Dual antiglioma action of metformin cell cycle arrest and mitochondria-dependent apoptosis.pdf

Cell. Mol. Life Sci. 64 (2007) 1290 – 1302

1420-682X/07/101290-13

DOI 10.1007/s00018-007-7080-4

Birkhuser Verlag, Basel, 2007

Cellular and Molecular Life Sciences

Research Article

Dual antiglioma action of metformin: cell cycle arrest and

mitochondria-dependent apoptosis

A. Isakovic a , L. Harhaji b , D. Stevanovic c , Z. Markovic d , M. Sumarac-Dumanovic e , V. Starcevic c , D. Micic e, * and

V. Trajkovic f, *

a Institute of Biochemistry, School of Medicine, University of Belgrade, Belgrade (Serbia)

b Institute for Biological Research, Belgrade (Serbia)

c Institute of Physiology, School of Medicine, University of Belgrade, Belgrade (Serbia)

d Vinca Institute of Nuclear Sciences, Belgrade (Serbia)

e Institute of Endocrinology, Diabetes and Diseases of Metabolism, School of Medicine, University of Belgrade,

Dr Subotica 13, 11000 Belgrade (Serbia), Fax: +381 11 3065081, e-mail : micicd@eunet.yu

f Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr Subotica 1, 11000

Belgrade (Serbia), Fax: +381 11 265 7258, e-mail : vtrajkovic@eunet.yu

Received 14 February 2007; received after revision 26 March 2007; accepted 3 April 2007

Online First 20 April 2007

Abstract. The present study reports for the first time a

dual antiglioma effect of the well-known antidiabetic

drug metformin. In low-density cultures of the C6 rat

glioma cell line, metformin blocked the cell cycle

progression in G 0 /G 1 phase without inducing signifi-

cant cell death. In confluent C6 cultures, on the other

hand, metformin causedmassive induction of caspase-

dependent apoptosis associated with c-Jun N-terminal

kinase (JNK) activation, mitochondrial depolariza-

tion and oxidative stress. Metformin-triggered apop-

tosis was completely prevented by agents that block

mitochondrial permeability transition (cyclosporinA)

and oxygen radical production (N-acetylcisteine),

while the inhibitors of JNK activation (SP600125) or

glycolysis (sodium fluoride, iodoacetate) provided

partial protection. The antiglioma effect of metformin

was reduced by compound C, an inhibitor of AMP-

activated protein kinase (AMPK), and was mimicked

by the AMPK agonist AICAR. Similar effects were

observed in the human glioma cell line U251, while rat

primary astrocytes were completely resistant to the

antiproliferative and proapoptotic action of metfor-

min.

Keywords. Metformin, cancer, cell cycle, apoptosis, mitochondrial depolarization, oxidative stress, c-Jun

N-terminal kinase, AMP-activated protein kinase.

Introduction

glucoregulatory properties of metformin are mainly

attributed to reduced hepatic glucose production and

augmented glucose uptake by the peripheral tissues

[1] . More recent studies have documented the ability

of metformin to stimulate adenosine monophosphate-

activated protein kinase (AMPK) [1, 2] , an intra-

cellular energy sensor that is activated by rising AMP

and acts by switching on ATP-generating catabolic

Metformin (1-(diaminomethylidene)-3,3-dimethyl-

guanidine) is an antihyperglycaemic drug commonly

used for the management of type-2 diabetes [1] . The

* Corresponding authors.

Cell. Mol. Life Sci. Vol. 64, 2007

Research Article

1291

pathways while switching off ATP-requiring processes

[3] . Since AMPK inhibits hepatic glucose production

and stimulates muscle glucose uptake [1] , it is

potentially an ideal mediator of metformin action. In

contrast to the progress in understanding the mech-

anisms underlying the therapeutic effect of metfor-

min, its biological actions that are not directly linked

to antidiabetic activity have rarely been investigated.

Interestingly, two independent clinical studies have

recently shown that metformin may reduce the risk of

cancer in patients with type II diabetes [4, 5] . This is

consistent with the ability of metformin to suppress

both the spontaneous development of mammary

adenocarcinomas in HER-2/neu-transgenic mice [6]

and the induction of pancreatic cancer in high fat-fed

hamsters [7] . It is not clear, however, whether the

observed effects were due to a direct action of the drug

on tumor cells or resulted from its effects on insulin

metabolism. Namely, it is known that hyperinsuline-

mia and associated metabolic alterations, which are

corrected by metformin treatment, may play a role in

the onset of some malignancies [8] . The capacity of

metformin to block proliferation of human breast

cancer cells [9] or cause apoptotic cell death in a

mouse insulinoma cell line [10] argues in favor of

direct antitumor action, but the underlying mecha-

nisms are not completely understood.

Gliomas are extremely aggressive neuroectodermal

tumors that represent the most common primary

malignancy in human central nervous system [11] .

Gliomas are incurable in most of the cases, and it has

been postulated that their relative resistance to

apoptosis may contribute to chemotherapy and radi-

ation resistance [11] . Cell motility apparently contrib-

utes to the invasive phenotype of malignant gliomas,

and interference with cell motility by different strat-

egies results in increased susceptibility of glioma to

apoptosis [11] . Interestingly, a recent study revealed

the ability of metformin to inhibit in vitro migration of

malignant glioma cells [12] , indicating its potential

usefulness in antiglioma therapy. Accordingly, glioma

cells express both AMPKa1 and AMPKa2 [13] , the

putative targets of metformin intracellular action, and

pharmacological activation or overexpression of

AMPK has been shown to reduce glioma cell growth

[14] . However, the effects of metformin on glioma cell

proliferation and viability have not been explored so

far.

The present study demonstrates for the first time that

metformin induces cell cycle arrest as well as mito-

chondrial depolarization- and oxidative stress-de-

pendent apoptosis in cultured glioma cells. Impor-

tantly, primary astrocytes were almost completely

resistant to the antiproliferative/cytotoxic effects of

the drug.

Materials and methods

Cell cultures. All chemicals were from Sigma (St. Louis, MO)

unless specifically stated. The rat glioma cell line C6 and the human

glioma cell line U251 were kindly donated by Dr. Pedro Tranque

(Universidad de Castilla-La Mancha, Albacete, Spain). Primary

astrocytes were isolated from brains of newborn Albino Oxford

rats as previously described [15]. The tumor cell lines and

astrocytes were maintained at 378C in a humidified atmosphere

with 5%CO 2 in aHepes (20 mM)-bufferedRPMI 1640 cell culture

medium supplemented with 5% fetal calf serum, 2 mM L-

glutamine, 10 mM sodium pyruvate, penicillin/streptomycin and

10 mM glucose. The cells were prepared for experiments using the

conventional trypsinization procedure with trypsin/EDTA. For the

measurement of cell number/mitochondrial activity or flow

cytometric analysis of the cell cycle in low-density cell cultures,

cells were incubated in 96-well flat-bottom plates (5 10 3 cells per

well) or 24-well flat-bottom plates (5 10 4 cells per well),

respectively. For the enzyme-linked immunosorbent assay

(ELISA)/Chou-Talalay analysis or cell viability/flow cytometric

examination of apoptotic events in high-density cell cultures, cells

were incubated in 96-well (2 10 4 cells per well) or 24-well (2 10 5

cells per well) flat-bottomplates, respectively. Cells were rested for

24 h when high-density cell cultures reached confluence and

treated with metformin hydrochloride (99.9%; Hemofarm,

Vrsac, Serbia) and/or different agents, as described in Results

and Figure legends. Suspension of nanocrystalline C 60 fullerene

( n -C 60 ) was prepared as previously described [16].

Determination of cell number and mitochondrial activity. The

number of adherent, viable cells was assessed using a crystal violet

assay, while mitochondrial dehydrogenase activity, as another

indicator of cell viability, was determined by mitochondria-

dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

tetrazolium bromide (MTT) to formazan. Both tests were per-

formed exactly as previously described [17] . The results are

presented as relative increase compared to the control value

(untreated cells) arbitrarily set to 1 or as % of control value.

Cell cycle and apoptosis analysis. The cell cycle was analyzed by

measuring the amount of propidium iodide (PI)-labeled DNA in

ethanol-fixed cells, exactly as previously described [18]. Apoptotic

and necrotic cell death were analyzed by double staining with

fluoresceinisothiocyanate (FITC)-conjugated annexin Vand PI, in

which annexin V bound to the apoptotic cells with exposed

phosphatidylserine, while PI labeled the necrotic cells with

membrane damage. Staining was performed according to the

manufacturers instructions (BD Pharmingen, San Diego, CA).

The green (FL1) and red (FL2) fluorescence of annexin/PI-stained

live cells and PI-stained fixed cells was analyzed with a FACSCa-

libur flow cytometer (BD, Heidelberg, Germany) using a peak

fluorescence gate to exclude cell aggregates during cell cycle

analysis. The numbers of viable (annexin – /PI – ), apoptotic (annex-

in + /PI – ) and necrotic (annexin + /PI + ) cells as well as the proportion

of cells in different cell cycle phases were determined with

CellQuest Pro software (BD). DNA fragmentation, as another

marker of apoptosis, was determined during cell cycle analysis by

counting the hypodiploid cells in the sub-G 0 /G 1 compartment [18].

Caspase activation. Activation of caspases was measured by flow

cytometry after labeling the cells with a cell-permeable, FITC-

conjugated pan-caspase inhibitor (ApoStat; R&D Systems, Min-

neapolis, MN) according to the manufacturers instructions.

Increase in green fluorescence (FL1) is a measure of caspase

activitywithin individual cells of the treated population. The results

are expressed as the percentage of cells containing active caspases.

Measurement of reactive oxygen species (ROS). Intracellular

production of ROS was determined by measuring the intensity of

green fluorescence emitted by the redox-sensitive dye dihydro-

rhodamine 123 (DHR; Invitrogen, Paisley, UK), which was added

to cell cultures (1 mM) at the beginning of treatment. At the end of

incubation, cells were detached by trypsinization and washed in

PBS, and the green fluorescence (FL1) of DHR-stained cells was

analyzed using a FACSCalibur flow cytometer.

1292 A. Isakovic et al.

Antiglioma action of metformin

Mitochondrial depolarization. The mitochondrial depolarization

was assessed using DePsipher (R&D Systems), a lipophilic cation

susceptible to changes inmitochondrial membrane potential. It has

the property of aggregating upon membrane polarization, forming

an orange-red fluorescent compound. If the potential is disturbed,

the dye can not access the transmembrane space and remains or

reverts to its green monomeric form. The cells were stained with

DePsipher as described by the manufacturer, and the green

monomer and the red aggregates were detected by flow cytometry.

The results are presented as a green/red fluorescence ratio

(geomean FL1/FL2), an increase of which reflects mitochondrial

depolarization.

Cell-based ELISA for mitogen-activated protein kinases (MAPK)

and glial fibrillary acidic protein (GFAP). A cell-based ELISAwas

used to measure activation of the MAPK family members p38

MAPK, extracellular signal-regulated kinase (ERK) and c-Jun-N-

terminal kinase (JNK), as well as activation of transcription factor

nuclear factor-kB(NF-kB) and expression of the astrocyte marker

GFAP. TheELISAwas performed using the appropriate antibodies

exactly as previously described [19]. The results are presented

relative to the control value, which was arbitrarily set to 1.

Mathematical analysis of cytotoxic interactions. To analyze the

type (additive, synergistic or antagonistic) of metformin interaction

with H 2 O 2 or n -C 60 , cells were treated with different doses of each

agent alone and appropriate combinations. Cell viability was

assessed using a crystal violet assay. The values of the combination

index, reflecting additive (=1), synergistic ( < 1) or antagonistic

( > 1) interactions, were calculated according to the method

Statistics. The statistical significance of differences was analyzed by

t -test or ANOVA followed by the Student-Newman-Keuls test. A

value of p < 0.05 was considered significant.

violet (Fig. 1D) andMTT tests (not shown). Microscopic

examination revealed a dramatic change in the mor-

phology of metformin-treated glioma cells, which be-

came larger and adopted a spindle-like shape with

markedly elongated fine tapering processes (Fig. 1E).

These morphological changes, however, were not asso-

ciated with glioma cell differentiation toward mature

astrocytes, as expression of the astrocyte marker GFAP

in C6 cells actually decreased after metformin treatment

to 61 5.9% of the value observed in untreated cells

( n =3, p < 0.05). Flow cytometric analysis showed that

metformin-treated glioma cells display higher FSC

values in comparison with control cells (Fig. 1F), thus

confirming the observed increase in cell size. Finally, the

proportion of cells in the G 0 /G 1 cell cycle phase was

significantly increased in metformin-treated glioma

cultures, while apoptotic cells with fragmented DNA

(sub-G 0 /G 1 ) could not be observed (Fig. 1G). Therefore,

the antiglioma effect of metformin is mainly a conse-

quence of cell cycle arrest. In primary astrocytes,

however, neither cell cycle alterations nor associated

morphological changes were discernible (data not

shown).

Metformin induces caspase-dependent apoptosis in

confluent glioma cells. We next investigated whether

metformin could affect confluent glioma cells, expecting

a weaker effect due to the reduced proliferation rate of

confluent cells. Surprisingly, the number of initially

confluent glioma cells after 48 h of incubation with

metformin (4 mM) was reduced to < 30% of control

values, which could not be attributed solely to a

proliferation block. Metformin-treated C6 cells lost

their polygonal morphology and became smaller, with

granular appearance and poorly defined margins

(Fig. 2A). Flow cytometric analysis revealed that met-

formin caused a large increase in numbers of apoptotic

(annexin + PI – ) glioma cells, while an increase in numbers

of annexin + PI + cells, which presumably underwent

secondary necrosis, was less pronounced (Fig. 2A).

Primary astrocytes, on the other hand, completely

preserved their normal morphology in the presence of

metformin and were largely resistant to its proapoptotic

action (Fig. 2B). Accordingly, a significant time-depend-

ent activation of caspases, apoptosis-executing cysteine

proteases with aspartate specificity, was observed in

metformin-treated C6 cells (Fig. 2C), while only a

marginal caspase induction was seen in primary astro-

cytes under the same cultivating conditions (Fig. 2D).

The increases in apoptotic cell number, caspase activa-

tion and DNA fragmentation were all efficiently pre-

vented by the pan-caspase inhibitor zVAD-fmk and the

protein synthesis inhibitor cycloheximide (CHX)

(Fig. 2E), thus confirming that metformin toxicity to

confluent glioma cells was due to induction of caspase-

Results

Metformin blocks proliferation of low-density glioma

cells. To assess the effect of metformin on glioma cell

proliferation, C6 and U251 glioma cells were seeded at

low density and their numbers determined by crystal

violet assay during 4-day incubation period. Metformin

inhibited the increase in glioma cell number in a dose-

dependent manner (Fig. 1A, C), and the highest con-

centration of the drug (8 mM) completely blocked the

proliferation of glioma cells. Similar results were ob-

tained with the MTTassay for mitochondrial respiration

(Fig. 1B). However, the values of mitochondrial respi-

ration per cell (expressed asMTT/crystal violet ratio) did

not significantly change in metformin-treated glioma

cultures (Fig. 1B), indicating that the mitochondrial

function was not affected. Therefore, the observed

reduction in cell number was due to an antiproliferative

rather than a cytotoxic effect of the drug. This was

consistent with the complete absence of apoptotic

(annexin + ) cells in glioma cell cultures exposed to

metformin (data not shown). The antiproliferative effect

was apparently reversible, as glioma cells incubated with

metformin (8 mM) for 48 h regained their proliferative

capacity after removal of the drug (data not shown). In

contrast to their transformed counterparts, rat primary

astrocytes were completely resistant to the antiprolifer-

ative action of metformin, as demonstrated by the crystal

Cell. Mol. Life Sci. Vol. 64, 2007

Research Article

1293

Figure 1. Metformin blocks proliferation of low-density

glioma cells. (A–D) Low-density C6 cells (A, B), U251

cells (C) or primary astrocytes (D) were incubated with

metformin, and cell number (crystal violet, CV) or

mitochondrial activity (MTT) was measured at the

indicated time points. A representative of three experi-

ments is presented, and the data are mean values of

triplicate observations (SD < 15% of the mean; * p < 0.05

refers to untreated cells). (E–G) After 96 h of treatment

with metformin (4 mM), the morphology of C6 cells was

examined by light microscopy (E), and cell size (F) and

cell cycle phase (G) were analyzed using flow cytometry.

Representative photographs and histograms are present-

ed. The numbers in (G) are mean SD values from three

independent experiments (* p < 0.05).

mediated, protein synthesis-dependent apoptosis. Re-

sults similar to those presented in Fig. 2A, C and E were

also obtained with U251 glioma cells (not shown).

decrease in the amount of red aggregates (Fig. 3A), a

pattern that is characteristic for disruption of mitochon-

drial membrane potential. This was accompanied by

significant generation of ROS, as concluded from a

green fluorescence shift that was readily observable in

metformin-treated C6 cells stained with the redox-

sensitive dye DHR (Fig. 3B). Results similar to those

presentedinFig.3AandBwerealsoobtainedwithU251

cells (data not shown). Cyclosporin A (CsA), an

inhibitor the mitochondrial permeability transition

(MPT) that precedes mitochondrial depolarization

[21] , and a well-known antioxidant agent, N-acetylcys-

teine (NAC) [22] , both prevented caspase activation,

DNA fragmentation and apoptotic cell death in C6

cultures (Fig. 3C). A time course analysis revealed that

Metformin-induced apoptosis is associated with mito-

chondrial depolarization and oxidative stress. To f u r t he r

elucidate the mechanisms underlying metformin-in-

duced apoptosis in confluent glioma cells, we assessed

if metformin could trigger mitochondrial depolarization

and production of ROS, two events that are frequently

associated with initiation of the apoptotic cascade.

Staining of C6 cells with the mitochondria-binding

fluorescent dye DePsipher revealed that metformin

treatment for 24 h caused an increase in the concen-

tration of the green monomer form with a concomitant

1294 A. Isakovic et al.

Antiglioma action of metformin

Figure 2. Metformin induces caspase-dependent

apoptosis in confluent glioma cells. (A–D) Confluent

C6 cells (A, C) or confluent primary astrocytes (B, D)

were incubated with metformin (4 mM). (E) Con-

fluent C6 cells were incubated with metformin

(4 mM) in the absence or presence of the pan-

caspase inhibitor zVAD-fmk (50 mM) or protein

synthesis blocker cycloheximide (CHX; 5 mg/mL).

Cell morphology (A, B), apoptosis (A, B, E; the

upper left quadrant in A, B contained < 5% cells)

and DNA fragmentation (E) were analyzed after

48 h using light microscopy or flow cytometry.

Caspase activation (C–E) was assessed at the indi-

cated time points (C) or after 36 h (D, E) by flow

cytometry. Representative photographs, dot plots

and histograms are presented. The data in (E) are

mean values SD from three separate experiments

(* p < 0.05 refers to both control and zVAD-fmk/

CHX-treated cells).

metformin-induced mitochondrial depolarization pre-

ceded ROS production by several hours (Fig. 3D).

Moreover, mitochondrial depolarization triggered by

metformin was actually associated with a decrease in

intracellular ROS levels (Fig. 3D), thus excluding the

possibility that disruption of mitochondrial membrane

potential was caused by oxidative stress. This was further

confirmed by the inability of either NAC or CHX to

prevent mitochondrial depolarization in glioma cells

(Fig. 3E), even though they efficiently reduced metfor-

min-induced ROS generation (Fig. 3F). On the other

hand, CsA readily hyperpolarized mitochondrial mem-

branes in metformin-treated C6 cells (Fig. 3E) but failed

to prevent production of ROS (Fig. 3F). Moreover, CsA

itself markedly increased ROS generation in C6 cells

even in the absence of metformin (Fig. 3F). These data

indicate that mitochondrial depolarization and oxidative

stress both participate in metformin-triggered apoptosis

of glioma cells but are apparently induced by partly

independent mechanisms. In accordance with the ob-

served resistance to metformin-induced apoptosis

(Fig. 2B, D), primary astrocytes did not display signifi-

cant mitochondrial depolarization or increased ROS

production following metformin treatment (not shown).

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika: