MOLECULAR MEDICINE REPORTS 18: 3429-3436, 2018

Isoliquiritigenin inhibits proliferation and metastasis of MKN28 gastric cancer cells by suppressing the PI3K/AKT/mTOR signaling pathway

XIU‑RONG ZHANG1,2, SHI‑YAO WANG3, WEN SUN4 and CHAO WEI5

1Department of Traditional Chinese Medicine, Maternal and Child Health Care of Shandong Province; 2Key Laboratory of Birth Regulation of National Health and Family Planning Commission of China, Jinan, Shandong 250014; 3Department of Chinese Medicine, The Second Clinical Medical College of Guangzhou University of Traditional Chinese Medicine, Guangzhou, Guangdong 510000; 4Department of Research, Beijing Splinger Medical Research Institute, Jinan, Shandong 250021; 5Department of Ophthalmology, The Second Hospital of Shandong University, Jinan, Shandong 250033, P.R. China

Received November 3, 2017; Accepted June 19, 2018

DOI: 10.3892/mmr.2018.9318

Abstract. Isoliquiritigenin (ISL) is a flavonoid extracted from following ISL pretreatment, thus suggesting that ISL triggered licorice root, which is known to serve important antitumor autophagy in MKN28 cells. In addition, the phosphorylation roles in numerous types of cancers; however, its effect on levels of protein kinase B (AKT) and mammalian target of gastric cancer remains to be elucidated. The present study rapamycin (mTOR) were significantly reduced following ISL aimed to explore the roles and underlying mechanisms of ISL treatment. These results indicated that ISL may influence in MKN28 gastric cancer cells. MKN28 cell proliferation apoptosis and autophagy in MKN28 cells by suppressing the was measured using the Cell Counting Kit‑8 (CCK8) assay. phosphoinositide 3‑kinase/AKT/mTOR signaling pathway. In A Transwell assay was used to determine the effects of ISL conclusion, the findings of the present study suggested that ISL on the migration and invasion of MKN28 cells. Apoptosis may inhibit MKN28 cell proliferation, migration and invasion was assessed by fow cytometry, and the expression levels of by inducing apoptosis and autophagy, implying potential as a apoptosis‑, autophagy‑ and signaling pathway‑related proteins therapeutic agent for gastric cancer. were detected by western blot analysis. The results of the CCK8 assay demonstrated that ISL significantly inhibited Introduction the proliferation of MKN28 cells (P<0.05). Transwell assays demonstrated that the migration and invasion of MKN28 Gastric cancer is the fourth most prevalent cancer world- cells were significantly inhibited following treatment with wide (1,2) and the second leading cause of cancer‑associated ISL (P<0.05). Flow cytometric analysis indicated that ISL mortality (3). Despite the important advances in cancer induced apoptosis of MKN28 cells. In addition, western blot therapy, gastric cancer remains a major malignancy and a analysis revealed that the ratio of microtubule‑associated serious threat to human health (4). Surgical, radiotherapeutic proteins 1A/1B light chain 3B (LC3)II/LC3I was upregulated, and chemotherapeutic strategies have recently become as was Beclin 1 expression; however, p62 was downregulated available for the treatment of gastric cancer (5); however, radiotherapy and chemotherapy exert severe adverse effects, and the development of drug resistance is common. Therefore, it is necessary to develop novel, effective and safe agents for the treatment of gastric cancer. Recently, natural Correspondence to: Dr Wen Sun, Department of Research, Beijing Splinger Medical Research Institute, 685 Jingsi Road, Huaiyin, products have attracted attention. Studies have reported Jinan, Shandong 250021, P.R. China that compounds from natural resources are suitable alterna- E‑mail: [email protected] tives for controlling cancer with minimal toxicity and high efficacy (6‑8). Dr Chao Wei, Department of Ophthalmology, The Second Hospital Isoliquiritigenin (ISL) is a flavonoid with a chalcone of Shandong University, 247 Beiyuan Road, Jinan, Shandong 250033, P.R. China structure that is derived from licorice compounds (9,10). It E‑mail: [email protected] is found ubiquitously in foods and beverages, and tobacco. ISL has been reported to exhibit a wide range of distinct Key words: isoliquiritigenin, MKN28 cells, proliferation, biological properties and pharmacological activities, including phosphoinositide 3‑kinase/protein kinase B/mammalian target of anti‑inflammatory, anti‑oxidation, antiplatelet aggregation, rapamycin, apoptosis, autophagy cardioprotective effects against ischemia‑reperfusion, and estrogenic properties (11‑13). ISL has also been reported to suppress the proliferation and to induce the apoptosis of 3430 ZHANG et al: ISL INHIBITS PROLIFERATION AND METASTASIS OF GASTRIC CANCER MKN28 CELLS numerous cancers in vitro and in vivo (14), including colon and and washed with PBS at room temperature. Subsequently, breast cancer (15‑18). images were captured and the cells in five random fields were At present, the anticancer effects and underlying mecha- counted manually using light microscopy. nisms of ISL on gastric cancer have not been fully elucidated. The migration assay was similar to the invasion assay, with The present study conducted a series of preliminary experiments the exception that the Transwell system was not coated with to suggest that ISL may inhibit the proliferation, migration and Matrigel, and the number of cells tested was 5,000. invasion of MKN28 gastric cancer cells, which may be associ- ated with phosphoinositide 3‑kinase (PI3K)/protein kinase B Apoptosis analysis. Following treatment with 20 µM ISL or (AKT)/mammalian target of rapamycin (mTOR) signaling 0.1% DMSO (control group) for 24 h, cells were collected and pathway‑mediated apoptosis and autophagy. digested with trypsin (Beijing Solarbio Science & Technology Co., Ltd.). Cells were then washed using precooled PBS (4˚C). Materials and methods The cell density was adjusted to 1‑5x106/ml by adding 1X binding buffer. Subsequently, 100‑µl cell suspension was placed Cell culture and treatment. The MKN28 human gastric cancer in a 5 ml fow tube, and 5 µl Annexin V/fuorescein isothio- cell line and the GES‑1 human gastric normal epithelial cyanate (FITC; BD Biosciences, Franklin Lakes, NJ, USA) was mucosa cell line were purchased from the Cell Bank of the added and mixed by ficking the tube for 5 min in the dark at Chinese Academy of Sciences (Shanghai, China). The cells room temperature, followed by incubation with 10 µl propidium were cultured in RPMI‑1640 medium supplemented with 10% iodide for 10‑15 min in the dark room at room temperature. (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Following this, 400 µl PBS was added and mixed, and the tubes Waltham, MA, USA), penicillin (100 U/ml) and streptomycin were subsequently placed on ice. The results were detected (0.1 mg/ml; both Sigma‑Aldrich; Merck KGaA, Darmstadt, using a flow cytometer (BD Biosciences) in 1 h and analyzed Germany) and incubated at 37˚C in a humidified atmosphere using FlowJo v10.0 software (FlowJo LLC, Ashland, OR, USA). containing 5% CO2. When the cells entered the logarithmic growth phase, they were washed with PBS three times, and Western blot analysis. To extract proteins, cells in the were then digested by trypsin (Beijing Solarbio Science & experimental and NC groups were added to 6‑well plates Technology Co., Ltd., Beijing, China). Once the cells became and treated with radioimmunoprecipitation assay lysis buffer rounded, the digestion was terminated and the cells were (CW Biotech, Beijing, China) plus protease inhibitor. Protein placed in culture medium, after which they were pipetted into concentration was measured using a Bicinchoninic Acid 6‑well plates for subsequent experiments. When cell confu- Protein Assay kit (CW Biotech), after which the proteins ence in the wells reached ~80%, the cells were treated with were heated for 5 min at 95˚C. Vertical electrophoresis was ISL (MedChemExpress, Monmouth Junction, NJ, USA) or performed with ~20 µg protein in each lane. The proteins 0.1% dimethyl sulfoxide (DMSO; Amresco, LLC, Solon, OH, were isolated by 10% SDS‑PAGE and transferred onto poly- USA) as a negative control (NC). vinylidene fuoride membranes. The membranes were blocked with 5% skim milk powder at room temperature for 1 h and Cell Counting Kit‑8 (CCK8) proliferation detection. CCK‑8 were incubated with primary antibodies overnight at 4˚C. (Beijing Solarbio Science & Technology Co., Ltd. Beijing, The following rabbit anti‑human primary antibodies were China) was used to assess the effects of ISL on MKN28 and used: AKT (1:1,000; cat. no. 9272; Cell Signaling Technology, GES‑1 cell proliferation. MKN28 or GES‑1 cells (100 µl) Inc., Danvers, MA, USA), phosphorylated (p‑)AKT (1:1,000; were seeded into 96‑well plates at a cellular density of cat. no. 4060; Cell Signaling Technology, Inc.), mTOR 1,000 cells/well. To the NC group, 0.1% DMSO was added, (1:1,000; cat. no. 2972; Cell Signaling Technology, Inc.), whereas 20 µM ISL was added to the experimental group. p‑mTOR (1:1,000; cat. no. 2971; Cell Signaling Technology, Cells were cultured for 0, 24, 48 and 72 h time intervals at Inc.), B‑cell lymphoma 2 (Bcl‑2; 1:1,000; cat. no. 4223; Cell 37˚C, and the cell viabilities at each time interval were subse- Signaling Technology, Inc.), caspase‑3 (1:1,000; cat. no. 9664; quently detected. Prior to detection, 10 µl CCK8 solution was Cell Signaling Technology, Inc.), Bcl‑2‑associated X protein added to each well for 1.5 h at 37˚C. The optical density (OD) (Bax; 1:1,000; cat. no. 5023; Cell Signaling Technology, value was measured at 450 nm using a microplate reader to Inc.), microtubule‑associated proteins 1A/1B light chain 3B obtain the proliferation curve. (LC3; 1:1,000; cat. no. 14600‑1‑AP; ProteinTech Group, Inc., Chicago, IL, USA), Beclin 1 (1:1,000; cat. no. 11306‑1‑AP; Transwell migration and invasion assays. For the invasion ProteinTech Group, Inc.), p62 (1:1,000; cat. no. 5114; Cell assay, the inner layer of a 24‑well Transwell system (EMD Signaling Technology, Inc.) and GAPDH (1:5,000; cat. Millipore, Billerica, MA, USA) was pre‑coated with Matrigel no. 10494‑1‑AP; ProteinTech Group, Inc.). The membranes (BD Biosciences, Franklin Lakes, NJ, USA) at a 1:6 dilution. were washed three times (5 min/wash) with Tris‑buffered Cells treated with 20 µM ISL or 0.1% DMSO for 24 h were saline containing 0.05% Tween‑20. The membranes were then suspended in serum‑free medium and 100‑µl cell suspension incubated with goat anti‑rabbit immunoglobulin G (H+L), (~1x104 cells) was added to the upper chambers. The lower horseradish peroxidase‑conjugated secondary antibody chambers were filled with culture medium. Following over- (1:5,000; cat. no. SA00001‑2; ProteinTech Group, Inc.) at 37˚C night incubation, cells on the surface of the upper chamber were for 1 h. After washing of the membranes, enhanced chemilu- removed with a cotton swab. Cells on the bottom were fixed in minescence (ProteinTech Group, Inc.) was used to detect the 4% paraformaldehyde for 0.5 h at room temperature, stained signals. Quantity One v4.6.9 software (Bio‑Rad Laboratories, with 0.1% crystal violet dye for 20 min at room temperature Inc., Hercules, CA, USA) was used to scan the gray value of MOLECULAR MEDICINE REPORTS 18: 3429-3436, 2018 3431 the blots, and GAPDH was used as an internal control. The ISL triggers autophagy in MKN28 cells. Autophagy in MKN28 relative expression levels were calculated by normalizing cells was detected by western blot analysis, which measured the target protein expression to the expression of the internal alterations in the protein expression levels of LC3I, LC3II, control. Beclin 1 and p62 (Fig. 4A). Compared with in the NC group, the ratio of LC3II/LC3I and the expression levels of Beclin 1 were Statistical analysis. Statistical analyses were performed clearly upregulated in response to ISL treatment, whereas p62 using SPSS software (version 18.0; SPSS, Inc., Chicago,