Download A Polyphenolic Fraction from Grape Seeds Causes Irreversible

Vol. 6, 2921–2930, July 2000
Clinical Cancer Research 2921
A Polyphenolic Fraction from Grape Seeds Causes Irreversible
Growth Inhibition of Breast Carcinoma MDA-MB468 Cells
by Inhibiting Mitogen-activated Protein Kinases Activation
and Inducing G1 Arrest and Differentiation1
Chapla Agarwal, Yogesh Sharma, Jifu Zhao, and
Rajesh Agarwal2
Center for Cancer Causation and Prevention, AMC Cancer Research
Center, Denver, Colorado 80214 [C. A., Y. S., J. Z., R. A.], and
University of Colorado Cancer Center, University of Colorado Health
Sciences Center, Denver, Colorado 80262 [R. A.]
ABSTRACT
In recent years, significant emphasis is being placed on
identifying naturally occurring cancer preventive and interventive agents. In this regard, a polyphenolic fraction isolated from grape seeds (hereafter referred as GSP) has
recently been shown by us and others to prevent tumorigenesis in mouse skin models. Chemical analysis of GSP has
shown that it is largely constituted with procyanidins that
are strong antioxidants. Breast cancer is the most common
invasive malignancy and the second leading cause of cancerrelated deaths in United States women. Accordingly, here we
investigated the effect of GSP on mitogenic signaling and
regulators of cell cycle and apoptosis as molecular targets
for the growth arrest, apoptotic death, and/or differentiation
of estrogen-independent MDA-MB468 human breast carcinoma cells. Treatment of cells with GSP (at 25-, 50-, and
75-␮g/ml doses for 1–3 days) resulted in a highly significant
inhibition (90% to complete, P < 0.001) of constitutive
activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase1/2 in a dosedependent manner after 72 h of treatment. Whereas GSP
treatment of cells did not show a conclusive effect on MAPK/
JNK1 activation, a moderate to highly significant inhibition
(15–70%, P < 0.1– 0.001) of constitutive activation of
MAPK/p38 was also observed in a dose-dependent manner
as early as 24 h of GSP treatment. GSP-treated cells also
showed a strong induction (1.7–2.7 fold, P < 0.001) of cyclindependent kinase inhibitor Cip1/p21 and a decrease (10 –
Received 11/23/99; revised 3/10/00; accepted 3/17/00.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
1
Supported in part by USPHS Grants CA64514 and CA83741, U. S.
Army Medical Research and Materiel Command Grant DAMD17-981-8588, and AMC Cancer Research Center Institutional Funds.
2
To whom requests for reprints should be addressed, at Center for
Cancer Causation and Prevention, AMC Cancer Research Center, 1600
Pierce Street, Denver, CO 80214. Phone: (303) 239-3580; Fax: (303)
239-3534; E-mail: [email protected].
50%, P < 0.1– 0.001) in cyclin-dependent kinase 4. Consistent with these findings, GSP-treated cells resulted in their
accumulation in G1 phase of the cell cycle in a dose-dependent manner. An irreversible growth inhibition (44 – 88%,
P < 0.001) was also observed in 50 and 75 ␮g/ml GSPtreated cells in a time-dependent manner. Additional studies
assessing the biological fate of GSP-treated cells showed that
they do not undergo apoptotic death, as evidenced by a lack
of DNA fragmentation, poly (ADP ribose) polymerase cleavage, and apoptotic morphology of the cells. A morphological
change suggestive of differentiation was observed in GSPtreated cells that was further confirmed by a significant
induction (1.7–2.6 fold, P < 0.001), in both a dose- and
time-dependent manner, in cytokeratin 8 protein level, a
marker of differentiation. An irreversible growth-inhibitory
effect of GSP possibly via terminal differentiation of human
breast carcinoma cells suggests that GSP and the procyanidins present therein should be studied more extensively to be
developed as preventive and/or interventive agents against
breast cancer in humans.
INTRODUCTION
Breast cancer is the most commonly diagnosed invasive
malignancy and a leading cause (after lung cancer) of cancer
deaths in American women (1). One approach to control this
malignancy is its prevention. In most of the cancer prevention
clinical trials including breast cancer, the term “prevention or
intervention” is also frequently used to chemopreventive suppression or reversal of premalignant lesions although the lesion
is not permanently eliminated (2, 3). Several studies in animal
tumor models including rat mammary tumors, followed by
epidemiological studies or vice versa, have convincingly established that consumption of yellow-green vegetables and fruits is
associated with a decreased risk of several malignancies, including breast cancer (4 –12). Accordingly, there has been an increased effort to define the mechanisms by which fruits and
vegetables afford protection against cancer and to identify the
nutritive and nonnutritive components of the fruits and vegetables that exert such effects. One group of cancer preventive
phytochemicals from fruits and vegetables that is receiving
increasing attention in recent years is polyphenolic antioxidants
(13–18).
Grapes (Vitis vinifera) are one of the most widely consumed fruits in the world. Grapes are rich in polyphenols, and
about 60 –70% of grape polyphenols exist in grape seeds as
dimers, trimers and other oligomers of flavan-3-ols commonly
known as procyanidins (or proanthocyanidins; Refs. 19 –21).
Commercial preparations of grape seed polyphenols are mar-
2922 GSP Causes Growth Inhibition in MDA-MB468 Cells
keted in the United States as GSE3, with 95% standardized
procyanidins as dietary supplement due to its health benefits,
particularly a very strong antioxidant activity of procyanidins. In
addition to grape seed, procyanidins are a diverse group of
polyphenolics that are widely distributed in fruits and vegetables
(22).
Several studies in recent years have shown the health
benefits of procyanidins as well as wine consumption, which is
also a source of procyanidins (23–26). In terms of its anticarcinogenic potential, oral feeding of 1% GSE in diet is shown to
inhibit adenomatous polyposis coli mutation-associated intestinal adenoma formation in MIN mice (27). Furthermore, recent
studies by us and others (28, 29) have shown that topical
application of a polyphenolic fraction isolated from grape seeds
(hereafter referred as GSP) or commercial GSE results in a
highly significant protection against phorbol ester-induced tumor promotion in chemical carcinogen-initiated mouse skin.
With regard to epidemiology, a case-control study showed that
increased consumption of grapes is associated with reduced
cancer risk (30). Together, these studies suggested that GSP and
the procyanidins present therein could also be effective in breast
cancer prevention and/or intervention.
In an effort to develop GSP and procyanidins as interventive agents against breast cancer, in this study we focused our
attention on the effect of GSP on MAPK activation and cell
cycle and apoptosis regulators. The selection of these molecular
targets was based on the fact that they are the major, possibly
causative, mitogenic and antiapoptotic signaling contributors to
the multifactorial mechanisms of uncontrolled breast cancer
growth. For example, enhanced expression of EGF receptor
family members [erbB1 (or EGFR) and Her-2/neu/erbB2] and
associated ligands (e.g., transforming growth factor ␣/EGF) has
been shown with high frequency in both estrogen-dependent and
-independent breast carcinomas and derived cells (31–36). This
high expression of growth factors and receptors leads to an
autocrine loop for both mitogenic and antiapoptotic signaling,
leading to autonomous growth and metastasis of breast cancer
(31–36). The activation of these and other signaling cascades
ultimately activate MAPKs that, following their translocation to
nucleus, activate transcription factors and command cell cycle
regulatory molecules for cell growth, proliferation, and differentiation (37– 47). Several studies have shown that MAPKs are
constitutively active in human breast carcinomas and derived
cell lines as well as in rat mammary tumors (48 –52). Together,
it can be appreciated that targeting the MAPK signaling pathway should be a useful strategy for the prevention and/or intervention of breast cancer. Using estrogen receptor-negative
breast carcinoma MDA-MB468 cells, in this study we demonstrate the irreversible inhibitory effect of GSP on cell growth
3
The abbreviations used are: GSE, grape seed extract; CDK, cyclindependent kinase; CDKI, CDK inhibitor; ERK1/2, extracellular signalregulated protein kinase 1/2; GSP, a polyphenolic fraction isolated from
grape seeds; IGF, insulin-like growth factor; IGF-1R, IGF-1 receptor;
MAPK, mitogen-activated protein kinase; PARP, poly(ADP ribose)
polymerase; ECL, enhanced chemiluminescence; EGF, epidermal
growth factor; JNK, c-jun-NH2-kinase; FACS, fluorescence-activated
cell-sorting; PI, propidium iodide.
and suggest that this effect is possibly via inhibition of MAPK
activation and modulation of cell cycle regulators involved in
G1 phase. The observed molecular effects of GSP on MDAMB468 cells result in a G1 arrest in cell cycle, followed by
terminal differentiation, not the apoptotic cell death.
MATERIALS AND METHODS
Cell Line and Other Reagents. The MDA-MB468 cell
line was obtained from the American Type Culture Collection
(Manassas, VA). Cells were grown in DMEM with 10% fetal
bovine serum, 100 units/ml penicillin, and 100 ␮g/ml streptomycin at 37°C in a 5% CO2 atmosphere. Anti-Cip1/p21 antibody was from Calbiochem (Cambridge, MA). Anti-cytokeratin
8 and anti-CDK4 antibodies were from Neomarkers, Inc. (Fremont, CA). Antibodies to CDK2, cyclin D1, and cyclin E and
rabbit antimouse immunoglobulin- and goat antirabbit immunoglobulin-horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Phospho- (and regular) MAPK/ERK1/2, JNK1, and
p38 antibodies were from New England Biolabs (Beverly, MA).
PARP antibody was from PharMingen (San Diego, CA). The
ECL detection system was from Amersham (Arlington Heights,
IL). The GSP preparation used was that described in detail
recently (29). Approximately 60% (w/w) of this total GSP
preparation has been identified and defined by us to contain
polyphenols, namely procyanidin B3 (3%), procyanidin B1
(0.7%), catechin (5%), procyanidin B4 (2%), procyanidin B2
(3.5%), epicatechin (6%), procyanidin C1 (6%), procyanidin
B5–3⬘-gallate (15%), and procyanidin B5 (19%), each w/w of
total GSP (29). For all of the studies, GSP was dissolved in
DMSO as a 10-mg/ml stock solution and diluted as desired
directly in the medium. Unless specified otherwise, the final
concentration of DMSO in culture medium during GSP treatment did not exceed 0.75% (v/v), and, therefore, the same
concentration of DMSO was present in control dishes.
GSP Treatment of Cells and Western Immunoblotting.
MDA-MB468 cells were grown in 100-mm dishes, as detailed
above, and at 70% confluency were treated with either DMSO
alone or varying concentrations of GSP. After 24, 48, and 72 h
of treatments, medium was aspirated, cells were washed two
times with cold PBS, and cell lysates were prepared as described
in detail recently (53). For Western immunoblotting, 40 –100 ␮g
of protein lysate per sample was denatured with 2⫻ sample
buffer, samples were subjected to SDS-PAGE on 12% gels, and
separated proteins were transferred onto membrane. The levels
of phospho- and regular ERK1/2, JNK1, p38, Cip1/p21, CDK4,
CDK2, cyclin D1, cyclin E, PARP, and cytokeratin 8 were
determined using specific primary antibodies, followed by peroxidase-conjugated appropriate secondary antibody and visualization by the ECL detection system, as described in detail
recently (53).
Cell Cycle Analysis. MDA-MB468 cells at 70% confluency were treated with either DMSO alone or varying concentrations of GSP. After 24, 48, and 72 h of treatments, medium
was aspirated, cells were quickly washed two times with cold
PBS and trypsinized, and cell pellets were collected. Approximately 0.5 ⫻ 106 cells in 0.5 ml of saponin/PI solution [0.3%
saponin (w/v), 25 ␮g/ml PI (w/v), 0.1 mM EDTA, and 10 ␮g/ml
Clinical Cancer Research 2923
RNase (w/v) in PBS] were left at 4°C for 24 h in the dark. Cell
cycle distribution was then analyzed by flow cytometry using
the FACS Analysis Core Services of the University of Colorado
Cancer Center (Denver, CO).
Cell Growth Assay. MDA-MB468 cells were plated at
5000 cells/cm2 density in 35-mm dishes under the culture conditions detailed above. After 24 h, cells were fed with fresh
medium and treated with either DMSO alone or varying concentrations of GSP. The cultures were fed with fresh medium
with or without the same concentrations of GSP every alternate
day up to the end of the experiment; each treatment and time
point had four plates. After 2, 4, and 6 days of treatments, cells
were trypsinized, collected, and counted using a hemocytometer. Trypan blue dye exclusion was used to determine cell
viability.
In another study, MDA-MB468 cells were plated at 5000
cells/cm2 density in 35-mm dishes and, after 24 h, treated with
DMSO alone or varying doses of GSP. After 72 h of treatments,
total cell growth was determined by cell counting. At this point,
in separate dishes, after 72 h of treatments with GSP, cultures
were washed several times with medium to remove GSP and
grown in fresh medium without GSP for another 24, 48, or 72 h.
The cell number was determined at these time periods.
DNA Ladder Assay. MDA-MB468 cells were grown in
100-mm dishes, as detailed above, and at 70% confluency were
treated with either DMSO alone or varying concentrations of
GSP. After 24, 48, and 72 h of treatments, trypsinized cells
(together with any floating cells) were collected and the DNA
ladder analysis was done as described in detail recently (54).
Morphological Analysis. MDA-MB468 cells were
grown in 100-mm dishes, as detailed above, and at 50% confluency were treated with either DMSO alone or 50- and 75␮g/ml concentrations of GSP. After 24, 48, 72, and 96 h of
treatments, pictures were taken using phase-contrast microscope
at ⫻200 magnification. The cell pellets were then collected for
cytokeratin 8 protein expression by Western immunoblotting, as
detailed above.
Densitometric and Statistical Analysis. Autoradiograms of the Western immunoblots were scanned using Adobe
Photoshop. The blots were adjusted for brightness and contrast
for minimum background, and the mean density for each band
was analyzed using Scanimage Program. As needed, a twotailed Student’s t test was used to assess statistical significance
of difference between vehicle- and GSP-treated samples. Until
and unless specified otherwise, the results shown in each case
are representative of three independent experiments with similar
findings.
RESULTS
GSP Inhibits Constitutive Activation of ERK1/2 and
p38, But Causes Moderate Effect on JNK1 in MDA-MB468
Cells. First, we focused our attention on the effect of GSP on
constitutive activation of MAPKs. Because the MAPK family
includes ERKs, JNK (stress-activated protein kinase), and p38
(HOG), which are functional units involved in three distinct
signaling pathways (37– 47), we assessed the effect of GSP on
all of these MAPKs. As shown in Fig. 1A, treatment of MDAMB468 cells with GSP resulted in a moderate to complete
Fig. 1 Effect of GSP on the constitutive activation of MAPK signaling in human breast carcinoma MDA-MB468 cells. Cells were
cultured in DMEM with 10% serum and at 70% confluency were
treated either with DMSO alone or 25-, 50-, and 75 ␮g/ml concentrations of GSP for 24, 48, and 72 h. Cell lysates were prepared and
subjected to SDS-PAGE, followed by Western blotting, as detailed in
“Materials and Methods.” The membranes were probed with antiphospho-ERK1/2, antiphospho-JNK1, and antiphospho-p38 MAPK
antibodies and then peroxidase-conjugated appropriate secondary
antibody. Visualization of proteins was done using ECL detection
system. A, phosphorylation of ERK1/2. B, phosphorylation of JNK1.
C, phosphorylation of p38. Treatments are as labeled in the figure.
IB, Western immunoblot. In each panel, the densitometric analysis
bars represent the mean ⫾ SE values (arbitrary units) of three
independent experiments. At each time point of the study, the
vehicle-treated control value is presented as 100%.
2924 GSP Causes Growth Inhibition in MDA-MB468 Cells
inhibition of constitutive activation of ERK1/2 in both a doseand time-dependent manner. The densitometric analysis of three
different blots showed that compared with DMSO control, after
24 h of GSP treatment, 25- and 50-␮g/ml doses were not
effective, but a 75-␮g/ml dose showed moderate (although
statistically not significant) inhibition (10%, P ⬍ 0.1) of constitutive ERK1/2 activation. However, a higher inhibition (20 –
25%, P ⬍ 0.05) was observed at both 50- and 75-␮g/ml doses
after 48 h of GSP treatment, and after 72 h these doses showed
complete inhibition (P ⬍ 0.001) of constitutive ERK1/2 activation; even a 25-␮g/ml dose resulted in 90% inhibition (P ⬍
0.001) after this treatment time (Fig. 1A). The observed decreases in constitutive ERK1/2 activation by GSP were not due
to a change in its protein levels following these treatments (data
not shown), suggesting that GSP impairs mitogenic signaling of
ERK1/2. These results are specifically important because of a
dose and time response where it could be suggested that a longer
treatment time with a lower dose(s) produce comparable efficacy to that with higher dose and shorter treatment time.
Unlike its effect on ERK1/2 activation, GSP did not show
a conclusive effect on JNK1 activation (Fig. 1B). For example,
24 h of treatment with 25-, 50-, and 75-␮g/ml doses did not
result in any change in constitutive activation of JNK1, whereas
after 48 h of treatment a 75-␮g/ml dose showed a strong
stimulation (Fig. 2B). Conversely, 50- and 75-␮g/ml doses of
GSP showed ⬃35% inhibition (P ⬍ 0.05) after 72 h of treatment
(Fig. 2B). Similar to ERK1/2, GSP did not show any change in
JNK1 protein expression following these treatments (data not
shown).
With regard to p38, as shown in Fig. 1C, compared with
DMSO control, GSP treatment of MDA-MB468 cells resulted in
a highly significant inhibition of p38 activation in both a doseand time-dependent manner. The observed effect of GSP on p38
was much stronger than ERK1/2 activation at both 24 and 48 h
at all three doses examined, but was less effective than ERK1/2
after 72 h of treatment (Fig. 1, C versus A). Quantitative analysis
of the blots showed that 24 h of GSP treatment at a 25-␮g/ml
dose was not effective, however, 50- and 75-␮g/ml doses resulted in 15% (P ⬍ 0.1) and 70% (P ⬍ 0.001) decrease,
respectively, in constitutive p38 activation. After 48 h of GSP
treatment of cells, as much as a 15% (P ⬍ 0.1), 68% (P ⬍
0.001), and 70% (P ⬍ 0.001) decrease was evident in p38
activation by these doses, respectively. No further effect, however, was observed following additional treatment time up to
72 h. An interesting observation was a strong increase in constitutive activation of p38 in control samples after 48 h, as
compared with those at 24 and 72 h (Fig. 1C). The reason for
this increase remains to be established. Taken together, these
findings suggest that GSP treatment of MDA-MB468 human
breast carcinoma cells results in a significant decrease in
ERK1/2 and p38 activation, the two MAPK pathways associated
with cell growth and differentiation (55), but has moderate, if
any, effect on JNK1, the MAPK pathway associated largely
with cell death (55–57).
GSP Induces Cip1/p21 Levels and Decreases G1 Phase
Regulators CDK4 and Cyclin D1 in MDA-MB468 Cells. A
controlled cell growth, proliferation, differentiation, and/or apoptosis are mediated via cell cycle progression that is governed
by cellular signaling (Refs. 58 – 62 and references therein).
Fig. 2 GSP induces Cip1/p21 levels and decreases G1 phase regulators
CDK4 and cyclin D1 in human breast carcinoma MDA-MB468 cells.
Cells were cultured in DMEM with 10% serum and at 70% confluency
were treated with either DMSO alone or 25-, 50-, and 75-␮g/ml concentrations of GSP for 24, 48 and 72 h. Cell lysates were prepared and
subjected to SDS-PAGE, followed by Western blotting, as detailed in
“Materials and Methods.” The membranes were probed with anti-Cip1/
p21, anti-CDK4, and anti-cyclin D1 antibodies and then peroxidaseconjugated appropriate secondary antibody. Visualization of proteins
was done using an ECL detection system. A, protein expression of
Cip1/p21. B, protein expression of CDK4. C, protein expression of
cyclin D1. Treatments are as labeled in the figure. IB, Western immunoblot. In each panel, the densitometric analysis bars represent the
mean ⫾ SE values (arbitrary units) of three independent experiments. At
each time point of the study, the vehicle-treated control value is presented as 100%.
Clinical Cancer Research 2925
However, cancer cells often display defects in the genes that
govern the cellular responses to a growth factor(s)-growth factor
receptor(s) interaction (61). Perturbations in cell cycle regulation have been demonstrated to be one of the most common
features of transformed cells (61), which could be associated
with gain of function for uncontrolled growth due to enhanced
expression of growth factor-receptor autocrine loop (31–36), a
lack of CDKI or loss in its function (58 – 63), and so on.
Accordingly, next we assessed the effect of GSP on cell cycle
regulators involved in G1 phase. As shown by data in Fig. 2A,
treatment of cells with GSP resulted in a significant induction of
Cip1/p21 in a dose-dependent manner. The densitometric analysis of blots showed that 24 h of GSP treatment caused a 1.7-,
2.3-, and 2.7-fold increase (P ⬍ 0.001) in Cip1/p21 at doses of
25, 50, and 75 ␮g/ml, respectively. Although 48 and 72 h of
GSP treatment also showed significant Cip1/p21 induction, it
was less profound than that at 24 h and amounted for 1.2–1.6fold increases (P ⬍ 0.1 to 0.001). Similar to its affect on
Cip1/p21, GSP treatment of cells also resulted in a significant
decrease in CDK4 protein levels (Fig. 2B). In this case, 24 h of
GSP treatment caused a 10% (P ⬍ 0.1), 30% (P ⬍ 0.05), and
50% (P ⬍ 0.001) decrease in CDK4 expression at 25-, 50-, and
75-␮g/ml doses, respectively. Similar effects of GSP were also
observed following its treatment for 48 and 72 h at identical
doses. Unlike its effect on Cip1/p21 and CDK4, GSP treatment
of cells for 24 h resulted in a marginal increase in cyclin D1
protein levels, however, 48 and 72 of treatment caused marginal
decrease (Fig. 2C); GSP treatment did not show any effect on
CDK2 and cyclin E protein expression (data not shown).
GSP Induces G1 Arrest in Cell Cycle Progression of
Human Breast Carcinoma MDA-MB468 Cells. On the basis of the results showing a strong induction in Cip1/p21 and a
decrease in CDK4 by GSP, we next examined its effect on cell
cycle progression. As shown in Fig. 3, FACS analysis of
DMSO-treated cells showed a cell cycle distribution that followed a proliferation pattern. However, a moderate to strong
difference in cell cycle progression was observed following
GSP treatment where all of the doses and time points examined
showed a G1 arrest in the cell cycle progression of MDAMB468 cells largely at the expense of S phase population (Fig.
3). Within 24 h after GSP treatment, compared with DMSOtreated control, 25- and 50-␮g/ml doses showed marginal accumulation of cells in G1 phase (55% and 58% of cells in G1,
respectively, compared with 53% in control; Fig. 3A). However,
as much as 71% of cells accumulated in G1 phase following a
75-␮g/ml dose of GSP treatment for 24 h (Fig. 3A). The increase
in G1 population by GSP at all of the doses was accompanied by
a significant decrease of the cells in S phase (Fig. 3B). An
accountable increase in G2-M population of the cells was also
observed following a 24-h treatment with GSP at these doses
(Fig. 3C). When MDA-MB468 cells were treated for a longer
time (i.e., 48 and 72 h) with GSP, even a lower dose (25 ␮g/ml)
resulted in a strong accumulation of the cells in G1 phase that is
moderately affected by increasing the doses to 50 and 75 ␮g/ml
(Fig. 3A). Once again, this increase was mainly due to a decrease in S phase population (Fig. 3B) with moderate alterations
in G2-M phase (Fig. 3C). Together, these results were consistent
with other findings, showing that (a) maximum effect of GSP on
Fig. 3 GSP induces G1 arrest in cell cycle progression of human breast
carcinoma MDA-MB468 cells. Cells were cultured in DMEM with 10%
serum and at 70% confluency were treated either with DMSO alone or
25-, 50-, and 75-␮g/ml concentrations of GSP for 24, 48 and 72 h. After
these treatments, cells were trypsinized and cell pellets were collected.
Approximately 0.5 ⫻ 106 cells were labeled with PI using 0.5 ml of
saponin/PI solution, at 4°C for 24 h in dark, as detailed in “Materials and
Methods.” Cell cycle distribution was then analyzed by flow cytometry
using Becton Dickinson FACS System. A, percentage of total cells in G1
phase. B, percentage of total cells in S phase. C, percentage of total cells
in G2-M phases. The data shown are representative of two independent
experiments (each done in duplicate) with ⬍5% variation.
Cip1/p21 was at 24 h (Fig. 2A) and (b) GSP modulates cell cycle
regulators associated with G1 phase (Fig. 2).
GSP Irreversibly Inhibits the Growth of MDA-MB468
Cells. The studies assessing the biological significance of the
observed effects of GSP showed that it causes the inhibition of
human breast carcinoma MDA-MB468 cells and that this inhibition was irreversible at higher doses examined. As shown by
data in Fig. 4A, GSP treatment of growing cells resulted in a
significant to almost complete inhibition of cell growth in both
a dose- and time-dependent manner. Compared with DMSOtreated controls, whereas a 10-␮g/ml dose was not effective, a
25-␮g/ml dose resulted in 20 –50% (P ⬍ 0.001) inhibition after
2– 6 days of treatment (Fig. 4A). A much higher inhibitory effect
was observed at 50- and 75-␮g/ml doses of GSP, accounting for
2926 GSP Causes Growth Inhibition in MDA-MB468 Cells
Fig. 4 GSP irreversibly inhibits anchorage-dependent growth of human breast carcinoma MDA-MB468 cells. A, cell growth inhibitory
effect of GSP. Cells were plated at 5000 cells/cm2 in 35-mm dishes.
After 24 h, cultures were treated with DMSO or GSP at the a concentration of 10 –100 ␮g/ml medium, and the total number of cells were
counted after 2, 4, and 6 days of these treatments. The cell growth data
shown are mean ⫾ SE of four independent plates; each sample was
counted in duplicate. B, washout study showing that the cell growth
inhibitory effect of GSP is irreversible. Cells were plated at 5000
cells/cm2 in 35-mm dishes and after 24 h were treated with indicated
doses of GSP, and total cell growth was determined after 72 h of
treatments. At this point, in separate dishes, 72 h after indicated treatments of GSP, cultures were washed several times with medium to
remove GSP and grown in fresh medium without GSP for the indicated
time. The cell number was determined by cell counting at the indicated
time after GSP washout. The cell growth data shown are mean ⫾ SE of
four independent plates; each sample was counted in duplicate. The bars
not clear in some data bars are due to their lower values.
40 – 65% and 63– 84% (P ⬍ 0.001) inhibition, respectively,
during 2– 6 days of treatment (Fig. 4A). A dose of 100 ␮g/ml
GSP resulted in 63%, 90%, and 92% (P ⬍ 0.001) inhibition of
anchorage-dependent cell growth after 2, 4, and 6 days of
treatment, respectively (Fig. 4A).
On the basis of the data shown in Fig. 4A, we next examined whether the observed inhibitory effect of GSP on cell
growth is due to its cytostatic effect, or is an irreversible
activity. For these studies, cells were treated with 25-, 50-, and
75-␮g/ml doses for 72 h and then washed several times with
medium to remove GSP from the culture. The cells were then
left in normal medium without GSP for their possible growth.
As shown in Fig. 4B, compared with DMSO-treated controls,
72 h of GSP treatment at a 25-␮g/ml dose, followed by a
washout study for 1–3 days, did not show any change in cell
growth. However, treatment of cells with 50 ␮g/ml GSP for 72 h
and then following their growth after 24, 48 and 72 h of washout
showed that there was 44%, 46%, and 52% inhibition (P ⬍
0.001) in cell growth, respectively (Fig. 4B). A much stronger
inhibition was evident in this washout study where 75 ␮g/ml
GSP led to a 77%, 86%, and 88% inhibition in cell growth after
24, 48 and 72 h of GSP washout, respectively (Fig. 4B). Because
another interpretation of the data shown in Fig. 4B leads to the
argument that the observed irreversible inhibition is due to the
fact that 72 h of GSP treatment at different doses already
reduced the total number of cells to begin with in washout
studies, we also followed cell growth pattern during 72 h after
GSP washout. As shown in Fig. 4B, in case of DMSO-treated
and 25 ␮g/ml GSP-treated cultures, after washout, the cell
growth pattern was comparable and was 205%, 172%, and
125% (compared with the previous 24 h) after 24, 48, and 72 h
of washout, respectively. However, under similar treatment conditions, these patterns were 140%, 164%, and 110% in case of
50 ␮g/ml GSP-treated cultures, and 140%, 104%, and 100% in
case of 75 ␮g/ml GSP-treated cultures (Fig. 4B). Together, these
results provide convincing evidence that the observed cell
growth inhibitory effects of GSP are not just cytostatic, but are
irreversible.
Irreversible Growth Inhibitory Effect of GSP Leads to
Differentiation of MDA-MB468 Cells. The observed irreversible growth inhibitory effect of GSP on MDA-MB468 cells
led to the question that what was the ultimate fate of the growth
arrested cells. Both apoptotic cell death and differentiation possibilities, therefore, were explored to answer this question. For
the apoptotic cell death studies, cultures were treated with 25-,
50-, and 75-␮g/ml doses of GSP for 24, 48 and 72 h, and both
floating and attached cells were subjected to several apoptosisassociated assays. In these studies, none of the GSP doses and
time points examined showed: (a) DNA ladder; (b) PARP
cleavage; (c) morphological changes suggestive of apoptosis;
(d) TUNEL staining; and (e) sub-G1 population in cell cycle
assay, as compared with positive findings with paclitaxel used
as a positive control (data not shown).
On the contrary, GSP-treated MDA-MB468 cells showed
unique morphological changes. As shown in Fig. 5, compared
with DMSO-treated control cultures, cells treated with 50 ␮g/ml
GSP for 4 days were much larger in their size with a change in
morphology to spindle shape (Fig. 5, A versus B). A more
profound effect of GSP was evident at a 75-␮g/ml dose for 4
days where cells became much larger in size with significant
changes in morphology because they were both elongated in
length and had spindle shape (Fig. 5, C versus A). Although less
profound, 48- and 72-h treatment time points at these doses of
GSP also showed morphological changes (data not shown).
These morphological changes were similar to that of epithelial
cell differentiation, suggesting that GSP induces differentiation
of MDA-MB468 cells. Following these morphological changes,
the cells started detaching from the culture dishes presumably
due to their death, suggesting that the observed morphological
changes with GSP are associated with terminal differentiation
that ultimately causes cell death. In other studies, as shown in
Clinical Cancer Research 2927
Fig. 5D, we found that GSP treatment of cells also results in a
significant induction of cytokeratin 8 protein expression, a
marker of epithelial cell differentiation (64). The densitometric
analysis of the blot showed that compared with DMSO, both 50and 75-␮g/ml doses of GSP resulted in a 1.7-fold increase (P ⬍
0.001) in cytokeratin 8 expression following a 48-h treatment.
Whereas no further increase was observed at these doses after
72 h of treatment, 96 h of GSP treatment at these doses showed
2.4- and 2.6-fold increases (P ⬍ 0.001) in cytokeratin 8 levels,
respectively.
DISCUSSION
The central finding in the present study is that a polyphenolic fraction isolated from grape seeds that is rich in procyanidins, inhibits constitutive activation of MAPK/ERK1/2 and
MAPK/p38, and causes an induction of CDKI Cip1/p21 and a
decrease in CDK4. These effects of GSP result in a G1 arrest in
cell cycle, followed by an irreversible inhibition of cell growth.
The GSP-treated cells unable to grow do not die by apoptosis
but undergo terminal differentiation, followed by cell death. Our
findings that GSP inhibits constitutive activation of ERK1/2 are
specifically significant because constitutive activation of this
pathway has been shown to be associated with human breast
carcinomas and derived cell lines for uncontrolled growth
(48 –52).
The MAPK family includes ERKs, JNK (stress-activated
protein kinase), and p38 (HOG), which are functional units
involved in three distinct signaling pathways (37– 47). Activated
MAPKs transport to the nucleus where they phosphorylate and
activate a series of transcription factors such as Elk-1, cMyc,
ATF-2, cJun, CREB, and so on (37– 47, 65– 67). The differential
responses of ERKs, JNKs, and p38 in terms of activation of
different transcription factors suggest that these signaling cascades are distinct (37– 47). Furthermore, several studies have
linked ERKs and p38 signaling to proliferation and differentiation, whereas the JNK pathway is largely associated with
apoptosis (55–57). Consistent with these reports, in the present
study we found that GSP inhibits constitutive activation of
ERK1/2 and p38, but had little (if any) effect on JNK activation.
These results were in accord with the biological effects of GSP
where it showed growth inhibitory and differentiation inducing
potential but did not cause apoptotic death. The observed inhibitory effect of GSP on ERK1/2 activation is similar to those
reported with other polyphenolic antioxidants, such as silymarin, genistein, quercetin, and others (54), however, to the best of
our knowledge, this is the first study showing the inhibitory
effect of a polyphenolic antioxidant fraction on p38 activation.
Earlier, it has been shown that erbB1 and other members of
Fig. 5 GSP induces both morphological changes and expression of
cytokeratin 8, suggesting differentiation of human breast carcinoma
MDA-MB468 cells. Cells were cultured in DMEM with 10% serum and
at 50% confluency were treated with either DMSO alone or 50- and
75-␮g/ml concentrations of GSP in DMSO. After 96 h of these treatments, pictures were taken using phase-contrast microscope at ⫻200
magnification. A, DMSO alone. B, 50 ␮g/ml GSP in DMSO. C, 75
␮g/ml GSP in DMSO. D, stimulatory effect of GSP on cytokeratin 8
protein levels. Cells at 50% confluency were treated either with DMSO
alone or 50- and 75-␮g/ml concentrations of GSP for 48, 72, and 96 h.
Cell lysates were prepared and subjected to SDS-PAGE, followed by
Western blotting, as detailed in “Materials and Methods.” The membranes were probed with anti-cytokeratin 8 antibody, followed by peroxidase-conjugated appropriate secondary antibody. Visualization of
proteins was done using ECL detection system. Treatments are as
labeled in the figure. IB, Western immunoblot.
2928 GSP Causes Growth Inhibition in MDA-MB468 Cells
the erbB family (Her-2/neu or erbB2) play an important role in
human breast cancer (31–36). In addition, IGF-1R has also been
shown to play an important role in the growth, proliferation, and
metastasis of breast cancer, which is also associated with a
decrease or loss of IGF-binding protein-3 that binds to IGF,
leading to an inhibition of the binding of this ligand to IGF-1R
(35). This impairs mitogenic and antiapoptotic signaling mediated by IGF-1/IGF-1R pathway causing a malignant cell growth
arrest and apoptotic cell death (68). These and other signaling
pathways ultimately activate MAPKs that then translocate to the
nucleus and activate transcription factors for cell growth, differentiation, and proliferation (37– 47, 68). Taken together,
these reports raise the question whether the observed inhibitory
effects of GSP on ERK1/2 and p38 activation are direct effect or
due to impairment of upstream signaling involving both receptor
tyrosine kinases and cytosolic signaling. This cause and effect is
an important issue that is being studied in an ongoing program.
Several studies have shown that mitogenic signaling must
be continually renewed, even if at quite low levels, throughout
much of the growth stimulating G1 phase of the mammalian cell
cycle (58 – 62). For example, a cell arrest or growth signal
determines whether the cells will be in a resting, nondividing
“G0 ” phase or whether they will enter the G1 phase of cell cycle
and thereby undergo cell replication and proliferation (58 – 62).
Cyclin D1 and its associated CDK4 (or CDK6) normally control
cell cycle events in early G1 phase, and cyclin E coupled with
CDK2 is involved in the transition from late G1 to S phase
(58 – 62). Alternatively, impairment of a growth-stimulatory signaling pathway leads to the induction of CDKIs (such as Cip1/
p21 and Kip1/p27) that inhibit the activity of cyclin-CDK complexes, leading to cell growth arrest (69). These studies suggest
that modulation of cell cycle regulatory molecules either directly or via impairment of mitogenic signaling that regulate
them, could be a practical and translation approach for the
prevention and/or intervention of human malignancies, including breast cancer. Consistent with these reports, in the present
study we found that GSP treatment of breast carcinoma MDAMB468 cells results in a significant induction of Cip1/p21. We
also found that GSP significantly inhibits CDK4 levels and
moderately alters cyclin D1 expression. Conversely, GSP was
not effective in inhibiting cyclin E and CDK2 levels. Together,
these data are consistent with the observed effect of GSP on G1
accumulation and suggest that GSP causes an early effect,
leading to arrest of cells in early G1 phase. These effects of GSP
on breast carcinoma cells are specifically important because an
overexpression of G1 phase cyclin D1 has been shown with high
frequency in human breast cancer (61).
An up-regulation of Cip1/p21 strongly correlates with cell
growth inhibition that ultimately decides the fate of cell between
differentiation and death. For example, inhibition of Cip1/p21
expression through transfection of Cip1/p21 antisense oligonucleotides has been shown to block growth factor-induced differentiation of SH-SY5Y neuroblastoma cells and resulted in
their death (70). Conversely, Cip1/p21 induction is shown in the
differentiation of a variety of cells such as myogenic, keratinocytic, promyelocytic (HL-60), and human melanoma cells (71–
73). Consistent with these findings, we observed that GSPcaused induction of Cip1/p21 and resultant G1 arrest did not
induce apoptosis but caused terminal differentiation of cells,
followed by their death. An irreversible cell growth inhibition
by GSP further supports this observation.
An analysis of the time kinetics of the observed effects of
GSP on cell cycle regulators suggests that possibly GSP directly
affects Cip1/p21 and CDK4 expression as an early response that
leads to cell growth inhibitory effect via G1 arrest. This growth
inhibitory effect of GSP then presumably leads to an impairment
of autocrine loop, causing a decrease in constitutive activation
of MAPK/ERK1/2 as a late response and a terminal differentiation of the cells. This presumption could be supported indirectly by the studies where at least in prostate carcinoma cells it
has been shown that an induction in MAPK/ERK1/2 activation
is associated with the reversal of cellular differentiation (74).
More studies, however, are needed to further establish this cause
and effect relationship.
In summary, based on the results of the present study, we
conclude that GSP and procyanidins present therein should be
studied in more detail to be developed as breast cancer preventive and/or interventive agents. In addition, studies are also
needed to establish whether the observed effective doses of GSP
in cell culture are achievable physiologically in breast cancer
patients and that such doses are both nontoxic and effective for
preventive intervention of this deadly malignancy.
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