Licochalcone B extracted from Glycyrrhiza uralensis Fisch induces

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Bioactive Constituents, Metabolites, and Functions

Licochalcone B extracted from Glycyrrhiza uralensis Fisch induces apoptotic effects in human hepatoma cell HepG2 Jun Wang, Ai-Mei Liao, Kiran Thakur, Jian-Guo Zhang, Ji-Hong Huang, and Zhao-Jun Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00324 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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Journal of Agricultural and Food Chemistry

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Licochalcone B extracted from Glycyrrhiza uralensis Fisch

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induces apoptotic effects in human hepatoma cell HepG2

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Jun Wang †, Ai-Mei Liao‡, Kiran Thakur †, Jian-Guo Zhang †, Ji-Hong Huang ‡,$*,

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Zhao-Jun Wei †,#*

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†

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230009, People’s Republic of China

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‡ College

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450001, People’s Republic of China

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$ Henan

School of Food and Biological Engineering, Hefei University of Technology, Hefei

of Biological Engineering, Henan University of Technology, Zhengzhou,

Cooperation Science and Technology Institute, Luoyang, 471000, People’s

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Republic of China

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# Anhui

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Qiangwang seasoning Food Co., Ltd., Jieshou 236500, People’s Republic of China

Province Key Laboratory of Functional Compound Seasoning, Anhui

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Jun Wang [email protected]

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Ai-Mei Liao, E-mail: [email protected]

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Kiran Thakur, E-mail: [email protected]

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Jian-Guo Zhang, E-mail: [email protected]

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Ji-Hong Huang, E-mail: [email protected]

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* Corresponding author, J.-H. Huang ([email protected]) & Z.-J. Wei

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([email protected]), Tel & Fax: 86-551-62901539.

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ACS Paragon Plus Environment


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Abstract: The present study explored the molecular mechanism by which

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Licochalcone B induces the cell cycle arrest and apoptosis in human hepatoma cell

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HepG2. Initial extraction and identification were performed by HPLC,

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UPLC-TOF-MS/MS, and NMR analysis, respectively. Licochalcone B inhibited the

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HepG2 growth with IC50 (110.15 μM) after 24 h, caused morphological distortion,

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seized the cell cycle in G2/M phase (cell arrest in G2/M:43.1±2.2% for 120 μM

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versus 23.7±1.2% for control), as well as induced apoptosis and intracellular ROS

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generation. Furthermore, exposure to Licochalcone B markedly affected the cell cycle

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(up/down regulation) at mRNA and protein levels. Apoptosis was induced through the

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activation of receptor-mediated and mitochondrial pathways. The inhibition of

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Caspase 8 and Caspase 9 proteins abolished the Licochalcone B induced apoptosis.

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The present work suggested that Licochalcone B may further be identified as a potent

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functional food component with specific health benefits.

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Keywords: Licochalcone B; Glycyrrhiza uralensis Fisch; Apoptosis; HepG2 cells;

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Mechanisms

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Introduction

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Primary liver cancers (PLC) are recognized as the most common malignancies

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worldwide, and among them, hepatocelluar carcinoma is the most prevalent type in

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East and Southeast Asia as well as Midwest Africa.1 Among malignant tumors,

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morbidity rate caused by PLC is the second highest in males and eighth highest in

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females. According to the previous meta research, nearly 746,000 deaths were marked

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due to liver cancer in 2012.2 Particularly in China, approximately 383,000 deaths

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were caused by liver cancer annually, which accounts for 51% of the world's

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mortality.3

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For the past few years, several herbal preparations and natural compounds have

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been examined for their anti-cancer activities.4 Glycyrrhiza uralensis Fisch is among

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the oldest traditional medicines 5recorded with 20 triterpenoids and 300 flavonoids.6

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Glycyrrhiza uralensis Fisch has many therapeutic advantages such as anti-cancer,7

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antiviral, antidiabetic,8 antimicrobial,9 antioxidant, anti-inflammatory10 and

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immunoregulatory effects.11 Licochalcone B (LCB), a chalcone existing in the root of

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Glycyrrhiza uralensis Fisch, is recognized with several health benefits such as

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cardioprotection,12 anti-Alzheimer’s disease (AD),13antioxidant and radical

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scavenging,9 and inducing cancer cell apoptosis.14,15 However, studies on the induced

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apoptotic effects of LCB are still in the preliminary stage, and its detailed mechanistic

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perspective warrants further research. In order to control the abnormally growing cells,

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cell cycle arrest and induced apoptosis may be suitable criteria to determine the

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possible anti-cancerous potential of any compound. 3



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Cell cycle is known to regulate the cellular growth and their proliferation during the

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cancer cells development. Previous studies showed that the advancement in cell cycle

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is mediated by sequential start and inactivation of diverse cellular proteins.16 Key

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mediators are the cyclin-dependent kinases (CDK), which are triggered at definite

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stage of cell cycle. Cyclin D associated with CDK4 and 6 are involved in cell cycle

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starting from G1 to M and the latter is mediated by CDK1 and Cyclin B1.17 Apoptosis

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is an important process representing the programmed cell death (PCD).18 Complex

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networks of regulatory factors are implicated in cell death. Among the complex

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networks, receptor-mediated pathway and mitochondrial pathway are the two most

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extensively studied pathways.19

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We aimed to unravel series of steps involved in underlying cellular and molecular

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mechanisms for the inhibitory effects of LCB on liver cancer cells. Since, our

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research group is focused on the natural plant-based components20-24 with a desire to

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expand their therapeutic values. Therefore, with the prior expertise, HepG2 a kind of

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hepatoma carcinoma cells were exposed to LCB in order to assess the cell growth,

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morphology, growth phase, apoptosis, and production of reactive oxygen species

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(ROS). Additionally, we were curious to study the underlying mechanism involved in

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cell cycle arrest and accelerated apoptosis process. The obtained findings elucidated

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that LCB may further be identified as a potent functional food component with

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specific health benefits.

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Chemicals and methods

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Chemicals

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Glycyrrhiza uralensis Fisch plants were purchased from Xinjiang ciconhabo

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pharmaceutical co. LTD at Xingjiang province, China. Chloroform, ethanol, ethyl

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acetate, and Tween 20 were purchased from Sinopharm Chemical Reagent Co.

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(Shanghai, China). HepG2 (Human hepatoma carcinoma cell line), HEK293 (human

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embryonic kidney cell line) and EAhy926 (human endothelial cell line) cells were

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obtained from Shanghai Wei Atlas Biological Technology Co., LTD. Cell culture

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growth medium and reagents such as 0.25% trypsin solution without EDTA and fetal

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bovine serum (FBS) were purchased from Invitrogen (Carlsbad, USA). Penicillin (100

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U/ml) and streptomycin (100 μg/ml) were purchased from Sigma Chemical Company

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(St. Louis, Missouri, USA). Dimethyl sulfoxide (DMSO) were purchased from

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AMRESCO (USA). All the antibodies used in this work were purchased from Cell

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Signaling Technology (Beverly, MA). Polyvinylidene difluoride (PVDF) were

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obtained from EMD Millipore Corporation (Billerica, MA, USA).

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Preparation of crude extract

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Roots of Glycyrrhiza uralensis Fisch (4.5kg) were sliced into small pieces and dried

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at 60 °C for 24h prior to milling into powders (40 meshes). Then the powders were

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extracted twice (2 × 60 min), each with 75% ethanol in an ultrasonic bath at 60°C.The

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extracts were combined and concentrated under reduced pressure with a YRE-2000E

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rotary evaporator (Yuhua Instrument Co., Gongyi, China). After evaporation of

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ethanol, the obtained extract was suspended with water and re-extracted with 5



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chloroform repeatedly for three times. Finally, the chloroform extract was

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decompressed and concentrated to approximately150 g.

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Purification and HPLC analysis of LCB

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The purification of crude extract was performed as per the methods described in the

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previous studies.23 The chloroform extract was fractionated on silica gel column (2.0

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kg, 100–200 mesh, 10×100 cm) with CHCl3–CH3OH (100:0-0:100, v/v) as the mobile

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phase. The eluate was identified by TLC (thin layer chromatography), and it was

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divided into nine components. The component 2 (36g) eluted with CHCl3–CH3OH

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=99:1(v/v) was subjected to silica gel column chromatography (200 g, 200–300 mesh,

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10×100 cm) with CHCl3–EtOAc as the mobile phase and resulted into light yellow

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crystal. Then after, Sephadex LH.20 column (50 g, 1.5×100 cm) was used to elute the

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crystal with methanol. The resulting extract (45mg) was subjected to High

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performance liquid chromatography (HPLC) using Hyper 0DS2 C18 column with

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ELSD detector. The High-performance liquid chromatography (HPLC) (AB SCIEX,

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USA), UPLCTOF-MS/MS (Waters, Milford, MA, USA), and nuclear magnetic

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resonance (NMR) (Bruker AVIII-600, USA) conditions were followed our previous

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study.24

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Cell lines and culture

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Previously cultured human hepatoma carcinoma cell line (HepG2) and two normal

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cell lines (HEK293 and EAhy926) were revived in modified DMEM medium at 37°C

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as per our previous study.24

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MTT assay for cell inhibition 6


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96-well plates were plated with 6×103 cells in each well and incubated overnight.

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Then after, cells were treated with different concentrations of LCB (40, 60, 80, 100,

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120, 140, 160, and 180 μM) for 24h. After cells were exposed to LCB, 20 μL of MTT

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reagent (5mg/mL) was added and incubated at 37°C for 3h. Finally, the medium was

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then removed and 150 μL of DMSO was added prior to absorbance measurement at

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510 nm. The inhibition of LCB on HepG2 were calculated by evaluating the cell

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viability of treated cells with that of DMSO treated control cells.

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Morphological Assay

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The morphological changes in HepG2 cells were noticed using inverted optic

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microscope (TS100, Nikon, Japan) due to inhibition ability of LCB treatment (40, 80,

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and 120 μM) for 12 or 24h.

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Cell cycle analysis

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For this, 3×105cells were treated with different concentration of LCB (40, 80 and 120

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μM) for 24 h. The treated cells were trypsinized and fixed with 70% ethanol before

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incubation at -20ºC for 24h. Upon washing with PBS washing, propidium iodide (PI)

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was added for 30 minutes and stored in the dark prior to Flow Cytometry (BD

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FACSCalibur, USA) to detect the cell cycle distribution. 24

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Apoptosis analysis by Flow Cytometry

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Annexin V-FITC/PI double staining assay (Beyotime, Shanghai, China) was used to

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determine the apoptosis of HepG2 cells. In brief, 3×105cells were exposed to LCB (40,

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80, and 120 μM) for 24h and resuspended with 500 μL of 1 × Annexin V binding

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buffer. The 5 μL Annexin V-FITC was added before incubation at 2–8 °C under dark 7



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conditions. After 15 minutes, 10 μL of PI was dripped to the mixture and allowed to

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stand for 10 minutes.24 The stained cells were examined using a flow cytometer

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(Beckman Coulter, USA).

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Assessment of intracellular ROS

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Accumulation of intracellular ROS was detected by Flow Cytometry using fluorescent

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probe 2′, 7′-dihydrofluorescein diacetate (DCFH-DA). Briefly, HepG2 cells were

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seeded at a density of 3×105 cells overnight prior to treatment with different

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concentrations of LCB (40, 80, and 120 μM) for 24h. Then after, the cells were

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harvested and stained with 10 μM DCFH-DA for 20 minutes at 37°C. Finally, the

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presence of ROS was evaluated by flow cytometry (Beckman Coulter, USA), and

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analysis was conducted by the WinMDI 2.8 software.24

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Reverse transcription and quantitative real-time PCR

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Cells (3×105 cells) treated with LCB (40, 80 and 120 μM) for 24h were used for total

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RNA isolation and cDNA synthesis. Real-time PCR (Applied Biosystems, Foster City,

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CA, USA) parameters were as follow: 95°C for 3 minutes, 40 cycles of 95°C for 10

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seconds, and 60°C for 20 seconds.24 The relative number of selected genes was

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calculated using the 2-ΔΔCt method. The list of primers is presented in Table 1.

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Western blotting

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The treated cells were mixed with cold PBS prior to lysis on ice with ice-cold RIPA

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buffer containing 2% PMSF (Sigma-Aldrich, USA) for 30 minutes. The lysed cells

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were centrifuged at 14,000 rpm/min and the supernatant fraction was collected.

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Proteins were separated using SDS–PAGE and placed onto a PVDF membrane. After 8


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blocking with 5% dried skim milk in blocking buffer [0.1%Tween 20 in 20 mmol/L

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PBST (pH 7.5)], the membrane was incubated with primary antibodies (Cell

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Signaling Technology, USA) at 4°C overnight followed by the addition of secondary

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antibody.24 Visualization was accomplished using ECL Plus Western Blotting

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Detection System (Thermo scientific, USA).

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Caspases inhibitor assay

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Cells (3×105 cells) were treated with Caspase 8 inhibitor(Z-IETD-FMK) and Caspase

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9 inhibitor(Z-LEHD-FMK) for 2 h prior to exposure or after exposure to 80 μM of

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LCB. The analysis was conducted by western blotting using Caspase 8 and Caspase 9

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antibodies.24

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Statistical analysis

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All analyses were carried out in triplicates and the data expressed as the mean ±

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standard deviation (SD). Statistical comparison between groups were analyzed using

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one-way ANOVA using SPSS 18 (SPSS, Inc., Chicago, IL, USA) followed by

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least-significant difference (LSD) post hoc test. Differences were considered to be

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significant for p < 0.05.

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Results

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LCB extraction from Glycyrrhiza uralensis Fisch

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The purity of extracted LCB was reported as 92.35% (Figure. 1A and B). The main

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peak was situated at 18.096 min (Figure. 1A). Subsequently, the quasi molecular ions

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of sample was reported as m/z = 121.0266 [M+H]+ (Figure. 1B). The further 9



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identification by NMR was presented in Figure. 2A and B.13 CNMR (151MHz)δ:

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190.28,164.95,152.67,151.63,141.40,141.23,133.95,132.63,122.62,122.17,121.58,118.4

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1,114.75,63.86,43.02,42.88,42.74,42.60,42.46,42.32,42.18.

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1H

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9.79 (s, 1H), 8.73 (s, 1H), 7.98 (d, J = 8.5 Hz, 2H), 7.82 (d, J = 15.6 Hz, 1H), 7.65 (d,

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J = 15.6 Hz, 1H), 7.31 (d, J = 8.6 Hz, 1H), 6.85 (d, J = 2.2 Hz, 1H), 6.61 (d, J = 8.5

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Hz, 1H), 3.74 (s, 3H), 2.49 – 2.45 (m, 1H).

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Effects of LCB on HepG2 cells survival

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After performing MTT assay, we found a significant growth inhibition in HepG2 cell

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in a concentration and time-dependent manner (Figure 3A). The IC50 of LCB on

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HepG2 cell was 110.15 μM at 24 h (Figure 3B). Moreover, the inhibitory effect of

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LCB on two normal cell lines (HEK293 and EAhy926) were investigated, while the

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IC50 values on two cells were more than 400 μM at 24 h (Figure 3C, D). The results

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demonstrate that LCB can remarkably inhibit the proliferation of HepG2 cells.

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Effect of LCB on HepG2 cells morphology

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HepG2 cells subjected to LCB for 12 and 24h undergone abnormal morphology

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changes (Figure 4). The normal cells in the control group were observed as irregular

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polygon on the cell wall with intact cytoskeleton. Whereas, the LCB treated cells

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were observed with protrusions and pyknosis. Some of the cells were even appeared

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to be stick together and rounded up. At higher LCB dose and treatment time, a

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reduction of cell numbers and greater destructive changes were observed in the cell

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morphology, which indicates the inhibition effect of LCB on HepG2 cells.

NMR (600 MHz , DMSO-d6) δ:1H NMR (600 MHz, DMSO-d6) δ:10.32 (s, 1H),

10


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Effects of LCB on HepG2 Cell Cycle

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After considerable growth inhibition of HepG2 cells by LCB, cell cycle progression

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was determined. The results in Figure 5A showed that LCB dose-dependently induced

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cell cycle block at G2/M phase. Figure. 5B represented the cell rate in subsequent

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growth phases. Taking into consideration the effects of LCB on cell cycle, molecular

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regulation in HepG2 cells after exposure to LCB were targeted studying key

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regulators in cycle-related genes. It was clear from Figure 6A, that CDK1, Cyclin B1,

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CHK2, CDC14B, and CDC7 mRNA expression were down regulated

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dose-dependently, while ZBTB17, CDC20, PKMYT1, GADD45A, GADD45B, SFN,

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CDKNIC, p21 and p53 were significantly up regulated.

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Based on above results, effect of LCB on the protein expression of selected genes

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was evaluated. The CDK1, Cyclin B1, and CHK2 protein levels in treated cells were

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lowered (Figure. 6B and C), which are the key protein of the G2/M phase. The

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phosphorylation of CHK2(p-CHK2) initiates check point of G2/M phase and further

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cell cycle is controlled through p53 (activator of p21and in return inhibitor of CDK1)

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dependent pathways.25 It was revealed that treatment with LCB resulted in improved

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protein expression of p21 and p53 (Figure. 6B and C).

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Effects of LCB on HepG2 cell apoptosis

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To confirm whether LCB inhibited cell growth was due to induction of apoptosis,

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flow cytometry was used. Figure. 7 showed that there was a concentration-dependent

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accumulation of apoptotic cells in LCB exposure group. Among the normal cells,

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approximately 5.08% of the cells were reported as apoptotic cells; on the other hand, 11



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LCB exposure increased the apoptotic cells to 18.29%, 30.14%, and 36.6%,

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respectively when exposed to 40, 80, and 120 μM for 24h which confirmed that

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apoptosis was induced in HepG2 cells.

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Effects of LCB on the expression of receptor-mediated pathway related genes

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Apoptosis is an active, signal-dependent process that can be influenced by many

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factors, such as toxins,26 drugs,27 and radical expoure.28 There are two major pathways

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(extrinsic and the intrinsic pathway) which regulate the accomplishment of apoptosis

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process. In case of external factors, firstly, mRNA expression of several genes (TNF,

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TNF-R1, Fas, FasL, Caspases 8, JUN, and FOS) and protein expression of TNF-R1,

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Fas and Caspases 8 after treatment with LCB for 24h were significantly up regulated

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(Figure.8). Furthermore, specific inhibitors were used to confirm the contribution of

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receptor-mediated pathway in LCB-induced apoptosis. The expression of Caspase 8

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was significantly down regulated upon treatment with Caspase 8 inhibitor

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(Z-IETD-FMK). While addition of LCB reduced the above inhibition to certain

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degree (Figure. 8D). These findings led to further focus on mitochondrial pathway

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responsible for LCB-induced apoptosis.

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Effects of LCB on the expression of mitochondrial pathway related genes

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In order to study the internal factors, it was revealed that LCB could increase the

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mRNA expression levels of Bid, Bak, PUMA, DIABLO, ENdoG, Caspase 9 and

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Caspases 3; while, decreased the Bcl-xl expression (Figure. 9A). Similarly, western

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blot analysis further confirmed that expression of Bak, Caspase 9, and Caspases 3 was

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increased except Bcl-xl (Figure. 9B and C). Subsequently, Caspase 9 inhibitor 12


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(Z-LEHD-FMK) remarkably lowered the LCB-induced rise of Caspase 9 (Figure. 9D),

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representing the involvement of mitochondrial pathway.

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Effects of LCB on the expression of other apoptosis related genes

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Involvement of other pathways in LCB-induced apoptosis was confirmed by down

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regulation of mRNA expression levels of PARP4 and BIRC3 as well as up regulation

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of ERN1, LMNA and DDIT3 (Figure. 10).

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Effects of LCB on reactive oxygen species generation

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Earlier studies supported the role of elevated ROS generated in cell apoptosis.29 As it

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can be seen in Figure. 11, ROS levels were clearly enhanced in HepG2 cells after

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treatment with LCB (40, 80, and 120 μM), which claims the role of oxidative stress in

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cell apoptosis. Based on our above results, comprehensive underlying mechanism for

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anti-cancer activity of LCB can be summarized in Figure. 12.

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Discussion

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Initially, activity-guided fractionation and repeated column chromatography of the

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chloroform fractions from Glycyrrhiza uralensis Fisch resulted in the isolation of

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LCB. An HPLC method was developed for the determination of LCB. The analysis

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was carried on the column of Hyper 0DS2 C18 column with the mobile phase

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consisted of acetonitrile and 0.5% acetic acid solution suspension. The purity of LCB

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was reported as 92.35% and the quasi molecular ions of sample was reported as m/z =

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121.0266 [M+H]+.The structure of LCB was elucidated by UPLC-TOF-MS/MS, 1H

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and 13C NMR and by comparison with these spectral data.1 And in the 1H NMR data, 13



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3.74 (s, 3H) was the OCH3 signal .7.82 (d, J = 15.6 Hz, 1H) and 7.65 (d, J = 15.6 Hz,

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1H) were the α- and β-H signals of chalcone . In the 13C NMR data, δ:190.28

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represented the C=O signal. Both the C=O and α-, β-H signals confirmed the mother

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nucleus of chalcone. Previous study claimed that LCB can be cytotoxic to several

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types of cancer cell lines, such as bladder cancer cell lines T24 , EJ14 and breast

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cancer cell line MCF-7.15 Then after, the molecular mechanisms of apoptotic effects

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of LCB treatment to human hepatoma cell HepG2 were performed in present

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research.

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Deregulation of the cell cycle is one of the most common alterations during cancer

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development.16 Therefore, cell cycle arrest is considered an effective strategy for

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various drugs. Our results provided convincing evidence that LCB induced significant

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cell cycle arrest in G2/M phase on HepG2 cells. However, in bladder cancer cells and

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breast cancer cells LCB induced S-phase arrest.14 In the former cell type, LCB

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induced cyclin A, Cdc25A, Cdc25B, Bcl-2, Bax, surviving and PARP gene

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expression changes.14 And in breast cancer cells LCB repressed the expression of

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cyclin A, CDK2 and CDC25A and activate p21, p53, Caspase 9 and Caspase 3.15 In

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our research, Real-time PCR analysis and western blot analysis revealed the

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molecular mechanism of the inhibition of LCB on HepG2 cells. CyclinB1, CDK1,

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and CHK2 are the key genes for G2/M phase. The phosphorylation of CHK2

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(p-CHK2) initiates check point of G2/M phase. CDK1 and Cyclin B1 can be formed

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into the mitotic (M)-phase promoting factor (MPF) during cell cycle progression.30

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P21, the main target for p53 is mainly responsible for DNA damage during cell cycle 14


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arrest.31 Our study showed the increased mRNA and protein levels for p21 and p53;

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while, downward trend was noticed for CHK2, Cyclin B1, and CDK1 in LCB treated

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HepG2 cells, which seized the G2/M phase. GADD45A and GADD45B are tumor

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suppressors which can activate downstream targets with the help of p53 to execute the

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G2/M block.32 ZBTB17 known as transcription factor MIZ1 is involved in cell-cycle

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regulation,33 and assisting ZBTB4 directly binds to the promoter of p21. SFN can

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participate in the transcriptional regulation of CDK-inhibitors, encodes a

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p53-regulated inhibitor of G2/M progression.34 CDKN1C also known as p57, together

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with p21 belongs to Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors.35

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Previously known, PKMYT1 arrested the cell cycle at G2/M transition and

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phosphorylated the Thr14 and Tyr15 in the CDK1-Cyclin B complex.36 Cdc20 served

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as a cell-cycle regulator 37 and CDC7 is a serine/threonine kinase, responsible for

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DNA replication initiation.38 CDC14B, an orchestrating participator in mitosis and

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cell division exit from M phase by disrupting G2/M cyclins.39 After treatment with

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LCB for 24 h, the mRNA expression of GADD45A, B, ZBTB17, SFN, CDKN1C,

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PKMYT1, and CDC20 were increased, while the mRNA expression of CDC7 and

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CDC14B were decreased.

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The receptor-mediated pathway and mitochondrial pathway are the major key

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players in apoptosis. LCB initiated the apoptosis in HepG2 through both

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receptor-mediated pathway and mitochondrial pathway. In receptor-mediated pathway,

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LCB improved the mRNA expression of TNF, TNF-R1, Fas, FasL, JUN, and FOS.

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TNF-R1 and Fas are two important death domain-containing receptors integrated with 15



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TNF and FasL, respectively.40,41 The protein expression of TNF-R1and Fas were also

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elevated in accordance with the mRNA expression. The FOS and JUN are

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immediate-early genes that encode for proteins which form a transcription factor

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AP-1 for apoptosis initiation when come in contact with extracellular stimuli.42

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Caspase 8 is the executioners of apoptosis and after activation it can trigger Caspase 3

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and results in apoptosis.43 The expression of Caspase 8 in HepG2 cells were elevated

328

in the mRNA and protein level after exposure to LCB for 24h. In mitochondrial

329

pathway, LCB up regulated the mRNA expression of Bid, Bak, PUMA, DIABLO,

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EndoG, and Caspase 9 except Bcl-xl. The protein expression of Bid, Bcl-xl, Bak, and

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Caspase 3 were consistent with their mRNA expression. The members of Bcl-2

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family such as Bcl-2, Bcl-xl, etc. are responsible for mitochondrial integrity, while the

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pro-apoptotic members such as Bak and Bax, etc. disrupt the integrity.23 After

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exposure to internal stimuli, Bid attaches to Bcl-2 family and Bak is released, and

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further release of cytochrome c helps in apoptosis induction, which activates the

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initiator Caspase 9. In return, activated Caspase 9 can process the downstream

337

caspases, such as Caspase 3. Caspase 3 worked as a key executioner during the latest

338

stages of apoptosis both by extrinsic and intrinsic pathways, which can intact with

339

both Caspase 8 and Caspase 9.44 The inhibition of Caspase 8 and Caspase 9 proteins

340

in receptor-mediated pathway and mitochondrial pathway abolished the LCB induced

341

apoptosis significantly. PUMA as an essential intermediate during p53-dependent

342

apoptosis can activate the Bax and mitochondrial outer membrane permeabilization.45

343

DIABLO attenuates the inhibitory effect of IAPs on caspases to accelerate the 16


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344

apoptosis.46. In addition to the above two main pathways, other genes and pathways

345

also have been involved in LCB induced apoptosis of HepG2. Previous studies

346

reported that DDIT3 and ENR1 as Endoplasmic Reticulum Stress(ER stress)

347

inducible genes are the key mediators to initiate the apoptosis process.47,48 Likewise,

348

BIRC3 as one of the inhibitors of apoptosis proteins, can suppress apoptosis through

349

inhibitory action on Caspase 3.48 PARP4 was suggested to contribute in the cell death

350

by reducing the supply of NAD and ATP.49 LMNA is required during DNA

351

replication and it also provides an attachment site for apoptotic signaling machinery.50

352

The cleavage of PARP and LMNA are known to be accomplished through Caspase

353

3.49,50

354

Previous research documented that accelerated ROS production and/or lowered

355

ROS scavenging ability can build up the cellular ROS which in return cause the cell

356

death.29 Due to their apoptotic responses, various natural products are recognized as

357

potent anti-cancerous agents by increasing the cellular ROS production. The LCB at

358

higher concentration could accelerate the ROS generation which can be associated

359

with HepG2 cell apoptosis.

360

Altogether, the inducing apoptotic effects of LCB in HepG2 cells were

361

accompanied by growth inhibition at IC50 (110.15 μM) for 24 h, morphological

362

disruption, and cell cycle arrest in G2/M phase. Going further, both the key pathways

363

regulating the cell death (intrinsic and extrinsic) possibly accelerated the

364

LCB-induced apoptosis. Other genes such as PARP4, BIRC3, DDIT3, LMNA, and

365

ERN1 also participate in the process of apoptosis. Furthermore, improved intracellular 17



366

ROS accretion as intermediate signaling molecules served as an important key for

367

apoptosis process in LCB treated cells.

368

369

Funding

370

This work was supported by the National Natural Science Foundation of China

371

(31850410476), the Major Projects of Science and Technology in Anhui Province

372

(1804b06020347, 17030701028, 18030701142, 18030701158), and Zhongyuan

373

scholars in Henan Province (192101510004).

374 375

There is no conflict of interest to declare.

376

377

References

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21



513

Figure captions

514

Figure 1 HPLC chromatograms and MS spectra of purified LCB. (A) HPLC

515 516 517

chromatograms. (B) MS spectra. Figure 2 NMR spectra of the prepared LCB. (A) 13C NMR spectra. (B) 1H NMR spectra.

518

Figure 3 Inhibitory action of LCB on HepG2 cells. (A) Inhibition rate of LCB on

519

HepG2 cells after 24 h. Inhibition curves of LCB on HepG2 cells (B), HEK296

520

cells (normal cells) (C), and EAhy926 cells (normal cells) (D) were investigated

521

after 24 h of treatment. The data were presented as means ± SD of three replicated

522

experiments.

523 524

Figure 4 Effect of LCB on morphological changes in HepG2 cells. The original magnification was × 200.

525

Figure 5 The cell counts in different cell cycle stages after LCB treatment. (A) The

526

cells in different cell cycle stages after 24h treatment. HepG2 cells were treated

527

with LCB at indicated concentrations, and evaluated with Annexin V/PI

528

dual-labeling technique. (B)The proportions of cells in different stages after 24h

529

treatment with LCB at indicated concentrations. Significant difference at p < 0.05

530

was shown with superscript a, b, c.

531

Figure 6 The expression of cell cycle related genes in HepG2 cell after LCB

532

treatment. (A) The mRNA level of related genes in HepG2 cells; (B) Western

533

blotting analyses of related genes in HepG2 cells; (C) The relative protein levels of

534

cell cycle related genes. HepG2 cells were treated with LCB at indicated 22


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535

concentrations (40 μM, 80 μM and 120 μM) for 24h, and used for expression

536

analysis compared with untreated cells. All the data were represented as means ±

537

SD of three replicated experiments. Significant difference at p < 0.05 was shown

538

with superscript a, b, c, d.

539

Figure 7 Apoptosis in HepG2 cells treated with LCB at indicated concentrations. The

540

apoptosis was assessed by Flow Cytometric analysis after staining with Annexin

541

V-PI after 24 h of treatment.

542

Figure 8 The relative expression level of death receptor pathway related genes in

543

LCB treated HepG2 cells. (A) The relative mRNA level of genes in HepG2 cells;

544

(B) Western blotting analyses of related genes; (C) The relative protein levels of

545

related genes; (D) The protein level of Caspase 8 gene in HepG2 cells treated with

546

or without Caspase 8 inhibitor (Z-IETD-FMK). HepG2 cells were treated with LCB

547

at indicated concentrations (40 μM, 80 μM and 120 μM) for 24h, and used for

548

expression analysis compared with untreated cells. The data were represented as

549

means ± SD of three replicated experiments. Statistical difference at p < 0.05 was

550

shown with superscript a, b, c, and d.

551

Figure 9 The relative expression level of mitochondrial pathway related genes in

552

LCB treated HepG2 cells. (A) The relative mRNA level of genes in HepG2 cells;

553

(B) Western blotting analyses of related genes; (C) The relative protein levels of

554

related genes; (D) The protein level of Caspase 9 gene in HepG2 cells treated with

555

or without Caspase 9 inhibitor (Z-LEHD-FMK). HepG2 cells were treated with

556

LCB at indicated concentrations (40 μM, 80 μM and 120 μM) for 24h, and used for 23



557

expression analysis compared with untreated cells. The data were represented as

558

means ± SD of three replicated experiments. Statistical difference at p < 0.05 was

559

shown with superscript a, b, c, and d.

560

Figure 10 The relative mRNA expression level of other apoptosis related genes in

561

LCB treated HepG2 cells. All the expression level of genes in treated HepG2 cells

562

with different concentrations of LCB (40 μM, 80 μM and 120 μM) was compared

563

with untreated cells. The data were represented as means ± SD of three replicated

564

experiments. Statistical difference at p < 0.05 was shown with superscript a, b, c,

565

and d.

566

Figure 11 Effects of LCB treatment on ROS generation in HepG2 cells. (A) untreated

567

cells; (B), (C), and (D) represent the cells treated with concentration of 40 μM, 80

568

μM and 120 μM for 24 h, respectively. (E) The ROS generation of HepG2 cells. (F)

569

The relative fluorescence intensity of ROS generation in LCB treated cells. The

570

data were represented as means ± SD of three replicated experiments. Statistical

571

difference at p < 0.05 was shown with superscript a, b, c.

572 573

Figure 12 Proposed mechanism of the induced apoptotic effects of LCB on HepG2 cells.

24


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574


Table 1 Primers used for real-time PCR Gene CDK1 CyclinB1 CHK2 CDC14B CDC7 ZBTB17 CDC20 PKMYT1 GADD45A GADD45B SFN CDKN1C p21 p53 TNF TNF-R1 Caspase 3 Caspase 8 Fas FasL FOS JUN Bid Bcl-xl Bak PUMA DIABLO

Primer Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward

Sequence TTGAAACTGCTCGCACTTGG TCCCGGCTTATTATTCCGCG CTGCTGGGTGTAGGTCCTTG TGCCATGTTGATCTTCGCCT GCAGACCCAGCTCTCAATGT TCAGGCGTTTATTCCCCACC GCCATTCTCTACAGCAGACCA TGTAAACCATTGCCAGATTGAGT AGTGCCTAACAGTGGCTGG CACGGTGAACAATACCAAACTGA GTGTGATGTGCGGTAAGGC TGGACTGGACGAATCTCTTGC GACCACTCCTAGCAAACCTGG GGGCGTCTGGCTGTTTTCA GCCTGCCAACATCTTCCTG CCCAGACTGAACACATCCGC GAGAGCAGAAGACCGAAAGGA CAGTGATCGTGCGCTGACT TACGAGTCGGCCAAGTTGATG GGATGAGCGTGAAGTGGATTT GACCATGTTGGAAGATTTGGGA TGCACCACTGAATGACCTTTT GCGGCGATCAAGAAGCTGT GCTTGGCGAAGAAATCGGAGA GCGGAACAAGGAGTCAGACA GAACCAGGACACATGGGGAG AGCACTGTCCAACAACACCA CTTCAGGTGGCTGGAGTGAG CCTCTCTCTAATCAGCCCTCTG GAGGACCTGGGAGTAGATGAG CTCTCCCCTCCTCTCTGCTT GGGTTGAGACTCGGGCATAG TGGACTGTGGCATTGAGACA CAGGTGCTGTGGAGTATGCA TATCCCGGATGGCTGACT GACATCGCTCTCAGGCTC AGATTGTGTGATGAAGGACATGG TGTTGCTGGTGAGTGTGCATT CTGCCACCCCTGAAGAAGAG GCCACTTTCCTCAGCTCCTT GGGGCAAGGTGGAACAGTTAT CCGCTTGGAGTGTATCAGTCA TCCAAGTGCCGAAAAAGGAAG CGAGTTCTGAGCTTTCAAGGT ATGGACCGTAGCATCCCTCC GTAGGTGCGTAGGTTCTGGT GCATTGTGGCCTTTTTCTCC GCTGCTGCATTGTTCCCATA ACTCTACCCCTGCTCCCATT CTTGGAGGCTTCTGACACGT ATGCCTGCCTCACCTTCATC TCAGCCAAAATCTCCCACCC CGCGCAGCGTAACTTCATTC 25



ENDOG Caspase 9 ERN1 PARP4 LMNA BIRC3 DDIT3 β-actin

Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

CCAAAGCCAATCGTCACAGTTTT GCAGCTACCAAAACGTCTATGT CACCTTGAAGAAGTGTGTGGG GCTCTTCCTTTGTTCATCTCC CATCTGGCTCGGGGTTACTGC CACAGTGACGCTTCCTGAAAC GCCATCATTAGGATCTGGGAGA ATGTGAGGCCCTTGTTGCAT CACGATCTTCCACTACTTTGGG AGCAGCGTGAGTTTGAGAGC AGACTGCCTGGCATTGTCC TTTCCGTGGCTCTTATTCAAACT GCACAGTGGTAGGAACTTCTCAT GGAAACAGAGTGGTCATTCCC CTGCTTGAGCCGTTCATTCTC TGTGATGGTGGGAATGGGTCAG TTTGATGTCACGCACGATTTCC

575

26


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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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Figure 11

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Figure 12

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Table of contents graphic

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Licochalcone B extracted from Glycyrrhiza uralensis Fisch induces

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