1:20) polyclonal
antibodies were obtained from New England BioLabs (Beverly, MA). The
monoclonal anti-proliferating cell protein Ki-67 antibody was obtained
from Sigma and used in a concentration of 7.5 µg/ml. Spheroids were washed in phosphate-buffered saline (PBS), fixed in either ice-cold methanol/acetone (7:3) or 4% paraformaldehyde (4 °C) and
permeabilized in PBS supplemented with 1% Triton X-100 (PBST) (Sigma).
Subsequently, they were incubated for 1 h in PBST 0.01%
containing 10% fat-free milk powder to reduce nonspecific binding and
for further 2 h with primary antibody. After washing three times
in PBST (0.1%), the spheroids were incubated for 60 min in PBST
(0.01%) supplemented with 10% milk powder and either a Cy5-conjugated
F(ab')2 fragment goat anti-mouse IgG (H+L) (concentration
3.25 µg/ml), a Cy5-conjugated F(ab')2 fragment goat
anti-rabbit IgG (H+L) (concentration 4.6 µg/ml), or a Cy5-conjugated
F(ab')2 fragment goat anti-rat (concentration 4.6 µg/ml)
(all from Dianova, Hamburg, Germany). Excitation was performed using a
633-nm helium-neon laser of the confocal setup. Emission was recorded
using a long-pass LP 655-nm filter set.
For quantitative immunohistochemistry, confocal images were recorded
from whole mount multicellular spheroids stained with only secondary
antibodies (background fluorescence image) and spheroids stained with
primary and secondary antibodies. The pinhole settings of the confocal
setup were adjusted to yield optical slices of 10-µm thickness. After
subtraction of background fluorescence, the fluorescence signal
(counts) was evaluated by the image analysis software of the confocal
setup in 3600-µm2 areas of interest and was routinely
exported for further analysis to the Sigma Plot graphic software.
Determination of the Intracellular Redox
Levels--
Intracellular redox levels were measured using the
fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate
(H2DCFDA) (Molecular Probes), which is a nonpolar compound
that is converted into a non-fluorescent polar derivative
(H2DCF) by cellular esterases after incorporation into
cells. H2DCF is membrane-impermeable and is rapidly
oxidized to the highly fluorescent 2',7'-dichlorofluorescein (DCF) in
the presence of intracellular ROS (17). For the experiments, multicellular tumor spheroids were incubated in E1 medium containing (in millimolar) NaCl 135, KCl 5.4, CaCl2 1.8, MgCl2 1, glucose 10, HEPES 10 (pH 7.4 at 23 °C), and 20 µM H2DCFDA dissolved in dimethyl sulfoxide
(Me2SO) was added. After 5, 10, 15, 20, and 30 min
intracellular DCF fluorescence (corrected for background fluorescence)
was evaluated in 3600-µm2 regions of interest using an
overlay mask. For fluorescence excitation, the 488-nm band of the argon
ion laser of the confocal setup was used. Emission was recorded using a
long-pass LP515-nm filter set.
Determination of Intracellular Glutathione--
Glutathione
levels were determined using a commercially available glutathione assay
kit (Calbiochem). Tumor spheroids were enzymatically dissociated in
0.1% trypsin/0.05% EDTA. An aliquot of 3.5 × 106
cells was lysed in 0.5 ml of meta-phosphoric acid, freeze-thawed, and
centrifuged (3000 × g) for 10 min. 50 µl of the
supernatant was used for the assay. The substitution product obtained
with reduced glutathione was transformed under alkaline conditions into
a chromophoric thione with a maximal absorbance at 400 nm. The
absorbance of the samples was determined using a Dynex (Reutlingen, Germany) microplate reader.
Reverse Transcriptase-Polymerase Chain Reaction
(RT- PCR)--
MDR-1 gene expression was monitored
by RT-PCR. After the indicated growth times for tumor
spheroids, culture medium was aspirated and spheroids were washed in
PBS. Subsequently the cells were lysed with 1 ml of RNA-clean (PqLab,
Erlangen, Germany) and processed according to the manufacturer's
protocol to obtain total cellular RNA. 1-µg aliquots were
electrophoresed through 1.2% agarose-0.67% formaldehyde gels and
stained with ethidium bromide to verify the quantity and quality of the
RNA. 1 µg of the isolated total RNA was reverse-transcribed using
random primers and Moloney murine leukemia virus reverse
transcriptase (Life Technologies, Inc.) for 60 min at 42 °C and 10 min at 70 °C. The single-stranded cDNA was amplified by
polymerase chain reaction using Taq DNA polymerase (Sigma).
35 cycles were performed under the following conditions: 20 s,
94 °C; 30 s, 60 °C; 60 s, 72 °C. The sequence for
hMDR-1 sense and antisense primers were 5'-CCCATCATTGCAATAGCAGG-3' and 5'-GTTCAAACTTCTGCTCCTGA-3' (18). PCR amplification gave a single 167-base pairs (bp) fragment originated from hMDR-1 mRNA. For semi-quantification, PCR conditions were chosen so that the reaction was within the linear exponential phase with respect to the amount of
cDNA template and number of cycles performed. Equal amounts of
RT-PCR products were loaded on 1.5% agarose gels, and optical densities of ethidium-bromide-stained DNA bands were quantified.
Immunoblotting--
Multicellular spheroids were lysed in 125 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerine, 20 mM dithiothreitol, 1 mM EDTA, 0.01% bromphenol blue. Equal amounts of proteins (15 µg per lane) were electrophoresed on 10% SDS-polyacrylamide gel electrophoresis gels.
Immunoblots of Pgp were prepared by electrophoretic transfer of
proteins from SDS-polyacrylamide gels to nitrocellulose by semi-dry
Western blotting. The nitrocellulose transfers were incubated for
1 h in blocking buffer (5% low-fat milk powder in PBS containing 0.1% Tween 20) and then probed for 1 h with 5 µg/ml polyclonal rabbit anti-mdr antibody (Ab-1) (Oncogene) in PBS, 0.1% Tween 20. As
secondary antibodies, a horseradish peroxidase-conjugated goat
anti-rabbit (dilution 1:2 × 104) antibody (Dianova)
was used.
Statistical Analysis--
Data are given as mean values ± S.D., with n denoting the number of experiments. In each
experiment at least 20 multicellular spheroids were analyzed.
Student's t test for unpaired data was applied as
appropriate. A value of p < 0.05 was considered significant.
 |
RESULTS |
Transient Expression of Pgp in Multicellular Tumor
Spheroids--
We have previously reported on the development of an
intrinsic Pgp-mediated MDR in multicellular prostate tumor spheroids of
the DU-145 cell line, which paralleled the induction of cell quiescence
in the depth of the multicellular tissue (7, 8). To evaluate whether
the transient expression of Pgp observed in DU-145 tumor spheroids
represents a general phenomenon of multicellular neoplastic tissues,
tumor spheroids of different origin, i.e. melanoma tumor
spheroids of the IGR cell line, hepatoma spheroids of the Hepa1 cell
line, and glioma spheroids of the Gli36 cell line were screened for the
expression of Pgp and compared with protein levels of Pgp in tumor
spheroids of the DU-145 cell line. It was observed that all cell lines
under investigation transiently expressed Pgp (Fig.
1, A-D) (n = 3). Owing to the cell-specific growth kinetics of tumor spheroids of
different origin (Fig. 2) the maximum Pgp
expression occurred at different times in DU-145, IGR, Hepa1, and Gli36
tumor spheroids (n = 3). In DU-145 prostate tumor
spheroids the expression of the MDR-1 gene in relation to tumor spheroid age was evaluated by RT-PCR and immunoblot analysis (Fig. 3). It was evidenced that
MDR-1 expression paralleled Pgp expression, i.e.
it was low in small tumor spheroids and increased with the development
of quiescent cell areas in 5- to 10-day-old tumor spheroids.
MDR-1 expression was decreased in large tumor spheroids,
which have been previously shown to contain central necrosis
(27-day-old) (n = 3) (19).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 1.
Expression of intrinsic Pgp during the growth
of multicellular tumor spheroids of different origin.
A, multicellular DU-145 prostate tumor spheroids;
B, multicellular IGR melanoma tumor spheroids; C,
multicellular Gli36 glioma tumor spheroids; and D,
multicellular Hepa1 tumor spheroids. The data were fitted by a Gaussian
algorithm.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2.
Growth kinetics of multicellular tumor
spheroids of different origin. The investigated tumor spheroids
were: glioma tumor spheroids of the Gli36 cell line, melanoma tumor
spheroids of the IGR cell line, prostate tumor spheroids of the DU-145
cell line, and hepatoma tumor spheroids of the Hepa1 cell line.
|
|

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 3.
Pgp expression and MDR-1
gene expression in respect to the cultivation time of
multicellular DU-145 tumor spheroids. A,
immunohistochemical analysis of Pgp expression in (from
left to right) whole mount small (diameter
100 ± 50 µm, days 3-6 of tumor spheroid culture), large
non-necrotic (diameter 300 ± 50 µm, days 10-15 of tumor
spheroid culture), as well as in large tumor spheroids containing
central necrosis (diameter 500 ± 100 µm, days 18-40 of tumor
spheroid culture). The bar represents 50 µm. B,
immunoblot analysis of Pgp expression in 1-, 5-, 11-, 16-, and
36-day-old tumor spheroids. Pgp expression obtained from the gel was
analyzed by densitometric analysis. Shown is a representative of three
independent experiments, which yielded comparable results.
C, RT-PCR analysis of MDR-1 gene expression in
respect to the cultivation time of multicellular tumor spheroids. After
different times of cultivation, as indicated, RNA was prepared from
tumor spheroids and the MDR-1-specific (167-bp) PCR products
were amplified and analyzed by gel electrophoresis, as described under
"Experimental Procedures." MDR-1 mRNA expression
obtained from the gel was analyzed by densitometric analysis. Shown is
a representative of three independent experiments which yielded
comparable results.
|
|
To correlate the increased expression of Pgp in DU-145 tumor spheroids
with the proliferation status of the tumor cells, the tissue levels of
the CDK inhibitors p27Kip1 and p21WAF-1 as well
as the expression of the proliferation-associated antigen Ki-67 were
analyzed. It was observed that p27Kip1 (Fig.
4A) and p21WAF-1
(Fig. 4B) were up-regulated in parallel to Pgp with
increasing size of tumor spheroids (n = 3). With the
onset of central necrosis, which occurs at a critical size of 350 ± 50 µm in DU-145 tumor spheroids (20) p27Kip1
expression declined to the level observed in small tumor spheroids, whereas p21WAF-1 expression remained on an elevated level.
Ki-67 expression was inversely correlated to the expression of Pgp and
declined with increasing age and size of tumor spheroids, indicating a
decrease in proliferative activity (n = 3).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
Expression of p27Kip1
(A), p21WAF-1 (B),
and Ki-67 (C) in DU-145 multicellular tumor
spheroids after different times of cell culture. Elevated
expression of p27Kip1 and p21WAF-1 was observed
in large, non-necrotic tumor spheroids. In large tumor spheroids
containing central necrosis a decline of p27Kip1 was
detected, whereas the expression of p21WAF-1 remained on an
elevated level. Ki-67 expression declined with increasing tumor
spheroid age. The data are presented as positive nuclei per 3600 µm2. Size classes of tumor spheroids are indicated by
dashed vertical lines.
|
|
The apparent correlation of cell quiescence and increased Pgp
expression was furthermore corroborated by investigation of the
intrinsic activity of the MAPK members ERK1,2, JNK, and p38 MAPK, which
are involved in mitogenic and stress-activated signaling pathways (Fig.
5). As expected, the activity of all
investigated MAPK members was low in large, non-necrotic DU-145 tumor
spheroids (diameter 300 ± 50 µm, days 14-15 of tumor spheroid
culture), which expressed maximum levels of Pgp (see Fig. 1). Increased JNK activity was observed in small tumor spheroids (diameter 100 ± 50 µm, days 3-6 of tumor spheroid culture), as well as large tumor spheroids containing central necrosis (diameter 500 ± 100 µm, days 18-40 of tumor spheroid culture) (n = 3),
whereas increased ERK1,2 activity was observed solely in exponentially
growing small tumor spheroids. A low basal activity of p38 MAPK was
observed in all size classes of tumor spheroids under investigation
(n = 3).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
Endogenous MAPK activity in DU-145 tumor
spheroids of different size classes. Phosphorylated JNK, ERK1,2,
and p38 were evaluated by immunohistochemistry. The immunofluorescence
of JNK in large, non-necrotic spheroids was set to 100%. Note that
MAPK activity was low in large, non-necrotic tumor spheroids, which
contain extended areas of cell cycle-inactive, quiescent cells. JNK
activity is resumed in large multicellular tumor spheroids containing
extended central necrosis correlating to an increased fraction of
proliferating cells in this size class of tumor spheroids. *,
p < 0.05, significantly different from MAPK activity
in small spheroids.
|
|
Endogenous ROS Generation and Glutathione Content in Multicellular
Tumor Spheroids--
Cancer cells endogenously generate ROS (21),
which are involved in signaling pathways maintaining cell proliferation
(22-24). Hence, it is expected that endogenous ROS generation is high
in small tumor spheroids consisting predominantly of proliferating cells, whereas it is low in large, non-necrotic, tumor spheroids, which
contain extended areas of cell cycle-inactive, quiescent cells. To
determine ROS generation in multicellular DU-145 tumor spheroids,
small, large non-necrotic as well as tumor spheroids containing
extended central necrosis were stained with the redox-sensitive dye
H2DCFDA, and the time course of ROS generation was
monitored. Indeed our data show that the generation of ROS (evaluated
after an incubation time of 20 min with H2DCFDA) was most
pronounced in small spheroids with 280 ± 25% of the fluorescence
increase observed in large, non-necrotic tumor spheroids (set to 100%) (n = 3). In tumor spheroids containing central
necrosis, the ROS generation was significantly resumed and amounted to
200 ± 25% of the ROS generation observed in large, non-necrotic
spheroids (n = 3) (Fig.
6A).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Characterization of the intracellular redox
state in multicellular DU-145 tumor spheroids. A,
intracellular ROS levels as determined by quantification of
intracellular DCF fluorescence. Small tumor spheroids (diameter
100 ± 50 µm, days 3-6 of tumor spheroid culture) (filled
circles), large non-necrotic tumor spheroids (diameter 300 ± 50 µm, days 10-15 of tumor spheroid culture) (filled
squares), as well as large tumor spheroids containing central
necrosis (diameter 500 ± 100 µm, days 18-40 of tumor spheroid
culture) (filled triangles) were incubated with the ROS
indicator H2DCFDA, and the time course of the generation of
fluorescent DCF was monitored. The data were fitted by linear
regression. B, expression of the NADPH oxidase subunits
p47phox and p67phox during cell culture of DU-145 tumor
spheroids. The data were obtained by quantification of p47phox
and p67phox immunofluorescence. C, evaluation of the
glutathione content in DU-145 tumor spheroids after different times of
cell culture. Note that the glutathione content is elevated in large
non-necrotic tumor spheroids (days 7-12). The data were fitted by a
Gaussian algorithm.
|
|
Intracellular ROS in DU-145 are presumably generated via an
NADPH-oxidase-like enzyme, which may be differently expressed during
the growth of tumor spheroids. Therefore, tumor spheroids were
immunostained for the NADPH-oxidase subunits p47phox and
p67phox. It was demonstrated that small tumor spheroids exerted
high levels of NADPH-oxidase expression, whereas in large non-necrotic tumor spheroids a significant down-regulation of NADPH oxidase expression was observed (Fig. 6B). In large tumor spheroids
containing central necrosis, p47phox and p67phox
expression was partially resumed which, however, did not reach statistical significance (n = 3).
Intracellular ROS levels are counterbalanced by the anti-oxidative
defense system. Therefore, the intracellular content of glutathione
which is an important cellular anti-oxidative thiol, was determined in
tumor spheroids after different times of tumor spheroid culture (Fig.
6C). Our data demonstrate that the glutathione content was
inversely correlated to ROS levels in tumor spheroids. Low levels of
glutathione were determined in small tumor spheroids (6.75 ± 0.09 mM on day 3 of cell culture, n = 3), which
generated ROS to significant amounts. However, elevated glutathione
levels were found in large, non-necrotic tumor spheroids with a maximum at day 12 of tumor spheroid cell culture (11 ± 0.85 mM, n = 3) corresponding to spheroid
diameters of ~250 µm. This size class of tumor spheroids
consequently generated low levels of ROS (see Fig. 6A).
Prolonged culture times (>12 days) resulted in a decline of the
glutathione content, which is in line with the observed elevated ROS
levels in large tumor spheroids containing extended central necrosis.
Effects of Intracellular ROS Elevation on Pgp Expression in
Multicellular Tumor Spheroids--
The working hypothesis of our
present study is based on the assumption that the intracellular redox
state of tumor spheroids regulates the expression of the MDR
transporter Pgp. To address this issue, intracellular glutathione was
reduced either by treatment of small tumor spheroids for 7 days,
i.e. from day 5 to day 12 of cell culture with 50 µM BSO, which is an irreversible inhibitor of
-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione biosynthesis (25) or by incubating tumor spheroids in glutamine-reduced cell culture medium (26). Both experimental conditions significantly increased ROS levels as compared with untreated control spheroids (Fig. 7)
(n = 3) and significantly stimulated tumor spheroid
growth (data not shown). The increased ROS levels could be completely
inhibited by addition of ebselen, which is an effective free radical
scavenger (27). Elevation of intracellular ROS in large, non-necrotic
multicellular spheroids by BSO and glutamine-reduced medium resulted in
a significant down-regulation of Pgp (Fig.
8A) from 405 ± 92% to
183 ± 31% and 194 ± 42%, respectively (n = 3). The effects of intracellular elevation of ROS on the MDR
phenotype were evaluated by doxorubicin retention experiments. It was
observed that incubation of tumor spheroids with BSO and in
glutamine-reduced medium resulted in a significant increase in
doxorubicin retention from 63 ± 1% to 94 ± 1.5% and
92 ± 6%, respectively (n = 3) (Fig.
8B). The modulatory effects of elevated ROS on the levels of
Pgp and on doxorubicin retention could be efficiently reversed when 1 µM of the free radical scavenger ebselen was
coadministered, indicating that the observed down-regulation of Pgp by
glutathione depletion was related to an increase of the intracellular
redox state.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of decreased intracellular
glutathione levels in large, non-necrotic tumor spheroids on the
intracellular ROS level. Treatment of tumor spheroids for 7 days
(i.e. from days 5 to 12) either with 50 µM BSO
(A) or incubation in glutamine-reduced medium (B)
significantly raised intracellular ROS as determined by the evaluation
of H2DCF oxidation to fluorescent DCF. The increase in the
oxidation kinetics was efficiently inhibited in the presence of the
free radical scavenger ebselen (1 µM). The data were
fitted by linear regression.
|
|

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of decreased intracellular
glutathione levels on the expression of Pgp (A) and
doxorubicin retention (B) in large, non-necrotic tumor
spheroids. Treatment of tumor spheroids for 7 days (days 5-12)
either with 50 µM BSO or incubation in glutamine-reduced
medium significantly reduced Pgp. The data represent the relative Pgp
expression (%) on day 12 of cell culture in relation to the Pgp
expression in tumor spheroids on day 5 of cell culture (see Fig. 1).
The decrease in Pgp consequently resulted in augmented doxorubicin
retention. The observed effects could be efficiently reversed by
coadministration of the free radical scavenger ebselen. *,
p < 0.05, significantly different from the untreated
control.
|
|
Redox-regulation of Pgp may be dose-dependent. To evaluate
the effects of increasing ROS concentrations on the expression levels
of Pgp, 6-day-old tumor spheroids displaying intermediate Pgp levels
were incubated in cell culture media containing increasing concentrations of H2O2 ranging from 1 to 750 µM. In parallel experiments cell lethality was assessed
by staining dead cells with the cell death marker SYTOX green.
Concentrations of 1 µM (n = 3) and 200 µM (n = 3) H2O2
significantly down-regulated Pgp expression to 128 ± 9% and
126 ± 18%, respectively, whereas Pgp expression was significantly increased to 285 ± 22%, when 750 µM
H2O2 was administered (Fig.
9A). However, under these
conditions cell lethality was significantly augmented (Fig.
9B), indicating that severe oxidative stress results in
up-regulation of Pgp, whereas nontoxic low levels of ROS induced
down-regulation of Pgp and reversal of the MDR phenotype.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of increasing concentrations of
H2O2 on the expression of Pgp
(A) and cell lethality (B).
Six-day-old tumor spheroids were treated for 24 h with either 1, 200, 500, or 750 µM H2O2. Pgp
expression was evaluated by quantitative immunohistochemisty in whole
mount tumor spheroids. Cell lethality was determined by analyzing
lethal SYTOX green-positive cell nuclei. Note that Pgp expression was
down-regulated upon treatment with non-toxic concentrations of
H2O2, whereas under conditions of increased
cell lethality an up-regulation of Pgp was observed. *,
p < 0.05, significantly different from the untreated
control.
|
|
Effects of Intracellular ROS Elevation on p27Kip1
Levels as Well as ERK1,2 and JNK
Activity--
H2O2 is known as a mediator of
cell cycle activity in multicellular tumor spheroids of the DU-145 cell
line (28, 29). The observed changes in the expression of Pgp may
therefore be related to cell cycle stimulation. To address this issue,
large non-necrotic tumor spheroids were either incubated with BSO or glutamine-reduced culture medium and the expression of either p27Kip1 (Fig.
10A) or the activity of
ERK1,2 (Fig. 10B) and JNK (Fig. 10C) was
assessed. Our data clearly indicate that elevation of intracellular ROS
by BSO resulted in a significant down-regulation of p27Kip1
from 383 ± 90% to 70 ± 50% (n = 3).
Comparable results were achieved following incubation with
glutamine-reduced medium (data not shown). Consequently, BSO treatment
resulted in an increase in phosphorylated ERK1,2 and JNK from 100 ± 25% to 200 ± 25% and 184 ± 46%, respectively (n = 3). The observed effects were efficiently
inhibited in the presence of 1 µM ebselen. Hence, our
data support the notion that ROS-mediated down-regulation of Pgp is
accompanied by a stimulation of ERK1,2 and JNK pathways.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 10.
Effects of decreased intracellular
glutathione levels on the expression of p27Kip1
(A), ERK1,2 (B), and JNK
(C) in large, non-necrotic tumor spheroids.
Treatment of tumor spheroids for 7 days (days 5-12) with 50 µM BSO reduced expression of p27Kip1 and
increased expression of ERK1,2 and JNK, which is both indicative for an
induction of cell cycle activity. The observed effects could be
efficiently reversed by coadministration of the free radical scavenger
ebselen (1 µM). *, p < 0.05, significantly different from the untreated control.
|
|
Involvement of Receptor Tyrosine Kinase-mediated Signaling
Pathways in the Regulation of Pgp Expression--
The data of the
present study suggest that the expression of Pgp is associated with
cell quiescence and can be down-regulated by mitogenic stimulation of
tumor spheroids with low levels of ROS. Furthermore, it has been
recently shown that hydrogen peroxide can activate signaling pathways
utilized by ROS-generating growth factors (30). We therefore
hypothesized that: 1) mitogenic stimulation by the use of EGF, which
has previously been shown to generate intracellular ROS (31), should
likewise down-regulate Pgp, 2) the down-regulation of Pgp expression by
BSO resulted from an activation of EGF-mediated signal transduction
pathways (Fig. 11). To verify these
assumptions large, non-necrotic tumor spheroids were either treated
with tyrphostin AG 1478 (10 µM), which selectively inhibits EGF receptor kinase, or incubated for 24 h in serum-free medium and in serum-free medium supplemented with EGF in a
concentration range of 2.5-50 ng/ml. The serum-free conditions were
chosen to avoid a possible influence of EGF present in the fetal calf
serum of the cell culture medium on the outcome of the experiments. Tyrphostin AG 1478 treatment as well as incubation in serum-free medium
resulted in a significant up-regulation of Pgp as compared with control
conditions. Upon treatment with 2.5, 5, 20, and 50 ng/ml EGF a
dose-dependent down-regulation of Pgp to 70 ± 15%, 61 ± 19%, 34 ± 7%, and 55 ± 10%, respectively, was
observed as compared with serum-free conditions (set to 100%)
(n = 3) (Fig. 11A). Coadministration of 20 ng/ml EGF and the free radical scavenger ebselen (1 µM)
totally abolished the down-regulation of Pgp observed by treatment with
EGF alone, indicating that the down-regulation of Pgp was mediated via
ROS. In a parallel set of experiments tumor spheroids were treated with
BSO in the presence of specific antagonists of the EGF signaling
pathway, i.e. the PKC inhibitors BIM-1 (1 µM)
and Ro-31-8220 (0.5 µM), the Ras antagonist FPT inhibitor III (1 µM), the Raf inhibitor ZM 336372 (1 µM), and the MEK1 inhibitor PD98059 (20 µM), respectively (n = 3) (Fig.
11B). The ROS-mediated down-regulation of Pgp by BSO
treatment was completely abolished upon inhibition of the Ras-mediated
tyrosine kinase signaling pathway, which clearly indicates that
EGF-mediated signal transduction pathways are negatively regulating Pgp
expression. None of the applied antagonists of receptor tyrosine kinase
pathways exerted significant effects on intracellular ROS levels (data
not shown).

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 11.
Regulation of Pgp expression in
multicellular DU-145 tumor spheroids by receptor tyrosine kinase
pathways. A, effects of tyrphostin AG 1478 (10 µM), which selectively inhibits EGF receptor kinase,
serum-free conditions, and different concentrations of EGF on the
expression of Pgp in large, non-necrotic tumor spheroids (diameter
300 ± 50 µm, day 14 of tumor spheroid culture). After 24 h
of incubation the tumor spheroids were fixed and Pgp expression was
quantified by immunohistochemistry. The data represent the relative Pgp
immunofluorescence (%) in relation to the Pgp immunofluorescence
obtained under serum-free conditions (set to 100%). Note, that the
down-regulation of Pgp by EGF was totally abolished upon
coadministration of the free radical scavenger ebselen. *,
p < 0.05, significantly different from serum-free
conditions. B, effects of antagonists of receptor tyrosine
kinase pathways on the down-regulation of Pgp by BSO. Note, that all
applied antagonists of receptor tyrosine kinase pathways totally
abolished the BSO-mediated down-regulation of Pgp. *, p < 0.05, significantly different from the untreated control.
|
|
 |
DISCUSSION |
The present study reports on the correlation of endogenous
intracellular ROS levels and the intrinsic expression of Pgp in multicellular prostate tumor spheroids of different size classes. The
transient expression of Pgp during the growth of tumor spheroids, and
its correlation with cell quiescence is apparently a general feature of
tumor tissues, because tumor spheroids cultivated from glioma,
melanoma, and hepatoma tumor cells displayed comparable features of
intrinsic expression of Pgp as observed in the DU-145 prostate cancer
cell line. The observation of a transient increase of Pgp-mediated drug
resistance during the growth of avascular micrometastases may be of
clinical significance, because the outcome of chemotherapeutic
treatment critically depends on the chemosensitivity of the treated
neoplastic tissue, which may be predicted from levels of Pgp expression
as well as the expression of cell cycle-related genes in tumor
biopsies. In DU-145 tumor spheroids the intrinsic Pgp expression was
clearly correlated to increased levels of the CDK inhibitors
p27Kip1 and p21WAF-1, which are increased
during cell cycle arrest. Consequently, tumor spheroids with elevated
levels of Pgp expression displayed minor endogenous activity of the
cell growth-associated MAPK-members ERK1,2 and JNK. There is increasing
evidence that the growth of neoplastic tissues is controlled by the
generation of ROS, which have been previously shown to arise in
Ras-mediated signaling pathways (23). The data of the present study are
in notion with these previous studies, because it is demonstrated that
increased ROS generation was observed in small tumor spheroids that
consist predominantly of exponentially growing Ki-67-positive cells as well as in large tumor spheroids containing extended central necrosis. The increase in Ki-67 expression was paralleled by an augmented activity of JNK in small spheroids and large spheroids containing central necrosis as well as an increased activity of ERK1,2 in small
tumor spheroids.
The correlation of elevated Pgp levels with reduced levels of ROS as
well as diminished activity of ERK1,2 and JNK led us to hypothesize
that raising intracellular ROS may down-regulate Pgp expression via
up-regulation of ERK1,2 and JNK. An increase in intracellular ROS was
achieved by: addition of hydrogen peroxide to the cell culture medium,
depletion of intracellular glutathione by the use of BSO and
glutamine-reduced cell culture medium, as well as treatment with EGF,
which has been previously demonstrated to raise intracellular ROS (31).
All these experimental procedures resulted in significant
down-regulation of Pgp levels, indicating that ROS are involved in the
signaling pathways that regulate the expression of Pgp. Interestingly,
with hydrogen peroxide concentrations exceeding 500 µM an
up-regulation of Pgp was observed. A rise in Pgp expression following
treatment of cells with hydrogen peroxide has been previously
demonstrated (15). In this study millimolar concentrations of
H2O2 were administered, resulting in a parallel up-regulation of poly-(ADP-ribose) polymerase, which is a nuclear enzyme induced by DNA strand breaks. Furthermore, stress-induced expression of Pgp has been recently evidenced under various conditions, i.e. UV radiation (32), low external pH and osmotic shock
(33) as well as heat stress (34). Most of these stress factors are accompanied by the generation of intracellular ROS. Under stress conditions the physiological role of Pgp may be related to the prevention of apoptosis (35), presumably by regulating
caspase-dependent apoptotic pathways (36). This notion is
furthermore supported by studies that demonstrated that overexpression
of Pgp is correlated with increased levels of the anti-apoptotic
Bcl-xL protein (37). ROS acting as signaling agents in the regulation
of cell proliferation are in the nanomolar to micromolar range and are
therefore significantly lower than the concentrations necessary to
induce DNA strand breakage (38). Hence, intracellular ROS may exert
differential effects on the regulation of MDR-related genes, which are
dependent on their intracellular concentration.
In the present study raising intracellular ROS by depletion of
glutathione resulted in a decline of p27Kip1 expression,
whereas activity of ERK1,2 and JNK was significantly elevated, which
indicates that in parallel to the down-regulation of Pgp expression a
stimulation of the cell cycle was achieved, suggesting that cell cycle
stimulation by ROS and the regulation of Pgp expression may share a
common signal transduction pathway. This assumption was supported by
experiments demonstrating that pharmacological inhibition of members of
the receptor tyrosine kinase pathway utilized by EGF, and involving PKC
and Ras/Raf as well as ERK1,2, abolished the observed down-regulation
of Pgp following elevation of intracellular ROS. Interestingly,
incubation of tumor spheroids in serum-free media and treatment with
tyrphostin, which inhibits protein tyrosine kinases, including
autophosphorylation of EGF receptor kinase, significantly increased the
expression of Pgp in multicellular tumor spheroids. These findings
support the notion that EGF present in the fetal calf serum used in the cell culture medium and/or EGF secreted by tumor cells may regulate Pgp
expression via the activation of receptor tyrosine kinase signaling pathways.
The apparent effects of ROS on Pgp expression levels and cell cycle
activity in multicellular tumor spheroids may be clinically utilized in
anti-cancer trials. In EMT-6 tumor spheroids down-regulation of
p27Kip1, which has been discussed as a regulator of MDR
(39), by antisense nucleotides increased cell proliferation and
sensitized tumor cells to 4-hydroperoxycyclophosphamide (39). A
comparable chemosensitization in EMT-6 tumor spheroids was observed by
treatment with hyaluronidase, which reduced intercellular communication
and activated cell proliferation (40). Furthermore, stimulation of cell
proliferation in noncycling hematopoietic progenitor cells and leukemic
blasts resulted in a down-regulation of Pgp-mediated MDR (41). An
elevation of ROS by depletion of intracellular glutathione sounds
reasonable, because BSO has successfully been applied in
vivo in cancer patients and was shown to increase the sensitivity
of cancer cells toward antineoplastic drugs (42). Because most
antineoplastic agents are acting against rapidly cycling cancer cells,
the concomitant cell cycle stimulation and down-regulation of Pgp by
ROS may offer new avenues for conventional chemotherapy. This may hold
especially true for hormone-independent metastatic prostate tumors,
which are characterized by a high fraction of cells resting in the
G0 phase of the cell cycle and a pronounced intrinsic
resistance to a broad range of anti-cancer agents (9).