Cancer Medicine

Open Access


The mTOR inhibition in concurrence with ERK1/2 activation is involved in excessive autophagy induced by glycyrrhizin in hepatocellular carcinoma Xuan Zhang1,a , Hua Yang2,a, Shuqiang Yue3,a, Guangbin He4,a, Shibin Qu1,a, Zhuochao Zhang1, Ben Ma1, Rui Ding1, Wei Peng1, Hongtao Zhang1, Zhaoxu Yang1, Kefeng Dou1, Kaishan Tao1 & Xiao Li1 1Department

of Hepatobiliary Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, China of Geriatrics, Xi’an No. 1 Hospital, Xi’an, Shaanxi, China 3Department of Oncological Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, China 4Department of Ultrasound Diagnosis, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, China 2Department

Keywords Autophagy, ERK1/2, glycyrrhizin, hepatocellular carcinoma, mTOR Correspondence Kefeng Dou, Kaishan Tao and Xiao Li, Department of Hepatobiliary Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, China. Tel: +86-029-84775255; Fax: +86-029-84775255; E-mails: [email protected]; [email protected]; [email protected] Funding Information No funding information provided. Received: 9 July 2016; Revised: 12 April 2017; Accepted: 18 May 2017 Cancer Medicine 2017; 6(8):1941–1951 doi: 10.1002/cam4.1127


authors contributed equally to this


Abstract Autophagy is a life phenomenon in which autophagosomes remove damaged or aging organelles and long-­ lived circulating proteins to maintain the cell’s stability. However, disorders of excessive autophagy are a response of cancer cells to a variety of anticancer treatments which lead to cancer cell death. The Akt/mammalian target of rapamycin (mTOR) and the extracellular signal-­ regulated kinase 1/2 (ERK1/2) pathways are both involved in nutrient-­induced autophagic phenomenon and exhibit vital relevance to oncogenesis in various cancer cell types, including hepatocellular carcinoma (HCC). However, the influence of autophagy for cancer cell death remains controversial and few scientists have investigated the variation of these two signaling pathways in cancer cell autophagic phenomenon induced by anticancer treatment simultaneously. Here, we explored the anticancer efficacy and mechanisms of glycyrrhizin (GL), a bioactive compound of licorice with little toxicity in normal cells. It is interesting that inhibition of Akt/mTOR signaling in concurrence with enhanced ERK1/2 activity exists in GL-­induced autophagy and cytotoxicity in HepG2 and MHCC97­H hepatocellular carcinoma cells. These results imply that the GL-­related anticancer ability might correlate with the induction of autophagy. The influence of induced autophagic phenomenon on cell viability might depend on the severity of autophagy and be pathway specific. In the subsequent subcutaneous xenograft experiment in vivo with MHCC97-­H cells, GL obviously exhibited its inhibitory efficacy in tumor growth via inducing excess autophagy in MHCC97-­H cells (P 95.0%, from Sigma-­ Aldrich, MO) was prepared in a 10 mmol/L stock solution with culture medium and was then diluted into required concentrations with culture medium for need. 3-­Methyladenine (3-­MA), a β type PI3K inhibitor which can combine with Vps34 to block the formation of autophagosome, and chloroquine, a proteolysis inhibitor, were purchased from Sigma-­Aldrich. Atg7 siRNA was used to silence autophagy-­essential gene Atg7 to verify the role of 3-­MA (Life Technologies, CA).

Determination of cell viability Cells were seeded into 96-­well plates at 3 × 103 cells per well and then administered with 0, 1, 2, and 4 mmol/L GL for 24, 48, and 72 h. Cell viability was detected using a CCK-­8 assay kit (Beyotime, Jiangsu, China) according to the manufacturer’s instructions. Cell viability was determined by measuring NADH production, resulting from dehydrogenase activity in viable cells. Briefly, each well was added with 10 μL of CCK-­ 8 solution and incubated at 37°C for 2 h. Thereafter, the optical density (OD) for each well was measured at 450 nm using a microplate reader (Bio-­ Rad Model 680, USA). All experiments were repeated three times, and the mean value was calculated. The cell survival equation: cell survival rate = (OD experiment − OD blank)/(OD control − OD blank) × 100%. To verify the role of excessive autophagy in glycyrrhizin-­ mediated cell death in vitro, autophagy was inhibited by 3-­MA (2 mmol/L) or Atg7 siRNA (50 nmol/L) before GL was administered. For autophagic flux, cells were treated with 10 μmol/L chloroquine.

siRNA transfection Atg7 siRNA was used to inhibit Atg7 expression. Transfection was conducted using Lipofectamine RNAi reagent (Life Technologies) according to the manufacturer’s instructions. A scrambled siRNA was transfected as the negative control. After transfection for 48 h, HCC cells were treated with GL for further analysis.

© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

X. Zhang et al.

Plate colony-­forming assay Cells in logarithmic growth phase were harvested and resuspended into a single cell suspension with DMEM supplemented with 10% FBS and then seeded in a six-­ well plate at a density of 300 cells per well in triplicate. They were cultured in growth medium supplemented with GL (0, 1, or 2 mmol/L) for 18 days. Then, the colonies were fixed in 95% ethanol and stained with a 4 g/L crystal violet solution, and colony formation was photographed and counted under a 100× magnification. Colonies containing over 50 cells were counted.

Cell migration assays Cell migration was detected using Transwell chambers (8  μm pore size; Millipore) without Matrigel matrix. In brief, cells in logarithmic growth phase were harvested and administered to GL in serum-­free medium at different concentrations (0, 1, or 2 mmol/L), 600 μL of complete medium was added to the bottom chamber and 200 μL of the cell suspension (containing 4 × 104 cells) was placed in the upper chamber. After 12 h, the cells on the top surface of the membrane were mechanically removed using a cotton swab, and the cells on the bottom surface of the membrane were fixed in 95% ethanol and stained with a 4 g/L crystal violet solution. Cells adhering to the bottom surface of the membrane were counted in five randomly selected areas under a 100× microscope field. Each experiment was repeated three times.

Acridine orange immunofluorescent staining The formation of acid vesicular organelles (AVOs) was assessed by acridine orange (AO) staining (Sigma Chemical Co., St. Louis, USA) and then observed with fluorescence microscopy. After treatment with glycyrrhizin for 12 h, cells were stained with 20 μg/mL AO in medium and incubated in the dark for 15 min at 37°C. Then, cells were washed with PBS four times, and an inverted fluorescence microscope (Olympus, Japan) was used to obtain fluorescent micrographs.

Western blot analysis Cells or tumor tissue extracts were digested in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL). The proteins were quantified using a BCA protein assay kit (Thermo Scientific) according to the manufacturer’s instructions. The total protein extracts were separated by 10% SDS-­PAGE and transferred to polyvinylidene difluoride

© 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Glycyrrhizin Induces Excessive Autophagy in HCC

membranes. The membranes were incubated with primary antibodies, including anti-­LC3B (Abcam, Cambridge, UK), anti-­ERK1/2 (Cell Signaling Technology CST, Danvers, MA), anti-­p-­ERK1/2(CST), anti-­mTOR (CST), anti-­p-­mTOR (CST), anti-­ p85 (Abcam), anti-­ p-­ p85 (Abcam), anti-­ Akt (CST), anti-­p-­Akt (CST), anti-­p70S6K (CST), anti-­p-­p70S6K (CST), anti-­ p62 (CST), anti-­ Craf (Abcam), anti-­ p-­ Craf (Abcam), anti-­ Atg7 (CST), anti-­ GAPDH and β-­actin (Abmart, China) overnight at 4°C. Then, the membranes were incubated with horseradish peroxidase (HRP)-­ conjugated secondary antibodies (Abcam) for 1 h at room temperature, and the protein bands were detected with a ChemiDoc™ XRS+ and Image Lab TM software (Bio-­Rad, Hercules, CA).

Subcutaneous xenografts in nude mice Nude mice (BALB/C nu/nu, 5-­ week-­ old, female) were purchased from HFK Bioscience Ltd. (Beijing, China) and maintained under specific pathogen-­free conditions in the laboratory animal center of Fourth Military Medical University (Xi’an, China). All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by The Research Animal Care and Use Committee of Fourth Military Medical University. For the tumorigenesis assay, MHCC97-­H cells (3 × 106) were suspended in a 100 μL mixture of equal volumes of medium and Matrigel and were implanted subcutaneously into the left flank of nude mice (n = 3 mice per group). When the tumors reached a volume of approximately 50–70 mm3, the mice were then randomly divided into two groups. The treatment group received a peritumoral administration of glycyrrhizin (100 mg/kg body weight in 80 μL olive oil, n = 3), whereas the vehicle control group received olive oil alone (n = 3). These treatments were carried out once daily for 40 days. The tumor volumes and the body weight of the mice were measured twice per week. The tumor volumes (mm3) were calculated with the following formula: tumor volume = (length × width2)/2. At the end of the experiment, the mice were sacrificed, and the tumors were harvested and weighed. The dissected tumors were frozen in liquid nitrogen for western blot analysis or fixed in formalin and embedded in paraffin for immunohistochemistry.

Immunohistochemistry The tissue samples were fixed in formalin, embedded in paraffin, and sectioned to a 4-­μm thickness. The sections were deparaffinized, hydrated, and boiled in 10 mmol/L citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidases were inactivated with 3% H2O2. After the 1943

Glycyrrhizin Induces Excessive Autophagy in HCC

X. Zhang et al.

Figure 1. Glycyrrhizin inhibits HepG2 and MHCC97-­H cell survival. (A) Structure of glycyrrhizin. (B) and (D) HepG2 and MHCC97-­H cells were exposed to the indicated concentrations of glycyrrhizin (0, 1, 2, or 4 mmol/L) for 24, 48, or 72 h. Cell viability was determined by a CCK-­8 solution cell viability assay. (C) Representative images of cell morphology in HepG2 and MHCC97-­H cells were taken after a 72-­h treatment with or without 2 mmol/L glycyrrhizin. Curves, mean (n = 3, in triplicate).

sections were blocked in goat serum, the sections were incubated with primary antibody (LC3B, Abcam, Cambridge, UK; Ki-­67, Boster, Wuhan, China) overnight at 4°C, incubated with biotinylated secondary antibody at room temperature for 1 h, and visualized with diaminobenzidine (DAB kit, ZSGB-­BIO, China). Hematoxylin was used to counterstain the nuclei.

Statistical analysis All data are presented mean ± SD. Intragroup comparisons were made by employing Student’s t-­test or an analysis of variance. All statistical analyses were performed using SPSS version 17.0 (IBM, Chicago, IL), and all figures were generated using GraphPad Prism 5.01 (GraphPad Software, La Jolla, CA). In all cases, a P 

2 activation is involved in excessive autophagy induced by glycyrrhizin in hepatocellular carcinoma.

Autophagy is a life phenomenon in which autophagosomes remove damaged or aging organelles and long-lived circulating proteins to maintain the cell's s...
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