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Introduction
Protein kinase C (PKC) is a family
of serine/threonine protein kinases that transduce signals for
tumorigenesis, tumor cell invasion, and metastasis, and has thus
been newly targeted for use in cancer treatment[1].
Activation of PKC augments tumor cell metastatic potential, whereas
suppression of PKC activity through PKC inhibitors reduces tumor
cell invasion and migration[1,2]. 7-Hydroxystaurosporine
(UCN 01) is a selective PKC inhibitor derived from the non-selective
protein kinase inhibitor staurosporine[3]. A number of
studies, including some conducted in our own labora-tories, have
revealed that UCN-01 holds promise for use as a single agent or in
combination with other chemotherapeutic agents, such as
camptothecin, 5-fluorouracil, tamoxifen, and ionizing radiation, in
inhibiting tumor cell growth in vitro and in vivo[3-9].
Our previous studies corroborate
other studies, which indicate that the anti-tumor activities of
UCN-01 are associated with arrest of cell cycle progression,
including G1/S and/or G2/M[4,5], apoptosis induction[5-7,10],
and the inhibition of DNA repair[11,12]. Most notably,
UCN-01 abrogates the S or G2 arrest caused by chemotherapeutic
agents[4]. It has also been found that UCN-01 inhibits
microvessel formation (angiogenesis), which is required for tumor
formation and growth[13,14]. Taken together, these
studies indicate that UCN-01 is a profound anti-tumor agent in
several different tumors, including glioma. Moreover, UCN-01 has
entered Phase I trials as a single agent in the USA and Japan[3,15].
In the present study, the effects of
UCN-01 on cell growth, invasion, and migration were investigated in
U-87MG, an invasive human glioblastoma cell line. The purpose of
this study is to explore whether UCN-01 can be used as a new
therapeutic agent in the treatment of glioblastoma growth and
invasion.
Materials and methods
Cell culture and chemicals
The human glioblastoma cell line
U-87MG was purchased from the American Type Culture Collection
(ATCC, Manassas, VA, USA) and maintained as monolayer cultures in
D-MEM supplemented with 10% fetal calf serum (FCS), glutamine 2
mmol/L , streptomycin 100 mg/L , and 100 kU/L benzylpenicillin G
(BioWhittaker, Walkersville, MD, USA). Cell number and viability
were determined by staining a small volume of cell suspension with
0.4% trypan blue saline solution and examining the cells using a
haemocytometer. The doubling time of U-87MG cell was approximately
30 h under our culture conditions. UCN-0 was obtained from the
Laboratory of Molecular Pharmaco-logy, Division of Basic Science,
NCI, NIH (Bethesda, MD, USA) and stored at -20 oC as a 10
mmol/L stock solution in 20% Me2SO. Phorbel
12-myristate-13-acetate (PMA) and ethanol (EtOH) were purchased from
Sigma Chemical Company (St Louis, MO, USA). PMA was dissolved in 20%
Me2SO and frozen at -20 oC for storage
purposes. UCN-01, PMA, and EtOH were further diluted in 2% medium
prior to use.
Expression vectors and
transfection A full-length BRCA1 or PTEN cDNA was expressed in a
pCMV-Tag2B vector (Stratagene, La Jolla, CA, USA), which allows for
the expression of proteins with an N-terminal FLAG sequ-ence, as
described previously[16]. Transfection of BRCA1 or PTEN
was performed using a transfection reagent, Lipofect-Amin 2000,
according to the manufacturer's instructions (Invitrogene,
Gaithersburg, MD, USA) as described previously[16]. The
efficiency of gene transfer was determined by a
¦Â-galactosidase
assay using co-transfection with plasmid pRSV-b-gal.
Measurement of PKC activity
Aliquots of 4¡Á106 untreated and treated cells were washed
with ice-cold Dulbecco's phosphate-buffered saline (PBS), suspended
in a homogenization buffer (0.05 mol/L Tris¯HCl, pH 7.5,
containing EDTA 5 mmol/L , EGTA 10 mmol/L ,
¦Â-mercaptoethanol 0.6
g/L , leupeptin 10 mmol/L , PMSF 1 mmol/L ), and broken by probe
sonication. The nuclei and unbroken cells were removed using
low-speed spin, and the resulting supernatant was spun at 100 000¡Ág
for 30 min. The nuclear pellet was resuspended in HB and was
referred to as a whole cell lysate. The 100 000¡Ág supernatant was
termed the soluble or cytosolic fraction. The 100 000¡Ág
pellet was washed with homogenization buffer, suspended by probe
sonication in homogenization buffer containing 0.1% Triton X-100,
incubated on ice for 1 h, and spun again at 100 000¡Ág. The
resulting supernatant was termed the particulate or membrane
fraction.
Total PKC activity in the three
fractions was estimated using a commercially available assay system
(Amersham Life Science, Arlington Heights, IL, USA) according to the
manufacturer's instructions. This assay is based on the
PKC-catalyzed transfer of the 32P-phosphate group from
[¦Ã-32P]ATP into a PKC-specific peptide substrate (amino
acids 65¯658 of the EGF receptor with the phosphorylation
site on Thr-654) in the presence of Ca2+
phosphatidylserine, and phorbol 12-myristate 13-acetate. All 3
fractions were diluted with 0.05 mol/L Tris-HCl (pH 7.5), and 25 mg
of each were added to equal volumes of reaction solution (consisting
of Tris¯HCl 0.05 mol/L, CaCl2 1.5 mmol/L,
dithiothreitol 7.5 mmol/L, PKC peptide substrate 45 mmol/L, and
dioleoyl-glycerol 82 mmol/L) as provided by the manufacturer.
Reactions were carried out at 37 oC for 15 min. After 15
min, the reactions were stopped using a stop reagent (provided by
the manufacturer); one hundred microlitre of each were blotted on
glass fiber filters and extensively washed with 0.1% orthophosphoric
acid. The bound radioactivity was counted using liquid scintillation
spectroscopy. The protein content in each of the 3 fractions was
measured. Radioactivity values resulting from the phosphorylation of
endogenous substrates were subtracted from all determinations.
Reactions were performed in triplicate and the values were averaged.
Each assay was performed at least 3 times.
Measurement of cell viability
Cell viability, an indicator of cytotoxicity, was evaluated using an
MTT assay as described previously[17]. Four sets of
experiments were performed in 10 wells for each treatment.
In vitro invasion assay
In vitro invasion assay was performed with a modified
Boyden chamber[17]. The surfaces of filter (0.8 mm pore
size) were coated with 25 mg Matrigel of uniform thickness for 1 h
at room temperature. The uniformity of the coating was checked by
Coomassie blue staining and low-power microscope observation. The
lower chamber was filled with 10% FCS medium containing fibronectin
(16 mg per chamber) as the chemoattractant. Cells (1¡Á108
cells/L) re-suspended in the medium containing 2% FCS and 50 or 100
nmol/L UCN-01 were carefully transferred onto the upper surface of
the filters in the chamber. After a 48-h incubation, the filter was
gently removed from the chamber. The cells on the upper surface were
removed with a cotton swab, cells that had passed through the
Matrigel and attached themselves to the lower surface of the filter
were fixed, stained with hematoxylin and eosin, and counted in 15
randomly selected microscopic fields (¡Á400) per filter. Experiments
were performed at least 3 times, independently. In the trials for
determining the effect of UCN-01 on the PMA- and EtOH-promoted cell
invasion, fibronectin was not added to the lower chamber.
Scratch wound assay The
spreading and migration capabilities of U-87MG cells were assessed
using a scratch wound assay[17] which measures the
expansion of a cell population on surfaces. The cells were seeded
into 6-well tissue culture dishes at a concentration of 2.5¡Á105
cells and cultured in medium containing 10% FCS to nearly confluent
cell monolayers, which were then carefully wound using 1-mL sterile
pipette tips. Any cellular debris was removed by washing with PBS.
The wounded monolayers were then incubated in 10% FCS medium
containing UCN-01 (50 or 100 nmol/L) for 24 h or 48 h, then
photographed under a light microscope (¡Á200). The experiments were
repeated in quadruplicate wells at least 3 times.
Immunoblot assay Protein
expression was assessed using an immunoblot assay as described
previously[16]. A monoclonal E-cadherin antibody was
purchased from Transduction Laboratories (Lexington, KY, USA). A
mouse monoclonal anti-FLAG antibody M2 (Stratagene, La Jolla, CA,
USA) was used to detect expression of the FLAG-tagged BRCA1 and PTEN
proteins. Equal protein loading and the protein transfer were
confirmed by immunoblotting for the determination of a-actin protein
using a polyclonal a-actin antibody (I-19, Santa Cruz, Hercule, CA,
USA) on the same Western blots stripped. A colored marker (Bio-Rad
Labora-tories, Hercules, CA, USA) was used as a molecular size
standard.
Reverse transcription-polymerase
chain reaction (RT-PCR) mRNA was assayed by RT-PCR as
described in previous studies[16]. cDNA was synthesized
from 2 µg of total RNA in a 30-µL reaction mixture containing
5¡Áreverse transcriptase reaction buffer (Life Technologies, Inc,
Gaithers-burg, MD, USA), dNTP 200 µmol/L , 100 µmol/L
solution of primers, 50 units of RNasin (Promega,
Madison, WI, USA), dithiotheithol 10 mmol/L , and 100
units of reverse transcriptase (Life Technologies, Inc).
The mixture was incubated at 37 ¡ãC for 60 min, heated to 95 ¡ãC for
10 min, and then chilled on ice. PCR was carried out in a
50-µL volume containing 10-20 ng cDNA, chelating buffer (Perkin-Elmer/Cetus,
Norwalk, CT, USA), 20 µmol/L dNTP mixture, 1.5 units of Taq
DNA polymerase (Perkin-Elmer/Cetus), and 0.5 µmol/L
of the following E-cadherin-specific primer pairs:
5'-CAATCTCA-AGCTCATGG-3' (forward) and 5'-CCATTCGTTCAAGTA-GTC-3'
(backward). The PCR was processed at 94 ¡ãC for 1 min, 54 ¡ãC for 1
min, and 72 ¡ãC for 1 min. To ensure that the RNA was of
sufficient purity to undergo RT-PCR, a PCR assay using
primers specific for the b-actin gene cDNA was performed for each
sample through the same PCR process. b-Actin specific
primer pairs were as follows: 5'-GTC AAC GGA TTT GGT CTG
TAT T-3' (forward); 5'-AGT CTT CTG GGT GGC AGT GAT-3' (backward).
Semi-quantitative PCR conditions for E-cadherin and b-actin are 28
cycles and 22 cycles, respectively. The PCR products were
electrophoresed on a 5% nondenaturing polyacrylamide gel.
The gel was then dried and exposed to an imaging plate,
and the radioactivity was determined using a Bioimage
Analyzer (Bas1000; Fuji, Kanagawa, Japan).
Cell-cell aggregation assay
Cells were harvested using HBSS buffer containing 0.01% trypsin and
centrifuged at 100 000¡Ág for 5 min. Cell pellets were
re-suspended in a HCMF buffer (NaCl 160 mmol/L, Na2HP4
0.6 mmol/L , 0.1%
w/v glucose, and 0.01% HEPES, pH 7.4) containing CaCl2 4
mmol/L at a cell density of 1¡Á105 cell/ml. Total 1 mL
aliquots of a single cell suspension were transferred to
microcentrifuge tubes. UCN-01 (50 or 100 nmol/L), anti-E-cadherin
antibody (0.5 mg/L, Transduction Laboratories, Lexington, KY, USA),
or a combination of both was added. The cell cultures were then put
on a shaker maintained at 37 oC for 60 min and 0.02 mL
aliquots from all cell cultures were removed and fixed in
0.060 mL of glutaraldehyde. The total number of particles (that is,
cells or aggregates) were counted by two investi-gators, using a
haemocytometer. The degree of aggregation is represented by the
aggregation index calculated by Nt=60/Nt=0,
where Nt=0 is the number of single cells before the
incubation and Nt=60 is the number of single cells after
the incubation for 60 min. Nt=x60/Nt=0>1=no
cell-cell adhesion; Nt=60/Nt=0<1=specific
cell-cell adhesion.
Statistical analysis Each
assay was performed at least 3 times. Statistical significance for
the results was assessed using Student's t-test. P<0.05
suggests a statistically significant difference.
Results
Effect of UCN-01 on proliferation of
U-87MG cells Using an MTT
assay, we determined the cell viability of
U-87MG glioblastoma cells after treatment with various doses of
UCN-01 for 24 or 48 h. As shown in Figure 1A, exposure to UCN-01
resulted in a loss of cell viability in a concentration- and
time-dependent manner. ID50, a dose at which 50% of the
cells lost their viability, was approximately 400 nmol/L and 260
nmol/L after the 24-h and 48-h treatments, respectively (P<0.01).
At UCN-01<100 nmol/L doses, no significant effect on cell viability
was observed at any exposure time points (P>0.01). UCN-01
doses of <100 nmol/L were thus selected for use in further studies.
We also determine the cytotoxicity of UCN-01.
Effect of UCN-01 on PKC activity
of U-87MG cells Subconfluent cells were left untreated or
treated with 100 nmol/L UCN-01 for 4 h and then harvested for assay
of PKC activity . The membranous fractions (404¡À23 pmol 32P/min,
P<0.01) and cytosolic factions (114¡À46 pmol 32P/min,
P<0.01) were significantly inhibited in the cells treated
with UCN-01 compared to the untreated control cells (885¡À42 pmol
32P/min for the membranous fractions, 289¡À65 pmol 32P/min
for the cytosolic fractions). Similarly, reduced PKC activity was
also observed after treatment with 50 nmol/L UCN-01 (data not
shown).
Effect of UCN-01 on invasion and
motility of U-87MG cells Cell invasion and migration are crucial
processes in tumor metastasis. A modified Boyden chamber assay was
performed in order to determine the ability of U-87MG cells to
invade through biological matrices in vitro, based on the
percentage of cells able to penetrate the reconstituted basement
membrane-coated filters and attach to the lower surface of filter.
As shown in Figure 1B, the invasion capacity of U-87MG cells was
markedly inhibited when UCN-01 was present in the upper chamber; a
reduction of approximately 12% and 25% (P<0.01) was observed
for 50 and 100 nmol/L UCN-01 after 48-h treatment, respectively. A
similar inhibition of cell invasion activity by UCN-01 was also
observed in U-373, another glioblastoma cell line (data not shown).
To examine whether UCN-01
anti-invasion potential is associated with its ability to suppress
cell spreading and migration, the effect of UCN-01 on U-87MG cell
motility was also analyzed using a scratch wound assay. As
demonstrated by the representative fields shown in Figure 1C, 50
nmol/L UCN-01 markedly inhibited the flattening and spread of both
cell lines along the edges of the wound compared to the untreated
control cells. When this experiment was repeated using a UCN-01
dosage of 100 nmol/L instead of 50 nmol/L, the anti-migration
observed was more significant.
Effect of UCN-01 on PMA- and
EtOH-stimulated cell invasion
We determined the effect of UCN-01 on
the stimulation of cell invasion caused by PMA and EtOH, two PKC
activators. U-87MG cells spontaneously penetrated through an
artificial basement membrane, and the number of invading cells
significantly increased when PMA or EtOH was included in the cell
suspension [invasion index 58¡À4 (PMA), 62¡À6 (EtOH) versus the
untreated control, 46¡À3; P<0.01]. However, both spontaneous
invasion and PMA- or EtOH-promoted cell invasion were markedly
inhibited by UCN-01(Figure 2A). The ability of UCN-01 to suppress
PMA- and EtOH-promoted cell invasion could not be attributed to
increased cytotoxicity, since UCN-01 did not significantly affect
the cytotoxicity of PMA or EtOH (Figure 2B).
Effect of BRCA1 and PTEN on
anti-invasion activity of UCN-01 To determine the effect of the
tumor suppressor genes BRCA1 and PTEN on the anti-invasion potential
of UCN-01, we compared the invasion behavior of U-87MG cells that
had been transiently transfected with the BRCA1 or PTEN gene in the
presence and absence of UCN-01 (100 nmol/L). The full-length BRCA1
or PTEN cDNA was expressed in a pCMV-Tag2B vector, which allows for
the expression of proteins with an N-terminal FLAG sequence.
Exponentially growing cells (3¡Á105 cell/well) in 6-well
tissue culture dishes were transfected with either the BRCA1 or PTEN
vector (5 mg/well) overnight, washed, and then incubated for 48 h.
Overexpression of the BRCA1 or PTEN transgene was confirmed by the
determination of BRCA1 or PTEN protein using an anti-FLAG antibody
(M2). Significant expression levels of the BRCA1 (220 kDa) and PTEN
(67 kDa) proteins were observed in U-87MG cells 48 h after
transfections (Figure 3A). The transfected cells were collected and
subjected to the in vitro invasion assay. As shown in Figure
3B, the transfection with the "empty" pCMV-Tag2B control vector had
no effect on UCN-01 modulation of cell invasion, whereas
overexpression of the BRCA1 or PTEN transgene significantly enhanced
the anti-invasion potential of UCN-01. BRCA1 or PTEN transfection
alone caused a 20% reduction in cell invasion; 100 nmol/L UCN-01
inhibited 30% of cell invasion. However, UCN-01 caused an 87% and a
90% inhibition in invasion in cells transfected with BRCA1 and PTEN,
respectively. These results suggest that a combination of UCN-01 and
BRCA1 or PTEN transgene synergistically inhibits the invasive
behavior of glioblastoma cells.
UCN-01 increases E-cadherin
expression To explore the mechanism(s) underlying the effect of
UCN-01 on U-87MG cell invasiveness and migration, we analyzed the
expression of E-cadherin protein, an adhesion molecule, by an
immunoblot assay, following treatment with UCN-01. As illustrated in
Figure 4A, U-87MG cells contained a low basal level of the
endogenous E-cadherin protein. Forty-eight hours exposure to UCN-01
resulted in a significant dose-dependent increase. UCN-01(50 and100
nmol/L resulted in approximate 2.2- and 6.3-fold increases in the E-cadherin
protein. Similar results were obtained for E-cadherin mRNA
expression in response to UNC-01 treatment (Figure 4C).
Effect of UCN-01 and anti-E-cadherin
antibody on formation of cell-cell aggregates To ascertain
whether E-cadherin plays an important role in the protection of
UCN-01 against the migration and invasion activity of U-87MG cells,
we examined the effect of UCN-01 on the formation of cell-cell
aggregates. UCN-01 100 nmol/L significantly inhibited the formation
of 3-dimensional cellular aggregates (aggrega-tion index=1.3, P<0.01)
compared to the untreated control U-87MG cells, which showed a
significant degree of cell-cell aggregation (aggregation
index=0.65). However, in the presence of the E-cadherin antibody,
the protective ability of UCN-01 against the formation of cell-cell
aggregates was markedly reduced (aggregation index=0.8). Similar
results were observed in another human glioblastoma cell line,
U-373 (data not shown). These results suggest that E-cadherin can
play a critical role in the anti-invasion potential of UCN-01.
Discussion
PKC is viewed as a new target for
suppressing tumor cell invasion and migration, and therefore
metastasis[1,2]. In the present study we found that in
human gliocarcinoma U-87MG cells, UCN-01 inhibits cell proliferation
in a concentration- and time-dependent fashion at higher doses (>100
nmol/L). Furthermore, at less cytotoxic doses (<100 nmol/L), UCN-01
reduces the PKC activity and suppresses the cell invasion and
migration capabilities. The anti-invasion and anti-migration
potential of UCN-01 was confirmed by further experiments in which
the PMA- or EtOH-promoted activities of cell invasion were blocked
by UCN-01. Both PMA and EtOH are potent activators of PKC[1,18].
These findings corroborate previous observations that PKC plays a
central role in the invasion of glioblastoma-derived cell lines[19].
Therefore, the profound inhibition of tumor cell invasion and
migration are novel features that contribute to the anti-tumor
properties of UCN-01.
Although the mechanisms by which
suppression of cell invasion and migration by UCN-01 have yet to be
discovered, our study has revealed an important finding: despite the
inhibition of PKC activity, up-regulated expression of the cell
adhesion E-cadherin protein accompanies UCN-01-modulated cell
invasion and migration activity. Moreover, a significant reduction
of UCN-01 resulted in increased formation of cell-cell aggregates by
the E-cadherin antibody. E-cad-herin, a transmembrane glycoprotein,
is a key mediator of cell-cell adhesion, which acts via the
formation of a complex with 3 major cytoplasmic catenins (a, b, and
g), and reportedly plays a key role in controlling the invasive and
metastatic progression of a variety of human carcinoma cells,
including glioblastoma[20]. Disruption of the E-cadherin/catenin
complex, due primarily to decreased or lost expression of E-cadherin,
is shown to be correlated with increased cell proliferation,
motility, and invasiveness associated with the progression of tumors[21].
Moreover, it is also found that tamoxifen, a PKC inhibitor, blocks
the invasion and migration of tumor cells by increasing the
expression and functions of E-cadherin/catenin complexes[1].
Therefore, E-cadherin is believed to be a critical mediator of the
anti-tumor invasion and migration potential of UCN-01, although
further experiments are needed for clarification of the relationship
between E-cadherin expression and PKC activity.
In addition, we also found that the
enforced expression of two tumor suppressor genes, BRCA1 or PTEN, by
a transient transfection assay significantly affects the inhibition
potential of UCN-01 on U-87MG glioblastoma cell invasion in a
synergistic manner. BRCA1 (Breast Cancer susceptibility gene 1) is
located at human chromosome 17q21, and mutations of BRCA1 are known
to confer an added risk for breast and ovarian cancers in women, and
for prostate cancer in men[22]. Increasingly, evidence
indicates that invasive breast tumors show decreased BRCA1 mRNA
expression and a loss of BRCA1 immunochemical staining relative to
non-invasive tumors and benign tissues[23]. A clinical
observation suggests that E-cadherin expression is potentially
correlated with BRCA1-associated breast cancer[24]. Our
recent studies also found that indole-3carbinol, a promising
phytochemical produced by cruciferous vegetables, results in
up-regulation of BRCA1 and E-cadherin/catenin complex expression,
which results in the suppression of tumor invasion and migration[17].
In addition, we also found that alcohol promotes tumor cell invasion
and migration and is associated with down-regulation of BRCA1[24].
Phosphatase and tensin homologue
deleted from chromosome 10 (PTEN) was identified in the 10q23
chromosome region and is often found in mutated forms in a wide
range of human malignancies, including glioblastoma[25].
The restoration of the PTEN gene into U-87MG cells has been found to
markedly suppress cell migration and invasion by negative regulation
of the signals generated at the focal adhesions, and by the direct
dephosphorylation and inhibition of FAK, which is involved in the
cell's interactions with extracellular matrix proteins responsible
for the migration and enhancement of cell spreading through
phosphorylation of its tyrosine[26]. Loss of PTEN
expression and inactivation of wild-type PTEN function can often be
observed in advanced or invasive tumors. Accordingly, these findings
suggest that both BRCA1 and PTEN can function as inhibitors of tumor
invasion and metastasis. Thus, our present findings provide in
vitro evidence to suggest that a combination of UCN-01 function
along with BRCA1 or PTEN gene therapy could be potentially useful in
the clinical prevention and treatment of glioblastoma, although it
is evident that for this to occur, further studies are required.
In conclusion, we discovered that
UCN-01, as a selective PKC inhibitor, suppressed the cell migration
and invasion and inhibits the PMA- and EtOH-promoted cell invasion
in human glioblastoma U-87MG cells. Moreover, this inhibition of
glioblastoma cell invasion and migration by UCN-01 is characterized
by an enhanced expression of cell adhesion molecule E-cadherin.
Therefore, UCN-01 can be a potent anti-tumor agent in the therapy of
glioblastoma, not only via the suppression of proliferation of
glioblastoma cells, but also via the inhibition of cell invasion and
metastasis. Our results, which support previous studies[19],
also indicate that the PKC signal transduction pathway can play an
important role in glioblastoma cell invasion and metastasis, and
that the suppression of this pathway may significantly impair the
malignant progression of human glioblastoma.
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