Lycopene Inhibits Human liver adenocarcinoma SK-Hep-1 Cells

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Lycopene Inhibits Human liver adenocarcinoma SK-Hep-1 Cells Metastasis by Down Regulation of NADPH Oxidase 4 Protein Expression Bo-Yi Jhou, Tuzz-YING SONG, Inn Lee, Miao-Lin Hu, and Nae-Cherng Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03036 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 21, 2017

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Journal of Agricultural and Food Chemistry

Lycopene Inhibits Human Liver Adenocarcinoma SK-Hep-1 Cells Metastasis by Down Regulation of NADPH Oxidase 4 Protein Expression Bo-Yi Jhou1, Tuzz-Ying Song2, Inn Lee4, Miao-Lin Hu1,3,* and Nae-Cherng Yang4,5,*

1

Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan

2

Department of BioIndustry Technology, Dayeh University, Changhua, Taiwan

3

Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan

4

Department of Nutrition, Chung Shan Medical University, Taichung, Taiwan

5

Department of Nutrition, Chung Shan Medical University Hospital, Taichung, Taiwan

*Corresponding Author: Nae-Cherng Yang, Ph.D., Tel: +886-4-24730022 ext.19009; E-mail address: [email protected] Or, Miao-Lin Hu, Ph.D., Tel: +886-4-2281-2363; Fax: +886-4-2281-2363; E-mail address: [email protected]

Running title: the anti-metastatic action of lycopene is via down-regulation of NOX4.

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ACS Paragon Plus Environment


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ABSTRACT

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NADPH oxidase 4 (NOX4) with the sole function to produce reactive oxygen species

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(ROS) can be a molecular target to disrupt cancer metastasis. Several studies have indicated

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that lycopene exhibited anti-metastatic actions in vitro and in vivo. However, the role of

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NOX4 in the anti-metastatic action of lycopene remains unknown. Herein, we first

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confirmed the anti-metastatic effect of lycopene (0.1-5 µM) on human liver adenocarcinoma

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SK-Hep-1 cells. We showed that lycopene significantly inhibited NOX4 protein expression

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with the highest inhibition of 64.3 ± 10.2 % (P < 0.05) at 2.5 µM lycopene. Lycopene also

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significantly inhibited NOX4 mRNA expression, NOX activity, and intracellular ROS levels

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in SK-Hep-1 cells. We then determined the effects of lycopene on transforming growth

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factor β (TGF-β)-induced metastasis. We found that TGF-β (5 ng/mL) significantly

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increased migration, invasion, adhesion, intracellular ROS level, matrix metalloproteinase

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(MMP)-9 and MMP-2 activities, NOX4 protein expression, and NOX activity. All these

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TGF-β-induced effects were antagonized by the incubation of SK-Hep-1 cells with

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lycopene (2.5 µM). Using transient transfection of siRNA against NOX4, we found that the

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down-regulation of NOX4 could mimic lycopene to inhibit the cell migration and the

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activities of MMP-9 and MMP-2 during the incubation with or without TGF-β on

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SK-Hep-1 cells. The results demonstrate that the down-regulation of NOX4 plays a crucial

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role in the anti-metastatic action of lycopene in SK-Hep-1 cells.

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KEYWORDS：：Lycopene / NADPH oxidase 4 / Metastasis / SK-Hep-1 cells / TGF-β

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INTRODUCTION

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Hepatocellular carcinoma (HCC), one of the most common malignant tumors, is the third

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common cause of death in cancer patients in world.1,2 Hepatectomy and liver transplantation

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are often used to improve the survival rate, but there is still a high incidence of recurrence

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because of intrahepatic and extrahepatic metastases.3 Tumor metastasis, the spread of tumor

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cells from the primary site to colonize distant organs, is the major cause of death in cancer

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patients. Metastasizing cells must first disseminate from the primary site, invade the

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surrounding tissue, intravasate and migrate in the circulation, extravasate into the blood

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vessels of distant tissue, colonize, initiate angiogenesis and finally grow at the new site.4

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Thus, inhibition of tumor metastasis can be regarded as a useful therapeutic strategy for

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alleviated cancer progression.

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Lycopene with a bright red carotene pigment is an acyclic and tetraterpene

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hydrocarbon compound containing eleven conjugated and two non-conjugated double

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bonds.5 Unlike other carotenes, such as β-carotene and α-carotene, lycopene has no

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pro-vitamin A activity because of lacking a β-ionone ring.6 Lycopene is the predominant

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carotenoid in tomatoes and can also be found in watermelons, guavas, papayas, and red

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grapefruits.7 In addition, lycopene is one of the major carotenoids in human plasma (about

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0.5 µM).5 Several studies have indicated that lycopene exhibits a variety of potential

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beneficial effects on human health, such as anti-oxidation,8,9 anti-inflammation,10

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immunomodulation,11 enhancement of gap junctional communication,12 induction of phase

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II enzymes,13 inhibition of cell proliferation,14 anti-angiogenesis,11,15 and

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anti-metastasis.16,17

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NADPH oxidases (NOX) are the only known enzyme family with the sole function of

48

producing reactive oxygen species (ROS).18,19 These multi-protein complexes are comprised 3



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of a catalytic, transmembrane-spanning subunit, and several structural and regulatory

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proteins localized in membrane and cytosol.20 The NOX family consists of seven members,

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including NOX1-5, and two dual oxidases (Duox), Duox1 and Duox2.21 Of the catalytic

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NOX subunits, NOX4, a 578-amino acid and six transmembrane domain ﬂavocytochrome,

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is the most widely distributed isoform in human and murine tissues, such as kidney, bone,

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vascular tissue, heart, liver, and lung tissues.22-24 At subcellular localization, NOX4 is not

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only localized in perinuclear regions and endoplasmic reticulum but also detected at the

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plasma membrane, focal adhesions, and mitochondria.25,26 NOX4 has been shown to act as

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electron transporter which can produce superoxide from cytosolic NADPH across biological

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membranes.27 In addition, NOX4 is expressed in a variety of cancer cell lines, such as

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prostate cancer, melanoma, bladder cancer, breast cancer, thyroid carcinoma, and ovarian

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cancer, with multi-functions including promotion of cell proliferation, cell transformation

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and angiogenesis.28 On the other hand, TGF-β has been shown to promote cancer metastasis

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through enhancement of invasive ability and inhibition of immune cell function both in

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vitro and in vivo.29 Several studies have demonstrated that TGF-β is a regulator of NOX4;

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for examples, the TGF-β-induced NOX4 activation has been implicated in osteoblast

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differentiation, fibroblast proliferation, endothelial cell cytoskeletal rearrangement, cell

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motility, epithelial-to-mesenchymal transition, pulmonary fibrosis, hepatitis C virus-induced

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hepatocyte oxidative stress and angiogenesis.30,31 Interestingly, it has been demonstrated

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that NOX4 plays an important role in TGF-β-induced 4T1 cell metastasis, suggesting that

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NOX4 can be a molecular target to disrupt cancer metastasis.28 However, it is unclear

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whether the TGF-β-induced NOX4 activation is involved in the anti-metastatic action of

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lycopene in SK-Hep-1 cells.

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SK-Hep-1 is an immortal, human cell line derived from the ascetic fluid of a patient with liver adenocarcinoma in 1971.32,33 SK-Hep-1 cells are not hepatocytes32,33 (note: 4


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hepatocytes are epithelial cells). Although the original paper33 reported that SK-Hep-1cells

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were endothelial cells, a following paper by Eun et al. (2014)32 demonstrated that SK-Hep-1

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cells did not express many of the endothelial markers, e.g., CD31. These authors further

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reported that SK-Hep-1 cells originated from mesenchymal cells but not from endothelia

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cells. In addition, liver cancers can be phenotypically divided into well-, moderate-, and

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poor-differentiated carcinomas, and liver specific genes can be detected in well- and

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moderate-differentiated hepatocarcinoma cells but not in poor-differentiated

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heptocarcinoma.32 Thus, SK-Hep-1 cells, which originate from mesenchyme without liver

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specific genes, should belong to the poor-differentiated heptocarcinoma.32 Because of their

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high metastatic capacities, SK-Hep-1 cells are suitable for cancer metastasis research and

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have been used wildly in the literature.

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The present study was aimed at revealing the role NOX4 in the anti-metastatic action

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of lycopene in a highly invasive SK-Hep-1 cells. We hypothesized that NOX4 is involved in

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the anti-metastatic action of lycopene in SK-Hep-1 cells. We cultured the cells in the

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medium with or without TGF-β to monitor the actions of lycopene on the metastasis of

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SK-Hep-1 cells. Furthermore, we used siRNA to knockdown the expression of NOX4 to

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mimic the effect of lycopene and to confirm the role of NOX4 inhibition in the effects of

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lycopene on the metastasis of SK-Hep-1 cell.

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MATERIALS AND METHODS

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Chemicals and Materials. All chemicals used in this study are of the highest grade.

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Tetrahydrofuran (THF) and butylated hydroxytoluene (BHT) as the solvents for lycopene

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were obtained from Merck (Darmstadt, Germany). The materials for cell culture, such as

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Dulbecco’s modified Eagles medium (DMEM), non-essential amino acid,

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penicillin/streptomycin, sodium pyruvate, fetal bovine serum (FBS), trypsin and Giemsa 5



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stain were obtained from Gibco/BRL (Grand Island, NY, USA). Sodium bicarbonate

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(NaHCO3), diphenyleneiodonium chloride (DPI), 2', 7'-dichlorofluorescein diacetate

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(DCFDA), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt

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hydrate (NADPH) and gelatin were purchased from Sigma-Aldrich (St. Louis, MO, USA).

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Transforming growth factor β (TGF-β) was purchased from Cell Signaling Technology

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(Beverly, MA, USA). Matrigel was purchased from BD Biosciences (San Jose, CA).

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Anti-NOX4 rabbit monoclonal antibody was purchased from Abcam (Cambridge, MA).

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siRNA against NOX4 (siGENOME Human NOX4 siRNA SMARTpool) and non-targeting

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siRNA control (siGENOME Non-Targeting siRNA Pool #1) were purchased from Thermo

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Scientific Dharmacon (USA). X-tremeGENE siRNA transfection reagent was purchased

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from Roche (Mannhein, Germany).

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Lycopene Preparation. Lycopene was purchased from Wako (Osaka, Japan) and the

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purity was > 90% as claimed by supplier. Lycopene was dissolved in THF/BHT to form a

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10 mM stock solution and stored at -80°C until used. The stock solution was then diluted

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with THF/BHT to form 0.05-2.5 mM working solution followed by diluted with FBS at 1:9

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ratio.34 THF/BHT-FBS-lycopene was added to the culture medium at a final concentration

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of 0.1-5 µM. The final concentration of THF/BHT and FBS in the medium was 0.2% (v/v)

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and 1.8% (v/v), respectively, which did not affect the assays described below.

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Cell Culture. The human liver adenocarcinoma SK-Hep-1 (BCRC No. 67005) cell line

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was purchased from Food Industry Research and Development Institute (Hsin Chu, Taiwan).

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SK-Hep-1 cells were cultured in DMEM medium containing 10% (v/v) FBS, 0.37% (w/v)

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NaHCO3, and 1% (v/v) antibiotic-antimycotic in a humidified incubator under 5% CO2 and

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95% air at 37°C.

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Cell Migration and Invasion Assay. Cell migration and invasion were determined using transwell chambers (Millipore) with 6.5 mm polycarbonate filters of 8 µm pore size 6


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according to the method reported by Repesh35 with minor modifications. The major

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difference between cell migration and invasion assay is that each filter for the invasion

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assay was pre-coated with 100 µL of a 1:20 diluted matrigel in cold DMEM to form a thin

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continuous film in the upper chamber. After pre-incubation with lycopene (0.1-5 µM) for 2,

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6 and 12 h, cells were adjusted to 1 × 105 cells/400 µL for migration and 5 × 104 cells/400

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µL for invasion in serum-free DMEM and placed in the upper chamber. The lower chamber

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was loaded with 600 µL of DMEM containing 10% FBS for an additional 6 h for migration

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and 24 h for invasion. Cells on the upper chamber were completely rubbed off using cotton

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swabs. Cells on the lower chamber were fixed in methanol, stained with Giemsa, and

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photographed under a microscope. For each replicate, the tumor cells in five random fields

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were selected, and the counts were averaged.

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Cell Adhesion Assay. The method reported by Yang et al.36 was used for the cell

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adhesion assay. In brief, the 24-well plates were pre-coated 100 µL of a 1:20 diluted

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matrigel in cold DMEM to form a thin continuous film and dried in a laminar hood

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overnight. After pre-incubation with lycopene (0.1-5 µM) for 2, 6 and 12 h, cells were

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adjusted to 5 × 104 cells/mL in DMEM containing 2% FBS and incubated for an additional

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2 h followed by incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide (MTT) for 1 h. The supernatant was removed, and dimethyl sulfoxide (DMSO)

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was added to dissolve the solid residue cells. The optical density at 570 nm of each well was

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measured using a microplate reader (FLUOstar OPTIMA, BMG Labtechnologies).

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Determination of Intracellular ROS Levels. Intracellular ROS levels were determined

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using the redox-sensitive probe DCFDA by spectrofluorometry. Cells (105 cells/mL) were

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seeded into six-well plates and incubated with different concentration of lycopene (0.1-5

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µM) for 0.5, 1, 2, 3, 6 h. After treatment, cells were incubated with 10 µM DCFDA for an

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additional 1 h in dark, and then washed with PBS and scraped into ddH2O followed by 7



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sonication. The fluorescence of H2-DCFDA stained cells were measured using

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spectrofluorometer (excitation wavelength 485 nm, emission wavelength 520 nm).

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Analysis of NADPH Oxidase Activity. NADPH oxidase activity was determined based

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on the published methods37 with minor modifications. Cells were incubated with lycopene

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(0.1-5 µM) in the absence or presence of TGF-β (5 ng/mL) for 2 h, and then collected by

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trypsinization followed by centrifuged at 2500 × g for 5 min at 4°C. After centrifugation,

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cells were treated with 250 µM NADPH and the rate of NADPH consumption was

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determined by the reduction in absorbance at 340 nm in 10 min using spectrophotometer

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(FLUOstar OPTIMA, BMG Labtechnologies). The absorption extinction coefficient used to

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calculate the amount of NADPH consumed was 6.22 mM-1cm-1. For analysis of specific

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oxidase activity, the DPI-inhibited rate of consumption of NADPH was measured by adding

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10 µM DPI for 30 min before the assays. An aliquot of cells was lysed by adding SDS, and

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protein concentration of cell lysates was determined.

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Gelatin Zymography. The activities of MMP-2 and MMP-9 in the culture medium were

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measured using gelatin zymography according to a protocol developed by Kleiner and

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Stetler-Stevenson38 with some minor modifications. Cells (105 cells/mL) were seeded into

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six-well plates and pre-incubated with lycopene (0.1-5 µM) for 2, 6, 12, 24 h in DMEM

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medium containing 2% (v/v) FBS, and then incubated with serum-free medium for an

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additional 24 h. A portion of the medium (20 µL) was collected and electrophoresed in 8%

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sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) containing 0.1%

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gelatin. After electrophoresis, gel was washed with 2.5% (v/v) Triton X-100 twice for 30

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min, and then incubated with reaction buffer [2 M Tris-HCl (pH 8.0), 1 M CaCl2 and

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1%NaN3] for 12-15 h at 37°C. The gel was stained with Coomassie brilliant blue R-250 for

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30 min, and then destained in solutions containing 10% (v/v) acetic acid and 50% (v/v)

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methanol. The relative MMP-2 and MMP-9 activities were quantified using 8


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MatroxInspector 2.1 software. Two bands appeared in this zymographic analysis of MMP;

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the upper one was MMP-9 activity and the lower was MMP-2.39

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Western Blotting. Protein expression of NOX4 was measured by western blotting. Cells

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were incubated with lycopene for the indicated time and lysed with cold

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radio-immuno-precipitaion assay (RIPA) buffer containing protease inhibitor cocktails

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followed by centrifugation at 12000 × g for 30 min at 4 °C. The proteins (50 µg) from the

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supernatant were resolved by SDS-PAGE electrophoresis and transferred onto a

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polyvinylidene fluoride (PVDF) membrane. After blocking with Tris Buffered Saline (TBS)

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buffer containing 5% non-fat milk, the membrane washed three times with TBS buffer

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containing 0.1% (v/v) tween-20 for 1 h, and then incubated with NOX4 primary antibodies

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at 4°C overnight. The membrane was incubated with fluorescein-conjugated secondary

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antibody for 1 h, and then detected with ECL chemiluminescent detection kit (Amersham

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Co, Bucks, UK). The relative density of the protein expression was quantified by

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AlphaEaseFC Analysis software.

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Real-time RT-PCR. NOX4 gene expression was detected by a quantitative real-time

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RT-PCR method following manufacturer’s instructions (Applied Biosystems, Foster City,

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CA). Briefly, total cellular RNA was extracted using the TRI Reagent (Applied Biosystems).

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cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied

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Biosystems). Quantitative real-time PCRs were performed using TaqMan Universal PCR

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Master Mix II (part #444043, Applied Biosystems) in a StepOne TM Real-Time PCR System

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(Applied Biosystems). TaqMan Gene Expression Assays for human NOX4 gene

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(Hs00276431 m1, Applied Biosystems) was used to detect relative mRNA expression and

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β-actin gene (Hs99999903 m1, Applied Biosystems) was used as an internal control.

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Amplification data were collected and analyzed with StepOne TM Software version 2.3

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(Applied Biosystems). 9



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Transient Transfection of siRNA Against NOX4. Cells (105 cells/mL) were seeded into

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six-well plates and grown overnight to a confluence of 70-90%. The stock solution (20 µM)

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of si-NOX4 was diluted with DEPC water to form 5 µM solutions at room temperature.

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X-tremeGENE siRNA transfection reagent was mixed with 5 µM si-NOX4 or non-targeting

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siRNA for 30 min, and then added to the culture medium at a final concentration of 25 nM.

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Cells were transfected with si-NOX4 or non-targeting siRNA (25 nM) for 48 h using

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X-tremeGENE siRNA transfection reagent according to the manufacturer’s instruction.

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Statistical Analysis. Values are expressed as means ± SD and analyzed using one-way

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analysis of variance (ANOVA) followed by Fisher’s protected least significant difference

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(LSD) test for comparisons of group means when the F ratio were significant (P < 0.05). All

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statistical analyses were performed using SPSS for Windows, version 10 (SPSS, Inc.); a P

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value < 0.05 is considered statistically significant.

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RESULTS

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Effects of Lycopene on Cancer Metastasis in SK-Hep-1 Cells Lycopene Inhibited Migration, Invasion and Adhesion in SK-Hep-1 Cells.

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Pre-incubation of SK-Hep-1 cells with lycopene (0.1-5 µM) for 2, 6 and 12 h significantly

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inhibited cell migration in a U-shaped manner, and the optimal concentration of lycopene

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was 2.5 µM, with an inhibition of 47.4% (P < 0.05) for 2 h, 39.8% (P < 0.05) for 6 h, and

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34.5% (P < 0.05) for 12 h (Figure 1A). The inhibitory effects of lycopene on cell invasion

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were similar to those on cell migration. Lycopene (0.1-5 µM) also significantly inhibited

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cell invasion in a U-shaped manner at 2, 6, and 12 h of incubation, and the strongest

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inhibition was 2.5 µM, with an inhibition of 42.8% (P < 0.05) for 2 h, 40.2% (P < 0.05) for

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6 h, and 36.5% (P < 0.05) for 12 h (Figure 1B). Similarly, lycopene (0.1-5 µM) significantly 10


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inhibited cell adhesion in a U-shaped manner at 2, 6 and 12 h, and the most effective

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inhibition was 2.5 µM, with an inhibition of 17.7% (P < 0.05) for 2 h, 27.2% (P < 0.05) for

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6 h, and 58.8% (P < 0.05) for 12 h (Figure 1C).

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Lycopene Inhibited Activities of MMP-9 and MMP-2 in Culture Medium of

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SK-Hep-1 Cells. SK-Hep-1 cells pre-treated with lycopene (2.5 µM) for 2-24 h resulted in

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a time-dependent inhibition of MMP-9 and MMP-2 activities, with an inhibition of 51.3%

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(P < 0.05) for MMP-9 and 71.3% (P < 0.05) for MMP-2 at 24 h of incubation (Figures 2A

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and 2B). We also found that lycopene (0.1-5 µM) significantly inhibited the activities of

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MMP-9 and MMP-2 in a U-shaped manner at 24 h of incubation, and the optimal

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concentration of lycopene was 2.5 µM, with an inhibition of 42.2% (P < 0.05) for MMP-9

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and 83.7% (P < 0.05) for MMP-2 (Figures 2C and 2D).

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Lycopene Inhibited NOX4 Protein and mRNA Expression in SK-Hep-1 Cells.

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SK-Hep-1 cells treated with lycopene (2.5 µM) for 0.5-6 h resulted in a significant

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inhibition of NOX4 protein expression, with the highest inhibition of 64.4% (P < 0.05) at 2

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h of incubation (Figures 3A and 3B). Lycopene at the concentration of 2.5 µM also

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significantly decreased NOX4 mRNA expression in a time-dependent manner, i.e.,

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lycopene maximally inhibited NOX4 mRNA expression at 1 h of pre-incubation (by 55%, P

241

< 0.05), then the effect weakened slightly afterwards (~33% inhibition at 6 h) (Figure 3C).

242

In addition, we found that incubation of SK-Hep-1 cells with lycopene (0.1-5 µM) for 2 h

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significantly inhibited NOX4 protein expression (Figures 4A and 4B) in a U-shaped manner,

244

with the highest inhibition of 64.3% (P < 0.05) at 2.5 µM lycopene.

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Lycopene Decreased the NADPH Oxidase Activity and Intracellular ROS Level in 11



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SK-Hep-1 Cells. Incubation of SK-Hep-1 cells with lycopene (0.1-5 µM) for 2 h

247

significantly decreased NADPH oxidase activity in a U-shaped manner (Figure 4C).

248

Similarly, incubation of SK-Hep-1 cells with lycopene (0.1-5 µM) for 2 h significantly

249

inhibited the intracellular ROS level in a U-shaped manner, with the strongest inhibition of

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71.6% (P < 0.05) at 2.5 µM lycopene (Figure 4D).

251 252 253

Effects of Lycopene on TGF-β-induced Cancer Metastasis in SK-Hep-1 Cells Lycopene Inhibited TGF-β-induced Migration, Invasion and Adhesion in SK-Hep-1

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Cells. Based on the above findings, we chose the most effective pre-incubation time (2 h for

255

migration and invasion, and 12 h for adhesion) and concentration (2.5 µM) to determine the

256

effects of lycopene on TGF-β-induced cancer cell metastasis. As we expected,

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pre-incubation of SK-Hep-1 cells with TGF-β (5 ng/mL) for 2 h significantly induced cell

258

migration and invasion, with an induction of 50.9% (P < 0.05) for migration (Figure 5A)

259

and 52.2% (P < 0.05) for invasion (Figure 5B). Lycopene (2.5 µM) significantly attenuated

260

TGF-β-induced migration (Figure 5A) and invasion (Figure 5B). Similarly, TGF-β (5

261

ng/mL) significantly induced cell adhesion at 12 h of incubation (Figure 5C), and lycopene

262

significantly inhibited TGF-β-induced cell adhesion (Figure 5C).

263

Lycopene Inhibited TGF-β-induced MMP-9 and MMP-2 Activities in Culture

264

Medium of SK-Hep-1 Cells. TGF-β (5 ng/mL) significantly induced activities of MMP-9

265

and MMP-2, with an induction of 41.7% (P < 0.05) for MMP-9 and 39.3% (P < 0.05) for

266

MMP-2 (Figure 5D), at 24 h of incubation. Lycopene (2.5 µM) significantly inhibited the

267

effect of TGF-β, with reduction of 22.0% (P < 0.05) for MMP-9 and 26.7% (P < 0.05) for

268

MMP-2 (Figure 5D), as compared with TGF-β treatment alone.

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Lycopene Inhibited TGF-β-induced NADPH Oxidase Activity and Intracellular ROS level in SK-Hep-1 Cells. TGF-β (5 ng/mL) significantly induced the NADPH oxidase 12


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activity and intracellular ROS level, with an induction of 88.5% (P < 0.05) (Figure 5E) and

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25.3% (P < 0.05) (Figure 5F), respectively. In contrast, lycopene (2.5 µM) significantly

273

inhibited TGF-β-induced NADPH oxidase activity and intracellular ROS level (Figure 5E

274

and 5F).

275

Lycopene Inhibited TGF-β-induced NOX4 Protein Expression in SK-Hep-1 Cells.

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TGF-β (5 ng/mL) significantly induced NOX4 protein expression (Figure 6A, Lane 2), with

277

an induction of 36.6% (P < 0.05), as compared with the vehicle control. In contrast,

278

lycopene significantly inhibited TGF-β-induced protein expression of NOX4 (Figure 6A,

279

Lane 4).

280 281

NOX4 Knockdown Mimicked the Effect of Lycopene on the Metastasis of SK-Hep-1

282

Cells

283

NOX4 Knockdown Inhibited NOX4 Protein Expression in SK-Hep-1 Cells Treated

284

with or without TGF-β. We found that the siNOX4 transfection dramatically abolished the

285

NOX4 expression in the group treated with (Figure 6A, Lane 6) or without TGF-β (Figure

286

6A, Lane 5), with an inhibition about 75.0%, as compared with the non-transfected control

287

group (Figure 6A, Lane 1). The results demonstrated that NOX4 knockdown could mimic

288

lycopene to inhibit the expression of NOX4 protein in SK-Hep-1 cells with a higher

289

inhibitory efficacy than that of lycopene. Because the siNOX4 transfection dramatically

290

abolished the expression of NOX4 proteins, the inhibited effect of lycopene on NOX4

291

proteins was not apparent (Figure 6A, Lane 7~8). In addition, our preliminary tests revealed

292

no inhibition on NOX-4 expression by the non-targeting siRNA control and no cytotoxicity

293

on SK-Hep-1 cells transfected with NOX-4 siRNA by the siRNA transfection reagent for 24

294

h (data not shown). These results validated our NOX-4 siRNA transfection system.

295

NOX4 Knockdown Inhibited Cell Migration in SK-Hep-1 Cells Induced with or 13



296

without TGF-β. We then tested the effect of NOX4 knockdown on the migration in

297

SK-Hep-1 cells induced with or without TGF-β. We found that the NOX4 knockdown

298

significantly inhibited cell migration, with an inhibition of 78.4% (P < 0.05), as compared

299

with the non-transfected group (Figures 7A and 7B). In addition, the TGF-β-increased

300

migration (as shown in Figure 5A) was completely abolished by siNOX4 transfection; these

301

results demonstrated the crucial role of NOX4 in the TGF-β-induced migration. Because the

302

expression of NOX4 was dramatically abolished, the attenuation of lycopene on cell

303

migration could not be seen (Figures 7A and 7B), suggesting that lycopene and the siNOX4

304

transfection act by the same mechanism.

305

NOX4 Knockdown Inhibited MMP-9 and MMP-2 Activities in Culture Medium of

306

SK-Hep-1 Cells Induced with or without TGF-β. It is known that MMP-9 and MMP-2

307

are related to the invasion ability of cancer cells, we also tested the effect of the NOX4

308

knockdown on the MMP-9 and MMP-2 activities in SK-Hep-1 cells induced with or

309

without TGF-β. We found that the NOX4 knockdown significantly inhibited MMP-9 and

310

MMP-2 activities, with an induction of 26.7% (P < 0.05) for MMP-9 and 29.0% (P < 0.05)

311

for MMP-2, as compared with the non-transfected group (Figures 7C and 7D). In addition,

312

the TGF-β-induced MMP-9 and MMP-2 activities (Figure 5D) were essentially eradicated

313

by the siNOX4 transfection (Figures 7C and 7D); the results showed a crucial role of NOX4

314

in the TGF-β-induced invasion. Under transient transfection with siNOX4, lycopene did not,

315

or only slightly, inhibit MMP-9 and MMP-2 activities when SK-Hep-1 cells were co-treated

316

with TGF-β and lycopene (Figures 7C and 7D), suggesting, again, that lycopene and the

317

siNOX4 transfection act by the same mechanism.

318 319 320

DISCUSSION 14


Page 14 of 42

Page 15 of 42


321 322

The main question addressed by this study was whether NOX4 is a potential target for

323

pharmacological intervention in lycopene-inhibited metastasis of SK-Hep-1 cells. Herein,

324

we showed that lycopene significantly suppressed the protein expression of NOX4. The

325

downstream signals of NOX4, i.e., NADPH oxidase activity and the ROS level, were also

326

decreased by lycopene in SK-Hep-1 cells. NOX4 mRNA expression was also suppressed by

327

lycopene, indicating that the inhibition of NOX4 protein expression by lycopene is due to

328

inhibition of NOX4 gene expression. We further found that lycopene dramatically

329

antagonized TGF-β-induced cell migration, invasion and adhesion, intracellular ROS levels,

330

and activities of MMP-9, MMP-2, and NADPH oxidase as well as the protein expression of

331

NOX4. Using transient transfection of siRNA against NOX4, we found that the

332

down-regulation of NOX4 could mimic lycopene to inhibit the cell migration and the

333

activities of MMP-9 and MMP-2 during incubation with or without TGF-β in SK-Hep-1

334

cells. The results demonstrate that down-regulation of NOX4 plays a crucial role in the

335

anti-metastatic action of lycopene in SK-Hep-1 cells. To the best of our knowledge, the

336

present study is the first to report that lycopene has strong ability to down-regulate NOX4

337

protein expression regardless the treatment TGF-β.

338

It is known that ROS are involved in numerous cell pathophysiological process and to

339

play an important role in a large number of disease processes.18,19,40 ROS also affect various

340

aspects of tumor biology, such as carcinogenesis, aberrant growth, metastasis and

341

angiogenesis.41 ROS also have been shown to work as second messengers in signal

342

transduction that regulate pivotal cellular signaling events involved in homeostasis, cell

343

proliferation and differentiation, inflammation, and immune responses.42 In mouse breast

344

cancer 4T1 cells, Zhang et al.28 demonstrated that NOX4 is involved in TGF-β-promoted

345

ROS production and cell migration. The present study revealed that the down-regulation of 15



346

NOX4 expression was likely result of decreased activity of NADPH oxidase, leading to

347

decreased intracellular levels of ROS in SK-Hep-1 cells. These results suggest that the

348

attenuation of lycopene on NOX4-mediated ROS production induced by TGF-β plays an

349

important role in the anti-metastatic action of lycopene.

350

In addition, intracellular ROS have been shown to activate the NF-κB signal cascades,

351

and antioxidants including carotenoids have been shown to block these cascades.43 We have

352

previously demonstrated that lycopene can inhibit NF-κB and Sp1 binding activity, and the

353

results suggest that inhibition of the binding activities is responsible for the decrease of the

354

MMPs activities by lycopene in SK-Hep-1 cells.43 Thus, lycopene may decrease

355

intracellular ROS by down-regulation of NOX4 protein which may then inhibit metastasis

356

of SK-Hep-1 cells by decreasing the binding activity of NF-κB and Sp1. Moreover, it

357

should be noted that the decrease in intracellular ROS levels induced by lycopene itself may

358

also play a role in its anti-metastatic action, as lycopene is a well-established antioxidant.44

359

Once lycopene crosses the cell membrane, it could also decrease intracellular ROS by its

360

antioxidant activity in the cytoplasm. Indeed, the antioxidant activity of lycopene has been

361

suggested to be related to its anti-invasion activity in rat ascites hepatoma cells.45

362

Furthermore, the results from our previous studies also suggested that the down-regulation

363

of mRNA and protein of nm23-H1, a tumor metastasis suppressor gene, were involved in

364

the antimetastic action of lycopene in SK-Hep-1 cells16 and in athymic nude mice17. Based

365

on these illustrations, a scheme regarding to the mechanism the mechanistic action of

366

lycopene is proposed herein in Figure 8. Further investigations are warranted to elucidate

367

the connection of ROS to signal molecules NF-κB and Sp1, as well as the tumor suppressor

368

gene nm23-H1 in the anti-metastatic action of lycopene. The effect of other carotenoids on

369

the down-regulation of NOX4 protein also needs further investigations.

370

As has been noted previously,16,43 the present results reveal that the concentration 16


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371

effects of lycopene on all markers were U- or bell-shaped, i.e., the effects of lycopene were

372

all lower at 5 µM than at 2.5 µM. A possible explanation for the U- or bell-shaped effects is

373

that the antioxidant activity of carotenoid may shift into pro-oxidant activity, depending on

374

the carotenoid concentration inside the cells and the cell redox status.46 In addition, it should

375

be noted that the most effective concentration of lycopene (2.5 µM) in the present study is

376

supraphysiological. The plasma lycopene concentration in healthy humans is around 0.5

377

µM47-49 and plasma levels of lycopene are increased from 653 to 739 nM after five servings

378

of vegetables and fruits after supplementation for 12 months50. Some limitations exist in this

379

study that may undermine the significance of our findings. One such limitation is that only

380

one cell line was used in the present study. Another limitation is that only the in vitro cell

381

culture system was used. Indeed, the process of cancer metastasis is much more complex in

382

vivo.

383

Although several papers have reported a crucial role of NOX4 activation in breast and

384

lung cancer cells,28,51 NOX4 may also play a dual role in liver tumorigenesis and metastasis.

385

For instance, it has been reported that the malignant progression of hepatocelluar carinoma

386

cells induced by transforming growth factor β-interacting factor depends on activation of

387

NOX4,52 suggesting that inhibition of NOX4 can be useful in fighting against liver tumor

388

metastasis. In contrast, an proapoptotic role of TGF-β in human hepatocellular carcinoma

389

cells has been reported to be mediated by NOX4 activation.53 NOX4 activation also

390

represses epithelial to amoeboid transition and efficient tumor dissemination.54 Furthermore,

391

NOX4 activation plays a crucial role in the TGF-β-induced senescence in hepatocellular

392

carcinoma cells and inhibits tumor growth.55 These results suggest that inhibition of NOX4

393

can exert a detrimental effect against liver tumorigenesis and aggressiveness. Thus, the

394

antimetastatic benefit of lycopene in hepatocarcinoma by NOX4 inhibition may be limited

395

in certain types of liver cancer cells such as SK-Hep-1 cells which have a high metastatic 17



396

activity from a mesenchymal origin. Further investigations are warranted to elucidate the

397

actual benefit of lycopene against the tumorigenesis and metastasis in vivo in cancers of

398

liver and other organs.

399

In summary, the present study demonstrates that NOX4 plays an important role in the

400

anti-metastatic action of lycopene in this cell line. By down-regulating NOX4 expression,

401

lycopene decreases the activity of NADPH oxidase and the intracellular ROS level, leading

402

to attenuation of TGF-β-induced signaling and cancer cell metastasis.

403 404 405

ACKNOWLEDGEMENT This research was supported in part by the Ministry of Education, Taiwan, under the

406

ATU plan and NSC101-2320-B-039-007-MY3 from the National Science Council,

407

Executive Yuan, Taiwan.

408 409 410

DISCLOSURE STATEMENT The authors declare no competing financial interest.

18


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health. Exp. Biol. Med. (Maywood) 2002, 227, 845-851. (50) Pierce, J. P.; Natarajan, L.; Sun, S.; Al-Delaimy, W.; Flatt, S. W.; Kealey, S.; Rock, C. L.; Thomson, C. A.; Newman, V. A.; Ritenbaugh, C.; Gold, E. B.; Caan, B. J. Women's Healthy Eating and Living Study Group. Increases in plasma carotenoid concentrations in response to a major dietary change in the women's healthy eating and living study. Cancer Epidemiol. Biomarkers Prev. 2006, 15, 1886-1892. (51) Zhang, C.; Lan, T.; Hou, J.; Li, J.; Fang, R.; Yang, Z.; Zhang, M.; Liu, J.; Liu, B. NOX4 promotes non-small cell lung cancer cell proliferation and metastasis through positive feedback regulation of PI3K/Akt signaling. Oncotarget. 2014, 5, 4392-43405. (52) Liu, Z. M.; Tseng, H. Y.; Tsai, H. W.; Su, F. C.; Huang, H. S. Transforming growth factor β-interacting factor-induced malignant progression of hepatocellular carcinoma cells depends on superoxide production from Nox4. Free Radic Biol Med. 2015, 84, 54-64. (53) Caja, L.; Sancho, P.; Bertran, E.; Fabregat I. Dissecting the effect of targeting the epidermal growth factor receptor on TGF-β-induced-apoptosis in human hepatocellular carcinoma cells. J Hepatol. 2011, 55, 351-358. (54) Crosas-Molist, E.; Bertran, E.; Rodriguez-Hernandez, I.; Herraiz, C.; Cantelli, G.; Fabra, À.; Sanz-Moreno, V.; Fabregat, I. The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination. Oncogene. 2017, 36, 3002-3014. (55) Senturk, S.; Mumcuoglu, M.; Gursoy-Yuzugullu, O.; Cingoz, B.; Akcali, K. C.; Ozturk, M. Transforming growth factor-beta induces senescence in hepatocellular carcinoma cells and inhibits tumor growth. Hepatology. 2010, 52, 966-974.

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Figure legends

Figure 1. Effects of lycopene on migration, invasion and adhesion in SK-Hep-1 cells. The cells were pre-incubated with lycopene (0.1-5 µM) for 2, 6 and 12 h for determination of migration (A), invasion (B), and adhesion (C). Values (mean ± SD, n=3) not sharing a common letter at the same incubation time are significantly different (P < 0.05).

Figure 2. Time-course and concentration effects of lycopene on activities of MMP-9 and MMP-2 in culture medium of SK-Hep-1 cells. For time-course analysis, the cells were pre-incubated with lycopene (2.5 µM) for 2, 6, 12 and 24 h, and the activities of MMP-9 and MMP-2 were measured using gelatin zymography. (A) A representative zymography result is shown. (B) The quantitative results of the gelatin zymography of MMP-9 and MMP-2 from three independent experiments are shown. For concentration-effect analysis, cells were pre-incubated with lycopene (0.1-5 µM) for 24 h and the gelatin zymography of MMP-9 and MMP-2 were measured. (C) A representative zymography result is shown. (D) The quantitative results of the gelatin zymography of MMP-9 and MMP-2 from three independent experiments are shown. Values (mean ± SD) not sharing a common letter are significantly different (P < 0.05).

Figure 3. Time-course effects of lycopene on NOX4 protein and mRNA expressions in SK-Hep-1 cells. The cells were incubated with lycopene (2.5 µM) for 0.5, 1, 2, 3 and 6 h and Western blotting analysis was conducted. (A) A representative data is shown. (B) The quantitative results of the expression of NOX4 by Western blot analysis (n=3) are shown. (C) The quantitative RT-PCR results of NOX4 mRNA expression (n=3) are shown. Values (mean ± SD) not sharing a common letter are significantly different (P < 0.05). 25



Figure 4. Concentration effects of lycopene on NOX4 protein expression, NADPH oxidase activity and intracellular ROS level in SK-Hep-1 cells. The cells were incubated with lycopene (0.1-5 µM) for 2 h for the determinations. (A) A representative experiment of NOX4 protein expression is shown. (B) The quantitative results of the expression of NOX4 by Western blot analysis (n=3) are shown. Figure (C) shows the results of NADPH oxidase activity (n=4) and (D) is intracellular ROS levels (n=4). Values (mean ± SD) not sharing a common letter are significantly different (P < 0.05).

Figure 5. Effects of lycopene on TGF-β-induced migration, invasion, adhesion, MMPs activities, intracellular ROS levels and NADPH oxidase activities in SK-Hep-1 cells. The cells were pre-incubated with lycopene (2.5 µM) and TGF-β (5 ng/mL) for appropriate time for determination of migration (pre-incubated for 2h) (A), cell invasion (pre-incubated for 2 h) (B), cell adhesion (pre-incubated for 12 h) (C), MMP-9 and MMP-2 activities (pre-incubated for 24 h) (D). The cells were also incubated with lycopene (2.5 µM) and TGF-β (5 ng/mL) for appropriate time for determination of NADPH oxidation activity (incubated for 2 h) (E) and intracellular ROS level (incubated for 3 h) (F).Values (mean ± SD, n=4) not sharing a common letter are significantly different (P < 0.05).

Figure 6. Effects of lycopene and the NOX4 knockdown on the NOX4 expression in SK-Hep-1 cells induced with or without TGF-β. Non-transfected and siNOX4-transfected cells were incubated with lycopene (2.5 µM) and/or TGF-β (5 ng/mL) for 2 h, and NOX4 were detected by Western blot (Fig 6A). The quantitative results of the expression of NOX 4 from three independent experiments are showed in Figure 6B. Values (mean ± SD) not sharing a common letter are significantly different (P < 0.05). 26


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Figure 7. Effects of NOX4 knockdown and lycopene on the migration in SK-Hep-1 cells and on MMP-9 and MMP-2 activities in culture medium induced with or without TGF-β. Non-transfected and siNOX4-transfected cells were pre-incubated with lycopene (2.5 µM) and/or TGF-β (5 ng/mL) for 2 h, and cell migration was measured. (A) A representative data of images of cell migration are shown. (B) The quantitative results of the migration from three independent experiments are shown. Non-transfected and siNOX4-transfected cells were pre-incubated with lycopene (2.5 µM) and/or TGF-β (5 ng/mL) for 24 h. (C) A representative data of the gelatin zymography of MMP-9 and MMP-2 are shown. (D) The quantitative results of the MMP-9 and MMP-2 activities from three independent experiments are shown. Values (mean ± SD) not sharing a common letter are significantly different (P < 0.05).

Figure 8. A proposed schematic diagram for the role of NOX4 in lycopene-mediated anti-metastasis in SK-Hep-1 cells.

：direct inhibition by lycopene;

: inhibition as a

result of the down-regulation of NOX4 by lycopene; dotted line or dotted arrow are suggestive or based on evidence from the literature. ⊕ : promotional effect of lycopene that is suggestive from the literature; Θ: inhibitory action of Nm23-H1 that is suggestive from the literature.

27



Figure 1

(A) Vehicle control Lycopene 0.1 µM Lycopene 0.5 µM Lycopene 1 µM Lycopene 2.5 µM Lycopene 5 µM

Cell migration (% of vehicle control)

120

100

a a

a

a

ab

b

b

b

80

b

bc

c c

c

60

c c c

c

d

40

20

0 2

12

6

Time (h)

(B)

Cell invasion (% of vehicle control)

120

100

a

a b

80

a

b

b

b c

c

b c

e

e

d

d

d

d

60

b

Vehicle control Lycopene 0.1 µM Lycopene 0.5 µM Lycopene 1 µM Lycopene 2.5 µM Lycopene 5 µM

40

20

0 2

12

6

Time (h)

28


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Figure 1 (cont’d)

(C)

Cell adhesion (% of vehicle control)

120

100

a b b

Vehicle control Lycopene 0.1 µM Lycopene 0.5 µM Lycopene 1 µM Lycopene 2.5 µM Lycopene 5 µM

a

a b c c

80

b bc

bc c bc b

60

c

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c

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0 2

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6

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29



Figure 2

(A)

(B)

30


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Figure 2 (cont’d)

(C)

(D)

31



Figure 3 (A)

(B)

NOX4 protein expression (% of 0 h)

120 a 100 b 80

b cd

60

c

d 40

20

0

0

0.5

2

1

6

3

Time (h)

(C)

NOX4 mRNA expression (fold of 0 h)

1.4 1.2

a

1.0 0.8

b

0.6

d

b,c c

b,c

0.4 0.2 0.0 0

1

2

3

4

5

6

7

Lycopene incubation time (h) 32


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Figure 4

(A)

(B)

33



Figure 4 (cont’d)

(C)

(D)

34


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Figure 5

(A)

Cell migration (% of vehicle control)

180 b 160 140 120 a

a

100 80 c 60 40 20 0 TGF-β β (5 ng/ml)

-

+

-

+

Lycopene (2.5 µM)

-

-

+

+

(B)

Cell invasion (% of vehicle control)

180

b

160 140 a

120 a

100 c

80 60 40 20 0 TGF-β β (5 ng/ml)

-

+

-

+

Lycopene (2.5 µM)

-

-

+

+

35



Figure 5 (cont’d)

(C)

180

Cell adhesion (% of vehicle control)

b 160 140 120

a a

100 80 c 60 40 20 0 TGF-β β (5 ng/ml)

-

+

-

+

Lycopene (2.5 µM)

-

-

+

+

(D)

36


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Figure 5 (cont’d)

NADPH oxidase activity (% of vehicle control)

(E) b

200

150 d a

100

50

c

0 TGF-β β (5 ng/ml) Lycopene (2.5 µM)

-

+

-

-

+

+ +

(F)

ROS level (% of vehicle control)

140

b

120

a a

100 c 80 60 40 20 0 TGF-β β (5 ng/ml)

-

+

-

+

Lycopene (2.5 µM)

-

-

+

+

37



Figure 6 (A)

(B)

38


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Figure 7

(A)

(B)

39



Figure 7 (cont’d) (C)

(D)

40


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Figure 8

41



TOC Graphic.

TGF-β β

NOX4

Metastasis of SK-Hep-1 cells Migration Invasion Adhesion

Lycopene

42


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Lycopene Inhibits Human liver adenocarcinoma SK-Hep-1 Cells

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