Effect of metformin on the proliferation, migration, and MMP-2 and -9 expression of human umbilical vein endothelial cells.
ABSTRACT Recent epidemiological studies have demonstrated that metformin lowers the risk of several types of cancer in diabetic patients. Matrix metalloproteinases (MMPs) play a crucial role in the degradation of the vascular basement membrane extracellular matrix proteins, thereby promoting endothelial cell invasion, migration and angiogenesis in the incidence and progression of tumors. The aim of this study was to investigate the effects of metformin on human umbilical vein endothelial cell (HUVEC) proliferation and migration, as well as on MMP-2 and MMP-9 expression. Cell proliferation was determined by cell counting and MTT colorimetric assays. Cell migration was assessed by the wound repair method. Quantitative real-time reverse transcription PCR was performed to quantify the mRNA expression of MMPs. Metformin at concentrations of 0.5-3.0 mM effectively reduced the number of endothelial cells by 5.5-55%, without being cytotoxic to the cells. Similarly, cell proliferation and migration were markedly inhibited by metformin. In addition, treatment with metformin demonstrated a strong (P<0.001) suppressive effect on the mRNA levels of MMP-2 and -9 in the endothelial cells. The inhibitory effects of metformin on endothelial cell number, migration, and MMP expression were reversed partially by compound C, which is an inhibitor of AMP-activated protein kinase (AMPK). The present study reports that metformin considerably inhibited the proliferation, migration, and MMP-2 and -9 expression of HUVECs, and the effect was partially AMPK-dependent. The obtained findings provide a molecular rationale, whereby metformin can exert anticancer effects.
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ABSTRACT: AMP-activated protein kinase (AMPK) is an evolutionary conserved energy-sensing enzyme that regulates cell metabolism. Emerging evidence indicates that AMPK also plays an important role in modulating endothelial cell function. In the present study, we investigated whether AMPK modulates endothelial cell growth. Treatment of cultured human umbilical vein endothelial cells with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 6,7-dihydro-4-hydroxy-3-(2'-hydroxy[1,1'-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662), or metformin inhibited cell proliferation and DNA synthesis. The antiproliferative action of AICAR was largely prevented by the adenosine kinase inhibitor 5'-iodotubercidin and mimicked by infecting endothelial cells with an adenovirus expressing constitutively active AMPK. In contrast, pharmacological blockade of endothelial nitric oxide synthase or heme oxygenase-1 activity failed to reverse the inhibition of endothelial cell growth by AICAR. Flow cytometry experiments revealed that pharmacological activation of AMPK arrested endothelial cells in the G₀/G₁ phase of the cell cycle, and this was associated with increases in p53 phosphorylation and p53, p21, and p27 protein expression and decreases in cyclin A protein expression and retinoblastoma protein phosphorylation. In addition, silencing p21 and p27 expression partially restored the mitogenic response of AMPK-activated cells. Finally, activation of AMPK by AICAR blocked the migration of endothelial cells after scrape injury and stimulated tube formation by endothelial cells plated onto Matrigel-coated plates. In conclusion, these studies demonstrate that AMPK activation inhibits endothelial cell proliferation by elevating p21 and p27 expression. In addition, they show that AMPK regulates endothelial cell migration and differentiation and identify AMPK as an attractive therapeutic target in treating diseases associated with aberrant endothelial cell growth.Journal of Pharmacology and Experimental Therapeutics 06/2012; 342(3):827-34. · 3.89 Impact Factor
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ABSTRACT: Breast cancer is the most frequently diagnosed tumor type and the primary leading cause of cancer deaths in women worldwide and multidrug resistance is the major obstacle for breast cancer treatment improvement. Emerging evidence suggests that metformin, the most widely used antidiabetic drug, resensitizes and cooperates with some anticancer drugs to exert anticancer effect. However, there are no data regarding the reversal effect of metformin on chemoresistance in breast cancer. In the present study, we investigated the resistance reversal effect of metformin on acquired multidrug-resistant breast cancer cells MCF-7/5-Fu derived from MCF-7 breast cancer cells and innate multidrug-resistant MDA-MB-231 breast cancer cells, and we found that metformin resensitized MCF7/5-FU and MDA-MB-231 to 5-fluorouracil (5-FU), adriamycin, and paclitaxel. We also observed that metformin reversed epithelial-mesenchymal transition (EMT) phenotype and decreased the invasive capacity of MCF7/5-FU and MDA-MB-231 cells. However, there were no significant changes upon metformin-treated MCF7 cells. Moreover, we found metformin treatment activated AMPK signal pathway in MCF7/5-FU and MDA-MB-231 cells and compound C, the AMPK inhibitor, could partly abolish the resensitization and EMT reversal effect of metformin. To the best of our knowledge, we are the first to report that metformin can resensitize multidrug-resistant breast cancer cells due to activating AMPK signal pathway. Our study will help elucidate the mechanism of chemoresistance and establish new strategies of chemotherapy for human breast cancer.Molecular and Cellular Biochemistry 10/2013; · 2.33 Impact Factor
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ABSTRACT: N6-isopentenyladenosine (iPA), an end product of the mevalonate pathway with an isopentenyl chain, is already known to exert a suppressor effect against various tumors. In this work, we investigated whether iPA also directly interferes with the angiogenic process, which is fundamental to tumor growth and progression. To this end, using human umbilical vein endothelial cells (HUVECs) as a suitable in vitro model of angiogenesis, we evaluated their viability, proliferation, migration, invasion, tube formation in response to iPA, and molecular mechanisms involved. Data were corroborated in mice by using a gel plug assay. iPA dose- and time-dependently inhibited all the neoangiogenesis stages, with an IC50 of 0.98 μM. We demonstrated for the first time, by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS), that iPA was monophosphorylated into 5'-iPA-monophosphate (iPAMP) by the adenosine kinase (ADK) inside the cells. iPAMP is the active form that inhibits angiogenesis through the direct activation of AMP-kinase (AMPK). Indeed, all effects were completely reversed by pretreatment with 5-iodotubercidin (5-Itu), an ADK inhibitor. The isoprenoid intermediate isopentenyl pyrophosphate (IPP), which shares the isopentenyl moiety with iPA, was ineffective in the inhibition of angiogenesis, thus showing that the iPA structure is specific for the observed effects. In conclusion, iPA is a novel AMPK activator and could represent a useful tool for the treatment of diseases where excessive neoangiogenesis is the underlying pathology.- Pisanti, S., Picardi, P., Ciaglia, E., Margarucci, L., Ronca, R., Giacomini, A., Malfitano, A. M., Casapullo, A., Laezza, C., Gazzerro, P., Bifulco, M. Anti-angiogenic effects of N6-isopentenyladenosine, an endogenous isoprenoid end-product, mediated by AMPK activation.The FASEB Journal 11/2013; · 5.70 Impact Factor
MOLECULAR MEDICINE REPORTS 5: 1068-1074, 2012
Abstract. Recent epidemiological studies have demonstrated
that metformin lowers the risk of several types of cancer in
diabetic patients. Matrix metalloproteinases (MMPs) play
a crucial role in the degradation of the vascular basement
membrane extracellular matrix proteins, thereby promoting
endothelial cell invasion, migration and angiogenesis in the
incidence and progression of tumors. The aim of this study
was to investigate the effects of metformin on human umbilical
vein endothelial cell (HUVEC) proliferation and migration, as
well as on MMP-2 and MMP-9 expression. Cell proliferation
was determined by cell counting and MTT colorimetric assays.
Cell migration was assessed by the wound repair method.
Quantitative real-time reverse transcription PCR was performed
to quantify the mRNA expression of MMPs. Metformin at
concentrations of 0.5-3.0 mM effectively reduced the number
of endothelial cells by 5.5-55%, without being cytotoxic to the
cells. Similarly, cell proliferation and migration were markedly
inhibited by metformin. In addition, treatment with metformin
demonstrated a strong (P<0.001) suppressive effect on the
mRNA levels of MMP-2 and -9 in the endothelial cells. The
inhibitory effects of metformin on endothelial cell number,
migration, and MMP expression were reversed partially by
compound C, which is an inhibitor of AMP-activated protein
kinase (AMPK). The present study reports that metformin
considerably inhibited the proliferation, migration, and MMP-2
and -9 expression of HUVECs, and the effect was partially
AMPK-dependent. The obtained findings provide a molecular
rationale, whereby metformin can exert anticancer effects.
Several epidemiological studies have found that diabetic
patients using metformin have a lower risk of cancer in
comparison to those using other anti-diabetic drugs. A case-
control study by Li et al (1) reported that the risk of pancreatic
cancer was 62% lower in diabetic patients who had been treated
with metformin than those who had never received the drug.
Other observational cohort studies demonstrated a decrease
of 25-37% in cancer cases in diabetic patients treated with
metformin (2,3). A study by Zhou et al (4) suggested that most
of the beneficial effects of metformin are mediated through its
ability to activate the AMP-activated protein kinase (AMPK).
AMPK is a key sensor of the cellular AMP/ATP ratio. AMPK is
activated by an increase in this proportion as a consequence of
the partial inhibition of the mitochondrial respiratory chain by
metformin (5). Various biological effects have been attributed
to the activation of AMPK by metformin. It interferes with
the action of the mammalian target of rapamycin (mTOR) that
functions as part of the cellular signaling processes regulating
cell growth, cell proliferation, cell motility, transcription and
protein synthesis (6,7). Furthermore, the upstream regulator of
AMPK is a protein kinase identified as LKB1 (8,9) which is a
well-known tumor suppressor. It has been suggested that LKB1
is a critical barrier to pulmonary tumorigenesis, differentiation
and metastasis (10). This fact further highlights the possible
role of AMPK activation in the anticancer effects of metformin.
Angiogenesis, an essential component of tumor progression,
is primarily achieved through the proliferation, survival, and
migration of endothelial cells (11). Angiogenesis is believed to
begin with matrix metalloproteinase (MMP)-mediated degra-
dation of the blood vessel basement membrane which contains
various extracellular matrix (ECM) proteins. Subsequently, it
is followed by sequential changes in vascular endothelial cells
(12). MMP-2 and -9, predominately expressed in the endothe-
lial cells, are directly involved in endothelial cell migration
and vascular remodeling during angiogenesis (13,14).
Tan et al (15) reported that metformin decreases angio-
genesis in women suffering from polycystic ovary syndrome
(PCOS) by increasing the anti-angiogenic thrombospondin-1.
In addition, metformin in a murine sponge model was found
to inhibit angiogenesis by decreasing vascularization, macro-
phage recruitment, collagen deposition and levels of the
Effect of metformin on the proliferation, migration, and MMP-2
and -9 expression of human umbilical vein endothelial cells
NILUFAR ESFAHANIAN1, YADOLLAH SHAKIBA2, BEHROZ NIKBIN2, HAMID SORAYA1,
NASRIN MALEKI-DIZAJI1, MAHMOOD GHAZI-KHANSARI3* and ALIREZA GARJANI1*
1Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz;
Departments of 2Immunology, and 3Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Received October 22, 2011; Accepted December 27, 2011
Correspondence to: Dr Alireza Garjani, Department of Pharmacology
and Toxicology, Faculty of Pharmacy, Tabriz University of Medical
Sciences, Tabriz, Iran
E-mail: firstname.lastname@example.org; email@example.com
Key words: metformin, matrix metalloproteinase, migration, prolif-
eration, endothelial cells
ESFAHANIAN et al: EFFECT OF METFORMIN ON HUVEC PROLIFERATION AND MIGRATION
transforming growth factor β1 (16). It can be proposed that
metformin controls and reduces the progression of cancer
through its anti-angiogenic effects. The effect of metformin on
human umbilical vein endothelial cells (HUVECs), an estab-
lished model for angiogenesis study, has not been elucidated
to date. The present study seeks to address whether metformin
interferes with endothelial cell functions in terms of prolif-
eration, migration and MMP expression. In addition, we also
speculated whether these effects are mediated by AMPK.
Materials and methods
Materials. HUVECs were purchased from the National Cell
Bank, Pasteur Institute of Iran. Metformin was provided by
the Osveh Pharmaceutical Laboratory (Tehran, Iran). Fetal
bovine serum (FBS), Dulbecco's modified Eagle's medium
(DMEM), TRIzol, and trypsin/EDTA 0.25% were obtained
from Invitrogen (USA). Compound C, DMSO and MTT
bromide] were obtained from Sigma-Aldrich (USA). The
Quantitect reverse transcription kit and Quantifast probe
PCR+Rox vial kit were obtained from Qiagen (USA). The
LDH cytotoxicity assay kit was purchased from Roche
(Germany). All the other reagents used in the experiments
were of analytical grade.
Cell culture. HUVECs were cultured in DMEM medium
supplemented with 10% FBS. The culture was carried out at
37˚C in 5% CO2. After the cells reached a confluence of 80%,
they were detached using 0.25% trypsin-EDTA. Subsequently,
the cells were subcultured once again.
Endothelial cell cytotoxicity assay. The experimental proce-
dure was conducted according to the method of Linford and
Dorsa (17) for measuring the cytotoxicity and cell lysis by
detecting lactate dehydrogenase (LDH) activity released from
the damaged cells. HUVECs were cultured in a 96-well culture
plate at a density of 1x104 cells/well in DMEM medium. After
24 h, metformin at different concentrations, compound C
(10 µM), and compound C plus 3 mM metformin were added
to the wells and the cells were incubated for an additional 72 h.
The plates were centrifuged at 200 x g. Then, 100 µl of the
LDH assay mixture was added, and the plates were incubated
at 37˚C for 30 min. The LDH release was estimated at 490 nm,
using ELISA (Behring ELISA Processor) and expressed
as a percentage of the control. All of the experiments were
performed in triplicate.
Endothelial cell proliferation assay. This assay aimed to deter-
mine whether metformin affects cell proliferation. HUVECs
were seeded at a density of 1x104 cells/well in a 12-well culture
plate and allowed to attach for 24 h. Next, the cells were washed
twice with PBS and treated with different concentrations of
metformin. HUVECs were treated with 10 µM compound C for
30 min alone or before adding metformin at the 3 mM concen-
tration [in the present series of experiments DMSO (0.8%)
was used as a vehicle]. After a 72-h incubation, the cells were
washed with PBS and harvested using trypsin-EDTA. The cell
count and viability were determined by trypan blue dye exclu-
sion assay. All the experiments were performed in triplicate.
MTT proliferation assay. The 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide (MTT) proliferation assay
is an index of cell viability and proliferation. The cells were
cultured in a 96-well culture plate at a density of 3x103 cells/
well for 24 h. After being treated with different concentrations
of metformin, compound C (10 µM), and compound C plus
3 mM of metformin for a further 72 h, the cells were washed
twice with PBS and subjected to the MTT assay. The cells
were incubated with the MTT solution at a final concentration
of 0.5 mg/ml for 3 h. Subsequently, the cells were lysed in
DMSO. The optical density was measured at 540 nm using an
ELISA reader (Behring ELISA Processor). All of the samples
were assayed in triplicate, and the mean value for each experi-
ment was calculated. The obtained results are expressed as a
percentage of the control, which is considered to be 100%.
Endothelial cell migration assay. HUVECs were cultured in a
6-well culture plate. A wound was made in the cell area using
a sterile yellow tip when the cells achieved 80-90% confluence.
The variation in the wound width within the experiments was
approximately 5%. First, the cells were washed with PBS.
Then, the cells were treated with a medium containing different
concentrations of metformin and 2% FBS (2% FBS allows cell
survival but not cell proliferation). After a 72-h incubation, the
cells were washed twice with PBS, fixed with methanol and
stained with Giemsa. The cell migration into the scratched area
was photographed at a magnification of x40. Subsequently, the
cell migration was quantified by calculating the difference in
the denuded area using a computerized planimetry package
(Landcalc, UK). The obtained data were expressed as a
percentage of the migration in the untreated endothelial cells.
RNA isolation and real-time quantitative PCR. The total
cellular RNA was extracted from the cultured cells (~1x105)
using TRIzol. The cells were lysed in 1 ml TRIzol and incu-
bated at room temperature for 5 min. Then, 200 ml chloroform
was added to the lysate, incubated for 3 min, and centrifuged
for 15 min at 12,000 x g at 4˚C. The aqueous layer was removed,
mixed with an equal volume of isopropanol and incubated for
Table I. Effects of metformin, compound C, and DMSO on
lactate dehydrogenase (LDH) activity in HUVECs.
Groups (n=4) LDH (% control)
Metformin (500 µM)
Metformin (1 mM)
Metformin (2 mM)
Metformin (3 mM)
Metformin (4 mM)
Metformin (5 mM)
Compound C (10 µM)
Metformin (3 mM) + compound C (10 µM)
DMSO (0.8%; vehicle)
Values are mean ± SD and expressed as the percentage of control
from four independent experiments.
MOLECULAR MEDICINE REPORTS 5: 1068-1074, 2012
1 h at 4˚C. The purified RNA was precipitated by centrifugation
at 12,000 x g for 15 min and was finally dissolved in 50 µl
diethylpyrocarbonate (DEPC)-treated water. One microgram of
the total-RNA was converted to cDNA using the Quantitect
reverse transcription kit (Qiagen). Real-time PCR was
performed by the Quantifast Probe PCR+Rox vial kit (Qiagen)
using the ABI Step One Plus Detection system (Applied
Biosystems, USA). The cycling conditions were 45 cycles in
two steps. An initial denaturation step at 95˚C for 3 min was
followed by denaturation at 95˚C for 3 sec, and annealing-
extension at 60˚C for 30 sec. For quantification, the target gene
was normalized to the internal standard gene 18S. The primers
were designed for detection of MMP-2 and -9 gene expression:
MMP-2, forward, 5'-TTGATGGCATCGCTCAGATC-3' and
reverse, 5'-TTGTCACGTGGCGTCACAGT-3'; MMP-9,
forward, 5'-GACGCAGACATCGTCATCCA-3' and reverse,
5'-CACAACTCGTCATCGTCGAAA-3'; 18S rRNA, forward,
5'-CGGCTACCACATCCAAGGAA-3' and reverse, 5'-GCT
Statistics. Data are presented as the mean ± SD. One-way
ANOVA was used to make comparisons between groups. A
Student-Newman-Keuls post test was performed to compare
the mean values between the treatment groups and the control
in case the ANOVA analysis indicated significant differences.
Differences between the groups were considered significant at
Effects of metformin on LDH release from endothelial cells.
HUVECs were incubated with different concentrations of the
drugs for 72 h to determine whether metformin, compound C,
and DMSO (as a vehicle) are cytotoxic against endothelial
cells. Subsequently, the lactate dehydrogenase (LDH) release
was measured. The LDH activity of the control and treated
groups is shown in Table I. The LDH values among the groups
were almost identical with no significant differences.
Effects of metformin on endothelial cell proliferation.
Incubation of the unstimulated human umbilical vein endo-
thelial cells with different concentrations of metformin
(0.5-3.0 mM) for 72 h induced a marked (P<0.05; P<0.001)
and dose-dependent reduction in the number of cells (Fig. 1).
Compound C was used as a pharmacological inhibitor of AMPK
for evaluating the role of the AMPK pathway in the metformin
anti-proliferation effects of HUVECs. Compound C, at a
concentration of 10 µM, caused a 16% reduction (P<0.001) in
the cell proliferation by itself. Metformin at the concentration
of 3 mM produced a strong (P<0.001) inhibition of HUVEC
proliferation both in the presence (37% inhibition) and absence
(55% inhibition) of compound C in comparison to the related
controls. However, the anti-proliferation effect of metformin
(3 mM) was significantly (18%; P<0.01), but not completely
reversed by compound C (Fig. 1). This inhibition did not result
from a cytotoxic effect, as assessed by the LDH release from
the control and the treated groups (Table I).
The anti-proliferative effects of metformin were confirmed
using an MTT proliferation assay (Fig. 2), with similar signifi-
cant (P<0.001) and concentration-dependent decreases noted
in endothelial cell proliferation. However, compound C did
not affect the cell viability in this set of experiments (Fig. 2).
Metformin at 3 mM produced a significant (P<0.001; 35%)
inhibition of endothelial cell proliferation in the presence of
compound C. However, the inhibitory effect was much lower in
comparison to the cells treated with metformin (3 mM) alone
(P<0.001; 52%). The results of the MTT assay also showed that
compound C partially blocked the anti-proliferative action of
metformin (Fig. 2), and this was comparable with that of the
cell counting experiments (Fig. 1).
Effects of metformin on endothelial cell migration. The
‘wound’ repair model of migration was used to evaluate the
effect of metformin on endothelial cell migration. Confluent
scrape-wounded HUVEC monolayers were incubated for 72 h
with metformin in the presence or absence of compound C.
Subsequently, the degree of closure of the ‘wound’ was
Figure 1. Effects of metformin, DMSO (as a vehicle for compound C experiments; 0.8%), compound C (Comp C), and metformin (Met; 3 mM) + Comp C
(10 µM) on the number of endothelial cells. HUVECs, seeded at 1x104 cells/well, were incubated at the indicated concentrations of drugs for 72 h. Data are
expressed as the mean ± SD number of cells/well (n=6). *P<0.05 and ***P<0.001 vs. 0 mM metformin (control); #P<0.01 and aP<0.001 vs. DMSO (control);
bP<0.01 vs. metformin 3 mM.
ESFAHANIAN et al: EFFECT OF METFORMIN ON HUVEC PROLIFERATION AND MIGRATION
assessed. It was observed that metformin at concentrations
of 0.5-3.0 mM induced a strong and significant (P<0.001)
concentration-dependent inhibition of ‘wound’ repair in
HUVECs from 31 to 80%, respectively (Fig. 3). Compound C
significantly inhibited the migration (P<0.001), which was
consistent with its effect on endothelial cell numbers. However,
Figure 2. Effects of metformin, DMSO (as a vehicle for compound C experiments; 0.8%), compound C (Comp C), and metformin (Met; 3 mM) + Comp C
(10 µM) on endothelial cell proliferation evaluated by the MTT assay. HUVECs, seeded at 3x103 cells/well, were incubated at the indicated concentrations of
drugs for 72 h and then exposed to the MTT solution. Data are expressed as the mean ± SD of the percentage of control values from six independent experi-
ments. ***P<0.001 vs. 0 mM metformin (control); aP<0.001 vs. DMSO (control); bP<0.01 vs. metformin 3 mM.
Figure 3. Metformin inhibits endothelial cell migration in a ‘wound’ repair model. (A) Photomicrographs (x40 magnification) showing confluent HUVECs
after mechanical scraping with a pipette tip and the migration of cells into the scraped area after treatment with the medium alone (control) or with metformin
(1-3 mM) or with metformin (3 mM) + compound C (10 µM). (B) Quantitation of the inhibition of HUVEC migration by metformin in the absence and presence
of compound C. Data are expressed as a percentage of the migration in the untreated endothelial cells (mean ± SD) from six independent experiments. ***P<0.001
vs. 0 mM metformin (control); #P<0.01 and aP<0.001 vs. DMSO (control); bP<0.01 vs. metformin 3 mM.
MOLECULAR MEDICINE REPORTS 5: 1068-1074, 2012
in comparison to the metformin alone-treated cells (3 mM),
compound C partially, but significantly (P<0.001) reversed the
anti-migratory effect of metformin.
Effects of metformin on MMP-2 and -9 expression in HUVECs.
HUVECs were incubated with different concentrations of
metformin for 72 h. Subsequently, the mRNA expression
of MMP-2 and -9 was examined. Metformin significantly
(P<0.001) decreased both the MMP-2 and -9 mRNA levels
in a concentration-dependent manner (Fig. 4). The most
marked decline in mRNA expression was noted with 3 mM of
metformin. DMSO, as a vehicle, or compound C, as an AMPK
inhibitor, had no effect on the mRNA expression of MMPs.
However, it was observed that compound C significantly
(P<0.05), but not completely, reversed the suppressive effect
of metformin (3 mM) on the MMP-2 and -9 mRNA expression
Angiogenesis is an integral part of tumor growth and metas-
tasis that has gained increased interest as a core component in
cancer therapy. Several case-control and observational cohort
clinical trials have reported that systemic treatment with the
anti-diabetic drug metformin considerably decreased the risk
of different types of cancer in diabetic patients (1-3,18-20).
A recent study on mice exposed to tobacco carcinogenesis
demonstrated that metformin decreased the tumor burden by
72%, which was correlated with decreased cellular prolifera-
tion and marked inhibition of mTOR in tumors (21). However,
it is necessary to clarify whether metformin exerts the
anticancer effect, at least in part, through an anti-angiogenic
effect. The present study found that metformin produced a
potent anti-proliferative and anti-angiogenic effect in vitro on
HUVECs, and that this effect was associated with reduced
mRNA expression of MMP-2 and -9.
It was clear from the experiments that metformin produced a
potent (P<0.001) and concentration-dependent inhibitory effect
on HUVEC proliferation. A concentration of 3 mM of metformin
reduced the endothelial cell numbers in culture by at least 55%.
The effect was also confirmed by an MTT proliferation assay. The
anti-proliferative effect of metformin was not due to cytotoxicity
due to the fact that treatment of endothelial cells with different
concentrations of metformin for extended periods of time neither
affected the integrity of the cell monolayer nor was it associ-
ated with increased LDH release, an indicator of cytotoxicity.
Furthermore, the obtained data showed that the migration of the
cells into the denuded area 72 h after the cultures were treated
with metformin was significantly (P<0.001) lower in comparison
to the untreated control. To the best of our knowledge, not many
studies have dealt with the effects of metformin on angiogenesis,
in particular the effects of metformin on the endothelial cell
proliferation and migration. A study conducted on PCOS women
treated with metformin reported that metformin decreased
angiogenesis by increasing the serum anti-angiogenic thrombo-
spondin-1 (15). Also, a recent in vivo study in a murine sponge
model demonstrated that metformin inhibited inflammatory
angiogenesis by decreasing the levels of transforming growth
factor-β1 (TGF-β1) (16). In addition, our findings revealed
that the MMP-2 and -9 expression in unstimulated HUVECs
was markedly downregulated, following the metformin treat-
ment of endothelial cells in a concentration-dependent manner.
Inhibitory action of metformin, as a pharmacological activator
of AMPK, on the MMP expression has been described in human
fibrosarcoma cells (22). However, no study has evaluated the
effect of the drug on MMP expression in vascular endothelial
cells. MMPs are involved in many endothelial cell processes,
such as cell migration and angiogenesis, as well as in tumor
invasion or metastasis. These enzymes play an important role
in physiological tissue remodeling, and also in pathological
remodeling associated with conditions such as wound healing
and tumor growth. In particular, MMP-2 and -9, the two
predominately expressed MMPs in endothelial cells, have been
directly implicated in the process of endothelial cell migration.
This is accomplished through proteolysis of the components of
the extracellular matrix (13,14).
Metformin has been used as an anti-diabetic drug since
1957 (23). The drug reduces blood sugar levels mainly through
Figure 4. Metformin inhibits MMP-2 and MMP-9 mRNA expression. HUVECs were starved in serum-free medium overnight before metformin treatment. The
cells were then incubated with metformin (0, 0.5, 1, 2 and 3 mM) or with metformin (3 mM) + compound C (10 µM) for 72 h. RNA was isolated and converted
to cDNA. The expression of MMP-2 or MMP-9 mRNA was analyzed by real-time PCR. Data are expressed as the mean ± SD of five independent experiments.
**P<0.001 and ***P<0.001 vs. the control group (0 mg/kg metformin); aP<0.001 vs. DMSO (control); bP<0.01 vs. metformin 3 mM.
ESFAHANIAN et al: EFFECT OF METFORMIN ON HUVEC PROLIFERATION AND MIGRATION
three mechanisms: decreased hepatic glucose production
(24,25), increased skeletal myocyte glucose uptake (26,27),
and reduction of hepatic lipids (28). AMP-activated protein
kinase (AMPK) provides a candidate target, which is capable
of mediating the beneficial metabolic effects of metformin
(4). AMPK is an important intracellular energy sensor,
which activates the catabolic pathways that generate ATP. In
addition, AMPK also inactivates ATP-consuming anabolic
pathways when the cellular AMP/ATP ratio is increased (29).
Proliferation and migration are ATP-consuming processes.
Thus, AMPK activity may be required for optimal cell
proliferation and survival, in particular under stress condi-
tions. It was observed that tumor xenografts prepared from
Ras-transformed mouse embryo fibroblasts lacking AMPK
lose their ability to grow in a hypoxic environment (30),
and 5-amino-4-imidazole carboxamide riboside (AICAR),
an AMPK agonist, increases the angiogenesis of endothelial
progenitor cells by phosphorylation of acetyl-coenzyme A
carboxylase (ACC) and eNOS (31). Furthermore, it has been
reported that the activation of AMPK signaling in endothelial
cells is essential for angiogenesis under hypoxic conditions
(32), but not in normoxia. On the contrary, a growing body of
evidence indicates that AMPK activation inhibits the growth
and/or survival of various cancer cell lines (33-38). It is now
obvious that AMPK is regulated by a well-recognized tumor
suppressor known as LKB1 (39), and that activation of AMPK
by metformin requires LKB1. Furthermore, AMPK activation
by metformin inhibits the mammalian target of rapamycin
(mTOR), a protein that plays a critical role in transcription,
cell growth, proliferation and migration (6,7).
It was observed that compound C, a cell-permeable
pyrazolopyrimidine derivative, acts as a potent and selective
ATP-competitive inhibitor of AMPK (4). In the present study,
we demonstrated that the anti-proliferative and anti-migratory
effects of metformin on endothelial cells as well as the inhibi-
tory effect of metformin on mRNA expression of MMP-1 and
MMP-2 were significantly but not completely blocked by
compound C. This indicates that the AMPK pathway is
involved, at least in part, in the anti-angiogenic action of
metformin. Surprisingly, compound C alone showed a slight
but significant anti-proliferative and anti-migratory action.
These paradoxical effects in the present study probably imply
the involvement of AMPK-dependent and AMPK-independent
mechanisms in metformin anti-angiogenic actions. The other
possibility is that both the activation and inhibition of AMPK
cause anti-proliferative effects through different downstream
pathways. Regarding the energy-saving and ATP-producing
roles of AMPK through enhancing fatty acid oxidation,
glycolysis and glucose uptake (30,40), especially in ATP
deprivation conditions, it is conceivable that AMPK inhibition
by compound C to some extent leads to the inhibitory effects
on HUVEC proliferation and migration. However, AMPK
activation has a wider role in reducing circulating levels of
insulin-like growth factor and inhibition of cell differentiation,
proliferation, and growth through the suppression of mTOR
(41), elongation factor-2 (42), and the cyclin (43) pathways.
In this study, comparison of the strong inhibitory effects of
metformin, as an AMPK activator, with the weak suppressive
effects of compound C, as an AMPK antagonist, on the prolif-
eration and migration of human umbilical vein endothelial cells
indicates the potentially beneficial effects of AMPK activation
in preventing angiogenesis and related diseases.
Many factors have been identified as stimulators of the
MMP expression in endothelial cells (44) but little is known
about the inhibitors of these elements in normal cells under
physiological conditions. Using an AMPKα-knockout mouse,
Morizane et al (45) reported that total AMPKα deletion signifi-
cantly elevated MMP-9 expression in embryonic fibroblast
cells. The authors also demonstrated that AMPK activation
by AICAR or by A769662 in wild-type fibroblasts suppressed
MMP-9 expression. Thus, it was concluded that both the
activity and the presence of AMPKα contribute as a regulator of
MMP-9 expression. Similarly, the present study demonstrated
that the expression of MMP-2 and -9 mRNA was decreased
in HUVECs incubated with metformin and this decrease was
reversed partially by compound C as an inhibitor of AMPK.
Collectively, the results of this study suggest that metformin
may have potential effects in arresting the progression of tumors
by inhibiting endothelial cell proliferation and migration
through the suppression of MMP-2 and -9 mRNA expression.
In addition, AMPK activity, at least in part, is required for the
above-mentioned effects. In conclusion, the results may clarify
the beneficial effect of metformin in reducing cancer incidence
in diabetic patients receiving the drug.
The present study was supported by grants from the Research
Vice Chancellors of the Tabriz University of Medical Sciences,
Tabriz, Iran and from the Research Vice Chancellors of the
Tehran University of Medical Sciences, Tehran, Iran.
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