Allicin Induces Anti-human Liver Cancer Cells through the p53 Gene

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Allicin induces anti-human liver cancer cells through p53 gene modulating apoptosis and autophagy Yung-Lin Chu, Chi-Tang Ho, Jing-Gung Chung, Raghu Rajasekaran, Yi-Chen Lo, and Lee-Yan Sheen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf403241s • Publication Date (Web): 23 Sep 2013 Downloaded from http://pubs.acs.org on October 5, 2013

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Journal of Agricultural and Food Chemistry

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Allicin induces anti-human liver cancer cells through p53 gene

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modulating apoptosis and autophagy

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Yung-Lin Chu†, Chi-Tang Ho†,‡, Jing-Gung Chung§, Rajasekaran Raghu‖, Yi-Chen Lo†, Lee-Yan

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Sheen*,†,│,├

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†

Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan

‡

Department of Food Science, Rutgers University, New Brunswick, NJ, USA

§

Department of Biological Science and Technology, China Medical University, Taichung, Taiwan

‖

Department of Horticulture and Landscape Architecture, National Taiwan University, Taipei,

Taiwan │

National Center for Food Safety Education and Research, National Taiwan University, Taipei,

Taiwan ├

Center for Food and Biomolecules, National Taiwan University, Taipei, Taiwan

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Running title: Allicin regulates apoptosis and autophagy via p53 in human liver cancer cells.

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* Corresponding author: Prof. Lee-Yan Sheen, Institute of Food Science and Technology,

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National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan. Tel: 886-2-

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33664129; Fax: 886-2-2362-0849; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer globally and

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ranks first among the cancer-related mortalities in Taiwan. This study aims to understand the

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modes of cell death mechanism induced by allicin, a major phytochemical of crushed garlic, in

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human hepatoma cells. Our earlier study indicated that allicin induced autophagic cell death in

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human HCC Hep G2 (p53wildtype) cells whereas in the present study allicin induced apoptotic cell

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death through caspase-dependent and caspase-independent pathways by ROS over-production in

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human HCC Hep 3B (p53mutation) cells. To gain insight into the cell death mechanism in p53

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knocked down Hep G2, we silenced p53 gene using siRNA mediated silencing. Allicin treatment

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induced apoptotic cell death in p53 knocked down Hep G2 cells similar to that of Hep 3B cells.

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These results suggest that allicin induced cell death in human hepatoma cells either through

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autophagy or apoptosis and might be a potential novel complementary gene therapeutic agent for

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the treatment of apoptosis resistant cancer cells.

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Key

words:

allicin,

apoptosis,

autophagy,

p53,


reactive

oxygen

species

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INTRODUCTION

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Phytochemicals present in human diet has attracted much attention for their health-related

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potential and chemoprevention of cancer. Garlic (Allium sativum), the legendary vegetable or

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spice is bestowed with numerous health beneficial functions such as antibiotic1, anti-virus2, anti-

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platelets aggregation3, anti-atherosclerotic4, anti-proliferative5 and anti-fatty liver properties6.

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The predominant component of garlic, allicin is bio-synthesized from its precursor alliin by

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alliinase. Allicin has been extensively studied for its wide-range of biological activity including

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anti-cancer7.

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Viral hepatitis can induce acute and chronic inflammation of the liver leading to cirrhosis, and is

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the major risk factor leading to liver cancer. Nearly 70% of liver carcinoma cases are induced by

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hepatitis B and C, and is the leading cancer deaths in Taiwan. In the present research we focus on

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the effect of allicin treatment on human hepatoma Hep 3B cells which contain an integrated

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hepatitis B virus genome and p53 gene mutation.

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Cell deaths include program cell death-I (PCD-I) also called apoptosis, program cell death-II

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(PCD-II) also called autophagy8, and non-program cell death normally referred as necrotic cell

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death. To validate p53 gene mediated regulation of PCD in human liver cancer cells, this study

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focuses on the effect of allicin treatment on p53-mutation (Hep 3B cells) and p53-wildtype (Hep

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G2 cells) human liver cancer cells.

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Reactive oxygen species (ROS) featured by uncoupling electrons including H2O2, O2•-, 1O2

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and •OH formed by endogenous sources and environmental factors. Normally, ROS maintains

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homeostasis in our body. Imbalance of cellular ROS leads to the injury of cellular structures.



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However, some studies suggest that tumor cells have their own threshold of ROS levels higher

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than that of normal cells. Upon drug treatment to tumor cells, it may induce ROS-production to

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exceed the survival threshold of tumor cells and trigger tumor death9.

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It has been well established that cancer cells have acquired an innate ability to escape apoptosis,

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and the number of genetic mutations have damaged their genes such as p53. There are over 50%

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cancer cells with p53 gene mutation, deletion or non-function. However, p53 gene is an

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important tumor suppressor gene which involves in many physiological functions such as cell

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death, stress, cell cycle, cell differentiation and immune response. Therefore, it will exert a great

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influence in suicide gene therapy for cancer cells with or without p53 gene function10. It has been

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reported that garlic could regulate some suicide gene in cancer treatment11. However, there is no

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study demonstrating how allicin mediates various cell death models by p53 gene. The objectives

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of this study are to elucidate the role of allicin in the regulation of different types of cell death in

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human liver cancer cells and to determine the potential of garlic in complementary suicide gene

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therapy in human liver cancer.

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MATERIALS AND METHODS

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Chemicals and Reagents

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Allicin, dimethyl sulfoxide (DMSO), propidium iodide (PI), RNase A, Tris-HCl, potassium

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phosphates, N-acetylcysteine (NAC), 3-methyladenine (3-MA), Triton X-100 and trypan blue

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were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). The fluorescent probe 2,7-

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dichlorodihydrofluorescein diacetate (DCFH-DA), Mito Tracker-Red, 4′,6-diamidino-2-

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phenylindole (DAPI), glycine, N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-


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yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-

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[2-[(acetyloxy)methoxy]-2-oxyethyl]-, (acetyloxy)methyl ester/ 121714-22-5 (Fluo-3/AM) and

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3,3′-dihexyloxacarbocyanine iodide (DiOC6) were purchased from Invitrogen Life Technologies

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(Carlsbad, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM) with 2 mM L-glutamine,

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fetal bovine serum, penicillin-streptomycin and trypsin-EDTA were obtained from Gibco BRL

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(Grand Island, NY, USA). Materials and chemicals for gel electrophoresis were from BioRad

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(Hercules, CA). Annexin V-FITC/PI Apoptosis Detection Kit I was procured from BD

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Biosciences (San Jose, CA, USA) and PolysineTM microscope adhesion slides (Thermo Fisher

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Scientific Inc., Waltham, MA, USA).

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The qualitative and quantitative analysis of allicin was performed as described in our previous

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study12.

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Cell Culture

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Human liver carcinoma Hep 3B (p53mutation) and Hep G2 (p53wildtype) cell lines were obtained

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from the Department of Medical Research and Education, Taipei Veterans General Hospital,

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Taipei, Taiwan. Hep 3B cells were placed into 10 cm2 dish with DMEM medium supplemented

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with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin, and

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grow under a humidified 5% CO2 and 95% air grown at 37 ˚C. Adherent cells were suspended by

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1 mL of 1% trypsin-EDTA for 5 min at 37 ˚C.

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Determination of cell morphology, viability, cell cycle and sub-G1 phase

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Hep 3B cells were seeded at a density of 1×105 cells/well in a 12-well plate. The seeded cells

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were incubated with 0, 15, 20, 25, 35, 40 and 50 µM of allicin for 24 h at 37 ˚C, 5% CO2 and



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95% air. The morphological changes were directly examined and photographed by phase-

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contrast microscope at 200× magnification. The percentage of viable cells was determined by

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flow cytometry (Becton-Dickinson, San Jose, CA, USA) after centrifugation of the cells and

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resuspension in PBS containing PI (5 µg/mL). For cell cycle distribution and sub-G1

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determination, isolated cells were fixed gently by 70% ethanol overnight at 4 ˚C and then re-

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suspended in PBS containing 50 µg/mL PI and 0.1 mg/mL RNase A and 0.1% Triton X-100 in

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dark room for 30 min at 37 ˚C, then were analyzed with a flow cytometer with Modfit LT

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Ver.2.0 (Verity software House, Topsham, ME, USA). Cells treated with 0.1% DMSO served as

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control.

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DAPI staining

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For DAPI staining, approximately 5×104 cells/mL of Hep 3B cells in 6-well plate were treated

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with 0, 20, 25, 35, 40 and 50 µM of allicin for 24 h. Cells were stained with DAPI, then

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examined and photographed using a fluorescence microscope as described elsewhere13. The

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nuclei was labeled by DAPI staining in which visualized by excitation at 330~385 nm, emission

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at 420 nm.

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Detections of ROS, and mitochondrial membrane potential (∆Ψm) in Hep 3B cells

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Approximately 1×105 cells/mL of Hep 3B cells were treated with 35 µM of allicin for 1, 3, and 6

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h, and the cells were re-suspended in 500 µL PBS with 10 µM DCFH-DA for ROS, and in 500

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µL PBS with 4 µmol/L DiOC6 for ∆Ψm. Cells were incubated at 37 ˚C in dark for 30 min before

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flow cytometry analysis.

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Apoptosis Assay

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To examine if allicin treatment induced apoptosis in Hep 3B cells, we seeded cells at a

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concentration of 1×105 cells/well in a 12-well plate and performed annexin V-FITC/PI double

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staining apoptosis assay. The cells were treated with 35 µM allicin for 0, 6, 12, and 24 h, and

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then washed twice with PBS buffer. Then we resuspended Hep 3B cells in 1X binding buffer

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with 1×106 cells/mL, and transferred 100 µL of 1X binding buffer with 1×105 cells/mL to a 5 mL

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FACs tube. The following steps were performed in the dark. To the FACS tubes 5 µL of annexin

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V-FITC and 10 µL PI were added. Finally after addition of 400 µL of 1X binding buffer samples

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were analyzed by flow cytometry within 1 h.

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Analysis of protein expression by Western blotting

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Hep 3B cells plated at a density of 1×107 cells on 10 cm2 dish were treated with 35 µM allicin for

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0, 3, 6, 12, 24 and 48 h for examining key proteins correlated with apoptosis, DNA damage, and

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ER stress signaling. For Western blot analysis, total proteins (25 µg) of cell lysates were

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separated on a 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) for

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90 min and transferred to nitrocellulose membrane (Millipore, Billerica, CA). The blot was

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soaked in blocking buffer (5% non-fat dry milk/0.05% Tween-20 in 20 mM TBS; pH=7.6) at

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room temperature for 1~1.5 h and incubated with individual primary monoclonal antibodies:

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HtrA2, Bcl-2, Bax, caspase-9, AIF (apoptotic induced factor), Endo G (endonuclease G),

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caspase-3, PARP, γ-H2AX and caspase-8 (Cell Signaling Technology, Danvers, MA, USA), in

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non-fat milk blocking buffer at 4 °C for over 4 h. After washing twice with TBS (Tris Buffered

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Saline), the blots were treated with horseradish peroxidase conjugated secondary antibody for

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detection by WesternBreeze chemiluminescent kit (Invitrogen) according to the manufacturer's



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instructions. Anti-β-actin (a rabbit monoclonal antibody) served as an internal control.

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Translocation for AIF and Endo G protein into nuclear detected by confocal laser scanning

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microscopy

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Hep 3B cells at density of 5×104 cells/well cultured on PolysineTM microscope adhesion slides

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(Cel-line, Thermo) were treated with or without 35 µM allicin for 24 h and fixed in 4%

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paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton-X 100 in PBS for 0.5 h

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with blocking of non-specific binding sites using 2% BSA as described14. The fixed cells were

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stained overnight with primary antibodies of AIF and Endo G (1:100 dilution) (green

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fluorescence). Subsequently they were washed twice with PBS and stained with secondary

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antibody (FITC conjugated goat anti-mouse IgG at 1:100 dilution) followed by staining with PI

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(red fluorescence) for DNA as described previously. All samples were visualized and

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photographed using a Leica TCS SP5 II confocal spectral microscope.

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γ-H2AX foci formation measured by immunostaining method

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Hep 3B cells were placed on PolysineTM microscope adhesion slides (Cel-line, Thermo) and

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treated with 35 µM allicin. At different time points (0, 3, 6, 12, and 24 h), cells were fixed in 4%

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paraformaldehyde/PBS and stained with anti-phospho γ-H2AX (ser139 clone, Cell Signaling)

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according to data sheet's dilution. γ-H2AX foci image was acquired on Leica TCS SP5 II

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confocal laser scanning microscope. All image data were quantified by Image J software, an

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image-inspired Java program from the National Institute of Health (NIH).

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Acridine orange staining for autophagy assays


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Acridine orange (AO) is the common method of detecting autophagy. It is used to measure

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proton pump driven lysosomal acidity generates a significant pH gradient resulting in the

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efficient concentration of AO within the lysosome organelles. The effectiveness of this AO

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concentration process is sufficient to create intra-lysosomal concentrations leading to

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precipitation of the AO into aggregated granules. Allicin-treated Hep 3B cells were stained with

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AO (1 µg/mL, Sigma) for 15 min at 37 °C and analysis by flow cytometry.

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Confocal Laser Scanning Microscopy for the Autophagosome of LC3-II-FITC Punctate

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Formation

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Hep 3B liver cancer cells at a density of 5×104 cells/well were seeded on PolysineTM microscope

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adhesion slides (Cel-line, Thermo) then were treated with or without 35 µM allicin. Then cells on

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the slides were fixed at various time points in 4% paraformaldehyde in PBS for 15 min, and

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permeabilized with 0.1% Triton-X 100 in PBS for 0.5 h with blocking of non-specific binding

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sites using 5% blocking buffer. The fixed cells were stained overnight at 4 ˚C with primary

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antibodies to LC3-II (Genetex, Irvine, CA, USA) (1:3000 dilution) (green fluorescence).

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Subsequently the slides were washed twice with PBS and stained with secondary antibody (FITC

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conjugated goat anti-rabbit IgG at 1:100 dilution), followed by nuclear staining with DAPI (blue

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fluorescence) as described previously. All samples were visualized using a Leica TCS SP5 II

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confocal spectral microscope. All image data were quantified by an image-inspired Java program,

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Image J software from the NIH.

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Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

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To validate the western results, qRT-PCR was performed. First strand cDNA was synthesized as



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a 20 µL reaction, with 1 µg of total RNA as the template using QuantiTect reverse transcription

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kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocol. The qPCR primers for

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Caspase 3 (F- TCGCTTCCATGTATGATCTTTG ; R - CTGCCTCTTCCCCCATTCT), AIF (F-

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TCTAGAGGAACATGCCATCG; R – GCTTTGAAGCAGAAGCTGGT) and β-actin (F -

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TGGCA CCACA CCTTC TACAA; R - GCAGC TCGTA GCTCT TCTCC) were chosen from

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qPrimerDepot primer database15, 16. β-actin was used as endogenous control. Real-time PCR was

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performed on an ABI StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City,

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CA, USA) using KAPA SYBR® FAST qPCR Kit Master Mix ABI Prism™ (KK4603, Kapa

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Biosystems, Woburn, MA, USA). The qPCR reactions were performed with 1 µL of 5 times

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diluted first strand cDNA, 1X qPCR master mix and 200 nM of each forward and reverse primer.

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The PCR conditions were 95 °C for 1 min followed by 40 cycles of 95 °C for 3 sec, 60 °C for 30

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sec and for verification of target gene amplifications, a dissociation stage at 95 °C for 15 sec, 60

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°C for 1 min, 95 °C for 15 sec. Gene expression was normalized to the endogenous control β-

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actin. Normalized gene expression of allicin group was expressed relative to control group. The

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expression levels were determined on four biological replicates, and each biological sample was

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replicated to obtain three technical replicates employing the 2-∆∆CT method for gene expression15.

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Transfection of siRNA-tumor protein 53 (siRNA-TP53)

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For transfection experiment, Hep G2 cells were seeded in 40-mm culture dishes and grown until

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80% confluence. Dharmacon ON-TARGETplus SMART pool siRNAs against Human TP53 at a

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final concentration of 25 nM were transfected to 1.5–2.5×105 Hep G2 cells using DharmaFECT-

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4 transfection reagent according to the manufacturers’ instructions. At 6 h post-transfection, the

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transfection reagent was replaced with complete medium. Allicin (35 µM) was added to the

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transfected cells and incubated for 24 hours at 37 oC in 5% CO2 incubator following which the

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cells were subjected to annexin V-FITC/PI assay. For Western blotting, the transfection was

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carried out in 10 cm2 plates.

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Statistical Analysis

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All results are reported as mean ± SD and the difference between the allicin-treated and control

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groups were analyzed by one way analysis of variance ANOVA and Duncan's multiple

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comparison tests (SAS Institute Inc., Cary, NC) to determine significant differences among

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treatments, p＜0.05.

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RESULTS AND DISCUSSION

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Garlic consumption may decrease the risk of cancer initiation and inhibit cell proliferation of

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gastric, colon, skin, and breast cancer cell lines17-20. It is well known that the cell death is

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regulated by apoptosis, necrosis, and autophagy21. Thus far some studies showed that garlic or its

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active components such as diallyl sulfides induce apoptosis in cancer cell lines22, 23. To the best



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of our knowledge, no studies demonstrated that garlic could regulate apoptosis, autophagy and

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necrosis in hepatocarcinoma cells. We are the first to report on the validation of allicin regulating

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different death models in liver cancer cells.

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Effect of allicin on viability and morphology of Hep 3B cells

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Allicin induced morphological changes in Hep 3B cells as observed under a phase-contrast

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microscope revealed cell shrinkage (Figure 1A). The viability of Hep 3B cells was assessed by

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flow cytometry. The IC50 of allicin was determined to be 35 µM in Hep 3B cells at 24 h and the

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number of viable cells changed (Figure 1B) in a dose-dependent manner after 24 h incubation. In

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other study, allicin treatment did not increase the leakage of lactate-dehydrogenase (LDH) of

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primary rat hepatocytes until 1 mM allicin treated with rat hepatocytes24. For this reason, allicin

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could be inferred as safe to normal liver cells.

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Allicin treatment increased distribution of apoptotic cells in Hep 3B cells

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Cell cycle is a key process in regulation of cells. Towards this we used flow cytometry to study

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the regulation of the cell cycle distribution in Hep 3B cells after allicin treatment. From Figure

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1C and supporting information Figure S1, it is quite evident that the percentage of sub-G1 phase

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distribution in whole cell cycle had increased indicating allicin induced genome destruction in

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Hep 3B cells significantly ( p

Allicin Induces Anti-human Liver Cancer Cells through the p53 Gene

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