Apoptosis DOI 10.1007/s10495-013-0929-0

ORIGINAL PAPER

Cryptotanshinone induces G1 cell cycle arrest and autophagic cell death by activating the AMP-activated protein kinase signal pathway in HepG2 hepatoma In-Ja Park • Woo Kyeom Yang • Sang-Hee Nam • Jongki Hong • Ki Ryeol Yang • Joungmok Kim • Sung Soo Kim • Wonchae Choe • Insug Kang • Joohun Ha

Ó Springer Science+Business Media New York 2013

Abstract AMP-activated protein kinase (AMPK) performs a pivotal function in energy homeostasis via the monitoring of intracellular energy status. Once activated under the various metabolic stress conditions, AMPK regulates a multitude of metabolic pathways to balance cellular energy. In addition, AMPK also induces cell cycle arrest or apoptosis through several tumor suppressors including LKB1, TSC2, and p53. LKB1 is a direct upstream kinase of AMPK, while TSC2 and p53 are direct substrates of AMPK. Therefore, it is expected that activators of AMPK signal pathway might be useful for treatment or prevention of cancer. In the present study, we report that cryptotanshinone, a natural compound isolated from Salvia miltiorrhiza, robustly activated AMPK signaling pathway, including LKB1, p53, TSC2, thereby leading to suppression of mTORC1 in a number of LKB1-expressing cancer cells including HepG2 human hepatoma, but not in LKB1deficient cancer cells. Cryptotanshinone induced HepG2 cell cycle arrest at the G1 phase in an AMPK-dependent manner, and a portion of cells underwent apoptosis as a

I.-J. Park
W. K. Yang
S.-H. Nam
S. S. Kim
W. Choe
I. Kang
J. Ha (&) Department of Biochemistry and Molecular Biology, Medical Research Center for Reactive Oxygen Species and Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea e-mail: [email protected] J. Hong
K. R. Yang College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea J. Kim Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Kyung Hee University, Seoul 130-701, Republic of Korea

result of long-term treatment. It also induced autophagic HepG2 cell death in an AMPK-dependent manner. Cryptotanshinone significantly attenuated tumor growth in an HCT116 cancer xenograft in vivo model, with a substantial activation of AMPK signal pathways. Collectively, we demonstrate for the first time that cryptotanshinone harbors the therapeutic potential for the treatment of cancer through AMPK activation. Keywords AMPK
Cryptotanshinone
Cell cycle arrest
Autophagy
HepG2 hepatoma

Introduction AMP-activated protein kinase (AMPK) plays a central role in cellular energy homeostasis. Under various metabolic stress conditions, it is activated by the accumulation of AMP as a result of ATP depletion [1], and is also phosphoactivated at Thr172 of a subunit by upstream kinases, including LKB1 [2], Ca2?-calmodulin-dependent kinase kinase b (CaMKKb) [3], and transforming growth factor-bactivated kinase 1 (TAK1) [4]. Once activated, AMPK regulates a multitude of metabolic pathways to protect cells from ATP-depleting stresses [1]. Indeed, AMPK controls glucose and lipid homeostasis, food intake, insulin signaling, and body weight as a central regulator, and thus, it is now considered a significant pharmacological target for the treatment of metabolic disorders such as obesity and type 2 diabetes [5, 6]. AMPK has been also implicated in cancer biology. Several tumor suppressors including LKB1 [7], p53 [8], and TSC2 [9], are associated with the AMPK signal network. The gene mutation of LKB1, a well characterized upstream kinase of AMPK, is associated with Peutz–Jeghers

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syndrome, an autosomal dominant disorder characterized by the growth of polyps in the gastrointestinal tract [10]. Tumor suppressor p53 and TSC2 are direct substrates of AMPK, and mutation of TSC2 genes can induce hamartomatous polys in various tissues and increases risk of cancers. The phosphoactivated TSC2 forms a complex with TSC1, and inhibits the activity of mTORC1, which plays a central role in cell proliferation and protein synthesis [11]. Therefore, activation of AMPK under various cancer microenvironments results in cell cycle arrest or apoptosis of cancer cell. AMPK signaling pathway is also central to the regulation of autophagy, an essential cellular degradation process for the clearance of damaged or superfluous proteins and organelles. Although autophagy is generally known as a defense mechanism in response to stressful conditions, it seemingly has dual roles in cancer [12, 13]. Metabolic stresses such as a poor nutrition and hypoxia robustly induce autophagy in caner, which serves as a back-up energy reserve, providing growth advantages to tumors. Paradoxically, autophagy also contributes to cancer cell death in many cases; cytotoxic drug treatment often triggers excessive cell damage, thereby promoting autophagic cell death [12, 13]. The inhibitory function of mTORC1 in autophagy is well established. AMPK promotes autophagy through the suppression of mTORC1 via multiple mechanisms; AMPK directly phosphorylates and activates TSC2, a negative regulator of mTORC1 [14]. AMPK also phosphorylates mTORC1 subunit Raptor, which then blocks the kinase activity of mTOR [15]. In addition, AMPK is able to directly promote autophagy by phosphorylating ULK1, a key regulator in autophagy initiation [16, 17]. A recent meta-analysis indicated that although cancer mortality is higher in diabetics, diabetic patients treated with metformin showed a substantially lowered cancer risk compared with diabetics on other treatment [18, 19]. It has been well established that AMPK mediates a large portion of the beneficial effects of metformin, the most widely prescribed anti-diabetic drug for type 2 diabetes. In the last decade, metformin has also gained special attention for its anti-cancer activity, and the main mechanism of this anticancer activity has been attributed to activation of AMPK signal pathways associated with LKB1, TSC2, and p53 [20, 21]. As demonstrated by the case of metformin, identification of a novel activator for AMPK may therefore significantly contribute to the treatment of metabolic syndrome and cancer. Previously, we reported cryptotanshinone, a natural compound isolated from Salvia miltiorrhiza, as an AMPK activator, describing the antidiabetic and anti-obesity effect [22]. Cryptotanshinone was also reported to have anti-cancer activities against diverse cancer cells [23–25], but the mechanisms of these activities remain to be fully understood. In the present study, we

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demonstrate that cryptotanshinone induces cell cycle arrest, apoptosis, and autophagic cancer cell death in vitro and in vivo in an AMPK-dependent manner, providing novel mechanisms for the anti-cancer property of cryptotanshinone. Similarities of underlying mechanisms of the pharmacological effects of cryptotanshinone and metformin are discussed.

Materials and methods Reagents The antibodies for phosphoactivated forms of AMPKThr172 (P-AMPK), ACC-Ser79 (P-ACC), LKB1-Ser428 (PLKB1), mTOR-Ser2448 (P-mTOR), TSC2-Ser1387 (P-TSC2), p53-Ser15 (P-p53) and Rb-Ser807/811 (P-Rb), and the antibodies against ACC, LKB1, LC3-I/II, and ATG12 were from Cell Signaling Technology (Beverly, MA). The antibodies against AMPK-a1, AMPK, Rb, p53, E2F1, CyclinD1, CyclinE, and p21 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). 3-Methyladenine was purchased from Sigma-Aldrich (St. Louis, MO), and cryptotanshinone and Compound C were obtained from TOCRIS (Bristol, UK). GFP-tagged LC3 was generously provided by Dr. Jae-Young Koh (Ulsan University, Korea), and E2F1-luc and E2F1-(DE2F)-luc were kindly provided by Dr. Young-Chae Chang (Daegu Catholic University, Korea). Cell lines Hepatoma (HepG2) and mouse embryonic fibroblast (MEF) cells were incubated in DMEM, and A549 (lung carcinoma), DU145 (prostate carcinoma), AGS (gastric carcinoma), and HCT116 (colorectal carcinoma) cells were maintained in RPMI medium 1,640, and all were supplemented with 10 % heat-inactivated FBS and antibiotics at 37 °C with 95 % air and 5 % CO2. AMPK WT and a knock out MEF cells were generously provided by Benoit Viollet (INSERM, France). Annexin V and propidium iodide (PI) double staining The ApoScanTM Annexin V-FITC apoptosis detection kit (BioBud, Seongnam, Korea) was used to detect apoptotic cells by flow cytometry (Beckman) according to the manufacturer’s instructions. Cells were trypsinized and were pelleted by centrifugation at 1,000 rpm for 5 min. They were then resuspended in binding buffer (500 lLs) and incubated with 1.25 lL of Annexin V-FITC (200 lg/mL) at room temperature for 15 min before staining with 10 lL of PI (30 lg/mL) at 4 °C in the dark.

Apoptosis

Fluorescence activated cell sorting (FACS) analysis Total cells were collected by centrifugation, and were fixed with 70 % ethanol and subsequently resuspended in PBS. Fluorescence intensity was measured by Beckman Coulter flow cytometry system using excitation and emission wavelengths of 488 and 525 nm, respectively. Cell viability assay The Vi-CELLTM XR Cell viability analyzer (Beckman Coulter) cell counter, which performs an automated trypan blue exclusion assay, was used to measure cell viability. The dead cells appear darker than the viable cells, allowing the contrast between live and dead cells to be used to determine cell viability. Miscellaneous analyses The level of ATP, reactive oxygen spices (ROS), luciferase, transient transfection, and adenovirus-mediated gene transfer were determined as previously described [24, 26]. Measurement of cellular AMP:ATP ratio via LC–ESI–MS/MS Cells were seeded on 10 cm culture dish, and were then treated with cryptotanshinone for 6 h. Adenosine phosphates were extracted from the cells with 1 mL of 0.6 mol/L perchloric acid in the ice bath for 1 min. After centrifugation, the supernatant was taken and quickly neutralized to pH = 6.5–6.8 with 1.5 mL of 1 mol/L KOH solution. The neutralized supernatant was then allowed to stand for 30 min in an ice bath to precipitate most of the potassium perchlorate, which was removed by paper filtration. Chromatographic separation of ATP and AMP in an LC system (Agilent 1200 series (Agilent Technologies, Palo Alto, CA, USA)) was performed using an RStech Hector column (150 9 4.6 mm, 3 lm; RStech Corporation, Daejeon, Korea). The flow rate was set at 400 ll/min. Mobile phase A was 10 mM ammonium carbonate buffer adjusted to pH 6 by formic acid in water and B was 100 % acetonitrile. The isocratic elution was 82 % A and 18 % B. All electrospray ionization (ESI)–tandem mass spectrometry (MS/MS) experiments were performed using an API 3200 instrument (MDS Sciex, Concord, ON, Canada). All parameters were optimized according to the manufacturer’s instruments. In ESI–MS experiments, mass spectrometric conditions were as follows: curtain gas, 20 psi; electron voltage, -4,500 V; temperature, 400 °C; nebulizing gas, 50 psi; and heating gas, 50 psi. The ESI–MS/MS experimental conditions were as follows: ATP, declustering potential (DP) -35 V, entrance potential (EP) -7 V,

collision energy (CE) -20 V, and collision cell exit potential (CXP) -6 V; AMP, DP -32 V, EP -5 V, CE -20 V, and CXP -12 V. The [M–H]- ions were selected as precursor ions in the negative-ion ESI mode. The MS/ MS fragmentations of ATP and AMP result in the product ions m/z 159 and 79 which correspond to the loss of a H2O molecule from diphosphate and phosphate, respectively. These were selected as multiple reaction-monitoring (MRM) transition ions. The dwell time of each MRM transition was 150 ms. Confocal microscopy Cells were cultured on poly-L-lysine-coated glass slides, and were then transiently transfected with GFP-LC3 plasmid. After treatment with cryptotanshinone, cells were stained with 1 lM Lyso Tracker Red (Invitrogen, Carlsbad, CA) for 15 min. Cells were then mounted and visualized under a confocal microscope (Zeiss Meta LSM 510 software, Germany). Animals and xenografts Five-week-old male Balb/c nu/nu mice were purchased from Central Laboratory Animal Inc. (Seoul, Korea) and were housed in sterile filter-topped cages. Animals were allowed to acclimatize for 1-week before being used. The experimental protocol (KHUASP (SE)-11031) was approved by the Institutional Animal Care and Use Committee of Kyung Hee University. HCT116 (6 9 106) cells were subcutaneously injected into mice (n = 8 mice/group). Mice were then injected intraperitoneally with or without cryptotanshinone at a concentration of 2.5 mg/kg every 2 days for 18 days. The control group received vehicle only (3 % dimethyl sulfoxide and 30 % polyethylene glycol). Tumor size was measured using a caliper and the volume was calculated by the modified formula ab2/2 (b is the smaller dimension). Immunohistochemistry analysis Paraffin sections were deparaffinized with xylene and were dehydrated with a series of ethanol of increasing concentration. Endogenous peroxidases were quenched with a short treatment of 3 % H2O2, and antigen retrieval in citrate buffer was used for enhancing the signal. The specimens were incubated overnight at 4 °C with phosphorAMPK-Thr172 (1:1,000) and the immunostained section was visualized with an EnVision detection kit (Dako, Glostrup, Denmark). Routine hematoxylin and eosin (H&E)-stained sections were examined to ensure the structural integrity of the tissues.

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Electron microscopy Cells were fixed in 2 % paraformaldehyde and 1 % OsO4 solution in 0.05 M sodium cacodylate buffer (pH 7.2) at 4 °C. Samples were stained in 0.5 % uranyl acetate for 30 min and were dehydrated in increasing concentrations of ethanol. Then, cells were infiltrated with Spurr’s resin and were dried in an oven at 70 °C for 24 h. The samples were sectioned with ultramicrotome (Tucson, USA) and were stained with 2 % uranyl acetated and Reynolds’ lead citrate. The samples were then observed with a transmission electron microscope (Carl Zeiss, Germany). Statistical analysis Statistical significance between groups was determined by Student’s t test analysis, and the data are presented as mean ± SE for three determinations in duplicate. Values of P \ 0.05 were considered significant (*).

Results Cryptotanshinone activates the AMPK signal pathway in HepG2 hepatoma We first examined the effect of cryptotanshinone (Fig. 1a) on the AMPK signal pathway in HepG2 hepatoma. AMPK was robustly activated by cryptotanshinone in a dose(Fig. 1b) and time-dependent manner (Fig. 1c), as assessed by the phosphorylation levels of LKB1-Ser428, AMPKaThr172, and those of downstream targets, including acetylCoA carboxylase (ACC)-Ser79, TSC2-Ser1387, and p53Ser15. In accordance with activation of TSC2, the activity of mTOR was suppressed by cryptotanshinone, as indicated by the phosphoactive level of mTOR-Ser2448. Under cryptotanshinone treatment, the expression level of p21cip1/waf1, a target gene of p53, was also increased. The AMPK inhibitor, compound C, significantly blocked the effect of cryptotanshinone on ACC, TSC2, p53, and mTOR, indicating that these molecules were indeed activated by cryptotanshinone in an AMPK-dependent manner (Fig. 1d). Collectively, these data indicate that cryptotanshinone activates the AMPK signal pathway in HepG2 cells. Cryptotanshinone induces cell cycle arrest and apoptosis of HepG2 cells in an AMPK-dependent manner As tumor suppressors, including LKB, TSC2, and p53, were activated by cryptotanshinone, we next investigated the effect of cryptotanshinone on cell cycle and apoptosis.

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Cryptotanshinone induced G1 phase arrest of HepG2 cells in a dose- and time-dependent manner (Fig. 2a), but the prolonged treatment eventually led to increases of the subG1 phase, indicating the induction of apoptosis (Fig. 2a, right panel). To present a complete apoptotic picture of cryptotanshinone in HepG2 cells, we performed FACS analysis of HepG2 cells, which were double-stained with AnnexinV/PI (Fig. 2b). The population of AnnexinV?/PI- cells, which indicates the early stages of apoptosis, increased approximately sixfold by cryptotanshinone in 24 h (3.9 % ? 23.1 %). The late stages of apoptosis, as indicated by changes in population of AnnexinV?/PI? cells, was also increased by cryptotanshinone. We also examined the change of various molecules involved in the regulation of the G1 phase. In quiescent cells, retinoblastoma protein (pRB) associates with E2F1 to repress E2F1responsive genes involved in cell cycle progression. During G1 to S phase transition, the phosphorylation of pRB by Cdk4/6-cyclin D or Cdk2-cyclin E results in the release of E2F1 transcription factor from E2F1-pRB complex, leading to expression of genes required for S phase transition [27]. In accordance with the cell cycle profile, cryptotanshinone treatment reduced the expression of cyclinD1, cyclinE, and E2F1 as well as the phosphorylated form of pRB (Fig. 2c). E2F1 is able to autoregulate the gene expression of E2F1 by directly binding to its own promoter [28]. To test the transcription activity of E2F1, HepG2 cells were transfected with E2F1 promoter construct containing luciferase reporter, and cryptotanshinone diminished E2F1 transcriptional activity, but the E2F1-(DE2F) promoter, in which the E2F1 responsive element was deleted, was not affected by cryptotanshinone (Fig. 2d). Next, to test the role of AMPK in cryptotanshinoneinduced G1 cell cycle arrest, we infected HepG2 cells with adenovirus expressing AMPK wild type (WT) or dominant negative form (DN). AMPK-DN significantly blocked cryptotanshinone-induced activation of AMPK and its downstream targets including ACC, p53, TSC2, as well as G1 cell cycle arrest (Fig. 2e), revealing the significant role of AMPK in G1 cell cycle arrest. Similarly, an AMPK inhibitor, compound C, effectively abrogated the G1 cell cycle arrest by cryptotanshinone (Fig. 2f). Moreover, cryptotanshinone induced cell cycle arrest and AMPK signal pathway in wild type MEF, but not in AMPKa knock out MEF (Fig. 2g). In addition to the regulation of cell cycle, prolonged cryptotanshinone treatment induced apoptosis, as illustrated in Fig. 2a, and the role of AMPK in apoptosis was also tested. Cryptotanshinone treatment for 24 h resulted in significant induction of apoptotic markers including poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavages in AMPKa wild type MEF, but these effects were not observed in AMPKa knock out MEF (Fig. 2H, upper panel). FACS analysis with Annexin V and

Apoptosis Fig. 1 Cryptotanshinone activates AMPK signal pathway in HepG2 hepatoma. a Chemical structure of cryptotanshinone. b, c HepG2 cells were treated with cryptotanshinone in dose- and time-dependent manners, and the indicated proteins were then examined by western blotting. d After pretreatment with compound C (15 lM) for 30 min, cells were exposed to cryptotanshinone for 12 h

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PI double staining revealed that cryptotanshinone significantly increased the early apoptotic cell population of AMPKa wild type MEF, but not AMPKa knock out MEF (Fig. 2h, bottom panel). These results show that cryptotanshinone induced cell cycle arrest at G1 phase, and finally led to apoptosis through AMPK activation in HepG2 cells. LKB1 determines the sensitivity of cancer cells to cryptotanshinone We next compared the efficacy of cryptotanshinone in a various cancer cells. Cryptotanshinone significantly induced G1 cell cycle arrest of HepG2, HCT116, AGS, but

P-TSC2 TSC2 P-mTOR

not that of A549 and DU145 cells (Fig. 3a). In accordance with cell cycle profile, AMPK signal pathway in HepG2, HCT116, and AGS cells was highly sensitive to cryptotanshinone, whereas cryptotanshinone failed to activate AMPK in A549 and DU145 cells (Fig. 3b). One of the critical features among the tested cancer cell lines is the presence or absence of LKB1; only cryptotanshinone-sensitive cells express LKB1. As demonstrated in Fig. 3c, the cell cycle profile and AMPK signal pathway of A549 cells, which lack LKB1 expression, were not affected by an increase in the concentration of cryptotanshinone. However, introduction of GFP-tagged LKB1 into A549 cells resulted in G1 cell cycle arrest and a distinctive activation of AMPK signal pathway by cryptotanshinone treatment

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Apoptosis b Fig. 2 Cryptotanshinone induces cell cycle arrest and apoptosis of

HepG2 cancer cells in an AMPK-dependent manner. a Cells were treated with cryptotanshinone (0, 5, 10 lM) for 12 h (left panel) and with cryptotanshinone (10 lM) for indicated time (right panel). Cell cycle distribution was then determined by FACS analysis. b HepG2 cells were treated with 10 lM cryptotanshinone for 24 h and were then double-stained with AnnexinV/PI, and FACS analysis was performed. c Under the same conditions with (a), cell cycle markers were determined by western blotting. d After transfection with E2F1luc and E2F1-(DE2F)-luc constructs, cells were exposed to the indicated concentration of cryptotanshinone for 12 h. e Cells were infected with adenovirus expressing AMPK-WT and -DN for 24 h. After exposure to vehicle or cryptotanshinone for 12 h, distribution of G1 phase was determined by FACS analysis, and the indicated proteins were detected by western blotting (below band of AMPKa1; endogenous, upper band of AMPKa1; exogenous). f After pretreatment with compound C (5 lM) for 30 min, cells were exposed to cryptotanshinone for 12 h. Distribution of G1 phase was then determined by FACS analysis. g AMPKa wild type and knock out MEF cells were treated with cryptotanshinone for 12 h. The cell cycle and protein file were investigated. h AMPK wild type and knock out MEF were treated with cryptotanshinone for 24 h, and apoptosis were then measured by western blotting (upper panel) and FACS analysis with Annexin V/PI double staining (bottom panel)

(Fig. 3d). These data again illustrate that the LKB1–AMPK signal axis is critical for cryptotanshinone-induced cancer cell cycle arrest. Cryptotanshinone activates LKB1–AMPK pathway by depleting cellular ATP in HepG2 cells In order to reveal the mechanisms for cryptotanshinone action, we next determined the cellular levels of ATP, a ratio of AMP:ATP, and reactive oxygen species (ROS) because AMPK is highly sensitive the intracellular AMP:ATP ratio [1] and ROS [29]. Cryptotanshinone rapidly depleted the ATP level of HepG2 cells (Fig. 3e) and increased the AMP:ATP ratio in a dose-dependent manner (Fig. 3f). Cryptotanshinone induced the generation of ROS approximately twofold in HepG2 cells and an anti-oxidant, N-acetyl cystein (NAC), almost completely blocked the ROS induction by cryptotanshinone (Fig. 3g). Direct treatment of H2O2 was used as a positive control of the production of ROS. NAC effectively blocked the activation of LKB1 and AMPK by H2O2 treatment, but failed to block LKB1 and AMPK activation induced by cryptotanshinone (Fig. 3h). These data suggest that increase in cellular AMP:ATP ratio, but not ROS generation, plays a major role in AMPK activation by cryptotanshinone. Cryptotanshinone induces autophagic cell death in HepG2 cells through AMPK activation The AMPK and mTOR signal pathway plays a central role in the regulation of autophagy [14, 15, 30], and so, we next

examined whether cryptotanshinone induces autophagy in HepG2 cells. During autophagy, microtubule-associated protein light chain 3 type I (LC3-I) form is converted to LC3 type II (LC3-II) form, which associates with the outer and inner membranes of the autophagosome [31]. Therefore, LC3 has been generally used as a marker of autophagy. Cryptotanshinone induced formation of LC3-II in a time-dependent manner, and this induction was almost completely blocked by the autophagy inhibitor, 3-methyladenine (3-MA) (Fig. 4a). Moreover, cryptotanshinone increased the number of punctuate GFP-LC3 structures, and double staining showed that GFP-LC3 was also stained with Lyso Tracker, indicating that some of GFP-LC3associated autophagosome had fused with lysosomes to form autolysosomes, and 3-MA significantly blocked this event (Fig. 4b). Next, we examined the autophagic flux in order to distinguish whether autophagosome accumulation induced by cryptotanshinone is the result of bona fide autophagy induction or a block in downstream steps. To this end, HepG2 cells were pretreated with bafilomycin A1, a specific inhibitor of vacuolar type H?-ATPase, which prevents autophagy at the late stage by inhibiting fusion between autophagosomes and lysosomes. The level of LC3-II was increased by treatment with bafilomycin A1 alone, and cryptotanshinone treatment resulted in more induction of LC3-II in the presence of bafilomycin A1 (Fig. 4c). Taken together, our data (Fig. 4) indicate that cryptotanshinone indeed induced autophagy in HepG2 cells by increasing autophagic flux. Autophagy plays a dual role in cancer, oncogenic function as well as tumor suppressive role [12, 13]. To clarify the role of autophagy induced by cryptotanshinone, we tested cell viability under cryptotanshinone condition in the presence of autophagy inhibitor. In our condition, the autophagy inhibitor 3-MA significantly blocked cell death induced by cryptotanshinone (Fig. 5a). Moreover, cryptotanshinone was able to induce the cell death of autophagyrelated gene 5 (Atg5) wild type MEF, but not of Atg5 knock out MEF (Fig. 5b); it has been well established that Atg5 is required for, and plays an important role in autophagy, as a conjugate to Atg12 [32, 33]. Taken together, our data indicated that autophagy induced by cryptotanshinone contributes to cell death. Our results further demonstrated that AMPK mediates autophagy under cryptotanshinone treatment in HepG2 cells; inhibition of endogenous AMPK activity by overexpression of its DN resulted in a significant block of LC3-II formation induced by cryptotanshinone (Fig. 5c). We further confirmed the role of AMPK in cryptotanshinone-induced autophagy via electron microscopy. At the ultrastructural level, an autophagosome is defined as a double-membraned structure containing undigested cytoplasmic contents, which has not fused with a lysosome. The autolysosome is a hybrid organelle

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Fig. 3 LKB1 determines the sensitivity of cancer cells to cryptotanshinone and cryptotanshinone rapidly depletes the intracellular ATP levels in HepG2 cells. The indicated cancer cells were treated with cryptotanshinone (10 lM) for 12 h, and the proportion of G1 phase (a) and the indicated proteins (b) were then determined by FACS and Western blotting, respectively. c A549 and HepG2 cells were treated with cryptotanshinone for 12 h, and then relative G1 phase and protein profile was determined. d A549 cells were transfected with GFP-tagged LKB1-WT and -DN constructs for 24 h, and cells were

then exposed to cryptotanshinone for 12 h. The relative G1 phase was detected by FACS analysis and proteins were determined via Western blotting. e, f Cells were treated with cryptotanshinone for 6 h, and then lysates were used for measurement of ATP levels (e) and AMP:ATP ratio (f). Glu-; glucose deprivation for 6 h. g Cells were pretreated with NAC (5 mM) for 30 min, and were exposed to cryptotanshinone or H2O2. The relative ROS levels were detected by FACS analysis. h Under the same conditions (g), the indicated proteins were detected by western blotting

generated by the fusion of an autophagosome and a lysosome, which has a single limiting membrane and contains cytoplasmic materials [34, 35]. Transmission electron

microscopy (TEM) revealed that cryptotanshinone evidently increased the number of autophagosomes (white arrowheads) and autolysosomes (black arrowheads), and its

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Fig. 4 Cryptotanshinone induces autophagy in HepG2 cells. a Cells were exposed to the indicated time and concentration, and the indicated proteins were then detected by western blotting (left panel). After pretreatment with 1 mM of 3-MA for 30 min, cells were treated with cryptotanshinone for 12 h (right panel). b Cells were transfected with GFP-tagged LC3 plasmid for 24 h, and then were pretreated with 1 mM

of 3-MA for 30 min and exposed to 10 lM of cryptotanshinone for 12 h. Fluorescent confocal for GFP-LC3 (green, left), LysoTracker (Red, middle) and the merged image (yellow, right) are shown. Scale bar represents 5 lm. c Cells were pretreated with bafilomycin A1 for 30 min, and were then exposed to cryptotanshinone. Indicated proteins were determined by western blotting (Color figure online)

induction was significantly blocked by AMPK inhibitor compound C (Fig. 5d). The inset of the middle figure shows each autophagic membrane structure. Cryptotanshinone significantly reduced the cell viability of AMPK wild type MEF with a concomitant induction of LC3-II, but these effects were not observed in AMPKa knock out MEF (Fig. 5e). These results indicate that cryptotanshinone induces autophagic cell death through AMPK activation in HepG2 hepatoma cells.

induced by cryptotanshinone treatment (Fig. 6b), and Western blot analysis showed that phosphorylation of AMPK, ACC, LKB1, p53 and TSC2 were increased, whereas that of Rb and mTOR were decreased by cryptotanshinone (Fig. 6c). Moreover, the expression of LC3-II was induced by cryptotanshinone (Fig. 6c). Together, these results suggest that cryptotanshinone showed anti-cancer properties via activation of AMPK signal pathway in vivo, as well as in cell culture system. The AMPK signal pathway under cryptotanshinone treatment is presented in the form of a diagram (Fig. 6d).

Cryptotanshinone attenuates the growth of tumor in vivo To determine the anti-cancer effects of cryptotanshinone on tumor growth in vivo, we injected HCT116 cancer cells into nude mice, and treated cryptotanshinone (2.5 mg/kg) by i.p. injection after average of tumor volume reached 100 mm3. The concentration of cryptotanshinone was chosen according to the previous report [36]. Cryptotanshinone effectively reduced tumor size (Fig. 6a) without affecting mouse body weight (data not shown). Immunohistochemistry analysis of tumor tissues revealed that phosphorylation level of AMPKa-Thr172 was markedly

Discussion Cryptotanshinone is one of the chief active ingredients isolated from dried roots of S. miltiorrhiza, which has been commonly used in oriental medicine for the treatment of circulatory disorders, cardiovascular disease, and chronic renal failure [37]. According to our current and previous study, cryptotanshinone appears to share strikingly similar properties and underlying mechanisms with metformin. In a previous study, we reported for the first time the

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Fig. 5 AMPK contributes to autophagic cell death of HepG2 cells induced by cryptotanshinone. a HepG2 cells were treated with cryptotanshinone for 24 h in the absence or presence of 3-MA (1 mM), and cell viability was then determined. b After exposing Atg5 wild type and knock out MEF to cryptotanshinone for 24 h, cell viability was measured. c HepG2 cells were infected with Adenovirus expressing AMPK-WT and -DN, and were then exposed to cryptotanshinone for 12 h. The indicated proteins were measured by western blotting. d After pretreatment with compound C (15 lM) for 30 min, cells were exposed to cryptotanshinone (10 lM) for 12 h. TEM

images shows extensive accumulations of autophagic structures in cryptotanshinone-treated cells. The inset middle figure shows each autophagic membrane structure. White arrowheads indicate autophagosomes with double membranes, and black arrowheads indicate autolysosomes with a single membrane. N nucleus, M mitochondria, Scale bars 0.5 lm, bar in inset 200 nm. e After AMPK wild type and knock out MEF were exposed to cryptotanshinone for 12 h, the level of LC3-II was determined. After treatment with cryptotanshinone for 24 h, cell viability was determined

anti-diabetic and anti-obesity effects of cryptotanshinone, and further demonstrated that AMPK mediates the effect of cryptotanshinone [22]. Here, we described the potential of cryptotanshinone as an anti-cancer reagent and revealed the

underlying mechanisms; cryptotanshinone induced cancer cell cycle arrest, apoptosis, and autophagic cell death in an AMPK-dependent manner in vivo as well as in vitro cell culture system.

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Fig. 6 Cryptotanshinone effectively inhibits the tumor growth in xenograft model. a HCT116 (6 9 106) cells were subcutaneously injected into 5 week-old male nude mice of eight per treatment group. Mice were intraperitoneally treated with cryptotanshinone 2.5 mg/kg every 2 days. Tumor volumes were calculated as described in ‘‘Materials and Methods’’. b After 18 days treatment, animals were

sacrificed and tumor tissues were subject to immunohistochemistry using phosphor-specific antibody for AMPK-Thr172. c Under the same conditions, homogenized cell lysates were prepared from the control and cryptotanshinone-treated tumor tissue from three different sets of nude mice, and were subjected to western blot assay. d The proposed signal pathway activated by cryptotanshinone in HepG2 hepatoma

Metformin suppresses hepatic glucose production, reduces insulin resistance, and enhances glucose uptake and utilization in skeletal muscle. It has been well established that AMPK mediates a large portion of these beneficial effects of metformin [20, 21]. Metformin has also gained widespread attention for its possible use in oncology in a last decade. Recent population studies

showed that metformin reduces cancer risk and cancerrelated mortality in diabetic patients [18, 19]. Among the characteristics of diabetes, hyperinsulinemia has been considered as a major player in cancer-related risk, because insulin can promote tumorigenesis by stimulating cell proliferation of epithelial tissues through the insulin receptor [20]. The two main mechanisms for

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tumor growth inhibition by metformin have been attributed to lowering insulin levels as well as activation of AMPK signal pathways, which lead to an inhibition of cell proliferation [20, 21, 38, 39]. Consequently, our data suggest that cryptotanshinone harbors very similar pharmacological effects to those of metformin, in terms of both its anti-cancer effect and its anti-diabetic effect through AMPK activation. Cryptotanshinone robustly activated AMPK signal pathway in a number of cancer cells (Figs. 1, 3) and in vivo (Fig. 6); cryptotanshinone activated tumor suppressor p53 and TSC2 in an AMPK-dependent manner, leading to a suppression of mTOR activity. The expression level of cyclin D1, cyclin E, phosphor-Rb, and E2F1 was concomitantly decreased by cryptotanshinone treatment (Fig. 2c). In accordance with the profile of these molecules, cryptotanshinone induced HepG2 cell cycle arrest at the G1 phase in an AMPK-dependent manner (Fig. 2a, e–g) and finally increased apoptosis (Fig. 2b, h). Numerous reports demonstrated that metformin also inhibited the growth of various cancer cells in vitro and in vivo via similar mechanisms to those described herein for the cryptotanshinone effect. For example, metformin induced breast cancer cell cycle arrest at the G1 phase via similar mechanisms including AMPK activation, down-regulation of cyclin D1, and reduction of E2F1 [40, 41]. Our results (Fig. 3a–d) indicate that LKB1 expression is essential for AMPK activation by cryptotanshinone, further demonstrating the functional requirement of AMPK signal pathway in the inhibition of tumor cell growth cryptotanshinone. Likewise, it was also reported that metformin does not inhibit cell growth of LKB1-deficient cancer cells, highlighting the significance of LKB1–AMPK signal axis for anti-cancer properties of metformin [42, 43]. The mechanism for AMPK activation by metformin was largely suggested to be inhibition of mitochondrial oxidative phosphorylation, leading to an increase in the AMP:ATP ratio [21], and we also observed that cryptotanshinone rapidly increased HepG2 cellular AMP:ATP ratio (Fig. 3f). Collectively, our data suggest that the AMPK signal pathway represents a common target for anti-cancer properties of cryptotanshinone and metformin. In addition, our study demonstrated that cryptotanshinone induced autophagy in HepG2 cells, as evidenced by the accumulation of LC-II, increase in a number of punctuate LC3 structures (Fig. 4a, b), TEM analysis (Fig. 5d), and increase of autophagy flux (Fig. 4c). Apparently, a dual role of autophagy has been observed in cancer. In general, autophagy is considered a pro-survival mechanism for tumor growth under stressful tumor microenvironments such as hypoxia and nutritional limiting conditions. However, the pro-survival role of autophagy contradicts the observation that loss-of-function

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mutations in autophagy processes are associated with increased tumorigenesis [44]. Moreover, cytotoxic drug treatment often results in the induction of autophagic cell death [13, 45]. Under our experimental conditions, autophagy induced by cryptotanshinone is likely to contribute to HepG2 cell death rather than survival, because autophagy inhibitor 3-MA significantly blocked cryptotanshinone-induced cell death (Fig. 5a). This notion was further supported by the observation that cryptotanshinone was not able to induce cell death in autophagy-defective Atg5 knock out MEF (Fig. 5b). We further demonstrated that AMPK was a critical player in cryptotanshinoneinduced autophagic cell death. First, the inhibition of AMPK activation by AMPK-DN (Fig. 5c) effectively blocked LC3-II accumulation by cryptotanshinone. Second, induction of autophagosome and autolysosome by cryptotanshinone was also significantly blocked by AMPK inhibitor (Fig. 5d). Finally, cryptotanshinone was not able to induce autophagy in AMPK knock out MEF without accompanying as significant cell death as in wild type MEF (Fig. 5e). The similar role of AMPK in autophagy was demonstrated in a recent report showing that metformin suppressed the growth of lymphoma via AMPK activation and enhanced cell sensitivity to anti-cancer agents via induction of autophagy [46]. Our data collectively suggest that induction of autophagic tumor cell death may provide a clinical benefit in cancer prevention. However, a better understanding of the conditions, mechanisms, and downstream signal pathways by which autophagy is induced to effect cell survival or cell death will be necessary for the design of a cancer treatment that can modulate autophagy. We confirmed the anti-cancer potential of cryptotanshinone in vivo as well as the significance of AMPK signal pathways; cryptotanshinone effectively reduced the tumor size of HCT116 xenograft in nude mice, and activated the upstream and downstream of AMPK (Fig. 6). Our data contained herein reveals the critical role of AMPK in the anti-cancer effect of cryptotanshinone. However, several other molecules were also reported for the action of cryptotanshinone; it inhibits the activation of hypoxia-inducible factor-1 (HIF-1) [47], TNF-alpha-induced matrix metalloproteinase-9 (MMP-9) production [23], enzyme activity of cyclooxygenase-2 (Cox-2) [48], and function of constitutive signal transducer and activator of transcription-3 (STAT-3) [49]. We also previously demonstrated that cryptotanshinone sensitized DU145 cells to Fas-mediated apoptosis; cryptotanshinone alone showed a marginal effect on DU145 cell death, but a combination treatment with agonistic anti-Fas antibody resulted in the marked induction of apoptosis of DU145 cells via suppression of Bcl-2 [25]. Therefore, studies on the crosstalk among AMPK and these molecules will allow us to better

Apoptosis

understand the potential of cryptotanshinone. In summary, our data suggest that cryptotanshinone deserves further investigation as an anti-cancer agent, implying that identification of a novel specific AMPK activator may significantly contribute to the treatment of cancer as well as metabolic disorders. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 20120009381). Conflict of interest

No conflict of interest is declared.

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