Marine natural product Methyl mycophenolate inhibits gastric cancer growth through regulating p53 and the downstream pathways

Abstract

Background

Gastric cancer (GC) is one of the most prevalent cancers and the fifth leading cause of cancer-related deaths globally. Methyl mycophenolate (MMP), a methyl ester derivative of mycophenolic acid, is derived from the marine fungus Phaeosphaeria spartinae, yet its role in GC remains unexplored.

Purpose

This study aims to investigate the therapeutic potential of MMP in GC and elucidate its underlying mechanisms.

Methods

We screened marine compounds for their inhibitory activity against GC cells using cell viability, colony formation assays, cell cycle analysis, and apoptosis detection. RNA sequencing and KEGG enrichment analysis identified key downstream pathways activated by MMP. Western blotting, qRT-PCR, and immunohistochemistry confirmed changes in the p53 signaling pathway. Protein stability was assessed through turnover and ubiquitination assays, while Co-IP verified the effect of MMP on p53 binding to MDM2. An in vivo tumorigenesis study evaluated MMP’s efficacy and safety in mice.

Results

MMP significantly inhibited GC cell proliferation and colony formation, induced apoptosis through the caspase pathway, and caused cell cycle G1 arrest by downregulating CDK4, CDK2 and upregulating p27. Mechanistically, MMP increased p53 protein levels and activated downstream targets (p21, PUMA, GADD45A) in a dose-dependent manner. It enhanced p53 stability by reducing ubiquitination. MMP injection in mice significantly inhibited tumor growth in a subcutaneous xenograft model.

Conclusion

MMP displays anti-GC activity by inducing apoptosis and cell cycle arrest via the p53 pathway. Our findings suggest MMP’s potential as a therapeutic agent for GC intervention.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-025-03835-6.

Introduction

Gastric cancer (GC) is one of the most common cancers and ranks as the fifth leading death of cancer worldwide [1]. The incidence and mortality rates are particularly high in East Asian countries [2]. Despite the improvement of GC treatment, including surgical resection, chemotherapy, radiotherapy, and targeted therapy, the prognosis of advanced stage patients remains poor [3–5]. Therefore, there is an urgent need to develop novel anticancer drugs and therapeutic targets to reduce drug resistance and treatment-related side effects, thereby improving GC patients’ outcomes.

The ocean is a complex open system that covers approximately 71% of the Earth’s surface area, and its organisms have evolved to thrive in specialized environments such as hypertonic conditions, low temperatures, or dim lighting, resulting in the unique genomes and metabolites. These distinctive adaptations have enabled marine organisms to synthesize many natural products with novel activities and potential for broad applications, which hold significant importance in the fields of medicine and biotechnology. As of the end of 2022, 17 marine-derived drugs have been approved for clinical use, including 12 drugs for the treatment of different types of cancer [6]. Currently, marine natural products have shown great anti-cancer activity in varies of cancers, including liver, breast, colorectal and prostate cancers [7–9], positioning them as a vital resource for the discovery and development of new drugs.

TP53 is a crucial tumor suppressor gene with low protein levels under non-stress conditions due to proteasomal degradation mediated by the E3 ubiquitin ligase MDM2 [10]. Under certain stress conditions, such as DNA damage, nutritional deficiency, or gene mutation, TP53 encodes proteins that respond to cellular stress through a cascade of phosphorylation events and other post-translational modifications. These modifications regulate the expression of target genes to induce DNA repair, or cell cycle arrest, senescence, and apoptosis [11]. However, under tumorigenic conditions, cancer cells often lose their stress response and the ability to monitor proliferation due to the dysfunction of p53 signaling pathway, which can be caused by mutations in the TP53 gene, dysregulation of its gene transcription or proteasomal degradation of the protein [12, 13].

Methyl mycophenolate (Mycophenolic Acid Methyl Ester, MMP) is a methyl ester derivative of mycophenolic acid (MPA), which is derived from the marine fungus Phaeosphaeria spartinae. Although MPA is clinically utilized as an immunosuppressant and is thought to have anti-cancer attributes, its application in cancer treatment has been limited by poor metabolic properties. These are marked by extensive glucuronidation at the phenolic ring, which leads to the formation of inactive metabolites. Additionally, the need for high doses to sustain efficacy, coupled with insufficient plasma concentrations to inhibit cancer cell proliferation, further impedes its use in cancer therapy [14, 15]. Here, based on our marine compounds screening, methyl ester form of MPA, MMP, exhibits immense potential as an anticancer agent in various GC cell lines and has not been reported so far. Therefore, the current study focused on investigating the inhibitory effects of MMP on gastric cancer and elucidating its molecular mechanism in detail.

In this study, we assessed the effects of MMP on GC cells, including cell proliferation, colony formation, apoptosis in vitro, and tumorigenesis in vivo. MMP demonstrated a significant inhibitory effect and was well tolerated. Additionally, we further elucidated that MMP inhibits GC growth by targeting the p53 signaling pathway, which involves the regulation of p53 protein stability and degradation. Finally, we confirmed that downstream pathways, including cell cycle regulation and apoptosis, were induced following MMP treatment. Collectively, for the first time, we have identified MMP as a potential therapeutic agent for human GC.

Methods

Cell culture and treatment

AGS and HEK-293T cells were obtained from ATCC. MKN-1, MKN-45, NUGC3 and GES-1 cells were obtained from MeisenCTCC. All the cells were maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA), except HEK-293T was maintained in DMEM medium (Gibco, Carlsbad, CA, USA). The cells were cultured in a medium containing 10% fetal bovine serum (NEWZERUM, UCR, Christchurch, NZ) and 100 U/ml penicillin and 100 µg/ml streptomycin, in 5% CO₂ at 37℃.

Drug preparation

The natural marine chemical library was obtained from TargetMol Company (TargetMol, #L6400, USA), MMP (C₁₈H₂₂O₆, molecular weight: 334.36, purity: 99.48%) (TargetMol, #T19424, USA), was dissolved in DMSO with a final concentration of 10mM and diluted by RPMI-1640 medium to prepare proper working concentrations.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from cells using RNAiso PLUS reagent (Takara, #9109, Osaka, Japan). Extracted RNA was reverse transcribed to cDNA using the PrimeScript™ RT Master Mix kit (Takara, #RR036A, Osaka, Japan) and diluted 6-fold to serve as a template, and three replicates were prepared for each set of samples. qPCR amplification was performed using the TB Green Premix Ex Taq II qPCR Quantification Kit (Takara, #RR820A, Osaka, Japan) according to the instructions. All the genes expression were normalized to β-actin. All primer sequences used in this study can be found in Additional file: Table S1.

Colony formation assay

Cells were seeded into 6-well plates at a density of 500 cells per well following the specified treatments. They were cultured to grow for 10–14 days, after which they were stained with 0.1% crystal violet fixed in methanol. Only colonies consisting of more than 50 cells were included in the count.

Cell apoptosis and cell cycle analysis

Cells were seeded into 6-well plates and treated with different concentration of MMP for 48 h. The percentages of cells in the G0/G1, S, and G2/M phases were determined using a Cell Cycle Analysis Kit (Beyotime, #C1052, China), 1µM Camptothecin (TargetMol, #T1123, USA) was used as a positive control for the cell cycle assay. Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, #C1062M, China), 1 µM Staurosporine (Enzo, #ENZ-51002, USA) was used as a positive control for apoptosis detection. Briefly, AGS cells were fixed in ice-cold 70% ethanol, at 4 °C overnight. Then the cells were incubated at 37 °C for 30 min with propidium working solution containing 10 µl of RNase A. Samples were analyzed using a CytoFlex flow cytometer (BD LSR Fortessa X-20), and 10,000 cells were collected per sample. Data were analyzed by the FlowJo v10 software.

Immunoblotting

Cells or tissues were lysed with SDS lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Bimake, #B15002). Total protein concentration was determined using a BCA protein assay kit (Applygen, #P1511, Beijing, China), then separated by SDS-PAGE gels (EpiZyme, #PG113, Shanghai, China) and transferred onto PVDF membranes (Merck Millipore, #ISEQ00010, Darmstadt, Germany). Next, PVDF membranes were blocked by Protein Free Rapid Sealing Solution for 10 min and incubation with primary antibodies. After overnight incubation of the primary antibody at 4℃, the membrane was subsequently washed in Tris buffered saline containing Tween-20 (Aladdin, #T104863, Croda England) and incubated with the suitable secondary antibodies conjugated with HRP at room temperature for 1 h. The membranes were analyzed by ECL kit and imaged with the imager (Amersham Imager600 UV, California, USA). Band density was quantified by ImageJ software (National Institutes of Health, Md, USA). The following antibodies were used in this study: p53 (1:1000; CST, #2527T, USA), p-p53 (1:1000; Proteintech, #28961, China), p21 (1:1000; CST, #2947, USA), GADD45A (1:1000; Zen BioScience, #201061, China), PUMA (1:1000; Zen BioScience, #R380526, China), CDK2 (1:1000; Zen BioScience, #R22532, China), p27 (1:1000; CST, #3686, USA), CDK4 (1:1000; Zen BioScience, #R23888, China), MDM2 (1:1000; Santa Cruz, #sc-965, USA), GAPDH (1:50000; Proteintech, #60004, China) and β-actin (1:5000; Affinity, #AF7018, China).

Cell viability assay

Cell viability was evaluated using the CCK8 kit (Dojindo, #CK04, JAPAN). Briefly, cells were seeded in 96-well plates (500 cells/well) and cultured in a 5% CO₂, 37℃ incubator overnight before treatment. Thereafter, the cells were treated with various concentrations of MMP for indicated times. At the test point, the cells were incubated with 100 µl medium containing 10 µl CCK8 at 37 °C for 2 h. The absorbance was measured using high-quality monochromator based UV/VIS spectrophotometer (Thermo Scientific Multiskan Go) at 450 nm. Cell viability and IC₅₀ were calculated using The GraphPad Prism software v. 8 (GraphPad, La Jolla, CA, USA).

Immunohistochemical (IHC) staining

Immunohistochemistry staining was performed as previously described [16]. Briefly, paraffin-embedded tissue Secti (4 μm) were deparaffinized, rehydrated, antigen retrieval, followed by hydrogen peroxide/ endogenous peroxidase. The sections were then incubated with primary antibodies overnight at 4 °C, followed by incubation with a horseradish-peroxidase-conjugated anti-mouse-rabbit secondary antibody and then visualized using diaminobenzidine. Finally, sections were counterstained with hematoxylin.

RNA sequencing (RNA-seq) and bioinformatics analysis

RNA purification, reverse transcription, library construction, and sequencing were executed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions provided by Illumina (San Diego, CA). Differential expression analysis was performed with the DESeq2 software. Genes exhibiting|log2FC| ≧ 1 and a false discovery rate (FDR) of ≤ 0.05 (based on DESeq2 outcomes) were categorized as significantly differentially expressed genes (DEGs). Additionally, functional enrichment analysis through the Kyoto Encyclopedia of Genes and Genomes (KEGG) was conducted to identify which DEGs were significantly linked to KEGG metabolic pathways, employing a Bonferroni-corrected P-value threshold of ≤ 0.05 in comparison to the whole-transcriptome background.

TUNEL assay

Apoptotic cells were quantified using the terminal deoxynucleotidyl transferase (TdT) dUTP nickel end labeling (TUNEL) assay (Elabscience, #E-CK-A325, China). Briefly, cells were transferred to 6-well plates and fixed with 4% paraformaldehyde for 20 min at ambient temperature, followed by infiltration with 0.25% Triton-X 100 for 10 min at 37 °C. The reaction was carried out using 50 µl of TUNEL reaction mixture in dark for 60 min at 37 °C, followed by the cell nucleuses staining with DAPI and the cells were immediately examined under a fluorescence microscope.

siRNA transfection

AGS cells were seeded in 6-well plates overnight. Transfection was performed using Lipofectamine RNAiMAX (ThermoFisher, #13778150, USA) according to the manufacturer’s protocol. Briefly, 75pmol p53 siRNA (GenePharma, China) or negative control siRNA (NC-siRNA, same concentration) was diluted in 150µL Opti-MEM serum-free medium. Separately, 9µL RNAiMAX reagent was mixed with 150µL Opti-MEM and incubated for 5 min at room temperature. The mixture was added dropwise to each well containing 1.5mL fresh complete medium. After 6 h of incubation, the medium was replaced with fresh complete medium. Cells were harvested 48 h post-transfection for downstream analysis.

Molecular Docking analysis

Molecular docking was performed using AutoDock Vina software. Initially, the 3D structure of MMP was downloaded and saved as an SDF file in PubChem (https://pubchem.ncbi.nlm.nih.gov). The protein structure of the p53 (PDB ID: 8F2H) in the “PDB” format was downloaded from the Protein Data Bank (PDB) database (https://www.rcsb.org/). Next, the protein was pretreated using PyMol software (https://www.pymol.org/) including dehydrogenation, hydrogenation, electron addition. In the final step, AutoDock software was employed for molecular docking and the binding potential was assessed by an affinity score. The optimal docking model was then selected and visualized using PyMol software.

Co-immunoprecipitation (Co-IP)

After indicated treatment, cells were lysed with cell lysis buffer (50 × 10^− 3M Tris-HCl PH 7.5, 150 × 10^− 3M NaCl, 1 × 10⁻³M EDTA, 1% NP-40) containing protease inhibitors cocktail and phosphatase inhibitors (Bimake, #B15002). For each lysate, after centrifugation, the supernatants were collected and incubated appropriate p53 antibodies overnight at 4 °C. Subsequently, the sample were incubated with protein A/G beads (Santa Cruz Biotechnology, #sc-2002, CA, USA) for 4 h. Following incubation, the beads were washed three times with cell lysis buffer. Then, propriate volume of 2× loading buffer was added to the beads and boiled for 15 min in 95 °C to elute the proteins. Afterward, immunoblot assays were conducted using specific antibodies.

Ubiquitination assay

Ubiquitinated plasmids were transfected into AGS cells using LIPOFECTAMINE 3000 (Invitrogen, #L3000015, Waltham, MA, USA) transfection reagent. Subsequently, after culturing the transfected plasmid with the aforementioned compounds for 48 h, the cells were treated with 10 µM MG132 (Sigma-Aldrich, #M8699-1MG, MO, USA) for 6 h before harvesting. Cells were lysed in denaturing buffer. 50 µL of nickel beads were added to the cell lysate and then incubated overnight at 4 °C with rotation. The protein complexes were washed and eluted for protein blotting analysis.

Protein turnover assay

Cells were incubated with the described compounds for 48 h and then treated with 5 ug/ml Cycloheximide (CHX). Cells were then harvested at the indicated times points after CHX treatment. Protein levels were analyzed by immunoblotting.

Animal studies

Animal experiments were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University (protocol no.2021-014). Female NSG mice aged at 4–6 weeks were purchased from Shanghai Model Organisms Center, Inc, maintained in the standard SPF animal house with free access to food and water. In total, 2 × 10⁶ AGS cells were suspended in 100 µl PBS and injected subcutaneously into the right hind limbs of the mice. When the tumors grew to 80 mm³, the mice were randomly separated into two groups (n = 7) and treated intraperitoneally (I.P.) with either vehicle (5% DMSO + 30% PEG300 + 5% Tween20 + 60% ddH₂O) or MMP (5% MMP + 30% PEG300 + 5% Tween20 + 60% ddH₂O, 30 mg/kg/3 day) for 21 days. The tumor volume and the weight of mice were measured every 3 days. At the end of treatment, all mice were sacrificed, and the weights of the tumor were recorded. The tumors and organs, including the heart, liver, kidney, and lung, were harvested at the end time point. The tumor size was calculated using the formula: (width)² × length/2.

Statistical analysis

In this study, data are presented as mean ± S.D. (standard deviation) from at least three independent experiments. Comparisons between two groups were analyzed using two-tailed unpaired Student’s t-test after confirming normal distribution (Shapiro-Wilk test) and homogeneity of variance (F-test). For multi-group comparisons, one-way ANOVA with Tukey’s post hoc correction was applied. The determination of half-maximal inhibitory concentration (IC₅₀) was conducted through non-linear curve fitting methods. Data analysis was carried out using GraphPad statistics software (GraphPad Software Prism 8.0, La Jolla, CA, USA). P-value lower than 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, n.s. indicates no significant difference).

Results

Screen and identify the marine compounds with anti-gastric cancer activity from the nature marine library

To identify marine compounds with anti-cancer activity against GC, we used human GC cell line AGS to screen 93 compounds from a commercial natural marine chemical library. We cultured AGS cells with the library at a final concentration of 10 µM for 48 h and determined the antiproliferative effect of each compound on the cells using the CCK8 assay (Fig. 1a). We screened four compounds among 93 compounds with higher than 80% inhibition of AGS cells, namely Methyl mycophenolate (MMP), Pyropheophorbide-α, Dolastatin 10, and Didemnin B (Fig. 1b). Since the limited tumor-accumulation ability of Pyropheophorbide-α, the application limitation of Dolastatin 10 and Didemnin B, both of which are potential toxic to patients, we chose to delve further into MMP, a compound that has not yet been studied but has great potential for application [17–22] (Fig. 1c). Subsequently, we tested the antiproliferative effects of MMP in AGS (TP53 wild-type) [23] and three other gastric cancer cell lines MKN-1, MKN-45 and NUGC3 (TP53-mutant) (Fig. 1d). The results showed that the viability of four GC cells were significantly inhibited after 48 h of treatment with MMP. The half-maximal inhibitory concentration (IC₅₀) of MMP was determined in these GC cell lines: 0.69 µM (AGS), 1.26 µM (MKN-1), 1.24 µM (MKN-45), and 1.75 µM (NUGC3) (Fig. 1e). The results indicated that MMP showed a wide range of growth-inhibitory activity against GC cells. With promising results showing antiproliferative activity of MMP in the four GC cell lines tested, we then investigated cytotoxicity in normal stomach epithelial cell GES-1 and 293T. The results showed that GES-1 and 293T both required higher doses of MMP (IC₅₀ value of 2.36µM and 2.63µM) compared to four GC cell lines, suggesting that MMP might be a suitable candidate as an anticancer drug for further study (Fig. 1e). Furthermore, as AGS is widely used in GC study and is the most sensitive cell line to MMP treatment, we chose AGS for subsequent experiment study.

Fig. 1.

Identification of Methyl mycophenolate as a novel anti-cancer compound against GC. a Schematic of the compound screening process. b AGS cells were treated with the compounds from the natural marine chemical library (10 µM) for 48 h, and then cell viability was assayed. The bar graph shows the inhibition of cell viability of the 93 compounds. c The Chemical structure of MMP. d AGS cells were treated with MMP (10µM) for 48 h and cell viability was analyzed using the CCK8 kit and their cell viability inhibition was calculated (n = 3). e Determination of IC₅₀ of the four GC cells as well as the GES-1 and 293T cell lines for 48 h (n = 3)

MMP induced caspase-dependent apoptosis in AGS

Due to the inhibitory effect of MMP on GC cell lines, we subsequently observed the morphology of AGS cells cultured with different concentrations of MMP using phase contrast microscopy, and the cells appeared to be reduced in size, wrinkled, rounded, and detached following increased concentration of MMP treatment. The post-treatment culture medium was concurrently collected, and floating cells were quantified, demonstrating a dose-dependent increase (Fig. 2a). Based on these altered cell morphology changes, we hypothesized that these cells might have undergone apoptosis, and therefore apoptosis was subsequently examined by Annexin V-FITC/PI double-staining assay. The results showed that MMP induced significant apoptosis in AGS cells at doses dependent manner. The percentage of apoptotic cells increased from 7.21 to 40.3% after MMP treatment (Fig. 2b). Furthermore, the TUNEL assay showed that in comparison to the control group, the apoptosis rates of AGS cells in the MMP treatment group were markedly higher (Fig. 2c). Consistent with the observations, Immunoblotting analysis showed a dose-dependent increase in the levels of caspase-3, cleaved-caspase-3, caspase-9, cleaved-caspase-9, and Bax, and a dose-dependent decrease in the level of the inhibitory apoptosis protein Bcl-2, in MMP treated GC cells (Fig. 2d). The above results suggest that MMP induces programmed apoptosis by activating the caspase-dependent cell death pathway.

Fig. 2.

MMP induced caspase-dependent apoptosis in AGS cell. a Morphological alterations of AGS cells following incubation with different concentration of MMP, the fold change in the number of floating cells and the percentage of cells with altered morphology was presented as a bar chart. b Apoptosis of AGS cells was detected by flow cytometry after treatment with MMP (along with 2 µM Staurosporine as a positive control for 8 h), and the apoptotic cell ratio was presented as a bar graph. c Apoptosis rate of AGS cells treated with 0.5 and 2 µM concentrations of MMP for 48 h assessed using the TUNEL assay. Left: Representative immunofluorescent images. Right: Bar charts displaying the average percentages of apoptotic cells. d Immunoblotting results of the expression of caspase-dependent apoptotic pathway proteins in MMP-treated AGS cells. Results are presented as the mean ± S.D. Significance is indicated by **P < 0.01, and ****P < 0.0001 vs. Con

MMP induced cell cycle arrest and significantly inhibited AGS cells growth in vitro

Next, we investigated the antiproliferative effect of MMP on AGS cell lines using the CCK8 assay. The results showed that the viability of AGS cells was significantly inhibited by MMP treatment under a dose-dependent manner. And AGS cells almost lost the growth activity when MMP concentration reach to 3µM (Fig. 3a). We also included gemcitabine, which has shown potent activity against GC cells in preclinical studies [24–26], as a positive control in the CCK8 assay to benchmark the efficacy of MMP (Fig.S3). Colony formation assays also indicated that increased doses of MMP treatment could significantly induce less and smaller colonies formation compared to the control group, which is consistent with the cell viability experiments results (Fig. 3b). To investigate whether MMP induces apoptosis and growth inhibition through the regulation of cell cycle arrest, we measured the cell cycle phases of AGS cells after MMP treatment using flow cytometry with propidium iodide (PI) staining. The results demonstrated that AGS cells exhibited significant cell cycle arrest in G1 phase after treatment with 1µM MMP (Fig. 3c). Moreover, to further explore the mechanisms underlying MMP-induced cell cycle arrest, we investigated key proteins regulating cell cycle by immunoblotting experiments. Our results demonstrate that MMP significantly increases p27 protein levels and reduces the expression of CDK2 and CDK4, which are the critical enzymes mediating the G1/S checkpoint (Fig. 3d). These results indicate that MMP could also inhibit GC cells proliferation through inducing GC cell cycle arrest.

Fig. 3.

MMP induced cell cycle arrest and significantly inhibited AGS cells growth in vitro. a AGS cells were treated with 0, 0.3, 1 and 3 µM MMP for 12, 24, 48, 72 h, and cell viability was analyzed using a CCK8 kit. b Representative images of colony formation in AGS cells treated with the indicated concentrations of MMP and statistical analysis of fold change in colony diameter (n = 3). c The effects of MMP on cell cycle arrest were measured by flow cytometry. AGS cells were treated with 1µM MMP for 48 h (n = 3), and 1µM Camptothecin was used as a positive control. The percentage of cells in various cell cycle phases was quantified using FlowJo v10 software. d Immunoblotting assay was used to detect the expression of p27, CDK2, and CDK4, and relative quantification with β-actin as standard. Results are presented as the mean ± S.D. Significance is indicated by **P < 0.01,***P < 0.001 and ****P < 0.0001 vs. Con

The p53 signaling pathway is involved in MMP induced gastric cancer cells apoptosis

To further investigate the detailed mechanism of MMP’s anticancer effect on GC cells, transcriptome analysis was performed in AGS cells following treatment with 1µM MMP or vehicle (control group). PCA analysis revealed good biological reproducibility within the control and MMP groups, while significant differences were observed between the two groups (Fig. 4a). With an adjusted p-value of 0.05 as cut-off, we identified a total of 704 genes that were differentially expressed in relation to MMP treatment, including 202 genes were downregulated and 502 genes were upregulated, simultaneously, the top 5 fold change upregulated and downregulated genes were identified (Fig. 4b). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed the activation of the p53 signaling pathway was the top rank changed pathway (Fig. 4c). The results of the Gene Set Enrichment Analysis (GSEA) demonstrated that most of the genes which were activated by p53 signaling pathway were enriched in the MMP treatment group (Fig. 4d). Cluster analysis confirmed that lots of the p53 signaling pathway regulated genes were increased after MMP treatment, such as TP53I3, BBC3, PIDD1, and GADD45A (Fig. 4e). Consequently, the sequencing results were further validated by qPCR assay, showing that the mRNA expression level of genes related to the p53 signaling pathway, such as TP53I3, BBC3 and GADD45A, were indeed increased in MMP treatment group (Fig. 4f). It is well known that the p53 signaling pathway plays a crucial role in regulating cell death and cell cycle to inhibit tumor progression in response to stress conditions like DNA damage, hypoxia, and abnormal oncogene expression. Therefore, we hypothesized that the p53 signaling pathway activation is essential for MMP-induced apoptosis.

Fig. 4.

The p53 signaling is involved in MMP induced gastric cancer cells apoptosis. a Principal Component Analysis (PCA) demonstrates biological differences between control and MMP groups (n = 3). b Volcano plot illustrating the distribution of differentially expressed genes (DEGs), with five genes exhibiting the most significant levels of upregulation and downregulation annotated. c KEGG enrichment analysis of DEGs showed that the p53 signaling pathway is one of the top rank pathways after MMP treatment. d GSEA analysis showed that most of the genes of p53 signaling pathway were enriched in MMP group compare to the control group. The p-value was 0.00 and the padjust value was 0.049. e The heatmap results generated from the clustering analysis of the differentially expressed genes in the p53 signaling pathway between control and MMP groups. f qPCR validation of the relevant DEGs in the p53 signaling pathway induced by MMP treatment (n = 3). Results are presented as the mean ± S.D. Significance is indicated by ****P < 0.0001 vs. Con

MMP induces apoptosis via the p53 signaling pathway

Based on the previous results and hypothesis, we first investigated the protein expression levels of the key downstream proteins in the p53 signaling pathway. Immunoblotting results indicated that the level of p53, p-p53, p21, GADD45A and PUMA (BBC3) were dose-dependently increased by MMP treatment (Fig. 5a). Subsequently, we treated AGS cells with the p53 inhibitor Pifithirn-α (PFA) and silenced p53 expression using siRNA to assess cell viability (Fig. 5b-c). The CCK8 and results showed that PFA and si-p53 could rescue the cell living ability which was reduced by MMP treatment (Fig. 5d-e). The colony formation assay revealed that PFA treatment restored clonogenic potential compared to the MMP treatment (Fig. 5f). Meanwhile, the results of Annexin V-FITC/PI double-staining assay showed that PFA and si-p53 could partially restore the apoptosis induced by MMP treatment (Fig. 5g-h). Collectively, these results demonstrated that MMP induce AGS cells apoptosis via activating the p53 signaling, establishing p53 as an indispensable mediator of this apoptotic process.

Fig. 5.

MMP induces apoptosis in gastric cancer cells via the p53 Signaling Pathway. a Immunoblotting was conducted to analyze the expression levels of key proteins associated with the p53 signaling pathway in AGS cells following 48 h of MMP treatment. b The expression levels of p-p53 in AGS cells were assessed via immunoblotting under conditions of treatment with 1 µM MMP and 20µM PFA. c Expression levels of p-p53 in AGS cells after treatment with 1 µM MMP and knockdown of p53 using siRNA. d The CCK8 assay measured the absorbance values of AGS cells cultured with MMP and PFA (n = 3). e The CCK8 assay detection of absorbance values in AGS cells treated with MMP and transfected with si-p53 (n = 3). f Addition of PFA to cultured AGS cells restored MMP-induced decreased clone-forming ability. The number of cloned cells in each group is counted on the right side (n = 3). g Flow cytometry detection of apoptosis in AGS cells cultured by PFA and MMP, representative images of apoptosis and statistical analysis are shown (n = 3). h Knockdown of p53 using siRNA after 24 h of MMP culture and detection of apoptosis levels by flow cytometry (n = 3). Results are presented as the mean ± S.D. Significance is indicated by **P < 0.01,***P < 0.001 and ****P < 0.0001 vs. Con

MMP activates p53 signaling pathway by inhibiting p53 protein ubiquitination and degradation

Since the p53 signaling pathway was activated by MMP treatment, we examined the mRNA expression level of p53 in the MMP-treated group and control group. The qPCR results demonstrated that there was no difference of p53 mRNA expression between two groups, while MMP increased p53 steady state protein expression on dose-dependent (Fig. 6a-b). Therefore, MMP might increase the p53 protein expression at post-translational level. Moreover, MG132 enhances MMP-mediated p53 protein expression levels (Fig. 6c), suggesting that MMP may regulate the proteasome-mediated p53 ubiquitination. In addition, MMP decelerates p53 protein turnover, and leads to reduced poly-ubiquitination level of p53 (Fig. 6d, f). On this basis we further investigated whether MMP treatment influences the actions of MDM2, which marks p53 for proteasomal degradation [27]. Our docking studies revealed that MMP may bind directly to the p53 protein, through the sites ASN-30, SER-33 and TYR-205 (K_D = -8.31 kcal/mol) (Fig. 6e). Notably, the amino acid residues of p53 involved in binding to MMP are structurally adjacent to the binding interface between p53 and MDM2 (Fig. S6). To validate the impact of MMP on the interaction between p53 and MDM2 proteins, we conducted the Co-IP experiment. The results showed that MDM2 binding to p53 was significantly reduced in the MMP-treated group (Fig. 6g). In conclusion, the above results suggest MMP treatment reduces the binding of p53 to MDM2, thereby attenuates ubiquitin-mediated degradation of p53, and enhancing its protein stability and expression.

Fig. 6.

MMP activates p53 signaling pathway by inhibiting p53 protein ubiquitination and degradation. a The mRNA expression level of p53 was measured by q-PCR between MMP treated group and control group (n = 3). b The immunoblotting experiment assessed the changes in expression levels of p53 and p-p53 proteins in cells cultured with increasing concentrations of MMP. c MG132 treatment enhances p53 protein expression levels compared to the control group without MG132 treatemnt. d MMP could decelerates the turnover rate of p53 protein. Quantitation was showed on the right panel. e The docking data indicated the interaction of MMP with p53 protein and the precise amino acids, bond lengths and binding energies involved. f AGS cells incubated with MMP and treated with 10 µM MG132 for 6 h were subjected to polyubiquitination assays. The cell lysates were pulled down by nickel beads and immunoblotted with an anti-p53 antibody. g Co-IP assay was used to validate the level of p53 binding to MDM2 protein

MMP effectively inhibits the growth of GC in vivo

To investigate the anti-tumor activity in vivo, mice bearing subcutaneous tumor xenografts were I.P. injected with MMP at a dose of 30 mg/kg/3 days based on previous study [28] (Fig. 7a). Compared to the control group, the volumes and the weights of the tumors were significantly decreased in MMP treatment group (Fig. 7b-d). The immunoblotting results of tumor tissue lysates showed that the p53, GADD45A and p21 were increased in MMP treatment group (Fig. 7e). TUNEL assays in the tumor tissues demonstrated that the TUNEL-positive cells were markedly enhanced upon MMP treatment (Fig. 7f), suggesting that MMP indeed promoted GC cell apoptosis in vivo. IHC staining showed a significant increase in the levels of p53, Bax, caspase-3 and caspase-9 proteins in the MMP - treated group compared to the control group, and the PCNA (a marker of proliferation) expression was significantly decreased after treatment with MMP (Fig. 7h). We further determined the safety of MMP in the xenograft model and found that there was no significant difference in body weight of the mice during MMP treatment and control group (Fig. 7g). Furthermore, histopathological examination of heart, liver, kidney, and lung revealed no obvious toxicity after MMP treatment (Fig. 7i). These findings suggest that MMP inhibits GC tumor growth through activation of the p53 signaling pathway and has a reliable safety profile in vivo.

Fig. 7.

MMP effectively inhibits GC tumor growth in vivo. a Treatment schedule of MMP is indicated. MMP (Vehicle) was I.P. injected every three days until the end of the experiments. b Macroscopic views of xenograft tumors at the endpoint of the experiment. Tumors were derived from AGS cells. c Xenograft tumor volumes were measured every three days (n = 7). d Average tumor weights from each group were calculated (n = 7). e The protein levels of p53, GADD45A, and p21 in tumor tissues were analyzed by immunoblotting. f Apoptotic cells were examined by TUNEL staining with red fluorescent, the scale bar is 100 μm. g Animal body weights were measured every three days (n = 7). h IHC staining results of PCNA, p53, Bax, caspase-3 and caspase-9 in the tumor tissues from the control and MMP treated tumor xenografts. The scale bar is 75 μm i H&E staining of the heart, liver, lung, kidney and tumor tissues in mice, the scale bar is 75 μm. Results are presented as the mean ± S.D. Significance is indicated by **P < 0.01 vs. Con

Discussion

As a vast ecosystem, the oceans harbor a rich and diverse array of biological resources, including many organic constituents and compounds with potential clinical value. This makes them a valuable area for research and a repository of resources [29]. In the present study, we screened a natural marine products library and found four of these products exhibited significant antiproliferative activity against the tested gastric cancer cells. Due to the limited application of the other three compounds, we chose to investigate the anticancer effects of MMP, the methyl ester form of MPA, and the underlying mechanisms involved [30, 31]. A couple of studies have shown that the growth inhibition of MMP and MPA on different cells. Makoto group’s research highlighted that MPA exhibited a stronger inhibitory effect on K562 leukemia cells, as evidenced by the lower IC₅₀ value compare to MMP (0.19 ± 0.01µM vs. 0.73 ± 0.04 µM) [32]. In contrast, Rungnapha’s study revealed that MMP showed greater sensitivity (lower IC₅₀) in FaDu (squamous cell carcinoma), A-549 (lung carcinoma), and SH-SY5Y (neuroblastoma) cells than MPA [33]. We also investigated the effects of MPA in AGS cells and found that MPA, similar to MMP treatment, effectively inhibit cell viability and colony formation while concurrently inducing apoptosis in a dose- and time-dependent manner (Fig.S2). However, our own investigation indicated that MMP possesses a more potent cell inhibitory effect relative to MPA in AGS (gastric cancer). Collectively, these findings suggest that the sensitivity of MMP and MPA to cell inhibition varies depending on the cell type. To further investigate the inhibitory effect of MMP on GC, we utilized four gastric cancer cell lines along with an in vivo animal model. For the first time, we demonstrated that MMP has strong inhibitory effects on GC growth, significantly suppressing tumor growth and decreasing tumor volume by more than 50% at a dose of 30 mg/kg compared to the control. Finally, we report that MMP inhibits the ubiquitination and degradation of p53 protein, stabilizing it and thereby activating downstream targets such as GADD45A and PUMA (BBC3) proteins, which promotes apoptosis in gastric cancer cells.

The fungi belonging to the genus Phaeosphaeria are capable of producing a variety of chemical compounds with multiple biological functions, which could potentially be applied in clinical settings. Phaeosphaeria spartinae, a species within the Phaeosphaeria genus, is an endophytic fungus of the marine algae Ceramium sp. Based on the carbon skeleton, the metabolites isolated from Phaeosphaeria spartinae can be mainly categorized into cyclohexanoids, naphthoquinones, anthraquinones, isocoumarins, isobenzofuran and terpenoide/steroidal compounds [34]. It has been reported that spartinoxide, a compound produced by Phaeosphaeria spartinae, exhibits antitumor activity [35]. However, whether other chemical compounds produced by Phaeosphaeria spartinae also possess antitumor effects remains to be further determined. In this study, we explored and identified MMP as a novel anticancer drug with great potential, capable of inducing cell cycle arrest and apoptosis in cancer cells through the activation of the p53 pathway. Although our current study and results are primarily focused on gastric cancer, we believe that MMP may have broad applicability to other types of tumors, warranting further study in the future.

Apoptosis is a crucial biological process that plays a significant role in various conditions. The two main signaling pathways that induce cell apoptosis are the extrinsic pathway and the intrinsic pathway, both of which are mediated by p53. In the extrinsic pathway, p53 triggers apoptosis by inducing the expression of cell death receptor such are Fas, death receptor 5, and TRAIL receptor, facilitating the formation of the death-inducing signaling complex (DISC). The DISC then recruits and activates caspase-8, which directly activates the executioner caspases. In the intrinsic pathway, p53 triggers the intracellular apoptotic pathway by activating the Bcl-2 family of pro-apoptotic genes, including BAX, Noxa, PUMA and BID. The interaction of these proteins promotes the multimerization and functional activation of BAX, leading to the release of cytochrome c and subsequent activation of caspase 9 [36]. Our results suggest that MMP likely induces apoptosis in GC cells through the intrinsic pathway, as evidenced by the upregulation of cleaved caspase 9, cleaved caspase 3, and Bax, as well as the downregulation of Bcl-2.

It has been demonstrated that the p53 molecule plays a pivotal role in cellular responses to DNA damage and other genomic aberrations, and it is closely linked to disease progression [36–38]. p53 interacts with downstream transactivators, such as PUMA/BBC3 and GADD45A, and other downstream transcriptional targets of apoptosis, serving as mediators of stress signaling [39, 40]. GADD45A has been shown to be involved in various cellular and biological processes, including DNA damage, cell proliferation, cell cycle regulation, and apoptosis [41], whereas PUMA/BBC3 is a key mediator of p53-dependent apoptosis [42, 43]. Therefore, investigating the functions and regulatory mechanisms of molecules like p53, PUMA/BBC3, and GADD45A is of great importance for cancer prevention and treatment. MMP can dose-dependently increase the expression of GADD45A, p21 and PUMA in GC cells. Moreover, treating GC cells with the p53 inhibitor PFA and si-p53 can rescue the inhibitory effects induced by MMP. Therefore, these results further support our findings that MMP induces apoptosis in GC cells through the p53 downstream pathway.

In our study, we observed an increase in the protein levels, but not mRNA levels, of p53 and p-p53 in GC cells following MMP treatment. The accumulation of p53 is an early and crucial step for p53 activation. Under normal physiological conditions, p53 is tightly regulated with a short half-life, partly due to the MDM2-p53 autoregulatory feedback loop [44–46]. MDM2 binds to the N-terminal transactivation domain of p53 via its p53-binding domain, and the RING domain of MDM2 catalyzes the ubiquitination of p53 [47]. The ubiquitinated p53 is subsequently degraded by the 26 S proteasomes in the cytoplasm or nucleus [48, 49]. In line with this regulatory mechanism, our results indeed revealed that MMP treatment stabilizes p53 protein levels and reduces the ubiquitination of p53. Furthermore, subsequent Co-IP experiments confirmed that MMP treatment decreases the binding of p53 to MDM2. In addition, the binding sites of p53 to MDM2 are located between residues 18 to 26, with Phe 19, Trp 23, and Leu 26 identified as the key binding residues [50]. Our molecular docking results showed that MMP could bind to p53 protein through the sites ASN-30, SER-33, and TYR-205, which are in proximity to the MDM2 binding sites (Fig. S6). This spatial arrangement may elucidate how MMP could interrupt the regulation between MDM2 and p53. Cancer is an extraordinarily heterogeneous disease. The most common spontaneous mutational inactivation event, or inhibition by a viral or bacterial etiology, in cancer is the inactivation of the p53 protein and the p53 pathway. The frequency of TP53 mutations in gastric cancer is nearly 50% [51]. Our results indicate that MMP can effectively inhibit the growth of AGS cells (with wild-type TP53) and in vivo tumorigenesis, suggesting a promising therapeutic application direction/strategy for TP53 wild-type GC patients. Notably, MMP also reduced cell viability by more than 60% in the TP53 mutation gastric cancer cell lines (NUGC3 and MKN-1). These findings suggest the existence of additional regulatory mechanisms underlying the effects of MMP treatment on TP53-mutated GC cells. Subsequent analyses revealed that MMP could elicit a modest increase rate of apoptosis in NUGC3 cells, but it failed to trigger apoptosis in MKN-1 cells. Intriguingly, MMP caused G1 cell cycle arrest in both cell lines, suggesting that MMP inhibit the viability of p53 mutant cell through cell cycle arrest rather than by inducing apoptosis via the p53 signaling pathway (Fig. S4). Furthermore, our findings revealed that MMP treatment significantly suppresses the PI3K/Akt/mTOR signaling pathway (Fig. S5 a) and enhances autophagy flux, as evidenced by increased levels of p-ULK1, p-beclin-1 and LC3I/II while concurrently decreasing p62 expression (Fig. S5 b). Therefore, MMP may inhibit the growth of TP53-mutated GC through these signaling pathway modulations, and the precise mechanism remains to be explored in future studies.

Sum up, this study provides a theoretical basis for determining the anticancer effects of MMP in the treatment of gastric cancer and its potential application in clinical studies and targeted therapies. Meanwhile, in vivo evaluations confirmed the safety of the compound. It is expected to provide new ideas for the development of novel anticancer drugs or drug combinations to improve therapeutic efficacy and to promote further exploration of marine biological resources in the field of cancer medicine.

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Abbreviations

GC

Gastric cancer

MMP

Methyl mycophenolate

MPA

Mycophenolic acid

CDK2

Cyclin-Dependent Kinase 2

CDK4

Cyclin-Dependent Kinase 4

Bcl-2

B-cell lymphoma 2

Bax

Bcl-2-associated X protein

TP53I3

Tumor protein p53 inducible protein 3

BBC3

Bcl-2 Binding Component 3

IHC

Immunohistochemistry

ECL

Enhanced chemiluminescence

CHX

Cycloheximide

Author contributions

H.Y. Yu: Study conceptualization, original draft preparation, funding acquisition, Methodology and manuscript review/editing. X.F. Liu: Validation, Visualization, formal analysis, and original draft writing. N. Xu: Methodology design and manuscript revision. J. Wang: Experimental investigations, data curation, and methodological development. K.N. Chen, H.W. Ke, Y.F. Xu: Data curation and methodological implementation. D.Y. Feng & L.S. Xiao: Experimental validation and verification. X.Q. Meng: Study conceptualization, methodology design, funding acquisition, and manuscript review/editing. S. Chen: Project administration, data coordination, and supervision. All authors critically reviewed the manuscript and approved the final version for publication.

Funding

This work was financially supported by the National Natural Science Foundation of China (82273133, 82102973); the Guangzhou Science and Technology Program Project (2025A03J4388); Guangdong Basic and Applied Basic Research Foundation (2023A1515030261).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Animal handling and experimental procedures were approved by the Animal Experimental Ethics Committee of the institute. Animal studies: No. 2021-014.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.