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Metformin inhibits breast cancer cell growth, colony

formation and induces cell cycle arrest in vitro
Irina N. Alimova, Bolin Liu, Zeying Fan, Susan M. Edgerton, Thomas Dillon, Stuart E. Lind &
Ann D. Thor
Published online: 15 Mar 2009.

To cite this article: Irina N. Alimova, Bolin Liu, Zeying Fan, Susan M. Edgerton, Thomas Dillon, Stuart E. Lind & Ann D. Thor
(2009) Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro, Cell Cycle, 8:6,
909-915, DOI: 10.4161/cc.8.6.7933

To link to this article: http://dx.doi.org/10.4161/cc.8.6.7933

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 [Cell Cycle 8:6, 909-915; 15 March 2009]; ©2009 Landes Bioscience

Report

Metformin inhibits breast cancer cell growth, colony formation and

induces cell cycle arrest in vitro
Irina N. Alimova,1,2 Bolin Liu,1 Zeying Fan,1 Susan M. Edgerton,1 Thomas Dillon,3 Stuart E. Lind1,4 and Ann D. Thor1,*

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UT
1Department of Pathology; 4Division of Medical Oncology; University of Colorado Denver School of Medicine; Aurora, Colorado USA; 2Department of Carcinogenesis and

Oncogerontology; Prof. N.N. Petrov Research Institute of Oncology; St. Petersburg, Russia; 3Science Department; Community College of Aurora; Denver, Colorado USA

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Abbreviations: RTK, receptor tyrosine kinase; ER, estrogen receptor; CDK, cyclin-dependent kinase; MAPK, mitogen-activated protein
kinase; PI-3K, phosphoinositide 3-kinase; AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; PARP, poly

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(ADP-ribose) polymerase; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis

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Key words: metformin, breast cancer, cell cycle, ER, erbB2

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The anti-diabetic drug metformin reduces human cancer inci- systems have been associated with an increased incidence of human

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dence and improves the survival of cancer patients, including those cancer.1-7 While the treatment of these important host factors is
with breast cancer. We studied the activity of metformin against typically multifactorial and complex, there is some evidence that
diverse molecular subtypes of breast cancer cell lines in vitro. the anti-diabetic biguanide drug metformin (1,1-dimethylbiguanide
Metformin showed biological activity against all estrogen receptor
O hydrochloride) reduces the incidence of several types of cancers,
Cell Cycle 2009.8:909-915.

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(ER) positive and negative, erbB2 normal and abnormal breast
cancer cell lines tested. It inhibited cellular proliferation, reduced
colony formation and caused partial cell cycle arrest at the G1
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including breast cancer. Like obesity and diabetes, breast cancer is
both highly prevalent and morbid and there is significant overlap in
the affected patient populations.8,9

checkpoint. Metformin did not induce apoptosis (as measured by Risk factors for breast cancer are multi-factorial, including female
DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 gender, older patient age, family history, estrogenic hormones (exog-
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subtype breast cancer cell lines. At the molecular level, metformin enous or endogenous), prior breast pathology, inherited genomic
treatment was associated with a reduction of cyclin D1 and E2F1 alterations, obesity and abnormal glucose metabolism.10-13 Cohort
expression with no changes in p27kip1 or p21waf1. It inhibited and case-control studies indicate that type II diabetes enhances the
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mitogen activated protein kinase (MAPK) and Akt activity, as relative risk (RR) of breast cancer by 10% to 20%.14 This relationship
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well as the mammalian target of rapamycin (mTOR) in both ER appears especially strong in post-menopausal females, where hyper-
positive and negative, erbB2-overexpressing and erbB2-normal insulinemia has been used as a marker of breast cancer risk.10,15-18 In
expressing breast cancer cells. In erbB2-overexpressing breast these patients, the use of the anti-diabetic agent metformin has been
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cancer cell lines, metformin reduced erbB2 expression at higher shown to decrease the incidence and improve the prognosis of breast
concentrations, and at lower concentrations within the therapeutic cancer.19,20 The off-patent Food and Drug Administration (FDA)
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range, it inhibited erbB2 tyrosine kinase activity evidenced by a approved drug, metformin, is the most widely prescribed anti-hyper-
reduction of phosphorylated erbB2 (P-erbB2) at both auto- and glycemic agent used worldwide. Metformin is best known as a growth
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Src- phosphorylation sites. These data suggest that metformin inhibitor which induces glucose stabilization, enhances insulin sensi-
may have potential therapeutic utility against ER positive and tivity and reduces circulating insulin levels without an associated
negative, erbB2-overexpressing and erbB2-normal expressing hypoglycemia. Patients treated with metformin also have shifts in
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breast cancer cells. fatty acid metabolism and may experience weight reduction.
Although metformin is widely prescribed, its biological and molec-
Introduction
09

ular activity against carcinogenesis is not well understood. In MCF-7

Abnormalities of glucose and fat metabolism are well known breast cancer cells, metformin induces AMP-activated protein kinase
20

to promote cardiovascular disease, diabetes, hypertension and (AMPK) activity and inactivates the mammalian target of rapamycin
stroke. More recently, abnormalities in these essential biological (mTOR), S6 kinase and mRNA translation.21 Because mTOR is a
central regulator of cellular response to mitogenic stimuli and envi-
©

ronmental cues, breast cancers with activating mutations of PIK3A

*Correspondence to: Ann D. Thor; Department of Pathology; University of Colorado
Denver School of Medicine; MS-B2116; P.O. Box 6511; Aurora, Colorado 80045
USA; Tel.: 303.724.3704; Fax: 303.724.3705; Email: ann.thor@uchsc.edu
Submitted: 10/28/08; Revised: 12/09/08; Accepted: 01/26/09
Previously published online as a Cell Cycle E-publication:
http://www.landesbioscience.com/journals/cc/article/7933
or a loss of PTEN may be particularly sensitive to metformin.22,23
Metformin and related compounds, which include phenformin
(1-phenylethylbiguanide), buformin (N-butylbiguanide), that coun-
terbalance aberrant glucose metabolism have been shown to inhibit
mammary tumorigenesis in rodent models (spontaneous or carcin-
ogen induced) as well.1-7,24 Metformin has most recently been shown

www.landesbioscience.com Cell Cycle 909

 Metformin inhibits cell growth

to inhibit mammary carcinogenesis in genetically at risk female

mice bearing the erbB2 (wt-rat-neu transgene).25 In aggregate, these
studies suggest that metformin may have anti-tumorigenic activity
against several of the molecular subtypes of human breast cancer. In
this report we describe in vitro studies of metformin action, using
breast cancer derived cell lines which represent the most common
subtypes of human breast cancer, luminal A (ER+, erbB2-), luminal
B (ER+, erbB2+) and Her2/erbB2 (ER-, erbB2+).

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Results

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Metformin inhibits breast cancer cell proliferation and reduces
colony formation in vitro. ER and erbB2 are important prognostic

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markers and therapeutic targets for breast cancer patients. To study the
effects of metformin in vitro, we used 4 breast cancer cell lines with

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variable expression levels of these two markers including: MCF-7,

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MCF-7/713 (MCF-7 transfected with erbB2, kindly provided by
Dr. C Benz), BT-474 and SKBR-3. These lines represent luminal

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A, luminal B and erbB2 subtypes of human breast cancers. Each of
these four cell lines showed growth inhibition with metformin treat-

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ment, in a dose dependent manner (Fig. 1A–D). At a concentration

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of 50 mM, BT-474 and SKBR-3 cells were 85% growth inhibited,
MCF-7 was 57% inhibited and MCF-7/713 cells were 50% growth
inhibited, as compared to untreated controls, in these 72 hours cell Figure 1. Metformin inhibits breast cancer cell proliferation. Human breast

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proliferation assays (Fig. 1). cancer cell lines (MCF7, MCF-7/713, BT474 and SKBR-3) were plated onto
Cell Cycle 2009.8:909-915.

96-well plates and incubated at 37°C with 5% CO2. After 24 hours, the
We next studied the ability of these 4 cell lines to form colonies
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culture medium was replaced with control (0.1 ml fresh medium contain-
on 6-well cell culture plates in the presence or absence of metformin ing 0.5% FBS) or same medium containing a series doses of metformin for
for 3 weeks. Metformin reduced colony formation at concentrations another 72 hours incubation. The percentages of surviving cells from each
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as low as 2 mM (Fig. 2A). The number of colonies formed was
reduced in a dose dependent manner, as shown in Figure 2B. At the
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cell line relative to controls, defined as 100% survival, were determined by
reduction of MTS. Data shows the representative of at least three indepen-
dent experiments. Bars, SD.8

highest concentration of metformin (50 mM), colony formation was

reduced over 90% as compared to the untreated controls. Of those
cell lines examined, metformin was somewhat less effective against promotes the G1-S cell cycle transition, a reduction in their expres-
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MCF-7/713 and SKBR-3 cells at moderate doses, as compared to sion would be growth inhibitory. The cyclin-dependent kinase
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MCF-7 and BT-474 cells. (CDK) inhibitor, p27kip-1 was reduced modestly in only one of the
Growth inhibition-induced by metformin does not involve cell lines (SKBR-3) with metformin treatment. Another CDK inhib-
apoptosis. To study the biological mechanisms by which metformin itor, p21waf-1 was not altered in any of the 4 cell lines in response to
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inhibited cell growth, we evaluated its ability to induce cell death via metformin (Fig. 5A).
apoptosis. Apoptosis was not identified in untreated cells or those Metformin alters signaling of erbB2 receptor tyrosine kinase
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treated with metformin, as compared to those treated with taxol (RTK) pathways. We next investigated receptor tyrosine kinase
(which exhibited significant cleavage of PARP), see Figure 3A. These (RTK) signaling changes in metformin treated, as compared to
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findings were also confirmed using a separate ELISA assay for apop- untreated breast cancer cells. The expression of erbB2 protein
tosis. As noted in the western blot analysis of PARP, taxol strongly and activated erbB2 (P-erbB2) were each reduced by metformin,
induced apoptosis whereas metformin and the untreated MCF-7, particularly in those lines with the highest levels of erbB2 expres-
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BT-474 and SKBR-3 cells showed no apoptosis by ELISA (Fig. sion. Metformin treatment also resulted in significant reductions
3B). These data indicate that metformin induces growth inhibition in the activation (phosphorylation) of mitogen-activated protein
09

through an apoptosis-independent mechanism in these breast cancer kinase (MAPK) but not MAPK expression (Fig. 5B). Similarly, Akt
cell lines. activation (P-Akt) was significantly reduced in each of the 4 lines,
20

Metformin induces cell cycle arrest at the G1 checkpoint. To although Akt protein expression was only modestly reduced. The
further study metformin-induced growth inhibition, we evaluated levels of mTOR and P-mTOR were decreased as well, although the
changes in cell cycle progression using flow cytometric methods. magnitude of these changes was not as significant as changes in the
©

Metformin treatment resulted in partial G1 cell cycle arrest, at a
concentration as low as 2 mM, in each of the 4 cell lines tested (Fig.
4A–D). This metformin associated G1 arrest was dose dependent.
We then determined whether metformin could induce changes in
molecular signaling associated with the G1 checkpoint. Both cyclin
D1 and E2F1 were significantly reduced in metformin treated cells,
as compared to controls (Fig. 5A). Because each of these proteins
proteins listed above (Fig. 5B).
Metformin induced changes in the expression or activity of erbB2
appear to be dose dependent. At low concentrations, metformin
reduced tyrosine phosphorylation without alteration of receptor
expression (Fig. 6A). These lower concentrations are within the
reported steady state plasma range (6 μM–30 μM) for patients taking
the drug for type II diabetes (http://www.rxlist.com/cgi/generic/

910 Cell Cycle 2009; Vol. 8 Issue 6

 Metformin inhibits cell growth

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Figure 2. Metformin inhibits colony formation of breast cancer cell lines. Human breast cancer cells (MCF7, MCF7/713, BT-474 and SKBR-3) were grown

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in six-well plates (1,000 cells/well) in triplicates. After 24 hours, the culture medium was replaced with fresh medium containing 0.5% FBS as control, or
Cell Cycle 2009.8:909-915.

same medium containing 2 mM, 10 mM or 50 mM metformin, and the culture medium was changed once every three days for three weeks. The pictures of
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six-well plates with colonies were taken by a digital camera on day 21 and the bar graph was obtained by calculating the percentages of colony numbers
from each cell line relative to controls, defined as 100%, measured by EAGLE EYETM II. Data shows the representative of three independent experiments.
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Bars, SD.
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fortamet_cp.htm). The reduction in phosphorylation occurred at

both autophosphorylation and Src phosphorylation sites of erbB2,
although the most profound inhibition occurred at the Src phospho-
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rylation site (Fig. 6B). At higher concentrations, metformin reduced

both the activity and expression of the erbB2 receptor. Collectively,
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our findings indicate that metformin induced significant inactiva-

tion of erbB2 signaling pathways. These effects appear to be more
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pronounced than the changes we observed in the downstream

signaling molecule mTOR (and P-mTOR) in these breast cancer
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cell lines.

Discussion
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The incidence and prevalence of type II diabetes has increased

worldwide, creating a health crisis of epic proportions in ‘western-
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ized’ societies. According to the American Diabetes Association, 20.8

million Americans have diabetes and that number is growing at ~8 %
per year. Untreated type II diabetes is characterized by an abnormal
09

glucose metabolism, hyperglycemia, hyperinsulinemia and relative

insulin resistance. These changes are associated with obesity, cardiovas-
20

Figure 3. Metformin does not induce apoptosis in tested breast cancer cells.
MCF7, MCF7/713, BT-474 and SKBR-3 cells were grown in 60 mm dishes. cular disease, peripheral vascular disease, diabetic neuropathy, cancer
After 24 hours, the culture medium was replaced with fresh medium contain- and other diseases. In women, the hormonal changes associated with
©

ing 0.5% FBS as control, or same medium containing either 20 mM met-
formin or 1 μM Taxol for another 24 hours incubation. The cell lysates were
prepared as described in the Materials and Methods. An equal amount of
protein for each sample was resolved by 8% SDS-PAGE, followed by western
blot analysis with specific antibodies directed against PARP or β-actin (A).
Cells from the same batch of cell lysates as prepared in (A) were subjected to
analysis of apoptosis determined using an ELISA that quantified cytoplasmic
DNA-histone complexes (B).
menopause often accelerate weight gain (obesity) and further promote
diabetic imbalance. These data suggest that with aging, critical inter-
actions between shifts in steroid hormone levels and glucose/fatty acid
metabolic imbalance may promote breast cancer.
The co-morbidities of type II diabetes and breast cancer are
increasingly drawing attention from the medical and lay communi-
ties. Breast cancer patients have over twice the incidence of type II

www.landesbioscience.com Cell Cycle 911

 Metformin inhibits cell growth

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Cell Cycle 2009.8:909-915.

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Figure 4. Effect of metformin on cell cycle progression in breast cancer cells. MCF-7, MCF-7/713, BT-474 and SKBR-3 cells were exposed to the indicated
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concentrations of metformin for 24 hours. Both adherent and non-adherent cells were harvested. Cell cycle distributions were analyzed by flow cytometry
as described in the Materials and Methods.
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diabetes (16%), than control adults in these same developed countries growth inhibitory effects of metformin are generally believed to
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(7%).14 The estimated excess relative risk of breast cancer in type II involve the activation of AMPK.28-30 AMPK is a serine/threonine
diabetes patients is 10–20% greater than non-diabetic women. protein kinase, which serves as an energy sensor in all eukaryotic cell
Patients with diabetes who develop breast cancer are also known to types. AMPK activation leads to G1 cell cycle arrest via upregulation
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have a worse prognosis and typically present with more aggressive of the p53-p21waf1 axis.31 In ER positive MCF-7 cells, metformin
disease (greater tumor stage and size, even when adjusted for body treatment resulted in a general decrease in mRNA translation.21
09

mass).13 Three mechanisms have been proposed that link diabetes to Metformin has recently been shown to induce apoptosis in p53 null
the pathogenesis of breast cancer, including: activation of the insulin colorectal tumor cells and autophagy in p53 wild-type colorectal
20

associated signaling pathways, activation of the insulin-like growth tumor cells.32 There is no report suggesting that mutant p53 would
factor pathway, and regulation of endogenous sex hormones.14 modulate metformin action on cancer cell proliferation and/or
Metformin is a widely prescribed oral drug available worldwide apoptosis.
©

at low cost. It reduces both basal and post-prandial glucose levels,
enhances insulin sensitivity (or reduces insulin resistance) and it does
not result in an increase in circulating insulin (hence it does not
induce hypoglycemia, the later which is associated with sulfonylurea
monotherapy).19,26,27 Metformin treatment has other beneficial
effects as well in many patients, including: a reduction in total body
weight, changes in fat metabolism and a decrease in adiposity. The
In this study, we sought to determine if cell lines representing
the major molecular subtypes of breast cancer luminal A, luminal
B and Her2/erbB2 would be sensitive to metformin. We found that
metformin induced similar growth inhibitory effects on both ER+
and ER- breast cancer cells. Considering the positive correlation
between type II diabetes and breast cancer as discussed above, and
the contributions of estrogenic hormones to the onset of diabetes

912 Cell Cycle 2009; Vol. 8 Issue 6

 Metformin inhibits cell growth

While metformin induced some reduction

in the expression of mTOR and P-mTOR
(as noted by others),21 this effect was less
quantitatively pronounced than the changes
we observed in the major signaling pathways
described above. Nonetheless, the nutrient-
sensing mTOR pathway is involved in cellular
and organismal aging, which may contribute

E.
to the concept that cancer is an age-related
disease.34,35 Thus, the ability of metformin to

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interfere with mTOR pathway supports the
hypothesis that metformin not only possesses

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anti-cancer activities, it could also be a poten-
tial anti-aging drug.35 In addition, mTOR

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is a key regulator of cellular responses to

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multiple stimuli, including amino acids and
growth factors. In cells with sufficient nutri-

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ents, mTOR relays signals to the translational
machinery leading to an upregulation of
mRNAs encoding proteins essential for cell

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growth and cell cycle progression.23 On the

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basis of our data, we cannot exclude mTOR
as a major regulator of the metformin-
induced effects we observe in these cell lines.
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Cell Cycle 2009.8:909-915.

Figure 5. Metformin reduces levels of cell cycle promoters (CyclinD1 and E2F1) and inhibits erbB-2 As reported by others, we did observe changes
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signaling in breast cancer cell lines. MCF7, MCF7/713, BT-474 and SKBR-3 cells were grown in 60 in AMPK activation with metformin (data
mm dishes. After 24 hours, the culture medium was replaced with fresh medium containing 0.5% FBS not shown) in each of these 4 cell lines.
as control, or same medium containing 2 mM, 10 mM or 50 mM metformin for another 24 hours
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In summary, we have shown a similar

incubation. The cell lysates were prepared as described in the Materials and Methods. 50 μg of total
cell lysates from each sample were used for western blot analyses performed with antibodies directed induction of G1 cell cycle arrest and a reduced
against Cyclin D1, E2F-1, p27kip1, p21waf-1 and b-actin (A); and P-erbB-2 (Phospho-erbB-2); erbB-2; clonogenicity in four diverse (ER+, ER-,
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P-mTOR (Phospho-mTOR); mTOR; P-Akt (Phospho-Akt, Ser473); Akt; P-MAPK (Phospho-p44/42 MAP erbB2 normal and abnormal) human breast
Kinase); MAPK; b-actin (B). cancer cell lines in response to metformin.
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Within the therapeutic dosage range for type

as well as breast cancer, our findings that the anti-type II diabetes II diabetes, metformin inhibited erbB2 tyrosine kinase activity and
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drug metformin inhibits the proliferation of breast cancer cells downstream signaling. These data suggest that metformin may have
independent of their ER status suggest that metformin may have anti-cancer activity involving important signaling pathways-beyond
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broad therapeutic efficacy in breast cancer treatment. Our data its effects on insulin and insulin-like growth factor associated mito-
also revealed that metformin inhibited cell growth through cell genesis.
cycle G1 arrest, regardless the p53 status of the breast cancer cells
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(MCF-7 and MCF-7/713 cells have wild-type p53, both SKBR-3 Materials and Methods
and B-T474 cells have mutant p53).33 Metformin treatment was Reagents. Metformin (1,1-dimethylbiguanide hydrochloride) was
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associated with a reduction of Cyclin D1 and E2F1 expression, purchased from Sigma Chemical Co., St. Louis, MO and diluted in
which mechanistically may have contributed to the G1 checkpoint
purified water. It was used across a range of concentrations at 2, 5, 10
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arrest. We found no significant changes in total protein levels of

waf1 kip1 and 50 mM diluted in media. Paclitaxel (Taxol) was obtained from
p21 or p27 with or without metformin treatment by western
Bristol-Myers Co/Mead Johnson Company, Princeton, NJ.
blot analysis (Fig. 5). It is not clear, however, if metformin treatment
Antibodies used for western blot analyses were from the
09

may lead to change the sub-cellular localization or translocation of

p21 waf1 kip1
or p27 . We have demonstrated that metformin at high following sources: Cyclin D1 (Cyclin D1, M-20), E2F1 (KH95),
p21waf1 (F-5) and p27kip1 (F-8) were obtained
20

concentrations resulted in decrease in erbB2 expression and activity, ERK2 (MAPK),

whereas at lower concentrations it induced changes in only erbB2 from Santa Cruz Biotechnology Inc., Santa Cruz, CA. P-MAPK
phosphorylation. These shifts in erbB2 activity were associated with (Phospho-p44/42 MAP Kinase Thr202/Tyr204), P-mTOR
©

reductions in downstream signaling through P-MAPK and P-Akt. (Phospho-mTOR Ser2448), mTOR, AKT, P-AKT (Phospho-AKT,
Most importantly, inhibition of erbB2 tyrosine kinase activity was Ser-473), P-erbB2 (Y1221/1222, and Y877) were purchased from
observed within the therapeutic dose range of metformin (Fig. 6) and Cell Signaling Technology, Danvers, MA. P-erbB-2/neu (PN2A,
involved both auto- and Src-associated phosphorylation sites. These Y1248) was from NeoMarkers, Fremont, CA. The erbB-2/neu
effects are of particular interest, because they suggest that the addi- (c-neu Ab-3) was from Oncogene Research Products, San Diego,
tion of metformin may synergistically enhance the anti-cancer effects CA. Poly (ADP-ribose) polymerase (PARP) monoclonal antibody
of the anti-erbB2 agents, like Herceptin. (C-2-10) was from BIOMOL Research Laboratories Inc., Plymouth

www.landesbioscience.com Cell Cycle 913

 Metformin inhibits cell growth

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Figure 6. Metformin at lower concentrations inhibits erbB2 tyrosine kinase activity without alteration of its protein levels. BT-474 and SKBR-3 cells were
Cell Cycle 2009.8:909-915.

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grown in 60 mm dishes. After 24 hours, the culture medium was replaced with fresh medium containing 0.5% FBS as control, or same medium containing
25 μM, 200 μM or 2,000 μM metformin for another 24 hours incubation. The cell lysates were prepared as described in the Materials and Methods. 50
μg of total cell lysates from each sample were used for western blot analyses performed with antibodies directed against erbB2, P-erbB2 (Y1248), P-erbB2
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(Y1221/1222), P-erbB2 (Y877) or b-actin. The experiments were carried out in triplicates (A); (B) The intensities of the signals on the western blots were
measured by a gel documentation system from Bio-Rad. The bar graph was obtained by calculating the percentages of the signals’ intensity from each
tyrosine phosphorylation site relative to controls, defined as 100%. Bars, SD.
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Meeting, PA. Monoclonal anti-β-actin (clone AC-75) was from Flow cytometric analysis. Flow cytometric analyses were
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Sigma Chemical Co., All other reagents were purchased from Sigma performed as described previously to define the cell cycle distribu-
Chemical Co., unless otherwise specified. tion for metformin treated and untreated cells.39 In brief, cells
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Cells and cell culture. The human breast cancer cell lines MCF-7, grown in 100-mm culture dishes (2 x 106 cells/dish) were harvested
BT-474 and SKBR-3 were obtained from the American Type Culture by trypsinization and fixed with 70% ethanol. Cells (1–2 x 106)
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Collection (ATCC, Rockville, MD). An MCF-7 subline transfected with were stained for total DNA content with a solution containing 50
c-erbB2 (MCF7/713) was a gift of Dr. C Benz (Buck Institute for Aging μg/ml propidium iodide and 100 μg/ml RNase I in PBS for 30
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Research, Novato, CA). All cell lines were maintained in Dulbecco’s min at 37°C. Cell cycle distribution was then analyzed at the Flow
Modified Eagle’s Medium: Nutrient Mix F-12 (D-MEM/F-12 1:1) Cytometry Core Facility of UCDHSC with a FACScan flow cytom-
(Invitrogen Corp., Grand Island, NY) supplemented with 10% fetal eter (Becton Dickinson, San Jose, CA, USA).
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bovine serum (FBS) (Invitrogen Corp.,), cultured in a 37°C humidified Clonogenic assay. Clonogenic assays were performed as described
atmosphere containing 95% air and 5% CO2 and split twice a week. previously.40 In brief, cells were seeded into 6-well plates in tripli-
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The ER and erbB2 status of these lines are as follows: MCF-7, ERα+, cates at a density of 1,000 cells/well in 2 ml of medium containing
erbB2 normal; MCF7/713, ERα downregulated, erbB2 transfected;36 10% FBS. After 24 hrs, cultures were replaced with fresh medium
BT-474, ERα+, erbB2 high and SKBR-3, ERα-, erbB2 high. containing 0.5% FBS (control) or same medium containing 2
09

Cell proliferation assay. A CellTiter96TM AQ non-radioactive mM, 10 mM or 50 mM metformin in a 37°C humidified atmo-
cell proliferation kit (Promega Corp., Madison, WI) was used to sphere containing 95% air and 5% CO2, and grown for 3 weeks.
20

determine cell viability as described previously.37,38 In brief, cells The cell clones were stained for 15 min with a solution containing
were plated onto 96-well plates at 3.5 x 104 cells well. After 24 hours 0.5% crystal violet and 25% methanol, followed by three rinses
©

in culture, the 10% FBS medium was removed and replaced by 0.5%
FBS in MEM/F12 with (treatment) or without (control) metformin
for 72 hours. After reading all wells at 490 nM with a micro-plate
reader, the percentages of surviving cells from each group relative to
controls, defined as 100% survival, were determined by reduction of
MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium, inner salt).
with tap water to remove excess dye. The colony numbers were
counted by gel documentation system EAGLE EYETM II (UVP,
LLC., Upland, CA).
Quantification of apoptosis. An apoptosis ELISA kit (Roche
Diagnostics Corp., Indianapolis, IN) was used to quantitatively
measure cytoplasmic histone-associated DNA fragments (mono-
nucleosomes and oligonucleosomes) as previously reported.40 This

914 Cell Cycle 2009; Vol. 8 Issue 6

 Metformin inhibits cell growth

photometric enzyme immunoassay was performed according to the
manufacturer’s instructions.
Western blotting analysis. Protein expression levels were deter-
mined by western blot analysis as previously described.37,38 In brief,
cells were lysed in a buffer containing 50 mM Tris, pH 7.4, 50 mM
NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylm-
ethylsulfonyl fluoride, 25 μg/ml leupeptin and 25 μg/ml aprotinin.
The lysates were centrifuged at full speed in a microcentrifuge for
21. Zakikhani M, Dowling R, Fantus IG, Sonenberg N, Pollak M. Metformin is an AMP
kinase-dependent growth inhibitor for breast cancer cells. Cancer Res 2006; 66:10269-73.
22. Hynes NE, Gullick W. Therapeutic targeting of signal transduction pathways and proteins
in breast cancer. J Mammary Gland Biol Neoplasia 2006; 11:1-2.
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124:471-84.
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A.D.T. mentor (Grant # PN 2006 02-25). The authors are grateful treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell
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to Ms. Lisa Litzenberger for her excellent assistance in arts prepara- 33. Concin N, Zeillinger C, Tong D, Stimpfl M, Konig M, Printz D, et al. Comparison of
tion, and to Drs. Vladimir N. Anisimov andLev M. Berstein for their p53 mutational status with mRNA and protein expression in a panel of 24 human breast
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