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    J Gastric Cancer. 2021 Dec;21(4):439-456. English.
    Published online Dec 31, 2021.
    https://doi.org/10.5230/jgc.2021.21.e40
    Copyright © 2021. Korean Gastric Cancer Association
    Original Article

    Vitexin Inhibits Gastric Cancer Growth and Metastasis through HMGB1-mediated Inactivation of the PI3K/AKT/mTOR/HIF-1α Signaling Pathway

    Peng Zhou,1,2 Zi-Han Zheng,2 Tao Wan,2 Jie Wu,2 Chuan-Wen Liao,2 and Xue-Jun Sun1
      • 1Department of General Surgery, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, People's Republic of China.
      • 2Department of Gastrointestinal Surgery, Jiangxi Provincial People’s Hospital, Nanchang, People's Republic of China.
    • Correspondence to Xue-Jun Sun. Department of General Surgery, First Affiliated Hospital of Xi’an Jiaotong University, No. 277, West Yanta Road, Xi’an, Shaanxi 710061, People's Republic of China. Email: xxueejuuss789@163.com
    Received July 21, 2021; Revised November 29, 2021; Accepted December 14, 2021.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    Purpose

    Gastric cancer (GC) has high morbidity and mortality and is a serious threat to public health. The flavonoid compound vitexin is known to exhibit anti-tumor activity. In this study, we explored the therapeutic potential of vitexin in GC and its underlying mechanism.

    Materials and Methods

    The viability, migration, and invasion of GC cells were determined using MTT, scratch wound healing, and transwell assays, respectively. Target molecule expression was determined by western blotting. Tumor growth and liver metastasis were evaluated in vivo using nude mice. Protein expression in the tumor tissues was examined by immunohistochemistry.

    Results

    Vitexin inhibited GC cell viability, migration, invasion, and epithelial-mesenchymal transition (EMT) in a dose-dependent manner. Vitexin treatment led to the inactivation of phosphatidylinositol-3-kinase (PI3K)/AKT/hypoxia-inducible factor-1α (HIF-1α) pathway by repressing HMGB1 expression. Vitexin-mediated inhibition in proliferation, migration, invasion and EMT of GC cells were counteracted by hyper-activation of PI3K/AKT/HIF-1α pathway or HMGB1 overexpression. Finally, vitexin inhibited the xenograft tumor growth and liver metastasis in vivo by suppressing HMGB1 expression.

    Conclusions

    Vitexin inhibited the malignant progression of GC in vitro and in vivo by suppressing HMGB1-mediated activation of PI3K/Akt/HIF-1α signaling pathway. Thus, vitexin may serve as a promising therapeutic agent for the treatment of GC.

    Keywords
    Epithelial-Mesenchymal transition; Gastric cancer; Hypoxia; Phosphatidylinositols; Vitexin

    INTRODUCTION

    Gastric cancer (GC) is the fifth most common malignancy worldwide, with 989,600 new cases/year reported in 2008. Furthermore, GC is the third leading cause of cancer-related death worldwide, with 783,000 deaths reported in 2018 [1, 2]. Notably, the survival rate of patients with GC in developed countries is poor [1]; however, in Korea, the 5-year survival rate of patients with GC, who typically obtain an early and comprehensive diagnosis, is nearly 90% [3, 4]. The high mortality rate of GC in other developed countries is mainly caused by the high rate of recurrence and metastasis; the high 5-year survival rates for patients with GC without lymph node metastasis after surgery drop sharply for patients with metastatic nodes [5]. Although there has been recent improvement in the survival rate with postoperative and perioperative chemoradiotherapy, the regional and distant recurrences remain to be resolved due to lack of effective treatment [6].

    Tumor metastasis is one of the major causes of GC recurrence [7]. Epithelial-mesenchymal transition (EMT) is an essential process that plays a pivotal role in GC metastasis, as well as normal development [8, 9]. EMT in tumor progression allows a polarized epithelial cell to acquire a mesenchymal cell phenotype that facilitates the intravasation of tumor cells into blood or lymph vessels and the subsequent distant metastasis [10]. Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) is one of the most essential intracellular signaling pathways involved in the regulation of EMT [11]. The hyper-activation of PI3K/AKT/mTOR signaling has been implicated in metastasis of multiple types of cancers including GC [12, 13]. Inhibition of PI3K/AKT/mTOR signaling, mediated by various inhibitors, effectively reduces tumor metastasis [14, 15], suggesting that PI3K/AKT/mTOR signaling can serve as a potential therapeutic target for GC.

    Vitexin, a bioactive flavonoid component derived from many traditional Chinese herbal medicines, has recently drawn growing attention due to its wide range of biological and pharmacological activities, including anti-cancer effects [16]. Vitexin inhibits glioblastoma and non-small cell lung cancer progression via inactivation of the PI3K/AKT/mTOR signaling pathway [17, 18]. However, the effect of vitexin and its detailed mechanisms in GC remain unclear. High-mobility group box 1 protein (HMGB1) is regarded as one of the essential activators of the PI3K/AKT/mTOR pathway and is known to facilitate cancer cell metastasis [19]. Hence, it is important to investigate whether vitexin affects GC metastasis by regulating the HMGB1-mediated PI3K/AKT pathway.

    A hypoxic microenvironment is a common feature of solid tumors due to the compact and avascular histological structure of tumor tissues [20]. Studies have shown that a hypoxic microenvironment may facilitate tumor cell metastasis [21, 22]. Hypoxia-induced expression of hypoxia-inducible factor-1α (HIF-1α) is a key promoter of EMT in various cancer cells [23, 24]. Interestingly, HIF-1α has been well documented as a downstream target of PI3K/AKT signaling pathway [25]. He et al. [26] have reported that HMGB1 facilitates angiogenesis and metastasis of breast cancer cells by modulating HIF-1α expression through the PI3K/AKT/mTOR pathway. Additionally, HMGB1 exerts a cardio-protective effect by enhancing HIF-1α expression in ischemic hearts by modulation of the PI3K/AKT pathway [27]. Furthermore, vitexin has been shown to act as an antagonist of HIF-1α to attenuate hypoxia-ischemia neonatal brain injury [28].

    These findings led us to investigate whether vitexin may inactivate PI3K/AKT/mTOR/HIF-1α pathway via HMGB1, which may delay GC progression. We investigated the suppressive effect of vitexin on GC cell proliferation, migration, invasion, and EMT in vitro and xenograft tumor growth and liver metastasis in vivo, and also clarified the underlying molecular mechanisms.

    MATERIALS AND METHODS

    Cell lines and reagents

    KRAS wild and mutant GC cell lines were used in this study. Human gastric adenocarcinoma cell line AGS with KRAS mutation (CRL-1739) and the normal human gastric epithelial cell line GES-1 were obtained from the American Type Culture Collection (Manassas, VA, USA). GC cell line SGC-7901 with wild type KRAS was obtained from the National Infrastructure of Cell Line Resource (Beijing, China). All cells were cultured in RPMI 1640 supplemented with 10% fetal bovine solution (GIBCO, Carlsbad, CA, USA) at 37°C with 5% CO2 supplement.

    Vitexin (Selleck Chemicals, Houston, TX, USA) was dissolved in dimethyl sulfoxide (Sigma, St. Louis, MO, USA) to prepare the stock solution (100 mM). Before experiments, the stock solution was diluted with phosphate-buffered saline (PBS) to the desired concentration.

    Cell transfection

    HMGB1 overexpression plasmid and pcDNA 3.1 vector were obtained from GenePharma (Shanghai, China). GC cells were transfected with the 2 plasmids using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. GC cells were subjected to subsequent experiments 48 hours after the transfection.

    Scratch wound healing assay

    For the scratch wound healing assay, treated GC cells were first cultured to confluence. Next, the cell monolayer was scratched with a pipet tip to create a “scratch wound.” The scratched cells and debris were removed by washing with pre-warmed PBS. Cells were maintained in a serum-free medium. Images were taken at 0 and 24 hours after scratch with a light microscope. The results were quantitatively analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

    Transwell invasion assay

    The transwell chamber was pre-coated with 70 μL Matrigel Matrix (356234; Corning Inc., Corning, NY, USA) at 37°C for 1 hour. Cells at a density of 5 × 104 suspended in 200 μL of serum-free media were seeded in the upper chamber. Five hundred microliters of complete medium with 10% fetal bovine solution was added to the lower chamber. After incubation for 24 hours at 37°C, the cells in the lower chamber were fixed and stained with 0.2% crystal violet, and the number of invasive cells were counted.

    MTT assay

    GC cells were seeded at a density of 2,000 cells per well into 96-well plates. At the indicated time points, MTT reagent (20 µL, 5 mg/mL; Abcam, Cambridge, UK) was added to each well. After incubation for 4 hours at 37°C, 150 μL of dimethyl sulfoxide was added to dissolve the formazan crystals. Absorbance was assessed at 492 nm using a microplate reader. The assays were performed in triplicate.

    Western blotting

    Total protein was extracted from tumor tissues or cells using RIPA Lysis Buffer (Beyotime, Shanghai, China). For detection of HMGB1, the cytosolic and nuclear extracts were obtained using the NE-PER nuclear and cytoplasmic extraction kit (Piece, Rockford, IL, USA) following the manufacturer’s instructions. Equal amount of protein (10 μg) was loaded onto 10% SDS-PAGE and then transferred to nitrocellulose membranes (0.2 µm; 1620112; Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% non-fat milk. Next, the membranes were probed with primary antibodies against E-cadherin (1:2,000; R&D, Minneapolis, MN, USA), HIF-1α (1:1,000, Abcam), HMGB1 (1:1000, CST, Trask Lane Danvers, MA, USA), MMP2 (1:1,000; CST), MMP9 (1:1,000; CST), mTOR (1:1,000; CST), N-cadherin (1:1,000; Abcam), PI3K (1:1,000; Abcam), p-PI3K (1:1,000; Abcam), p-AKT (1:1,000; CST), AKT (1:2,000, CST), p-mTOR (1:1,000; CST), and β-actin (1:1,000; CST), followed by incubation with the secondary antibody (1:10,000; LI-COR Biosciences, Lincoln, NE, USA). The protein band signals were captured and analyzed using an Odyssey CLx Imaging System (LI-COR Biosciences). β-actin was used as an endogenous reference.

    In vivo experiments

    All experimental protocols were approved and conducted under the supervision of the Institutional Animal Care and Use Committee of Xi’an Jiaotong University. Athymic nude mice (6-week-old females) were provided by the Laboratory Animal Center of the Institute of Genetics (Beijing, China). The mice were maintained in a specific pathogen free facility at 26°C with 12/12 hours light/dark cycles. The mice were assigned into the following groups (n=5 per group): control (0.1% DMSO), low dose (intraperitoneal, 1 mg/kg vitexin, once a day for 4 weeks), high dose (intraperitoneal, 2 mg/kg vitexin, once a day for 4 weeks), high dose + pcDNA 3.1 (AGS cells stably transfected with pcDNA 3.1), and high dose + HMGB1 (AGS cells stably transfected with HMGB1 overexpression plasmid).

    To generate xenograft tumors from GC cells, 1×106 AGS cells were suspended in 50 μL PBS and injected into one side of the posterior flank of nude mice subcutaneously. Tumor volume was determined weekly, by measuring the length, width, and thickness of tumors with a caliper. Four weeks post-injection, the xenografted tumors were collected and weighed. To evaluate GC metastasis in vivo, 1×107 AGS cells suspended in 200 μL PBS suspension were injected into the tail vein of nude mice [29, 30]. Four weeks later, liver tissues were isolated and fixed in 4% paraformaldehyde for routine hematoxylin and eosin staining. The liver tissues were imaged and macroscopic nodules in the tissues were quantified.

    Immunohistochemistry

    The xenograft tumor tissues were fixed in 10% formalin for 24 hours and embedded in paraffin. Tissue blocks were cut into 5 μm-thick sections using Leica RM2155 Microtome. The sections were dewaxed in xylene and rehydrated in an ethanol gradient. For the antigen retrieval step, sections were immersed in 0.01% sodium citrate buffer and heated till the boiling point using a microwave oven. Sections were blocked with 5% normal goat serum for 1 hour and incubated with primary antibodies against E-cadherin (1:200; R&D), HMGB1 (1:100; CST), Ki67 (1:200; CST), and N-cadherin (1:100; Abcam) overnight at 4°C. Subsequently, sections were developed using the biotin-streptavidin HRP detection system (ZSGB-BIO, Beijing, China) followed by 3,3′-diaminobenzidine staining. The sections were imaged using a Zeiss imaging system (Zeiss, Oberkochen, Germany).

    Statistical analyses

    All data from at least 3 biological replicates were expressed as mean±standard deviation. Data were analyzed using Student’s t-test or analysis of variance using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Statistical significance was set at P<0.05.

    RESULTS

    Vitexin suppresses GC cell viability in a dose-dependent manner

    To determine the effect of vitexin on GC cell viability, GC cells were treated with different concentrations of vitexin (0, 10, 20, 40, 80, and 160 μM) for 24 hours and 48 hours. The MTT assay demonstrated that exposure to vitexin for 24 hours and 48 hours led to a significant dose- and time-dependent decline in GC cell viability (Fig. 1A). However, treatment with vitexin for 48 hours did not affect the viability of the normal human gastric epithelial cell line GES-1 (Fig. 1B). The chemical structure of vitexin is shown in Fig. 1C.

    Fig. 1
    Vitexin suppresses gastric cancer cell viability in a dose-dependent manner. (A) The viability of SGC-7901 and AGS cells treated with different concentrations (0, 10, 20, 40, 80, 160 μM) of vitexin for 24 hours and 48 hours was determined using an MTT assay. (B) The viability of GES-1 cells treated with different dosages of vitexin (0, 10, 20, 40 μM) for 48 hours was determined using an MTT assay. (C) Chemical structure of vitexin.
    **P<0.01; ***P<0.001.

    Vitexin suppresses the migration, invasion, and EMT of GC cells

    As migration and invasion are 2 distinct features of cancerous cells, we investigated whether vitexin could affect the migration and invasion of GC cells. Scratch wound healing assays indicated that vitexin treatment remarkably reduced the migration rate of SGC-7901 and AGS cells in a dose dependent manner (Fig. 2A). Similarly, transwell invasion assays showed that an increase in vitexin concentration was coupled with a decline in the number of invasive cells (Fig. 2B). The protein levels of EMT markers closely related with the migratory and invasive behaviors were also assessed. In both GC cell lines, treatment with vitexin significantly increased E-cadherin expression, but decreased N-cadherin, MMP9 and MMP2 levels in a dose-dependent manner (Fig. 2C). These observations indicated that the migration, invasion, and EMT of GC cells was attenuated by vitexin treatment.

    Fig. 2
    Vitexin inhibits the migration, invasion, and epithelialmesenchymal transition of GC cells. SGC-7901 and AGS cells were treated with 10 μM and 40 μM vitexin for 48 hours. (A) Effect of vitexin on GC cell migration was measured using a scratch wound healing assay. (B) Effect of vitexin on GC cell invasion was measured using a transwell invasion assay. (C) Protein levels of E-cadherin, N-cadherin, MMP9, and MMP2 in response to vitexin treatment were detected through western blotting.
    *P<0.05; **P<0.01; ***P<0.001.

    Vitexin inhibits the activation of PI3K/AKT/mTOR/HIF-1α pathway

    In order to explore the underlying mechanism of the inhibitory effect of vitexin on EMT, we investigated the PI3K/AKT/mTOR/HIF-1α pathway. After exposure to vitexin for 48 hours, no change was detected in the total protein levels of PI3K, AKT, and mTOR (Fig. 3A). However, treatment with vitexin resulted in a decline in the phosphorylation of PI3K, AKT, and mTOR in a dose-dependent fashion (Fig. 3A). HIF-1α, a downstream effector of the PI3K/AKT/mTOR pathway, was also downregulated by vitexin treatment (Fig. 3A).

    Fig. 3
    Vitexin inhibits the activation of PI3K/AKT/mTOR/HIF-1α pathway in GC cells. (A) SGC-7901 and AGS cells were incubated with 10 μM and 40 μM vitexin for 48 hours. The protein levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, and HIF-1α were detected through western blotting. B. SGC-7901 and AGS cells were treated with 40 μM vitexin for 0, 3, 6, 12, 24, and 48 hours. Protein levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR and HIF-1α were assessed through western blotting.
    PI3K = phosphatidylinositol-3-kinase; mTOR = mammalian target of rapamycin; HIF-1α = hypoxia-inducible factor-1α; GC = gastric cancer.

    *P<0.05; **P<0.01; ***P<0.001.

    Next, the kinetics of vitexin-mediated inhibition of PI3K/AKT/mTOR/HIF-1α pathway was assessed. Decreased levels of p-PI3K, p-AKT, p-mTOR, and HIF-1α were first observed at 6 hours after treatment with 40 μM vitexin (Fig. 3B), and continued to decline in a time-dependent fashion up to 48 hours. These results suggested that the PI3K/AKT/mTOR/HIF-1α pathway may be involved in vitexin-mediated inhibition in EMT of GC cells.

    Hyper-activation of PI3K/AKT/mTOR/HIF-1α pathway abolished the anti-cancer effects of vitexin

    To further assess the role of PI3K/AKT/mTOR/HIF-1α pathway in the inhibitory effect of vitexin on the malignant phenotypes of GC cells, we included an AKT agonist, SC79, along with the vitexin treatment. As assessed by MTT assay, the decreased cell viability in vitexin-treated SGC-7901 and AGS cells was alleviated with the addition of SC79 (Fig. 4A). Treatment with SC79 significantly increased the migration rate of SGC-7901 and AGS cells exposed to vitexin (Fig. 4B). Similarly, SC79 treatment dramatically increased the number of invasive GC cells in the presence of vitexin (Fig. 4C). Western blotting analysis confirmed that the decrease in p-AKT and p-mTOR levels observed in vitexin-treated GC cells was recovered after addition of SC79. Furthermore, the vitexin-mediated downregulation of HIF-1α, N-cadherin, MMP9, and MMP2 and upregulation of E-cadherin were partly reversed by SC79 treatment (Fig. 4D). Thus, forceful activation of PI3K/AKT/mTOR/HIF-1α pathway reversed the anti-tumor effects of vitexin on GC cells.

    Fig. 4
    Hyper-activation of the PI3K/AKT/mTOR/HIF-1α pathway reversed the inhibitory effects of vitexin on GC cell viability, migration, invasion, and EMT. SGC-7901 and AGS cells were treated with 10 μM and 40 μM vitexin, or a combination of SC79 (AKT agonist) for 48 hours. (A) Viability of GC cells was determined using an MTT assay. (B) Migration of GC cells was determined using the scratch wound healing assay. (C) Invasion of GC cells was measured using a transwell invasion assay. (D) Protein levels of p-AKT, AKT, mTOR, p-mTOR, E-cadherin, N-cadherin, MMP9, and MMP2 in GC cells with various treatments were detected through western blotting.
    PI3K = phosphatidylinositol-3-kinase; mTOR = mammalian target of rapamycin; HIF-1α = hypoxia-inducible factor-1α; GC = gastric cancer; EMT = epithelial-mesenchymal transition.

    *P<0.05; **P<0.01; ***P<0.001.

    HMGB1 participates in the inhibition of vitexin in GC cell growth, migration, invasion, and EMT

    To determine whether HMGB1 was responsible for the inhibitory effect of vitexin on GC cell malignant properties, we examined the protein expression of HMGB1 in GC cells in response to various concentrations of vitexin. As determined by western blotting, in both SGC-7901 and AGS cells, vitexin strikingly decreased HMGB1 protein levels in a dose-dependent manner (Fig. 5A).

    Fig. 5
    HMGB1 inhibition is involved in the anti-cancerous effect of vitexin. (A) HMGB1 protein level in GC cells exposed to 10 μM and 40 μM vitexin for 48 hours was detected by western blotting. (B) The mRNA expression of HMGB1 in GC cells after transfection with pcDNA3.1 or HMGB1 overexpression plasmid for 48 hours was examined through qPCR. SGC-7901 and AGS cells were treated with 40 μM vitexin, or a combination with pcDNA3.1/HMGB1 overexpression plasmid transfection for 48 hours. (C) Viability of GC cells was determined using an MTT assay. (D) Migration was determined using a scratch wound healing assay. (E) The invasive ability of GC cells was measured using a transwell invasion assay. (F) Protein levels of E-cadherin, N-cadherin, MMP9, and MMP2 were measured through western blotting.
    GC = gastric cancer; HMGB1 = high-mobility group box 1 protein; qPCR = quantitative polymerase chain reaction.

    *P<0.05; **P<0.01; ***P<0.001.

    GC cells were transfected with an overexpression plasmid of HMGB1, resulting in a significant increase in HMGB1 mRNA expression in the cells (Fig. 5B). Vitexin-mediated suppression in GC cell viability (Fig. 5C), migration (Fig. 5D), and invasion (Fig. 5E) were partly rescued by the overexpression of HMGB1. Likewise, the increased expression of E-cadherin, but decreased expression of N-cadherin, MMP9, and MMP2 in vitexin-treated GC cells were counteracted by the overexpression of HMGB1 (Fig. 5F). These results suggested that vitexin inhibited GC cell growth, migration, invasion, and EMT by downregulating HMGB1.

    Vitexin restrained the activation of PI3K/AKT/mTOR/HIF-1α pathway through HMGB1

    Next, we sought to investigate the involvement of HMGB1 in vitexin-mediated inactivation of PI3K/AKT/mTOR/HIF-1α pathway. Western blotting of nuclear and cytosolic fractions of SGC-7901 and AGS cells indicated that treatment with vitexin promoted the translocation of HMGB1 from the cytoplasm to the nucleus. The cytoplasmic level of HMGB1 was increased after overexpression of HMGB1 (Fig. 6A). Furthermore, vitexin-mediated downregulation of p-PI3K, p-AKT, p-mTOR, and HIF-1α in GC cells was recovered by overexpression of HMGB1; however, there was no significant change in total PI3K, AKT, and mTOR levels among the different groups (Fig. 6B). These results indicated that vitexin repressed the activation of PI3K/AKT/mTOR/HIF-1α pathway in GC cells by inhibiting cytosolic HMGB1 expression.

    Fig. 6
    Vitexin restrains the activation of PI3K/AKT/mTOR/HIF-1α pathway through HMGB1. SGC-7901 and AGS cells were incubated with 40 μM vitexin, or a combination with pcDNA3.1/HMGB1 overexpression plasmid transfection for 48 hours. The protein levels of cytoplasmic/nuclear HMGB1 (A), PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR and HIF-1α (B) in SGC-7901 and AGS cells were detected through western blotting.
    HMGB1 = high-mobility group box 1 protein; PI3K = phosphatidylinositol-3-kinase; mTOR = mammalian target of rapamycin; HIF-1α = hypoxia-inducible factor-1α.

    *P<0.05; **P<0.01; ***P<0.001.

    Vitexin inhibits xenograft tumor growth and liver metastasis in vivo

    To determine the effect of vitexin on in vivo solid tumor growth, we established a xenograft mouse model. The tumor volume and weight were inhibited in a dose-dependent manner by the administration of 1 mg/kg and 2 mg/kg vitexin (Fig. 7A-C). In addition, the protein levels of HMGB1, p-PI3K, p-AKT, p-mTOR, and HIF-1α in the xenograft tumor tissues were decreased by vitexin in a dose-dependent manner (Fig. 7D). Immunohistological staining further showed that the expression levels of Ki67, HMGB1 and N-cadherin was decreased, while the expression level of E-cadherin was increased in the vitexin treatment groups (Fig. 7E).

    Fig. 7
    Vitexin inhibits tumor growth and liver metastasis in vivo. To generate tumor xenografts, 1×106 AGS cells were subcutaneously injected into one side of the posterior flank of nude mice. The mice in low-dose and high-dose groups were intraperitoneally injected with 1 mg/kg and 2 mg/kg vitexin, respectively. The tumor tissues were collected 28 days after the injection. (A) Growth curves of the xenografted tumors were plotted. (B) Weight of xenografted tumors. (C) Representative photographs of GC xenografted tumors. (D) Protein levels of HMGB1, PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR and HIF-1α in tumor tissues were detected through western blotting. (E) The expression of Ki67, HMGB1, E-Cadherin, and N-Cadherin in tumor tissues was examined through immunohistochemistry staining. (F) To evaluate GC metastasis in vivo, 1×107 AGS cells were injected into the tail vein of nude mice. Four weeks later, the liver tissues were imaged. Macroscopic nodules in the liver tissues were quantified. (G) Liver metastasis was determined by hematoxylin and eosin staining.
    GC = gastric cancer; HMGB1 = high-mobility group box 1 protein; PI3K = phosphatidylinositol-3-kinase; mTOR = mammalian target of rapamycin; HIF-1α = hypoxia-inducible factor-1α.

    *P<0.05; **P<0.01; ***P<0.001.

    The metastatic potential of GC cells was also evaluated using a nude mouse model in vivo. As shown in Fig. 7F, the number of metastatic nodules in liver tissues was reduced by treatment with 1 mg/kg and 2 mg/kg vitexin. The histological examination of liver tissues indicated that the area of metastatic tumor cells significantly decreased in mice treated with vitexin (Fig. 7G). Thus, vitexin exerted an inhibitory effect on in vivo GC tumor growth and liver metastasis.

    Overexpression of HMGB1 attenuated the anti-cancer activity of vitexin in vivo

    To verify the involvement of HMGB1 in the anti-cancer activity of vitexin in vivo, AGS cells stably transfected with pcDNA 3.1 or HMGB1 plasmid were subcutaneously injected into the nude mice. Overexpression of HMGB1 reversed the 2 mg/kg vitexin-induced decrease in tumor size and weight 28 days after the injection (Fig. 8A-C). Western blotting results indicated that the reduced levels of HMGB1, p-PI3K, p-AKT, p-mTOR and HIF-1α in 2 mg/kg vitexin-treated tumor tissues were partly reversed by HMGB1 overexpression (Fig. 8D). Additionally, treatment with 2 mg/kg vitexin resulted in a decrease in Ki67, HMGB1, and N-cadherin expression and an increase in E-cadherin expression; these changes could be prevented by HMGB1 overexpression (Fig. 8E). Overexpression of HMGB1 also counteracted the inhibitory effect of 2 mg/kg vitexin on liver metastasis (Fig. 8F and G). These results suggested that vitexin suppressed tumor growth and liver metastasis in vivo by inhibiting HMGB1 expression.

    Fig. 8
    Overexpression of HMGB1 counteracts the anti-cancer activity of vitexin in vivo. AGS cells (1×106) stably transfected with pcDNA3.1/HMGB1 overexpression plasmid were subcutaneously injected into nude mice. The mice in vitexin groups were intraperitoneally injected with 2 mg/kg vitexin. The tumor tissues were collected 28 days after the injection. (A) Growth curves of xenografted tumors were plotted. (B) Weight of xenografted tumors. (C) Representative photographs of GC xenografted tumors. (D) Western blotting was used to assess the protein levels of HMGB1, PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, and HIF-1α in tumor tissues. (E) Immunohistochemistry staining was conducted to determine the expression of Ki67, HMGB1, E-cadherin, and N-cadherin in tumor tissues. (F) AGS cells (1×106) stably transfected with pcDNA3.1/HMGB1 overexpression plasmid were injected into the tail vein of nude mice to evaluate GC metastasis in vivo. Four weeks later, the liver tissues were imaged. Macroscopic nodules in the liver tissues were quantified. (G) Liver metastasis was observed through hematoxylin and eosin staining.
    GC = gastric cancer; HMGB1 = high-mobility group box 1 protein; PI3K = phosphatidylinositol-3-kinase; mTOR = mammalian target of rapamycin; HIF-1α = hypoxia-inducible factor-1α.

    *P<0.05; **P<0.01; ***P<0.001.

    DISCUSSION

    GC is one of the most life-threatening forms of cancers and is a severe threat to public health [31]. The high recurrence rate observed in patients with GC after surgical treatment is the leading cause of short survival in these patients [32]. Metastasis is a major contributor in GC recurrence and EMT plays an important role during metastasis [7]. Vitexin is considered a therapeutic candidate for GC because of its inhibition of EMT in multiple types of cancers [17, 33] and also suppression of HIF-1α expression, which plays an important role in EMT-mediated metastasis in cancer [28]. In the present study, vitexin suppressed the viability, migration, invasion, and EMT of GC cells. Mechanistically, vitexin restrained HMGB1 expression and the activation of the downstream PI3K/AKT/mTOR/HIF-1α signaling pathway. More importantly, vitexin significantly inhibited tumor growth and liver metastasis in vivo in a dose-dependent manner. Thus, this study may provide a theoretical foundation for the feasibility of vitexin as a potential therapeutic agent in the treatment of GC.

    The inhibitory effect of vitexin in cancer progression has been documented in various types of cancers including glioblastoma, lung cancer, and leukemia [17, 18, 34]. Vitexin exerts anti-tumor effects by the promotion of apoptosis and autophagy and the suppression of proliferation and migration through several signaling pathways, including increased caspase-3, caspase-9, and Hsp90 expression, inhibition of HIF-1α and Bcl-2 expression, as well as binding to transferrin receptor [16]. Additionally, mitogen-activated protein kinase, protein kinase C, PI3K/AKT, and β-catenin pathways are the molecular targets for the anti-cancer activity of vitexin [16]. However, the effect of vitexin on GC has rarely been studied.

    In the present study, we used 2 different GC cell lines (SGC-7901 and AGS) and one normal non-transformed gastric cell line (GES-1) to evaluate the anti-tumor potential of vitexin. Vitexin exhibited a significant inhibitory effect on the viability of the 2 GC cell lines, but did not affect the growth of GES-1 cells. The 2 GC cell lines used in the study differ in terms of their genetic background and origination. SGC-7901 cells are derived from the metastatic lymph node of an East-Asian female patient [35]. In contrast, AGS cells were obtained from the resected stomach adenocarcinoma of an untreated Caucasian female [36]. Additionally, AGS cells carry a KRAS mutation, while this mutation is absent in SGC-7901 cells. Its inhibitory effect on these 2 different GC cell lines indicated that vitexin may be effective in patients with GC with different stages of development and diverse genetic background and genomic characteristics.

    This is the first study to report the effect of vitexin on GC cell migration, invasion, and EMT. In this study, the concentrations of vitexin were chosen based on previous studies focusing on other types of cells [17, 28]. We demonstrated that vitexin inhibited the migration, invasion, and EMT of both SGC-7901 and AGS cells in a dose-dependent fashion. In several other studies, the anti-cancer effects of vitexin were mainly attributed to its effect on cell proliferation and apoptosis [18, 33, 37, 38]. The findings of this study help broaden our understanding of the anti-tumor mechanism of vitexin. Based on the results from GES-1 cells, vitexin did not exhibit obvious toxicity to normal tissues, further establishing its potential as an ideal therapeutic drug.

    The rapid growth of tumor cells may result in hypoxia even with an abundant supply of oxygen, and the insufficient vascularization in solid tumors further amplifies the hypoxia response within tumor cells [39]. In response to hypoxia, cancer cells exhibit modified expression of many genes regulated by HIF-1α, which is the major component of hypoxia signaling pathways [40]. Most of the HIF-1α-mediated changes are observed in the expression of genes associated with proliferation, EMT, and metastasis [41], which allows cancer cells to survive in the hypoxic environment. Consistent with these studies, our results indicated that vitexin inhibited HIF-1α expression in GC cells in a dose-dependent manner. The activation of tyrosine kinases, such as IGFR and EGFR, stimulates the activation of PI3K/AKT/mTOR pathway, which may result in increased HIF-1α expression [42]. The PI3K/AKT/mTOR pathway is essential for the regulation of cell survival and apoptosis [43], and abnormal activation of this signaling pathway is commonly observed in cancer, including GC [44]. Hence, the present study investigated the effect of vitexin on PI3K/AKT/mTOR signaling pathway in GC. Our data suggested that vitexin treatment significantly restrained the activation of the PI3K/AKT/mTOR signaling pathway. Hyper-activation of the PI3K/AKT/mTOR pathway reversed the anti-cancerous effects of vitexin on GC cells through suppression of HIF-1α expression. These findings revealed that vitexin delayed the progression of GC cells by inactivating the PI3K/AKT/mTOR/HIF-1α pathway.

    The role of vitexin in HMGB1 regulation in GC cells is another fundamental discovery of this study. HMGB1, a highly conserved nuclear protein, acts as a pivotal regulator of apoptosis and cell viability [45]. A previous study has shown that HMGB1 expression is strongly correlated with higher stage of GC [46], and inhibition of HMGB1 expression inhibits the malignant development of GC [47]. Previous studies have reported that HMGB1 is a contributor to EMT. Upregulation of HMGB1 promotes EMT and angiogenesis during liver cancer progression [48]. Ma et al. [49] have previously revealed that HMGB1 aggravates the development of silicosis by facilitating EMT. Hence, we also evaluated the involvement of HMGB1 in the inhibition of vitexin during EMT of GC cells. This is the first study to report that vitexin downregulates HMGB1 expression in GC cells, and the overexpression of HMGB1 could counteract the inhibitory effects of vitexin on growth, migration, invasion, and EMT of GC cells. More importantly, vitexin promoted the nuclear translocation of HMGB1, thereby suppressing the activation of PI3K/AKT/mTOR pathway. Thus, cytoplasmic HMGB1 was responsible for the activation of the PI3K/AKT/mTOR pathway. However, the mechanism by which vitexin may downregulate HMGB1 in GC cells remains unclear. There are several possible underlying mechanisms for this effect. Firstly, vitexin may directly bind to the HMGB1 protein and promote its degradation. Secondly, vitexin may inhibit HMGB1 expression by upregulating a series of miRNAs that target HMGB1. These speculations will be tested in our future studies.

    This study investigated the connection between HMGB1 and PI3K/AKT/mTOR/HIF-1α pathway in GC cells. In a previous study, HMGB1 was demonstrated to be a modulator of HIF-1α via PI3K/AKT/mTOR pathway during breast cancer progression [26]. Recent studies have also indicated that HMGB1 modulates the PI3K/AKT/mTOR signaling pathway in myocardial ischemia/reperfusion injury and LPS-induced acute lung injury [50, 51]. Yao et al. [27] have documented that HMGB1 upregulates HIF-1α via the PI3K/AKT pathway, attenuating acute myocardial ischemia/reperfusion injury in rats. In the present study, overexpression of HMGB1 promoted the expression of p-PI3K, p-AKT, and p-mTOR in vitexin-treated GC cells, suggesting the activation of the PI3K/AKT/mTOR pathway. Vitexin inhibited cytoplasmic translocation of HMGB1 and subsequent activation of PI3K/AKT/mTOR pathway in GC cells. Therefore, vitexin repressed the development of GC cells by inhibiting HMGB1-mediated activation of PI3K/AKT/mTOR/HIF-1α pathway.

    In summary, our findings demonstrated that vitexin suppressed the in vitro and in vivo growth and metastasis of GC cells. Additionally, vitexin effectively suppressed the activation of PI3K/AKT/mTOR pathway by inhibiting HMGB1, which led to a decrease in HIF-1α expression. Thus, based on the findings of this study, vitexin may be considered as a novel effective agent for the treatment of GC.

    Notes

    Author Contributions:

    • Conceptualization: Z.P.

    • Data curation: Z.H.Z.

    • Formal analysis: W.T., W.J.

    • Writing - original draft: L.C.W.

    • Writing - review & editing: S.X.J.

    Conflict of Interest:No potential conflict of interest relevant to this article was reported.

    ACKNOWLEDGMENTS

    We would like to give our sincere gratitude to the reviewers for their constructive comments.

    References

      1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69–90.
      1. Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, Rosso S, Coebergh JW, Comber H, et al. Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer 2013;49:1374–1403.
      1. Hong S, Won YJ, Lee JJ, Jung KW, Kong HJ, Im JS, et al. Cancer statistics in Korea: incidence, mortality, survival, and prevalence in 2018. Cancer Res Treat 2021;53:301–315.
      1. Guideline Committee of the Korean Gastric Cancer Association (KGCA), Development Working Group & Review Panel. Korean practice guideline for gastric cancer 2018: an evidence-based, multi-disciplinary approach. J Gastric Cancer 2019;19:1–48.
      1. Wang J, Wang L, Li S, Bai F, Xie H, Shan H, et al. Risk factors of lymph node metastasis and its prognostic significance in early gastric cancer: a multicenter study. Front Oncol 2021;11:649035
      1. Information Committee of the Korean Gastric Cancer Association. Korean Gastric Cancer Association-led nationwide survey on surgically treated gastric cancers in 2019. J Gastric Cancer 2021;21:221–235.
      1. Okugawa Y, Mohri Y, Tanaka K, Kawamura M, Saigusa S, Toiyama Y, et al. Metastasis-associated protein is a predictive biomarker for metastasis and recurrence in gastric cancer. Oncol Rep 2016;36:1893–1900.
      1. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009;139:871–890.
      1. Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest 2009;119:1420–1428.
      1. Pastushenko I, Blanpain C. EMT transition states during tumor progression and metastasis. Trends Cell Biol 2019;29:212–226.
      1. Chiu HC, Li CJ, Yiang GT, Tsai AP, Wu MY. Epithelial to mesenchymal transition and cell biology of molecular regulation in endometrial carcinogenesis. J Clin Med 2019;8:439.
      1. Zhang Y, Qu X, Hu X, Yang X, Hou K, Teng Y, et al. Reversal of P-glycoprotein-mediated multi-drug resistance by the E3 ubiquitin ligase Cbl-b in human gastric adenocarcinoma cells. J Pathol 2009;218:248–255.
      1. Tan X, Chen S, Wu J, Lin J, Pan C, Ying X, et al. PI3K/AKT-mediated upregulation of WDR5 promotes colorectal cancer metastasis by directly targeting ZNF407. Cell Death Dis 2017;8:e2686
      1. Boffa DJ, Luan F, Thomas D, Yang H, Sharma VK, Lagman M, et al. Rapamycin inhibits the growth and metastatic progression of non-small cell lung cancer. Clin Cancer Res 2004;10:293–300.
      1. Hill EE, Kim JK, Jung Y, Neeley CK, Pienta KJ, Taichman RS, et al. Integrin alpha V beta 3 targeted dendrimer-rapamycin conjugate reduces fibroblast-mediated prostate tumor progression and metastasis. J Cell Biochem 2018;119:8074–8083.
      1. Ganesan K, Xu B. Molecular targets of vitexin and isovitexin in cancer therapy: a critical review. Ann N Y Acad Sci 2017;1401:102–113.
      1. Zhang G, Li D, Chen H, Zhang J, Jin X. Vitexin induces G2/M-phase arrest and apoptosis via Akt/mTOR signaling pathway in human glioblastoma cells. Mol Med Rep 2018;17:4599–4604.
      1. Liu X, Jiang Q, Liu H, Luo S. Vitexin induces apoptosis through mitochondrial pathway and PI3K/Akt/mTOR signaling in human non-small cell lung cancer A549 cells. Biol Res 2019;52:7.
      1. Pan B, Chen D, Huang J, Wang R, Feng B, Song H, et al. HMGB1-mediated autophagy promotes docetaxel resistance in human lung adenocarcinoma. Mol Cancer 2014;13:165.
      1. Al-Ostoot FH, Sherapura A, V V, Basappa G, H K V, B T P, et al. Targeting HIF-1α by newly synthesized Indolephenoxyacetamide (IPA) analogs to induce anti-angiogenesis-mediated solid tumor suppression. Pharmacol Rep 2021;73:1328–1343.
      1. Higgins DF, Kimura K, Bernhardt WM, Shrimanker N, Akai Y, Hohenstein B, et al. Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition. J Clin Invest 2007;117:3810–3820.
      1. Zuo J, Wen J, Lei M, Wen M, Li S, Lv X, et al. Hypoxia promotes the invasion and metastasis of laryngeal cancer cells via EMT. Med Oncol 2016;33:15.
      1. Srivastava C, Irshad K, Dikshit B, Chattopadhyay P, Sarkar C, Gupta DK, et al. FAT1 modulates EMT and stemness genes expression in hypoxic glioblastoma. Int J Cancer 2018;142:805–812.
      1. Park SJ, Kim JG, Kim ND, Yang K, Shim JW, Heo K. Estradiol, TGF-β1 and hypoxia promote breast cancer stemness and EMT-mediated breast cancer migration. Oncol Lett 2016;11:1895–1902.
      1. Liu Z, Sun T, Piao C, Zhang Z, Kong C. METTL13 inhibits progression of clear cell renal cell carcinoma with repression on PI3K/AKT/mTOR/HIF-1α pathway and c-Myc expression. J Transl Med 2021;19:209.
      1. He H, Wang X, Chen J, Sun L, Sun H, Xie K. High-mobility group box 1 (HMGB1) promotes angiogenesis and tumor migration by regulating hypoxia-inducible factor 1 (HIF-1α) expression via the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in breast cancer cells. Med Sci Monit 2019;25:2352–2360.
      1. Yao HC, Zhou M, Zhou YH, Wang LH, Zhang DY, Han QF, et al. Intravenous high mobility group box 1 upregulates the expression of HIF-1α in the myocardium via a protein kinase B-dependent pathway in rats following acute myocardial ischemia. Mol Med Rep 2016;13:1211–1219.
      1. Min JW, Hu JJ, He M, Sanchez RM, Huang WX, Liu YQ, et al. Vitexin reduces hypoxia-ischemia neonatal brain injury by the inhibition of HIF-1alpha in a rat pup model. Neuropharmacology 2015;99:38–50.
      1. Liu J, Wang G, Zhao J, Liu X, Zhang K, Gong G, et al. LncRNA H19 promoted the epithelial to mesenchymal transition and metastasis in gastric cancer via activating Wnt/β-catenin signaling. Dig Dis. 2021
        Forthcoming.
      1. Zhou H, Hu X, Li N, Li G, Sun X, Ge F, et al. Loganetin and 5-fluorouracil synergistically inhibit the carcinogenesis of gastric cancer cells via down-regulation of the Wnt/β-catenin pathway. J Cell Mol Med 2020;24:13715–13726.
      1. Hundahl SA, Phillips JL, Menck HR. The National Cancer Data Base Report on poor survival of U.S. gastric carcinoma patients treated with gastrectomy: Fifth Edition American Joint Committee on Cancer staging, proximal disease, and the “different disease” hypothesis. Cancer 2000;88:921–932.
      1. Macdonald JS, Smalley SR, Benedetti J, Hundahl SA, Estes NC, Stemmermann GN, et al. Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. N Engl J Med 2001;345:725–730.
      1. He JD, Wang Z, Li SP, Xu YJ, Yu Y, Ding YJ, et al. Vitexin suppresses autophagy to induce apoptosis in hepatocellular carcinoma via activation of the JNK signaling pathway. Oncotarget 2016;7:84520–84532.
      1. Lee CY, Chien YS, Chiu TH, Huang WW, Lu CC, Chiang JH, et al. Apoptosis triggered by vitexin in U937 human leukemia cells via a mitochondrial signaling pathway. Oncol Rep 2012;28:1883–1888.
      1. Long-Bao W, Bo-Wen Q, Yan-Xing X. Establishment of human gastric cancer cell line (SGC-7901) intraperitoneally transplantable in nude mice. In: Takahashi T, editor. Recent Advances in Management of Digestive Cancers. Tokyo: Springer; 1993. pp. 416-418.
      1. Barranco SC, Townsend CM Jr, Casartelli C, Macik BG, Burger NL, Boerwinkle WR, et al. Establishment and characterization of an in vitro model system for human adenocarcinoma of the stomach. Cancer Res 1983;43:1703–1709.
      1. Tan Z, Zhang Y, Deng J, Zeng G, Zhang Y. Purified vitexin compound 1 suppresses tumor growth and induces cell apoptosis in a mouse model of human choriocarcinoma. Int J Gynecol Cancer 2012;22:360–366.
      1. Yang SH, Liao PH, Pan YF, Chen SL, Chou SS, Chou MY. The novel p53-dependent metastatic and apoptotic pathway induced by vitexin in human oral cancer OC2 cells. Phytother Res 2013;27:1154–1161.
      1. Li X, Wang M, Li S, Chen Y, Wang M, Wu Z, et al. HIF-1-induced mitochondrial ribosome protein L52: a mechanism for breast cancer cellular adaptation and metastatic initiation in response to hypoxia. Theranostics 2021;11:7337–7359.
      1. Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell 2012;148:399–408.
      1. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003;3:721–732.
      1. O’Donnell JL, Joyce MR, Shannon AM, Harmey J, Geraghty J, Bouchier-Hayes D. Oncological implications of hypoxia inducible factor-1alpha (HIF-1alpha) expression. Cancer Treat Rev 2006;32:407–416.
      1. Lee TJ, Sartor O, Luftig RB, Koochekpour S. Saposin C promotes survival and prevents apoptosis via PI3K/Akt-dependent pathway in prostate cancer cells. Mol Cancer 2004;3:31.
      1. Baghery Saghchy Khorasani A, Pourbagheri-Sigaroodi A, Pirsalehi A, Safaroghli-Azar A, Zali MR, Bashash D. The PI3K/Akt/mTOR signaling pathway in gastric cancer; from oncogenic variations to the possibilities for pharmacologic interventions. Eur J Pharmacol 2021;898:173983
      1. Zhu J, Wang FL, Wang HB, Dong N, Zhu XM, Wu Y, et al. TNF-α mRNA is negatively regulated by microRNA-181a-5p in maturation of dendritic cells induced by high mobility group box-1 protein. Sci Rep 2017;7:12239.
      1. Suren D, Arda Gokay A, Sayiner A. High Mobility Group Box 1 (HMGB1) expression in gastric adenocarcinomas. J BUON 2018;23:422–427.
      1. Tian L, Wang ZY, Hao J, Zhang XY. miR-505 acts as a tumor suppressor in gastric cancer progression through targeting HMGB1. J Cell Biochem 2019;120:8044–8052.
      1. Zhang Y, Ren H, Li J, Xue R, Liu H, Zhu Z, et al. Elevated HMGB1 expression induced by hepatitis B virus X protein promotes epithelial-mesenchymal transition and angiogenesis through STAT3/miR-34a/NF-κB in primary liver cancer. Am J Cancer Res 2021;11:479–494.
      1. Ma J, Xu Y, Li W, Zhou Y, Wang D, Yang M, et al. High-mobility group box 1 promotes epithelial-to-mesenchymal transition in crystalline silica induced pulmonary inflammation and fibrosis. Toxicol Lett 2020;330:134–143.
      1. Li R, Zou X, Huang H, Yu Y, Zhang H, Liu P, et al. HMGB1/PI3K/Akt/mTOR signaling participates in the pathological process of acute lung injury by regulating the maturation and function of dendritic cells. Front Immunol 2020;11:1104.
      1. Zhou YH, Han QF, Gao L, Sun Y, Tang ZW, Wang M, et al. HMGB1 protects the heart against ischemia-reperfusion injury via PI3K/AkT pathway-mediated upregulation of VEGF expression. Front Physiol 2020;10:1595.
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    MeSH Terms
    Animals
    Blotting, Western
    Cell Survival
    Epithelial-Mesenchymal Transition
    Exhibition
    Flavonoids
    HMGB1 Protein
    Heterografts
    Hypoxia
    Immunohistochemistry
    In Vitro Techniques
    Liver
    Mice
    Mice, Nude
    Mortality
    Neoplasm Metastasis*
    Phosphatidylinositols
    Proteins
    Public Health
    Respect
    Stomach Neoplasms*
    Wound Healing
    Metrics
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