ORIGINAL RESEARCH ARTICLE

Journal of

Critical Role of AMPK/FoxO3A Axis in Globular AdiponectinInduced Cell Cycle Arrest and Apoptosis in Cancer Cells

Cellular Physiology

ANUP SHRESTHA,1 SAROJ NEPAL,1 MI JIN KIM,1 JAE HOON CHANG,1 SANG-HYUN KIM,2 GIL-SAENG JEONG,3 CHUL-HO JEONG,3 GYU HWAN PARK,4 SUNGHEE JUNG,5 JAECHEONG LIM,5 EUNHA CHO,5 SOYOUNG LEE,5 AND PIL-HOON PARK1* 1

College of Pharmacy, Yeungnam University, Gyeongsan, Republic of Korea

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Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea

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College of Pharmacy, Keimyung University, Daegu, Republic of Korea

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College of Pharmacy, Kyungpook National University, Daegu, Republic of Korea

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Radioisotope Research Division, Department of Research Reactor Utilization, Korea Atomic Energy Research Institute, Daejeon, Republic of Korea

Adiponectin predominantly secreted from adipose tissue has exhibited potent anti-proliferative properties in cancer cells via modulating cell cycle and apoptosis. FoxO3A, a Forkhead box O member of the transcription factor, plays a critical role in modulating expression of genes involved in cell death and/or survival. In this study, we investigated the role of FoxO3A signaling in anti-cancer activities of adiponectin. Herein, we have shown that treatment with globular adiponectin (gAcrp) increases p27 but decreases cyclinD1 expression in human hepatoma (HepG2) and breast (MCF-7) cancer cells. Gene ablation of FoxO3A prevented gAcrp-induced increase in p27 and decreased in cyclin D1 expression, and further ameliorated cell cycle arrest by gAcrp, indicating a critical role of FoxO3A in gAcrp-induced cell cycle arrest of cancer cells. Moreover, treatment with gAcrp also induced caspase-3/7 activation and increased Fas ligand (FasL) expression in both HepG2 and MCF-7 cells. Transfection with FoxO3A siRNA inhibited gAcrp-induced caspase-3/7 activation and FasL expression, suggesting that FoxO3A signaling also plays an important role in gAcrp-induced apoptosis of cancer cells. We also found that gene silencing of AMPK prevented gAcrp-induced nuclear translocation of FoxO3A in HepG2 and MCF-7 cells. In addition, suppression of AMPK also blocked gAcrp-induced cell cycle arrest and further attenuated gAcrp-induced caspase-3/7 activation, indicating that AMPK signaling plays a pivotal role in both gAcrp-induced cell cycle arrest and apoptosis via acting as an upstream signaling of FoxO3A. Taken together, our findings demonstrated that AMPK/FoxO3A axis plays a cardinal role in anti-proliferative effect of adiponectin in cancer cells. J. Cell. Physiol. 231: 357–369, 2016. © 2015 Wiley Periodicals, Inc.

Adipose tissue is considered as a dynamic endocrine organ as it secrets a wide range of hormone-like substances, collectively called adipokines. Among the various adipokines, adiponectin is the most abundant in the plasma (Swarbrick and Havel, 2008) and possesses diverse biological functions. In addition to its well-known functions of lipid metabolism, insulin sensitization (Yamauchi et al., 2002) and anti-inflammation (Ouchi and Walsh, 2007), a growing body of evidence highlights adiponectin possesses potent anti-tumor activities (Barb et al., 2007) through various mechanisms, including inhibition of metastasis, angiogenesis, and proliferation of cancer cells (Brakenhielm et al., 2004). In particular, recent studies have shown that adiponectin suppresses growth of cancer cells through different mechanisms (Kelesidis et al., 2006). For example, adiponectin causes cell cycle arrest, as well as induces apoptosis in cancer cells. However, the molecular mechanisms underlying these anti-proliferative effects of adiponectin in cancer cells are still largely unknown. Forkhead box O (FoxO) transcription factor family is involved in a number of biological activities (Salih and Brunet, 2008). Among the various FoxO members, FoxO3A has been shown to play an important role in regulation of cancer cell proliferation by inducing transcription of the target genes involved in cell death and/or survival of cancer cells. Transcriptional activity of FoxO3A is regulated by subcellular localization and DNA-binding property, as well as by protein © 2 0 1 5 W I L E Y P E R I O D I C A L S , I N C .

expression. Previous studies have demonstrated that various post-transcriptional modifications play a critical role in the regulation of transcriptional activity of FoxO3A (Calnan and Brunet, 2008; Zhao et al., 2011). For example, phosphorylation by Akt or SGK (serum and glucocorticoid-induced kinase) causes the localization of FoxO3A in the cytoplasm (Biggs et al., 1999), in contrast, phosphorylation by MST1 (mammalian Ste20-like kinases) and JNK1 causes nuclear translocation

Contract grant sponsor: National Research Foundation of Korea (NRF); Contract grant number: 2013R1A1A4A01011110. Contract grant sponsor: KAERI Major Project, Development of Radioisotope Production and Application Technology; Contract grant number: 525140-15. *Correspondence to: Pil-Hoon Park, College of Pharmacy, Yeungnam University, Gyeongsan, Republic of Korea. E-mail: [email protected] Manuscript Received: 14 February 2015 Manuscript Accepted: 15 June 2015 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 18 June 2015. DOI: 10.1002/jcp.25080

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(Lehtinen et al., 2006), suggesting that FoxO3A localization is determined by the specific residue(s) phosphorylated by a certain type of kinases. Likewise, acetylation by p300 causes activation, while deacetylation by SIRT1 induces repression of the FoxO3A signaling (Motta et al., 2004). In addition, protein expression level of FoxO3A is adjusted by the proteasomal degradation triggered by polyubiquitination (Yang et al., 2008). Previous studies have also shown that post-transcriptional modifications, including phosphorylation and acetylation modulate DNA-binding property of FoxO and thus its transcriptional activity (Zhang et al., 2002). In addition to the critical role in energy homeostasis, AMPactivated protein kinase (AMPK) contributes to the diverse biological responses via phosphorylating set of target molecules. Recent studies have shown that AMPK induces phosphorylation and subsequent activation of FoxO3A, and further suggest that AMPK/FoxO3A axis plays a critical role in the regulation of cancer cell growth (Chiacchiera and Simone, 2009, 2010). It has been also reported that globular adiponectin (gAcrp) treatment causes nuclear translocation of FoxO3A in HepG2 cells, which in turn leads to autophagy induction, suggesting that FoxO3A would be a promising target mediating the physiological functions of gAcrp (Nepal and Park, 2013). Although previous studies have suggested that FoxO3A plays an important role in the regulation of cancer cell proliferation and adiponectin activates FoxO3A, the role of FoxO3A signaling in adiponectin-induced modulation of cancer cell proliferation has not been determined. Cell proliferation is a highly sophisticated phenomenon involving a number of regulatory proteins, including cyclins, cyclin-dependent kinases (Cdk), their substrate proteins, and the Cdk inhibitors (CKI) (Golias et al., 2004). In neoplasia, genes regulating cell proliferation, apoptosis, and differentiation are altered and control mechanisms are lost. Among the various genes regulating cell cycle, cyclins bind with Cdks and form cyclin–Cdk complex which activates cell cycle via phosphorylation of subset of target genes. In particular, Cyclin D (D1, D2, and D3) modulates G1/S-phase transition by activating the cyclin-dependent kinases leading to the phosphorylation of retinoblastoma protein. Cyclin D– Cdk4/6 complexes activates cyclin E/Cdk2 through titration of the Cdk inhibitors p21Cip1 and p27Kip1 resulting in progression of cell cycle phase from G0–G1 to S phase (Massague, 2004). Therefore, amplification or overexpression of cyclin D has been frequently observed in the occurrence of different types of cancer (Diehl, 2002). In addition, p27, a member of the Kip/Cip family of CKIs, binds to and prevents the activation of cyclin E-CDK2, thereby controlling the cell cycle progression at G1 phase (Sherr and Roberts, 1999). Therefore, p27 has been considered as a tumor suppressor gene. Dysfunction of p27 causes over-activation of the cell cycle and contributes to the development of various tumors (Fero et al., 1996). Proliferation of cancer cell can be also regulated by apoptosis, as well as by controlling cell cycle. Extrinsic pathway of apoptosis is initiated by binding of Fas ligand (FasL) or TNF-a with their cognate receptor, while intrinsic pathway is initiated by release of cytochrome C from mitochondria into the cytosol. After the activation of series of signaling cascade, both pathways finally result in activation of caspase-3, the final executioner of apoptotic cell death (Elmore, 2007). Recent evidences have suggested that FoxO3A signaling is associated with induction of apoptosis via modulation of the factors involved in intrinsic and extrinsic pathways of apoptosis (Zhang et al., 2011). For instance, FoxO3A signaling is implicated in free fatty acid-induced apoptosis of hepatocytes mainly via induction of Bim, a pro-apoptotic member of the BCL-2 protein family (Barreyro et al., 2007), while it activates extrinsic pathway via modulation of TRAIL and DR-4 and DR-5, which JOURNAL OF CELLULAR PHYSIOLOGY

play a critical role in resveratrol-induced apoptosis in prostate cancer cells (Chen et al., 2010). Adiponectin is considered as a promising endogenous molecule to regulate cancer progression through a number of different mechanisms. However, the underlying molecular mechanisms are not clearly understood. In the present study, we have investigated the critical role of FoxO3A signaling in gAcrp-induced both inhibition of cancer cell proliferation and induction of apoptosis. We have demonstrated the evidence that AMPK/FoxO3A pathway plays a critical role in adiponectin-induced inhibition of cell proliferation by decreasing cyclin D1 and increasing p27 expression, and also induction of apoptosis by inducing Fas ligand expression both in human hepatoma and breast cancer cells. Materials and Methods Materials All the cell culture reagents were purchased from Hyclone Laboratories (South Logan, UT). Recombinant human globular adiponectin (gAcrp) was procured from Peprotech, Inc. (Rocky Hill, NJ). Cell Titer 96 AQueous one solution cell proliferation assay kit (MTS) and caspase-3/7 activity assay kit and caspase-8 activity assay kit were purchased from Promega Corporation (Madison, WI). CyclinD1, p21, cyclin E polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FoxO3A, phospho-AMPK, total AMPK, phospho-Liver kinase B1 (LKB1), total LKB1, FasL, p27 Kip1, and b-actin antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Secondary antibodies were from Pierce biotechnology (Rockford, IL). Cycle test plus DNA reagent kit was purchased from BD (San Jose, CA). Cell culture Both human hepatoma cancer cells (HepG2) and human breast cancer cells (MCF-7) were purchased from American Type Culture Collection (ATCC, Rockville, MD). The HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 1% penicillin–streptomycin, and 0.1% amphotericin. MCF-7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37°C and 5% CO2 in an incubator with a humidified atmosphere. Cell viability measurement For the determination of cell viability, cells were seeded at the density of 4
104 cells per well in 96-well plates. After overnight incubation, cells were washed with phosphate-buffered saline (PBS) once and then treated with globular adiponectin with the indicated concentrations for the indicated time period. Then, 20 ml of MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) solution was added and then cells were incubated for 2 h at 37°C. The resultant cell viability was determined by measuring absorbance at 490 nm using Versamax microplate reader (Sunnyvale, CA). Caspase-3/7 and caspase-8 activity assay Caspase-3/7 and caspase-8 activities were assessed by using Caspase-Glo 3/7 and Caspase-Glo 8 assay kits (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. In brief, cells were seeded at a density of 4
104 cells per well in 96-well plate in duplicate. After overnight incubation, cells were treated with indicated concentrations of globular adiponectin for indicated time periods. Finally, the luminescence was measured from the cleavage of luminogenic substrate AcDEVD-pNA and LETD sequence for caspase-3/7 and caspase-8,

FoxO3A IN CANCER CELL DEATH BY ADIPONECTIN

TABLE 1. Sequences of primers used in quantitative RT-PCR Target gene GAPDH p27 CyclinD1 p130 CyclinE1 p21 Fas ligand

Primer

Nucleotide 50 -ACCACAGTCCATGCCATCAC-30 50 -TCCACCACCCTGTTGCTGTA-30 50 - AGATGTCAAACGTGCGAGTG-30 50 -TCTCTGCAGTGCTTCTCCAA-30 50 -CCTAAGTTCGGTTCCGATGA-30 5’-ACGTCAGCCTCCACACTCTT-3’ 50 -AGAACCTGGAAAGGGCAGAT-30 50 -CTGGTGGGGAGCTGTACCTA-30 50 -ATCCTCCAAAGTTGCACCAG-30 50 -AGGGGACTTAAACGCCACTT-30 5’-ATGAAATTCACCCCCTTTCC-3’ 50 -CCCTAGGCTGTGCTCACTTC-30 50 -CCAGCTTGCCTCCTCTTGAG-30 50 -TCCTGTAGAGGCTGAGGTGTCA-30

F R F R F R F R F R F R F R

respectively, with a micro-plate reader (Flurostar Optima, BMG Labtech, Ortenberg, Germany). Quantitative real time-PCR (qPCR) Total RNA was isolated using Qiagen lysis solution according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed for the synthesis of cDNA. Real time-PCR amplification was then performed with a Roche LightCycler 2.0 (Mannheim, Germany) using the absolute qPCR SYBR green capillary mix AB gene system (Thermo scientific, Glasgow, UK) at 95°C for 15 min followed by 40 cycles at 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec. The primer sequences used for amplification of target genes are listed in Table 1. Transient transfection with small interfering RNA (siRNA) Cells were transfected with corresponding siRNA for target gene or scrambled control siRNA with Hiperfect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The siRNA duplexes used for this study were chemically synthesized by Bioneer (Daejeon, South Korea) and are listed in Table 2. Gene silencing efficiency was assessed by Western blot analysis after 48 h of transfection. Western blot analysis Total cellular extracts were prepared by lysis of cells in RIPA buffer containing halt protease inhibitor cocktail (Thermoscientific, Rockford, IL). The nuclear and cytosolic fractions of the cells were prepared by lysis of the cells using subcellular fractionation buffer (250 mM sucrose, 20 mM HEPES pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and halt protease inhibitor cocktail). The cellular lysates were then passed through 25G needle 10–15 times for homogenization followed by centrifugation at 720g for 5 min at 4°C. The remaining supernatants were centrifuged at 10,000g for 15 min at 4°C. The

TABLE 2. Sequences of small interfering RNA used in transfection Target gene FoxO3A AMPKa LKB1 Scrambled Control

Primer F R F R F R F R

Nucleotide 5 -GACGAUGAUGCGCCUCUCU-30 50 -AGAGAGGCGCAUCAUCGUC-30 50 -CUGAGUUGCAUAUACUGUA-30 50 -UACAGUAUAUGCAACUCAG-30 50 -CGUGUGUAUGAACGGCACA-30 50 -UGUGCCGUUCAUACACACG-30 50 -CCUACGCCACCAAUUUCGU-30 50 -ACGAAAUUGGUGGCGUAGG-30

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cellular debris was then removed and the obtained supernatants were taken as cytosolic fractions. The nuclear fraction was prepared by lysing the nuclear pellet using RIPA buffer followed by centrifugation at 10,000g for 15 min and the obtained supernatants were taken as nuclear fraction. All the procedures mentioned above were carried out in ice. For immunoblot analysis, 30 mg of solubilized protein obtained was separated by SDS–PAGE and transferred to PVDF membrane. The membrane was then blocked with 5% skimmed milk, or 5% BSA was used for detecting phosphorylated protein. After that, the membrane was incubated with the respective primary antibodies (all antibodies are diluted 1:1,000 in 3% BSA) for overnight and then incubated with secondary horseradish peroxidase labeled antibodies. Chemiluminescent images of the blots were finally obtained using a Fujifilm LAS-4000 mini (Fujifilm, Tokyo, Japan). The membranes were then stripped and reprobed with b-actin as the loading control. Cell cycle analysis Cell cycle analysis was performed using Cycle test plus DNA reagent kit (BD, San Jose, CA) according to the manufacturer’s instructions. Briefly, cells were seeded at a density of 2
105 cells per 35 mm dish. After the required duration of globular adiponectin treatment, the cells were washed with PBS and collected by centrifugation for 5 min at 300g at room temperature. Buffer solutions, solution A and solution B, were sequentially added according to the instruction. Finally, 200 ml of solution C (Propidium Iodide) was added and incubated for 10 min in the dark. DNA content of the stained cells was then analyzed by a flow cytometer (BD FACSVerse). The distribution of cells in each cell cycle phase was determined using Flow Jo X software. Statistical analysis Values are presented as mean  SEM of at least three independent experiments. Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests using GraphPad prism software version 5.01 (CA, USA). Differences between groups were considered to be significant at P < 0.05. Results Globular adiponectin decreases cell viability in human hepatic and breast cancer cells

Previous studies have shown that adiponectin inhibits proliferation of many different cancer cells (Dalamaga et al., 2012). Before investigating the potential mechanisms underlying, we first confirmed inhibitory effect of globular adiponectin (gAcrp) on the viability of human hepatoma (HepG2) and breast cancer cells (MCF-7) in our experimental condition. We found that gAcrp treatment decreased cell number in both HepG2 (Fig. 1A) and MCF-7 cells (Fig. 1B) in a time-dependent manner consistent with previous reports. Globular adiponectin induces cell cycle arrest and modulates expression of p27 and cyclin D1

The rate of cancer cell viability can be determined by cell cycle progression and apoptosis. To further analyze the underlying cause of the decrease in the cell viability, we first examined the effect of gAcrp on cell cycle. As shown in Figure 2, treatment of cells with gAcrp gradually increased the number of cells in G0– G1 phase, while the number in S and G2-M phase gradually decreased both in HepG2 (Fig. 2A) and MCF-7 (Fig. 2B) cells as expected, indicating that gAcrp arrests the cell growth in G0– G1 phase of cell cycle. Cell cycle is a complex process that requires the proper coordination of various genes (Vermeulen et al., 2003). In order to further elucidate the mechanisms

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Fig. 1. Effects of globular adiponectin on cell viability in human hepatic and breast cancer cells. HepG2 cells (A) and MCF-7cells (B) were treated with gAcrp (1 mg/ml) for the indicated time periods. MTS assay was carried out as described in the materials and methods. The values represent fold change of the viable cells compared to the cells not treated with adiponectin. All data are expressed as the mean  SEM of three independent experiments.  P < 0.05 compared to the control group.

underlying cell cycle arrest by gAcrp, we examined the effects of gAcrp on the expression of genes regulating cell cycle. As shown in Figure 2, treatment of HepG2 cells with gAcrp increased expression of p27, an inhibitor of cyclin-dependent kinase, in HepG2 cells at both mRNA (Fig. 2C) and protein (Fig. 2D) level in a time-dependent manner. Treatment with gAcrp also enhanced expression level of p27 in MCF-7 cells both at mRNA and protein level (Fig. 2E and F), while it decreased expression of cyclin D1 both in HepG2 (Fig. 2G) and MCF-7 cells (Fig. 2H). However, we observed that gAcrp did not significantly affect expression of other cell cycle regulating genes, including p21 (cyclin-dependent kinase inhibitor 1A), p130 (retinoblastoma-like 2), and cyclin E1 in both HepG2 and MCF-7 cells (supplementary Fig. S2), indicating that gAcrp regulates expression of the genes involved in cell cycle in a gene-specific manner. FoxO3A signaling is involved in gAcrp-induced cell cycle arrest

FoxO3A, originally reported as a tumor suppressor gene, is known to modulate transcription of the genes regulating cell cycle and apoptosis of cancer cells (Huang and Tindall, 2007). We have previously shown that gAcrp treatment causes nuclear translocation of FoxO3A required for autophagy induction (Nepal and Park, 2013). With this information in consideration, we examined if FoxO3A signaling plays a role in the gAcrp-induced cell cycle arrest and further modulation of p27 and cyclin D1 expression. As depicted in Figure 3A, treatment with gAcrp caused nuclear translocation of FoxO3A consistent with previous observations. Furthermore, gene silencing of FoxO3A by transfection with siRNA restored gAcrp-induced decrease in cell number both in HepG2 (Fig. 3B) and MCF-7 cells (Fig. 3D). Knocking down of the FoxO3A gene also restored gAcrp-induced G0–G1 cell cycle phase arrest in HepG2 (Fig. 3C) and MCF-7 cells (Fig. 3E), providing a critical evidence for the role of FoxO3A signaling in gAcrp-induced cell cycle arrest. To further investigate the molecular mechanisms underlying, we examined the modulatory role of FoxO3A in the expression of p27 and cyclinD1. Gene silencing of FoxO3A by siRNA transfection in HepG2 cells resulted in inhibition of gAcrp-induced p27 mRNA expression (Fig. 3F). Likewise, JOURNAL OF CELLULAR PHYSIOLOGY

transfection with siRNA targeting FoxO3A resulted in restoration of decrease in cyclin D1 expression by gAcrp both in HepG2 (Fig. 3G) and MCF-7 cells (Fig. 3H), indicating the critical role of FoxO3A signaling in modulating the expression of the genes controlling cell cycle in cancer cells treated with gAcrp. FoxO3A signaling is implicated in gAcrp-induced apoptosis

Apoptosis and cell cycle are the two main factors dictating the rate of cell viability. Therefore, we also examined effects of gAcrp on apoptosis of cancer cells. As shown in Figure 4A and B, prolonged treatment with gAcrp caused activation of caspase-3/7 in HepG2 and caspase-7 in MCF-7 cells consistent with previous observations. However, gene silencing of FoxO3A by siRNA transfection significantly decreased the apoptotic effect of gAcrp in HepG2 cells (Fig. 4C), suggesting the pivotal role of FoxO3A signaling in gAcrp-induced apoptosis. Fas ligand (FasL), acting as an inducer of extrinsic pathway of apoptosis, is under the transcriptional regulation by FoxO3A (Brunet et al., 1999). To further elucidate the mechanisms underlying apoptosis induction by gAcrp, we examined the potential role of FoxO3A in the expression of Fas ligand. As depicted in Figure 4D and F, gAcrp treatment induced significant increase in the expression of FasL at mRNA level both in HepG2 and MCF-7 cells. We also observed increase in protein expression of FasL in HepG2 (Fig. 4E) and MCF-7 (Fig. 4G) cells. Furthermore, gAcrp treatment induced activation of caspase-8, acting as an intermediate for extrinsic pathways of apoptosis, in both HepG2 and MCF-7 cells (Fig. 4I), confirming the induction of extrinsic pathway of apoptosis by gAcrp. Gene silencing of FoxO3A blocked gAcrp-induced increase in FasL mRNA expression (Fig. 4H), suggesting that FoxO3A signaling plays a critical role in gAcrp-induced apoptosis probably via modulation of FasL gene expression. AMPK/FoxO3A signaling is required for gAcrp-induced cell cycle arrest in HepG2 cells

50 adenosine monophosphate-activated protein kinase (AMPK) is well known as a signaling mediator for wide range

FoxO3A IN CANCER CELL DEATH BY ADIPONECTIN

Fig. 2. Effects of globular adiponectin on cell cycle of HepG2 and MCF-7 cells and expression of p27 and cyclin D1. HepG2 cells (A) and MCF-7 cells (B) were treated with gAcrp (1 mg/ml) for indicated time periods. Cell cycle analysis was performed as mentioned in the materials and methods. Values are the percentage of cells in each of the three cell cycle phases and are expressed as mean  SEM (n ¼ 3) (upper part). Images are the representative of three independent experiments that showed similar results (lower panel).  P < 0.05 compared to control. HepG2 cells (C) and MCF-7 cells (E) were treated with gAcrp (1 mg/ml) for indicated time period. p27 mRNA expression level was determined by qRT-PCR and normalized to GAPDH mRNA as stated in materials and methods. Values represent fold changes compared to control and are expressed as mean  SEM, n ¼ 4.  P < 0.05 compared with control group. HepG2 cells (D and G) and MCF-7 cells (F and H) were treated with gAcrp (1 mg/ml) for the indicated time periods. p27 (D and F) and cyclin D1 (G and H) protein level was determined by Western blot analysis as described in materials and methods (upper part). Images are the representative of three independent experiments that showed similar results. Quantitative analysis of p27 & b-actin and cyclin D1 & b-actin expression was performed by densitometric analysis and shown in the below part. Values are presented as mean  SEM (n ¼ 3).  P < 0.05 compared with control group.

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of biological responses by adiponectin. Recent studies have shown that activation of AMPK signaling inhibits tumor growth and metastasis (Krishan et al., 2014). In a continuing study to identify the upstream signaling molecule mediating

FoxO3A activation and cell cycle arrest, we examined the involvement of AMPK signaling. For this, we first confirmed that treatment with gAcrp led to a rapid phosphorylation of AMPK in HepG2 (Fig. 5A) and MCF-7 cells (Fig. 5B). Furthermore, gene silencing of AMPK prevented gAcrpinduced nuclear translocation of FoxO3A in HepG2 (Fig. 5C) and in MCF-7 (Fig. 5D) in our experimental condition consistent with the previous report (Nepal and Park, 2013). The purity of cytoplasmic fraction was ascertained by measuring the level of lamin B1, a marker of the nuclear fraction. Lamin B1 was not detected to the significant level in the cytoplasmic fraction (Fig. 5D). We next investigated the functional role of AMPK signaling in gAcrp-induced cell cycle arrest. As shown in Figure 5E and F, gAcrp treatment caused increase in the percentage of cells in G0–G1 phase, while decrease in S and G2-M phase consistent with previous observations. However, cell cycle arrest induced by gAcrp treatment in HepG2 cells was significantly restored by transfection with siRNA targeting AMPK (Fig. 5E), suggesting the critical role of AMPK signaling in gAcrp-induced cell cycle arrest. Pretreatment with compound C, a pharmacological inhibitor of AMPK, showed the similar results to those from gene silencing of AMPK (Fig. 5F). Similarly, knockdown of AMPK gene also restored the suppression of cyclin D1 expression by gAcrp (Fig. 5G) and suppressed the gAcrp-induced increase in p27 mRNA expression (Fig. 5H). All these results indicate that AMPK/FoxO3A axis plays a crucial role in the gAcrpinduced cell cycle arrest in cancer cells probably via modulation of cyclinD1 and p27 expression.

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Fig. 3. Continued.

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Fig. 3. Role of FoxO3A signaling in gAcrp-induced cell cycle arrest in HepG2 and MCF-7 cells. (A) HepG2 cells were incubated with gAcrp (1 mg/ml) for the indicated time periods. Cytosolic and nuclear fractions were prepared as described in materials and methods and expression level of FoxO3A in each fraction was determined by Western blot analysis. Images are representative of three independent experiments that showed similar results. HepG2 cells (B) and MCF-7 cells (D) were transfected with siRNA targeting FoxO3A or scrambled control siRNA. After 36 h incubation, cells were lysed for the determination of FoxO3A siRNA transfection efficiency via Western blot analysis of FoxO3A protein expression (upper part). Cells were then treated with gAcrp (1 mg/ml) for 24 h and cell viability was determined by MTS assay as described previously. Values represent fold change compared with the cells not treated with globular adiponectin and are expressed as mean  SEM, n ¼ 3 (lower part).  P < 0.05 compared with the control group; #P < 0.05 compared with the cells treated with gAcrp and not transfected. HepG2 cells (C) and MCF-7 cells (E) were transfected with FoxO3A or scrambled control siRNA. Cells were then treated with gAcrp (1 mg/ml) for 24 h and cell cycle was analyzed using flow cytometer as described in materials and methods. Values are the percentage of cells in each of the three cell cycle phases and are expressed as mean  SEM (n ¼ 3) (upper part). Images are the representative of three independent experiments that showed similar results (lower panel).  P < 0.05 compared to control; #P < 0.05 compared with cells treated with gAcrp but not transfected. (F) HepG2 cells were transfected with FoxO3A or scrambled control siRNA and treated with gAcrp (1 mg/ml) for 8 h. p27 mRNA expression level was determined by qRT-PCR and normalized to GAPDH mRNA. Values represent fold changes compared to control and are expressed as mean  SEM (n ¼ 4).  P < 0.05 compared to control group; #P < 0.05 compared to cells treated with gAcrp and not transfected. HepG2 cells (G) and MCF-7 cells (H) were transfected with FoxO3A siRNA or scrambled control siRNA and treated with gAcrp (1 mg/ml) for 8 h. CyclinD1 protein expression level was determined by Western blot analysis as described previously (upper part). Images are representative of three independent experiments that showed similar results. Quantitative analysis of the cyclin D1 expression was performed by densitometric analysis and shown in the below part. Values are presented as mean  SEM (n ¼ 3).  P < 0.05 compared to control group; #P < 0.05 compared to cells treated with gAcrp and not transfected.

FoxO3A IN CANCER CELL DEATH BY ADIPONECTIN

Fig. 4. Involvement of FoxO3A signaling in gAcrp-induced apoptosis in HepG2 and MCF-7 cells. HepG2 cells (A) and MCF-7 cells (B) were treated with gAcrp (1 mg/ml) for the indicated time periods then caspase-3/7 activity was measured as described in the materials and methods. Values represent the fold change compared with the control group and are expressed as mean  SEM (n ¼ 4).  P < 0.05 compared to cells not treated with gAcrp. (C) HepG2 cells transfected with FoxO3A or scrambled control siRNA were treated with gAcrp (1 mg/ml) for 48 h and caspase-3/7 activity was determined as indicated in materials and methods. Values represent the fold change compared with control group and are expressed as mean  SEM (n ¼ 3).  P < 0.05 compared to cells not treated with gAcrp. #P < 0.05 compared to cells treated with gAcrp and not transfected. HepG2 cells (D) and MCF-7 cells (F) were treated with gAcrp (1 mg/ml) for the indicated time periods. Fas ligand (FasL) mRNA expression level was determined by qRT-PCR and normalized to GAPDH mRNA. Values represent fold changes compared to control group and are expressed as mean  SEM (n ¼ 4).  P < 0.05 compared with control group. HepG2 cells (E) and MCF-7 cells (G) were treated with gAcrp (1 mg/ml) for the indicated time periods. Cells were then lysed to determine the FasL protein expression level by Western blot analysis (upper part). Images are representative of three independent experiments that showed similar results. Quantitative analysis of the FasL expression was performed by densitometric analysis and shown in the lower part. Values are presented as mean  SEM (n ¼ 3).  P < 0.05 compared with control group. (H) HepG2 cells transfected with FoxO3A or scrambled control siRNA were treated with gAcrp (1 mg/ml) for 24 h. Fas ligand (FasL) mRNA expression level was then determined by qRT-PCR and normalized to GAPDH mRNA. Values shown are the results of four independent experiments and are expressed as mean  SEM.  P < 0.05 compared to control; #P < 0.05 compared to cells treated with gAcrp and not transfected. (I) HepG2 cells and MCF-7 cells were treated with gAcrp (1 mg/ml) for the indicated time periods. Caspase-8 activity was then measured as mentioned in materials and methods. Values represent the fold change compared with the control group and are expressed as mean  SEM (n ¼ 3).  P < 0.05 compared to cells not treated with gAcrp.

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AMPK/FoxO3A signaling is required for gAcrp-induced apoptosis

We next examined the implication of AMPK signaling in gAcrpinduced apoptosis of HepG2 cells. As shown in Figure 6A,

gAcrp-induced activation of caspase-3/7 in HepG2 cells was blocked by transfection of siRNA targeting AMPK. Similar results were also observed by pretreatment with compound C (Fig. 6B), indicating the involvement of AMPK signaling in apoptosis of cancer cells by gAcrp. In addition, gene silencing of AMPK also significantly suppressed gAcrp-induced FasL expression in HepG2 cells (Fig. 6C). All these results indicate that AMPK/FoxO3A axis plays an important role in the regulation of cancer cell proliferation by gAcrp through induction of apoptosis, as well as through cell cycle arrest. LKB1 acts as an upstream signaling molecule of AMPK phosphorylation in HepG2 and MCF-7 cells treated with gAcrp

LKB1 is known to act as a signaling molecule leading to the activation of AMPK (Hawley et al., 2003). To further identify the signaling pathways involved in gAcrp-induced activation of AMPK/FoxO3A axis, we investigated if LKB1 is required for AMPK activation. As presented in Figure 7, gAcrp treatment led to a very rapid phosphorylation of LKB1 at Ser428 in HepG2 cells (Fig. 7A) and MCF-7 cells (Fig. 7B), which preceded phosphorylation of AMPK. Gene ablation of LKB1 by transfection with siRNA almost completely inhibited gAcrpinduced phosphorylation of AMPK in HepG2 cells (Fig. 7C),

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Fig. 5. Continued.

JOURNAL OF CELLULAR PHYSIOLOGY

Fig. 5. Role of AMPK/FoxO3A signaling in gAcrp-induced cell cycle arrest in HepG2 cells. HepG2 cells (A) and MCF-7 cells (B) were treated with gAcrp (1 mg/ml) for the indicated time period. Cells were then lysed to determine the expression level of phosphorylated AMPKa (Thr172) by Western blot analysis (upper part). Images are representative of three independent experiments that showed similar results. Quantitative analysis of the p-AMPK expression was performed by densitometric analysis and shown in the below part. Values are presented as mean  SEM (n ¼ 3).  P < 0.05 compared to cells not treated with gAcrp. HepG2 cells (C) and MCF-7 cells (D) were transfected with siRNA specific for AMPKa or scrambled control and then treated with gAcrp (1 mg/ ml). After 24 h incubation, cells were lysed for the determination of AMPKa siRNA transfection efficiency via Western blot analysis of AMPKa protein expression (upper part). Cytosolic and nuclear fractions were separated as explained in materials and methods and the expression level of FoxO3A in each fraction was determined by Western blot analysis (lower part). Images are representative of three independent experiments that showed similar results. (E) HepG2 cells transfected with siRNA for AMPKa or scrambled control were treated with gAcrp (1 mg/ml) for 24 h. Cell cycle was then analyzed using flow cytometer as described in materials and methods. Values are the percentage of cells in each of the three cell cycle phases and are expressed as mean  SEM (n ¼ 3) (upper part). Images are the representative of three independent experiments that showed similar results (lower part).  P < 0.05 compared to cells not treated with gAcrp; #P < 0.05 compared with cells treated with gAcrp but not transfected. (F) HepG2 cells were pretreated with compound C (10 mM) followed by treatment with gAcrp (1 mg/ml) for 24 h. Cell cycle was then analyzed using flow cytometer. Values are the percentage of cells in each of the three cell cycle phases and are expressed as mean  SEM (n ¼ 3) (upper part). Images are the representative of three independent experiments that showed similar results (lower part).  P < 0.05 compared to cells not treated with gAcrp; #P < 0.05 compared with cells treated with gAcrp but not transfected. (G) HepG2 cells transfected with siRNA targeting AMPKa or scrambled control were treated with gAcrp (1 mg/ml) for 8 h. Cyclin D1 protein level was then determined by Western blot analysis (upper part). Images are the representative of three independent experiments that showed similar results. Quantitative analysis of the cyclin D1 expression was performed by densitometric analysis and shown in the below panel. Values are presented as mean  SEM (n ¼ 3). (H) HepG2 cells transfected with AMPKa or scrambled control siRNA were treated with gAcrp (1 mg/ml) for 8 h. Messenger RNA expression level of p27 was determined by qRTPCR and normalized to GAPDH mRNA. Values represent fold change compared to control and are expressed as mean  SEM (n ¼ 4).  P < 0.05 compared to cells not treated with gAcrp; #P < 0.05 compared to cells treated with gAcrp and not transfected.

FoxO3A IN CANCER CELL DEATH BY ADIPONECTIN

Fig. 6. Role of AMPK/FoxO3A signaling in gAcrp-induced apoptosis in HepG2 cells. (A) HepG2 cells were transfected with AMPKa siRNA or scrambled control siRNA and treated with gAcrp (1 mg/ml) for 48 h. Caspase-3/7 activity was then measured as indicated in materials and methods. Values shown are the results of three independent experiments and are expressed as mean  SEM (n ¼ 3).  P < 0.05 compared to cells not treated with gAcrp; #P < 0.05 compared with cells treated with gAcrp and not transfected. (B) HepG2 cells were pretreated with compound C (10 mM), a pharmacological inhibitor of AMPK, and then treated with gAcrp (1 mg/ml) for additional 48 h. Caspase-3/7 activity was then measured as indicated in materials and methods. Values represent fold change compared with the cells not treated with gAcrp and are expressed as mean  SEM (n ¼ 3).  P < 0.05 compared with the cells not treated with gAcrp; #P < 0.05 compared with cells treated with gAcrp and not transfected. (C) HepG2 cells transfected with AMPKa or scrambled control siRNA were treated with gAcrp (1 mg/ml) for 24 h. Fas ligand (FasL) mRNA expression level was determined by qRT-PCR and normalized to GAPDH mRNA. Values represent fold changes compared to control. Values shown are the results of three independent experiments and are expressed as mean  SEM.  P < 0.05 compared to cells not treated with gAcrp; #P < 0.05 compared with cells treated with gAcrp but not transfected.

verifying that LKB1 plays an important role in AMPK activation and thereby in the inhibition of cell viability by gAcrp. Discussion

Adiponectin has been increasingly reported to possess antitumor activities. The anti-proliferative capacity of adiponectin has been demonstrated in wide range of cells such as prostate (Bub et al., 2006), endometrium (Cong et al., 2007), breast cancer cells (Kang et al., 2005), etc. Adiponectin exhibits antitumor activity through various mechanisms, including inhibition of cell proliferation via regulating cell cycle and/or apoptosis (Kelesidis et al., 2006). The anti-tumor activity can also be achieved by its anti-angiogenic and anti-metastatic capacity (Brakenhielm et al., 2004; Saxena and Sharma, 2010). However, its molecular mechanisms are yet not clearly understood. In this study, we explored the possible underlying mechanism for the anti-tumor activity of adiponectin. Herein, we demonstrated the critical evidence that AMPK/FoxO3A signaling plays a crucial role in globular adiponectin (gAcrp)induced anti-proliferative activity in cancer cells. JOURNAL OF CELLULAR PHYSIOLOGY

In the present study, we presented that globular adiponectin inhibits the viability of human hepatoma (HepG2) and breast cancer (MCF-7) cells. This effect was shown in a dosedependent manner. In particular, 1 mg/ml of globular adiponectin produced prominent effect, while no significant effect was shown at lower concentration (supplementary Fig. S1A and B). Therefore, 1 mg/ml of globular adiponectin was used in further investigations. A number of studies have also found the anti-proliferative and apoptotic effect of adiponectin in cancer cells similar to our findings, such as preventing the proliferation of myelomonocytic lineage cells by induction of apoptosis (Yokota et al., 2000) and hepatoma cell line via modulation of mTOR pathway (Saxena et al., 2010). However, many other reports have also demonstrated the proproliferative capacity of adiponectin. For instance, adiponectin treatment induced proliferation of HT-29 colon epithelial cancer cells (Ogunwobi and Beales, 2006) and human embryonic kidney cells (HEK 293) (Lee et al., 2008). Landskroner-Eiger et al. (2009) has described that adiponectin facilitates mammary tumor growth by promoting angiogenesis in an in vivo study. Adiponectin has been also shown to protect

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Fig. 7. Role of LKB1 in phosphorylation of AMPK by gAcrp in HepG2 cells. HepG2 cells (A) and MCF-7 cells (B) were treated with gAcrp (1 mg/ml) for time period as indicated. Phosphorylated level of LKB1 at Ser428 was determined by Western blot analysis (upper part). Images are representative of three independent experiments that showed similar results. Quantitative analysis of p-LKB1 and total LKB1 expression was performed by densitometric analysis and shown in the below part. Values are presented as mean  SEM (n ¼ 3).  P < 0.05 compared with cells not treated with gAcrp. (C) HepG2 cells were transfected with siRNA targeting LKB1 or scrambled control siRNA. After 24 h incubation, cells were lysed for the determination of LKB1 siRNA transfection efficiency via Western blot analysis of LKB1 protein expression (upper left part). Cells were then stimulated with gAcrp (1 mg/ml) for 60 min. The level of phosphorylated AMPKa (Thr172) was determined by Western blot analysis (lower part). Images are representative of three independent experiments that showed similar results. Quantitative analysis of AMPKa expression was performed by densitometric analysis and is shown in the lower panel. Values are presented as mean  SEM (n ¼ 3).  P < 0.05 compared with cells not treated with gAcrp; #P < 0.05 compared with cells treated with gAcrp but not transfected.

the cells from apoptosis by ethanol (Nepal et al., 2012). These contradictory findings could be due to the different experimental conditions. Cell type-specific responses and differences in experimental models may explain these discrepancies. For example, in a mouse model, adiponectin was found to promote tumor growth by enhancing angiogenesis via modulation of T-cadherin, a different type of adiponectin receptor from adipoR1 and adipoR2, and not by directly influencing cell proliferation (Denzel et al., 2009). In addition, use of trimeric, hexameric, or higher molecular weight complex of adiponectin might have resulted into variable biological outcomes. Moreover, concentration of adiponectin used can also play a role in this ambiguity. Higher concentration of adiponectin showed proliferative effect in a study conducted by Chen et al. (2012), while our findings were observed in a comparatively lower concentration. In consideration of the potent anti-tumor activity of adiponectin, future studies clarifying the conflicting effects of adiponectin would be JOURNAL OF CELLULAR PHYSIOLOGY

valuable to provide further insights into the mechanisms underlying the regulation of tumor progression. In particular, adiponectin has been shown to induce proliferation of MCF-7 cells in the presence of 17-b-estradiol, which is opposite to the results from the present study, while it triggered cellular apoptosis of MDA-MB-231 breast cancer cells (Pfeiler et al., 2008). These results indicate that cross-talk between estrogen receptor signaling and adiponectin would play a critical role in determining the fate of breast cancer cells treated with adiponectin. Irrespective of the controversial effects on cancer cell growth, a growing body of evidence demonstrated the potent anti-tumor activities of adiponectin in various cancer cells and adiponectin, therefore, has been considered as a promising endogenous molecule regulating cancer development and/or growth. Herein, we have also found that gAcrp treatment causes both cell cycle arrest and apoptosis induction in human hepatoma (HepG2) and breast cancer cells (MCF-7). Although

FoxO3A IN CANCER CELL DEATH BY ADIPONECTIN

previous studies have suggested the potent anti-proliferative effects of adiponectin, detailed molecular mechanisms underlying are not clearly understood. In the current study, we have presented AMPK/FoxO3A axis plays a crucial role in gAcrp-induced increase in the expression of genes involved in cell cycle regulation and further inhibition of cancer cell viability. The cell cycle is regulated via complicated mechanisms involving various genes. In the present study, we evaluated the effects of gAcrp on the expression of various genes involved in cell cycle regulation and found that gAcrp affected the expression of cyclinD1 (Fig. 2G and H) and p27 (Fig. 2C–F) via FoxO3A/AMPK-dependent manner. However, we did not observe any significant change in the expression of p21, cyclinE1, and p130 (retinoblastoma family protein) at mRNA and protein level (supplementary Fig. S2) in the present study. Previous studies have reported that adiponectin decreases cyclin D1 expression via modulation of glycogen synthase kinase-3b/b-catenin axis in nude mice bearing breast cancer cells (Wang et al., 2006) and via regulation of c-Myc in MCF-7 cells (Dieudonne et al., 2006). In the present study, we also found that gAcrp decreased cyclinD1 protein expression in both HepG2 and MCF-7 cells (Fig. 2G and H). However, gAcrp did not significantly affect the expression at mRNA level (supplementary Fig. S2), suggesting that gAcrp regulates cyclinD1 expression at post-transcriptional level, even if it is regulated via FoxO3A transcription factor-dependent manner (Fig. 3G and H). Cyclin D1 gene expression can be regulated at transcriptional, translational, or post-translational (e.g., protein stability) level. In this study, we observed that genetic ablation of FoxO3A by siRNA restored the gAcrp-suppressed cyclin D1 expression (Fig. 3G and H), suggesting involvemet of FoxO3A signaling in cyclin D1 expression. FoxO3A has been shown to modulate cyclinD1 expression through various different mechanisms. For example, Schmidt et al. (2002) has reported that FoxO3A regulates cyclin D as a transcriptional repressor. Additional studies have also shown that FoxO3A induces BCL6 which causes the transcriptional repression of cyclinD2 (Fernandez de Mattos et al., 2004; Glauser and Schlegel, 2009). Chen et al. (2010) has reported FoxO3A to be a transcriptional regulator of cyclin D1. However, in the present study, gAcrp decreased cyclin D1 protein level without significant change in mRNA level of cyclinD1, ruling out the possibility that FoxO3A controls cyclin D1 expression as a direct transcriptional regulator. Based on this observation, we speculated that FoxO3A decreases cyclinD1 expression via indirect manner, for example, inducing expression of the genes which negatively regulates cyclinD1 expression at post-transcriptional level. It has been reported that cyclinD1 expression is regulated through proteasomal degradation pathway (Feng et al., 2007). To identify the molecular mechanisms underlying regulation of cyclinD1 expression by FoxO3A, we investigated the effect of gAcrp on the proteasomal degradation and found that gAcrp treatment increased overall proteasomal activity in HepG2 cells (data not shown), raising a possibility that gAcrp and FoxO3A signaling regulate cyclinD1 expression via proteasome-dependent pathway. Although at this stage, we cannot provide the detailed molecular mechanisms underlying suppression of cyclinD1 expression via FoxO3A signaling, ubiquitination-mediated degradation of cyclin D1 by gAcrp and involvement of FoxO3A signaling are being investigated. In contrast to the complicated manner for the regulation of cyclinD1 expression, globular adiponectin modulates expression of p27 under direct control of FoxO3A. Previous studies have demonstrated that adiponectin increases p27 expression in hepatic stellate cells (Adachi and Brenner, 2008), colon cancer cells (Kim et al., 2010), and vascular smooth cells (Hirai et al., 2013) via multiple mechanisms. However, to the best of our knowledge, this is the first report to demonstrate involvement of FoxO3A in the regulation of p27 expression. JOURNAL OF CELLULAR PHYSIOLOGY

In the present study, we observed the apoptotic effect of gAcrp in HepG2 cells (Fig. 4A) and MCF-7 cells (Fig. 4B), which was previously reported in different types of cancer cell lines, including endothelial cells (Brakenhielm et al., 2004), endometrial carcinoma cell (Cong et al., 2007), and other human breast cancer cells (Kang et al., 2005). In order to explore the molecular mechanism underlying gAcrp-induced apoptosis, we looked into the effects of gAcrp on mRNA expression of various genes modulating intrinsic pathway of apoptosis, including BCL2-like 11 (BIM), BCL2/adenovirus E1B 19kDa interacting protein 3-like (BNIP3L), and genes mediating the extrinsic pathway of apoptosis, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors DR-4 and DR-5, Fas ligand (FasL) and its receptor (FasR). We also studied the effect of gAcrp on the mRNA expression of phosphatase and tensin homolog (PTEN), which acts as a tumor suppressor by negatively regulating Akt/PKB signaling pathway. However, in our experimental conditions, gAcrp treatment did not produce significant effect on the mRNA expression of those genes (data not shown), except FasL (Fig. 4D–G). Previous studies have shown that FoxO3A is a transcription factor responsible for the expression of Fas ligand through different mechanisms. For example, Yang et al. (2006) has shown that radiation induces translocation of FoxO3A into nucleus, thereby up-regulates the expression of target genes, such as FasL and Bim, resulting in cell death of osteosarcoma cell. In addition, nuclear translocation of FoxO3A by doxorubicin was found to transcriptionally repress miR21 which suppressed the translation of FasL in A549 human lung cancer cell (Wang and Li, 2010). In accordance with the previous reports, we also observed that gAcrp-induced expression of FasL in HepG2 is mediated through FoxO3Adependent manner (Fig. 4H). With regard to the apoptotic effects of adiponectin, a large number of studies focused on increment of pro-apoptotic genes such as Bax and/or decrease of anti-apoptotic Bcl-2 genes (Dieudonne et al., 2006). For example, adiponectin was found to induce apoptosis in murine myeloid cell lines M1 by decreasing anti-apoptotic Bcl-2 gene without significant effect on pro-apoptotic genes including Bax, Bak, and p53 (Yokota et al., 2000). In addition, adiponectin induced caspase-mediated apoptosis in murine pancreatic cells both in vitro and in vivo (Kato et al., 2014). Data presented here suggest that adiponectin causes apoptotic effect via activation of FoxO3A and subsequently inducing FasL expression in HepG2 and MCF-7 cells. To the best of our knowledge, this is the first report demonstrating the involvement of Fas ligand in adiponectin-induced apoptosis of cancer cells. In addition to the well-known metabolic functions, recent studies have shown that AMPK signaling also plays a critical role in the regulation of cancer cell proliferation via induction of apoptosis and cell cycle arrest (Motoshima et al., 2006). A number of tumor suppressor genes, such as p53, mTOR, and p27, are considered as the downstream signaling molecules of AMPK activation (Okoshi et al., 2008; Luo et al., 2010). For instance, AMPK activation causes apoptosis via p53-dependent manner (Okoshi et al., 2008). Similarly, AICAR, a pharmacological activator of AMPK, suppresses cell growth of human hepatic tumor cells (Imamura et al., 2001). Moreover, metformin has recently received attention as a anti-tumor agent, since it induces inhibition of cancer cell proliferation via activation of AMPK signaling (Zhuang and Miskimins, 2008). In fact, Dieudonne et al. (2006) reported that human recombinant adiponectin reduced growth of MCF-7 cells through AMPK activation and inactivation of p42/p44 MAPK. In the present study, we have further demonstrated the detailed downstream signaling of AMPK, including nuclear activation of FoxO3A and further modulating expression of genes involved in cell cycle and regulation of apoptosis via modulation of caspase-3/7

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Fig. 8. Proposed model for the role of AMPK/FoxO3A signaling in the gAcrp-induced cell cycle arrest and apoptosis in cancer cells. Globular adiponectin generates anti-proliferative effects in human hepatoma and breast cancer cells via both induction of cell cycle arrest and apoptosis. Treatment with globular adiponectin leads to a rapid phosphorylation of LKB1 which in turn activates AMPK. Activation of AMPK signaling causes translocation of FoxO3A into the nucleus. The AMPK/FoxO3A axis plays a critical role in gAcrp-induced cell cycle arrest at G0–G1 phase through induction of p27 expression and suppression of cyclin D1 expression. In addition to cell cycle arrest, AMPK and FoxO3A signaling also plays a crucial role in gAcrp-induced apoptosis in cancer cells via induction of Fas ligand expression which in turn activates caspase-8 followed by activation of caspase-3/7. Detailed mechanisms underlying decrease in cyclinD1 expression by FoxO3A remains to be determined.

activity and FasL gene expression. Herein, we clearly showed that AMPK signaling plays a critical role in adiponectin-induced cell cycle arrest and apoptosis via activation of FoxO3A. As described earlier, FoxO3A activity is determined via various post-translational modifications, including phosphorylation, acetylation, and ubiquitination (Calnan and Brunet, 2008). Activation of AMPK signaling has been shown to cause the nuclear shuttling of FoxO3A resulting in the expression of various target genes (Chiacchiera et al., 2009; Li et al., 2009), indicating a critical role of AMPK signaling in FoxO3A activation. In addition, during the preparation of the manuscript, Queiroz et al. (2014) recently reported that metformin, an anti-diabetic drug acting via AMPK activation, induced cell cycle arrest and apoptosis via oxidative stress, AMPK and FoxO3A in MCF-7 cells, suggesting a crucial role of AMPK/FoxO3A in the regulation of cancer cell growth by metformin which showed similar mechanisms to those from gAcrp-induced inhibition of cancer cell proliferation. Although previous studies have also indicated the involvement of AMPK signaling in cell cycle arrest and apoptosis, the detailed molecular mechanisms has not been demonstrated. Herein, we clearly demonstrated that AMPK/FoxO3A axis plays a pivotal role in gAcrp-induced cell cycle arrest via modulation of cyclinD1 and p27 expression. Increasing evidence has recently demonstrated that obesity is closely associated with incidence of cancer (Ungefroren et al., 2015). In particular, development of colon, breast, and hepatic cancer has close relationship with obesity. Among the various mechanisms proposed for the relationship between obesity and cancer incidence, alterations in the plasma level of adipokines have been getting much attention lately. In particular, blood adiponectin level is reduced in obese people (Arita et al., 1999; Ungefroren et al., 2015). As shown in the current study and previous reports, adiponectin inhibits proliferation of cancer cells. Therefore, decrease in blood JOURNAL OF CELLULAR PHYSIOLOGY

adiponectin level in obesity could be a potential mechanism underlying development and/or progression of breast and hepatic cancer. Unraveling the molecular mechanisms underlying anti-tumor activity by gAcrp would generate promising strategies for the treatment of cancer associated with obesity. In the present study, we have demonstrated the involvement of AMPK and FoxO3A signaling in adiponectininduced inhibition of cell viability and provided novel mechanistic insight into its anti-proliferative effects. In conclusion, here, we have demonstrated for the first time that the anti-proliferative effect of globular adiponectin is mediated by AMPK/FoxO3A axis via inducing cell cycle arrest and apoptosis in human hepatoma (HepG2) and breast cancer cells (MCF-7). Moreover, induction of cell cycle arrest is accompanied by decrease in cyclin D1 protein expression and increase in p27 expression, and apoptosis induction is associated with increase in Fas ligand expression. Furthermore, LKB1 acts as an upstream molecule leading to the activation of AMPK in HepG2 and MCF-7 cells treated with gAcrp (Fig. 8). The present study suggests that AMPK/FoxO3A axis would be a novel mechanism for the anti-tumor activity of adiponectin in various cancer cells and further modulation of AMPK/FoxO3A axis would be a promising therapeutic strategy in obesityassociated cancer development.

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A4A01011110) and the KAERI Major Project, Development of Radioisotope Production and Application Technology (525140-15). All authors have no conflict of interest.

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