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John Wayne Cancer Institute at Saint John’s Health Center, Santa Monica, CA 90404, USA
1Correspondence: John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404, USA. E-mail: Cabot{at}jwci.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: glucosylceramide synthase • antisense • breast cancer • apoptosis • chemotherapy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Accumulation of glucosylceramide (GC) is
a characteristic of some multidrug-resistant (MDR) cancer cells of
breast, ovarian, colon, and epithelioid carcinomas (3
4
5)
.
Drug resistance induced by MDR1 transfection is accompanied
by increased levels of GC (6)
. Further, preliminary
clinical studies show that GC levels are elevated in tumor specimens
from patients with breast cancer and melanoma who demonstrated poor
response to chemotherapy (7)
. The enzyme glucosylceramide
synthase (GCS, ceramide glucosyltransferase,
UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC
2.4.1.80) transfers glucose from UDP-glucose to ceramide and produces
GC. An increased capacity for ceramide glycosylation has been revealed
in multidrug-resistant human breast carcinoma cells (8)
,
and enhanced expression of GCS by gene targeting confers resistance to
adriamycin and to tumor necrosis factor 
in breast cancer cells
(9
, 10)
. In an opposing scenario, GCS antisense
transfection can reverse adriamycin resistance in drug-resistant breast
cancer cells (11)
. As ceramide glycosylation emerges as a
novel mechanism of adriamycin resistance (3
4
5
, 8
, 9
, 11)
,
we hypothesized that decreasing the potential for ceramide
glycosylation would be one avenue to overcome cellular resistance to
several classes of anticancer drugs. To identify the role of GCS in
multidrug resistance, and to explore a novel approach to reverse
resistance, we introduced GCS sense and GCS antisense cDNA into
adriamycin-resistant human breast carcinoma cells and assessed the
influence of GCS on the nature of multidrug resistance, using classical
antitumor agents belonging to the anthracycline, Vinca
alkaloid, and taxane classes. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell lines and culture conditions
The human breast adenocarcinoma cell line, MCF-7-AdrR, which is
resistant to adriamycin (12)
, was kindly provided by Dr.
Kenneth Cowan (UNMC Eppley Cancer Center, Omaha, Neb.) and Dr. Merrill
Goldsmith (National Cancer Institute, Bethesda, Md.). Cells were
maintained in RPMI 1640 medium containing 10% (v/v) FBS, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 584 mg/liter L-glutamine.
Cells were cultured in a humidified 5% CO2
atmosphere tissue culture incubator and subcultured weekly using
trypsin-EDTA (0.05%, 0.53 mM) solution. The stably transfected cells,
MCF-7-AdrR/GCS and MCF-7-AdrR/asGCS, were cultured in RPMI 1640 medium
containing 400 µg/ml G418 (geneticin) in addition to the above
components.
Giemsa staining was performed as described (11)
. Cells
were seeded in 60-mm dishes (105 cells per dish)
in 10% FBS RPMI 1640 medium and grown for 2 days at 37°C. After
fixing, cells were stained with KaryoMAX Giemsa solution (Gibco BRL)
and photomicrographed.
pcDNA 3.1/his-GCS and pcDNA 3.1/his A-asGCS expression vectors and
transfection
pCG-2, a Bluescript II KS containing GlcT-1(ref.13 terminology
for GCS) in the EcoRI site, was kindly provided by Dr.
Shinichi Ichikawa and Dr. Yoshio Hirabayashi (The Institute of Chemical
and Physical Research, Saitama, Japan). The full-length cDNA of human
GCS was subcloned into the EcoRI site in the pcDNA 3.1/His A
with Xpress tag peptide (Invitrogen) in the upstream region. Xpress tag
fuses at the NH2 terminus of the cloned gene;
therefore, GCS will be expressed as Xpress-GCS. The antisense- and
sense-orientation of GCS cDNA was analyzed with Vector NTI 4.0 and
doubly checked by restriction digestion. When MCF-7-AdrR cells reached
20% confluence, pcDNA 3.1-asGCS (GCS antisense) or pcDNA 3.1-GCS (10
µg/ml, 100-mm dish) were introduced by co-precipitation with calcium
phosphate (Mammalian Transfection Kit; Stratagene, La Jolla, Calif.).
The transfected cells were selected in RPMI 1640 medium containing 10%
FBS and 400 µg/ml G418. Each G418-resistant clone, isolated using
cloning cylinders, was propagated and later screened by GCS enzyme
assay. pcDNA 3.1/his A plasmid was used in control transfections.
GCS assay
To determine the levels of GCS in the G418-resistant clones, a
modified radioenzymatic assay was used (9
, 14)
. Cells were
homogenized by sonication in lysis buffer (50 mM Tris-HCl, pH 7.4, 1.0
µg/ml leupeptin, 10 µg/ml aprotinin, 25 µM PMSF). Microsomes were
isolated by centrifugation (129,000 g, 60 min). The enzyme
assay, containing 50 µg microsomal protein, in a final volume of 0.2
ml, was performed in a shaking water bath at 37°C for 60 min. The
reaction contained a liposomal substrate composed of
C6-ceramide (1.0 mM), phosphatidylcholine (3.6
mM), and brain sulfatides (0.9 mM). Other reaction components included
sodium phosphate buffer (0.1 M) pH 7.8, EDTA (2.0 mM),
MgCl2 (10 mM), dithiothreitol (1.0 mM), ß-NAD
(2.0 mM), and [3H]UDP-glucose (0.5 mM).
Radiolabeled and unlabeled UDP-glucose were diluted to achieve the
desired radiospecific activity (4,700 dpm/nmol). To terminate the
reaction, tubes were placed on ice and 0.5 ml isopropanol and 0.4 ml
Na2SO4 were added. After
brief vortex mixing, 3 ml t-butyl methyl ether was added,
and the tubes were mixed for 30 s. After centrifugation, 0.5 ml of
the upper phase that contained GC was withdrawn and mixed with 4.5 ml
EcoLume for analysis of radioactivity by liquid scintillation
spectroscopy.
RNA analysis
Cellular mRNA was purified using a mRNA isolation kit
(Boehringer Mannheim, Indianapolis, Ind.). Equal amounts of mRNA (5.0
ng) were used for reverse transcription polymerase chain reaction
(RT-PCR), as described previously (11)
. Under upstream
primer (5'-CCTTTCCTCTCCCCACCTTCCTCT-3') and downstream primer
conditions (5'-GGTTTCAGAAGAGAGACACCTGGG-3'), a 302-bp fragment in the
5'-terminal region of the GCS gene was produced using the ProSTAR HF
single-tube RT-PCR system (High Fidelity, Stratagene) in a thermocycler
(Mastercycler Gradient, Eppendorf). mRNA’s were reverse transcribed
using MMLV-reverse transcriptase at 42°C for 15 min. DNA was
amplified with TaqPlus Precision DNA polymerase in a
40-cycle PCR reaction using the following conditions: denaturation at
95°C for 30 s, annealing at 60°C for 30 s, and elongation
at 68°C for 120 s. RT-PCR products were analyzed by 1% agarose
gel electrophoresis stained with ethidium bromide. ß-actin was used
as a control for consistent loading of gels.
Cytotoxicity assay
Assays were performed as described previously (9)
.
Briefly, cells were seeded in 96-well plates
(2x103 cells per well) in 0.1 ml RPMI 1640
medium containing 10% FBS and cultured at 37°C for 24 h before
addition of drug. Drugs were added in FBS-free medium (0.1 ml), and
cells were cultured at 37°C for the indicated periods. Drug
cytotoxicity was determined using the Promega 96 Aqueous cell
proliferation assay kit (Promega, Madison, Wisc.). Absorbance at 490 nm
was recorded using a Microplate Fluorescent Reader, model FL600
(Bio-Tek, Winooski, Vt.).
Colony formation in soft agar
The influence of GCS on cell growth in soft agar was analyzed by
[3H]thymidine incorporation (15
, 16)
. Cells (2x104) were suspended in 0.5
ml RPMI 1640 medium (supplemented with 2 U/ml insulin and 10% FBS),
containing 0.25% agarose III and the indicated concentrations of
adriamycin. The mixture was added over a layer of 0.35% agar (0.5 ml)
in RPMI 1640 medium in 24-well plates. After 72 h, 1.0 µCi
[3H]thymidine was added to each well in 0.1 ml
RPMI 1640 medium, and plates were maintained in the incubator an
additional 48 h. Cells were harvested by heating (5 ml PBS,
100°C, 40 min) and centrifugation (1,000 g, 15 min), and
lysed with KOH (0.075 M, 0.3 ml, 60 min). Cell lysates were mixed with
4 ml EcoLume for analysis of radioactivity by liquid scintillation
spectroscopy.
Apoptotic cell death detection by ELISA and DNA fragmentation
The presence of mono- and oligonucleotides, a feature of cells
undergoing apoptosis (17
, 18)
, was evaluated by Cell Death
Detection ELISA, performed following kit instructions (Boehringer
Mannheim). Briefly, cells were treated without or with the indicated
concentration of adriamycin for 48 h, and
104 cells from each sample were lysed in 200 µl
lysis buffer. After centrifugation (1000 g, 10 min), a
20-µl aliquot of lysate supernatant (103 cells
per tube) was incubated with DNA-histone antibody and anti-DNA
conjugated antibody for 2 h at 24°C and then incubated with
substrate for 15 min. Absorbance was measured at 405 nm.
DNA fragmentation analysis was performed as described previously
(10)
. Briefly, 0.5 x 106 cells
were seeded in 10-cm dishes in medium containing 5% FBS. After
attachment, cells were treated with 2 µM adriamycin for 72 h.
After harvest by trypsin-EDTA and centrifugation, cells were digested
with lysis buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 25 mM EDTA,
0.5% SDS, 0.3 mg/ml proteinase K). DNA was extracted with
phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) and precipitated by
incubating in one-half volume 7.5 M ammonium acetate plus two volumes
100% ethanol at -20°C overnight, followed by centrifugation (10,000
g, 20 min, 4°C). Contaminating RNA was digested in
RNA-digestion buffer (10 mM Tris-HCl, 0.1 mM EDTA, 0.1% SDS, 100 U/ml
RNase mixture). Re-extracted DNA (2–10 µg) was analyzed by
electrophoresis on a 2% agarose gel in TAE buffer (40 mM Tris acetate,
1 mM EDTA, pH 8.3) and was visualized with ethidium bromide under UV
light.
Ceramide and glucosylceramide analysis
Analysis was performed as described previously (3
, 9)
. Briefly, cells were seeded in 6-well plates
(6x104 cells per well) in 10% FBS RPMI 1640
medium. After 24 h, cells were shifted to 5% FBS medium with or
without agents and grown for the indicated times. Cellular lipids were
radiolabeled by adding [3H]palmitic acid (2.5
µCi/ml culture medium) for 24 h. After removal of medium, cells
were rinsed twice with PBS (pH 7.4), and total lipids were extracted as
described (9)
. The organic lower phase was withdrawn and
evaporated under a stream of nitrogen. Lipids were resuspended in 100
µl of chloroform/methanol (1:1, v/v), and aliquots were applied to
TLC plates. Ceramide was resolved using a solvent system containing
chloroform/acetic acid (90:10, v/v), and GC was resolved using a
solvent system containing chloroform/methanol/ammonium hydroxide
(70:20:4, v/v). Commercial lipid standards were co-chromatographed.
After development, lipids were visualized with iodine vapor staining
and identified by migration. The ceramide and GC area was scraped into
0.5 ml water. EcoLume counting fluid (4.5 ml) was added, the samples
were mixed, and radioactivity was quantitated by liquid scintillation
spectrometry. Radiochromatograms were sprayed with EN
(3)
HANCE (DuPont/NEN) and exposed for 2–3 days.
Caspase-3 assay
Caspase-3 activity was assayed by DEVD-AFC cleavage, using the
ApoAlert Caspase-3 assay kit (Clontech, Palo Alto, Calif). The assay
was performed as described previously (10)
. Cells were
seeded in 100-mm dishes (5x106 cells per dish)
in 10% FBS RPMI 1640 medium. After 24 h, cells were shifted to
5% FBS RPMI 1640 medium without or with adriamycin and grown for an
additional 24 or 48 h. After harvest, cells
(106 per vial) were lysed (50 µl lysis buffer
on ice, 10 min), and cell debris was removed by centrifugation (4°C,
10,000 g, 5 min). The soluble fraction was incubated with 50
µM conjugated substrate DEVD-AFC in a 100 µl reaction volume at
37°C, for 60 min. The free AFC fluoresce was measured at
excitation 400 nm and
emission 505 nm using a FL600 Microplate
Fluorescence Reader. The caspase-3 inhibitor,
acetyl-Asp-Glu-Val-Asp-aldehyde was used to exclude nonspecific
background in the enzymatic reaction.
Western blot analysis
Western blots were performed using a modified procedure
(9
, 10
, 19)
. Confluent cell monolayers were washed twice
with PBS containing 1.0 mM PMSF and detached with trypsin-EDTA
solution. Cells, pelleted by centrifugation, were solubilized in 1.0 ml
cold TNT buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1.0% Triton
X-100, 1.0 mM PMSF, 1.0% aprotinin) for 60 min with shaking. Insoluble
debris was excluded by centrifugation at 12,000 g for 45 min
at 4°C. The detergent soluble fraction was loaded in equal aliquots,
based on protein concentration, and resolved using 4–20% gradient
SDS-PAGE. The transferred blot was blocked (3% fat-free milk powder in
10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) and
immuno-blotted with GCS antiserum (1:1000) in binding solution (0.5%
BSA in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl) at 4°C for 18 h. To
detect Xpress tag and P-glycoprotein, the antibodies of anti-Xpress tag
(1:500) and C219 (5 µg/ml) were used in place of GCS antiserum.
Detection using enzyme-linked chemiluminescence was performed using ECL
(Amersham).
Rhodamine assay
The rhodamine assay, as a functional test for P-glycoprotein
efflux activity, was performed as described previously, with
modification (20
, 21)
. MCF-7 cell variants were harvested
using trypsin and washed with RPMI 1640 medium. Cells, 2.5 x
106 in 1.0 ml 5% FBS RPMI 1640 medium, were
incubated with rhodamine-123 (0.1 mg/ml) for 30 min at 37°C. After
centrifugation at 500 g for 15 min, supernatants were
discarded, and the cells were washed twice in RPMI 1640 medium. Uptake
of rhodamine-123 was measured by adding 200 µl culture medium
containing 0.02% SDS to the cells, and fluorescence was measured at
excitation 485
nm/emission 530 nm using the FL-600
fluorescent microplate reader. For efflux measurements, 200 µl of 5%
FBS RPMI 1640 medium was added, and the cells were incubated at 37°C
for another 60 min. After three washes, cell fluorescence was measured
using SDS as above. The efflux was calculated by the difference in cell
fluorescence after the 60-min incubation compared with initial cell
uptake parameters.
For fluorescence photomicrographs, cells were incubated with 0.1 mg/ml rhodamine-123 in 5% FBS RPMI 1640 medium for 30 min at 37°C. After rinsing, cells were fixed with cold acetic acid/methanol (1:3, v/v) and photomicrographed using an Olympus IX70 fluorescence microscope equipped with a photomicrographic system.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. MCF-7-AdrR cells, which were selected by treatment of
MCF-7 cells with adriamycin, exhibit multidrug resistance (8
, 11
, 22
, 23)
, have a higher level of GC, and show higher GCS activity
compared with MCF-7 cells (3
, 8
, 11
, 24
, 25)
. In the
present study, MCF-7-AdrR cells were transfected with either pcDNA
3.1/his-GCS or pcDNA 3.1/his-asGCS. The full-length GCS (1.7 kb) was
excised from pCG-2 (13)
and inserted in the
EcoRI site of pcDNA 3.1/his A (Fig. 1
). The orientations of sense and antisense were selected using double
restrictive mapping (HindIII and XhoI plus
NotI). The pcDNA 3.1/his-GCS was used to develop the
MCF-7-AdrR/GCS cell line, and pcDNA 3.1/his-asGCS was used to develop
the MCF-7-AdrR/asGCS cell line. As illustrated in Fig. 1
(bottom),
because GCS sense confers cellular resistance, it was hypothesized that
GCS antisense would sensitize cells to anticancer agents. After
co-precipitation with calcium phosphate, the stable expression clones
were selected using G-418 and screened using the GCS in
vitro radioenzymatic assay. We found that the number of
G418-resistant clones in MCF-7-AdrR asGCS-transfected cells was much
lower (54/106), compared with MCF-7-AdrR cells
transfected with pcDNA3.1/his A (251/106) or
pcDNA3.1/his-GCS vectors (240/106). Among these
clones, Clone 8 in pcDNA3.1/his-GCS transfected MCF-7-AdrR cells stably
displayed the highest GCS activity, and Clone 30 in pcDNA3.1/his-asGCS
transfected cells exhibited the lowest GCS activity when assayed in
more than four different experiments. Another six antisense-transfected
clones displayed a 5–20% reduction in GCS activity, compared with
empty-vector transfected MCF-7-AdrR clones. Clone 8 was designated
MCF-7-AdrR/GCS, and Clone 30 was designated MCF-7-AdrR/asGCS cells.
Cells at passages 5–16 were used in the experiments.
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MCF-7-AdrR/GCS cells expressed higher GCS levels as measured by RT-PCR,
Western blotting, and radioenzymatic assays, compared with MCF-7-AdrR
cells (Fig. 2
, see Fig. 3
). MCF-7-AdrR/GCS cells expressed 168% greater mRNA levels
as shown by RT-PCR (Fig. 2A
). Western blot analysis using
anti-Xpress antibody showed a strong GCS-Xpress tag band in
MCF-7-AdrR/GCS cells (Fig. 2B
, middle). GCS protein in
MCF-7-AdrR/GCS (Fig. 2C
, middle) was likewise greater
(145%) than that of MCF-7-AdrR cells (Fig. 2C
, left). GCS
in vitro enzyme activity in MCF-7-AdrR/GCS cells was 184%
greater compared with the parent cell line, MCF-7-AdrR, or the empty
vector (TC) transfected cells (50.5±3.1 vs. 27.37±2.2 pmol GC/h/µg
protein, P<0.001, Fig. 3A
). Collectively, these data clearly demonstrate that the
GCS-transfected cell line MCF-7-AdrR/GCS expresses higher levels of
GCS.
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MCF-7-AdrR/asGCS cells showed lower levels of GCS compared with parent
MCF-7-AdrR cells. MCF-7-AdrR/asGCS cells expressed 30% lower mRNA
(Fig. 2A
). In Western blots, using anti-Xpress antibody, we
did not find a GCS-Xpress tag band in MCF-7-AdrR/asGCS cells (Fig. 2B
, right) but rather a higher molecular weight form of
Xpress-tag. As shown by Western blot, using GCS antibody, 50% less GCS
protein was present in MCF-7-AdrR/asGCS cells (Fig. 2C
,
right). GCS in vitro enzyme activity in MCF-7-AdrR/asGCS
cells was 30% lower (19.7±1.0 vs. 27.37±2.2 pmol GC/h/µg protein,
P<0.001, Fig. 3A
) compared with parent cells or
empty-vector transfection controls. These results indicate that the
expressed GCS antisense mRNA effectively reduces GCS translation and
GCS activity in MCF-7-AdrR/asGCS cells.
The morphology of the three cell lines is shown in Fig. 3B
.
MCF-7-AdrR/GCS cells are more globular, growing in clusters.
MCF-7-AdrR/asGCS cells, including the nuclei, are flatter and larger
compared with the dome-shaped, more stellate MCF-7-AdrR cells. The
MCF-7-AdrR/asGCS cell line is also cuboidal with less dense cytoplasm
compared with MCF-7-AdrR cells, and the cells have a lower
nucleus/cytoplasm ratio compared with the other cell lines. The
population doubling times for three cell lines were similar: 32, 30,
and 30 h for MCF-7-AdrR/asGCS, MCF-7-AdrR/GCS, and MCF-7-AdrR
cells, respectively.
Anticancer drug response of MCF-7-AdrR, AdrR/GCS, and AdrR/asGCS
cells
GCS transfection upgraded resistance to adriamycin and other
antineoplastic drugs in MCF-7-AdrR cells, a well known
multidrug-resistant cell line (8
, 11
, 22
23
24
25)
that
displays high levels of GC (3)
. As shown in Fig. 4A
, viability of MCF-7-AdrR/GCS cells was significantly higher
than MCF-7-AdrR cells when exposed to increasing concentrations of
adriamycin (P<0.01). GCS-transfected cells were threefold
more resistant to adriamycin compared with MCF-7-AdrR cells
(EC50, 37.3 vs. 12.4 µM, Table 1
) and twofold more resistant to daunorubicin
(EC50, 1.7 vs. 0.9 µM) and actinomycin D
(EC50, 0.15 vs. 0.08, Table 1
, Fig. 4
). However,
an increase in cellular resistance to other anthracyclines,
Vinca alkaloids, taxanes, or cisplatin did not accompany GCS
transfection (Table 1)
. As hypothesized, transfection of GCS
effectively upgraded MCF-7-AdrR cellular resistance to several natural
product chemotherapeutic agents, including adriamycin, daunorubicin,
and actinomycin D.
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In contrast, the antisense-transfected model, MCF-7-AdrR/asGCS,
displayed a marked increase in sensitivity to several different classes
of anticancer drugs. As shown in Fig. 4
, MCF-7-AdrR/asGCS cell
viability in response to adriamycin treatment was significantly reduced
compared with MCF-7-AdrR cells, even at concentrations as low as 0.5
µM (P<0.001), where survival dropped below 50%.
Adriamycin sensitivity in antisense transfected cells increased 28-fold
(Table 1)
. The EC50 of adriamycin in
MCF-7-AdrR/asGCS was 0.44 versus 12.4 µM in MCF-7-AdrR and 37 µM in
MCF-7-AdrR/GCS cells. For comparison from previous studies, the
EC50 of adriamycin in MCF-7 cells was 0.37 µM
(9)
. Thus, GCS antisense transfection of MCF-7-AdrR cells
restored adriamycin sensitivity to the level displayed by MCF-7 cells.
MCF-7-AdrR/asGCS cells were also 11- and 7-fold more sensitive to
daunorubicin (EC50, 0.08 vs. 0.91 µM) and
idarubicin (EC50, 0.06 vs. 0.42 µM),
respectively, compared with MCF-7-AdrR cells (Table 1)
. Interestingly,
GCS antisense transfection markedly potentiated cellular sensitivity to
Vinca alkaloids, taxanes, and actinomycin D. The degree of
increased sensitivity to taxol and vinblastine in GCS antisense cells
is shown in Fig. 5A
, B
, respectively. GCS antisense transfection
diminished the cytotoxic threshold of taxol and vinblastine from the
micromolar to nanomolar range. Cellular sensitivity to vinblastine,
vincristine, taxol, taxotere, and actinomycin D increased by 115-, 50-,
241-, 66-, and 9-fold, respectively (Table 1)
. In contrast, sensitivity
to 5-flurouracil increased only 3.6-fold in MCF-7-AdrR/asGCS cells, and
the EC50 of cisplatin in the three cell lines
remained unchanged (Table 1)
. These data demonstrate that the
expression of GCS antisense reverses multidrug resistance, and that
reversal is specific for certain drug classes.
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Colony formation in soft agar under drug-free conditions was also
evaluated, and a difference in the three cell lines was not
demonstrated. However, under adriamycin stress, GCS sense cDNA
transfectants exhibited enhanced colony formation (Fig. 6
), whereas GCS antisense cDNA transfectants showed a marked decrease in
colony formation (Fig. 6)
. With 2.5 µM adriamycin, colony formation
was <10% in MCF-7-AdrR/asGCS cells, compared with 70% in MCF-7-AdrR
and 99% in MCF-7-AdrR/GCS cells. Thus, adriamycin toxicities in the
GCS antisense-transfected cells were on a par, whether treated under
anchorage-dependent (Table 1)
or anchorage-independent growth
conditions (Fig. 6)
.
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Resumption of ceramide signal in MCF-7-AdrR/asGCS cells potentiates
drug cytotoxicity
To further elucidate the dynamics of ceramide metabolism in
anthracycline sensitivity, we measured ceramide generation in the three
cell lines and found that adriamycin elevated ceramide levels in GCS
antisense transfected cells. As shown in Fig. 7A
, adriamycin at 0.5 µM increased the levels of ceramide in
MCF-7-AdrR/asGCS cells by 184% compared with MCF-7-AdrR cells (632±67
vs. 344±25 cpm) and 227% (857±24 vs. 376±16 cpm) at 2.5 µM
(P<0.001). In contrast, MCF-7-AdrR/GCS cells did not
display a ceramide response when challenged with adriamycin (Fig. 7A
). Another agent, actinomycin D, also potentiated cellular
ceramide generation in GCS antisense transfected cells (Fig. 7B
), with no significant changes occurring in either
MCF-7-AdrR or MCF-7-AdrR/GCS cells.
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We next examined GC levels in the three cell lines under basal
conditions and under adriamycin exposure. As shown in Fig. 8A
by TLC autoradiography, GCS antisense transfection reduced
cellular levels of GC (lane 3), compared with MCF-7-AdrR cells (lane
1). When evaluated by scintillation spectroscopy, MCF-7-AdrR/asGCS
cells exhibited an 
27% decrease in GC compared with parent cells
(1854±54 vs. 2546±133 cpm, Fig. 8B
, minus adriamycin). GC
was slightly elevated in GCS sense transfectants (Fig. 8A
,
lane 2); however, the difference was not statistically significant
compared with MCF-7-AdrR cells (2819±174 vs. 2546±133 cpm, Fig. 8B
, minus adriamycin). Although adriamycin exposure elicits
ceramide increases in MCF-7-AdrR/asGCS cells (Fig. 7A
),
exposure to this drug did not alter GC metabolism in any of the cell
lines (Fig. 8B
).
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Increases in effector caspase activity in ceramide-governed apoptotic
signaling are consistent with changes in ceramide levels. With 10 µM
adriamycin (EC50 in MCF-7-AdrR cells), caspase-3
activity in MCF-7-AdrR/asGCS cells increased 3- (1.99±0.005 vs.
0.63±0.005 nmole AFC/106 cells,
P<0.001) and 3.6-fold (3.6±0.04 vs. 1.02±0.03 nmole
AFC/106 cells, P<0.001), at 24- and
48-h treatment, respectively, compared with MCF-7-AdrR cells
(Fig. 9A
). Under like conditions, caspase-3 activity in
MCF-7-AdrR/GCS cells had no significant decrease, compared with
activity in MCF-7-AdrR cells at 48 h. Further characterization
revealed that adriamycin elicited apoptosis only in the GCS
antisense-transfected cells (Fig. 9B
, 9C
). The
levels of apoptosis in GCS antisense cells increased with increasing
concentration of adriamycin. The apoptotic index was 370 (0.44±0.01
vs. 0.12±0.005 OD) and 700% (0.84±0.04 vs. 0.12±0.005 OD) of
untreated MCF-7-AdrR/asGCS cells at 0.5 and 5.0 µM adriamycin,
respectively (Fig. 9B
). Using DNA fragmentation analysis,
adriamycin treatment (5.0 µM, 72 h) only induced apoptosis in
MCF-7-AdrR/asGCS cells (Fig. 9C
, lane 6). Overall, these
data show that reduced glycosylation potential enhances cellular
buildup of free ceramide under adriamycin stress and potentiates the
progression of programmed cell death via caspase.
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P-glycoprotein expression and efflux parameters
P-glycoprotein–modulated drug efflux is the most widely
characterized drug resistance mechanism (26)
, and it is
highly expressed in MCF-7-AdrR cells (9
, 22)
. However,
adriamycin resistance produced by enforced expression of GCS is not
accompanied by increased P-glycoprotein in MCF-7/GCS cells
(9)
. To assess the role of the MDR1 phenotype
in enhanced drug sensitivity expressed by GCS antisense transfectants,
we analyzed rhodamine-123 efflux and P-glycoprotein expression in the
MCF-7 cell variants (Fig. 10
). Enhanced rhodamine-123 efflux was observed in all cell lines, except
MCF-7 (Fig. 10A
). The enhanced efflux correlated with
overexpression of P-glycoprotein in the MCF-7-AdrR cell variants (Fig. 10B
). Therefore, the up- or down-regulation of GCS activity
by transfection had little influence on either rhodamine-123 efflux or
P-glycoprotein expression (Fig. 10A
, 10B
). This
suggests that P-glycoprotein is not a major mechanism involved in
chemotherapy sensitization that accompanies GCS antisense transfection.
Interestingly, we found enhanced uptake of rhodamine-123 in
MCF-7-AdrR/asGCS cells, compared with MCF-7-AdrR and MCF-7-AdrR/GCS
cells. The degree of enhancement was similar with the fluorescence
intensity observed in wild-type MCF-7 cells (Fig. 11A
). Quantitative fluorescence measurement of intracellular
rhodamine-123 showed that MCF-7 cells took up 41% of the total
(7,043±57/1.7x104 FU, [fluorescence units])
on a parallel with MCF-7-AdrR/asGCS (41%,
7,077±518/1.7x104 FU). Rhodamine-123 uptake in
GCS antisense transfectants was fivefold greater than uptake in
MCF-7-AdrR cells (7,077±518 vs. 1,331±213 FU) and 10-fold greater
than uptake in MCF-7-AdrR/GCS cells (7,077±518 vs. 669±106 FU)
(Fig. 11B
).
|
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 22
, 23)
.
Although there is overexpression of P-glycoprotein and
glutathione-S-transferase in MCF-7-AdrR cells (22
, 27)
,
there have been no reports showing that antisense transfection with
either P-glycoprotein or glutathione-S-transferase reverses multidrug
resistance in these cells. Our study shows that drug resistance can be
reversed by antisense manipulation of ceramide metabolism, and the work
reinforces the doctrine that drug resistance in cancer is
multifactorial (28)
.
Through gene targeting, it has been shown that GCS sense cDNA
transfection induces resistance to adriamycin by 11-fold in MCF-7 cells
(9)
. Although MCF-7-AdrR cells constitutively display high
GCS activity and elevated GC levels (3
, 8)
, introducing
GCS cDNA into these multidrug-resistant cells upgraded, albeit only
severalfold, the resistance threshold for adriamycin (Fig. 4
, Table 1
),
daunorubicin, and actinomycin D (Table 1)
. Compared with MCF-7 cells,
the high basal levels of GC (sixfold more) and enhanced GCS enzyme
activity (8)
, which contribute to multidrug resistance,
may interfere with the efficacy of GCS transfection for further
enhancement of drug resistance in MCF-7-AdrR cells. On the other hand,
GCS antisense, which effectively reintroduced ceramide/caspase
signaling (Figs. 7
and 8)
, substantially restored cellular sensitivity
to many anticancer drugs, including anthracyclines, taxanes,
Vinca alkaloids, and actinomycin D (Table 1)
. All of these
agents are substrates for P-glycoprotein (26)
, whereas
5-flurouracil and cisplatin, whose toxicities were not greatly modified
by GCS antisense, are not classified as pump drugs. This raises the
question of selectivity of GCS antisense for reversal of drug
resistance, a topic that is currently being pursued. GCS antisense also
sensitized cells that were grown in soft agar to adriamycin (Fig. 5)
and sensitized cells grown under hypoxic conditions
(O2<5%, data not shown). These results suggest
that GCS antisense is promising for restoring drug sensitivity under
conditions that mimic tumor in vivo environments. Overall,
these data indicate that GCS is a cause of multidrug resistance in
MCF-7 breast cancer cells.
Overexpression of P-glycoprotein reduces intracellular accumulation of
chemotherapeutic agents and produces multidrug resistance (26
, 29)
. However, the role that P-glycoprotein exerts in breast
cancer has been difficult to define (30
31
32)
. Kim et al.
reported that P-glycoprotein (MDR-1) is a minor determinant
of drug resistance in MCF-7-AdrR cells (32)
. We did not
observe increased P-glycoprotein expression in MCF-7/GCS cells in which
adriamycin resistance was induced by GCS overexpression
(9)
. We also did not observe decreased expression of
P-glycoprotein in MCF-7-AdrR/asGCS cells. When we analyzed
rhodamine-123 efflux and P-glycoprotein expression in MCF-7 cell lines
(Fig. 8)
, our observations were much in line with previous work showing
that MCF-7-AdrR cells have an enhanced efflux capacity, complementing
the enhanced levels of P-glycoprotein (12
, 22
, 23)
.
Although GCS antisense transfection sensitized resistant cells to
several different types of drugs, antisense did not modulate either
P-glycoprotein expression or efflux parameters. This suggests that
P-glycoprotein is not the major mechanism involved in chemotherapy
sensitization that accompanies GCS antisense transfection.
Ceramide is a lipid second messenger in the apoptotic pathway,
participating in cell death initiated by anticancer drugs, cytokines,
and ionizing radiation (33
, 34)
. The apoptotic impact of
adriamycin, daunorubicin, and actinomycin D depends, in part, on
cellular ceramide generation (9
, 24
, 35
, 36)
. Increased
ceramide levels enhance the efficacy of vinblastine (37)
and taxol (38)
. In this study, we show that impairment of
ceramide glycosylation increases intracellular ceramide levels and
propels apoptotic signaling in response to antineoplastic drugs. In
addition to glycosylation, ceramide can be converted to sphingomyelin
(via sphingomyelin synthase), galactosylceramide (via
galactosylceramide synthase), or undergo deacylation to form
sphingosine (via ceramidase) (39)
. However, as a route to
remove ceramide, up-regulating the conversion of ceramide to
sphingomyelin, through sphingomyelin synthase, may be harmful to the
cell, as the levels of sphingomyelin are tightly regulated. Therefore,
although sphingomyelin synthase regulates intracellular ceramide levels
(40)
, GCS appears to be more influential over cell growth
and apoptosis. GCS converts cytotoxic ceramide to GC, an essential
building block of more complex cell membrane components. Various works
show that GC stimulates cell proliferation (41
, 42)
and
tumor growth (43
, 44)
, whereas GCS inhibitors arrest cell
division (45
, 46)
, exhibit anticarcinogenic properties
(46)
, and reduce metastasis (47)
. Complex
derivatives of GC, such as gangliosides GM3,
GM2, and GD2, are also
involved in cell growth, tumor metastasis, and drug resistance
(48
49
50)
. Therefore, lowering glycosphingolipid synthesis
by modifying GC metabolism appears to be a novel strategy in cancer
chemotherapy (10
, 51
52
53)
.
In the present study, GCS antisense markedly sensitized breast cancer
cells to several well-known antitumor agents. Taking into account the
30% decrease in GCS activity in MCF-7-AdrR/asGCS cells (Fig. 3)
,
multifold increases in sensitivity to the various drugs (Table 1)
are
striking. In addition to modulating cellular ceramide and GC levels
under stress, GCS catalyzes the first glycosylation step in the
biosynthesis of glycosphingolipids, lipids which are present on
virtually all mammalian cell plasma membranes (54
, 55)
.
Glycosphingolipids are integral components of plasma membrane
microdomains, such as rafts, caveolae, and
GM3-enriched microdomains (56
, 57)
.
Microdomains are involved in drug resistance (58
, 59)
,
mediating membrane trafficking and signal activity, and coupling cell
adhesion interactions with signaling (60
, 61)
. A GCS
knockout study in mice showed that the consequence of homozygosity was
embryonic lethality, revealing a vital role for GCS during development
and differentiation (62)
. Lowering GCS by
1-phenyl-2-decanoylamino-3-morpholino-propanol exposure brings about
morphological change in Chinese hamster ovary cells (63)
,
in cultured cortical neuron (64)
, and in PC12 cells
(65)
. Ganglioside, GQ1b, is essential for synapse
formation and synaptic activity (64)
. We also found GCS
antisense cDNA markedly altered MCF-7-AdrR cell morphology (Fig. 2)
and
greatly increased the uptake of rhodamine-123 (Fig. 10)
. This suggests
that alteration of membrane glycosphingolipid composition, by enforced
expression of GCS antisense, effects cell morphology and membrane
functional activities, including drug transport. Enhanced drug import,
which may partially underlie the increased sensitivity to anticancer
drugs observed in MCF-7-AdrR/asGCS cells, is being further
investigated.
|
ACKNOWLEDGMENTS |
|---|
We thank Dr. Shinichi Ichikawa and Dr. Yoshio Hirabayashi (Laboratory for Cellular Glycobiology, The Institute of Chemical and Physical Research, RIKEN, Saitama, Japan) for providing the ceramide glucosyltransferase cDNA, pCG 2, and Dr. D. L. Marks and Dr. R. E. Pagano (Mayo Clinic and Foundation) for GCS antiserum. We appreciate the advice of Dr. James Hardin, John Wayne Cancer Institute, in the review of this work.
Received for publication April 27, 2000.
Revision received August 7, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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