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Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer globally and ranks first among the cancer-related mortalities in Taiwan. Thi...
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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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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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,

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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