metformin amplifies chemotherapy-induced ampk …...cancer therapy: preclinical metformin amplifies...

Cancer Therapy: Preclinical

Metformin Amplifies Chemotherapy-Induced AMPK Activation andAntitumoral Growth

Guilherme Z. Rocha1, Mar��lia M. Dias1, Eduardo R. Ropelle1, Felipe Os�orio-Costa1, Franco A. Rossato2,Anibal E. Vercesi2, Mario J.A. Saad1, and Jos�e B.C. Carvalheira1

AbstractPurpose: Metformin is a widely used antidiabetic drug whose anticancer effects, mediated by the

activation of AMP-activated protein kinase (AMPK) and reduction of mTOR signaling, have become

noteworthy. Chemotherapy produces genotoxic stress and induces p53 activity, which can cross-talk with

AMPK/mTOR pathway. Herein, we investigate whether the combination of metformin and paclitaxel has

an effect in cancer cell lines.

Experimental Design: Human tumors were xenografted into severe combined immunodeficient

(SCID)mice and the cancer cell lines were treated with only paclitaxel or onlymetformin, or a combination

of both drugs. Western blotting, flow cytometry, and immunohistochemistry were then used to char-

acterize the effects of the different treatments.

Results: The results presented herein show that the addition of metformin to paclitaxel leads to

quantitative potentialization of molecular signaling through AMPK and a subsequent potent inhibition of

the mTOR signaling pathway. Treatment with metformin and paclitaxel resulted in an increase in the

number of cells arrested in the G2–M phase of the cell cycle, and decreased the tumor growth and increased

apoptosis in tumor-bearing mice, when compared with individual drug treatments.

Conclusion: We have provided evidence for a convergence of metformin and paclitaxel induced

signaling at the level of AMPK. This mechanism shows how different drugs may cooperate to augment

antigrowth signals, and suggests that target activation of AMPK by metformin may be a compelling ally in

cancer treatment. Clin Cancer Res; 17(12); 3993–4005. �2011 AACR.

Metformin is an oral hypoglycemiant agent used as first-line therapy for type 2 diabetes, which is now prescribed toalmost 120 million people in the world. There are a largenumber of epidemiologic studies indicating that diabeticshave an increased risk of cancer and cancer mortality (1, 2).Increasing evidence also supports a decreased risk of cancermortality associated with metformin use in patients withtype 2 diabetes (3–6). Furthermore, metformin has beenshown to inhibit the growth of cancer cells in vitro and invivo (7–12) and, while there are still no randomized controltrials of metformin as a therapy for cancer, there is intri-guing evidence that metformin may enhance chemother-apy for established tumors (13, 14).Metformin has been found to activate AMP-activated

protein kinase (AMPK) signaling (15), and this has become

an important focus of interest in carcinogenesis, becauseAMPK has been implicated in the regulation of mTORactivity, which is frequently activated in cancer (16–20).AMPK is the downstream component of the tumor sup-pressor, LKB1, which acts as a sensor of cellular energycharge, being activated by increasing AMP, coupled withfalling ATP (21). The AMP/LKB1-dependent activation ofAMPK results from pathologic stresses such as heat shock,hypoxia, glucose deprivation, and metformin administra-tion (15, 21). AMPK is also activated through Caþ2/calmo-dulin (CaM)-dependent protein kinase kinase (CaMKK),which in contrast to that mediated by AMP/LKB1, ismediated by calcium increases and functions indepen-dently of AMP (22, 23). Once activated, AMPK phosphor-ylates acetyl-CoA carboxylase (ACC) and switches onenergy-producing pathways at the expense of energy-depleting processes (24).

Another direct consequence of AMPK activation is theinhibition of the mTOR kinase signaling pathway. mTORcatalytic activity is halted by AMPK activation of the TSC1–TSC2 complex, which inactivates the Rheb GTPase (25,26). In addition, mTOR activity is positively regulated bygrowth factors and nutrients (amino acids). PI3K/Aktsignaling regulates mTOR through phosphorylation/inac-tivation of mTOR’s negative regulator, TSC2 (17, 27).mTOR activation results in the phosphorylation of the

Authors' Affiliations: Departments of 1Internal Medicine and 2ClinicalPathology, FCM, Universidade Estadual de Campinas (UNICAMP), Cam-pinas, SP, Brazil

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Jos�e B.C. Carvalheira, Departament of InternalMedicine, FCM–UNICAMP, Cidade Universit�aria Zeferino Vaz, Campinas,SP, Brazil, 13083-970. Phone: 55-19-35218950; Fax: 55-19-35218950;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-2243

�2011 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 3993

Cancer Research. on October 26, 2020. © 2011 American Association forclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 4, 2011; DOI: 10.1158/1078-0432.CCR-10-2243

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serine/threonine kinase p70S6K and the translationalrepressor eukaryotic initiation factor (eIF) 4E bindingprotein (4E-BP1), which have an essential role in regulatingcell growth and proliferation by controlling mRNA transla-tion and ribosome biogenesis (28).

To achieve normal cell growth and proliferation, it iscritical for cells to have robust antigrowth signaling sys-tems. AMPK has a major role as an antigrowth signal,because it is activated by p53, a sensor of DNA damagestress (29). Recently, the genotoxic stress effect was furtherevaluated and it has been suggested that the inhibition ofmTOR activity occurs through the p53-dependent upregu-lation of sestrins (SESN1 and SESN2) and consequentactivation of AMPK (30). These observations indicate thatmetformin acts synergistically with chemotherapeuticdrugs that increase genotoxic stress through a convergentsignaling of metformin-mediated LKB-1/AMPK activationand chemotherapeutic drug activation of SESNs, culminat-ing in an increased AMPK activation andmTOR inhibition.Thus, this study was designed to investigate whether met-formin potentiates paclitaxel antitumor effects, a well-known chemotherapeutic drug frequently used in breastand lung cancer patients (31, 32), and to observe whetherthese drugs share common intracellular signal transductionpathways and to determine whether these signaling sys-tems modulate each other’s actions in different cancer celllineages and in xenografted tumor cells in mice.

Materials and Methods

Antibodies, chemicals, and buffersAll the reagents were from Sigma-Aldrich unless other-

wise specified. Paclitaxel was obtained from Laborat�orioQu��mico Farmaceutico Bergamo Ltda. Anti–phospho-mTOR, anti-mTOR, anti–phospho-p70S6K, anti-p70S6K,

anti–phospho-4E-BP1, anti–4E-BP1, anti–phospho-AMPKa, anti-AMPKa, anti–b-actin, anti–acetyl-lys379-p53, anti–phospho-p53, anti–phospho-ACC, anti–caspase3, anti–cleaved caspase 3, anti-p27, and anti–phospho-Rbantibodies for immunoblotting were from Cell SignalingTechnology; anti-p53 and anti-SESN2 antibodies forimmunoblotting were from Santa Cruz Biotechnology;and anti-SESN1 and anti-SESN3 antibodies for immuno-blotting were from Abcam.

Cell cultureThe human breast cancer cell lineMCF-7 (LKB1-positive)

and human lung cancer cell line A549 (LKB1-negative)were obtained from ATCC. MCF-7 and A549 cells werecultured in Dulbecco’s Modified Eagle’s Medium contain-ing 10% FBS with the addition of antibiotics or fungicides.Both cell lines were maintained at 37�C in a humid atmo-sphere and 5% CO2.

TransfectionA total of 3 � 105 cells were seeded in a tissue culture

plate in complete growth medium and incubated over-night. On the day of transfection, 200 pmol of siRNA wasdiluted into OPTI-MEM (Life Technologies) and mixedwith 10 mL of Lipofectamine 2000 (Life Technologies)according to supplier’s protocol. The transfection mediumwas then replaced by complete medium and after 24 hourscells were treated with metformin (10 mmol/L) and pacli-taxel (1 mmol/L) and incubated for an additional 6 hours.siRNA for AMPK was 50-AAUUACUUCUGGUGCAG-CAUAGCGG-30 forward and 50-CCGCUAUGCUGCACCA-GAAGUAAUU-30 reverse; for SESN1, 50-GAACCUUCUCA-GAGUGCUUGAACUG-30 forward and 50-CAGUUCAAG-CACUCUGAGAAGGUUC-30 reverse; and for SESN2,50- GGAUAGCGAGUAGCCAUGGUCUUCC-30 forwardand 50-GGAAGACCAGGGCUACUCGCUAUCC-30 reverse.

Cell viability assayCells were seeded at a density of 2� 104 cells/well in 24-

well plates containing 1 mL of complete medium in tri-plicate. Cells were allowed to attach overnight beforetreating with the indicated dose of metformin and pacli-taxel for 24 hours. Subsequently, viable cells were countedby using trypan blue staining or they were treated with 0.3mg/mL of MTT for 4 hours and MTT-formazan conversionwas analyzed by spectrophotometry at 570 nm after culturemedium was removed and ethanol was added.

Cell-cycle analysisCells were trypsinized, washed in PBS, centrifuged, and

pellets were fixed in 200 mL of 70% ethanol and stored at�20�C until use. Cells were centrifuged and pellets resus-pended in 200 mL of PBS, and 10 mg/mL of RNAse A wasincubated for 1 hour at 37�C. Subsequently, cells wereresuspended in propidium iodide solution (0.1% sodiumcitrate,0.1%TritonX-100,and50mg/mLpropidiumiodide).Cell-cycle analysiswas carried out by flow cytometry (FACS-calibur). Data were analyzed by ModFit LT software.

Translational Relevance

Targeted therapies are being increasingly investigatedas new treatment options in oncology. Metformin is awidely used antidiabetic drug whose anticancer effectsrepresent a promising and novel approach for the treat-ment of cancer. Chemotherapy produces genotoxicstress and induces p53 activity, which can cross-talkwith AMPK/mTOR pathway through sestrins. We testedthe hypothesis that the combination of metformin andpaclitaxel could show a synergistic effect in cancer celllines. The findings presented here suggest that com-bined treatment is more effective in arresting cells inthe G2–M phase of the cell cycle, decreasing tumorgrowth and increasing apoptosis in tumor-bearing micethrough a signaling convergence of metformin andpaclitaxel at the level of AMP-activated protein kinase(AMPK). Our findings, therefore, show that differentdrugs may cooperate to increase antigrowth signals, andsuggests that target activation of AMPK may be analternative therapeutic strategy in cancer treatment.

Rocha et al.

Clin Cancer Res; 17(12) June 15, 2011 Clinical Cancer Research3994




Complex I oxygen consumptionMeasurement of oxygen consumption by MCF-7 and

A549 after treatment for 24 hours with metformin (10mmol/L), paclitaxel (1 m/mol/L), or the combination ofboth drugs was carried out by a Oxygraph equipped with aClark-type electrode (Hansatech Instruments Limited) in aclosed chamber equipped with magnetic stirrer and tem-perature control at 37�C. Approximately 2.5� 106 of viableMCF-7 cells/mL, and 4 � 106 of viable A549 cells/mL,permeable with 10 mmol/L of digitonin, were added in2 mL of reaction medium containing 125 mmol/L sucrose,65 mmol/L KCl, 10 mmol/L HEPES, 2.0 mmol/L K2HPO4,1.0 mmol/L MgCl2 (pH 7.2), 50 mmol/L EGTA, and com-plex I substrates (2.0 mmol/L malate, 1.0 mmol/L a-keto-glutarate, 1.0 mmol/L pyruvate, and 1.0 mmol/Lglutamate). Analyses of oxidative phosphorylation andrespiratory activity of mitochondria were made by sequen-tial additions of 100 mmol/L ADP, 2 mg/mL chloramphe-nicol acetyltransferase, 100 nmol/L carbonylcyanidep-trifluoromethoxyphenylhydrazone, 5 mmol/L succinate,0.5 mmol/L antimycin, and 200 mmol/L N,N,N0,N0-tetra-methyl-p-phenylenediamine/ascorbate. The data werereproduced and calculated by the device’s specific software.

Human tumor xenograft modelsFour-week-old male severe combined immunodeficient

(SCID) mice were provided by the State University of Cam-pinas—Central Breeding Center. Animals were inoculated inthe dorsal region, subcutaneously, with 1 � 106 A549 cells.The mice had ad libitum access to food and water. Oncetumors became palpable, tumor volume (V) was calculateddaily by measuring the length (L) and width (W) of thetumorwith calipers byusing the formula:V¼ [W� L� (WþL)/2)] � 0.52. Each group contained 15 animals.Treatments began when tumors reached 50 to 100 mm3.

Metformin was given daily by gavage at 500 mg/kg bodyweight. Paclitaxel was given once a week by intraperitoneal(i.p.) injection of 10 mg/kg body weight. All experimentswere approved by the Ethics Committee of the State Uni-versity of Campinas.

Tissue extractsMice were anesthetized with sodium amobarbital (15

mg/kg body weight, i.p.). Tumors were removed, mincedcoarsely and homogenized in extraction buffer [1% Triton-X 100, 100 mmol/L Tris (pH 7.4), containing 100 mmol/Lsodium pyrophosphate, 100 mmol/L sodium fluoride, 10mmol/L EDTA, 10 mmol/L sodium vanadate, 2 mmol/LPMSF and 0.1 mg of aprotinin/mL]. The extracts werecentrifuged at 11,000 rpm and 4�C and the supernatantsof these tissues were used.

Protein analysis by immunoblottingWhole tissue extracts and cell pellets were homogenized

in extraction buffer, treated with Laemmli sample buffercontaining 100 mmol/L DTT and heated in a boiling waterbath. For total extracts, similar-sized aliquots (50 mg pro-tein) were subjected to SDS-PAGE. Proteins were resolved

on 8% to 15% SDS gels and blotted onto nitrocellulosemembranes (Bio-Rad). Band intensities were quantified byoptical densitometry of developed autoradiographs byusing Scion Image software (Scion Corporation).

ImmunohistochemistryTo detect Ki-67 and cleaved caspase 3, microwave post-

fixation was carried out by a domestic oven which wasdelivered to slides immersed in 0.01 mol/L citrate buffer,pH 6.0, in two 7-minute doses separated by a 2-minutebreak. Sections were then incubated at 4�C overnight withprimarymonoclonalmouse anti-human Ki-67 cloneMIB-1fromDako (diluted 1:100) and anti-cleaved caspase 3 fromCell Signaling Technology. The slides were then incubatedwith avidin–biotin complex LSABþ Kit from Dako Cyto-mation for 30 minutes followed by the addition of diami-nobenzidine tetrahydrochloride as a substrate-chromogensolution. After hematoxylin counterstaining and dehydra-tion, the slides were mounted in Entellan from Merck.

Terminal deoxynucleotidyl transferase–mediateddUTP nick end labeling assay

Terminal deoxynucleotidyl transferase–mediated dUTPnick end labeling (TUNEL) staining was done by a com-mercial apoptosis detection kit (Roche), according to therecommendations of the manufacturer. Analysis and doc-umentation of results were carried out by a Leica FW4500 Bmicroscope.

Statistical analysisData are presented as mean � SEM of at least 3 inde-

pendent experiments. All groups were studied in paralleland differences between groups were analyzed by ANOVA,as appropriate, and Bonferroni post hoc tests for multipleunpairwise comparisons of means. The level of significanceadopted was P < 0.05.

Results

Metformin activates AMPK and inhibits mTOR inMCF-7 breast cancer cells and A549 lung cancer cells

To examine the effect of metformin on cancer cellgrowth, MCF-7 breast cancer and A549 lung cancer celllines were treated with various concentrations of metfor-min (1–50 mmol/L) for different periods of time (0–8hours). Metformin treatment resulted in the activation ofAMPK, with increased phosphorylation of AMPKa at Thr-172 in a time- and dose-dependent manner. Activation ofAMPK is associated with decreased activation of mTOR andp70S6K, a critical translational pathway for protein synth-esis (10). Metformin treatment resulted in attenuatedactivation of mTOR, as shown by the decreased phosphor-ylation ofmTOR, p70S6K, and 4E-BP1, in a time- and dose-dependent manner in treated cancer cells, compared withuntreated cells (Fig. 1A–D).

We also observed the effect of 2-deoxy-D-glucose (2-DG),another AMPK activator, in both cell lines at various con-centrations, and for different periods of time. As observed

Metformin Sensitizes Cancer Cells to Paclitaxel

www.aacrjournals.org Clin Cancer Res; 17(12) June 15, 2011 3995




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Figure 1. Metformin and paclitaxel activate AMPK and inhibit mTOR in MCF-7 and A549 cells. MCF-7 cells were treated (A) with 10 mmol/L metforminfor the indicated time and (B) for 6 hours with the indicated doses. The lysates were immunoblotted (IB) with the indicated antibodies. A549 cells weretreated (C) with 10 mmol/L metformin for the indicated time and (D) for 6 hours with the indicated doses. The lysates were immunoblotted with the indicatedantibodies. MCF-7 cells were treated (E) with 1 mmol/L paclitaxel for the indicated time and (F) for 6 hours with the indicated doses. The lysates wereimmunoblotted with the indicated antibodies. A549 cells were treated (G) with 1 mmol/L paclitaxel for the indicated time and (H) for 6 hours with the indicateddoses. The lysates were immunoblotted with the indicated antibodies. Data are representative of at least 3 experiments.

Rocha et al.





for the metformin treatment, 2-DG led to activation ofAMPK and inactivation of the mTOR signaling pathway(Supplementary Fig. S1A–D).A549 lung cancer cell line is negative for LKB1 and recent

reports have shown that LKB1 deficiency in hepatocytesimpairs metformin action (33). However, in this study weshow that AMPK activation in A549 cells after metformintreatment is independent of LKB1 and is dose-responsive,starting from 1 to 100 mmol/L with ACC phosphorylationand mTOR and p70S6K inactivation (SupplementaryFig. S2). The same pattern of AMPK and ACC activationand mTOR and p70S6K inactivation is seen on MCF-7 cellline, which is LKB1 normal (Supplementary Fig. S2).

Paclitaxel activates AMPK and inhibits mTOR inMCF-7 breast cancer cells and A549 lung cancer cellsTo investigate the mechanisms underlying the antipro-

liferative effects of paclitaxel, we characterized the effects ofpaclitaxel on AMPK and the mTOR pathway. As recentlyreported, genotoxic stress increases the amount of SESNs,and this effect leads to AMPK activation (30). Our resultsshow that paclitaxel treatment increased the acetylation ofp53 at Lys-379, the phosphorylation of p53 at Ser 15,which are known markers of genotoxic stress (34, 35),and the amount of SESN1, SESN2, and SESN3 in a time-and dose-dependent manner in both cell lines. Paclitaxeltreatment also resulted in increased phosphorylation ofAMPKa at Thr-172, in a time- and dose-dependentmanner.The increased activation of AMPK led to inactivation ofmTOR as evidenced by diminished phosphorylation ofmTOR, p70S6K, and 4E-BP1 also in a time- and dose-dependent manner (Fig. 1E–H).

Effect of combined treatment of AMPK activatorsand paclitaxel on cancer cell linesWe next sought to determine the effects of the combined

treatment of AMPK activators with paclitaxel. In MCF-7cells, as shown in Figure 2A, paclitaxel treatment led to ahigher increase in acetyl-Lys 379 p53 than metformin or 2-DG treatment, as well as an increase in the amount ofSESN2 (Fig. 2B). This increase in SESN2 in paclitaxel-treated cells was followed by an increase in the phosphor-ylation of Thr-172 of AMPK (Fig. 2C) and inhibition ofmTOR (Fig. 2D), p70S6K (Fig. 2E), and 4E-BP1 (Fig. 2F),when compared withmetformin or 2-DG treatments alone.Even though metformin or 2-DG treatment do not increaseSESN2 (Fig. 2B), we observe an increase in AMPK phos-phorylation (Fig. 2C) and inhibition of mTOR (Fig. 2D),p70S6K (Fig. 2E), and 4E-BP1 (Fig. 2F), when comparedwith vehicle-treated cells. In A549 cells, we also observedan increase in acetyl-Lys379 p-53 and in the amount ofSESN2 only in the paclitaxel-treated cells (Fig. 2G and H),when compared with control and metformin and 2-DGalone, and this was correlated with an increase in the Thr-172 phosphorylation of AMPK and decrease in the activa-tion of mTOR, p70S6K, and 4E-BP1 when compared withcontrol. Once again, metformin and 2-DG treatments didnot increase SESN2 (Fig. 2H) but they were able to increase

AMPK phosphorylation (Fig. 2I) and inhibit mTOR(Fig. 2J), p70S6K (Fig. 2K), and 4E-BP1 (Fig. 2L), whencompared with vehicle-treated cells.

Metformin and paclitaxel inhibit cell viabilityTo examine the effects of metformin and paclitaxel on

cancer cell growth, we treated MCF-7 and A549 cell lineswith metformin or paclitaxel alone or in combinationand cell viability was determined. As shown in Figure 3Aand B, both metformin and paclitaxel inhibited cellviability, as related to vehicle-treated cells. The metformintreatment was statistically significant at 10 mmol/L forthe 48- and 72-hour treatment (Fig. 3A) in MCF-7 cellsand that a 1 mmol/L dose of metformin was capable ofreducing A549 cells proliferation by approximately 20%to 30% for both the 48- and 72-hour treatments. Thepaclitaxel treatment was effective only at the dose of 10nmol/L for both 48- and 72-hour treatments (Fig. 3B).Figure 3C and D shows that in the combined treatmentmetformin potentiates paclitaxel action on MCF-7(Fig. 3C) and A549 (Fig. 3D) cells, as a dose of1 nmol/L of paclitaxel is statistically different from themetformin and vehicle-treated cells for both the 48- and72-hour treatments. At 10 nmol/L of paclitaxel, metfor-min does not further potentiate paclitaxel treatment onMCF-7 cells. However, in A549 cells, we observed thatat the 48-hour treatment metformin (10 mmol/L) and10 nmol/L paclitaxel is more effective on reducing cellgrowth than paclitaxel-alone treatment (Fig. 3D). Wethen analyzed cell viability by trypan blue staining ofboth cell lines (Fig. 3E), which showed that both met-formin and paclitaxel inhibited cell viability, as related tovehicle-treated cells, and the combined treatment wasmore effective than either treatment alone.

Effect of metformin, 2-DG, and paclitaxel on cell cycleTo evaluate the mechanism of growth inhibition by

metformin, 2-DG, and paclitaxel, the cell-cycle profilewas analyzed by flow cytometry after treatment with met-formin, 2-DG, or paclitaxel alone, or the combination of thedrugs. Vehicle treatment presented the majority of cells intheG1 phase of the cell cycle (MCF-7, 68.8%; A549, 71.2%),a small part in theG2–Mphase (MCF-7, 12.4%;A549 8.6%)and the rest of the cells were found to be in the S-phase(MCF-7 18.8%, A549, 20.2%). 2-DG treatment resulted in aslight increase in cells in G1 phase arrest (MCF-7, 72.1%;A549, 77.4%), with a decrease in S-phase (MCF-7, 15.3%;A549, 15.4%) and no significant alteration in G2–M phase(MCF-7, 12.6%; A549, 7.2%). Metformin treatmentresulted in an increase in the number of cells in the G1

phase (MCF-7, 80.6%; A549, 81.2%) with almost similarnumber of cells in the G2–M phase of MCF-7 cells (MCF-7,9.5%; A549, 8.7%) and a reduction in the number of cells inthe S-phase in both cell lines (MCF-7, 9.9%; A549, 10.2%).Metformin combined with 2-DG (MET þ 2-DG) treatmentresulted in a decrease in the number of cell in G1 phase(MCF-7, 53.5%; A549, 52.1%), and increase in cells in theG2–M phase arrest (MCF-7, 33%; A549, 35.3%) and a






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Figure 2. Effect of combined treatment with 2-DG, metformin, and paclitaxel on MCF-7 and A549 cells. Cells were treated with 10 mmol/L metformin,10 mmol/L 2-DG, and 1 mmol/L paclitaxel for 6 hours, and cells were prepared as described in Materials and Methods. The lysates of MCF-7 cells wereimmunoblotted (IB) with (A) acetyl-Lys-379 p53 and p53, (B) SESN2 and b-actin, (C) pAMPKa and AMPKa, (D) pmTOR and mTOR, (E) pp70S6K andp70S6K, and (F) p4E-BP1 and 4E-BP1. The lysates of A549 cells were immunoblotted (IB) with (G) Acetyl-Lys-379 p53 and p53, (H) SESN2 and b-actin,(I) pAMPKa and AMPKa, (J) pmTOR and mTOR, (K) pp70S6K and p70S6K, and (L) p4E-BP1 and 4E-BP1. Data (mean � SEM; n ¼ 3 experiments in triplicate)are presented as relative to control. *, P � 0.05 vs. control; #, P � 0.05 vs. paclitaxel; z, P � 0.05 vs. metformin.

Rocha et al.





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Figure 3. Metformin (MET) and paclitaxel (PTX) inhibit cell viability. A, cell viability of MCF-7 and A549 cells treated with increasing doses of metforminfor 48 or 72 hours measured by MTT assay. B, cell viability of MCF-7 and A549 cells treated with increasing doses of paclitaxel for 48 or 72 hours measured byMTT assay. Data (mean � SEM; n ¼ 3 experiments in triplicate) are presented as relative to vehicle. *, P � 0.05 vs. vehicle. C, cell viability of MCF-7cells treated with increasing doses of paclitaxel or associated with metformin (10 mmol/L) for 48 or 72 hours measured by MTT assay. D, cell viability of A549cells treated with increasing doses of paclitaxel or associated with metformin (10 mmol/L) for 48 or 72 hours measured by MTT assay. Data(mean� SEM; n¼ 3 experiments in triplicate) are presented as relative to vehicle. *,P� 0.05 vs. vehicle; #,P� 0.05 vs. metformin. E, cell viability ofMCF-7 andA549 cells treated with metformin (10 mmol/L), paclitaxel (1 umol/L), or a combination, as measured by trypan blue staining. Cells were treated for48 hours with respective drugs. Data (mean � SEM; n ¼ 3 experiments in triplicate) are presented as relative to vehicle. *, P � 0.05 vs. vehicle; #, P �0.05 vs. metformin; z, P � 0.05 vs. paclitaxel. F, cell-cycle analysis of MCF-7 and A549 cells treated with metformin (10 mmol/L), paclitaxel (1 mmol/L), or acombination, as measured by flow cytometry (FACScalibur). Cells were treated for 24 hours with respective drugs. G, MCF-7 and A549 cells were treatedwith the indicated drugs for 24 hours and cell lysates were immunoblotted (IB) with the indicated antibodies. H, complex I oxygen consumption rates ofMCF-7 and A549 cells treated for 24 hours with metformin (10 mmol/L), paclitaxel (1 mmol/L), or a combination, as measured by a Oxygraph equipped withClark-type electrode. Data (mean � SEM; n ¼ 3 experiments) are presented as relative to vehicle. *, P � 0.05 vs. vehicle; z, P � 0.05 vs. paclitaxel.






decrease in cells in S-phase (MCF-7, 13.4%;A549, 12.6%)asrelated to vehicle-treated cells. Paclitaxel treatment, asexpected, caused an increase in the number of cells in theG2–M phase (MCF-7, 21.5%; A549, 18.8%) with a reduc-tion in the number of cells in the G1 phase (MCF-7, 64%;A549, 64.7%) and in the S-Phase (MCF-7, 14.5%; A549,16.5%) in both cell lines (Fig. 3F).

The combined treatment of 2-DG and paclitaxel, ofmetformin and paclitaxel and the triple therapy resultedin a synergistic effect of G2–M cell-cycle arrest. Cells treatedwith 2-DG and paclitaxel showed a reduction in G1 phase(MCF-7, 57.1%; A549, 60.4%), there was no significantalteration in the number of cells in S-phase (MCF-7, 15.1%;A549, 16.9%); however, there was an increase in G2–Mphase (MCF-7, 27.8%; A549, 22.7%) compared with eithertreatment alone. Cells treated with metformin and pacli-taxel showed a reduction in G1 phase arrest, compared witheither treatment alone (MCF-7, 54.6%; A549, 45.5%). Thistreatment resulted inno significant alteration in thenumberof cells in the S-phase comparedwith either treatment alone(MCF-7, 13.5%; A549, 12.8%). Additionally, when weanalyzed theG2–Mphase,weobserveda significant increasein the number of cells in this phase in the metformincombined with paclitaxel treatment, as compared witheither treatment alone (MCF-7, 31.9%; A549, 41.8%).Finally, the triple therapy resulted in a decrease in thenumber of cells in G1 phase as compared with MET þ 2-DG treatment or paclitaxel treatment alone (MCF-7, 40.9%;A549, 39.7%), a slight decrease in the number of cells in S-phase (MCF-7, 10.9%; A549, 12.1%) and a significantincrease in the number of cells in G2–M phase (MCF-7,48.1%; A549, 48.2%). Thus, Figure 3F shows an increase incell-cycle arrest in the G2–M phase, during the combinedtreatment of MET+2-DG, 2-DG and paclitaxel, metforminand paclitaxel, and the triple therapy and a decrease in theG1 phase, indicating that cells submitted to these combinedtreatments were not further undergoing division.

We then examined the protein levels of caspase 3 andcleaved caspase 3, of cyclin D1, of p27, and of phosho-Rbin the cells. After 24 hours, caspase 3 was slightly decreasedin the metformin and paclitaxel treatments and notablyreduced in both cell lines treated with the combination ofmetformin and paclitaxel (Fig. 3G). Cleaved caspase 3 wasslightly increased with metformin or paclitaxel treatments,and strongly increased in metformin and paclitaxel com-bined treatment after 24 hours in both cell lines (Fig. 3G).Cyclin D1 levels were only reduced inmetformin treatmentin both cell lines (Fig. 3G). p27 expression was increased inmetformin treatment whereas phosphorylation of Rb wasreduced in metformin treatment (Fig. 3G).

Cancer cell metabolism is unaffected by paclitaxel andhampered by metformin

To determine whether metformin, paclitaxel, or thecombined treatment affect cancer cell metabolism we ana-lyzed their effects on mitochondrial complex I oxygenconsumption in MCF-7 and A549 cell lines. Metformindecreased complex I oxygen consumption by 58% in MCF-

7 and by 92% in A549, whereas paclitaxel had a modesteffect in both cell lines (Fig. 3H). The combined treatmentshowed no significant difference from the metformin treat-ment alone (Fig. 3H).

AMPK is implicated in the synergistic effect ofmetformin and paclitaxel

Figure 4A and B shows that p53 is activated with pacli-taxel treatment and that further stimulation with metfor-minþ paclitaxel, 2-DGþ paclitaxel (double therapy), or acombination of metformin þ 2-DG þ paclitaxel (tripletherapy) does not increase its activation. The same patternis seen in SESN2 expression.On the contrary, AMPK showsa further increase in activation after double or tripletherapies compared with paclitaxel only and vehicle treat-ments. Additionally, mTOR and its direct substratesp70S6K and 4EBP-1 are inhibited with paclitaxel treat-ment and further inhibition is observedwith the double ortriple treatments.

To further evidence the role of AMPK and SESNs in thecombination treatment, we treated MCF-7 and A549 cellswith siRNA toAMPKand to SESN1andSESN2andanalyzedSESN1 and SESN2 expressions and AMPK, mTOR, p70S6K,and 4E-BP1 phosphorylation. Figure 4C and D show thattreatment with SESN1 and SESN2 siRNA dampers SESN1and SESN2 expressions, respectively, and reduces AMPKphosphorylation, with an increase in mTOR, p70S6K, and4E-BP1 phosphorylation. Treatment with AMPK siRNAdoesnot reduce SESN1or2 expressionsbut abolishesAMPKphosphorylation and expression, which increases mTOR,p70S6K, and 4E-BP1 phosphorylation.

Thus, these results clearly show an essential role for SESNand AMPK in the activation of AMPK and inhibition ofmTOR, respectively, after the combined treatment of met-formin and paclitaxel.

The effect of metformin and paclitaxel on A549 tumorgrowth in SCID mice

Xenografted SCID mice were treated with control vehi-cle, metformin, paclitaxel, or metformin and paclitaxel.Treatments began when the tumors presented an averagesize of 50 mm3 and tumor growth rate was measureddaily after the beginning of the treatment. Figure 5Ashows that metformin and paclitaxel is clearly moreeffective in reducing tumor growth, as compared witheither paclitaxel alone, metformin alone, or the control.For the entire experiment, the animals treated with com-bination of metformin and paclitaxel presented almostno tumor growth, with the final tumor volume of 71 mm3

being very close to the tumor volume at the beginning ofthe treatment, as compared with the final volumes of thecontrol (377 mm3), metformin (203 mm3), and pacli-taxel (137 mm3) as shown in Figure 5B. We observed that2-DG and paclitaxel combination yielded similar resultsto metformin and paclitaxel combination. However, wedid not observe an additive effect with the triple therapywhen compared with the double therapy (SupplementaryFig. S3A and B).

Rocha et al.





The reduced tumor growth following metformin andpaclitaxel treatment is due to the reduced proliferationof tumor cells, as shown by Ki-67 staining and quantifica-tion (Fig. 5C and D) and increased apoptosis, as quantifiedby TUNEL staining (Fig. 5E and F) and cleaved caspase 3staining (Fig. 5G and H). In the control group, Ki-67–positive cells were 25.5% (�2.8) of the total, in metforminthese cells were 13.4% (�1.1), whereas in paclitaxel thesecells were 10.8% (�1.2) and metformin and paclitaxelpresented 7.2% (�0.4) Ki-67–positive cells (Fig. 5C andD). The results of the TUNEL staining experiments showthat the control group presented a 9.5% (�1.1) apoptosis,whereasmetformin apoptosis was 16.7% (�4.5), paclitaxelapoptosis was 31.8% (�1.8), andmetformin and paclitaxelapoptosis was 35.9% (�4.8) (Fig. 5E and F). Similarly, inthe cleaved caspase 3 staining experiments, the controlgroup presented a 6.5% (�1.0) positive cells, whereasmetformin presented 22.3% (�0.8) positive cells, pacli-taxel presented 27.4% (�2.1) positive cells, andmetforminand paclitaxel combined presented 39.9% (�3.6) cellspositive for cleaved caspase 3 (Fig. 5G and H). These dataindicate a reduced proliferation and increased apoptosis inthe combined treatment and are consistent in showing thatthere is a significant advantage in the use of combinationtreatment with metformin and paclitaxel, as comparedwith treatment with either agent alone.

Effect of metformin, paclitaxel, and metformin andpaclitaxel treatment on AMPK and the mTOR pathwayin A549 xenografts

As treatment with metformin and paclitaxel inhibitedtumor growth, we sought to determine the AMPK/mTORpathway activation status in the tumor tissue of animalstreated with metformin, paclitaxel, and the combination ofmetformin and paclitaxel. Figure 6A shows that bothtreatments with paclitaxel resulted in a higher acetylationof Lysine 379 of p-53 and increased quantity of SESN2, ascompared with control or metformin (Fig. 6B). The phos-phorylation of AMPK at Thr172 was also higher in themetformin and paclitaxel treatment, when compared withpaclitaxel alone, metformin alone, or control (Fig. 6C).Both treatments with paclitaxel and metformin also acti-vated AMPK, as compared with the control. Similarly,phosphorylation of mTOR (Fig. 6D), and its direct sub-strates p70S6K (Fig. 6E) and 4E-BP1 (Fig. 6F), were reducedfollowing metformin and paclitaxel treatments.

Discussion

In this study, we show that the combination of metfor-min and paclitaxel has a major antitumor effect in vivo andinduces massive cell-cycle arrest in vitro. These effects arecorrelated with a potent activation of AMPK. Our results

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Figure 4. AMPK involvement in the synergistic effect of metformin (MET) and paclitaxel (PTX). A, MCF-7 cells were treated with paclitaxel alone (1 umol/L), withmetformin/paclitaxel (10 mmol/L/1 mmol/L), with 2-DG/paclitaxel (10 mmol/L/1 mmol/L), or the triple therapy for 6 hours. The cell lysates were thenimmunoblotted with the indicated antibodies. B, A549 cells were treated with paclitaxel alone (1 mmol/L), with metformin/paclitaxel (10 mmol/L/1 umol/L), with2-DG/paclitaxel (10 mmol/L/1 mmol/L), or the triple therapy for 6 hours. The cell lysates were then immunoblotted (IB) with the indicated antibodies. C, MCF-7cells were transfected with siRNA for AMPKa, for SESN1, or for SESN2, and than treated with metformin/paclitaxel (10 mmol/L/1 mmol/L) for 6 hours. The celllysates were then immunoblotted with the indicated antibodies. D, A549 cells were transfected with siRNA for AMPKa, for SESN1, or for SESN2, and thantreated with metformin/paclitaxel (10 mmol/L/1 mmol/L) for 6 hours. The cell lysates were then immunoblotted with the indicated antibodies.






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Figure 5. Metformin (MET) and paclitaxel (PTX) synergize in vivo to reduce A549 tumor growth. A549 cells (1.0 � 106) were injected subcutaneouslyinto the flank of SCID mice. Once the tumor reached 50 to 100 mm3, treatments were initiated, as indicated in Materials and Methods. Data are presentedas mean � SEM. A, tumor growth was measured daily after beginning treatment. B, tumor volume after 3 weeks of treatment. C, representativemicrophotograph of Ki-67 staining on tumor sections (arrows indicate positive Ki-67 staining). D, graph of percentage of Ki-67–positive cells per field;4 fields per tumor section; mean � SEM. E, representative microphotograph of TUNEL staining on tumor sections. F, graph of percentage of TUNEL-positivenuclei of cells per field; 4 fields per tumor section; mean � SEM. G, representative microphotograph of cleaved caspase 3 staining on tumor sections.H, graph of percentage of cleaved caspase 3–positive nuclei of cells per field; 4 fields per tumor section; mean � SEM. *, P � 0.05 vs. control; #, P � 0.05 vs.paclitaxel; z, P � 0.05 vs. metformin.

Rocha et al.





show that metformin, which induces a moderate decreasein ATP levels (36), is able to produce molecular activationof AMPK and inactivation of mTOR signaling in breast andlung cancer cells, whereas paclitaxel, through activation ofp53 and SESNs, yields similar effects to metformin. Com-bined treatment with metformin and paclitaxel leads to aquantitative increase in AMPK activation and a drasticreduction of molecular signaling through the mTOR path-way. Likewise, the combination of paclitaxel with 2-DG,which like metformin, leads to intracellular ATP depletion(37, 38), severely inhibited the mTOR signaling pathway.It was initially shown that metformin was capable of

reducing proliferation in different types of cancer including,prostate, colon, andbreast cancer cell lines. Subsequently, invivo experiments with metformin resulted in tumor growthinhibition of up to 55% (12, 39). In accordance with thesedata,we herein show thatmetformin treatment resulted in a

reduction of A549 and MCF-7 cell viability and a decreasedtumor volume (approximately 50%) of A549 tumor whenxenografted in SCIDmice. These effects were paralleled by adecrease in the central regulator of cell growth and survival,mTOR signaling pathway, as measured by p70S6K and4EBP-1 phosphorylation.

The mechanisms by which cells protect their geneticmaterial during genotoxic stress include the alert of check-point proteins and arrest of cell growth and proliferation(40, 41). The major cellular stress-sensing molecule is p53,which halts cell growth and proliferation by increasingSESNs, thus leading to activation of AMPK and inhibitionofmTOR(29, 30).Herewe show that paclitaxel induces p53activation in the cancer cells and activates AMPK. AMPKactivation resulted indecreasedmTORpathway activity; thiseffectmaybe related to the reductionof cellmetabolism thatis observedduringprolongedmitosis, inducedbypaclitaxel.

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Figure 6. Metformin (MET) and paclitaxel (PTX) activate AMPK and inhibit mTOR in vivo. Mice bearing A549 xenografts were treated with only metforminor paclitaxel, or a combination, as described in Materials and Methods. The A549 tumor lysates were immunoblotted (IB) with (A) acetyl-Lys-379 p53and p53, (B) SESN2 and b-actin, (C) pAMPKa and AMPKa, (D) pmTOR and mTOR, (E) pp70S6K and p70S6K, and (F) p4E-BP1 and 4E-BP1. Data (mean �SEM; n ¼ 8) are presented as relative to control. *, P � 0.05 vs. control; #, P � 0.05 vs. paclitaxel; z, P � 0.05 vs. metformin.






Intracellular interactions between different signalingsystems may function as mechanisms for enhancing orcounter-regulating signaling pathways. In the case ofmetformin, the cross-talk with paclitaxel-induced signal-ing pathways resulted in direct interactions between thesedrug-induced signaling systems at the level of AMPK.Simultaneous treatment with both drugs led to increasedphosphorylation of AMPK and a drastic reduction ofmTOR signaling pathway. Furthermore, there was noincrease of the effects of the combination of metforminand paclitaxel compared with only metformin on tumorcell metabolism. These results suggest that the positivecross-talk between metformin and paclitaxel-induced sig-naling was due to additive effects on AMPK activation.Further inhibition of mTOR pathway with the tripletherapy does not change the antineoplastic effect ofmetformin and paclitaxel combination.

The mTOR pathway is a crucial pathway, downstream ofseveral growth factor receptors including epidermal growthfactor, platelet-derived growth factor, KIT, and insulin likegrowth factor I receptor, which coordinate tumor growth(42, 43). The deregulated mTOR pathway is very frequentin human cancer. These alterations include mutationalactivation of the p110a subunit of phosphoinositide 3-kinase (PI3K), loss of PTEN function, overexpression ofPI3K, Akt, eIF4E, and p70S6K, as well as inactivation oftuberous sclerosis 1 or 2 (42, 43). It was also establishedthat the mTOR pathway can be inactivated by AMPK (44),which acts through a PI3K-independent mechanism.

The susceptibility of cancer cells to PI3K inhibitors ishighly determined by the presence of mutations in com-ponents of the PI3K/Akt/mTOR pathway (45). In contrast,our results showed that a drastic reduction of the mTORpathway, elicited by the combination of metformin andpaclitaxel, yields decreased cell viability in both MCF-7,which has a mutational activation of the PI3K catalyticsubunit, and A549 cells, which does not harbor geneticalterations in the PI3K/Akt/mTOR pathway. As the mTORpathway is essential to cell metabolism and growth, anddelayed mitosis induced by paclitaxel is associated with areduction in gene transcription, our data suggest that thecancer cells may be "pathway addicted," independently ofharboring a mutation in the PI3K/Akt/mTOR pathwayduring paclitaxel-induced cell-cycle arrest. It is interestingto note that the susceptibility of cancer cells to metforminand paclitaxel combination occurred in a LKB1 indepen-

dent manner. These data are in accordance with Sanli andcolleagues (46) that recently showed that metformin canactivate AMPK, probably through action of a metabolitederived from complex I inhibition. Thus, our data suggestthat metformin antineoplastic effects are effective evenwhen LKB1 is suppressed.

Toxicity elicited by paclitaxel has been linked to irrever-sible tubulin polymerization, a cell-cycle block at the meta-phase–anaphase transition, and cell death (47, 48). On thecontrary, in addition to the metabolic activity of AMPK,there is growing evidence that AMPKhas a crucial role in theestablishment of cell division, and it has been suggested thatAMPK may be essential in the coordination between thesensing of energy resources and genome division (49, 50).Our results show that the combination of metformin andpaclitaxel has an additive effect on cell viability and, inaccordancewith aprevious study that combined2 activatorsof AMPK, metformin and 2-DG, we observed a compellingaccumulation of cells in G2–M (36). As gene transcription issilenced during mitosis and paclitaxel plus metformininduced a more prolonged division, our results suggest thatthis event leads to a greater decrease in cell viability.

In conclusion, we observed a convergence of paclitaxel-and metformininduced signaling at the level of AMPK.This mechanism illustrates how different drugs may coop-erate to augment antigrowth signals, suggesting that targetactivation of AMPK bymetforminmay be a compelling allyin cancer treatment.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Nicola Conran for English language editing. We alsothank Luiz Janeri, J�osimo Pinheiro, and Gerson Ferraz for technicalassistance.

Grant Support

This study was supported by grants from Fundac~ao de Amparo �a Pesquisa doEstado de S~ao Paulo (FAPESP) and Conselho Nacional de desenvolvimentocient��fico e tecnol�ogico (CNPq).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 20, 2010; revised February 28, 2011; accepted April 20,2011; published OnlineFirst May 4, 2011.

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2011;17:3993-4005. Published OnlineFirst May 4, 2011.Clin Cancer Res Guilherme Z. Rocha, Marília M. Dias, Eduardo R. Ropelle, et al. Activation and Antitumoral GrowthMetformin Amplifies Chemotherapy-Induced AMPK

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