Sauchinone exerts anticancer effects by targeting AMPK signaling in hepatocellular carcinoma cells

Accepted Manuscript Sauchinone exerts anticancer effects by targeting AMPK signaling in hepatocellular carcinoma cells Young Woo Kim, Eun Jeong Jang, Chang-Hyun Kim, Ju-Hee Lee PII:

S0009-2797(16)30594-4

DOI:

10.1016/j.cbi.2016.11.016

Reference:

CBI 7859

To appear in:

Chemico-Biological Interactions

Received Date: 13 June 2016 Revised Date:

8 November 2016

Accepted Date: 16 November 2016

Please cite this article as: Y.W. Kim, E.J. Jang, C.-H. Kim, J.-H. Lee, Sauchinone exerts anticancer effects by targeting AMPK signaling in hepatocellular carcinoma cells, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.11.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Chemico-Biological Interactions Original Research Article

RI PT

Sauchinone exerts anticancer effects by targeting AMPK signaling in hepatocellular carcinoma cells

a

M AN U

SC

Young Woo Kim a, Eun Jeong Jang a, Chang-Hyun Kim b, Ju-Hee Lee c, *

College of Korean Medicine, Daegu Haany University, Gyeongsan 38610, Republic of

Korea b

Department of Medicine, College of Medicine, Dongguk University, Goyang 10326,

Republic of Korea

TE D

College of Korean Medicine, Dongguk University, Goyang 10326, Republic of Korea

EP

c

* Corresponding authors: Dr. JH Lee, E-mail: [email protected], College of Korean

AC C

Medicine, Dongguk University, Goyang, Gyeonggi-do 10326, Korea. Tel: +82-31-961-5839; Fax: +82-31-961-5835

1

ACCEPTED MANUSCRIPT Abbreviations ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; BCA, bicinchoninic acid; Bcl-2, B-cell lymphoma 2; DAPI, 4,6-diamidio-2-phenylindole; DMEM, Dulbecco’s Eagle’s

medium;

4E-BP1,

eIF4E-binding

protein

1;

ECL,

enhanced

RI PT

modified

chemiluminescence; ERK, extracellular signal-regulated kinases; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;

SC

GSK-3β, glycogen synthase kinase-3β; HCC, hepatocellular carcinoma; HFD, high fat diet; HIF-1α, hypoxia-inducible factor-1α; HRP, horseradish peroxidase; JNK, c-jun amino-

M AN U

terminal kinase; LKB1, liver kinase B1; MAPKs, mitogen-activated protein kinases; MMP, mitochondrial membrane potential; mTOR, mammalian target of rapamycin; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; Nrf2, nuclear factor-erythroid 2related factor-2; PAI-1, plasminogen activator inhibitor-1; PARP, poly (ADP-ribose)

TE D

polymerase; PI, propidium iodide; PKCδ, protein kinase Cδ; PVDF, polyvinylidene fluoride; Rh123, rhodamine 123; RIPA, radioimmunoprecipitation assay; RPMI-1640, Roswell Park Memorial Institute Media 1640; RT-PCR, reverse transcriptional polymerase chain reaction;

EP

S6K1, ribosomal protein S6 kinase 1; SC, Saururus chinensis; SD, standard deviation; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIRT1, sirtuin 1; SREBP-

AC C

1c, sterol regulatory-element-binding protein-1c; TUNEL, terminal deoxynucleotidyl transferase–mediated nick end labeling; VEGF, vascular endothelial growth factor

2

ACCEPTED MANUSCRIPT

ABSTRACT Sauchinone is a pharmacologically active compound isolated from Saururus chinensis, which has been used as a traditional Oriental medicine to treat fever, jaundice, and various

RI PT

inflammatory diseases. In this study, we investigated the effect of sauchinone against hepatocellular carcinoma (HCC) and sought to elucidate the mechanism involved. Cell viability was measured by an MTT assay. Cell cycle distributions and the mitochondrial

SC

membrane potential were analyzed using flow cytometry. Cell death was analyzed by annexin V assay, 4',6-diamidino-2-phenylindole staining, and terminal deoxynucleotidyl transferase

M AN U

dUTP nick-end labeling assay. Protein and mRNA levels were assessed by western blot and real-time PCR, respectively. Malignant properties were investigated by a wound healing migration assay and invasion assay. Sauchinone suppressed the proliferation of human HCC cells in a dose-dependent manner. Moreover, it induced the G0/G1 phase cell cycle arrest and

TE D

mitochondrial dysfunction and then triggered the apoptosis by activating the JNK/p38 pathway in Huh-7 cells. In addition, sauchinone induced the activation of the AMP-activated protein kinase (AMPK) pathway, and compound C (an AMPK inhibitor) blocked the

EP

sauchinone-induced mitochondrial dysfunction. The AMPK activation by sauchinone

AC C

inhibited the phosphorylation of the mammalian target of rapamycin (mTOR) and its downstream targets, such as ribosomal protein S6 kinase 1 and eIF4E-binding protein 1. Furthermore, sauchinone attenuated key proangiogenic factors, including hypoxia-inducible factor-1α, vascular endothelial growth factor, and plasminogen activator inhibitor-1, resulting in decreased migration and invasion of HCC cells. These results provide evidence for sauchinone to be considered as a potent anticancer agent by targeting of the AMPK-mTOR pathway in HCC. 3

ACCEPTED MANUSCRIPT

Key words: Sauchinone, Apoptosis, Anti-cancer, AMPK, Hepatocellular carcinoma

RI PT

1. Introduction

Hepatocellular carcinoma (HCC) is a primary malignancy of hepatocytes and one of the

SC

most common causes of cancer death worldwide. HCC is the end point of liver disease progression and frequently develops in the cases of liver cirrhosis or chronic liver disease [1].

M AN U

The early diagnosis of HCC is difficult, and thus, many patients are diagnosed with later stage disease, which usually has a poor prognosis. Surgery is the only curative therapy; although chemotherapy is administered in the cases in which surgery cannot be performed, the response rates are low [2]. Therefore, many researchers have been trying to identify new

TE D

therapeutic targets to improve responses to chemotherapy.

AMP-activated protein kinase (AMPK) has been recently presented as a possible target for cancer prevention and treatment [3-5]. It is an energy-sensing enzyme that plays a key

EP

role in the maintenance of energy balance by monitoring the cellular ratios of AMP:ATP and/or ADP:ATP [6]. AMPK is activated at high AMP:ATP ratios caused by stressors, such as

AC C

glucose deprivation, hypoxia, and oxidative stress [7]. AMPK activation promotes catabolic pathways to generate ATP, which inhibits ATP-consuming anabolic pathways including cell growth and proliferation [5, 8]. The relevance of AMPK to HCC has been demonstrated through its tumor suppressor function. Zheng et al. [9] have reported that low AMPK activity is associated with aggressive phenotypic features of HCC, its poor outcomes, and recurrence. It has been suggested that inactivation of AMPK promotes the pathogenesis of HCC via the 4

ACCEPTED MANUSCRIPT sirtuin 1/p53 pathway [10]. Accordingly, AMPK activators, such as metformin, are considered potential anti-cancer agents for HCC. Herbal medicine has received much attention owing to its efficacy and safety [11].

RI PT

Preclinical studies have shown that some phytochemicals, such as curcumin, resveratrol, and quercetin, derived from herbal medicines, inhibit the proliferation and induce the apoptosis via AMPK activation in various cancers [12-14]. During our efforts to identify anticancer

SC

candidates from herbal medicines, we have found that sauchinone upregulates the phosphorylation of AMPK [15].

M AN U

Sauchinone is a pharmacologically active compound isolated from Saururus chinensis, which is used in traditional Oriental medicine to treat fever, jaundice, and various inflammatory diseases, such as edema and asthma [16]. A number of studies have reported that sauchinone has cytoprotective, anti-inflammatory, anti-oxidant and anti-photoaging

TE D

activities [17-19]. In a previous study, we have found that sauchinone protects the liver against excess oxidative stress and hepatic injury induced by iron deposition [15]. Subsequently, it was found that the hepatoprotective effect of sauchinone on acetaminophen-

EP

induced toxicity was due to Nrf2 activation through the PKCδ-GSK3β pathway [20]. In another study, we have shown that sauchinone ameliorates hepatic steatosis by inhibiting

AC C

SREBP-1c-dependent lipogenesis and high fat diet-induced oxidative stress [21]. More recently, we have reported that sauchinone has potential utility for the prevention of liver fibrosis [22]. However, the effects of sauchinone on cancer have not been previously investigated.

Based on the ability of sauchinone to active AMPK and protect the liver against various liver diseases, we investigated whether sauchinone had tumor-suppressing effects and then

5

ACCEPTED MANUSCRIPT sought to identify the responsible mechanisms using HCC cell lines.

RI PT

2. Materials and methods 2.1. Chemicals and reagents

Sauchinone was isolated from the n-hexane fraction of Saururus chinensis, as previously

SC

described, and confirmed by a spectroscopic analysis [15]. Anti-PARP, anti-caspase3, antiBcl-2, anti-p-AMPK, anti-AMPK, anti-p-ACC, anti-p-LKB1, anti-LKB1, anti-p-mTOR, anti-

M AN U

p-S6K1, anti-p-4E-BP1, anti-p-JNK, anti-JNK, anti-p-p38, anti-p38, anti-p-ERK and antiERK antibodies were supplied by Cell Signaling Technologies (Danvers, MA, USA). Antihypoxia-inducible factor-1α (HIF-1α) and anti-cyclin D1 antibody were obtained from BD Biosciences (SanJose, CA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit

TE D

and goat anti-mouse IgGs were obtained from Zymed Laboratories (San Francisco, CA, USA). Compound C and Z-VAD-FMK were purchased from Calbiochem (San Diego, CA, USA). RNase A was purchased from Qiagen (Valencia, CA), and anti-β-actin antibody, 3-

EP

(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), propidium iodide (PI),

AC C

rhodamine 123 (Rh123), CoCl2, and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Cell culture

Huh-7 cells (a human HCC cell line) were purchased from the Health Science Research Resources Bank (Osaka, Japan), and other human HCC cell lines, HepG2 and Hep3B, and normal human Chang liver cells were obtained from the American Type Culture Collection 6

ACCEPTED MANUSCRIPT (ATCC, Manassas, VA). Huh-7 cells were cultured in Roswell Park Memorial Institute Media 1640 (RPMI-1640), whereas HepG2, Hep3B, and Chang liver cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum

RI PT

(FBS) and 1% penicillin/streptomycin. Cultures were maintained at 37°C in a CO2 incubator in a controlled humidified atmosphere composed of 95% air and 5% CO2. FBS, cell culture media, penicillin/streptomycin, and all other reagents used for the cell culture studies were

SC

purchased from Hyclone (Gaithersburg, MD, USA).

M AN U

2.3. Cell viability assay

Cell viabilities were evaluated using a MTT assay. Cells were plated at 3–5×103 cells per well in 96-well plates and treated with either dimethyl sulfoxide (DMSO) as the control or with various concentrations (10–50 µM) of sauchinone for 24 h or 48 h. After incubation,

TE D

viable cells were stained with MTT solution (0.2 mg/ml, 4 h) and the formazan crystals so obtained were dissolved by adding 200 µl of DMSO. The absorbance was measured at 570 nm using a multimode microplate reader (Tecan, Research Triangle Park, NC, USA). The

EP

analysis was conducted in triplicate.

AC C

2.4. Cell cycle analysis

Huh-7 cells were plated in 100 mm-diameter culture dishes. On the following day, cells were treated with various concentrations of sauchinone (10–50 µM) or 0.1% DMSO for 24 h. Floating and adherent cells were collected and fixed overnight in cold 70% ethanol at 4°C. After washing with PBS, cells were stained with 50 µg/ml PI and 100 µg/ml RNase A for 1 h in the dark and then subjected to a flow cytometric analysis to determine the percentage of

7

ACCEPTED MANUSCRIPT cells at specific phases of the cell cycle. Flow cytometry was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 488 nm argon laser. Events were evaluated for each sample and cell cycle distributions were obtained using Cell

RI PT

Quest software (Becton Dickinson). The results are presented as numbers of cells versus amounts of DNA as indicated by the intensities of fluorescence signals. All experiments were

SC

conducted in triplicate.

2.5. Annexin V assay

M AN U

Huh-7 cells were treated with sauchinone for 24 h, and 5×105 cells were then removed, washed twice with cold PBS, and double stained with annexin V-fluorescein isothiocyanate (FITC) and PI in annexin binding buffer. Percentages of stained cells were determined using a Partec GmbH FACS Calibur flow cytometer (Münster, Germany). To avoid non-specific

TE D

fluorescence from dead cells, live cells were gated using a forward and side scatter.

2.6. DAPI staining and TUNEL assay

EP

Huh-7 cells were plated onto 18-mm cover glasses in RPMI-1640 medium at approximately 70% confluence for 24 h. Cells were then treated with 50 µM sauchinone for

AC C

24 h, fixed in ice-cold 2% para-formaldehyde, washed with PBS, and stained with 2 µg/mL 4,6-diamidio-2-phenylindole (DAPI) for 20 min at 37°C. Stained cells were observed under a fluorescence microscope (Carl Zeiss, Germany) with a peak excitation wavelength of 340 nm. Terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) was performed using a TUNEL kit (Chemicon, Temecula, CA) following the manufacturer’s protocol.

8

ACCEPTED MANUSCRIPT 2.7. Measurement of mitochondrial membrane potentials Mitochondrial membrane potentials (MMPs) were measured by flow cytometry using rhodamine 123 (Rh123), a membrane-permeable cationic fluorescent dye [23]. Huh-7 cells

RI PT

were stained with 0.05 µg/ml of Rh123 for 1 h, and harvested by trypsinization. After washing with PBS containing 1% FBS, changes in MMPs were assessed by measuring fluorescence intensities using a Partec GmbH FACS Calibur flow cytometer (Münster,

SC

Germany).

M AN U

2.8. Western blots

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, Rockford, IL, USA), containing protease/phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA), and protein concentrations were then assessed using a bicinchoninic acid (BCA)

TE D

assay [24]. Proteins (30–50 µg) were separated by a 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Protein bands were detected

EP

using enhanced chemiluminescence (ECL) system (Amersham Biosciences, Piscataway, NJ, USA). Equal loading of proteins was verified by β-actin immunoblottings. At least three

AC C

separate experiments were performed to confirm the changes. The band intensities were quantified using ImageJ 1.42 software (NIH Bethesda, USA).

2.9. Real-time RT-PCR

Total RNA was isolated from Huh-7 cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA (1 µg) was reverse-transcribed using a reverse transcriptional polymerase chain

9

ACCEPTED MANUSCRIPT reaction (RT-PCR) kit (Promega, Madison WI, USA) to obtain cDNA. Real-time PCR was carried out using the 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Cp values of vascular endothelial growth

RI PT

factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) were normalized versus that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using 7500 System SDS Software Version 1.2 (Applied Biosystems). Melting curve analysis was performed after amplification

VEGF,

5´-AGGAGGGCAGAATCATCACG-3´

TCGTCCAGCGGGATCTGA-3´

(reverse);

(forward) human

and

5´-

PAI-1,

5´-

M AN U

CAAGGCCCACAGGGATTTTCT-3´

SC

to verify the accuracy of the amplicon. The following specific primers were used: human

(forward)

and

5´-CCTGGTCATGTTGCCTTTC-3´

(reverse); human GAPDH, 5´-GAAGGTGAAGGTCGGAGTC-3´ (forward) and 5´-

TE D

GAAGATGGTGATGGGATTTC-3´ (reverse).

2.10. Wound healing migration assay

HepG2 cells and Huh-7 cells were plated separately on 6-well plates at 90% confluence.

EP

Cells were wounded with a razor blade and injury lines were marked. After wounding, detached cells were removed with serum-free medium and the plates were further incubated

AC C

in DMEM or RPMI-1640 supplemented with 5% FBS containing various concentrations of sauchinone. After 48 h, these cells were rinsed with serum-free medium and fixed in absolute methanol. Cell migratory behavior was then observed under phase-contrast microscopy (Olympus, Tokyo, Japan) and documented.

2.11. Transwell invasion assay

10

ACCEPTED MANUSCRIPT Matrigel invasion assay was performed as previously described [25]. In brief, diluted 1:20 matrigel (1 mg/ml) (BD Biosciences, Beit-HaEmek, Israel) in serum free culture media was added to the upper chamber of a 24-well transwell plate, and incubated at 37°C for 3–4 h to

RI PT

cause gelling. Huh-7 cells were harvested from tissue culture flasks, washed, and resuspended in 1% FBS in RPMI-1640 medium, which were then added to the upper wells at a density of 5×104 cells/well in 200 µl medium containing 10–50 µM sauchinone or 0.1%

SC

DMSO; lower wells were filled with 500 µl medium containing 5% FBS. Plates were incubated at 37°C for 36–48 h, and non-invading cells on the upper surfaces of transwell

M AN U

inserts were removed with a cotton swab. Cells that had travelled to lower surfaces were fixed with absolute methanol, stained with hematoxylin, and observed under a light microscope.

TE D

2.12. Statistical analysis

Results are presented as the mean ± standard deviations (SDs) of three independent experiments. Student’s t-test was used to determine the significances of intergroup differences.

AC C

EP

Statistical significance was accepted for p values of < 0.05 or 0.01, as indicated.

3. Results

3.1. Sauchinone inhibited proliferation of HCC cells To investigate the anticancer effects of sauchinone on the human HCC cell lines, we first evaluated its effects on cell viability using the MTT assay. HepG2, Huh-7, and Hep3B cells were treated with various concentrations (0–50 µM) of sauchinone for 24 h or 48 h. As shown 11

ACCEPTED MANUSCRIPT in Fig. 1A–C, sauchinone inhibited the growth of all three HCC cell lines in both dose- and time-dependent manners. Among the cell lines tested, Huh-7 cells were found to be the most sensitive to sauchinone, and thus, were used in subsequent experiments. To determine

RI PT

whether sauchinone was cytotoxic to normal hepatocytes, cell viability of normal human Chang liver cells was assessed in the presence of various concentrations of sauchinone. The results showed that Chang liver cells treated with 10 and 30 µM sauchinone maintained cell

SC

viability at a level similar to that in the untreated control. Treatments of the normal human liver cells with 50 µM sauchinone for 24 h and 48 h decreased the cell viability by 10.9% and

normal human Chang liver cells.

M AN U

14%, respectively (Fig. 1D). These results demonstrated low cytotoxicity of sauchinone to

To investigate the relationship between the anti-proliferative effect of sauchinone and cell cycle progression, we examined the cell cycle distribution of Huh-7 cells treated with

TE D

sauchinone or vehicle. Huh-7 cells were found to accumulate in the G0/G1 phase after the treatment with 30 µM sauchinone, whereas 50 µM sauchinone increased the number of cells in the subG1 phase indicative of dead cells (Fig. 2A). In addition, sauchinone dose- and time-

EP

dependently down-regulated the expression of cyclin D1, a checkpoint protein involved in the regulation of the G1/S transition, whereas it up-regulated that of the p27Kip1 and p21Waf1

AC C

cyclin-dependent kinase inhibitors (Fig. 2B and C).

3.2. Sauchinone induced apoptosis of HCC cells Since 50 µM sauchinone increased the percentage of cells in the subG1 phase by 2.6– 14.2%, we investigated the ability of sauchinone to induce apoptosis. As shown in Fig. 3A, sauchinone increased the PARP cleavage and decreased the protein levels of the caspase-3

12

ACCEPTED MANUSCRIPT proform and Bcl-2 in Huh-7 and Hep3B cells. Furthermore, the flow cytometry based annexin V assay showed that apoptotic cells, including early and late apoptotic cells (annexin V-positive/PI-negative or -positive) were induced by the treatment with 10 µM sauchinone

RI PT

(18.2%), and their numbers increased in a dose-dependent manner (Fig. 3B). We further confirmed the sauchinone-induced apoptosis by visualizing the nuclear morphology with DAPI staining (data not shown) and by detecting DNA fragmentation using a TUNEL assay

SC

(Fig. 3C).

Early apoptosis is always accompanied by the disruption of the mitochondrial membrane,

M AN U

resulting in a rapid collapse of the electrochemical gradient [26]. Therefore, we measured the effect of sauchinone on the alternation of MMP, which was shown to correlate with the intrinsic pathway of apoptosis, by flow cytometric analysis using Rh123, a mitochondrial specific fluorescent dye. Figure 3D illustrates that sauchinone treatment significantly

TE D

increased the number of Rh123-negative cells, indicating mitochondrial dysfunction. To verify the importance of caspase-3 in the sauchinone-induced apoptosis of Huh-7 cells, the cells were pretreated with Z-VAD-FMK, a cell-permeable and irreversible pan-caspase

EP

inhibitor, for 1 h prior to the treatment with 50 µM sauchinone. Pretreatment with Z-VADFMK significantly suppressed the sauchinone-induced mitochondrial dysfunction in Huh-7

AC C

cells, indicating that the sauchinone-induced cell death was mediated by caspase activation (Fig. 3E). These results clearly suggest that sauchinone induces intrinsic apoptosis through mitochondrial dysfunction.

3.3. Sauchinone induced AMPK activation in Huh-7 cells

13

ACCEPTED MANUSCRIPT To understand the molecular mechanisms responsible for the inhibition of cell growth, we analyzed the protein levels of AMPK and those of proteins upstream and downstream of AMPK. As expected, sauchinone treatment led to the phosphorylation of AMPK, LKB1, and

RI PT

ACC in a dose-dependent manner, without altering their total protein levels (Fig. 4A). To determine whether the inhibitory effect of sauchinone was dependent on the activation of AMPK, we treated Huh-7 cells with compound C, an AMPK-specific inhibitor. We observed

SC

that the inhibition of AMPK greatly reduced the MMP loss induced by sauchinone (Fig. 4B). To explore the relationship between AMPK and mitogen-activated protein kinases (MAPKs)

M AN U

in the induction of apoptosis, we assessed the changes in the protein levels of major kinases, such as ERK, JNK, and p38. Western blot analyses showed that sauchinone upregulated the phosphorylation of JNK and p38 and downregulated that of ERK in a dose-dependent manner, without affecting their total levels (Fig. 4C). Since AMPK/mTOR signaling pathways are

TE D

involved in the regulation of cancer cell growth, proliferation, and survival, we also analyzed the effects of sauchinone on the protein levels of mTOR and its downstream targets S6K1 and 4E-BP1 and found that sauchinone effectively inhibited the phosphorylation of all three

EP

proteins (Fig. 4D). We also confirmed that the key kinases were suppressed in Hep3B cells,

AC C

another HCC cell line (Supplementary Fig. 1).

3.4. Sauchinone suppressed malignant properties of HCC cells Hypoxia is a condition that often exists in solid tumors and is known to induce biological responses crucial for tumor growth (i.e., cell proliferation, angiogenesis, and metastasis) through increasing the expression of HIF-1α [27]. Therefore, we examined the effects of sauchinone on the HIF-1α protein levels under hypoxic conditions. Huh-7 cells were treated

14

ACCEPTED MANUSCRIPT with 100 µM CoCl2, a well-known chemical hypoxia inducer, for 24 h. As expected, CoCl2 induced HIF-1α, but this induction was suppressed in a dose-dependent manner by sauchinone in Huh-7 cells (Fig. 5A). VEGF and PAI-1, target genes of HIF-1, are critical

RI PT

factors involved in angiogenesis, invasion, and hemostasis [28, 29]. Thus, we determined the effects of sauchinone on the mRNA levels of VEGF and PAI-1. As shown in Fig. 5B and C, while CoCl2 treatment upregulated VEGF and PAI-1 mRNA, co-treatment with sauchinone

SC

significantly downregulated both.

Cancer cell migration and invasion are key properties of tumor growth, angiogenesis, and

M AN U

metastasis [30]. To examine the effect of sauchinone on the ability of cells to move in a chemoattractant gradient, wound healing migration assays were performed using HepG2 and Huh-7 cells. Sauchinone was found to effectively inhibit the HepG2 and Huh-7 cell migration induced with FBS as an attractant in a dose-dependent manner (Fig. 6A). To assess the effect

TE D

of sauchinone on the cell invasive capacity, we performed a transwell invasion assay using Huh-7 cells. As shown in Fig. 6B, sauchinone dramatically suppressed the invasiveness of the cells toward the gradient of FBS used as an attractant in a dose-dependent manner. In fact,

EP

slight cytotoxic activity was also observed at the highest concentration (50 µM) of sauchinone in both the migration and invasion assays in Huh-7 cells. These data indicated

AC C

that sauchinone inhibited the malignant properties of HCC cells.

4. Discussion

Beneficial effects of sauchinone, a pharmacologically active compound found in S. chinensis, have been described in various preclinical studies [15, 20-22]. In the present study, we investigated the antitumor activity of sauchinone using HCC cells and explored the 15

ACCEPTED MANUSCRIPT underlying molecular mechanisms with a focus on AMPK. The following new findings are reported in this study: 1) sauchinone inhibits the proliferation or growth of HCC cells by inducing AMPK activation, leading to G0/G1 arrest, and subsequent apoptosis; and 2)

RI PT

sauchinone inhibits the angiogenic and metastatic properties of HCC cells through the HIF1α/VEGF pathway. To the best of our knowledge, the present study demonstrates, for the first time, that sauchinone inhibits properties associated with malignancy, such as angiogenesis

SC

and metastasis, in HCC cells. Additionally, our results provide the first evidence that the sauchinone-induced apoptosis occurs via the activation of AMPK in HCC cells.

M AN U

Several studies have investigated the activity of S. chinensis or its major components against various cancers. Kim et al. [31] have found that a methylene chloride fraction of S. chinensis inhibited the proliferation and induced apoptosis of LNCaP cells (a human prostate cell line) and MCF-7 cells (a breast cancer cell line). Furthermore, neolignans derived from

TE D

an 80% ethanol extract of S. chinensis showed a potent anti-proliferative activity against human cervical and lung cancer cells [32]. Recently, Jeong et al. [33] have showed that S. chinensis and its components caused human stomach cancer cells to undergo apoptosis. More

EP

recently, an S. chinensis extract has been shown to induce endoplasmic reticulum stress, leading to the apoptosis caused by mitochondrial damage in HepG2 HCC cells [34]. However,

AC C

the mechanisms responsible for the anti-cancer activities of S. chinensis extracts or its major components have not been fully elucidated. Because we have found in our previous studies that sauchinone induces AMPK activation in hepatocytes and because its effect on cancer has been poorly investigated, we investigated the effects of sauchinone against HCC. Consistent with previous studies, our results demonstrated that sauchinone showed an anti-proliferative activity, inducing apoptosis through mitochondrial dysfunction in HCC cells.

16

ACCEPTED MANUSCRIPT AMPK has been considered an important target for cancer prevention and treatment over the last decade [5]. AMPK activation has been reported to suppress the proliferation of various cancers via multiple mechanisms, which include the regulation of cell cycle

RI PT

progression, apoptosis, autophagy, and inhibition of protein synthesis and de novo fatty acid synthesis [35]. Indeed, many natural product-derived compounds, such as resveratrol, quercetin, curcumin, and epigallocatechin gallate, have been reported to exhibit anticancer

SC

activity by activating AMPK [12-14, 36]. In the present study, we focused on AMPK as a potential molecular target for anticancer activity of sauchinone. Our results showed that

M AN U

sauchinone strongly upregulated the AMPK phosphorylation, significantly attenuated the HCC cell growth and viability, and induced apoptosis through mitochondrial dysfunction. To examine the relationship between the sauchinone-induced AMPK activation and HCC cell apoptosis, we inhibited AMPK activity in Huh-7 cells using compound C (an AMPK specific

TE D

inhibitor) and observed that AMPK inhibition prevented the sauchinone-induced MMP loss. Sauchinone also induced the cell cycle arrest in the G0/G1 phase, which is consistent with the AMPK-induced G1 cell cycle arrest via upregulation of the p53-p21 axis [35].

EP

AMPK is also linked to MAPK signaling pathways, and the ERK, JNK, and p38 protein kinases are often deregulated in various types of human cancers [37]. It has been generally

AC C

accepted that ERK is involved in the regulation of cell proliferation, while activation of the JNK and p38 kinases is related to apoptosis [38-40]. In a previous study, it has been reported that AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleotide increased the production of reactive oxygen species, activated JNK and caspase-3, and resulted in the apoptosis of prostate cancer cells [41]. AMPK has also been shown to phosphorylate p53 and p38 MAPK, which enhances apoptotic responses [42]. In our study, sauchinone induced the

17

ACCEPTED MANUSCRIPT activation of JNK and p38 in a dose-dependent manner and thus, contributed to the apoptosis of Huh-7 cells. On the other hand, sauchinone was found to inhibit the ERK phosphorylation and thus inhibited the Huh-7 cell proliferation.

RI PT

Recent studies have implicated AMPK/mTOR signaling pathways in the regulation of cancer cell growth, proliferation, and survival. In particular, the activation of AMPK suppresses cancer cell growth by inhibiting mTOR signaling pathways [43]. In this study,

SC

mTOR and its downstream targets, such as S6K1 and 4E-BP1, were effectively downregulated by sauchinone. These findings suggest that the anti-proliferative activity of

M AN U

sauchinone is in part associated with the activation of AMPK in HCC cells. mTOR signaling also plays a critical role in HCC progression by regulating the tumor cell motility, invasion, angiogenesis, and metastasis [44]. In particular, increasing evidence demonstrates that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in the regulation of cell

TE D

motility and invasion [45]. Rapamycin, an mTORC1 inhibitor, suppressed the phosphorylation of S6K1 and 4E-BP1, resulting in decreased migration and invasion of osteosarcoma cells [46]. Moreover, mTORC1 induces the HIF-1α synthesis and thereby

EP

mediates the production of many angiogenic growth factors, including VEGF, which enhance the tumor angiogenesis through multiple signaling pathways [47]. Consistent with these

AC C

studies, we showed that sauchinone inhibited the HIF-1α protein synthesis, and this diminished the VEGF and PAI-1 expression in HCC cells and their abilities to migrate and invade.

In conclusion, our results suggest that the anti-cancer effects of sauchinone on HCC cells are caused by regulation of the AMPK-mTOR axis and that sauchinone may be considered a candidate therapeutic for the treatment of HCC.

18

ACCEPTED MANUSCRIPT

Conflicting Interests

RI PT

The authors declare that there are no conflicts of interest

Acknowledgements

SC

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No.2012R1A5A2A42671316; No.2014R1A2A2

M AN U

A01007375; No.2015K1A3A1A59069800) and by the Korea government (Ministry of Education) (No.2015R1D1A1A01060340), and also by the Grant K16830 awarded to Korea Institute of Oriental Medicine (KIOM) from Korea Ministry of Education, Science and

AC C

EP

TE D

Technology (MEST).

19

ACCEPTED MANUSCRIPT

References [1] U. Saran, B. Humar, P. Kolly, J.F. Dufour, Hepatocellular carcinoma and lifestyles, J Hepatol, 64 (2016) 203-214.

RI PT

[2] M. Schwartz, S. Roayaie, M. Konstadoulakis, Strategies for the management of hepatocellular carcinoma, Nat Clin Pract Oncol, 4 (2007) 424-432.

[3] I. Kim, Y.Y. He, Targeting the AMP-Activated Protein Kinase for Cancer Prevention and

SC

Therapy, Front Oncol, 3 (2013) 175.

[4] H.S. Kim, J. Lim, Y. Lee da, J.H. Ryu, J.S. Lim, Kazinol C from Broussonetia kazinoki

M AN U

activates AMP-activated protein kinase to induce antitumorigenic effects in HT-29 colon cancer cells, Oncol Rep, 33 (2015) 223-229.

[5] W. Li, S.M. Saud, M.R. Young, G. Chen, B. Hua, Targeting AMPK for cancer prevention and treatment, Oncotarget, 6 (2015) 7365-7378.

TE D

[6] D.G. Hardie, M.L. Ashford, AMPK: regulating energy balance at the cellular and whole body levels, Physiology (Bethesda), 29 (2014) 99-107. [7] M.M. Mihaylova, R.J. Shaw, The AMPK signalling pathway coordinates cell growth,

EP

autophagy and metabolism, Nat Cell Biol, 13 (2011) 1016-1023.

AC C

[8] D.G. Hardie, AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy, Nat Rev Mol Cell Biol, 8 (2007) 774-785. [9] L. Zheng, W. Yang, F. Wu, C. Wang, L. Yu, L. Tang, B. Qiu, Y. Li, L. Guo, M. Wu, G. Feng, D. Zou, H. Wang, Prognostic significance of AMPK activation and therapeutic effects of metformin in hepatocellular carcinoma, Clin Cancer Res, 19 (2013) 5372-5380. [10] C.W. Lee, L.L. Wong, E.Y. Tse, H.F. Liu, V.Y. Leong, J.M. Lee, D.G. Hardie, I.O. Ng, Y.P. Ching, AMPK promotes p53 acetylation via phosphorylation and inactivation of 20

ACCEPTED MANUSCRIPT SIRT1 in liver cancer cells, Cancer Res, 72 (2012) 4394-4404. [11] J. Mann, Natural products in cancer chemotherapy: past, present and future, Nat Rev Cancer, 2 (2002) 143-148.

RI PT

[12] W. Pan, H. Yang, C. Cao, X. Song, B. Wallin, R. Kivlin, S. Lu, G. Hu, W. Di, Y. Wan, AMPK mediates curcumin-induced cell death in CaOV3 ovarian cancer cells, Oncol Rep, 20 (2008) 1553-1559.

SC

[13] J.T. Hwang, D.W. Kwak, S.K. Lin, H.M. Kim, Y.M. Kim, O.J. Park, Resveratrol induces apoptosis in chemoresistant cancer cells via modulation of AMPK signaling pathway,

M AN U

Ann N Y Acad Sci, 1095 (2007) 441-448.

[14] Y.K. Lee, S.Y. Park, Y.M. Kim, W.S. Lee, O.J. Park, AMP kinase/cyclooxygenase-2 pathway regulates proliferation and apoptosis of cancer cells treated with quercetin, Exp Mol Med, 41 (2009) 201-207.

TE D

[15] Y.W. Kim, S.M. Lee, S.M. Shin, S.J. Hwang, J.S. Brooks, H.E. Kang, M.G. Lee, S.C. Kim, S.G. Kim, Efficacy of sauchinone as a novel AMPK-activating lignan for preventing iron-induced oxidative stress and liver injury, Free Radic Biol Med, 47

EP

(2009) 1082-1092.

[16] B.S. Chung, M.G. Shin, Dictionary of Korean folk medicine, Young Lim Sa, Seoul,

AC C

Korea, 1990.

[17] G.S. Jeong, D.S. Lee, B.H. Park, K.B. Kwon, Y.C. Kim, Sauchinone protects pancreatic beta cells against cytokine-mediated toxicity, Toxicol In Vitro, 25 (2011) 505-512. [18] B. Li, D.S. Lee, H.G. Choi, K.S. Kim, D.G. Kang, H.S. Lee, G.S. Jeong, Y.C. Kim, Sauchinone suppresses pro-inflammatory mediators by inducing heme oxygenase-1 in RAW264.7 macrophages, Biol Pharm Bull, 34 (2011) 1566-1571.

21

ACCEPTED MANUSCRIPT [19] G. Park, H.G. Kim, Y. Sim, S.H. Sung, M.S. Oh, Sauchinone, a lignan from Saururus chinensis, protects human skin keratinocytes against ultraviolet B-induced photoaging by regulating the oxidative defense system, Biol Pharm Bull, 36 (2013) 1134-1139.

RI PT

[20] H.Y. Kay, Y.W. Kim, H. Ryu da, S.H. Sung, S.J. Hwang, S.G. Kim, Nrf2-mediated liver protection by sauchinone, an antioxidant lignan, from acetaminophen toxicity through the PKCdelta-GSK3beta pathway, Br J Pharmacol, 163 (2011) 1653-1665.

SC

[21] Y.W. Kim, Y.M. Kim, Y.M. Yang, T.H. Kim, S.J. Hwang, J.R. Lee, S.C. Kim, S.G. Kim, Inhibition of SREBP-1c-mediated hepatic steatosis and oxidative stress by sauchinone,

M AN U

an AMPK-activating lignan in Saururus chinensis, Free Radic Biol Med, 48 (2010) 567578.

[22] J.H. Lee, E.J. Jang, H.L. Seo, S.K. Ku, J.R. Lee, S.S. Shin, S.D. Park, S.C. Kim, Y.W. Kim, Sauchinone attenuates liver fibrosis and hepatic stellate cell activation through

TE D

TGF-beta/Smad signaling pathway, Chem Biol Interact, 224C (2014) 58-67. [23] S.H. Choi, Y.W. Kim, S.G. Kim, AMPK-mediated GSK3beta inhibition by isoliquiritigenin contributes to protecting mitochondria against iron-catalyzed oxidative

EP

stress, Biochem Pharmacol, 79 (2010) 1352-1362. [24] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano,

AC C

E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Measurement of protein using bicinchoninic acid, Anal Biochem, 150 (1985) 76-85. [25] A. Valster, N.L. Tran, M. Nakada, M.E. Berens, A.Y. Chan, M. Symons, Cell migration and invasion assays, Methods, 37 (2005) 208-215. [26] M. Bras, B. Queenan, S.A. Susin, Programmed cell death via mitochondria: different modes of dying, Biochemistry (Mosc), 70 (2005) 231-239.

22

ACCEPTED MANUSCRIPT [27] P. Vaupel, The role of hypoxia-induced factors in tumor progression, Oncologist, 9 Suppl 5 (2004) 10-17. [28] J.A. Forsythe, B.H. Jiang, N.V. Iyer, F. Agani, S.W. Leung, R.D. Koos, G.L. Semenza,

RI PT

Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1, Mol Cell Biol, 16 (1996) 4604-4613.

[29] T. Kietzmann, U. Roth, K. Jungermann, Induction of the plasminogen activator inhibitor-

SC

1 gene expression by mild hypoxia via a hypoxia response element binding the hypoxiainducible factor-1 in rat hepatocytes, Blood, 94 (1999) 4177-4185.

M AN U

[30] G. Bozzuto, P. Ruggieri, A. Molinari, Molecular aspects of tumor cell migration and invasion, Ann Ist Super Sanita, 46 (2010) 66-80.

[31] H.Y. Kim, T.W. Choi, H.J. Kim, S.M. Kim, K.R. Park, H.J. Jang, E.H. Lee, C.Y. Kim, S.H. Jung, B.S. Shim, K.S. Ahn, A methylene chloride fraction of Saururus chinensis

TE D

induces apoptosis through the activation of caspase-3 in prostate and breast cancer cells, Phytomedicine, 18 (2011) 567-574.

[32] Y.J. Lee, J. Kim, J.M. Yi, S.M. Oh, N.S. Kim, H. Kim, D.S. Oh, O.S. Bang, J. Lee, Anti-

EP

proliferative neolignans from Saururus chinensis against human cancer cell lines, Biol Pharm Bull, 35 (2012) 1361-1366.

AC C

[33] H.J. Jeong, B.S. Koo, T.H. Kang, H.M. Shin, S. Jung, S. Jeon, Inhibitory effects of Saururus chinensis and its components on stomach cancer cells, Phytomedicine, 22 (2015) 256-261.

[34] A.Y. Lee, Y.A. Han, J.E. Kim, S.H. Hong, E.J. Park, M.H. Cho, Saururus chinensis Baill induces apoptosis through endoplasmic reticulum stress in HepG2 hepatocellular carcinoma cells, Food Chem Toxicol, 83 (2015) 183-192.

23

ACCEPTED MANUSCRIPT [35] H. Motoshima, B.J. Goldstein, M. Igata, E. Araki, AMPK and cell proliferation--AMPK as a therapeutic target for atherosclerosis and cancer, J Physiol, 574 (2006) 63-71. [36] J.T. Hwang, J. Ha, I.J. Park, S.K. Lee, H.W. Baik, Y.M. Kim, O.J. Park, Apoptotic effect

(2007) 115-121.

RI PT

of EGCG in HT-29 colon cancer cells via AMPK signal pathway, Cancer Lett, 247

[37] L. Min, B. He, L. Hui, Mitogen-activated protein kinases in hepatocellular carcinoma

SC

development, Semin Cancer Biol, 21 (2011) 10-20.

[38] C.S. Hill, R. Treisman, Transcriptional regulation by extracellular signals: mechanisms

M AN U

and specificity, Cell, 80 (1995) 199-211.

[39] Z. Xia, M. Dickens, J. Raingeaud, R.J. Davis, M.E. Greenberg, Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis, Science, 270 (1995) 1326-1331. [40] N.L. Johnson, A.M. Gardner, K.M. Diener, C.A. Lange-Carter, J. Gleavy, M.B. Jarpe, A.

TE D

Minden, M. Karin, L.I. Zon, G.L. Johnson, Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death, J Biol Chem, 271 (1996) 3229-3237.

EP

[41] H. Sauer, S. Engel, N. Milosevic, F. Sharifpanah, M. Wartenberg, Activation of AMPkinase by AICAR induces apoptosis of DU-145 prostate cancer cells through generation

AC C

of reactive oxygen species and activation of c-Jun N-terminal kinase, Int J Oncol, 40 (2012) 501-508.

[42] S. Cardaci, G. Filomeni, M.R. Ciriolo, Redox implications of AMPK-mediated signal transduction beyond energetic clues, J Cell Sci, 125 (2012) 2115-2125. [43] K. Inoki, T. Zhu, K.L. Guan, TSC2 mediates cellular energy response to control cell growth and survival, Cell, 115 (2003) 577-590.

24

ACCEPTED MANUSCRIPT [44] A. Villanueva, D.Y. Chiang, P. Newell, J. Peix, S. Thung, C. Alsinet, V. Tovar, S. Roayaie, B. Minguez, M. Sole, C. Battiston, S. Van Laarhoven, M.I. Fiel, A. Di Feo, Y. Hoshida, S. Yea, S. Toffanin, A. Ramos, J.A. Martignetti, V. Mazzaferro, J. Bruix, S.

RI PT

Waxman, M. Schwartz, M. Meyerson, S.L. Friedman, J.M. Llovet, Pivotal role of mTOR signaling in hepatocellular carcinoma, Gastroenterology, 135 (2008) 1972-1983, 1983 e1971-1911.

metastasis, Curr Protein Pept Sci, 12 (2011) 30-42.

SC

[45] H. Zhou, S. Huang, Role of mTOR signaling in tumor cell motility, invasion and

M AN U

[46] X. Wan, A. Mendoza, C. Khanna, L.J. Helman, Rapamycin inhibits ezrin-mediated metastatic behavior in a murine model of osteosarcoma, Cancer Res, 65 (2005) 24062411.

[47] K.M. Dodd, J. Yang, M.H. Shen, J.R. Sampson, A.R. Tee, mTORC1 drives HIF-1alpha

TE D

and VEGF-A signalling via multiple mechanisms involving 4E-BP1, S6K1 and STAT3,

AC C

EP

Oncogene, 34 (2015) 2239-2250.

25

ACCEPTED MANUSCRIPT

Figure captions Fig. 1. Effect of sauchinone on proliferation of human HCC cells. The inhibitory effects of sauchinone on the proliferation of HepG2 (A), Huh-7 (B), Hep3B (C), and Chang liver cells

RI PT

(D) were assessed using an MTT assay. Results are expressed as the percentage of cell proliferation relative to that in the vehicle-treated control. Values represent the mean ± SD of

SC

triplicate wells.

Fig. 2. Effect of sauchinone on Huh-7 cell cycle distribution. (A) Huh-7 cells were treated

M AN U

with sauchinone for 24 h, then stained with PI, and analyzed using a FACSCalibur flow cytometer (upper panel). The histogram indicates the percentages of total cells in each phase of the cell cycle (lower panel). (B and C) Western blots show that sauchinone decreased the cyclin D1 protein levels in a dose- and time-dependent manner. Significant vs. the vehicle-

TE D

treated control, *p < 0.05, **p < 0.01

Fig. 3. Effect of sauchinone on mitochondrial dysfunction and apoptosis of Huh-7 cells. (A)

EP

Expression of apoptosis-related proteins was assayed by western blotting in cells treated with sauchinone at the indicated concentrations for 24 h. (B) Apoptosis was determined by flow

AC C

cytometric analysis using annexin V-FITC and PI staining. (C) Induction of apoptosis by sauchinone (50 µM) was assessed using a TUNEL assay. The degree of apoptosis was quantified by counting TUNEL-positive cells. (D) Mitochondrial damage caused by sauchinone was investigated using rhodamine 123. (E) Reversal of the effect of sauchinone on MMP by Z-VAD-FMK. Significant vs. the vehicle-treated control, **p < 0.01; significant vs. sauchinone treatment, ##p < 0.01 26

ACCEPTED MANUSCRIPT

Fig. 4. Effect of sauchinone on the activation of AMPK and its downstream targets in Huh-7 cells. (A) Western blotting of AMPK signaling-related proteins. (B) Reversal of the effect of

RI PT

sauchinone on MMP by compound C. Significant vs. vehicle-treated control, **p < 0.01; significant vs. sauchinone treatment, ##p < 0.01 (C) Western blotting of major kinases of the

SC

MAPK signaling pathway. (D) Western blotting of mTOR and its downstream effectors.

Fig. 5. Effect of sauchinone on the HIF-1α/VEGF pathway in Huh-7 cells. (A) Induction of

M AN U

HIF-1α protein expression by sauchinone in hypoxia-induced Huh-7 cells. (B) Western blotting of components of the mTOR signaling pathway. (C) Relative mRNA expression levels of HIF-1α target genes in Huh-7 cells, determined by qPCR. Huh-7 cells were incubated with 100 µM CoCl2 for 24 h in the presence or absence of the indicated

TE D

concentrations of sauchinone.

Fig. 6. Effect of sauchinone on the migration and invasion of HCC cells. (A) A wound

EP

healing migration assay was used to investigate the motility of HepG2 cells (upper panel) and Huh-7 cells (lower panel). (B) A transwell invasion assay was performed to determine the

AC C

invasive ability of Huh-7 cells using matrigel. Original magnification ×100.

Supplementary Fig. 1. Effect of sauchinone on the activation of AMPK and its downstream targets in Hep3B cells. Hep3B cells were treated with sauchinone at the indicated concentrations for 24 h. Western blotting shows that sauchinone increased the phosphorylation of AMPK and ACC, whereas that of mTOR and ERK was decreased.

27

ACCEPTED MANUSCRIPT

B)

HepG2

*

**

**

**

**

D)

TE D

Hep3B

**

**

**

EP

**

AC C

C)

M AN U

SC

**

Huh-7

RI PT

A)

Fig. 1. Kim et al.

**

** **

Chang

** **

A)

ACCEPTED MANUSCRIPT Sau 10 Sau 30

Con M1 M2 M3 M4

M1 M2 M3 M4

M1 M2 M3 M4

M AN U

SC

RI PT

M1 M2 M3 M4

Sau 50

C)

B)

p27 p21

AC C

β-actin

30 50

TE D

Cyclin D1

Con 10

EP

Sau (μM)

Sau 30 μM

Time (h)

0

6

12

18

24

Cyclin D1 p27 p21 β-actin

*

**

** **

Fig. 2. Kim et al.

**

ACCEPTED MANUSCRIPT Hep3B Huh-7

A) Sau (μM)

Con 10 30 50

Con 10

30

50

PARP Caspase-3

RI PT

Bcl-2

C) ** **

AC C

D)

EP

TE D

**

M AN U

B)

SC

β-actin

Con

**

Sau

E)

**

**

**

**

##

z-vad

Fig. 3. Kim et al.

ACCEPTED MANUSCRIPT

A)

B) Sau (μM)

Con 10

30

50

p-ACC

**

RI PT

p-AMPK AMPK

##

SC

p-LKB1

β-actin

C)

M AN U

LKB1

CC

D)

JNK p-p38

Sau (μM) p-mTOR p-S6K1

p-4E-BP1 β-actin

AC C

p38

50

TE D

p-JNK

Con 10 30

EP

Sau (μM)

p-ERK ERK

β-actin

Fig. 4. Kim et al.

Con 10

30

50

ACCEPTED MANUSCRIPT

A)

-

-

CoCl2 (100 μM)

-

+

10 30 +

50

+

+

RI PT

Sau (μM)

HIF-1α β-actin

SC

B)

M AN U

* #

##

TE D

##

Sau (μM)

CoCl2

AC C

EP

C)

**

#

#

Sau (μM) CoCl2

Fig. 5. Kim et al.

ACCEPTED MANUSCRIPT

A)

Con

Sau 10

Con

Sau 10

Sau 30

SC

RI PT

HepG2

M AN U

Huh-7

EP

TE D

Sau 30

AC C

B)

Sau 50

Fig. 6. Kim et al.

Sau 50

ACCEPTED MANUSCRIPT

Highlights Sauchinone inhibited proliferation of human HCC cells in a dose-dependent manner.

Sauchinone suppressed the malignant properties of HCC cells.

RI PT

It induced G0/G1 cell cycle arrest, mitochondrial dysfunction, and apoptosis.

AC C

EP

TE D

M AN U

SC

Anti-cancer effects of sauchinone are caused by regulation of the AMPK-mTOR axis.