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. 2025 Sep 29;15:33429. doi: 10.1038/s41598-025-18912-z

18β-Glycyrrhetinic acid inhibits the proliferation and metastasis of gastric cancer by inhibiting the TCTP/AKT/P53 signaling pathway

Jun-Fei Zhang 1,5,✉, Ya-Hong Li 2, Zhao-Zhao Wang 3, Shu-Min Jia 3, Peng Yang 2, Rui-Nan Wang 3, Jia-Nan Zhao 2, Ling Yuan 4,✉, Yi Nan 2,3,✉
PMCID: PMC12480900  PMID: 41023076

Abstract

18β-Glycyrrhetinic acid (18β-GA) has a therapeutic effect on gastric cancer (GC), but its mechanism of action is still unclear. We analyzed the effects of 18β-GA on the proliferation, migration and invasion of GC cells using CCK technology, colony formation assay, wound healing assay and Transwell assay. BALB/c nude mice were used to establish subcutaneous transplantation models, orthotopic transplantation models and tail vein Liver metastasis models, and GC proliferation and metastasis models were constructed using zebrafish. After continuous treatment with 18β-GA, the inhibitory effect of drug on tumor was observed in vivo. Finally, western blot, immunohistochemistry (IHC) and immunofluorescence (IF) were used to detect the expression of key proteins in the TCTP/AKT/P53 signaling pathway and the key proteins in the cell apoptosis, cell cycle and EMT signaling pathways. Our in vitro and in vivo functional experiments showed that 18β-GA treatment could inhibit GC proliferation and metastasis, and the effect of 18β-GA alone was similar to that of TCTP knockdown, while the inhibition effect of 18β-GA combined with TCTP knockdown was the best. In GC, the expression of TCTP, p-AKT and Ki67 protein was significantly decreased after treatment with 18β-GA, while the expression of P53 protein was increased. In addition, 18β-GA treatment significantly increased the protein expression levels of Bax, P27, P21 and E-Cadherin, and decreased the protein expression levels of Bcl-2, Cyclin D1, N-Cadherin and Vimentin. Therefore, 18β-GA can promote cell apoptosis by inhibiting the TCTP/AKT/P53 signaling pathway, arrest the cell cycle and inhibit the proliferation and metastasis of GC.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-18912-z.

Keywords: 18β-Glycyrrhetinic acid, Gastric cancer, TCTP/AKT/P53 signaling pathway

Subject terms: Cancer, Gastric cancer

Introduction

Gastric cancer (GC) is a common malignant tumor of the digestive tract occurring in the gastric epithelium, which seriously threatens human health. According to the “Global Cancer Statistics Report 2020: estimates of incidence and mortality of 36 cancers in 185 countries” : GC ranks the fifth in the incidence of cancer and the fourth in the mortality of cancer in the world, and it ranks the third in both the incidence and mortality of cancer in China1. Even if patients are treated with surgery, they also need drug adjuvant therapy to reduce recurrence and metastasis, which brings great health hazards and heavy economic burden to patients and their families. Moreover, the effect of existing GC clinical treatment drugs is not satisfactory2. Therefore, the search for new drugs for GC treatment has become a research hotspot. In recent years, the anti-tumor effect of traditional Chinese medicine has attracted more and more attention. Especially, the natural components of traditional Chinese medicine have shown strong anti-tumor pharmacological activity, and have become a popular choice for the development of new anti-tumor drugs in the future3. Building on prior research, we identified 18β-Glycyrrhetinic acid (18β-GA)—a key licorice-derived compound—as a potent inhibitor of GC cell proliferation with significant therapeutic implications, yet its exact mechanism of action awaits further elucidation4.

Translationally Controlled Tumor Protein (TCTP/Tpt1) is a highly conserved protein in a variety of species. Many current studies have shown that TCTP is highly expressed in a variety of tumors and is associated with poor prognosis of patients5–8. Accumulating studies have further demonstrated its central role in promoting tumorigenic processes, including cellular proliferation and metastatic dissemination9,10. Su et al.11 indirectly demonstrated that TCTP was highly expressed in GC in vitro. In addition, our previous study has clinically demonstrated that high expression of TCTP in GC is associated with poor prognosis of patients, and it is a key regulator of GC proliferation and metastasis. Current studies have shown that through negative feedback regulatory loop interaction between P53 and TCTP12TCTP can inhibit P53 transcription, thereby inhibiting apoptosis and promoting cancer. In addition, studies have shown that TCTP plays a key physiological role in cell survival and migration through the Akt pathway13. Our study also demonstrated that in GC, TCTP inhibited cell apoptosis, promoted cell cycle and EMT by phosphorylating AKT and inhibiting P53, leading to GC proliferation and metastasis. In addition, our previous study found that 18β-GA could promote cell apoptosis and arrest cell cycle during the inhibition of GC14,15. Therefore, we hypothesized that 18β-GA might inhibit GC proliferation and metastasis by interfering with the TCTP/AKT/P53 signaling pathway to promote cell apoptosis, arrest cell cycle and EMT signaling pathway. Hence, we used GC cell Lines, orthotopic transplantation and metastasis models of gastric cancer, and zebrafish proliferation and metastasis models to investigate the effect of 18β-GA on GC proliferation and metastasis in vitro and in vivo. Western blot, immunohistochemistry and immunofluorescence techniques were used to further explore its possible mechanism of action. To lay the foundation for further drug development and utilization in the future.

Materials and methods

Cell culture and transfection

AGS (CL-0022) and MKN-45 (CL-0292) gastric cancer cell lines (Wuhan Pricella Biotechnology, China) were STR-authenticated and cultured at 37 °C/5% CO₂ in RPMI-1640 (MKN-45) or Ham’s F-12 (AGS) medium with 10% FBS (Servicebio, China). Lentiviral vectors (TCTP, shTCTP, and controls) were sourced from Shanghai Genechem (China).

CCK-8 assay

AGS (8 × 103/well) and MKN-45 (6 × 103/well) cells were seeded in 96-well plates at 37 °C with 5% CO₂. After incubating the cells in shNC, shTCTP, shNC + 18β-GA, and shTCTP + 18β-GA groups for 1, 2, 3, 4, and 5 days, CCK-8 solution (10 µL) was added to each well and incubated for 2 h. The optical density values at 450 nm were measured using a microplate reader (Molecular Devices Sunnyvale, CA, USA).

Clonogenic assay

Post-transfection (shNC/shTCTP), AGS (1.5 × 103/well) and MKN-45 (1 × 103/well) cells were cultured in 6-well plates for 10 days. For 18β-GA treatment, four groups (shNC, shTCTP, shNC + 18β-GA, shTCTP + 18β-GA) were analyzed. Colonies (≥ 50 cells/colony) were methanol-fixed, crystal violet-stained, and counted microscopically (n = 3).

Transwell assay

Chambers (Millipore) in 24-well plates were used, with Matrigel-coated (invasion) or uncoated (migration) membranes. Lower chambers contained 10% FBS medium (500 µL), while upper chambers held transfected cells (shNC/shTCTP) in serum-free medium ± 18β-GA (200 µL). After 24 h, membranes were fixed (methanol), stained (crystal violet), and imaged (Leica microscope; 10×). Cells in 8 random fields/group were quantified via ImageJ (v1.4.3.67; triplicates).

Wound healing assay

Transfected (shNC/shTCTP) AGS/MKN-45 cells were cultured in 6-well plates (37 °C). At confluency, scratches were created (sterile tip), and baseline images (0 h) were acquired (Olympus microscope). 18β-GA was added post-imaging. After 24 h/48 h incubation, cells were PBS-washed (3×) for debris removal.

Western blot analysis

Cell lysates were homogenized in RIPA buffer (Servicebio, China) supplemented with protease/phosphatase inhibitors using a magnetic bead grinder (JXFSTPRP-CL, Jing Xin, China). Protein concentrations were quantified via BCA assay (Omni-Easy, China), and equal amounts (15 µg/lane) were resolved on 7.5–12.5% SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad system, CAVOY, China).The membranes were then blocked with 5% skim milk powder at room temperature for 2 h and incubated at 4 °C overnight with antibodies against TCTP (cat. 10824-1-AP, 1:3000), phosphorylated (p)-AKT (cat. 66444-1-Ig, 1:5000), Akt (cat. 60203-2-Ig, 1:10000), P53 (cat. 2355, 1:1000), Bax (cat. AF0120, 1:2000), Bcl-2 (cat. AF6139, 1:2000), P27 (cat. 25614-1-AP, 1:4000), P21 (cat. 10355-1-AP, 1:2000), Cyclin D1 (cat. AF0931, 1:1000), E-Cadherin (cat. ab40772, 1:25000), N-Cadherin (cat. 22018-1-Ig, 1:5000), Vimentin (cat. 60330-1-Ig, 1:50000) and GAPDH (cat. T0004; 1:10,000) followed by incubation at room temperature for 1 h with goat anti-rabbit secondary antibodies (cat. S001; 1:10,000) or goat anti-mouse secondary antibodies (cat. AS014; 1:10,000; Abclonal). Protein bands were detected with an enhanced chemiluminescence kit (KeyGen BioTECH, Jiangshu, China) by capturing light sources with an ultrasensitive multifunction imager (Amersham lmager 680RGB) and were semiquantified via ImageJ software (Rawak Software, Inc., Germany).

Immunohistochemistry (IHC) and immunofluorescence (IF) staining

For IHC, paraffin-embedded tissues were Subjected to deparaffinization and rehydration, and then stained with targeted antibodies at 4℃ overnight. The tissues were treated with a secondary antibody, followed by incubation with 3,3′-diaminobenzidine (DAB). Subsequently, the tissues were counterstained with hematoxylin and observed under a Light microscope. The expression levels of target proteins in tumor tissues were determined via IF. The fixed tumor tissue was permeabilized with 0.5% Triton X-100 for 20 min. The samples were blocked with 5% BSA blocking solution at room temperature for 60 min and then washed with PBS. The membrane was Subsequently incubated with the target antibody overnight at 4 °C and further treated with a goat anti-rabbit IgG H&L (AlexaFluor® 594) secondary antibody (cat. ZF-0516, 1:100). The tumor tissues were then stained with 4’,6-diamino-2-phenylindole (DAPI, C0060, Solarbio, China) and observed with a fluorescence microscope (MF43-N, Mshot, China) to obtain representative fluorescence images. All the sections were scanned with a digital scanner (Aperio Versa 8, Leica, Germany), and images were presented and captured with Aperio ImageScope - Pathology Slide Viewing Software (version 12.4.3) (https://www.leicabiosystems.com/us/digital-pathology/manage/aperio-imagescope/) (Leica, Germany).

Protein docking

The AutoDockTools Vina (http://autodock.scripps.edu/) was utilized for molecular docking to analyze the mechanism of binding of 18β-GA with TCTP. Firstly, the mol2 format of 18β-GA was obtained from the TCMSP data (https://old.tcmsp-e.com/tcmsp.php), and high-resolution protein crystal structures of TCTP was selected as the receptors from the RCSB PDB database (https://www.rcsb.org/), and the PYMOL software was used to remove water molecules and heteromolecules in the receptors, the AutoDock Tools software was used to add hydrogen atoms for receptors. Then, with 18β-GA as the ligand and protein as the receptor, the docking box was set by AutoDockTools software, the molecular docking was performed by Vina program, and the results with the lowest docking energy were saved for visualization. Finally, PYMOL software was used to visualize the docking results with the lowest energy, and the names of the amino acid residues and the sizes of the hydrogen bonds were shown at the receptor and ligand binding sites.

BALB/c nude mouse tumor model

All BALB/c nude mice were purchased from Vital River. For the Subcutaneous tumor model, 5-week-old male BALB/c nude mice were randomly divided into the shNC (n = 4), shTCTP (n = 4), shNC + 18β-GA (n = 4) and shTCTP + 18β-GA (n = 4) groups. MKN-45 cells (~ 3 × 106) were resuspended in medium containing 40% Matrigel (BD Biosciences) and Subsequently injected into the right flank of each mouse. The cells were transfected with the corresponding lentivirus according to the grouping requirements before inoculation. At 5 days after subcutaneous inoculation, the shNC + 18β-GA and shTCTP + 18β-GA groups were intraperitoneally injected with 50 mg/kg, and the shNC and shTCTP groups were given normal saline. The drug was administered once every other day. Tumor size was measured weekly from the time of inoculation, fixed at 9–11 am, and the procedure time was controlled to within 1 min. During the first 3 weeks, the activity status of mice was observed every 3 days. In the fourth week, the observation rate was controlled once a day. The tumor volume was calculated based on the following formula: Length x width2/2. Tumors were required to be < 2 cm in maximum diameter at the end of the experiment. An orthotopic GC model was established by first growing subcutaneous tumors in mice (n = 4) until reaching volumes of 100–200 mm3. Tumors were excised and dissected into 1-mm3 tissue blocks, and optimal samples were selected for implantation. Male BALB/c nude mice (5 weeks old) were divided into two groups: shNC (n = 6) and shNC + 18β-GA (n = 6). Mice were fasted for 12 h, anesthetized with isoflurane (2%) and Surgically implanted with tumor tissue. A 1.5-cm incision was made below the xiphoid to expose the stomach, where a small groove was created in the gastric wall. Tumor blocks were placed into the groove and fixed with bioglue, and the incision was closed. Post-surgery, mice received saline injection and recovery support on a thermal blanket. Tumor growth was monitored via bioluminescence imaging (FUSIONFX system) after intraperitoneal injection of D-luciferin (150 mg/kg), with data analyzed using Fusion software. At 3 days after surgery, the shNC + 18β-GA group was intraperitoneally injected with 50 mg/kg and the shNC group was given normal saline. The drug was administered once every other day. For the tail vein metastasis model, luciferase labeling was performed in advance and lentivirus was transfected into MKN-45 cells according to the group. MKN-45 cells (~ 3 × 105) stably expressing firefly luciferase were injected into the tail vein of 5-week-old male BALB/c nude mice, which were randomly divided into the shNC group (n = 6) and the shNC + 18β-GA group (n = 6). At 1 week after tail vein injection, the shNC + 18β-GA group was intraperitoneally injected with 50 mg/kg and the shNC group was given normal saline. The drug was administered once every other day. Bioluminescence imaging was performed for the tumor-bearing mice in each group using a FUSIONFX imaging system (Vilber Lourmat). After the mice were anesthetized with isoflurane gas (2%; the maintenance and induction concentrations were the same), D-luciferin potassium salt dissolved in PBS (150 mg/kg) was intrabitoneally injected (Beyotime Institute of Biotechnology), and bioluminescence imaging was performed 5–10 min later. All the images were collected and analyzed using Fusion software (Vilber Lourmat). Tissue sampling was performed after euthanasia with CO₂ (Maintained at 40% for 1 min to rapidly lose consciousness and then adjusted to 100% for 10 min) at the end of all animal experiments. All experimental procedures strictly complied with the ARRIVE 2.0 guidelines.

Zebrafish GC model

Zebrafish were purchased from Fujian Anrui Biotechnology Co., LTD. Healthy zebrafish embryos (AB strain) were cultured in E3 solution at 28.5˚C until 48 h post-fertilization. MKN-45 GC cells were genetically modified using lentivirus and labeled with a red fluorescent dye (5 µM DiI). After the embryos reached 48 hpf, the larvae were anesthetized (MS-222 (40 mg/l)) and placed in grooves of AGAR plates. The stained cells were then transplanted into the central yolk sac or ventral yolk space of zebrafish larvae using a microinjection instrument. Each experimental group included at least 30 larvae (minimum 10 per subgroup). At 2 and 48 h post-injection, 10 larvae were randomly selected, and fluorescent tumor areas were imaged under a microscope. To determine the safe dose of 18β-GA, embryos were treated with six concentrations (2–8 µg/ml) for 48 h. The half-lethal concentration (LC50) was calculated based on mortality rates, and half of the LC50 was used as the safe working concentration. At the end of the experiment, the zebrafish were euthanized by the MS-222 anesthesia method.

Statistical analysis

All quantitative data are expressed as mean ± SD. Intergroup differences were analyzed by one-way ANOVA, with post hoc comparisons (Tukey’s test for homoscedastic data or Tamhane’s T2 test for heteroscedastic data) based on variance homogeneity assessment. Statistical analyses were performed using SPSS 24.0 (IBM, USA), and a two-tailed significance threshold of P < 0.05 was applied.

Results

18β-GA inhibits the proliferation, metastasis and invasion of gastric cancer cells in vitro

To further investigate the mechanism by which 18β-GA inhibits GC proliferation, metastasis and invasion, we first examined the effect of 18β-GA on GC cell proliferation using CCK-8 (Fig. 1A-B) and clone forming assays (Fig. 1C-D). The results showed that 18β-GA alone could significantly inhibit the proliferation of GC cells, and the effect was similar to that of TCTP knockdown, while the inhibition effect of 18β-GA combined with TCTP knockdown on GC in AGS and MKN-45 cells was significantly better than that of 18β-GA alone (P < 0.05) (Fig. 1A-D). We further evaluated the inhibitory effect of 18β-GA on GC metastasis using wound healing assay and Transwell migration and invasion assay after treatment with 18β-GA in AGS and MKN-45 cells (Fig. 1E-J). The results showed that 18β-GA treatment significantly inhibited the wound healing ability of AGS and MKN-45 cells, and the therapeutic effect was similar to that of TCTP knockdown, while the inhibition ability of 18β-GA combined with TCTP knockdown was significantly better than that of 18β-GA alone (Fig. 1E-F). In addition, we performed cell migration and invasion assays on AGS and MKN-45 cells, and the results showed that the migration (Fig. 1I-J) and invasion (Fig. 1G-H) of AGS and MKN-45 cells were significantly inhibited after 18β-GA silencing treatment, and the effect was similar to that after TCTP knockdown, and the inhibitory effect was most obvious after combined application. These results indicate that 18β-GA exhibits a good potential to inhibit the proliferation and metastasis of gastric cancer cells in vitro.

Fig. 1.

Fig. 1

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18β-GA inhibits the proliferation, metastasis and invasion of gastric cancer cells in vitro. A-B: Cell proliferation in the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups of AGS (A) and MKN-45 (B) cells was analyzed via a CCK8 assay for 5 consecutive days. The data are expressed as the mean ± SD (n = 3/group), # indicates p < 0.05 (shNC + 18β-GA vs. shNC), Y indicates p < 0.05 (shTCTP + 18β-GA vs. shNC), and & indicates p < 0.05(shTCTPA vs. shNC). C-D: ShNC group, shNC + 18β-GA group, shTCTP group and shTCTP + 18β-GA group. Representative images (top) of colony formation and colony quantification (bottom) in AGS (C) and MKN-45 (D) cells. E-F: Representative images (left) of the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups and statistical analysis (right) of the wound healing ability of AGS (E) and MKN-45 (F) cells. G-H: Representative images (top) and statistical map (bottom) of cell invasion capacity in AGS (G) and MKN-45 (H) cells in the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups. I-J: Representative images (top) and statistical map (bottom) of the migration of AGS (I) and MKN-45 (J) cells in the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups. Data are expressed as the mean ± SD (n = 3/group), P value legend: ns > 0.05, *<0.05, **<0.01, ***<0.001.

18β-GA inhibits the proliferation and metastasis of gastric cancer in vivo

Furthermore, we used MKN-45 cells to establish a Subcutaneous xenograft model, an orthotopic transplantation model and a tail vein injection metastasis model to observe the inhibitory effect of 18β-GA on the proliferation and metastasis of GC. The results showed a significant reduction in tumor size and weight after continuous treatment with 18β-GA in the subcutaneous xenograft model compared with the shNC group (Fig. 2A-C). The results of in vivo imaging tumor fluorescence intensity detection in the orthotopic transplantation model of gastric cancer showed that the tumor fluorescence intensity was significantly reduced after continuous treatment with 18β-GA compared with the shNC group at 21 days after surgery (Fig. 2D). Finally, in the tail vein injection metastasis model, we also found that the tumor fluorescence intensity was significantly reduced after continuous treatment with 18β-GA compared with the shNC group (Fig. 2E). To further explore the effects of 18β-GA on the proliferation and metastasis of gastric cancer in zebrafish models, the median lethal concentration (LC50) of 18β-GA in zebrafish needs to be explored to determine the Subsequent drug treatment concentration. We configured 18β-GA into concentrations of 2 µg/mL, 4 µg/mL, 5 µg/mL, 6 µg/mL, 7 µg/mL, and 8 µg/mL, and selected 20 fish for each concentration for the study and set up three replicates. The LC50 was calculated as 5 µg/mL by probit method. The experimental treatment was given at a concentration of 1/2LC50. The results showed that 18β-GA treatment could significantly inhibit GC proliferation (Fig. 2F-G) and metastasis (Fig. 2H-I) in zebrafish, and the therapeutic effect was similar to that after TCTP knockdown. These results indicate that the proliferation and metastatic capacity of GC can be significantly inhibited after serial treatment with 18β-GA in vivo.

Fig. 2.

Fig. 2

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18β-GA inhibits the proliferation and metastasis of gastric cancer in vivo. A: Subcutaneous xenograft tumor images of shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups (n = 4/group). B: Tumor volume changes in the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups over 4 consecutive weeks. The data are expressed as the mean ± SD (n = 4/group), # indicates p < 0.05 (shNC + 18β-GA vs. shNC group), ￥indicates p < 0.05 (shTCTP vs. shNC group), and & indicates p < 0.05 (shTCTP + 18β-GA vs. the shNC group). C: Statistical charts of the tumor weights of the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups. D: The mean photon counts of shNC group and shNC + 18β-GA group were measured on the 3rd and 21 st day after operation. E: Statistical map of the mean photon counts of gastric cancer liver metastases in the shNC group and shNC + 18β-GA group. F-G: Representative images (F) and statistical graphics (G) of tumor cell fluorescence in the yolk sacs of zebrafish in the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups postinjection. H-I: Representative images (H) and statistical graphics (I) of the fluorescent MKN-45 cells in the ventral vitelline spaces of zebrafish in the hNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups postinjection. Data are expressed as the mean ± SD (n = 10/group), P value legend: *<0.05 (vs. shNC).

18β-GA inhibits the TCTP/AKT/P53 signaling pathway in GC

It has been demonstrated that 18β-GA can inhibit the proliferation and metastasis of GC in vitro and in vivo, but the exact mechanism remains unclear. Therefore, we intend to further investigate the mechanism of 18β-GA on GC proliferation and metastasis. Firstly, we used molecular docking technology to predict the molecular docking of 18β-GA and TCTP protein. The results showed that the binding energy of 18β-GA-TCTP complex was − 8.56 kcal/mol (Fig. 3A), indicating that 18β-GA could well combine with TCTP protein, which may be its therapeutic target for GC. We then examined the expression levels of TCTP, p-AKT and P53 proteins using western blots in AGS and MKN-45 cells after 18β-GA treatment. The results showed that compared with the shNC group, treatment with 18β-GA alone significantly inhibited the expression of TCTP (Fig. 3B-C) and p-AKT (Fig. 3D-E) proteins and increased the expression of P53 (Fig. 3F-G) protein in AGS and MKN-45 cells, and the effect was similar to that of TCTP knockdown. The effect of 18β-GA combined with TCTP knockdown group was significantly better than that of single use group. At the same time, we performed IF detection of TCTP, p-AKT and P53 proteins in subcutaneous xenograft tumor tissues of the shNC group, shNC + 18β-GA group, shTCTP group and shTCTP + 18β-GA group, and the results were consistent with GC cells (Fig. 3H). The results of Ki67 also showed that compared with the shNC group, 18β-GA treatment alone could significantly reduce the expression level of ki67 protein in tumor tissues, and the effect was similar to that after TCTP knockdown, and the inhibition of ki67 protein expression was most obvious after 18β-GA combined with TCTP knockdown (Fig. 3H). The results showed that 18β-GA treatment significantly inhibited the expression of TCTP, p-AKT, P53 and ki67 proteins in both orthotopic gastric cancer xenograft tissues and liver metastatic gastric cancer tissues compared with the shNC group. The expression levels of p-AKT and ki67 proteins, while increasing the expression level of P53 protein (Fig. 3I). The results of all experiments were consistent. Taken together, our results indicate that 18β-GA exerts an inhibitory effect on the TCTP/AKT/P53 signaling pathway in GC.

Fig. 3.

Fig. 3

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18β-GA inhibits the TCTP/AKT/P53 signaling pathway in GC. A: 3D crystal structure for 18β-GA in the 18β-GA-TCTP complex. B-C: The expression levels of TCTP in the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups were analyzed by Western blotting in AGS (B) and MKN-45 (C) cells. Representative images (top) of the Western blot analyses and relative band intensity (bottom) of TCTP protein expression. D-E: The expression levels of p-AKT in the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups were analyzed by Western blotting in AGS (D) and MKN-45 (E) cells. Representative images (top) of the western blot analyses and relative band intensity (bottom) of p-AKT protein expression. F-G: The expression levels of P53 in the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups were analyzed by Western blotting in AGS (F) and MKN-45 (G) cells. Representative images (top) of the western blot analyses and relative band intensity (bottom) of P53 protein expression. H: The expression of TCTP, P53, p-AKT and ki67 in the subcutaneous tumor tissues of the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups was analyzed via IF. The target protein fluoresced green. I: The expression of TCTP, P53, p-AKT and ki67 in orthotopic gastric cancer tissues or hepatic metastases of mice with gastric cancer after 18β-GA treatment was analyzed via IHC.

Treatment with 18β-GA induced cell apoptosis, halted cell cycle progression, and inhibited EMT

By measuring the levels of Bcl-2, Bax, P27, P21, Cyclin D1, N-Cadherin, E-Cadherin and Vimentin in GC cell, the molecular mechanism involves the therapeutic function of 18β-GA through the regulation of apoptosis, cell cycle and EMT was further studied. The results of western blot showed that 18β-GA treatment significantly increased the expression of Bax protein and decreased the expression of Bcl-2 protein compared with the shNC group, and the effect of 18β-GA treatment was similar to that of TCTP knockdown (Fig. 4A-C). In addition, our study also found that 18β-GA treatment could significantly increase the expression of P27 and P21 proteins and decrease the expression of Cyclin D1 compared with the shNC group, and the effect of 18β-GA treatment was similar to that of TCTP knockdown, while the effect of 18β-GA combined with TCTP knockdown was better than that of 18β-GA alone (Fig. 4A and D-F). The expressions of P27, P21 and Cyclin D1 proteins in orthotopic gastric cancer xenografts and liver metastases were detected by IHC. The results were consistent with those of western blot (Fig. 4J). Finally, we examined the expression of N-Cadherin, E-Cadherin and Vimentin, the key proteins in EMT signaling pathway (Fig. 4A and G-I). Compared with the shNC group, 18β-GA treatment could significantly increase the expression of E-Cadherin protein. However, the expression of N-Cadherin and Vimentin was decreased. The expressions of Bcl-2, Bax, P27, P21, Cyclin D1, N-Cadherin, E-Cadherin and Vimentin were detected by IHC in orthotopic gastric cancer xenografts and liver metastases, and the results were consistent with those of western blot (Fig. 4J).

Fig. 4.

Fig. 4

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Treatment with 18β-GA induced cell apoptosis, halted cell cycle progression, and inhibited EMT A-I: Representative images (A) and relative intensity of western blot analysis of Bcl-2 (B), Bax (C), Cyclin D1 (D), P27 (E), P21 (F), N-Cadherin (G), E-Cadherin (H) and Vimentin (I) protein expression in the shNC, shNC + 18β-GA, shTCTP and shTCTP + 18β-GA groups. Data are expressed as mean ± SD (n = 3/group), P-value legend: ns > 0.05, *<0.05, **<0.01, ***<0.001,****<0.0001. J: The expression of Bax, Bcl-2, P27, P21, Cyclin D1, E-cadherin, N-cadherin and Vimentin in orthotopic gastric cancer tissues or hepatic metastases of gastric cancer after 18β-GA treatment was analyzed via IHC.

Discussion

GC ranks as the fifth most prevalent malignancy globally and the fourth leading cause of cancer-associated mortality16. Due to the proliferation and metastasis characteristics of GC, the optimal time for surgery is missed when GC is diagnosed, and adjuvant therapy such as drugs is needed16–18. However, there is still no specific drug for GC treatment. Current studies have demonstrated that traditional Chinese medicine assumes an important role in the treatment of advanced gastric cancer, particularly in the adjuvant therapy of gastric cancer chemotherapy and the management of patients with non-surgical indications19. It possesses the characteristics of fewer side effects, significantly enhancing the quality of Life of patients and prolonging the Survival time of patients. Previous studies found that 18β-GA in licorice showed good efficacy in the treatment of GC4,14,15. In addition, in lung cancer, 18β-GA can indirectly regulate the expression of MAPK/STAT3/NF-κB pathway proteins by promoting the production of ROS, promote the apoptosis of lung cancer cell A549, inhibit its metastasis and arrest the cell cycle at G2/M20. In prostate cancer, 18β-GA is capable of regulating the transcription of the androgen receptor (AR), suppressing the expression of AR and its target genes TMPRSS2, PSA, and NKX3.1, and strengthening the inhibitory effect of anti-androgens bicalutamide and furalutamide on LNCaP cells21. In GC studies, it was discovered that 18β-GA could enhance the expression of P21 and P27, restrain the proliferation of the GC cell line BGC823, and cause the cell cycle to arrest at the G0/G1 phase21. In another study, 18β-GA was capable of inhibiting the migration and invasion of GC cells SGC-7901 by up-regulating the expression of E-cadherin and down-regulating the expression of MMP2 and MMP9 in a dose-dependent manner via the ROS/PKC-α/ERK signaling pathway22. In vivo experiments have shown that 18β-GA can inhibit the proliferation of GC cells in mice, promote the differentiation of gastric mucosal epithelial cells, and effectively curb the development of GC and the production of inflammation in mice by regulating Wnt and COX-2 signaling pathways23. These studies Suggest that 18β-GA May be a valuable monomeric component in tumor therapy including GC. However, the specific mechanism of 18β-GA in the treatment of GC is still unclear.

Kim et al. found that TCTP was highly expressed in the blood of patients with gastric cancer, and it was associated with the poor prognosis of the patients24. Nishiyama et al. conducted TCTP protein expression detection on surgical samples from patients with gastric cancer and found that TCTP was highly expressed in gastric cancer tissues and was associated with poor prognosis of the patients25. Furthermore, Our previous study found that TCTP protein can act as a key regulator of GC proliferation and metastasis, and TCTP can inhibit downstream cell apoptosis by regulating AKT/P53 signaling pathway, promote cell cycle and EMT, and further promote GC proliferation and metastasis. To determine whether 18β-GA can inhibit the proliferation and metastasis of GC by Suppressing the expression of TCTP protein in GC, the effects of 18β-GA on the proliferation, migration and invasion of GC cells were analyzed through CCK technology, colony formation assay, wound healing assay and Transwell assay. The results showed that 18β-GA significantly inhibited the proliferation and metastasis of GC cells, which was consistent with the effect of TCTP knockout, and the inhibition effect of 18β-GA combined with TCTP knockout on the proliferation and metastasis of GC cells was better than that of 18β-GA alone. Furthermore, we used BALB/c nude mice to establish subcutaneous transplantation models, orthotopic transplantation models and tail vein Liver metastasis models, and used zebrafish to construct GC proliferation and metastasis models. After continuous treatment with 18β-GA, the tumor inhibitory effect of the drug was observed in vivo. The results also showed that the proliferation and metastasis models established by BALB/c nude mice and zebrafish showed a good inhibitory effect after 18β-GA treatment. These results laid the foundation for the development and application of 18β-GA in clinical GC treatment. Therefore, we further investigated the mechanism of 18β-GA inhibiting GC proliferation and metastasis. We found that 18β-GA treatment could significantly inhibit the expression of TCTP protein in AGS and MKN-45 cells, and TCTP protein expression levels in GC tissues were detected by IF and IHC. The results also showed that 18β-GA treatment could inhibit the expression of TCTP protein in GC tissues. This suggests that TCTP protein May be an effective target of 18β-GA in GC.

Current studies have shown that TCTP can promote cell proliferation by regulating the AKT/P53 signaling pathway axis. Du et al.26 confirmed that in A549 lung cancer cells under stress conditions, TCTP can regulate the AKT signaling pathway and inhibit the expression of P53, so that lung cancer cells maintain a higher metabolic level and protect cancer cells from apoptosis induced by external stress. A feedback signaling pathway can be formed between TCTP and P53, which contributes to the formation and progression of tumors12. In addition, in the study of HT29 colon cancer cells, sertraline could directly bind to TCTP, leading to the reversal of TCTP-induced P53 ubiquitination and preventing TCTP from binding to MDM2, restoring MDM2 self-ubiquitination and inhibiting cell proliferation27. The AKT/P53 signaling pathway refers to the activation of the serine/threonine protein kinase AKT by various factors, which can promote the phosphorylation of MDM2 through related regulatory mechanisms. It inhibits the activity of P53 by promoting P53 nuclear export and targeting P53 as an E3 ubiquitin Ligase for 26 S proteasome degradation, and then plays a role in cell growth control. It is implicated in fundamental physiological activities such as cell cycle arrest, DNA repair, metabolic alterations, antioxidant effects, anti-angiogenesis effects, autophagy, senescence, and apoptosis28. Meanwhile, our previous study demonstrated for the first time that the activation of the TCTP/AKT/P53 signaling pathway in GC inhibits cell apoptosis and promotes cell cycle and EMT, which constitutes an important mechanism for GC proliferation and metastasis. In our in vitro and in vivo studies, 18β-GA treatment significantly inhibited the expression of p-AKT protein, increased the expression of P53 protein, promoted the expression of apoptotic proteins Bax and Cleaved-Caspase-3, and decreased the expression of anti-apoptotic protein Bcl-2. Regarding the key regulatory proteins of the cell cycle, 18β-GA treatment elevated the expression levels of P27 and P21 proteins, but reduced the expression levels of Cyclin D1 proteins. Moreover, 18β-GA increased the expression of E-Cadherin protein, but decreased the expression of N-Cadherin and Vimentin protein. Taken together, our results indicated that 18β-GA in Glycyrrhiza radix could inhibit the proliferation and metastasis of GC by promoting cell apoptosis, arresting the cell cycle and EMT through regulating the TCTP/AKT/P53 signaling pathway (Fig. 5).

Fig. 5.

Fig. 5

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Diagram of the mechanism by which 18β-GA inhibits GC proliferation and metastasis.

Conclusion

In the field of tumor research, including gastric cancer, the focus has shifted towards identifying specific targets and developing drugs targeting those targets, which has become a prominent trend in tumor research29,30. Our results Suggested that 18β-GA in Glycyrrhiza radix could inhibit the proliferation and metastasis of GC by promoting cell apoptosis, arresting cell cycle and EMT via regulating the TCTP/AKT/P53 signaling pathway. However, whether 18β-GA can directly inhibit the proliferation and metastasis of gastric cancer by targeting and inhibiting the expression of TCTP protein still needs to be further verified through techniques Such as pull-down, MST, BLI, and SPR. Moreover, after establishing TCTP knockdown and overexpression mouse models using specific gene editing technology, and establishing spontaneous gastric cancer animal models, the therapeutic effect of 18β-GA can be further observed to lay the foundation for its clinical development.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (1.7MB, pptx)

Acknowledgements

Thanks to the Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education (Ningxia Medical University) for providing experimental equipment. We would like to thank Fujian Anburui Biotechnology Co., Ltd., for the technical support provided in the zebrafish experimental research. We thank Ningxia Keji Biological Co., Ltd., for providing technical support in the histopathological experiments.

Abbreviations

18β-GA

18β-Glycyrrhetinic acid

GC

Gastric cancer

IHC

Immunohistochemistry

IF

Immunofluorescence

TCTP

Translationally controlled tumor protein

Author contributions

ZJF, YL and NY designed the study. LYH, WZZ, JSM and YP performed multiple experiments and wrote the manuscript. WRN and ZJN analyzed the data.

Funding

The study was supported by the grants from National Natural Science Foundation of China (No. 82260879, 82374261, 82205116) and Innovation and entrepreneurship project of Ningxia(Letter of Ningrenshe [2024] No.4).

Data availability

The datasets used and/or analysed during the current study available from the author (Jun-Fei Zhang E-mail: zhangjunfei007@126.com) on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All animal procedures were carried out in accordance with the regulations of the Animal Protection Committee of Ningxia Medical University, and all experimental procedures were approved by the Ethics Committee of the General Hospital of Ningxia Medical University(KYLL-2023-0063).

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Jun-Fei Zhang, Email: 2021011068@nxmu.edu.cn.

Ling Yuan, Email: 20080017@nxmu.edu.cn.

Yi Nan, Email: 20080011@nxmu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (1.7MB, pptx)

Data Availability Statement

The datasets used and/or analysed during the current study available from the author (Jun-Fei Zhang E-mail: zhangjunfei007@126.com) on reasonable request.

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