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5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro and in Vivo via AMP-activated Protein Kinase *

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 47, pp. 39582–39593, November 25, 2005 Printed in the U.S.A. 5-Aminoimidazole-4-carboxamide-1--D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro and in Vivo via AMP-activated Protein Kinase Received for publication, July 8, 2005, and in revised form, September 21, 2005 Published, JBC Papers in Press, September 21, 2005, DOI 10.1074/jbc.M507443200 ‡ ‡ § ‡1 Ramandeep Rattan , Shailendra Giri , Avtar K. Singh , and Inderjit Singh ‡ § From the Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425 and the Department of Pathology and Laboratory Medicine, Ralph Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425 5-Aminoimidazole-4-carboxamide-1--4-ribofuranoside (AICAR) phorylates and inactivates a number of metabolic enzymes involved in is widely used as an AMP-kinase activator, which regulates energy ATP-consuming pathways like fatty acid, cholesterol synthesis, and homeostasis and response to metabolic stress. Here, we investigated protein synthesis that include enzymes like acetyl-Co enzyme A carbox- the effect of AICAR, an AMPK activator, on proliferation of various ylase (ACC), fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA cancer cells and observed that proliferation of all the examined cell reductase, and mammalian target of rapamycin (mTOR) and activates lines was significantly inhibited by AICAR treatment due to arrest ATP-generating process like fatty acid oxidation and glucose uptake (8). in S-phase accompanied with increased expression of p21, p27, and The mechanisms of activating AMPK include direct allosteric binding p53 proteins and inhibition of PI3K-Akt pathway. Inhibition in in of AMP to the  subunits and phosphorylation, catalyzed by an vitro growth of cancer cells was mirrored in vivo with increased upstream AMP kinase (AMPKK), recently identified to be LKB1 expression of p21, p27, and p53 and attenuation of Akt phosphoryl- (STK11) (9–11). Recent studies have demonstrated that AMPK can also ation. Anti-proliferative effect of AICAR is mediated through acti- be activated by other stimuli that do not cause a detectable change in the vated AMP-activated protein kinase (AMPK) as iodotubericidin AMP/ATP ratio, like hyperosmotic stress and pharmacological agents and dominant-negative AMPK expression vector reversed the like thiazolidinediones, metformin, and 5-aminoimidazole-4-carbox- AICAR-mediated growth arrest. Moreover, constitutive active amide-1--D-ribofuranoside (AICAR) (11–14). AMPK arrested the cells in S-phase by inducing the expression of Activation of AMPK has been related with protection from injury and p21, p27, and p53 proteins and inhibiting Akt phosphorylation, sug- apoptosis caused by myocardial ischemia (15, 16) and apoptosis due to gesting the involvement of AMPK. AICAR inhibited proliferation in metabolic stress (17–19). In these scenarios, AMPK has been proposed both LKB and LKB knock-out mouse embryo fibroblasts to similar as an anti-apoptotic molecule. However, recent reports have indicated extent and arrested cells at S-phase when transfected with domi- anti-proliferative and pro-apoptotic action of activated AMPK using nant negative expression vector of LKB. Altogether, these results pharmacological agents or AMPK overexpression. AMPK activation indicate that AICAR can be utilized as a therapeutic drug to inhibit has been shown to induce apoptosis in human gastric cancer cells (20), cancer, and AMPK can be a potential target for treatment of various lung cancer cells (21), prostate cancer (22), pancreatic cells (23), and cancers independent of the functional tumor suppressor gene, LKB. hepatic carcinoma cells (24) and enhance oxidative stress induced apo- ptosis in mouse neuroblastoma cells (25), by various mechanisms that includes inhibition of fatty acid synthase pathway and induction of AMP-activated protein kinase (AMPK) is a highly conserved serine/ stress kinases and caspase 3. threonine protein kinase. It is a heterotrimer containing a catalytic () AMPK is an anti-growth molecule because of its relationship with and two regulatory subunits ( and ), each of which have at least two two tumor suppressor genes: LKB and TSC2 (tuberous sclerosis com- isoforms (1). AMPK is called the “fuel gauge” of the biological system, plex 2). LKB functions as an upstream kinase (AMPKK) that activates because it is activated under conditions that deplete cellular ATP and AMPK (26). LKB mutations result in Peutz-Jeghers syndrome, which elevate AMP levels, such as glucose deprivation, heat shock, hypoxia, results in predisposition to cancers of the colon, pancreas, breast, and and ischemia (2, 3), and also by hormones like leptin (4), adiponectin (5), other sites (27–29). Mutations of LKB1 typically occur in the catalytic catecholamine (6), and interleukin-6 (7). Upon activation, AMPK phos- domain, leading to loss of its kinase activity and presumably a failure to phosphorylate and activate AMPK (30). TSC2 forms a complex with TSC1 and inhibits mTOR, leading to inhibition in protein synthesis and * This work was supported by National Institutes of Health Grants NS-22576, NS-34741, NS-40810, NS-37766, and NS-40144. The costs of publication of this article were negative regulation of cell size and growth (31). Mutations of defrayed in part by the payment of page charges. This article must therefore be TSC1TSC2 causes tuberous sclerosis, which is associated with hamar- hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tomatous polyps in multiple tissues and an increased risk of cancers To whom correspondence should be addressed: Children Research Institute, Medical (32). University of South Carolina, 173 Ashley Ave., 5th Floor, Charleston, SC 29425. Tel.: 843-792-7542; Fax: 843-792-7130; E-mail: [email protected]. In the present study we have investigated the effect of AICAR on cell The abbreviations used are: AMPK, AMP-activated protein kinase; ACC, acetyl-CoA car- proliferation in vivo and in vitro in various cancer cell lines. AICAR is boxylase; mTOR, mammalian target of rapamycin; AMPKK, AMPK kinase; AICAR, converted to its triphosphorylated form ZMP, inside the cell, by an 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside; TSC2, tuberous sclerosis complex 2; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; adenosine kinase (14), which acts as an AMP analogue and activates TdR, thymidine ribotide; PBS, phosphate-buffered saline; PCNA, proliferating nuclear AMPK and its upstream kinase LKB without affecting the ATP:AMP antigen; GFP, green fluorescent protein; eGFP, enhanced GFP; AS, antisense; MS, mis- sense; PI3K, phosphatidylinositol 3-kinase; DN, dominant negarive; CA, constitutively ratio in the cell (14). AICAR-mediated AMPK activation was found to active; MEF, mouse embryo fibroblasts; JNK, c-Jun NH -terminal kinase; MTT, 3-(4,5- be a proficient inhibitor of cell proliferation and the mechanism of its dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ZMP, AICA riboside mono- phosphate metabolite. anti-proliferative effect may be mediated via inhibition of PI3K-Akt 39582 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 47 •NOVEMBER 25, 2005 This is an Open Access article under the CC BY license. AMPK as a Potential Target for Treatment of Cancer FIGURE 1. AICAR inhibits proliferation of cancer cells. Specified numbers of cells (C6, MCF-7, PC3, CEM, and K562) were plated, treated with AICAR (0.25–1 mM), followed by exposure to [ H]thymi- dine for 6 h and subsequent counts. The data is representation of three separate experiments done in triplicates. ***, p  0.001 compared with control; **, p  0.01 compared with control, NS, non-significant compared with control. pathway and increased expression of cell cycle inhibitory proteins p21, expression vector (D157A) was a kind gift from Dr. David Carling (MRC p27, and p53, thereby exhibiting potential as an anti-cancer drug. Clinical Sciences Centre, London, UK), and AMPK1 and 2 constitu- tive expression vectors were kind gifts from Dr. Jin-Zhong Zhang (Case MATERIALS AND METHODS Western Reserve University, Cleveland, OH) and Dr. Benoit Viollet Reagents and Cell Culture—DMEM/F-12, DMEM/4.5 g of glucose (Rene´ Descartes University, Paris, France), respectively. LKB wild type, medium, fetal bovine serum (FBS), and Hanks’ balanced salt solution LKB dominant negative (kinase dead), STRAD and MO25 expres- were obtained from Invitrogen as was RPMI 1640. AICAR was pur- sion vectors were kind gifts from Dr. Dario R. Alessi (Wellcome Trust chased from Toronto Research Chemicals (Ontario, Canada). Iodotu- Biocenter, University of Dundee, Dundee, UK). bericidin was obtained from Calbiochem. [ H]Thymidine ribotide Cell Culture—C6 glioma cells, T98G, U87MG, and PC-3 were main- ([ H]TdR) was purchased from PerkinElmer Life Sciences. Propium tained in DMEM/F-12 medium supplemented with 10% FBS and anti- iodide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide biotics. MCF-7 cells were maintained in DMEM/4.5 g of glucose with (MTT), and transfection reagent, FuGENE, were purchased from Roche 10% FBS. CEM and K-562 were maintained in RPMI 1640 supple- Applied Science. The enhanced chemiluminescence (ECL) detecting mented with 10% FBS. LKB knock-out and wild-type mouse embryo reagent was from Amersham Biosciences, and the luciferase assay sys- fibroblasts (MEFs) were a kind gift from Dr. Tomi P. Makela (Institute of tem was from Promega (Madison, WI). C glioma, T98G astrocytoma, Biomedicine and Helsinki University Central Hospital, Biomedicum U87MG astrocytoma, MCF-7 breast cancer, and PC-3 prostrate carci- Helsinki, University of Helsinki, Finland) and were maintained in noma cell lines were obtained from ATCC (Rockville, MD), hematolog- DMEM/4.5 g of glucose with 10% FBS, essential amino acids, and anti- ical cancer cell lines (CEM T-lymphoblast cells, K-562 chronic myelog- biotics. All treatments were done in the presence of serum. enous leukemia cells) were a kind gift from Dr. J. Barredo (Medical Thymidine Incorporation—Proliferation of cells was determined by 3 3 6 University of South Carolina). Primary antibodies, p21, p27, and p53 [ H]thymidine ribotide ([ H]TdR) incorporation into DNA. 1  10 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti- cells per well of adherent cell lines (C6, MCF-7, and PC3) and 0.25 10 bodies against phosphospecific as well as pan-Akt, mTOR, and AMPK cells/well of suspension cell lines (NALM-6, CEM, CEMP, and K562) were from Cell Signaling (Beverly, MA). AMPK -dominant negative were plated in respective medias. Cells were incubated for 18–24 h with NOVEMBER 25, 2005• VOLUME 280 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 39583 AMPK as a Potential Target for Treatment of Cancer or without the presence of AICAR at the indicated concentrations. Each group was exposed to 37 kBq/ml [methyl- H]thymidine in the same medium for 6 h. The adherent cells were fixed by 5% trichloroacetic acid and lysed in SDS/NaOH lysis buffer overnight. Radioactivity was meas- ured by Beckman LS3801 liquid scintillation counter (Canada). Suspen- sion cell culture was harvested by cell harvester (Packard instrument Co., Meriden, CT), and radioactivity was measured by 1450 microbeta liquid scintillation counter (PerkinElmer Life Sciences). Clonogenic Assay—Cells were treated with AICAR for 18–24 h, trypsinized, counted, and 300 cells/100-mm plate were plated. The cells were allowed to form colonies, and media was changed every third day for 2–3 weeks. The colonies were stained with MTT and enumerated (33). Flow Cytometry Assessment of Cell Cycle—Cellular DNA content was assessed by flow cytometry. Cells were cultured in 6-well plates and treated with AICAR or transfections were performed. Cells attached to the plate were collected with trypsin, washed, and resuspended in 100 l of PBS, and 5 ml of 70% ethanol was added slowly while continuous vortexing of cells and were fixed overnight. Next day, cells were spun, washed, and suspended in 400 l of PBS with addition of 10 mg/liter FIGURE 2. AICAR inhibits clonogenic potential of transformed cells. C6 glioma and RNase A and 75 M propidium iodide. Cells were acquired by flow PC3 prostate cells were treated with AICAR for 18 h, trypsinized and 300 cells/100-mm cytometry (BD Biosciences FACSCalibur flow cytometer) using Modfit plate were plated to form colonies. The data are a representation of three separate LT software. experiments done in triplicates. ***, p  0.001 compared with control; **, p  0.01 compared with control. Immunoblot—After a stipulated time of incubation in the presence or absence of AICAR, cells were scraped, washed with Hanks’ buffer, and antibody (1:100) followed by tyramide signal enhancement technique sonicated in 50 mM Tris-HCl (pH 7.4) containing protease inhibitors (1 (Renaissance TSA for Immunocytochemistry, PerkinElmer Life Sci- mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, 5 g/ml antipain, ences) per manufacturer’s instructions. After washing, slides were air- 5 g/ml pepstatin A, and 5 g/ml leupeptin). Proteins (50 g/lane) were dried and mounted with aqueous mounting media (Vectashield, Vector resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Laboratories). The sections were examined under a fluorescence micro- The membranes were blocked for1hin5% nonfat dry milk in TTBS (20 scope (Olympus BX-60) with an Olympus digital camera (Optronics, mM Tris, 500 mM NaCl, and 0.1% Tween 20, pH 7.5) and incubated Goleta, CA) using a dual band pass filter. Images were captured and overnight in primary antibody (p21, p27, p53, Akt, -actin, mTOR, processed using Adobe Photoshop 7.0. PCNA at 1:2000 dilution) containing 5% nonfat dry milk for non-phos- Transfection Studies—Plasmids were purified using the endotoxin- pho antibodies and containing 5% albumin for phospho-antibodies free plasmid midi prep kit (Qiagen). For transient transfections, C gli- (Akt-p, mTOR-p at 1:1000 dilution). The blots were washed four times oma cells were seeded in 6-well plates and grown to 60–80% confluence with TTBS (5 min/wash) and incubated for 45 min at room temperature in DMEM/F-12 plus 5% FBS without antibiotics and transfected using with respective horseradish peroxidase-conjugated secondary antibody FuGENE reagent. 1–3 gofAMPKDNorAMPK1CA or AMPK2 (1:5000). The blots were washed three times in TTBS and once in 0.1 M CA expression vector along with 1 g of eGFP expression vector or PBS (pH 7.4) at room temperature; protein expression was detected insertless expression vector (pcDNA3.1) were used for transfecting. with ECL. Cells were treated with AICAR for 24 h and processed for GFP-gated Animals—Adult male Wistar rats weighing 200–250 g were pur- DNA analysis by flow cytometry. Similarly, LKB1 wild type (1 g) and chased from Charles-River Laboratories. Animals were maintained, and dominant negative (1 g) along with STRAD (0.5 g) and MO25 (0.5 all protocols were approved by the animal use committees of the Med- g) expression vectors were used for transfection studies. ical University of South Carolina in compliance with the Guide for the Antisense Experiments—To decrease the levels of endogenous Care and Use of Laboratory Animals (National Institutes of Health). AMPK, C6 glioma cells were transfected for 48 h with 25 M phospho- Tumor Implantation—C6 glioma cells were prepared fresh from cul- thiorated antisense (AS) oligonucleotide (5-CGCCCGTCGTCGT- ture to ensure optimal viability of cells during tumor inoculation. The GCTTCTGC-3) directly against both the 1- and 2-subunits of cells were trypsinized, and 10 tumor cells prepared in 100 lofPBS AMPK (36, 37) and a missense (MS) oligonucleotide (5-CTCCCG- were injected subcutaneously in the lateral side of the right hind leg of GCTTGCTGCCGT-3) along with eGFP expression vector (36). Oli- the rats, after shave and sterile preparation. On the 5th day of implan- gonucleotides were transfected with FuGENE reagent per the manufac- tation 100 mg/kg body weight/day of AICAR was given intra-peritoneal turer’s instructions. The cells were then treated with AICAR for 24 h until the 14th day, when the animals were sacrificed and the tumor was and analyzed for cell cycle analysis by flow cytometry. excised, weighed, and fixed in formalin (34). PI3K Activity—After2hof AICAR (1 mM) treatment, cells were lysed Immunohistochemistry—Tumor sections were processed as previ- with ice-cold lysis buffer containing 1% v/v Nonidet P-40, 100 mM NaCl, ously described (35). In brief, deparaffinized and rehydrated sections 20 mM Tris (pH 7.4), 10 mM iodoacetamide, 10 mM NaF,1mM sodium were microwaved for 10 min in antigen unmasking fluid (Vector Labo- orthovanadate, and protease inhibitors (Sigma-Aldrich). Lysates were ratories, Burlingame, CA), treated with 3% hydrogen peroxide in meth- incubated at 4 °C for 15 min, followed by centrifugation at 13,000 g for anol to eliminate endogenous peroxidase activity and blocked to reduce 15 min. The supernatant was precleared with protein A/G-Sepharose nonspecific staining. Sections were incubated overnight with primary beads (Amersham Biosciences) for1hat4°C, followed by the addition 39584 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 47 •NOVEMBER 25, 2005 AMPK as a Potential Target for Treatment of Cancer FIGURE 3. AICAR causes cell cycle arrest in S-phase. C6 glioma and U87MG astrocytoma cells were treated with AICAR at indicated concentrations. After overnight fixation cells were suspended in PBS with RNase A and propidium iodide and acquired for DNA content by flow cytometry using Modfit LT software. The first peak represents the cells in G /G phase, 0 1 the second peak with slashed bars represents the cells accumulated in S-phase, and the third peak represents cells in the M-phase. The data are also graphically represented as percentage of cells in S-phase and M-phase. The data are a representative of three separate experiments. ***, p  0.001 compared with control; **, p  0.01 compared with control. NOVEMBER 25, 2005• VOLUME 280 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 39585 AMPK as a Potential Target for Treatment of Cancer FIGURE 4. AICAR inhibits cell proliferation in vivo. A, weight of excised tumor implanted in lateral side of the right flank of rats (n  8) from vehicle (saline) and AICAR (100 mg/kg body weight/day) treated. AICAR significantly reduced the tumor mass as compared with vehicle treated animals. *, p  0.05 compared with vehicle (Bi). Immunofluorescent microscopy images showing decreased PCNA expression in AICAR-treated tumor sections as compared with vehicle-treated rats, stained as described under “Materials and Methods.” The number of enumerated cells is depicted graphically (Bii). Data are mean  S.D. of 10 fields from three different experiments. ***, p  0.001 compared with control. C, Western blot depicting decreased expression of PCNA in the AICAR-treated tumor tissue from 2 different sets of animals as compared with vehicle-treated data. Each set had n  6. of 1 g/ml p85 mAb. After 2-h incubation at 4 °C, protein G-Sepharose TLC and visualized by exposure to iodine vapor and autoradiography beads were added, and the resulting mixture was further incubated for (38). 1 h at 4 °C. The immunoprecipitates were washed twice with lysis buffer, Statistical Analysis—The data were statistically analyzed by perform- once with PBS, once with 0.5 M LiCl and 100 mM Tris (pH 7.6), once in ing the Student-Newman-Keuls Test. water, and once in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl , RESULTS and 0.25 mM EDTA). PI3K activity was determined using a lipid mixture of 100 l of 0.1 mg/ml phosphatidylinositol and 0.1 mg/ml phosphati- AICAR Inhibits Proliferation of Cancer Cells—To investigate the dylserine dispersed by sonication in 20 mM HEPES (pH 7.0) and 1 mM effect of AICAR on the growth of various cancer cell lines, namely PC-3 EDTA. The reaction was initiated by the addition of 20 Ci of (human prostate cancer cell), MCF-7 (human breast cancer cell line), [- P]ATP (3000 Ci/mmol, PerkinElmer Life Sciences) and 100 M C6 glioma (rat transformed brain glial cells), U87MG (human astrocy- ATP, and terminated after 15 min by the addition of 80 lof1 N HCl and toma cell line), K-562 (human chronic myelogenous leukemia cells), and 200 l of chloroform:methanol (1:1). Phospholipids were separated by CEM (human T-lymphoblast cells), cells were plated in their respective 39586 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 47 •NOVEMBER 25, 2005 AMPK as a Potential Target for Treatment of Cancer FIGURE 5. AICAR induces AMPK and ACC phos- phorylation in vitro and in vivo. A, C6 glioma and PC3 prostate cells were treated with AICAR at indi- cated concentrations and harvested at specified time points, cell lysates were processed for the detection of phospho-AMPK (p-Thr-172) and phospho-ACC by immunoblot as discussed under “Materials and Methods.” The blots are represen- tatives of three individual experiments done. B, cell lysates were prepared from the vehicle- and AICAR-treated tumor tissues from two different set of animals (each set had n  6) and processed for the detection of phospho-AMPK (p-Thr-172) and phospho-ACC as above. medium for growth and treated with different concentrations of AICAR liferating cell nuclear antigen), a marker for proliferating cells. AICAR (0.25–1 mM) for 24 h, and cell proliferation was examined by [ H]thy- significantly reduced the expression and number of cells exhibiting midine uptake. AICAR inhibited the proliferation of all cell lines tested PCNA expression, indicating that the number of proliferating cells is significantly in a dose-dependent manner (Fig. 1). All tested cell lines reduced by AICAR treatment in vivo as demonstrated by immunohis- underwent significant proliferation inhibition, indicating that this phe- tochemistry and its expression by Western blot (Fig. 4, B and C). Thus, nomenon is widespread and not limited to a specific cell type/line. To the anti-proliferative effect of AICAR is effective in vivo as well and can further confirm this observation, a clonogenic assay was performed, be exploited for applications in attenuating cancer cell growth. where cells were treated with AICAR for 24 h, trypsinized and plated at AICAR Mediates Its Anti-proliferative Action via AMP-activated Pro- a density of 300 cells/100-mm plate without AICAR. After 3 weeks, tein Kinase—AICAR, is the earliest known AMPK activator, and most of formed colonies were counted by staining the live cells with MTT. its effects have been shown to be because of AMPK activation, although AICAR treatment significantly reduced the number of colonies being few reports of its AMPK-independent effects exist (22). To investigate if formed as compared with the untreated cells (Fig. 2), suggesting that a AMPK activation is responsible for the anti-proliferative effects single treatment of AICAR treatment is sufficient to cause a sustained observed by AICAR treatment, the phosphorylation of AMPK and its inhibition of proliferation in different cancer cell lines. downstream target, ACC, an enzyme in the fatty acid synthesis pathway, AICAR Causes Cell Cycle Arrest in S-phase—Inhibition in prolifera- was taken as an indicator of AMPK activation. AICAR induced the tion would indicate an anomaly in the cell cycle. To examine this, cells phosphorylation of AMPK and ACC in a dose- and time-dependent were treated with AICAR (0.5–1 mM), and phases of cell cycle were manner as demonstrated in C6 glioma and PC3 prostate cell lines (Fig. 5, analyzed by flow cytometry. Treatment of cells with AICAR resulted in A and B). Similar phosphorylation of ACC and AMPK was observed in accumulation of cells in S-phase (peak with slashed bars), with almost vivo, in the AICAR-treated excised tumor tissue (Fig. 5C). Iodotuberi- no cells detected in M-phase (third peak) suggesting that inhibition cidin is an inhibitor of adenosine kinase and inhibits the conversion of in proliferation by AICAR is due to the arrest of cell cycle at S-phase AICAR to its activated form ZMP inside the cell and thus inhibits acti- (Fig. 3). vation of AMPK by AICAR. Cells were pretreated with iodotubericidin AICAR Inhibits Proliferation in Vivo—To investigate whether the 30 min before the addition of AICAR (0.5–1 mM), and proliferation was anti-proliferative effects of AICAR extends to the in vivo system, we measured after 16 h by [ H]thymidine uptake. Iodotubericidin treat- utilized the rat flank tumor model (34). Wherein, C6 glioma cells (1  ment inhibited the proliferation arrest caused by AICAR thus indicating 10 ) were implanted aseptically in the right flank of the rat, and after 5 the involvement of AMPK (Fig. 6A). To further confirm the role of days of tumor formation, animals were treated with 100 mg/kg body AMPK, C6 glioma cells were transiently transfected with dominant neg- weight of AICAR intraperitoneally. On day 14, animals were sacrificed, ative (DN) and constitutive active (CA) forms of AMPK along with and the tumors were excised, weighed, and fixed. Weight of the tumors eGFP expression vector. The cells were treated with AICAR for 18 h, was taken as an index of tumor development and progression. AICAR and GFP-positive cells were analyzed by flow cytometry for DNA con- treatment was able to reduce the growth of tumors in animals signifi- tent to determine the cells in S-phase. C6 glioma cells transfected with cantly (50%) when compared with untreated animals (Fig. 4A). To AMPK dominant negative were not able to undergo S-phase arrest examine the status of proliferating cells in vivo, immunohistochemistry when treated with AICAR (Fig. 6B). Inversely, C6 glioma cells trans- was performed on the sections of excised tumor tissues for PCNA (pro- fected with expression vector of constitutive active AMPK1 were NOVEMBER 25, 2005• VOLUME 280 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 39587 AMPK as a Potential Target for Treatment of Cancer FIGURE 6. AICAR mediated its anti-proliferative action via AMPK. A, cells (CEM, K-526, and PC-3) were pretreated with iodotubericidin (0.1 M) before addition of AICAR and assayed for [ H]thymidine incorporation. Iodotubericidin reversed the AICAR-induced proliferation block. The data are representative of three separate experiments performed in triplicates. ***, p 0.001 compared with control; ###, p 0.001 compared with AICAR (B and C) C6 glioma cells were transiently co-transfected with 2 g of AMPK dominant negative (DN)(B) or AMPK constitutive active (CA)(C) and 1 g of eGFP expression vector. The DNA content was normalized by pcDNA3. AICAR was added where indicated, and after 18 h cells were fixed overnight and analyzed for arrest in S-phase as detailed under “Materials and Methods.” AICAR was not able to arrest the cells in the presence of AMPK DN (B), whereas AMPK CA expression was sufficient to arrest the cells in S-phase and showed additive effect with AICAR (C). The data are representative of three separate experiments. ***, p 0.001 compared with control; ###, p 0.001 compared with AICAR; ##, p 0.01 compared with AICAR; #, p 0.05 compared with AICAR; NS, non-significant compared with control. D, cells were transfected with AMPK antisense (AS) and missense (MS) oligonucleotides along with eGFP expression vector and treated with AICAR. The level of AMPK protein was reduced by the transfection of AS, whereas MS had no effect (inset) at 72 h. The cells were fixed and processed for GFP-gated S-phase arrest. Antisense of AMPK abolished the AICAR-mediated S-phase arrest, whereas MS oligonucleotide had no effect. These data are representative of 3 separate experiments. ***, p  0.001 compared with control; ###, p  0.001 compared with AICAR; NS, non-significant compared with AICAR. 39588 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 47 •NOVEMBER 25, 2005 AMPK as a Potential Target for Treatment of Cancer FIGURE 7. AICAR inhibits PI3K-Akt Pathway. A, C6 cells were treated with AICAR (1 mM) for 2 h and processed for PI3K activity as described under “Materials and Methods.” AICAR significantly reduced the PI3K activity as assessed by inositol 1,4,5-bisphosphate levels. The inositol 1,4,5- bisphosphate levels were measured by densitom- etry analysis. The blot is representative of three separate experiments. **, p 0.01 compared with control (B) C6 cells were treated with AICAR for increasing time points as indicated, cells lysates were prepared and analyzed for Akt (Ser-473) and mTOR (Ser-1448) phosphorylation by Western blot as detailed under “Materials and Methods.” C, cell lysates were prepared from the treated tumor tissue from two different set of animals and pro- cessed for the detection of phospho-Akt, which was reduced by AICAR treatment. FIGURE 8. AICAR regulates the expression of cdk inhibitors via AMPK. Protein expression of cell cycle inhibitors p21, p27, and p53 was increased by AICAR treatment as analyzed by immunoblot in C6 cells treated with AICAR (A) and tumor tissue (B). C, immunofluorescent microscopy images of tumor sections from vehicle and treated rats, stained with p21, p27, and p53 antibodies as described under “Materials and Methods.” D,C6 glioma cells were transiently transfected with 2 g of AMPK dominant negative (DN) or AMPK consti- tutive active (CA) with DNA normalization done with pcDNA and treated with AICAR where indi- cated. Cell lysates were prepared and assessed by immunoblot for p21, p27, and phospho-Akt expressions. AICAR induced p21 and p27 expres- sion and down-regulated phospho-Akt as before (lane 2), which was reversed by AMPK DN expres- sion (lane 3) and had no effect with AICAR treat- ment (lane 4). AMPK CA 1 and 2 overexpression was able to induce the p21 and p27 expression and attenuate phospho-Akt by itself (lanes 5 and 6). The blots are representatives of three individual experiments done. NOVEMBER 25, 2005• VOLUME 280 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 39589 AMPK as a Potential Target for Treatment of Cancer found to be arrested at S-phase similar to AICAR-treated cells (Fig. 6C). Further, we used the antisense (AS) approach to knock out the expres- sion of AMPK. C6 glioma cells were transfected with AMPK antisense (AS) and missense (MS) oligonucleotides, and levels of AMPK were observed 48 h post transfection. The level of AMPK protein was atten- uated by AS, whereas MS had no effect (Fig. 6D, inset). Moreover, trans- fection with antisense of AMPK along with GFP expression vector in C6 glioma cells significantly reduced the AICAR-mediated S-phase arrest; however, MS oligonucleotide did not affect the potential of AICAR to arrest cells in S-phase (Fig. 6D). Taken together, these evidences point strongly toward a role for AMPK as an effective anti-proliferative system. AICAR/AMPK Inhibits the PI3K-Akt Pathway—Because AICAR inhibits cell proliferation and PI3K-Akt is one of the most important pathways regulating proliferation, we examined the effect of AICAR on the PI3K-Akt pathway. C6 glioma cells were treated with AICAR (1 mM) for 2 h, and PI3K activity was assessed using phosphoinositol as a sub- strate, and we observed that AICAR treatment significantly reduced the PI3K activity (Fig. 7A). One of the downstream effectors of PI3K, Akt is the main mediator regulating proliferation (39). AICAR also reduced the phosphorylation of Akt in vitro and in vivo (Fig. 7, Bi and C). It also inhibited the phosphorylation (Ser-1448) of mTOR (Fig. 7Bii), which is a downstream target of Akt and regulates protein synthesis and cell growth (40). Thus, attenuation of the PI3K-Akt pathway may be one of the mechanisms by which AMPK negatively regulates growth. AICAR Regulates the Expression of Cyclin-dependent Kinase Inhibi- tors via AMPK—Because AICAR inhibits the cell proliferation by arresting cells at S-phase in vitro as well as in vivo, we examined the expression of cyclin-dependent kinase (cdk) inhibitors, which bind to cyclin-cdk complexes and inhibit the progression of cell cycle. AICAR induced the expression of p21 and p27, the cip/kip protein cdk inhibi- tors in a time-dependent manner (Fig. 8A). It also induced the expres- sion of p53, which is known to regulate the cell cycle as well as p21 expression (41). The expression of p21, p27, and p53 proteins, were also increased in vivo, as assessed by immunohistochemistry of tissue sec- tions and by Western blot analysis of protein isolated from excised tumor tissue (Fig. 8, B and C). The effect of AICAR on the expression of growth regulators is mediated via activation of AMPK, because trans- FIGURE 9. LKB (AMPKK) status does not affect AICAR mediated growth arrest. A, LKB / / fected AMPK DN abolished the AICAR-mediated induction of p21, knock-out (LKB ) and wild-type (LKB ) mouse embryo fibroblast were treated with AICAR and analyzed by immunoblot for AMPK and ACC phosphorylation. The blots are whereas the CA form of AMPK1 and 2 induced the expression by representatives of three individual experiments done. B, LKB knock-out (LKB ) and itself (Fig. 8D). In case of p27, AMPK DN reduced the AICAR-induced wild-type (LKB ) mouse embryo fibroblast were treated with varying concentrations of AICAR and assayed for [ H]thymidine incorporation. The data are representative of expression, but AMPK1 and 2 CA forms were only able to induce p27 three separate experiments. ***, p  0.001 compared with control. C, C6 glioma cells protein marginally compared with AICAR. In case of Akt, DN were transiently transfected with 1 gof LKB dominant negative (DN)or LKB wild type AMPK2-transfected cells did not respond to the AICAR-mediated (WT) with STRAD (0.5 g) and MO25 (0.5 g) expression vectors, eGFP, and pcDNA and treated with AICAR where indicated. After 18 h cells were fixed overnight and ana- inhibition in Akt phosphorylation, whereas, in CA-transfected cells, lyzed for arrest in S-phase as detailed under “Materials and Methods.” there was significant inhibition (Fig. 8E), indicating that AMPK activa- tion is responsible for increase in cdk inhibitor protein expressions and inhibition of Akt phosphorylation. S-phase arrest observed when C6 cells were transiently transfected with LKB (AMPKK) Status Does Not Affect AICAR-mediated Growth LKB dominant negative and wild-type expression vector along with Arrest—LKB is a recently discovered upstream target of AMPK (AMPK expression vectors of its cofactors, STRAD and MO25, and treated kinase, AMPKK), which phosphorylates AMPK at Thr-172 for its full with AICAR (Fig. 9C). These data indicate that AMPK activation by activation. LKB itself is a tumor suppressor gene and inactivation of LKB AICAR is sufficient to cause growth arrest and does not require activa- results in predisposition to various cancers (27–29). It is being hypoth- tion by LKB. esized that the anti-tumor effects of LKB are due to AMPK activity. To DISCUSSION examine the possible involvement of LKB in AICAR/AMPK-induced growth arrest, we utilized LKB knock-out (LKB ) and LKB WT In this study we have demonstrated that AMPK activation by AICAR (LKB ) MEF cell lines. AICAR was able to induce the phosphoryla- results in growth arrest at S-phase due to inhibition of PI3K-Akt path- tion of AMPK and ACC to a similar extent in both MEFs (Fig. 9A). way and up-regulation of cdk inhibitors, independent of its upstream AICAR was able to inhibit proliferation in both knock-out and wild-type kinase LKB. This inference is based on the following observations: 1) MEFs to a similar extent (Fig. 9B). This was further supported by similar Treatment of various cancer cell lines by AICAR attenuated the prolif- 39590 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 47 •NOVEMBER 25, 2005 AMPK as a Potential Target for Treatment of Cancer FIGURE 10. A schematic representation of effect of AICAR on proliferation of cancer cells. AICAR, upon entering the cell, is converted to ZMP, which activates AMPK. Upon activation AMPK increases the expression of p21, p27, and p53 proteins, which may be responsible for the S-phase arrest being observed. On the other hand it inhibits the PI3K activity and Akt phosphorylation, which results in inhibition of mTOR and speculative reg- ulation of other targets like MDM2, Bad, and caspase 9, resulting in a proliferation and cell growth block. The overall signaling taking place results in a sustained proliferation arrest, which can ultimately lead to loss of viability due to onset of senescence or apoptotic pathways. eration both in vitro and in vivo studies. 2) The attenuation of cell These evidences point strongly toward AMPK activation being the proliferation was due to the activation of AMPK as evident from aden- major cause of growth arrest. Thus, AMPK can be considered as a neg- osine kinase inhibitor studies (iodotubericidin), the use of expression ative regulator of proliferation and can modulate protein expression to vectors (dominant negative and constitutive active) and AMPK anti- this effect, classifying it as a tumor suppressor system that can be sense experiments. 3) The growth arrest is mediated by inhibition of exploited for attenuation of cancers. PI3K activity and Akt phosphorylation and up-regulation of cell-cycle Activation of AMPK by AICAR, metformin, or thiazolidinediones or inhibitor proteins p21, p27, and p53. 4) Activation of AMPK in the expression of constitutively active mutants has been shown to cause absence of LKB also results in growth arrest. We show here the direct death or attenuate the growth of cancer cells. AICAR- and rosiglita- relation between AMPK activation and growth inhibition in vitro and in zone-mediated AMPK activation caused proliferation block and cell vivo. In addition, these observations strongly indicate AICAR, an AMPK death by inhibiting fatty acid and protein synthesis pathways and activator to be an efficient anti-proliferative agent in vitro and in vivo. increasing p21 expression in prostate cells (22). Adenosine-induced Being a pharmacological activator of AMPK, AICAR has been used AMPK was shown to cause apoptosis in gastric cancer cells (20), and extensively to study its role in physiology (13, 14). It has recently been activation of AMPK by AICAR and its CA form was shown to cause shown to have anti-inflammatory properties that were reported to be apoptosis in pancreatic cells by inducing JNK pathway (23). Similarly, mediated by AMPK activation (36, 42), although its AMPK-independ- AMPK induced JNK and caspase 3 activity resulting in apoptosis in liver ent effects have also been reported (22). In our study, AICAR mediates cells (24). AMPK activation was also demonstrated to enhance H O - 2 2 its effect via activation of AMPK, which is supported by both pharma- mediated apoptosis in neuroblastoma cells by inducing NF- and p38- cological (iodotubericidin) as well as molecular approaches (DN, CA, JNK pathways (25). These studies, along with the present study, suggests and AS AMPK transfections). Treatment of iodotubericidin, inhibitor AMPK as an efficient growth inhibitor and apoptosis inducer. On the of conversion of AICAR to ZMP, abrogated the S-phase arrest of cells other hand, it also has been shown to have a protective effect on stress- (Fig. 6A). Moreover, inhibition of AMPK either by its DN expression injured cells in heart ischemia and reperfusion injury model (15, 18). vector or AS oligonucleotide also resulted in the reversal of AICAR- AMPK activation protects primary astrocytes from fatty acid-induced mediated growth arrest, whereas AMPK CA expression was able to death by inhibiting de novo ceramide synthesis (17) and protects human accumulate cells in S-phase (Fig. 6, B–D). AMPK DN also blunted the umbilical vein endothelial cells from hyperglycemia by inhibition of elevated expression of p21 and p27, whereas AMPK CA itself was able to caspase 3 and Akt activation (18) and by similar mechanism in thymo- induce their expression (Fig. 8). The induction of p27 by AMPK CA 1 cytes (19). In pancreatic cancer cells, AMPK was shown to bestow tol- and 2 seems to be marginal as compared with p21. The observation erance toward nutrient deprivation (43). These studies presented represented here is consistent and reproducible. Right now we do not AMPK as a protective agent. The reason for these apparently opposing have an explanation for this disparity. One of the possible explanations effects of AMPK activation in cell survival and cell death is not known, could be that p27 regulation is related to the differential localization of but it can be speculated that in actively dividing cancer cells, the inhibi- the 1 (cytosol) and 2 (nuclear) isoforms of the catalytic subunits of tion of ATP-consuming processes by AMPK may be less compatible AMPK, which is yet to be established in terms of p27 regulation. AICAR with their survival, whereas in non-dividing cells, where the protective seems to affect the PI3K-Akt proliferation pathway, because AICAR and effects of AMPK have been observed under acute stress, the shutdown AMPK CA inhibited PI3K activity and Akt phosphorylation (Fig. 7). of ATP-consuming pathways may not alter the balance for survival. 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Hemminki, A., Markie, D., Tomlinson, I., Avizienyte, E., Roth, S., Loukola, A., Bignell, G., Warren, W., Aminoff, M., Hoglund, P., Jarvinen, H., Kristo, P., Pelin, K., Ridanpaa, M., Salovaara, R., Toro, T., Bodmer, W., Olschwang, S., Olsen, A. S., Stratton, M. R., de Acknowledgments—We thank Dr. J. Barredo (Medical University of South la Chapelle, A., and Aaltonen, L. A. (1998) Nature 391, 184–187 Carolina, SC) for his kind gift of CEM T-lymphoblast and K-562 chronic 28. Jenne, D. E., Reimann, H., Nezu, J., Friedel, W., Loff, S., Jeschke, R., Muller, O., Back, myelogenous leukemia cell lines. We thank Dr. David Carling (MRC Clinical W., and Zimmer, M. (1998) Nat. Genet. 18, 38–43 Sciences Centre, London, UK) for his kind gift of AMPK -dominant negative 2 29. Nakanishi, C., Yamaguchi, T., Iijima, T., Saji, S., Toi, M., Mori, T., and Miyaki, M. expression vector (D157A). We thank Dr. Jin-Zhong Zhang (Case Western (2004) Oncology 67, 476–479 Reserve University, Cleveland, OH) and Dr. Benoit Viollet (Rene´ Descartes 30. Forcet, C., Etienne-Manneville, S., Gaude, H., Fournier, L., Debilly, S., Salmi, M., Baas, A., Olschwang, S., Clevers, H., and Billaud, M. (2005) Hum. Mol. Genet. 14, University, Paris, France) for their kind gift of AMPK1 and 2 constitutive 1283–1292 active constructs, respectively. We thank Dr. Dario R. Alessi (Wellcome Trust 31. Inoki, K., Zhu, T., and Guan, K. L. (2003) Cell 115, 577–590 Biocentre, University of Dundee, and Dundee, UK) for the kind gift of LKB wild 32. Li, Y., Corradetti, M. N., Inoki, K., and Guan, K. L. (2004) Trends Biochem. Sci. 29, type, LKB dominant negative (kinase-dead), STRAD, and MO25 expression 32–38 vectors. We thank Dr. Tomi P. Makela (Institute of Biomedicine and Helsinki 33. Giocanti, N., Hennequin, C., Rouillard, D., Defrance, R., and Favaudon, V. (2004) Br. J. 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(2001) J. Mol. Neurosci. 17, 175, 566–574 45–58 43. Kato, K., Ogura, T., Kishimoto, A., Minegishi, Y., Nakajima, N., Miyazaki, M., and 38. Giri, S., Rattan, R., Singh, A. K., and Singh, I. (2004) J. Immunol. 173, 5196–5208 Esumi, H. (2002) Oncogene 21, 6082–6090 39. Brazil, D. P., Yang, Z. Z., and Hemmings, B. A. (2004) Trends Biochem. Sci. 29, 44. Kimura, N., Tokunaga, C., Dalal, S., Richardson, C., Yoshino, K., Hara, K., Kemp, B. E., 233–242 Witters, L. A., Mimura, O., and Yonezawa, K. (2003) Genes Cells 8, 65–79 40. Harrington, L. S., Findlay, G. M., and Lamb, R. F. (2005) Trends Biochem. Sci. 30, 45. Bolster, D. R., Crozier, S. J., Kimball, S. R., and Jefferson, L. S. (2002) J. Biol. Chem. 277, 35–42 23977–23980 41. el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, 46. Song, G., Ouyang, G., and Bao, S. (2005) J. Cell Mol. Med. 9, 59–71 NOVEMBER 25, 2005• VOLUME 280 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 39593 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro and in Vivo via AMP-activated Protein Kinase *

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American Society for Biochemistry and Molecular Biology
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 47, pp. 39582–39593, November 25, 2005 Printed in the U.S.A. 5-Aminoimidazole-4-carboxamide-1--D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro and in Vivo via AMP-activated Protein Kinase Received for publication, July 8, 2005, and in revised form, September 21, 2005 Published, JBC Papers in Press, September 21, 2005, DOI 10.1074/jbc.M507443200 ‡ ‡ § ‡1 Ramandeep Rattan , Shailendra Giri , Avtar K. Singh , and Inderjit Singh ‡ § From the Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425 and the Department of Pathology and Laboratory Medicine, Ralph Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425 5-Aminoimidazole-4-carboxamide-1--4-ribofuranoside (AICAR) phorylates and inactivates a number of metabolic enzymes involved in is widely used as an AMP-kinase activator, which regulates energy ATP-consuming pathways like fatty acid, cholesterol synthesis, and homeostasis and response to metabolic stress. Here, we investigated protein synthesis that include enzymes like acetyl-Co enzyme A carbox- the effect of AICAR, an AMPK activator, on proliferation of various ylase (ACC), fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA cancer cells and observed that proliferation of all the examined cell reductase, and mammalian target of rapamycin (mTOR) and activates lines was significantly inhibited by AICAR treatment due to arrest ATP-generating process like fatty acid oxidation and glucose uptake (8). in S-phase accompanied with increased expression of p21, p27, and The mechanisms of activating AMPK include direct allosteric binding p53 proteins and inhibition of PI3K-Akt pathway. Inhibition in in of AMP to the  subunits and phosphorylation, catalyzed by an vitro growth of cancer cells was mirrored in vivo with increased upstream AMP kinase (AMPKK), recently identified to be LKB1 expression of p21, p27, and p53 and attenuation of Akt phosphoryl- (STK11) (9–11). Recent studies have demonstrated that AMPK can also ation. Anti-proliferative effect of AICAR is mediated through acti- be activated by other stimuli that do not cause a detectable change in the vated AMP-activated protein kinase (AMPK) as iodotubericidin AMP/ATP ratio, like hyperosmotic stress and pharmacological agents and dominant-negative AMPK expression vector reversed the like thiazolidinediones, metformin, and 5-aminoimidazole-4-carbox- AICAR-mediated growth arrest. Moreover, constitutive active amide-1--D-ribofuranoside (AICAR) (11–14). AMPK arrested the cells in S-phase by inducing the expression of Activation of AMPK has been related with protection from injury and p21, p27, and p53 proteins and inhibiting Akt phosphorylation, sug- apoptosis caused by myocardial ischemia (15, 16) and apoptosis due to gesting the involvement of AMPK. AICAR inhibited proliferation in metabolic stress (17–19). In these scenarios, AMPK has been proposed both LKB and LKB knock-out mouse embryo fibroblasts to similar as an anti-apoptotic molecule. However, recent reports have indicated extent and arrested cells at S-phase when transfected with domi- anti-proliferative and pro-apoptotic action of activated AMPK using nant negative expression vector of LKB. Altogether, these results pharmacological agents or AMPK overexpression. AMPK activation indicate that AICAR can be utilized as a therapeutic drug to inhibit has been shown to induce apoptosis in human gastric cancer cells (20), cancer, and AMPK can be a potential target for treatment of various lung cancer cells (21), prostate cancer (22), pancreatic cells (23), and cancers independent of the functional tumor suppressor gene, LKB. hepatic carcinoma cells (24) and enhance oxidative stress induced apo- ptosis in mouse neuroblastoma cells (25), by various mechanisms that includes inhibition of fatty acid synthase pathway and induction of AMP-activated protein kinase (AMPK) is a highly conserved serine/ stress kinases and caspase 3. threonine protein kinase. It is a heterotrimer containing a catalytic () AMPK is an anti-growth molecule because of its relationship with and two regulatory subunits ( and ), each of which have at least two two tumor suppressor genes: LKB and TSC2 (tuberous sclerosis com- isoforms (1). AMPK is called the “fuel gauge” of the biological system, plex 2). LKB functions as an upstream kinase (AMPKK) that activates because it is activated under conditions that deplete cellular ATP and AMPK (26). LKB mutations result in Peutz-Jeghers syndrome, which elevate AMP levels, such as glucose deprivation, heat shock, hypoxia, results in predisposition to cancers of the colon, pancreas, breast, and and ischemia (2, 3), and also by hormones like leptin (4), adiponectin (5), other sites (27–29). Mutations of LKB1 typically occur in the catalytic catecholamine (6), and interleukin-6 (7). Upon activation, AMPK phos- domain, leading to loss of its kinase activity and presumably a failure to phosphorylate and activate AMPK (30). TSC2 forms a complex with TSC1 and inhibits mTOR, leading to inhibition in protein synthesis and * This work was supported by National Institutes of Health Grants NS-22576, NS-34741, NS-40810, NS-37766, and NS-40144. The costs of publication of this article were negative regulation of cell size and growth (31). Mutations of defrayed in part by the payment of page charges. This article must therefore be TSC1TSC2 causes tuberous sclerosis, which is associated with hamar- hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tomatous polyps in multiple tissues and an increased risk of cancers To whom correspondence should be addressed: Children Research Institute, Medical (32). University of South Carolina, 173 Ashley Ave., 5th Floor, Charleston, SC 29425. Tel.: 843-792-7542; Fax: 843-792-7130; E-mail: [email protected]. In the present study we have investigated the effect of AICAR on cell The abbreviations used are: AMPK, AMP-activated protein kinase; ACC, acetyl-CoA car- proliferation in vivo and in vitro in various cancer cell lines. AICAR is boxylase; mTOR, mammalian target of rapamycin; AMPKK, AMPK kinase; AICAR, converted to its triphosphorylated form ZMP, inside the cell, by an 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside; TSC2, tuberous sclerosis complex 2; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; adenosine kinase (14), which acts as an AMP analogue and activates TdR, thymidine ribotide; PBS, phosphate-buffered saline; PCNA, proliferating nuclear AMPK and its upstream kinase LKB without affecting the ATP:AMP antigen; GFP, green fluorescent protein; eGFP, enhanced GFP; AS, antisense; MS, mis- sense; PI3K, phosphatidylinositol 3-kinase; DN, dominant negarive; CA, constitutively ratio in the cell (14). AICAR-mediated AMPK activation was found to active; MEF, mouse embryo fibroblasts; JNK, c-Jun NH -terminal kinase; MTT, 3-(4,5- be a proficient inhibitor of cell proliferation and the mechanism of its dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ZMP, AICA riboside mono- phosphate metabolite. anti-proliferative effect may be mediated via inhibition of PI3K-Akt 39582 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 47 •NOVEMBER 25, 2005 This is an Open Access article under the CC BY license. AMPK as a Potential Target for Treatment of Cancer FIGURE 1. AICAR inhibits proliferation of cancer cells. Specified numbers of cells (C6, MCF-7, PC3, CEM, and K562) were plated, treated with AICAR (0.25–1 mM), followed by exposure to [ H]thymi- dine for 6 h and subsequent counts. The data is representation of three separate experiments done in triplicates. ***, p  0.001 compared with control; **, p  0.01 compared with control, NS, non-significant compared with control. pathway and increased expression of cell cycle inhibitory proteins p21, expression vector (D157A) was a kind gift from Dr. David Carling (MRC p27, and p53, thereby exhibiting potential as an anti-cancer drug. Clinical Sciences Centre, London, UK), and AMPK1 and 2 constitu- tive expression vectors were kind gifts from Dr. Jin-Zhong Zhang (Case MATERIALS AND METHODS Western Reserve University, Cleveland, OH) and Dr. Benoit Viollet Reagents and Cell Culture—DMEM/F-12, DMEM/4.5 g of glucose (Rene´ Descartes University, Paris, France), respectively. LKB wild type, medium, fetal bovine serum (FBS), and Hanks’ balanced salt solution LKB dominant negative (kinase dead), STRAD and MO25 expres- were obtained from Invitrogen as was RPMI 1640. AICAR was pur- sion vectors were kind gifts from Dr. Dario R. Alessi (Wellcome Trust chased from Toronto Research Chemicals (Ontario, Canada). Iodotu- Biocenter, University of Dundee, Dundee, UK). bericidin was obtained from Calbiochem. [ H]Thymidine ribotide Cell Culture—C6 glioma cells, T98G, U87MG, and PC-3 were main- ([ H]TdR) was purchased from PerkinElmer Life Sciences. Propium tained in DMEM/F-12 medium supplemented with 10% FBS and anti- iodide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide biotics. MCF-7 cells were maintained in DMEM/4.5 g of glucose with (MTT), and transfection reagent, FuGENE, were purchased from Roche 10% FBS. CEM and K-562 were maintained in RPMI 1640 supple- Applied Science. The enhanced chemiluminescence (ECL) detecting mented with 10% FBS. LKB knock-out and wild-type mouse embryo reagent was from Amersham Biosciences, and the luciferase assay sys- fibroblasts (MEFs) were a kind gift from Dr. Tomi P. Makela (Institute of tem was from Promega (Madison, WI). C glioma, T98G astrocytoma, Biomedicine and Helsinki University Central Hospital, Biomedicum U87MG astrocytoma, MCF-7 breast cancer, and PC-3 prostrate carci- Helsinki, University of Helsinki, Finland) and were maintained in noma cell lines were obtained from ATCC (Rockville, MD), hematolog- DMEM/4.5 g of glucose with 10% FBS, essential amino acids, and anti- ical cancer cell lines (CEM T-lymphoblast cells, K-562 chronic myelog- biotics. All treatments were done in the presence of serum. enous leukemia cells) were a kind gift from Dr. J. Barredo (Medical Thymidine Incorporation—Proliferation of cells was determined by 3 3 6 University of South Carolina). Primary antibodies, p21, p27, and p53 [ H]thymidine ribotide ([ H]TdR) incorporation into DNA. 1  10 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti- cells per well of adherent cell lines (C6, MCF-7, and PC3) and 0.25 10 bodies against phosphospecific as well as pan-Akt, mTOR, and AMPK cells/well of suspension cell lines (NALM-6, CEM, CEMP, and K562) were from Cell Signaling (Beverly, MA). AMPK -dominant negative were plated in respective medias. Cells were incubated for 18–24 h with NOVEMBER 25, 2005• VOLUME 280 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 39583 AMPK as a Potential Target for Treatment of Cancer or without the presence of AICAR at the indicated concentrations. Each group was exposed to 37 kBq/ml [methyl- H]thymidine in the same medium for 6 h. The adherent cells were fixed by 5% trichloroacetic acid and lysed in SDS/NaOH lysis buffer overnight. Radioactivity was meas- ured by Beckman LS3801 liquid scintillation counter (Canada). Suspen- sion cell culture was harvested by cell harvester (Packard instrument Co., Meriden, CT), and radioactivity was measured by 1450 microbeta liquid scintillation counter (PerkinElmer Life Sciences). Clonogenic Assay—Cells were treated with AICAR for 18–24 h, trypsinized, counted, and 300 cells/100-mm plate were plated. The cells were allowed to form colonies, and media was changed every third day for 2–3 weeks. The colonies were stained with MTT and enumerated (33). Flow Cytometry Assessment of Cell Cycle—Cellular DNA content was assessed by flow cytometry. Cells were cultured in 6-well plates and treated with AICAR or transfections were performed. Cells attached to the plate were collected with trypsin, washed, and resuspended in 100 l of PBS, and 5 ml of 70% ethanol was added slowly while continuous vortexing of cells and were fixed overnight. Next day, cells were spun, washed, and suspended in 400 l of PBS with addition of 10 mg/liter FIGURE 2. AICAR inhibits clonogenic potential of transformed cells. C6 glioma and RNase A and 75 M propidium iodide. Cells were acquired by flow PC3 prostate cells were treated with AICAR for 18 h, trypsinized and 300 cells/100-mm cytometry (BD Biosciences FACSCalibur flow cytometer) using Modfit plate were plated to form colonies. The data are a representation of three separate LT software. experiments done in triplicates. ***, p  0.001 compared with control; **, p  0.01 compared with control. Immunoblot—After a stipulated time of incubation in the presence or absence of AICAR, cells were scraped, washed with Hanks’ buffer, and antibody (1:100) followed by tyramide signal enhancement technique sonicated in 50 mM Tris-HCl (pH 7.4) containing protease inhibitors (1 (Renaissance TSA for Immunocytochemistry, PerkinElmer Life Sci- mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, 5 g/ml antipain, ences) per manufacturer’s instructions. After washing, slides were air- 5 g/ml pepstatin A, and 5 g/ml leupeptin). Proteins (50 g/lane) were dried and mounted with aqueous mounting media (Vectashield, Vector resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Laboratories). The sections were examined under a fluorescence micro- The membranes were blocked for1hin5% nonfat dry milk in TTBS (20 scope (Olympus BX-60) with an Olympus digital camera (Optronics, mM Tris, 500 mM NaCl, and 0.1% Tween 20, pH 7.5) and incubated Goleta, CA) using a dual band pass filter. Images were captured and overnight in primary antibody (p21, p27, p53, Akt, -actin, mTOR, processed using Adobe Photoshop 7.0. PCNA at 1:2000 dilution) containing 5% nonfat dry milk for non-phos- Transfection Studies—Plasmids were purified using the endotoxin- pho antibodies and containing 5% albumin for phospho-antibodies free plasmid midi prep kit (Qiagen). For transient transfections, C gli- (Akt-p, mTOR-p at 1:1000 dilution). The blots were washed four times oma cells were seeded in 6-well plates and grown to 60–80% confluence with TTBS (5 min/wash) and incubated for 45 min at room temperature in DMEM/F-12 plus 5% FBS without antibiotics and transfected using with respective horseradish peroxidase-conjugated secondary antibody FuGENE reagent. 1–3 gofAMPKDNorAMPK1CA or AMPK2 (1:5000). The blots were washed three times in TTBS and once in 0.1 M CA expression vector along with 1 g of eGFP expression vector or PBS (pH 7.4) at room temperature; protein expression was detected insertless expression vector (pcDNA3.1) were used for transfecting. with ECL. Cells were treated with AICAR for 24 h and processed for GFP-gated Animals—Adult male Wistar rats weighing 200–250 g were pur- DNA analysis by flow cytometry. Similarly, LKB1 wild type (1 g) and chased from Charles-River Laboratories. Animals were maintained, and dominant negative (1 g) along with STRAD (0.5 g) and MO25 (0.5 all protocols were approved by the animal use committees of the Med- g) expression vectors were used for transfection studies. ical University of South Carolina in compliance with the Guide for the Antisense Experiments—To decrease the levels of endogenous Care and Use of Laboratory Animals (National Institutes of Health). AMPK, C6 glioma cells were transfected for 48 h with 25 M phospho- Tumor Implantation—C6 glioma cells were prepared fresh from cul- thiorated antisense (AS) oligonucleotide (5-CGCCCGTCGTCGT- ture to ensure optimal viability of cells during tumor inoculation. The GCTTCTGC-3) directly against both the 1- and 2-subunits of cells were trypsinized, and 10 tumor cells prepared in 100 lofPBS AMPK (36, 37) and a missense (MS) oligonucleotide (5-CTCCCG- were injected subcutaneously in the lateral side of the right hind leg of GCTTGCTGCCGT-3) along with eGFP expression vector (36). Oli- the rats, after shave and sterile preparation. On the 5th day of implan- gonucleotides were transfected with FuGENE reagent per the manufac- tation 100 mg/kg body weight/day of AICAR was given intra-peritoneal turer’s instructions. The cells were then treated with AICAR for 24 h until the 14th day, when the animals were sacrificed and the tumor was and analyzed for cell cycle analysis by flow cytometry. excised, weighed, and fixed in formalin (34). PI3K Activity—After2hof AICAR (1 mM) treatment, cells were lysed Immunohistochemistry—Tumor sections were processed as previ- with ice-cold lysis buffer containing 1% v/v Nonidet P-40, 100 mM NaCl, ously described (35). In brief, deparaffinized and rehydrated sections 20 mM Tris (pH 7.4), 10 mM iodoacetamide, 10 mM NaF,1mM sodium were microwaved for 10 min in antigen unmasking fluid (Vector Labo- orthovanadate, and protease inhibitors (Sigma-Aldrich). Lysates were ratories, Burlingame, CA), treated with 3% hydrogen peroxide in meth- incubated at 4 °C for 15 min, followed by centrifugation at 13,000 g for anol to eliminate endogenous peroxidase activity and blocked to reduce 15 min. The supernatant was precleared with protein A/G-Sepharose nonspecific staining. Sections were incubated overnight with primary beads (Amersham Biosciences) for1hat4°C, followed by the addition 39584 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 47 •NOVEMBER 25, 2005 AMPK as a Potential Target for Treatment of Cancer FIGURE 3. AICAR causes cell cycle arrest in S-phase. C6 glioma and U87MG astrocytoma cells were treated with AICAR at indicated concentrations. After overnight fixation cells were suspended in PBS with RNase A and propidium iodide and acquired for DNA content by flow cytometry using Modfit LT software. The first peak represents the cells in G /G phase, 0 1 the second peak with slashed bars represents the cells accumulated in S-phase, and the third peak represents cells in the M-phase. The data are also graphically represented as percentage of cells in S-phase and M-phase. The data are a representative of three separate experiments. ***, p  0.001 compared with control; **, p  0.01 compared with control. NOVEMBER 25, 2005• VOLUME 280 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 39585 AMPK as a Potential Target for Treatment of Cancer FIGURE 4. AICAR inhibits cell proliferation in vivo. A, weight of excised tumor implanted in lateral side of the right flank of rats (n  8) from vehicle (saline) and AICAR (100 mg/kg body weight/day) treated. AICAR significantly reduced the tumor mass as compared with vehicle treated animals. *, p  0.05 compared with vehicle (Bi). Immunofluorescent microscopy images showing decreased PCNA expression in AICAR-treated tumor sections as compared with vehicle-treated rats, stained as described under “Materials and Methods.” The number of enumerated cells is depicted graphically (Bii). Data are mean  S.D. of 10 fields from three different experiments. ***, p  0.001 compared with control. C, Western blot depicting decreased expression of PCNA in the AICAR-treated tumor tissue from 2 different sets of animals as compared with vehicle-treated data. Each set had n  6. of 1 g/ml p85 mAb. After 2-h incubation at 4 °C, protein G-Sepharose TLC and visualized by exposure to iodine vapor and autoradiography beads were added, and the resulting mixture was further incubated for (38). 1 h at 4 °C. The immunoprecipitates were washed twice with lysis buffer, Statistical Analysis—The data were statistically analyzed by perform- once with PBS, once with 0.5 M LiCl and 100 mM Tris (pH 7.6), once in ing the Student-Newman-Keuls Test. water, and once in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl , RESULTS and 0.25 mM EDTA). PI3K activity was determined using a lipid mixture of 100 l of 0.1 mg/ml phosphatidylinositol and 0.1 mg/ml phosphati- AICAR Inhibits Proliferation of Cancer Cells—To investigate the dylserine dispersed by sonication in 20 mM HEPES (pH 7.0) and 1 mM effect of AICAR on the growth of various cancer cell lines, namely PC-3 EDTA. The reaction was initiated by the addition of 20 Ci of (human prostate cancer cell), MCF-7 (human breast cancer cell line), [- P]ATP (3000 Ci/mmol, PerkinElmer Life Sciences) and 100 M C6 glioma (rat transformed brain glial cells), U87MG (human astrocy- ATP, and terminated after 15 min by the addition of 80 lof1 N HCl and toma cell line), K-562 (human chronic myelogenous leukemia cells), and 200 l of chloroform:methanol (1:1). Phospholipids were separated by CEM (human T-lymphoblast cells), cells were plated in their respective 39586 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 47 •NOVEMBER 25, 2005 AMPK as a Potential Target for Treatment of Cancer FIGURE 5. AICAR induces AMPK and ACC phos- phorylation in vitro and in vivo. A, C6 glioma and PC3 prostate cells were treated with AICAR at indi- cated concentrations and harvested at specified time points, cell lysates were processed for the detection of phospho-AMPK (p-Thr-172) and phospho-ACC by immunoblot as discussed under “Materials and Methods.” The blots are represen- tatives of three individual experiments done. B, cell lysates were prepared from the vehicle- and AICAR-treated tumor tissues from two different set of animals (each set had n  6) and processed for the detection of phospho-AMPK (p-Thr-172) and phospho-ACC as above. medium for growth and treated with different concentrations of AICAR liferating cell nuclear antigen), a marker for proliferating cells. AICAR (0.25–1 mM) for 24 h, and cell proliferation was examined by [ H]thy- significantly reduced the expression and number of cells exhibiting midine uptake. AICAR inhibited the proliferation of all cell lines tested PCNA expression, indicating that the number of proliferating cells is significantly in a dose-dependent manner (Fig. 1). All tested cell lines reduced by AICAR treatment in vivo as demonstrated by immunohis- underwent significant proliferation inhibition, indicating that this phe- tochemistry and its expression by Western blot (Fig. 4, B and C). Thus, nomenon is widespread and not limited to a specific cell type/line. To the anti-proliferative effect of AICAR is effective in vivo as well and can further confirm this observation, a clonogenic assay was performed, be exploited for applications in attenuating cancer cell growth. where cells were treated with AICAR for 24 h, trypsinized and plated at AICAR Mediates Its Anti-proliferative Action via AMP-activated Pro- a density of 300 cells/100-mm plate without AICAR. After 3 weeks, tein Kinase—AICAR, is the earliest known AMPK activator, and most of formed colonies were counted by staining the live cells with MTT. its effects have been shown to be because of AMPK activation, although AICAR treatment significantly reduced the number of colonies being few reports of its AMPK-independent effects exist (22). To investigate if formed as compared with the untreated cells (Fig. 2), suggesting that a AMPK activation is responsible for the anti-proliferative effects single treatment of AICAR treatment is sufficient to cause a sustained observed by AICAR treatment, the phosphorylation of AMPK and its inhibition of proliferation in different cancer cell lines. downstream target, ACC, an enzyme in the fatty acid synthesis pathway, AICAR Causes Cell Cycle Arrest in S-phase—Inhibition in prolifera- was taken as an indicator of AMPK activation. AICAR induced the tion would indicate an anomaly in the cell cycle. To examine this, cells phosphorylation of AMPK and ACC in a dose- and time-dependent were treated with AICAR (0.5–1 mM), and phases of cell cycle were manner as demonstrated in C6 glioma and PC3 prostate cell lines (Fig. 5, analyzed by flow cytometry. Treatment of cells with AICAR resulted in A and B). Similar phosphorylation of ACC and AMPK was observed in accumulation of cells in S-phase (peak with slashed bars), with almost vivo, in the AICAR-treated excised tumor tissue (Fig. 5C). Iodotuberi- no cells detected in M-phase (third peak) suggesting that inhibition cidin is an inhibitor of adenosine kinase and inhibits the conversion of in proliferation by AICAR is due to the arrest of cell cycle at S-phase AICAR to its activated form ZMP inside the cell and thus inhibits acti- (Fig. 3). vation of AMPK by AICAR. Cells were pretreated with iodotubericidin AICAR Inhibits Proliferation in Vivo—To investigate whether the 30 min before the addition of AICAR (0.5–1 mM), and proliferation was anti-proliferative effects of AICAR extends to the in vivo system, we measured after 16 h by [ H]thymidine uptake. Iodotubericidin treat- utilized the rat flank tumor model (34). Wherein, C6 glioma cells (1  ment inhibited the proliferation arrest caused by AICAR thus indicating 10 ) were implanted aseptically in the right flank of the rat, and after 5 the involvement of AMPK (Fig. 6A). To further confirm the role of days of tumor formation, animals were treated with 100 mg/kg body AMPK, C6 glioma cells were transiently transfected with dominant neg- weight of AICAR intraperitoneally. On day 14, animals were sacrificed, ative (DN) and constitutive active (CA) forms of AMPK along with and the tumors were excised, weighed, and fixed. Weight of the tumors eGFP expression vector. The cells were treated with AICAR for 18 h, was taken as an index of tumor development and progression. AICAR and GFP-positive cells were analyzed by flow cytometry for DNA con- treatment was able to reduce the growth of tumors in animals signifi- tent to determine the cells in S-phase. C6 glioma cells transfected with cantly (50%) when compared with untreated animals (Fig. 4A). To AMPK dominant negative were not able to undergo S-phase arrest examine the status of proliferating cells in vivo, immunohistochemistry when treated with AICAR (Fig. 6B). Inversely, C6 glioma cells trans- was performed on the sections of excised tumor tissues for PCNA (pro- fected with expression vector of constitutive active AMPK1 were NOVEMBER 25, 2005• VOLUME 280 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 39587 AMPK as a Potential Target for Treatment of Cancer FIGURE 6. AICAR mediated its anti-proliferative action via AMPK. A, cells (CEM, K-526, and PC-3) were pretreated with iodotubericidin (0.1 M) before addition of AICAR and assayed for [ H]thymidine incorporation. Iodotubericidin reversed the AICAR-induced proliferation block. The data are representative of three separate experiments performed in triplicates. ***, p 0.001 compared with control; ###, p 0.001 compared with AICAR (B and C) C6 glioma cells were transiently co-transfected with 2 g of AMPK dominant negative (DN)(B) or AMPK constitutive active (CA)(C) and 1 g of eGFP expression vector. The DNA content was normalized by pcDNA3. AICAR was added where indicated, and after 18 h cells were fixed overnight and analyzed for arrest in S-phase as detailed under “Materials and Methods.” AICAR was not able to arrest the cells in the presence of AMPK DN (B), whereas AMPK CA expression was sufficient to arrest the cells in S-phase and showed additive effect with AICAR (C). The data are representative of three separate experiments. ***, p 0.001 compared with control; ###, p 0.001 compared with AICAR; ##, p 0.01 compared with AICAR; #, p 0.05 compared with AICAR; NS, non-significant compared with control. D, cells were transfected with AMPK antisense (AS) and missense (MS) oligonucleotides along with eGFP expression vector and treated with AICAR. The level of AMPK protein was reduced by the transfection of AS, whereas MS had no effect (inset) at 72 h. The cells were fixed and processed for GFP-gated S-phase arrest. Antisense of AMPK abolished the AICAR-mediated S-phase arrest, whereas MS oligonucleotide had no effect. These data are representative of 3 separate experiments. ***, p  0.001 compared with control; ###, p  0.001 compared with AICAR; NS, non-significant compared with AICAR. 39588 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 47 •NOVEMBER 25, 2005 AMPK as a Potential Target for Treatment of Cancer FIGURE 7. AICAR inhibits PI3K-Akt Pathway. A, C6 cells were treated with AICAR (1 mM) for 2 h and processed for PI3K activity as described under “Materials and Methods.” AICAR significantly reduced the PI3K activity as assessed by inositol 1,4,5-bisphosphate levels. The inositol 1,4,5- bisphosphate levels were measured by densitom- etry analysis. The blot is representative of three separate experiments. **, p 0.01 compared with control (B) C6 cells were treated with AICAR for increasing time points as indicated, cells lysates were prepared and analyzed for Akt (Ser-473) and mTOR (Ser-1448) phosphorylation by Western blot as detailed under “Materials and Methods.” C, cell lysates were prepared from the treated tumor tissue from two different set of animals and pro- cessed for the detection of phospho-Akt, which was reduced by AICAR treatment. FIGURE 8. AICAR regulates the expression of cdk inhibitors via AMPK. Protein expression of cell cycle inhibitors p21, p27, and p53 was increased by AICAR treatment as analyzed by immunoblot in C6 cells treated with AICAR (A) and tumor tissue (B). C, immunofluorescent microscopy images of tumor sections from vehicle and treated rats, stained with p21, p27, and p53 antibodies as described under “Materials and Methods.” D,C6 glioma cells were transiently transfected with 2 g of AMPK dominant negative (DN) or AMPK consti- tutive active (CA) with DNA normalization done with pcDNA and treated with AICAR where indi- cated. Cell lysates were prepared and assessed by immunoblot for p21, p27, and phospho-Akt expressions. AICAR induced p21 and p27 expres- sion and down-regulated phospho-Akt as before (lane 2), which was reversed by AMPK DN expres- sion (lane 3) and had no effect with AICAR treat- ment (lane 4). AMPK CA 1 and 2 overexpression was able to induce the p21 and p27 expression and attenuate phospho-Akt by itself (lanes 5 and 6). The blots are representatives of three individual experiments done. NOVEMBER 25, 2005• VOLUME 280 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 39589 AMPK as a Potential Target for Treatment of Cancer found to be arrested at S-phase similar to AICAR-treated cells (Fig. 6C). Further, we used the antisense (AS) approach to knock out the expres- sion of AMPK. C6 glioma cells were transfected with AMPK antisense (AS) and missense (MS) oligonucleotides, and levels of AMPK were observed 48 h post transfection. The level of AMPK protein was atten- uated by AS, whereas MS had no effect (Fig. 6D, inset). Moreover, trans- fection with antisense of AMPK along with GFP expression vector in C6 glioma cells significantly reduced the AICAR-mediated S-phase arrest; however, MS oligonucleotide did not affect the potential of AICAR to arrest cells in S-phase (Fig. 6D). Taken together, these evidences point strongly toward a role for AMPK as an effective anti-proliferative system. AICAR/AMPK Inhibits the PI3K-Akt Pathway—Because AICAR inhibits cell proliferation and PI3K-Akt is one of the most important pathways regulating proliferation, we examined the effect of AICAR on the PI3K-Akt pathway. C6 glioma cells were treated with AICAR (1 mM) for 2 h, and PI3K activity was assessed using phosphoinositol as a sub- strate, and we observed that AICAR treatment significantly reduced the PI3K activity (Fig. 7A). One of the downstream effectors of PI3K, Akt is the main mediator regulating proliferation (39). AICAR also reduced the phosphorylation of Akt in vitro and in vivo (Fig. 7, Bi and C). It also inhibited the phosphorylation (Ser-1448) of mTOR (Fig. 7Bii), which is a downstream target of Akt and regulates protein synthesis and cell growth (40). Thus, attenuation of the PI3K-Akt pathway may be one of the mechanisms by which AMPK negatively regulates growth. AICAR Regulates the Expression of Cyclin-dependent Kinase Inhibi- tors via AMPK—Because AICAR inhibits the cell proliferation by arresting cells at S-phase in vitro as well as in vivo, we examined the expression of cyclin-dependent kinase (cdk) inhibitors, which bind to cyclin-cdk complexes and inhibit the progression of cell cycle. AICAR induced the expression of p21 and p27, the cip/kip protein cdk inhibi- tors in a time-dependent manner (Fig. 8A). It also induced the expres- sion of p53, which is known to regulate the cell cycle as well as p21 expression (41). The expression of p21, p27, and p53 proteins, were also increased in vivo, as assessed by immunohistochemistry of tissue sec- tions and by Western blot analysis of protein isolated from excised tumor tissue (Fig. 8, B and C). The effect of AICAR on the expression of growth regulators is mediated via activation of AMPK, because trans- FIGURE 9. LKB (AMPKK) status does not affect AICAR mediated growth arrest. A, LKB / / fected AMPK DN abolished the AICAR-mediated induction of p21, knock-out (LKB ) and wild-type (LKB ) mouse embryo fibroblast were treated with AICAR and analyzed by immunoblot for AMPK and ACC phosphorylation. The blots are whereas the CA form of AMPK1 and 2 induced the expression by representatives of three individual experiments done. B, LKB knock-out (LKB ) and itself (Fig. 8D). In case of p27, AMPK DN reduced the AICAR-induced wild-type (LKB ) mouse embryo fibroblast were treated with varying concentrations of AICAR and assayed for [ H]thymidine incorporation. The data are representative of expression, but AMPK1 and 2 CA forms were only able to induce p27 three separate experiments. ***, p  0.001 compared with control. C, C6 glioma cells protein marginally compared with AICAR. In case of Akt, DN were transiently transfected with 1 gof LKB dominant negative (DN)or LKB wild type AMPK2-transfected cells did not respond to the AICAR-mediated (WT) with STRAD (0.5 g) and MO25 (0.5 g) expression vectors, eGFP, and pcDNA and treated with AICAR where indicated. After 18 h cells were fixed overnight and ana- inhibition in Akt phosphorylation, whereas, in CA-transfected cells, lyzed for arrest in S-phase as detailed under “Materials and Methods.” there was significant inhibition (Fig. 8E), indicating that AMPK activa- tion is responsible for increase in cdk inhibitor protein expressions and inhibition of Akt phosphorylation. S-phase arrest observed when C6 cells were transiently transfected with LKB (AMPKK) Status Does Not Affect AICAR-mediated Growth LKB dominant negative and wild-type expression vector along with Arrest—LKB is a recently discovered upstream target of AMPK (AMPK expression vectors of its cofactors, STRAD and MO25, and treated kinase, AMPKK), which phosphorylates AMPK at Thr-172 for its full with AICAR (Fig. 9C). These data indicate that AMPK activation by activation. LKB itself is a tumor suppressor gene and inactivation of LKB AICAR is sufficient to cause growth arrest and does not require activa- results in predisposition to various cancers (27–29). It is being hypoth- tion by LKB. esized that the anti-tumor effects of LKB are due to AMPK activity. To DISCUSSION examine the possible involvement of LKB in AICAR/AMPK-induced growth arrest, we utilized LKB knock-out (LKB ) and LKB WT In this study we have demonstrated that AMPK activation by AICAR (LKB ) MEF cell lines. AICAR was able to induce the phosphoryla- results in growth arrest at S-phase due to inhibition of PI3K-Akt path- tion of AMPK and ACC to a similar extent in both MEFs (Fig. 9A). way and up-regulation of cdk inhibitors, independent of its upstream AICAR was able to inhibit proliferation in both knock-out and wild-type kinase LKB. This inference is based on the following observations: 1) MEFs to a similar extent (Fig. 9B). This was further supported by similar Treatment of various cancer cell lines by AICAR attenuated the prolif- 39590 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 47 •NOVEMBER 25, 2005 AMPK as a Potential Target for Treatment of Cancer FIGURE 10. A schematic representation of effect of AICAR on proliferation of cancer cells. AICAR, upon entering the cell, is converted to ZMP, which activates AMPK. Upon activation AMPK increases the expression of p21, p27, and p53 proteins, which may be responsible for the S-phase arrest being observed. On the other hand it inhibits the PI3K activity and Akt phosphorylation, which results in inhibition of mTOR and speculative reg- ulation of other targets like MDM2, Bad, and caspase 9, resulting in a proliferation and cell growth block. The overall signaling taking place results in a sustained proliferation arrest, which can ultimately lead to loss of viability due to onset of senescence or apoptotic pathways. eration both in vitro and in vivo studies. 2) The attenuation of cell These evidences point strongly toward AMPK activation being the proliferation was due to the activation of AMPK as evident from aden- major cause of growth arrest. Thus, AMPK can be considered as a neg- osine kinase inhibitor studies (iodotubericidin), the use of expression ative regulator of proliferation and can modulate protein expression to vectors (dominant negative and constitutive active) and AMPK anti- this effect, classifying it as a tumor suppressor system that can be sense experiments. 3) The growth arrest is mediated by inhibition of exploited for attenuation of cancers. PI3K activity and Akt phosphorylation and up-regulation of cell-cycle Activation of AMPK by AICAR, metformin, or thiazolidinediones or inhibitor proteins p21, p27, and p53. 4) Activation of AMPK in the expression of constitutively active mutants has been shown to cause absence of LKB also results in growth arrest. We show here the direct death or attenuate the growth of cancer cells. AICAR- and rosiglita- relation between AMPK activation and growth inhibition in vitro and in zone-mediated AMPK activation caused proliferation block and cell vivo. In addition, these observations strongly indicate AICAR, an AMPK death by inhibiting fatty acid and protein synthesis pathways and activator to be an efficient anti-proliferative agent in vitro and in vivo. increasing p21 expression in prostate cells (22). Adenosine-induced Being a pharmacological activator of AMPK, AICAR has been used AMPK was shown to cause apoptosis in gastric cancer cells (20), and extensively to study its role in physiology (13, 14). It has recently been activation of AMPK by AICAR and its CA form was shown to cause shown to have anti-inflammatory properties that were reported to be apoptosis in pancreatic cells by inducing JNK pathway (23). Similarly, mediated by AMPK activation (36, 42), although its AMPK-independ- AMPK induced JNK and caspase 3 activity resulting in apoptosis in liver ent effects have also been reported (22). In our study, AICAR mediates cells (24). AMPK activation was also demonstrated to enhance H O - 2 2 its effect via activation of AMPK, which is supported by both pharma- mediated apoptosis in neuroblastoma cells by inducing NF- and p38- cological (iodotubericidin) as well as molecular approaches (DN, CA, JNK pathways (25). These studies, along with the present study, suggests and AS AMPK transfections). Treatment of iodotubericidin, inhibitor AMPK as an efficient growth inhibitor and apoptosis inducer. On the of conversion of AICAR to ZMP, abrogated the S-phase arrest of cells other hand, it also has been shown to have a protective effect on stress- (Fig. 6A). Moreover, inhibition of AMPK either by its DN expression injured cells in heart ischemia and reperfusion injury model (15, 18). vector or AS oligonucleotide also resulted in the reversal of AICAR- AMPK activation protects primary astrocytes from fatty acid-induced mediated growth arrest, whereas AMPK CA expression was able to death by inhibiting de novo ceramide synthesis (17) and protects human accumulate cells in S-phase (Fig. 6, B–D). AMPK DN also blunted the umbilical vein endothelial cells from hyperglycemia by inhibition of elevated expression of p21 and p27, whereas AMPK CA itself was able to caspase 3 and Akt activation (18) and by similar mechanism in thymo- induce their expression (Fig. 8). The induction of p27 by AMPK CA 1 cytes (19). In pancreatic cancer cells, AMPK was shown to bestow tol- and 2 seems to be marginal as compared with p21. The observation erance toward nutrient deprivation (43). These studies presented represented here is consistent and reproducible. Right now we do not AMPK as a protective agent. The reason for these apparently opposing have an explanation for this disparity. One of the possible explanations effects of AMPK activation in cell survival and cell death is not known, could be that p27 regulation is related to the differential localization of but it can be speculated that in actively dividing cancer cells, the inhibi- the 1 (cytosol) and 2 (nuclear) isoforms of the catalytic subunits of tion of ATP-consuming processes by AMPK may be less compatible AMPK, which is yet to be established in terms of p27 regulation. AICAR with their survival, whereas in non-dividing cells, where the protective seems to affect the PI3K-Akt proliferation pathway, because AICAR and effects of AMPK have been observed under acute stress, the shutdown AMPK CA inhibited PI3K activity and Akt phosphorylation (Fig. 7). of ATP-consuming pathways may not alter the balance for survival. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Nov 25, 2005

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