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Experimental Therapeutics, Preclinical Pharmacology |
Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan [Y. L., M. B., S. A., F. H. S.], and Developmental Therapeutics Program, National Cancer Institute, Bethesda, MD [A. M. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apoptosis is one of the important pathways through which chemotherapeutic agents inhibit the growth of cancer cells. Thus, it is important to investigate whether the induction of apoptosis and alterations of apoptosis-related gene expression are associated with the molecular mechanism by which flavopiridol may exerts its biological effects on breast cancer cells. The induction of apoptosis is partly mediated intracellularly by several genes, such as Bcl-2 and Bax (8) . Bcl-2 functions as a suppressor of apoptotic death triggered by a variety of signals (9) , whereas a predominance of Bax over Bcl-2 accelerates apoptosis upon apoptotic stimuli (10) .
Breast cancer is the most common cancer in women in the United States,
and it remains the second leading cause of cancer-related female deaths
in this country (11)
. Several genes have been implicated
in breast cancer aggressiveness. c-erbB-2 is a key molecule
for breast cancer metastasis. Overexpression of the c-erbB-2
gene has been found in 
20–30% of human breast cancers and has been
positively correlated with invasion and metastasis in cancers
(12
, 13)
. It has been found that transfection of
c-erbB-2 into breast cancer cells increased their ability
for invasion and metastasis and that an increase of MMPs was detected
in c-erbB-2 transfected cells (14)
. MMPs are
believed to be key molecules for cancer invasion and metastasis
(15, 16, 17)
. Furthermore, overexpression of
c-erbB-2 has been shown to be correlated with an increase in
MMP secretion and metastatic potential of breast cancer cells in
experimental metastasis assays (14)
.
In this study, we investigated whether flavopiridol could inhibit the growth of breast cancer cell lines, MDA-MB-435 and 435.eBs (established by transfecting c-erbB-2 cDNA into MDA-MB-435), and whether c-erbB-2 overexpression may affect the sensitivities of these cells to flavopiridol. In addition, we were interested in elucidating the molecular mechanism by which flavopiridol may induce apoptotic cell death in these isogenic cell lines. We also investigated the effect of flavopiridol on MMP3 secretion and invasion, which was previously shown to be affected by c-erbB-2 transfection. Our data show that flavopiridol inhibits the growth of breast cancer cells irrespective of c-erbB-2 overexpression, induces apoptosis, regulates the expression of genes, and inhibits invasion and, thus, may inhibit metastasis of breast cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Growth Inhibition.
The MDA-MB-435 cells, 435.neo cells, and 435.eB cells were seeded at a
density of 5 x 104/well in a six-well
culture dish. After 24 h, the cells were treated with 70
nM, 150 nM, and 300 nM of
flavopiridol or DMSO (vehicle control). Cells treated with flavopiridol
or DMSO for 1–3 days were harvested by trypsinization, stained with
0.4% trypan blue, and counted using a hemocytometer.
Protein Extraction and Western Blot Analysis.
The breast cancer cells (MDA-MB-435, 435.neo, and 435.eB) were plated
and cultured in complete medium and allowed to attach for 24 h
followed by the addition of 150 nM or 300 nM
flavopiridol and incubation for 24, 48, and 72 h. Control cells
were incubated in the medium with DMSO using the same time points.
After incubation, the cells were harvested by scraping the cells from
culture dishes using a scraper and collected by centrifugation. Cells
were resuspended in Tris-HCl buffer, sonicated for 2 x 10 s,
and lysed using an equal volume of 4% SDS. Protein concentration was
then measured using protein assay reagents (Pierce, Rockford,
IL). Cell extracts were boiled for 10 min and chilled on ice,
subjected to 14 or 10% SDS-PAGE, and electrophoretically transferred
to a nitrocellulose membrane. Each membrane was incubated with
monoclonal Bcl-2, c-erbB-2 (1:500, Oncogene, Cambridge,
MA), Bax (1:5000; Biomol, Plymouth Meeting, PA), and
rabbit polyclonal ß-actin (1:5000, Sigma, MO) antibodies,
washed with Tween 20 in Tris-buffered saline, and incubated with
secondary antibody conjugated with peroxidase. The signal was then
detected using the chemiluminescent detection system (Pierce).
Densitometric Analysis for c-erbB-2 Expression.
Autoradiograms of the Western blots for c-erbB-2 and actin
protein expression were scanned with the Gel Doc 1000 image scanner
(Bio-Rad, Hercules, CA). The bidimensional optical densities of
c-erbB-2 and actin proteins on the films were quantified and
analyzed with Molecular Analyst software (Bio-Rad). The Ratios of
c-erbB-2/actin were calculated by standardizing the ratios
of each control to the unit value.
Zymography of MMP Activity.
Gelatin zymography was performed according to the methods published
previously (14
, 19)
, with a slight modification. The
breast cancer cells (MDA-MB-435, 435.neo, and 435.eB) were plated and
cultured in complete medium and allowed to attach overnight. The cells
were washed three times with DMEM/F12 and incubated in conditioned
medium for 24, 48, and 72 h. The conditioned media were DMEM/F12
without serum and phenol red and with 150 or 300 nM
flavopiridol or DMSO. The culture supernatants were collected, and cell
debris was spun off. The supernatants were concentrated using spin
columns (Amicon, Beverly, MA), and the protein concentration was
measured using protein assay reagents (Pierce). The samples with equal
proteins were then subjected to 10% nonreducing SDS-PAGE (containing
0.1% gelatin). After electrophoresis, the gels were washed with 2.5%
Triton X-100 for 30 min and with developing buffer (10 mM
Tris base, 40 mM Tris-HCl, 200 mM NaCl, 5
mM CaCl2, and 0.02% Briji 35) for
1 h. After washing, the gels were incubated with fresh developing
buffer overnight at 37°C. The gels were then stained with Coomassie
solution (0.5% Coomassie blue in 10% methanol, 5% acetic acid) for
1 h and de-stained with same solution without Coomassie blue.
DNA Ladder Formation.
Cellular cytoplasmic DNA from cells treated with 300 nM
flavopiridol for 24, 48, 72 h or with DMSO for 24 h (as
control) was extracted using 10 mM Tris (pH 8.0), 1
mM EDTA, and 0.2% Triton X-100. The lysate was centrifuged
for 15 min at 13,000 x g to separate the fragment DNA (soluble)
from intact chromatin (nuclear pellet). The supernatant from the lysate
was treated with RNase, followed by SDS-Proteinase K digestion, phenol
chloroform extraction, and isopropanol precipitation. DNA was separated
through a 1.5% agarose gel. After electrophoresis, gels were stained
with ethidium bromide, and the DNA was visualized by UV light.
Analysis of Cleavage of CPP32 and PARP.
Cells treated with 100 nM flavopiridol or with DMSO (as
control) for 24 and 48 h were lysed in lysis buffer [10
mM Tris-HCl (pH 7.1), 50 mM sodium chloride, 30
mM sodium pyrophosphate, 50 mM sodium fluoride,
100 µM sodium orthovanadate, 2 mM
iodoacetic acid, 5 µM ZnCl2, 1
mM phenylmethylsulfonyl fluoride, and 0.5% Triton X100].
The lysates were kept on ice for 30 min and vigorously vortexed before
centrifugation at 12,500 x g for 20 min. Fifty micrograms of
total proteins were resolved on 14% or 10% SDS-PAGE and then
transferred to nitrocellulose membrane. The membrane was incubated with
primary monoclonal antihuman CPP32 antibody (1:250, Santa Cruz, CA) or
PARP antibody (1:5000; Biomol), washed with TTBS, and incubated with
secondary antibody conjugated with peroxidase. The signal was then
detected using the chemiluminescent detection system (Pierce).
Cell Invasion Assay.
The invasion assay was carried out following standard methods using the
matrigel invasion assay procedure as described below. Matrigel was
purchased from Becton Dickinson Labware (Bedford, MA), and
Costar transwell culture inserts were purchased from Fisher scientific
(Itasca, IL). The transwell was coated with 100 µl of
matrigel, which were diluted in ice cold DMEM at a final concentration
of 400 µg/ml and incubated at 37°C for 3 h to allow the
matrigel to polymerize. DMEM was allowed to evaporate under sterile
conditions under the culture hood. The matrigel-coated inserts were
rehydrated by adding 100 µl of serum-free medium and incubating for
2 h at 37°C. The breast cancer cells were cultured as described
earlier, trypsinized, collected by centrifugation, and resuspended in
serum-free medium, and the cell numbers were calculated by using the
trypan blue exclusion method using the hemocytometer. A different
number of cells in 0.5 ml was dispensed into each culture inserts, and
the cells were incubated in a tissue culture incubator for 18 h
without and with 150 and 300 nM flavopiridol. After the
incubation, the medium from the culture inserts was discarded, and the
inner surface was wiped with cotton wipes to remove any noninvading
cells through the matrigel. The inserts were fixed for 3 min in Hema 3
fixative, then consecutively stained with Hema 3/Eosin and Hema
3/methylene blue obtained from Fisher Scientific (Itasca, IL). The
inserts were washed in distilled water, allowed to air dry, and then
examined under a light microscope counting four random fields from each
insert. The percent inhibition of cell invasion relative to controls
was plotted using graph pad prism software.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The treatment of MDA-MB-435, 435.neo, 435.eB1, and 435.eB4 breast
cancer cells for 1–3 days with 70 nM, 150 nM,
and 300 nM of flavopiridol resulted in inhibition of cell
proliferation, which was dose-dependent. However, the dose-dependent
growth inhibition was not influenced by the status of c-erbB-2
overexpression (IC50s for all three cell lines was about 70
nM). The data on the inhibition of cell growth in parental
and 435.neo was found to be identical; hence, Fig. 1
represents
parental cells as the control. Furthermore, in all our subsequent
experiments, we could not find any difference between the parental cell
line and 435.neo; therefore, all subsequent data have been presented
with 435 cells as the control, except Fig. 6
, in which 435.neo has been
presented. The overall inhibition of cell proliferation could be due to
the induction of apoptosis elicited by flavopiridol. We, therefore,
investigated whether flavopiridol could induce apoptosis in these
breast cancer cells.
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. The induction of apoptosis
was pronounced at 24 h of treatment, and it is directly correlated
with the inhibition of cell growth. Western blot analysis revealed that
inactive CPP32 (32 kDa) was cleaved to yield an active fragment (17
kDa) after flavopiridol treatment for 24 h (Fig. 2B).
Furthermore, PARP cleavage analysis showed that the full-size PARP (116
kDa) protein was also cleaved to yield an 85-kDa fragment after
treatment with flavopiridol for 24 h (Fig. 2B)
in all
cell lines tested, corresponding with the activation of CPP32. These
three independent methods of measuring apoptosis provided strong
evidence that apoptosis was induced in all cell lines treated with
flavopiridol and that the apoptosis inducing activity of flavopiridol
was found to be independent of c-erbB-2 overexpression. To explore the
mechanisms by which flavopiridol induces apoptosis, we investigated the
alterations in the expression of genes, which are known to be involved
in the apoptotic pathway.
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Furthermore, the expression of Bax was also found to be modestly
up-regulated after 24 h of 150 nM or 300
nM flavopiridol treatment (Fig. 3B).
We could not find any substantial difference in Bcl-2 and Bax
expression among these 435 cell lines, suggesting that the modulation
in Bax and Bcl-2 by flavopiridol is not dependent on
c-erbB-2 expression.
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; however, the
down-regulation was much more pronounced in parental cells compared to
those overexpressing c-erbB-2. It is important to note that to detect
erbB-2 expression, the blots with parental cells were exposed for a
longer period of time as compared to those with erbB-2 overexpression.
To obtain a quantitative value for the protein expression of
c-erbB-2, absorbance measurement was conducted as described
under "Materials and Methods." The ratios of c-erbB-2 to
actin protein expression revealed that cells treated with flavopiridol
showed a 55–90% decrease in c-erbB-2 compared to the
untreated control (Fig. 5)
.
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These data suggested that increased c-erbB-2 expression in
435.eB cells can lead to increased secretion of MMPs as reported
earlier (14)
and that flavopiridol can inhibit the
expression of c-erbB-2 and, therefore, decrease the
secretion of MMPs, which may be important for the decrease in invasion
and metastatic ability of breast cancer cells exposed to flavopiridol.
However, the decline in MMPs 2 and 9 could be a direct effect on MMPs
and not necessarily dependent on the down-regulation of c-erbB-2, which
requires further investigation. Whether flavopiridol could have any
effect on their ability to invade through matrigel was
subsequently investigated as described below.
Inhibition of Cell Invasion by Flavopiridol.
It has been reported earlier that c-erbB-2 transfected MDA-MB-435
(435.eB1 and 435.eB4) cells secret MMPs, and these cells acquire
invasion and metastatic potential compared to parental cells
(435) or cells transfected with control plasmid (435.neo;
Ref. 14
). We have performed cell invasion assays using
these breast cancer cells treated with flavopiridol, which showed
inhibition of cell invasion through matrigel within 18 h (Fig. 6)
, suggesting that the down-regulation
of c-erbB-2 followed by the down-regulation of MMPs may play a direct
role in the invasion characteristic of these cells when exposed to
flavopiridol. The percent inhibition in cell invasion was found to be
higher in parental cells, which secret less MMPs compared to 435.eB1,
which secrets higher amounts of MMPs and, thus, the residual amount of
MMPs observed in flavopiridol treated 435.eB1 remains relatively higher
compared to parental cells, which could explain the differential effect
on cellular invasion among these cell lines. Collectively, these
results provide strong evidence for the antitumor as well as
anti-invasive properties of breast cancer cells when exposed to
flavopiridol irrespective of c-erbB-2 overexpression, suggesting
broader application of flavopiridol in breast cancer.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been demonstrated that Bcl-2 and Bax play a major role in determining whether cells will undergo apoptosis under experimental conditions that promote cell death. Bcl-2 protects cells from apoptosis, whereas increased expression of Bax can induce apo-ptosis (9 , 10) . It has also been found that the ratio of Bax:Bcl-2, rather than Bcl-2 alone, is important for the survival of drug-induced apoptosis in leukemia cell lines (10) . In our study, a decrease in Bcl-2 expression and up-regulation of Bax expression were observed in breast cancer cells after treatment with flavopiridol for 24 h, corresponding with the induction of apoptosis after flavopiridol treatment. These changes, however, were not related with the status of c-erbB-2 expression. Our results suggest that up-regulation of Bax and down-regulation of Bcl-2 may be one of the molecular mechanisms through which flavopiridol induces apoptosis. However, further in-depth studies are warranted given the importance and potent biological activity of flavopiridol. It is important to note that Konig et al. (21) have found a significant down-regulation of Bcl-2 only after long exposure and a higher concentration of flavopirodol, but two other studies could not find any changes in the levels of Bcl-2 (20 , 23) . Our studies show some modest down-regulation of Bcl-2, which could either be due to the differences in the cell lines used or this modest effect could be due to the concentration of flavopiridol and the duration of treatment. Further studies with higher concentrations and longer exposure time may provide results similar to the findings obtained by Konig et al. (21) . The modulation of cdk as observed previously (1 , 3 , 26) and its association with the induction of apoptosis is still unknown, but the apparent modest down-regulation of Bcl-2 and up-regulation of Bax may be important for the induction of apoptosis elicited by flavopiridol.
Overexpression of c-erbB-2 has been found in breast
cancers, and it has been correlated with lymph node metastasis in
cancer patients (29
, 30)
. Tan et al.
(14)
introduced the human c-erbB-2 gene into
the very low c-erbB-2-expressing MDA-MB-435 human breast
cancer cells and established 435.eB transfectants that express high
levels of c-erbB-2. They compared the metastatic phenotypes
of parental MDA-MB-435 cells and the 435.eB transfectants and found
that overexpression of c-erbB-2 can enhance the MMP
secretion and metastatic potential of MDA-MB-435 human breast cancer
cells. In our study, we found that flavopiridol significantly inhibited
c-erbB-2 expression (55% to 90%) in the 435.eB
transfectants and parental MDA-MB-435 cells, including 435.neo. Because
MMPs were previously found to be up-regulated by c-erbB-2
transfection and overexpression, we also compared the secretion of MMPs
in 435.eB transfectants and MDA-MB-435 with and without flavopiridol
treatment. Higher levels of MMPs in 435.eB transfectants than in
parental MDA-MB-435 were observed, corresponding with a higher
metastatic potential of 435.eB transfectants as previously reported
(14)
. After treatment with flavopiridol, the levels of
MMPs decreased in 435.eB transfectants and MDA-MB-435 parental cells.
The decrease in MMPs may be responsible for the inhibition of cell
invasion observed in this system (Fig. 6)
. It is important to note the
we were able to observe inhibition of cell invasion in all cell lines
within 18 h in our assay system, suggesting that the inhibition of
cell invasion may be directly responsible for the biological effect of
flavopiridol. The inhibition of cell invasion appears not to be due to
cell growth inhibition or apoptosis as suggested by the duration of the
experiment and also based on parallel experiments where the initial
plating cell density was compensated for these variables. Our results
suggest that flavopiridol can inhibit the expression of
c-erbB-2 and, in turn, decrease the secretion of MMPs in
breast cancer cells, which, in turn, inhibits cell invasion and further
suggests that flavopiridol may also inhibit metastatic properties of
breast cancer cells. However, the down-regulation of MMPs could be
independent of c-erbB-2 in flavopiridol-treated breast cancer cells;
this requires further in depth investigation.
In conclusion, our results demonstrated that flavopiridol inhibited the growth of breast cancer cells, regulated the expression of apoptosis-related genes, and induced apoptosis in MDA-MB-435 breast cancer cells. Furthermore, flavopiridol inhibited the expression of c-erbB-2, the secretion of MMPs, and cell invasion, suggesting that flavopiridol may also inhibit metastasis of breast cancer. Flavopiridol, thus, may be an effective chemotherapeutic or preventive agent against breast cancer. However, much remains to be studied about the molecular mechanisms of flavopiridol as an anticancer agent in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was partly funded by the George
Puschelberg foundation. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, Wayne State University School of
Medicine, 9374 Scott Hall, 540 East Canfield Avenue, Detroit, MI 48201.
Phone: (313) 966-7279; Fax: (313) 577-0057 or (313) 966-7558; E-mail: fsarkar{at}med.wayne.edu
3 The abbreviation used is: MMP, matrix
metalloproteinase.
Received 8/ 3/99; revised 10/ 1/99; accepted 10/ 4/99.
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: Bcl-2 ratio modulates the response to dexamethasone in leukaemic cells and is highly variable in childhood acute leukaemia. Int. J. Cancer, 71: 959-965, 1997.[CrossRef][Medline]
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