4-Acetylantroquinonol B Suppresses Tumor Growth and Metastasis of

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4-Acetylantroquinonol B suppresses tumor growth and metastasis of hepatoma cells via blockade of translationdependent signaling pathway and VEGF production Chien-Hsin Chang, Tur-Fu Huang, Kung-Tin Lin, Chun-Chieh Hsu, Wei-Luen Chang, Shih-Wei Wang, Feng-Nien Ko, Hui-Chin Peng, and Ching-Hu Chung J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504434v • Publication Date (Web): 10 Dec 2014 Downloaded from http://pubs.acs.org on December 12, 2014

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Journal of Agricultural and Food Chemistry

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4-Acetylantroquinonol

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metastasis

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translation-dependent signaling pathway and VEGF production

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Chien-Hsin Chang1, Tur-Fu Huang1, Kung-Tin Lin1, Chun-Chieh Hsu1, Wei-Luen

5

Chang1, Shih-Wei Wang2, Feng-Nien Ko3, Hui-Chin Peng1 and Ching-Hu Chung2*

6

1. Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei,

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Taiwan.

8

2. Department of Medicine, Mackay Medical College, New Taipei City, Taiwan.

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3. Oneness Biotech Co., Ltd. Taipei, Taiwan.

of

B

suppresses

hepatoma

cells

tumor via

growth

and

blockade

of

10 11

*To whom correspondence should be addressed:

12

Ching-Hu Chung, Ph.D., Department of Medicine, Mackay Medical College,

13

No. 46, Sec. 3, Jhong-Jheng Rd., Sanzhi Dist., New Taipei City, Taiwan.

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TEL: 886-2-26360303; FAX: 886-2-26361295;

15

E-mail: [email protected]

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ACS Paragon Plus Environment


1

ABSTRACT

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Hepatocellular carcinoma (HCC) has become one of most common malignancy and

3

leading cause of cancer mortality worldwide. Previous study has shown that

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4-acetylantroquinonol B (4AAQB) isolated from Antrodia cinnamomea (or

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niu-chang-chih) was observed to inhibit HepG2 cell proliferation via affecting cell

6

cycle. However, the in vivo effects and anti-metastatic activity of 4AAQB has not yet

7

been addressed. Here, we found that 4AAQB inhibited HepG2 and HuH-7 hepatoma

8

cell growth in both in vitro and in vivo models, and exhibited pronounced inhibitory

9

effects on HuH-7 tumor growth in xenograft and orthotopic models. 4AAQB

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efficiently inhibited the phosphorylation of mTOR and its upstream kinases and the

11

downstream effectors, and decreased the production of VEGF and activity of Rho

12

GTPases in HuH-7 cells. Furthermore, 4AAQB inhibited in vitro HuH-7 cell

13

migration and in vivo pulmonary metastasis. Our results suggested that 4AAQB is a

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potential candidate for HCC therapy.

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Running title: Anti-cancer effect of 4-acetylantroquinonol B

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Key word: Hepatocellular carcinoma, Antrodia cinnamomea, 4AAQB, RhoGTPase,

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mTOR, metastasis, VEGF

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Page 3 of 38


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Introduction

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HCC has become one of most common malignancy and leading cause of cancer

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mortality worldwide. It causes more than a million fatalities each year worldwide and

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it is prevalent in Asia, including Taiwan; however the increasing incidence of HCC

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has also been reported in Western countries (1, 2). The predominant risk factor in Asia

6

and Africa is chronic hepatitis B viral (HBV), however which in Western countries

7

and Japan is chronic hepatitis C viral (HCV) infection (3, 4). Although surgery is the

8

most effective therapeutic approach for HCC, tumor invasiveness, intrahepatic spread

9

and extrahepatic metastasis are the key factors to affect the HCC prognosis (5).

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Therefore, it is important to elucidate the molecular pathogenesis of HCC to develop

11

new strategies against this devastating disease.

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There are many signalling pathways are involved in tumor cell growth and metastasis

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(6). Phosphatidylinositol 3-kinase (PI3K)/Akt pathway was demonstrated to play a

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major role in these processes. PI3K can then activate Akt that subsequently

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phosphorylates several other cytoplasmic proteins and mammalian target of

16

rapamycin (mTOR). mTOR phosphorylates the translational regulators p70S6 kinase

17

(p70S6K) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1),

18

resulting in cell growth promotion and cell cycle progression (3, 7). The

19

PI3K/Akt/mTOR signalling pathway has recently become a promising target owing to 3



1

its frequent activation and dysregulation in HCC. The mTOR pathway is activated in>

2

50% patients with HCC, and phosphorylation of Akt has also been detected in up to

3

71% HCC samples. This aberrant signalling is associated with invasion and metastasis

4

of HCC and correlated with poor outcome (8).

5

Antrodia cinnamomea (niu-chang-chih) is a well-known medicinal mushroom and

6

only grows naturally in Taiwan. It has been applied in traditional Chinese medicine

7

for the treatment of diarrhea, food poisoning, drug intoxication, abdominal pain, itchy

8

skin, hypertension, and cancer therapy (9, 10). The biological activities of A.

9

cinnamomea have been described, including anti-oxidant, anti-inflammatory,

10

hepatoprotective, and anti-tumor activities (11-13). A. cinnamomea has recently

11

become more popular as a therapeutic agent for several human cancers (9, 11, 14).

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Several pure compounds in the fruiting body and mycelium of A. cinnamomea with

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anti-tumor activity have been isolated and identified (12), particularly for hepatoma

14

via the inhibition of cell growth and induction of apoptosis in human liver cancer cell

15

lines (15, 16). Lin et al. isolated 4-acetylantroquinonol B (4AAQB) from the

16

mycelium of A. cinnamomea, which potently inhibited the proliferation and growth of

17

the hepatocellular carcinoma cell line (IC50 0.1µg/ml) (17). A previous study showed

18

that 4AAQB exerted inhibitory effect on tumor growth by decreasing the expression

19

of CDK2 and CDK4 and increasing the expression of p27, p53 and p21, resulting in 4


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cell cycle arrest (18). However, to the best of our knowledge, there have been no

2

studies reporting that 4AAQB and other related antroquinonol compounds exhibit

3

anti-cancer efficacy in in vivo models. In this study, we found that 4AAQB inhibited

4

thePI3K/Akt/mTOR pathways in the human hepatocellular carcinoma cell line HuH-7.

5

In addition, 4AAQB displayed prominent anti-tumor and anti-metastatic effects in

6

xenograft and lung metastasis models.

5



1

Materials and methods

2

Materials

3

The 4-acetylantroquinonol B was isolated from the mycelium of Antrodia

4

cinnamomea as described previously (17). Lactate dehydrogenase (LDH) assay

5

reagents were purchased from Promega (Madison, WI). Antibodies to phospho-Akt

6

(Ser473), phospho-mTOR (Ser2448) were purchased from Epitomics (Burlingame,

7

CA).Antibody tophospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-p70S6K

8

(Thr389) and β-actin were purchased from Cell Signaling Technologies (Boston,

9

MA).

10

Cell culture

11

Human hepatocellular carcinoma cell line HepG2 and HuH-7 were obtained from the

12

American Type Culture Collection. HepG2 and HuH-7 cells were cultured at 37oC in

13

HEPES-buffered Dulbecco’s modified Eagle’s medium (DMEM), containing 10%

14

fetal bovine serum (Gibco) supplemented with NaHCO3, glutamax I (Gibco), 100

15

IU/mL penicillin G (sodium salt), 100 µg/mL streptomycin and 0.25µg/mL

16

amphotericin B (antibiotic-antimitotic solution, Gibco).

17

Cell viability assay

18

Hepatoma cells (HepG2 and HuH-7, 5x103cells/well) were sub-cultured onto 96-well

19

plates and starved with serum-free DMEM for 48 h. Then hepatoma cells were 6


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incubated in DMEM with 10% FBS in the absence or presence of various

2

concentrations of 4AAQB (0.1, 0.3, 1, 3, and 10 µg/ml) for 48h. After treatment, the

3

medium was discarded firstly. In order to fix the adherent cells, 100µl of

4

trichloroacetic acid (10% (w/v)) were adding to each well and incubating at 4°C for 1

5

hour. Then the plates were then washed with deionized water and dried in the air.

6

Each well were added with 50µl of Sulforhodamine B (SRB) solution (0.4% w/v in

7

1% acetic acid) and incubated for 5 min at room temperature. To remove unbound

8

SRB in the plates, the plates were washed with 1% acetic acid and then air dried. The

9

residual bound SRB was solubilized with 100µl of 10 mM Tris base buffer (pH 10.5),

10

and then read using a micro titer plate reader at 495 nm.

11

Cell cycle analysis

12

HuH-7 cells were pretreated with vehicle and different concentrations of 4AAQB for

13

24 hours. Cell cycle experiments were performed with HuH-7 cells treated with

14

vehicle as a control. According to the manufacturer’s protocol (Apoptosis, and Cell

15

Proliferation Kit; BD Biosciences, San Jose, CA), the experiment was conducted and

16

analyzed by Flow cytometry.

17

Oligonucleotide microarray analysis

18

HuH-7 cells were pretreated with vehicle or 4AAQB (3µg/ml) for 24 hours.

19

Microarray experiments were performed with HuH-7 cells treated with vehicle as a 7



Page 8 of 38

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control. According to the manufacturer’s guidelines, the synthesized cDNA was

2

synthesized by bye and hybridized onto Human OneArray microarrays (Phanlanx

3

Biotech Group, Hsinchu, Taiwan).Data analysis was conducted also according to the

4

manufacturer’s

5

Eberwine-based amplification method with Amino Allyl Message Amp II aRNA

6

Amplification Kit (Ambion, AM1753) to generate amino-allyl antisense RNA.

7

Labeled aRNA coupled with NHS-CyDye was prepared and purified prior to

8

hybridization. Purified coupled aRNA was quantified using NanoDrop ND-1000; All

9

array results are verified by pass the CyDye incorporation efficiency at > 10 dye

10

molecular/1000nt.After duplicate sample analysis, there are 1,177 up-regulated genes

11

and 791 down-regulated genes.

12

Western blotting

13

Western blotting was performed as previously described (19). HuH-7 cells were

14

pretreated with vehicle of various concentrations of 4AAQB (0.3, 1, 3, 10 µg/ml) for

15

12 hours. For the Rho/Rac1 pull-down assay, cells were harvested as described above

16

and processed according to the manufacturer's protocol (Rho/Rac1 Activation Assay

17

kit; Millipore, Billerica, MA). The binding active Rho/Rac1 proteins were detected by

18

western blotting.

19

ELISA

guidelines.

Target

preparation

was

8


performed

using

an

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HuH-7 cells (105cells/well in 96well) were treated with various concentrations of

2

4AAQB or vehicle for 24hr, and then the supernatant was collected to examine the

3

amount of vascular endothelial growth factor (VEGF) protein. VEGF Human ELISA

4

Kit (Novex®, Invitrogen) was used according to the manufacturer’s instructions.

5

Migration assay

6

Migration assay of HuH-7 cells was measured by Coaster Transwells (polycarbonate

7

filter, 8µm pore size) which were coated with 0.2% gelatin. HuH-7 cells

8

(5x104cell/well) were treated with vehicle or different concentrations of 4AAQB (1, 3,

9

10µg/ml), and then cells were seeded into the upper chamber. The bottom chamber

10

was added DMEM medium supplemented with 10% FBS. After incubation at 37℃

11

for 16 hr, all non-migrant cells were removed from the upper face of the Transwell

12

membrane with a cotton swab, and theHuH-7 cells that had transmigrated through the

13

micropore and that were still bound to the membrane were fixed with 4%

14

paraformadehyde in PBS solution and stained with 0.5% toluidine blue in 4%

15

paraformadehyde. Migration was quantified by counting the number of stained cells

16

on the membrane under a light microscope (Nikon, Japan) at a magnification of 200×

17

in 3 random fields, and then photographed. All determinations were obtained by

18

replication in at least three independent experiments.

19

Animals 9



1

8-10 weeks-old male NOD SCID mice were used in all studies. All the experimental

2

protocols regarding animal study have been approved by the Institutional Animal Care

3

and Use Committee at College of Medicine, Tzu-Chi University.

4

Subcutaneous xenograft models

5

HepG2 or HuH-7 (2 x 106) cells in a volume of 100 µl were injected subcutaneously

6

into the right flank of the male NOD SCID mice. After nine days, when tumors

7

reached 2 mm in diameter, the mice were randomly given with vehicle or 4AAQB at a

8

dosage of 0.2 or 2 mg/kg for the HepG2 model and 0.5 or 2 mg/kg for the HuH-7

9

model once daily by intraperitoneal injection. Tumor size and body weight were

10

measured every day. The tumor volume was calculated according to the following

11

formula: V= (length x width2/2). In accordance with Animal Ethics Committee, the

12

experiments should be stop when unbearable situation was appeared in animals.

13

Immunohistochemistry Study

14

Paraffin embedded tumor were cut in 25 µm sections and then immunolocalized with

15

ROCK1. The sections were incubated with 1% H2O2and washed three times in PBS

16

containing Triton X-100 (0.4%) and 2% BSA. Blocking was performed with 10%

17

BSA and then incubated on rocking table with Rock1 monoclonal antibody at 4 °C

18

overnight. Sections were washed with PBS, incubated with the biotinylated

19

anti-mouse IgG, washed with PBS again, and then incubated with 0.2% ABC solution 10


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(Vector Laboratories, Burlingame, CA). After washed with PBS, sections were treated

2

with DAB (Sigma) for 15 min. Sections were mounted on gelatinized slides, air-dried

3

overnight and cover slipped.

4

Orthotopic tumor models

5

An orthotopic tumor model was created by intrahepatically injecting 2 x106 HuH-7

6

cells in a volume of 100 µl into 8-10-wk-old male NOD SCID mice. Seven days after

7

implantation, mice were randomly divided into two groups (vehicle and 4AAQB).

8

4AAQB-treated groups received 2 mg/kg by intraperitoneal injection every day. The

9

tumor-bearing mice were sacrificed by CO2 asphyxiation on Day 25 after

10

administration of 4AAQB, and the livers were excised. Tumor-bearing liver lobes

11

were fixed in formalin (10%). The orthotopic hepatocarcinoma area of liver surface

12

was white and background is dark red. White area was quantified using ImageJ

13

software

14

Experimental metastasis analysis

15

Metastasis analysis was performed as previously described (20). Briefly, HuH-7 cells

16

(2 x 106 cells) were slowly injected into the lateral tail vein of NOD SCID mouse to

17

initiate tumor metastasis. 4AAQB (0.5 or 2 mg/kg) was intraperitoneally administered

18

to mice everyday from 3 days before tumor cell injection to 21 days after tumor cell

19

injection (the day sacrified). 11



1

Statistical analysis

2

All values are presented as mean ± SEM. Differences between groups were assessed

3

by one-way ANOVA and Newman–Keuls multiple comparison test where appropriate.

4

P values less than 0.05 (P < 0.05) were considered to be significantly different.

12


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Results

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Effects of 4AAQB on the inhibition ofHepG2 and HuH-7 hepatoma cell

3

proliferation

4

The structure of 4AAQB is shown in Fig. 1A. A previous study reported that 4AAQB

5

displayed a great growth inhibitory effect on HepG2 cells (17). Since the

6

Hepatocellular carcinoma is the major type in hepatoma and HepG2 is a

7

hepatoblastoma, we used another hepatocellular carcinoma cell line HuH-7 to

8

examine the inhibitory effects of 4AAQB in this study. We first determined the effect

9

of 4AAQB on hepatoma cell proliferation. HepG2 and HuH-7 cells were treated with

10

4AAQB for 48 h, followed by SRB assay. As shown in Fig. 1B-1C, 4AAQB inhibited

11

the proliferation of HuH-7 cells in a concentration dependent manner, with a similar

12

potency in HepG2 cells (IC50= 0.57 µg/ml, ≈ 1.9 µΜ in HuH-7 and 0.365 µg/ml, ≈

13

1.22 µΜ in HepG2).

14

Effects of 4AAQB on HuH-7 cell cycle

15

In light of a previous study showing that 4AAQB causes cell cycle arrest in HepG2

16

cells, we further examined the effects of 4AAQB on cell cycle and apoptosis in

17

HuH-7 cells. Here we found that after treating the cells with 4AAQB, the proportion

18

of cells in the G0/G1 phase is increased, and that in the S phase decreased (Fig. 1D).

19

However, 4AAQB did not induce cell apoptosis in HuH-7 cells (data not shown). 13



1

Anti-tumor effects of 4AAQB in in vivo models

2

To assess the anti-tumor effects of 4AAQB in vivo, HepG2 and HuH-7 cells were

3

subcutaneously injected into the right flanks of NOD/SCID mice in order to establish

4

a xenograft model. Tumor-bearing mice were intraperitoneally administered with

5

4AAQB at a dosage of 0.2 or 2 mg/kg/day for the HepG2 model and 0.5 or 2

6

mg/kg/day for the HuH-7 model. Tumor volume rapidly increased in the vehicle

7

group in both the models. The administration of 4AAQB significantly suppressed

8

tumor growth in a dose-dependent manner in both the models (Fig. 2). In addition,

9

ROCK-1 expression was studied by DAB staining. 4AAQB treated group

10

significantly decreased ROCK1 expression in the HuH-7 tumor cell (Fig 2C).

11

Therefore, we examined whether 4AAQB elicits anti-tumor activity when HuH-7

12

tumor cells are orthotopically transplanted. Seven days after tumor transplantation,

13

mice were intraperitoneally administered with vehicle or 4AAQB at a dosage of 2

14

mg/kg/day. In this intrahepatic model, 4AAQB also exhibited a pronounced inhibitory

15

effect on tumor growth (Fig. 3). These results clearly showed that 4AAQB displayed

16

anti-tumor activity in vivo.

17

Effects of 4AAQB on mRNA expression

18

To determine whether 4AAQB treatment induces transcriptomic changes, microarray

19

using Human OneArray system were performed. The top 10 enrichment pathway 14


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terms from gene set enrichment analysis are shown in Supplementary Table 1. The

2

top-scoring network was linked with the ECM pathway. In this pathway, there were 6

3

genes that were significantly down- or up-regulated (MAPK1, TGB1, RAF1, ROCK1,

4

ARHGAP5 and GSN). These genes are known to be associated with cell shape

5

changes during cell spreading or migration which both ERK and PI3Kare known to be

6

important. In addition, we found that the expression of epithelial-mesenchymal

7

transition

8

(Supplementary Table 2).

9

4AAQB inhibits the PI3K/Akt/mTOR and ERK pathways in HCC cells

(EMT)-associated

genes

was

altered

after

4AAQB

treatment

10

Given that the cell cycle was partly affected by 4AAQB and that the microarray data

11

showed that the expression of EMT-associated genes was altered after 4AAQB

12

treatment, we validated this observation to further understand the underlying

13

mechanism of action of 4AAQB. The PI3K/Akt/mTOR and RAS/RAF/MAPK

14

pathways, well-known signaling pathways, are important in HCC development. Thus,

15

follow-up experiments to determine whether 4AAQB affects the phosphorylation of

16

these proteins in HuH-7 cells were performed. Western blot experiments showed that

17

4AAQB suppressed the phosphorylation of PI3K, Akt, mTOR, p70S6K and ERK (Fig.

18

4). However, total expression of these proteins was not affected after 4AAQB

19

treatment. These results indicated that the anti-cancer activity of 4AAQB against 15



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HuH-7 cells is associated with the PI3K/Akt/mTOR and ERK pathways. We further

2

compare the 4AAQB’s inhibitory activity on cell proliferation with the

3

well-characterized inhibitors of these pathways to determine their correlation in

4

HuH-7 cells. HuH-7 cells were treated with either 20µM PD98059 (ERK-specific

5

inhibitor), 10µM LY294002 (PI3K-specific inhibitor), 10µMU73122 (PLC inhibitor)

6

or 1µM rapamycin (mTOR inhibitor), followed by SRB assays. As shown in Fig. 4B,

7

PD98059, LY294002 and rapamycin resulted inapproximately 50 % inhibition of cell

8

growth, indicating that ERK, PI3K, and mTOR are involved in HuH-7 cell growth,

9

while 4AAQB (1µg/ml) resulted in a inhibition to a greater extent. However, U73122

10

resulted in only a 25%-30 % inhibition.

11

4AAQB inhibits VEGF release

12

As per previous study, high VEGF expression is associated with hepatocellular

13

carcinoma progression (21). The PI3K/Akt/mTOR pathway increases the synthesis of

14

transcript factors to regulate the expression and production of VEGF. Therefore, we

15

examined the effect of 4AAQB on VEGF release in HuH-7 cells. After a 24-h

16

treatment with various concentrations of 4AAQB, the supernatant was collected for

17

analysis.

18

concentration-dependent manner in HuH-7 cells (Fig. 5A).

19

Effects of 4AAQB on Rho and Rac1 activation

4AAQB

was

observed

to

suppress

16


VEGF

release

in

a

Page 17 of 38


1

The Rho GTPase signalling cascade, including Rho and Rac which are activated by

2

VEGF, is a key regulator of cytoskeletal dynamics and affects various cellular

3

processes such as vesicle trafficking, cytoskeleton regulation and migration (22). As

4

shown in Fig. 5B, we found that 4AAQB inhibited the activation of Rho and Rac1 in

5

a concentration dependent manner, indicating that Rho GTPases are involved in the

6

inhibitory actions of 4AAQB.

7

4AAQB inhibits in vitro cell migration and in vivo pulmonary metastasis in

8

HuH-7 cells

9

Because of the EMT-associated genes that were altered after 4AAQB treatment

10

(Supplementary Table 2), we further examined whether 4AAQB affects cell migration

11

in HuH-7 cells. As shown in Fig. 6A, HuH-7 cell migration was inhibited by 4AAQB

12

in a concentration-dependent manner. Finally, we investigated the effect of 4AAQB in

13

the HuH-7 lung metastasis model in NOD/SCID mice. In this model, a single

14

intravenous bolus injection of 2x106 HuH-7 cells resulted in lung metastases. 4AAQB

15

(0.5 or 2 mg/kg) was intraperitoneally administered to the mice from 3 days before

16

tumor cell injection. Twenty-one days later, the mice were sacrificed, and lungs were

17

removed. Mice treated with 4AAQB result in a significant dose-dependent reduction

18

in lung metastases (Fig. 6B).

17



1

Discussion

2

A.cinnamonea has been widely used to treat cancer and inflammation. Various

3

components isolated from the fruiting bodies and mycelia of A. cinnamomea have

4

been reported to exhibit some biological activity (23). Antroquinonol, a ubiquinone

5

derivative isolated from the mycelia of A. cinnamomea shows potent anti-cancer

6

activity against various human malignant carcinoma cell lines; it is particularly potent

7

against Hep3B (IC500.13+0.02µM) (24). Morever, it has been reported to display

8

hepato-protective effects both in vitro and in vivo (25). Lin & Chiang further

9

investigated the mechanism of action of 4AAQB and found that it potently inhibits

10

HepG2 cell proliferation by exerting various effects on cell cycle regulators (18). The

11

present study reveals that 4AAQB significantly suppresses HuH-7 cell proliferation,

12

with a similar inhibitory effect on HepG2 cells, and exhibits potent anti-tumor effects

13

in vivo. To the best of our knowledge, this is the first study to demonstrate the

14

anti-tumor effect of 4AAQB in vivo.

15

Aberrant activation and deregulation of the PI3K/Akt/mTOR pathway was occurred

16

in various human cancers. This pathway has become one of the most important

17

therapeutic targets for anti-cancer drug development because of its integral role in the

18

regulation of various important cellular processes (3, 7, 26). Currently, targeting

19

mTOR signalling pathways for chemotherapy for cancer, including HCC, is 18


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investigated because of its aberrant activation in approximately 50% patients (27).

2

Microarray data also showed that various genes were affect by 4AAQB treatment,

3

especially linking with the ECM pathway (Supplementary Table 1), indicating that the

4

effect of 4AAQB may be associated with this pathway. We further found that 4AAQB

5

effectively inhibited the phosphorylation of mTOR and its upstream kinases, PI3K

6

and Akt, and the downstream effectors (p70S6K) in HuH-7 cells (Fig. 4). It has been

7

shown that p70S6K activity is essential for G1 progression (28). This result may be

8

supported by the fact that 4AAQB induced G1 arrest in HepG2 cell in a previous

9

study (18) and in HuH-7 cells in our study. However, 4AAQB did not induce

10

apoptosis in HuH-7 cells, indicating that its anti-proliferative activity excluded an

11

apoptotic effect. The mTOR signaling pathway is also regulated by a

12

PI3K/Akt-independent pathway, such as ERK, p38 MAPK and AMPK (29). (Fig. 4).

13

We also use these kinase inhibitors to evaluate the importance of these protein kinases

14

involved in cancer cell growth. Since 4AAQB significantly inhibited the

15

phosphorylation of ERK/PI3K/mTOR signaling pathway and ERK/PI3K/mTOR

16

inhibition are correlated with cancer cell growth inhibition, the anticancer effect of

17

4AAQB may dependent by PI3K/Akt/mTOR pathway blockage.

18

In addition, mTOR plays a key role in angiogenesis, i.e. the formation of new blood

19

vessels to provide oxygen and nutrients to growing and dividing cells. mTOR 19



1

activation through the PI3K/Akt pathway is associated with increased translation of

2

hypoxia-inducible factor (HIF)-1α, therefore driving the expression of angiogenic

3

growth factors (30). VEGF is spontaneously produced by hepatoma cells; after a 24-h

4

treatment with 4AAQB, VEGF production decreased in a concentration-dependently

5

in HuH-7 cells (Fig. 5A), indicating that a downstream effector of mTOR was also

6

inhibited by 4AAQB. VEGF, acting directly through the VEGF receptor (VEGFR),

7

leads to the activation of downstream signalling pathway and phenotypic changes

8

such as an increase in cell migration and invasion (31). A growing body of evidence

9

indicated that one of the signalling cascades activated by VEGF, Rho GTPases

10

including Rho, Rac1 and Cdc42, play a crucial role in controlling a variety of cellular

11

processes such as cell proliferation, actin cytoskeleton reorganization, and gene

12

expression (32, 33). The aberrant activity of GTPases has been implicated in cancers

13

and other diseases (34, 35). In the present study, 4AAQB inhibited the activation of

14

Rho and Rac1 in a concentration-dependent manner (Fig. 5B).

15

Various therapeutic approaches, including resection, hepatic artery injection

16

sclerotherapy, ethanol injections and ablation therapy have had remarkable effects on

17

primary lesions in HCC. However, HCC prognosis remains poor because of tumor

18

invasiveness, intrahepatic spread, and extrahepatic metastase (36). Pulmonary lesions

19

are the most common extrahepatic HCC metastases, accounting for 50%–60% of the 20


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1

total metastases that occur (37, 38). Therefore, it needs to consideration of more

2

aggressive treatments for extrahepatic metastases. In the in vivo pulmonary metastasis

3

model, 4AAQB dose-dependently reduced lung metastasis foci (Fig. 6B), indicating

4

that 4AQQB exhibited not only anti-tumor but also anti-metastatic activities towards

5

HCC.

6

In conclusion, we demonstrated that 4AAQB inhibited tumor growth in vitro by

7

inducing cell cycle arrest and blocking the PI3K/Akt/mTOR and ERK pathways. The

8

production of VEGF and activity of Rho GTPase were also decreased after 4AAQB

9

treatment. More importantly, 4AAQB exhibited anti-tumor activity in vivo, including

10

xenograft and orthotopic tumor models in addition to pulmonary metastasis. Taken

11

together, our results suggest that 4AAQB is a potential anti-cancer agent for treating

12

HCC. While no clinical studies have been conducted with 4AQQB, there are only two

13

Antrodia cinnamomea have been approved and with results. One study was aimed to

14

determine the MTD/DLTs and safety/tolerability profiles of antroquinonol (pure

15

compound purified from Antrodia cinnamomea), there are no treatment-emergent

16

adverse events (TEAEs) were reported as determined (39). There are only mild side

17

effects including stomach upset, dry mouth and diarrhea, which can be alleviated by

18

stopping use, or taking the supplement with meals to relieve stomach upset and

19

diarrhea. Further clinical trials are required for a full evaluation of this valuable 21



1

medicinal mushroom for potential clinical applications.

22


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1

Abbreviations

2

HCC: Hepatocellular carcinoma

3

4AAQB: 4-acetylantroquinonol B

4

PI3K: phosphatidylinositol 3-kinase

5

mTOR: mammalian target of rapamycin

6

p70S6K:p70S6 kinase

7

SRB: Sulforhodamine B

8

VEGF: vascular endothelial growth factor

9 10

Acknowledgments

11

This study was supported by research grants from the Ministry of Science and

12

Technology of Taiwan (102-2320-B-715-002-MY2) awarded to Ching-Hu Chung.

13 14

Conflict of Interest Statement

15

All authors have no financial or personal relationships with other people or

16

organizations that could inappropriately influence our work.

17 18

Author Contributions

19

Conceived and designed the experiments: Tur-Fu Huang and Ching-Hu Chung

20

Performed the experiments: Chien-Hsin Chang, Kung-Tin Lin, Hui-Chin Peng and 23



1

Ching-Hu Chung

2

Analyzed the data: Chien-Hsin Chang, Chun-Chieh Hsu, Shih-Wei Wang and

3

Ching-Hu Chung

4

Contributed materials purified/analysis tools: Wei-Luen Chang and Feng-Nien Ko

5

Wrote the manuscript: Chien-Hsin Chang and Ching-Hu Chung

24


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Figure Legends

2

Figure 1. The structure of 4AAQB and its effect on hepatoma cells viability and

3

cell cycle. The structure of 4AAQB is shown in (A). HepG2 (B) and HuH-7 (C) cells

4

were seeded onto plates for 24hr and starved for another 48 hr with serum-free

5

DMEM. Quiescent cells were cultivated with DMEM containing 10% FBS in the

6

absence or presence of various concentrations of 4AAQB. After 48 h, cell viability

7

was detected by the SRB assay. The results are presented as percentages of inhibition

8

(mean ± SEM, n = 4). (D) HuH-7 cells were treated with vehicle or 4AAQB (1 and 3

9

µg/ml) for 24 hr and analyzed by flow cytometry. Cell cycle distribution is shown as

10

percentage in cell cycle phase.

11

Figure 2. Effect of 4AAQB on HepG2 and HuH-7 tumor xenograft in vivo. SCID

12

mice were s.c. inoculated with 2 x 106 cells of HepG2 (A) or HuH-7 (B). When

13

tumors reached 2 mm in diameter, mice were administered with vehicle or indicated

14

dosage of 4AAQB by intraperitoneal injection once daily (QD). The tumor volume of

15

HepG2 and HuH-7 xenograft models were measured and plotted daily in

16

4AAQB-treated or control mice. Points, mean tumor volume (mm3); (C) Expression

17

of Rock1 in HuH-7 tumor xenograft was studied and the ROCK 1 immunoreactive

18

(IR) cells were counted. Scale Bar=10µm, SE;*p

4-Acetylantroquinonol B Suppresses Tumor Growth and Metastasis of

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