
==== Front
J Pharm Anal
J Pharm Anal
Journal of Pharmaceutical Analysis
2095-1779
2214-0883
Xi'an Jiaotong University

S2095-1779(24)00118-7
10.1016/j.jpha.2024.101021
101021
Original Article
Terpene extract from the stem of Celastrus orbiculatus inhibits actin cytoskeleton remodelling in gastric cancer cells by regulating the protein interaction between PTBP1 and ACTN4
Chu Zewen ab1
Zhu Miao ab1
Luo Yuanyuan ab1
Hu Yaqi ab
Feng Xinyi ab
Shen Jiacheng ab
Wang Haibo whbosy@163.com
ab⁎⁎⁎
Sunagawa Masataka suna@med.showa-u.ac.jp
c⁎⁎
Liu Yanqing yzumpi@163.com
ab⁎
a Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou, Jiangsu, 225001, China
b The Key Laboratory of Syndrome Differentiation and Treatment of Gastric Cancer of the State Administration of Traditional Chinese Medicine, Yangzhou, Jiangsu, 225001, China
c Department of Physiology, School of Medicine, Showa University, Tokyo, 142-8555, Japan
⁎ Corresponding author. Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou, Jiangsu, 225001, China. yzumpi@163.com
⁎⁎ Corresponding author. suna@med.showa-u.ac.jp
⁎⁎⁎ Corresponding author. Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou, Jiangsu, 225001, China. whbosy@163.com
1 These authors contributed equally to this work.

13 6 2024
8 2024
13 6 2024
14 8 1010212 3 2024
2 6 2024
11 6 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Adjuvant chemoradiotherapy, molecular targeted therapy, and immunotherapy are frequently employed to extend the survival of patients with advanced gastric cancer (GC). However, most of these treatments have toxic side effects, drug resistance, and limited improvements in survival and quality of life. Therefore, it is crucial to discover and develop new medications targeting GC that are highly effective and have minimal toxicity. In previous studies, the total terpene extract from the stem of Celastrus orbiculatus demonstrated anti-GC activity; however, the specific mechanism was unclear. Our research utilising co-immunoprecipitation-mass spectrometry (Co-IP-MS), polypyrimidine tract binding protein 1 (ptbp1) clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9)-knockout (KO) mouse model, tissue microarray, and functional experiments suggests that alpha actinin-4 (ACTN4) could be a significant biomarker of GC. PTBP1 influences actin cytoskeleton restructuring in GC cells by interacting with ACTN4. Celastrus orbiculatus stem extract (COE) may directly target ACTN4 and affect the interaction between PTBP1 and ACTN4, thereby exerting anti-GC effects.

Graphical abstract

Image 1

Highlights

• COE inhibits the malignant biological behavior of GC cells and regulate actin cytoskeleton remodelling in GC cells.

• COE may target and bind to ACTN4, affecting the interaction between PTBP1 and ACTN4.

• ACTN4 knockdown suppresses GC cell proliferation and motility.

• COE is a potential new anti-tumour TCM.

Keywords

Traditional Chinese medicine
Polypyrimidine tract binding protein 1
Alpha actinin-4
Gastric cancer
Actin skeleton remodelling
==== Body
pmc1 Introduction

Gastric cancer (GC) is one of the most prevalent forms of cancer worldwide, holding the fifth highest incidence position in both occurrence and fatality rates [1]. Currently, surgery is the primary treatment for GC. Despite recent progress in the diagnosis and treatment, the prognosis for patients with GC remains grim because of tumour recurrence and metastasis rate [2]. Similar to other cancer types, growth and advancement of GC are influenced by intricate connections between various oncogenic and tumour-suppressor pathways [[3], [4], [5]]. Therefore, studying these signalling pathways and understanding the molecular mechanisms underlying GC development is crucial for identifying diagnostic markers and developing new anti-cancer drugs [6].

The complex system of actin filaments throughout the cell is crucial for internal movement, structural organisation, compartmentalisation, and cell shape and movement [7]. Actin filaments are critical components of the cytoskeleton [8], and their dynamic remodelling provides the structural basis and driving force for tumour cell morphological changes and metastasis [9,10]. The cytoskeleton is completely restructured during metastasis of GC cells [[11], [12], [13]]. Therefore, studying methods to prevent changes in the actin cytoskeleton of GC cells is crucial for tackling issues such as tumour recurrence and metastasis.

Traditional Chinese medicines (TCM) have been used in China for several millennia. Significant advancements have been made in the prevention and treatment of GC metastasis with TCM, owing to ongoing experimental research and clarification of the molecular biological mechanisms of anti-cancer herbs. Many herbal monomers and compound prescriptions are effective against the malignant biological behaviour of GC, and numerous herbal preparations have been used in clinical applications. TCM can effectively inhibit angiogenesis [14] and lymphangiogenesis [15], suppress epithelial-mesenchymal transition (EMT) [16,17], and regulate oncogenes and tumour suppressor genes [18,19].

Celastrus orbiculatus Thunb., a vine-like plant belonging to the Celastraceae family, possesses the therapeutic effects of dispelling wind, eliminating dampness, dredging meridians to relieve pain, and activating blood circulation for detoxification. Our research team conducted extensive studies on the anti-tumour mechanisms and safety of Celastrus orbiculatus Thunb.. The extraction method and application of Celastrus orbiculatus stem extracts (COE) have been patented in China (Patent No.: 200710025343.3) [20]. Our previous research demonstrated that COE can prevent changes in the actin cytoskeleton [21] and hinder EMT [22], but the exact mechanisms remain unknown. In an earlier study, we employed transcriptome sequencing to identify potential targets of COE in GC cells. By comparing different genes and conducting experiments, we identified and verified the importance of polypyrimidine tract binding protein 1 (PTBP1) in GC [23,24], as well as its role in preventing GC cell metastasis [25]. However, PTBP1 is not a cytoskeleton-related protein; thus, we hypothesised that it interacts with cytoskeleton proteins, thereby influencing actin cytoskeleton remodelling. This study aimed to enhance our prior discoveries, broaden our research focus, and further investigate the precise mechanisms through which PTBP1 controls actin cytoskeleton restructuring in GC cells and the influence of COE on these mechanisms. We aimed to uncover new molecular pathways that cause the inhibitory effect of COE on GC metastasis, providing a basis for understanding its multi-target inhibition and clinical use.

2 Experimental

2.1 Cell strains and cell cultivation

Human GC cell lines HGC-27, AGS, and MKN28 and GES-1 human gastric epithelial cell lines were purchased from Wuhan Procell Life Science & Technology Co., Ltd. (Wuhan, China). The Roswell Park Memorial Institute (RPMI) 1640 medium used for growth was supplemented with 10% foetal bovine serum (FBS) (TransGen Biotech, Beijing, China) and 1% penicillin-streptomycin solution (double antibiotics) (Beyotime Biotechnology, Shanghai, China). The environmental settings were maintained at 37 °C with a 5% CO2 atmosphere. For cryopreservation, the cells were stored in a freezing medium containing 55% RPMI 1640 (Hyclone, Logan, UT, USA), 40% FBS, and 5% dimethyl sulfoxide (DMSO) (BioFroxx, Aachen, Germany) at −80 and −150 °C.

2.2 Preparation of COE and analysis of its effective active compounds

Celastrus orbiculatus Thunb. (Serial No.: 170812) was purchased from Guangzhou Zhixin Pharmaceutical Company (Guangzhou, China). According to the method described in the Chinese National Invention Patent (Patent No.: ZL200710025343.3), COE was prepared, ground into fine powder, and 0.016 g of fine COE powder was placed in a centrifuge tube. Then, 100 μL of DMSO was added and the powder was fully dissolved into a dark-brown viscous liquid. Subsequently, 10 mL of serum-free medium was added, and the mixture was sterilised using a 0.22-μm microporous membrane filter to obtain COE stock solution, which was stored at 4 °C. According to the experimental protocol, the serum-free medium was diluted to the desired concentration (final concentration of DMSO <0.1%).

In the early stages of our research, we used the high performance liquid chromatography with diode array detector (HPLC-DAD) method to identify and characterise the compounds in COE [26]. A total of 26 compounds with different structural classes were isolated from COE, consisting of 6 phenylpropanoid compounds, 12 diterpenoid compounds, and 8 triterpenoid compounds. During the initial screening, certain compounds, such as hederagonic acid, betulonic acid, 3β-oleanolic acid, and 28-hydroxy-3-oxo-olean-12-en-29-oic acid, were shown to inhibit the proliferation of GC cells [27].

2.3 Screening of PTBP1 interacting proteins in AGS cell by co-immunoprecipitation-mass spectrometry (Co-IP-MS)

The Co-IP-MS experiment was conducted by Wuhan Genecreate Biological Engineering Co., Ltd. (Wuhan, China). Briefly, a clean blade was used to dig out a specific strip. The strip was cut into 0.5–0.7 mm cubes. After decolorising with test stain/silver staining decolorising solution, the rubber block with was washed three times with 500 μL of acetonitrile solution until the rubber particles were white. Five-hundred microliters of 10 mM dithiothreitol (DTT) was inserted in a tube, placed in a water bath at 56 °C for 30 min, and centrifuged for reduction reaction; subsequently, 500 μL of decolorising solution was added and mixed at room temperature for 5–10 min, then the gel spots were washed, centrifuged, and the supernatant was discarded. Five-hundred microliters of 55 mM iodoacetamide (IAM) was quickly added, then the tubes were placed in a dark room at room temperature for 30 min. The solution was then centrifuged at low speed for an alkylation reaction, and 500 μL of decolorising solution was added and mixed at room temperature for 5–10 min. The gel spots were washed, centrifuged at low speed, and the supernatant was discarded; subsequently, 500 μL of acetonitrile was added until the colloidal particles were white, and then vacuum dried for 5 min. Trypsin (0.01 μg/μL) was added according to the volume of the gel, the tubes were placed in an ice bath for 30 min, an appropriate amount of 25 mM NH4HCO3 (pH 8.0) enzymatic hydrolysis buffer was added and allowed to stand overnight at 37 °C. The next day, 300 μL of extract was added, sonicated for 10 min, centrifuged, and the supernatant was collected; this procedure was repeated twice and the obtained extracts were combined and vacuum dried.

The sample was dissolved with 10–20 μL of 0.2% trifluoroacetic acid, centrifuged at 10,000 rpm for 20 min, and the Ziptip was moistened 15 times with a wetting solution. A balance solution was used to equilibrate the sample 10 times. The solution was inhaled for 10 cycles and blown eight times with rinse liquid. A 100 μL aliquot of flushing solution was added to each tube and a separate tube was used for each sample to avoid cross-contamination. Fifty microliters of eluent was added to a clean Eppendorf tube and pipetted repeatedly to elute the peptide. Then, the sample was drained.

The peptide samples were diluted to 1 μg/μL on the machine buffet. The sample volume was set at 5 μL and scanned for 60 min. The peptides were scanned at a mass-to-charge ratio (m/z) of 350–1200 in the sample. MS data were collected using a TripleTOF™ 5600+ LC-MS system (AB SCIEX, Framingham, MA, USA). The peptide samples were dissolved in 2% acetonitrile and 0.1% formic acid and analysed using the TripleTOF™ 5600 plus mass spectrometer coupled with an Eksigent nanoLC system (AB SCIEX). The peptide solution was added to the C18 capture column (350 μm × 0.5 mm, 3 μm; AB SCIEX), and the C18 analytical column (75 μm × 150 mm, 3 μm) was applied with a 60-min time gradient and a flow rate of 300 nL/min (Welch Materials, Inc., Milford, MA, USA) for gradient elution. The two mobile phases used were buffer A (2% acetonitrile, 0.1% formic acid, and 98% H2O) and buffer B (98% acetonitrile, 0.1% formic acid, and 2% H2O). For information dependent acquisition (IDA), the MS spectrum was scanned with an ion accumulation time of 250 ms, and the MS spectrum of 30 precursor ions was acquired with an ion accumulation time of 50 ms. The MS1 spectrum was collected in the range of m/z 350–1200, and the MS2 spectrum was collected in the range of m/z 100–1500. The precursor ion dynamic exclusion time was set to 15 s.

Raw MS/MS files from the mass spectrometer were submitted to ProteinPilot (https://sciex.com.cn/products/software/proteinpilot-software) for data analyses. The Paragon algorithm in ProteinPilot was used to identify proteins by searching the UniProt database. The settings included using a TripleTOF™ 5600 instrument (AB SCIEX), modifying cysteine with IAM, and selecting biological modifications as the focus of identification. The protein results were filtered based on the following criteria: peptides with an unused score >1.3 (confidence level >95%) were deemed reliable, and proteins with at least one unique peptide were retained (Supplementary data).

2.4 Bioinformatics analysis

The QIAGEN protein database was used to predict the downstream genes of ptbp1 (Supplementary data). The gene expression profiling interactive analysis (GEPIA) [28] was used to compare cytokeratin 19 (ck19), alpha actinin-4 (actn4), tropomyosin 3 (tpm3), non-muscle myosin heavy chain IIA (myh9), myosin heavy chain 14 (myh14), villin 1 (vil1), myosin 1D (myo1d), keratin 17 (ck17), myosin 1C (myo1c), and cortactin (cttn) expressions in healthy individuals and patients with GC.

2.5 Co-IP

The cell culture liquid was carefully removed and the cells were rinsed twice with phosphate-buffered saline (PBS) (Beyotime Biotechnology). After removing the PBS, pre-chilled (at 4 °C) IP lysis was added (Thermo Fisher Scientific Inc., Waltham, MA, USA) to the cells according to the recommended volume specified in the kit instructions. The cells were incubated on ice and mixed several times. The lysate was transferred to a fresh tube and centrifuged to collect cell debris. The supernatant was transferred to a fresh tube and the protein concentration was determined using a bicinchoninic acid assay (BCA) protein assay kit (Beyotime Biotechnology). The cell lysate from each sample was combined with 10 μg of the IP antibody in a centrifuge tube. The antibody/lysate mixture was diluted to 500 μL with an IP lysis buffer. The tube was placed on a vortex mixer and stirred at 4 °C overnight to ensure thorough mixing. The magnetic beads in the bottle were thoroughly mixed by inversion, gentle vortexing, or using a rotator. Pierce protein A/G magnetic beads (25 μL (0.25 mg)) were placed in a centrifuge tube. IP lysis (175 μL) was added to the beads and gently mixed. The tube was placed on a magnetic rack to collect the beads against the side of the tube and the liquid on the top was disposed. The tube was inverted several times after adding 1 mL of lysis/wash buffer to mix the solution. The beads were collected using the DynaMag™-2 Magnet (Thermo Fisher Scientific Inc.) and the supernatant was discarded. The antigen sample and antibody mixture were combined in a tube with beads, and the tube was placed on a vortex mixer and incubated at room temperature for 1 h. IP lysis buffer was added to the tube and gently stirred. The beads were then gathered and the liquid above them was removed. This step was repeated twice. Distilled water (500 μL) was poured into the tube and gently stirred. The beads were collected using the DynaMag™-2 Magnet and the supernatant was discarded. Elution solution (100 μL) was added to the tube. The tubes were mixed and incubated at room temperature for 10 min. The beads were separated magnetically and the supernatant containing the target antigen was retained. To counteract the acidic pH, 10 μL of neutralising buffer was added for every 100 μL of eluate. Subsequently, the samples were analysed by Western blotting.

2.6 RNA binding protein immunoprecipitation (RIP) assay

RNA was extracted from the cells. The magnetic beads were prepared and 900 μL of RIP IP buffer was added to a centrifuge tube. Cell lysate supernatant (100 μL) was added to the magnetic bead-antibody complex to a volume of 1 mL, and 10 μL of lysate marked as “Input” was added. The solution was incubated overnight for 3 h. The solution was centrifuged, the centrifuge tube was placed on a magnetic rack, and the supernatant was discarded. RIP wash buffer (500 μL) was added, vortexed, the tube was placed on the magnetic rack again, and the supernatant was discarded. This washing step was repeated 3–5 times. The RNA was purified according to the kit instructions. The supernatant was carefully aspirated, air-dried, dissolved in 10–20 μL of diethyl pyrocarbonate water, and stored at a low temperature. RNA reverse transcription and complementary DNA (cDNA) verification were then performed. Using cDNA as a template, enrichment was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The number of cycles required for the fluorescence signal in each reaction tube to reach a specified threshold was defined as the average cycle threshold (CT) value for detecting target gene expression. The relative expression levels of messenger RNA (mRNA) was denoted as 2-ΔΔCT values. The ΔCT of the target gene is calculated by subtracting the CT value of the reference gene in the same sample from the CT value of the target gene. The relative cycle number (ΔΔCT value) of the target gene is determined by subtracting the average ΔCT of the target gene in the control group from the average ΔCT of the target gene in the test group. The expression in the control group was normalised.

2.7 Molecular docking

The crystal structures of ACTN4 (6OA6) [29], PTBP1-RNA recognition motif (RRM)1 (1SJQ), PTBP1-RRM2 (1SJR) [30], and RNA binding domains 3 and 4 of PTBP1 (2EVZ) [31] were selected for molecular docking simulations. The crystal structures were obtained from the Protein Data Bank (PDB) (https://www.rcsb.org/). We utilised AutoDockTools (Olson Laboratory, La Jolla, CA, USA) to manually optimise the two protein structures by removing water molecules and adding hydrogen atoms. Subsequently, the GRAMM docking server (https://gramm.compbio.ku.edu/) was used to perform protein-protein docking. The resulting protein-protein complexes were manually optimised through similar procedures such as water removal and hydrogen addition. Finally, PyMOL software (Schrödinger, Cambridge, MA, USA) was used to predict protein interactions and generate interaction maps of protein-protein complexes.

2.8 Lentiviral transfection

AGS, MKN28, and HGC-27 cells with ACTN4 knockdown/AGS cell with PTBP1 knockdown were transfected according to the “Lentiviral Vector User Manual” provided by GeneChem (Shanghai, China) (Tables S1 and S2). A suspension of AGS and MKN28 cells at a density of 2 × 104–3 × 104 cells/mL was prepared in RPMI 1640 complete medium. The suspension (500 μL) was inoculated into each well of a 24-well plate and cultured until the cell confluence reached 20%–30%. The original medium was aspirated, and 20 μL of high trans G infection enhancement solution (25×; GeneChem) was added. The corresponding amount of virus was added based on the cell multiplicity of infection (MOI) and viral titre. After incubation for 8–16 h, the virus was removed and RPMI 1640 complete medium was added. Changes in cell morphology were observed during this period. The medium was replaced intermittently to maintain cell viability. Approximately 72 h after infection, the infection efficiency was observed using an inverted fluorescence microscope. Infected cells were digested and continuously cultured in petri dishes. Puromycin was used to screen and amplify the infected cells. The cells were collected for Western blot analysis. Cells that showed normal identification results were cryopreserved at −80 and −150 °C for future use.

2.9 Western blotting

Total cellular proteins were extracted using a lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) (Beyotime Biotechnology), whereas cytoskeletal proteins were isolated using a subcellular protein extraction kit (GeneChem). After calculating the loading volume, the protein samples were mixed with 5× loading buffer (Beyotime Biotechnology) at a 1:4 ratio and heated to ensure complete denaturation. Sodium dodecyl-sulphate polyacrylamide electrophoresis (SDS-PAGE) gels were prepared based on the molecular weights of the target proteins, and the samples were loaded for electrophoresis. The proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA) and blocked. The primary antibodies were diluted according to the manufacturer's instructions and incubated with the membrane overnight at 4 °C with gentle shaking to ensure complete immersion. The membranes were then incubated with the corresponding diluted secondary antibodies for 2 h at room temperature. The luminescent reagent was prepared according to the instructions provided with the ECL Chemiluminescence Kit (New Cell & Molecular Biotech, Suzhou, China). The membrane was placed in a Molecular Imager® ChemiDoc™ XRS + gel imaging system (Bio-Rad, Hercules, CA, USA), and a luminescent reagent was applied to ensure complete immersion for imaging. The resulting images were processed and quantitatively analysed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The antibodies used in this study are listed in Table S3.

2.10 Cell Counting Kit-8 (CCK-8)

According to the instructions of the CCK-8 assay (New Cell & Molecular Biotech), cells in good growth condition (divided into untreated, short hairpin negative control (shNC), and shACTN4 groups based on the experiment) were harvested, counted, and seeded into a 96-well plate. The cells were incubated, and 200 μL of sterile PBS was added to the surrounding empty wells to prevent evaporation. For COE-related experiments, after 24 h of cell culture in the 96-well plate, the medium was replaced with fresh medium containing different concentrations of COE (20, 40, 80, 160, and 200 μg/mL), and the cells were incubated for another 24 h. After the incubation period, 10 μL of CCK-8 solution was added to each well, and the plate was incubated again for 1 h. Finally, the plate was removed from the incubator and placed in a multi-functional microplate reader (PerkinElmer, Waltham, MA, USA) to measure the absorbance at 450 nm.

2.11 Cell cycle assay

Cell pellets were then washed with ice-cold PBS. The cell suspension was centrifuged and the supernatant was removed to retain cell pellets. Ice-cold 75% ethanol (1 mL) was added to the collected cell pellet and the cells were gently pipetted to ensure thorough mixing. The cells were then fixed in a refrigerator at 4 °C for 24 h. Subsequently, the cells were centrifuged to remove the ethanol fixative, while the cell pellet was retained. Cells were dispersed by gently tapping the bottom of the centrifuge tube. They were then resuspended, washed with PBS, and centrifuged again. After tapping the bottom of the tube, a working solution (Wuhan Servicebio Biotechnology Co., Ltd., Wuhan, China) was added, and the cells were gently pipetted to mix them with the staining solution. The cells were then incubated in the dark at 37 °C for 30 min before flow cytometer analysis. The flow cytometer detected red fluorescence at an excitation wavelength of 488 nm with light scattering. Cell DNA content and light scattering were analysed using FlowJo software (Treestar, Ashland, OR, USA).

2.12 5-Ethynyl-2′-deoxyuridine (EdU) cell proliferation assay

A 2× EdU working solution (Abbkine, Wuhan, China) was prepared by diluting EdU (10 mM) to 1:500 ration with complete RPMI 1640 medium. An equal volume of pre-warmed 2× EdU working solution (20 μM) was added at 37 °C to a 6-well plate, resulting in a final EdU concentration of 1×. The cells were incubated for 2 h at 37 °C. After EdU labelling was complete, the medium was aspirated and the cells were fixed. The fixative was removed and the cells were washed three times with 1 mL of wash buffer for 3–5 min each. Next, the wash buffer was aspirated and 1 mL of Triton X-100 permeabilization solution was added to each well and incubated for 10–15 min at room temperature. The permeabilization solution was removed and the cells were washed with 1 mL of wash buffer for 5–6 min. Click reaction mixture (0.5 mL) was added to each well and then shaken gently to ensure uniform sample coverage. The mixture was incubated in the dark at room temperature. The click reaction mixture was aspirated and the cells were washed with wash buffer for 5–6 min. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and the cells were observed under an inverted fluorescence microscope (Olympus, Tokyo, Japan).

2.13 Transmission electron microscopy (TEM)

To better observe the remodelling of cytoskeletal actin, transforming growth factor-β (TGF-β) (PeproTech, Cranbury, NJ, USA) was added to GC cells at a concentration of 15 ng/mL. Subsequent experiments were conducted under the same condition. After discarding the culture medium from the cells treated with culture/COE (cell density ≤70%), trypsin was added for digestion. We observed whether the cell clumps were approximately the size of mung beans. The supernatant was discarded and an electron microscope fixative (Wuhan Servicebio Biotechnology Co., Ltd.) was added at room temperature. The cell clumps were dispersed and resuspended. The cells were fixed in the dark for 30 min before being stored at 4 °C. Subsequent TEM experiments were conducted by Wuhan Servicebio Biotechnology Co., Ltd.. Images were acquired using a TEM (Hitachi, Ltd., Tokyo, Japan).

2.14 Cytoskeleton staining and immunofluorescence (IF)

The cells were fixed with 4% paraformaldehyde, then permeabilised with 0.1% Triton X-100. Blocking solution was applied for 30 min. For labelling, tetramethylrhodamine isothiocyanate-conjugated phalloidin (Merck, Darmstadt, Germany) was incubated for 1 h at room temperature. Nuclei were counterstained by incubating the cells with DAPI/Hoechest for 3 min, and fluorescent images were visualised using a laser confocal microscope (Hitachi, Ltd.) or fluorescence microscope (Olympus).

For IF staining, ptbp1 Cas9-knockout (KO) mouse models were created [24]. After successful identification, three mice were selected from the wild-type (WT) and KO groups. The mice were sacrificed, and the target organs were carefully dissected and fixed in 4% formalin. The fixed organs were processed into paraffin sections. The paraffin sections were subjected to a series of treatments, including dewaxing, rehydration, antigen retrieval, and blocking with hydrogen peroxide. Subsequently, the sections were incubated with 3% bovine serum albumin (BSA) to prevent nonspecific binding. Next, the sections were incubated with the primary antibody diluted in PBS and incubated at 4 °C overnight. The sections were then washed with PBS and incubated with the corresponding horseradish peroxidase (HRP)-labelled secondary antibody for 50 min in the dark. The above steps were repeated for the second primary antibody, followed by incubation with the corresponding HRP-labelled secondary antibody for another 50 min. After washing with PBS, the sections were dried and incubated with DAPI for 15 min in the dark. Sections were then washed again with PBS, dried, and mounted with rhamsan gum (Shanghai Yuanye Bio-Technology, Co., Ltd., Shanghai, China). Finally, the immunostained sections were observed using a fluorescence microscope (Olympus). For the cellular immunofluorescence assay, the cells were treated and fixed; all other methods were identical.

2.15 High-content cell imaging system

After 24 h of incubation at 37 °C, the samples (4000–8000 per well) were transferred to the PerkinElmer Operetta CLS high-content imaging system (PerkinElmer). The dissection level was adjusted based on the bright-field images, and once set, the cell motility detection module was selected. Temperature, recording intervals, and culture conditions were programmed into the system. The cells were continuously monitored for an additional 12 h, during which the high-content imaging system automatically tracked cell division, proliferation, and motility. Data collection and analysis were performed using Harmony software (PerkinElmer).

2.16 Subcutaneous tumour xenografts and in-vivo imaging system (IVIS) living-image technology

BALB/c nude mice were raised under normal conditions until they were 6–7 weeks of age. GC cells in good growth conditions were expanded, digested, and centrifuged to prepare a cell suspension in Dulbecco PBS (D-PBS) (Beyotime Biotechnology). Corning Matrigel basement membrane matrix gel (Corning Inc., Corning, NY, USA) was added at a ratio of 1:6 to 1:8 and mixed thoroughly. Nude mice were randomly divided into shNC and shACTN4 groups, with five mice in each group. For COE-related experiments, nude mice were randomly divided into two groups with six mice in each group. After tumour formation, the treatment group was administered COE (40 mg/kg) by gavage (the selected dose is based on previous in vivo experiments in nude mice) [22,32], whereas the control group received the same volume of sterile saline. Under sterile conditions, 0.2 mL of a cell suspension was injected subcutaneously into the right axilla of the mice to form a small wheal. Tumour formation was observed approximately a week later. After approximately three weeks, IVIS living-image technology (PerkinElmer) was used to observe changes in the fluorescence expression of GC cells in vivo. Mice were placed in an anaesthesia box with isoflurane gas and transferred to an in vivo imaging system. The filter was adjusted to capture the images. After the mice were sacrificed, the subcutaneously transplanted tumours were dissected, and the mice were anatomised to observe whether there was tumour metastasis in the organs. Photographs of the tumours were taken, and their weights were measured and recorded.

2.17 Tissue microarray chip and multiple IF staining

Tissue microarray chips (Cat. No.: HStmA180Su17) were purchased from Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). The chips were placed in an oven and baked at 63 °C. Subsequently, the 10× repair solution was diluted to a 1× working solution and boiled in a microwave for 3 min. The slides were added and the solution was microwaved at low power for continued repair. After cooling to room temperature, the slides were rinsed with pure water. The slides were then removed, placed in a moist chamber, treated with H2O2 for 10 min, and washed with tris-buffered saline with Tween 20 (TBST). The slides were then placed in a moist chamber again, and blocking buffer was added and incubated for 10 min. After removing the blocking buffer, a diluted primary antibody working solution was added and incubated for 1 h. The slides were placed in the moist chamber and the secondary antibody was added and incubated for 10 min. Subsequently, the slides were placed in the moist chamber again and an opal dye diluent was added (dilution ratio of 1:100; PerkinElmer), incubated at room temperature for 10 min, and washed with TBST. The repair process was repeated by diluting the 10× repair solution to 1× working solution, boiling in a microwave for 3 min at high power, adding the slides, and continuing the repair for 15–20 min at low power. This staining process was repeated until all the markers were labelled. Finally, the slides were placed in a moist chamber again, DAPI working solution was added and incubated at room temperature for 5 min, and then mounted with fluorescent anti-fade mounting medium. The slides were then transferred to an automated quantitative pathological imaging system (TissueGnostics, Vienna, Austria) for quantitative analysis and photography.

2.18 Cellular thermal shift assay (CETSA)

After digestion, the supernatant was aspirated, and the cells were resuspended in PBS containing PMSF. They were then subjected to repeated freezing and thawing cycles between liquid nitrogen and a 37 °C water bath. Subsequently, the cell lysate was centrifuged at 14,000 g for 20 min at 4 °C to collect the supernatant containing the cellular components. The supernatant was divided into two portions. One portion was treated with 100 μg/mL of COE, and the other was treated with an equal volume of serum-free medium. Both solutions were incubated at room temperature for 30 min. Subsequently, the treated cell lysates were divided into six aliquots and subjected to heat denaturation at different temperatures (45, 48, 51, 54, 57, and 60 °C) for 3 min each. The denatured samples were analysed by Western blotting to detect specific proteins.

2.19 Duolink proximity ligation assay (PLA)

The Duolink® In Situ Red Starter Kit mouse/rabbit (Sigma-Aldrich, St. Louis, MO, USA) was used. Two GC cell lines (divided into COE-untreated and COE-treated groups) were deposited on glass slides and pre-treated by fixation, retrieval, and/or permeabilization. One drop of the blocking solution was added to each sample. The slides were incubated in a heated humidified chamber for 60 min at 37 °C. Primary antibodies (PTBP1 and ACTN4) were diluted to suitable concentrations in the antibody diluent. The blocking solution was removed from the slides and the primary antibody solution was added to each sample. The slides were then incubated in the humidified chamber. The optimal incubation temperature and time for the primary antibodies were determined. The primary antibody solution was removed from the slides, and they were washed twice for 5 min in 1× wash buffer A at room temperature. Excess wash buffer was removed, and the PLA probe solution was applied. The slides were incubated in a pre-heated humidified chamber for 1 h at 37 °C. The PLA probe solution was removed from the slides, and they were washed twice for 5 min in 1× wash buffer A at room temperature. Excess buffer was removed and ligation solution was applied. The slides were incubated in a pre-heated humidified chamber for 30 min at 37 °C, then the ligation solution was removed. Subsequently, the slides were washed twice for 5 min in 1× wash buffer A at room temperature. Excess wash buffer was removed and amplification solution was applied. The slides were incubated in a pre-heated humidified chamber for 100 min at 37 °C, then the amplification solution was removed, and the slides were washed twice for 10 min in 1× wash buffer B at room temperature. Excess wash buffer was removed from the slides. The nuclei were stained with DAPI and allowed to rest for 30 min before they were analysed using a fluorescence microscope (Olympus) [33].

2.20 Immunohistochemistry (IHC)

Wuhan Servicebio Technology Co., Ltd. prepared the paraffin sections following the instructions of the IHC kit (absin, Shanghai, China). Briefly, the paraffin sections were placed in a 60 °C oven for drying, followed by dewaxing and hydration in sequence. Antigen retrieval was performed on the tissue sections by placing the hydrated sections in sodium citrate and heating them in a microwave oven. After washing with PBS, a histological pen was used to draw a circle on the sections, and 100 μL of endogenous peroxidase blocker was added for incubation. The sections were washed again with PBS, followed by overnight incubation with diluted primary antibody at 4 °C. After washing with PBS, 100 μL of HRP-labelled anti-mouse/rabbit secondary antibody polymer was added under dark conditions for incubation for 30 min. The freshly prepared diaminobenzidine (DAB) chromogen solution was incubated at room temperature for 5 min, then the slides were rinsed with tap water. Haematoxylin (100 μL) was added, the slides were incubated for 3 min, and then rinsed with tap water for 10 min to achieve blue coloration. The sections were dehydrated and sequentially cleared. Rhamsan gum was dropped onto the slides for sealing, which were then fixed overnight before observation and photography using a bright-field microscope (Olympus).

2.21 qRT-PCR

The RNA Easy Fast Animal Tissue/Cell Total RNA Extraction Kit (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China) was used for RNA extraction. Briefly, the samples were processed by adding 350 μL of lysis buffer to every 10–20 mg of tissue and thoroughly homogenising the tissue using an electric homogenizer. Subsequently, 10 μL of proteinase K was added, mixed thoroughly, and incubated at room temperature for 5 min. The RNA solution was extracted according to the subsequent steps and its concentration was measured. For reverse transcription of total RNA to synthesize cDNA, the loading volume of 2 μg was calculated based on the RNA concentration. The PCR tubes were pre-cooled on ice and 2 μg of RNA, 12 μL of ddH2O, and 1 μL of oligo(dT) were added and mixed thoroughly. The mixed PCR tubes were heated in a water bath at 65 °C for 5 min and then immediately placed on ice. ribonucleases (RNase) inhibitor (1 μL), 5× reaction buffer (4 μL), and deoxynucleoside triphosphate (dNTP) mix (2 μL) were added to the cooled PCR tubes and mixed thoroughly. The PCR tubes were then incubated at 42 °C for 60 min and at 70 °C for 5 min. Subsequently, fluorescent quantitative PCR amplification (Roche, Basel, Switzerland) was performed. The results were recorded, and data processing and analysis were conducted as described in Section 2.6. The relevant primer sequences are listed in Table S4.

2.22 Colony formation assay and cell adhesion assay

For the clone formation assay, AGS GC cells were digested, counted, and reduced to a specific number based on their proliferation rate and seeded in a 6-well culture plate (Corning Inc.). The plate was incubated for 24 h, followed by 7–10 days of drug treatment. To prevent the medium from drying out during long-term culture, it was changed midway through the treatment and fresh drug was added. After the culture period, the cells were fixed and stained with a crystal violet solution. Clonal cell counting was performed using ImageJ software (National Institutes of Health). For the cell adhesion assay, the Matrigel gel (Corning Inc.) was prepared at a 1:8 ratio with serum-free RPMI 1640 medium and spread on a 24-well plate (Corning Inc.). The gel was solidified by placing it in an incubator at 37 °C. AGS cells were seeded in the Matrigel-coated 24-well plates. COE was added after 24 h and the mixture was incubated for another 24 h. Non-adherent cells were washed away with PBS, then the adherent cells were fixed and stained with crystal violet. Images were captured using a microscope (Olympus). Cell counting was performed using ImageJ software (National Institutes of Health).

2.23 Ethical statement

The study was approved by the Research Ethical Committee of Shanghai Outdo Biotech Co., Ltd., China (Approval No.: SHYJS-CP-1807021), with a waiver of the consent requirement. All animal experiments adhered strictly to the Guidelines of the Experimental Animal Care and Use System endorsed by the Animal Care and Use Committee of Yangzhou University, China (Approval No.: YXVLL-2022-93).

2.24 Statistical analysis

All experiments, except the in vivo experiments, were performed at least three times. All data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Groups were compared using an unpaired two-tailed Student's t-test or one-way analysis of variance (ANOVA). The P-value <0.05 was considered to be statistically significant.

3 Results

3.1 PTBP1 may interact with ACTN4 in GC cells

We used Co-IP-MS to screen for proteins that interact with PTBP1 in GC cell AGS (Figs. 1A and B). By comparing the identified proteins with the cluster of orthologous groups (COG) of proteins database, we predicted their potential functions and categorised them accordingly. Our findings suggest that PTBP1 may bind to cytoskeletal proteins (Fig. 1C). Subsequently, we classified the identified proteins, considering those with an unused score > 1.3 and unique peptides > 1 as confidently identified, with higher scores indicating greater confidence. We then selected the top 10 cytoskeletal proteins most likely to interact with PTBP1 [[34], [35], [36], [37], [38], [39], [40], [41], [42], [43]]. Using GEPIA to analyse the expression of these 10 genes in patients with GC versus healthy individuals (Fig. 1D), and after reviewing the relevant literature, we chose ACTN4 as our focus, hypothesising that PTBP1 may interact with ACTN4. This hypothesis was supported by predictions from the QIAGEN protein database (Fig. 1E) and validated by Co-IP experiments (Fig. 1F). RIP experiments further confirmed that PTBP1 could bind to ACTN4 RNA (Fig. 1G), and that knockdown of PTBP1 in GC cells also reduced the expression of ACTN4 (Fig. 1H). Additionally, by searching the PDB database and performing molecular docking simulations, we predicted protein-protein interactions between the four domains of PTBP1 and ACTN4, generating interaction maps that indicate the potential interaction between PTBP1 and ACTN4 (Fig. 1I).Fig. 1 Polypyrimidine tract binding protein 1 (PTBP1) may interact with alpha actinin-4 (ACTN4) in gastric cancer (GC) cells. (A) Screening of PTBP1 interacting proteins in AGS cells using co-immunoprecipitation-mass spectrometry (Co-IP-MS). (B) 217 proteins were identified, of which 41 were identified by two samples simultaneously. The number of unique proteins identified by IP and IgG was 164 and 12, respectively. (C) The proteins are identified by comparing with the Cluster of Orthologous Groups (COG) database, and their functional classification are predicted through statistical analysis. Identification results were compared to the statistical map of the COG database. The abscissa represents the number of proteins, and the ordinate represents the annotated COG entries. (D) The Gene Expression Profiling Interactive Analysis (GEPIA) website was used to analyse the Cancer Genome Atlas (TCGA) database for data on patients with GC. (E) The possible downstream target genes of PTBP1 were predicted by QIAGEN protein database analysis. (F) Co-IP assay confirmed that PTBP1 protein binds to ACTN4 protein. (G) RNA binding protein immunoprecipitation (RIP) assay confirmed that PTBP1 protein binds to ACTN4 RNA. (H) Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the changes in ACTN4 after PTBP1 knockdown in GC cells. (I) The protein-protein interaction figure was generated using PyMOL. ∗P < 0.05 and ∗∗P < 0.01. M: marker; TPM: transcripts per million; STAD: stomach adenocarcinoma; CK19: cytokeratin 19; TPM3: tropomyosin 3; MYH9: non-muscle myosin heavy chain IIA; MYH14: myosin heavy chain 14; VIL1: villin 1; MYO1D: myosin 1D; K17: keratin 17; MYO1C: myosin 1C; CTTN: cortactin; shNC: short hairpin negative control; NLS: nuclear localization signal; NES: nuclear export signal; RRM: RNA recognition motif.

Fig. 1

3.2 ACTN4 regulates the proliferation of GC cells

To investigate ACTN4 expression in GC cells, we performed Western blotting to detect ACTN4 levels in three human GC cell lines and one human gastric epithelial cell line. Compared with the gastric epithelial cells, GC cells exhibited higher ACTN4 expression, with AGS cells line showing the highest expression (Fig. 2A). Subsequently, we knocked down ACTN4 in both AGS and MKN28 GC cell lines to generate stable ACTN4-knockdown cells. CCK-8 assays demonstrated that ACTN4 knockdown suppressed the proliferative capacity of both GC cell lines compared to that of the control (Fig. 2B). Cell cycle analysis revealed an increase in the G0/G1 phase and a relative decrease in the S phase, indicating that cell cycle arrest occurs at the G0/G1 phase following ACTN4 knockdown in GC cells (Fig. 2C). Consistent with these findings, EdU assays showed that cell proliferation was inhibited by ACTN4 knockdown (Fig. 2D). Taken together, these results suggest that ACTN4 knockdown suppresses the proliferation of GC cells.Fig. 2 Alpha actinin-4 (ACTN4) regulates the proliferation of gastric cancer (GC) cells. (A) ACTN4 expression in three human GC cell lines and one human gastric mucosal epithelial cell line was detected by Western blotting; statistical plots are shown on the right. (B) Cell Counting Kit-8 (CCK-8) assay was used to detect the proliferation of two GC cell lines with stable knockdown of ACTN4 expression, and the measured absorbance values (absorption of optical density 450 (OD450)) were used to draw line charts. (C) Cell cycle assay was used to detect the percentage of cells in G1/G0, S, and G2/M phases; statistical plots are shown on the right. (D) 5-Ethynyl-2′-deoxyuridine (EdU) staining was used to detect the DNA replication activity of the two GC cell lines after ACTN4 knockdown. Data are representative of three independent experiments. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. ns: no statistical significance. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; shNC: short hairpin negative control; DAPI: 4′,6-diamidino-2-phenylindole.

Fig. 2

3.3 The actin cytoskeleton remodelling of GC cells is affected by ACTN4 knockdown

To better observe actin cytoskeleton remodelling, we induced significant cytoskeletal changes in cells by adding TGF-β. The TEM results showed that ACTN4 knockdown reduced the number of microfilaments and disrupted their arrangement in both GC cell lines (Fig. 3A). In addition, cytoskeleton staining revealed that ACTN4 knockdown decreased F-actin expression, resulting in reduced filopodia and lamellipodia formation, indicating suppressed actin cytoskeleton remodelling and migratory capacity (Fig. 3B). To elucidate the relationship between ACTN4 and the signalling pathways involved in actin cytoskeleton remodelling, we used Western blotting to assess the expression of relevant proteins in ACTN4-knockdown GC cells. Following ACTN4 knockdown, the key phosphorylation sites of ezrin/radixin/moesin (ERM) and vasodilator-stimulated phosphoprotein (VASP) were downregulated (Figs. 3C and D), which promote actin cytoskeleton remodelling. These findings suggest that ACTN4 knockdown impairs actin cytoskeleton remodelling in GC cells, indicating that ACTN4 plays a regulatory role in this process.Fig. 3 The actin cytoskeleton remodelling of gastric cancer (GC) cells is affected by alpha actinin-4 (ACTN4) knockdown. (A) Transmission electron microscopy (TEM) was used to observe the changes of microfilaments in the two GC cell lines. After the actin cytoskeleton remodelling induced by transforming growth factor-β (TGF-β), the number of microfilaments in the short hairpin (sh)ACTN4 group was less than that in the short hairpin negative control (shNC) group, and the arrangement of microfilaments tended to be disordered. Red arrows indicate the cellular microfilaments. (B) Cytoskeleton staining was used to observe the fluorescence and morphological changes of F-actin in the two GC cell lines. After adding TGF-β to induce cell actin skeleton remodelling, F-actin fluorescence was weakened, and the number of pseudopodia was relatively reduced in the shACTN4 group compared with the control group. White arrows indicate cellular filopodia and lamellipodia. (C, D) Western blotting was used to detect the expression levels of ACTN4, phospho-ezrin, phospho-radixin, phospho-moesin, ezrin, radixin and moesin, phospho-vasodilator-stimulated phosphoprotein (VASP) (Ser157), phospho-VASP (Ser239), and VASP in GC cells (C) and the quantified statistical plot drawn after statistical analysis (D). Data are representative of three independent experiments. ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. ns: no statistical significance; DAPI: 4′,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Fig. 3

3.4 The migration ability and EMT process of GC cells were inhibited after ACTN4 knockdown

We employed a high-content imaging system to observe cell motility more effectively. Using the cell segmentation, localization, and tracking module, we analysed the movement trajectories and displacement distances of the two GC cell lines over a 12 h period (Figs. 4A and B). Compared with the control group, the mean square displacement of both GC cell lines was reduced upon ACTN4 knockdown, indicating suppressed cell motility. EMT-related signalling pathways play a crucial role in GC cell migration, as epithelial cells acquire mesenchymal characteristics, leading to enhanced cell motility and migratory capacity. Therefore, Western blotting was used to assess the expression of EMT-related signalling proteins in GC cells (Fig. 4C). The results showed that ACTN4 knockdown suppressed the expression of EMT-related signalling proteins compared to the control group. In addition, we conducted a subcutaneous xenograft tumour experiment in nude mice. The volume and weight of the subcutaneous xenograft tumours in the shACTN4 group were smaller than those in the shNC control group (Figs. 4D−F), indicating that ACTN4 knockdown inhibits GC cell proliferation. We also performed IF staining for paxillin, and the results showed that paxillin fluorescence intensity in the areas of enrichment was reduced upon ACTN4 knockdown (Fig. 4G), suggesting that focal adhesions-mediated cell migration was affected. Taken together, these findings suggest that ACTN4 knockdown suppressed GC cell motility and EMT.Fig. 4 The migration ability and epithelial-mesenchymal transition (EMT) process of gastric cancer (GC) cells were inhibited after alpha actinin-4 (ACTN4) knockdown. (A) The mean square displacement vs. observation time from well level data. (B) Visualisation of cell displacement. Current displacement Y was plotted against current displacement X. Each point corresponds to the displacement of a cell at a given time point. Mean square displacement: squared norm of a vector from the first point to the current observation point; averaged over all cells per well. Current displacement X (μm): X component of a vector from the first point to the current observation point; averaged over all cells per well. Current displacement Y (μm): Y component of a vector from the first point to the current observation point; averaged over all cells per well. (C) Western blotting was used to detect the expression levels of proteins involved in EMT signalling pathway (E-cadherin, N-cadherin, vimentin, matrix metalloproteinase 9 (MMP9), and matrix metalloproteinase 2 (MMP2) in GC cells. The right panel shows the quantified statistical plot drawn after statistical analysis. (D–F) After 21 days, the mice were sacrificed, photographed (D), the tumours volume were measured (E) and the tumours were weighed (F) (n = 5). (G) Immunofluorescence (IF) was used to observe the fluorescence expression of paxillin in GC cells. White arrows indicate regions where paxillin is more enriched. Data are representative of three independent experiments. ∗P < 0.05 and ∗∗P < 0.01. shNC: short hairpin negative control.

Fig. 4

3.5 PTBP1 is co-expressed with ACTN4 and they are highly expressed and correlated in patients with GC

To explore the underlying mechanisms at the animal level, we investigated the correlation between PTBP1 and ACTN4 in a ptbp1 Cas9-KO mouse model. After euthanising the mice from both the WT and ptbp1 Cas9-KO groups, we performed IF experiments on their dissected and fixed internal organs. We selected several organs in which GC was most likely to metastasize (stomach, liver, and lungs) for IF co-staining of ACTN4 and PTBP1. The fluorescence expression of ACTN4 and PTBP1 was significantly decreased in the stomach, liver, and lungs of ptbp1 Cas9-KO mice compared to that in the WT group. Additionally, the fluorescent expression patterns of ACTN4 and PTBP1 were similar, indicating a correlation between the two proteins (Figs. 5A and B). These findings suggest that PTBP1 and ACTN4 are co-expressed in the ptbp1 Cas9-KO mouse model.Fig. 5 Polypyrimidine tract binding protein 1 (PTBP1) is co-expressed with alpha actinin-4 (ACTN4) and both are highly expressed and correlated in patients with gastric cancer (GC). (A) After the mice were sacrificed, the internal organs were removed, and the internal organs were fixed and sectioned for immunofluorescence (IF) experiments. ACTN4, PTBP1, and 4′,6-diamidino-2-phenylindole (DAPI) stainings were performed. (B) A brief schematic of creating the ptbp1 clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9)-knock out (KO) mouse model. (C) Representative images for multiplexed IF staining (ACTN4, green; PTBP1, pink; cytokeratin (CK), red; and DAPI, blue). (D, E) The number different labelled cells were counted separately to obtain their distribution in the tumour (D) and the proportional distribution of different cells corresponding to each sample (E). Each vertical bar represents a sample, and different colours represent different cells. (F) Differential expression analysis between cancer and adjacent tissues. The differences of PTBP1+CK+, ACTN4+CK+, and PTBP1+ACTN4+CK+ in GC tissues and their corresponding adjacent tissues were analysed and plotted (n = 75). (G) A cancer correlation matrix diagram; the features displayed on the X-axis and Y-axis are shown in the graph. Orange-red indicates a positive correlation, and blue indicates negative correlation. The darker the colour, the greater the correlation. In the association network diagram, the association was calculated based on Spearman's correlation analysis, and the co-expression relationship was mirrored by the selected threshold based on the selected significance identification drawing method and correlation coefficient. The red lines represent positive correlations. ∗P < 0.05 and ∗∗∗P < 0.001. WT: wild type; ATG: initiation codon; TAG: termination codon; sgRNA: single guide RNA.

Fig. 5

We conducted multiplex IF staining (PTBP1/ACTN4/DAPI/cytokeratin (CK)) on tissue microarrays from patients with GC to investigate the co-expression of PTBP1 and ACTN4. By analysing the proportions of both proteins in positively stained cells, we found that PTBP1 and ACTN4 were highly expressed in GC tissues compared with adjacent non-cancerous tissues (Fig. 5C). Furthermore, by analysing the number and proportion of positively co-expressed cells with CK-labelled gastric epithelial cells, we observed a higher positivity rate for PTBP1 and ACTN4 expression in GC epithelial cells than in the adjacent tissues (Figs. 5D and E). The positivity rates for PTBP1 and ACTN4 co-staining were also higher (Fig. 5F). Co-expression analysis revealed a correlation between PTBP1 and ACTN4 in both CK-positive and CK-negative cells (Fig. 5G). These results suggest that PTBP1 and ACTN4 were highly expressed and correlated in patients with GC.

3.6 COE inhibits the malignant biological behavior of GC cells

A CCK-8 kit was used to assess the effects of COE on the proliferation of GC cells (HGC-27, AGS, and MKN28). The results showed that COE had a significant inhibitory effect on the proliferation of the three GC cell lines, demonstrating a concentration-dependent trend. The 24 h half maximal inhibitory concentration (IC50) values for the three cell lines were as follows: HGC-27, 137.1 μg/mL; AGS, 101.1 μg/mL; and MKN28, 129.2 μg/mL. Therefore, in subsequent experiments, a dose of 100 μg/mL was selected for COE. Additionally, to observe the proliferative effects of COE on human gastric epithelial cells, we conducted experiments using GES-1 cells. The results revealed a 24 h IC50 of 203.5 μg/mL for GES-1 cells, which was significantly higher than that of the three GC cell lines. This suggests that COE has an inhibitory effect on the proliferation of GC cells while exhibiting relatively low toxicity towards human gastric epithelial cells (Fig. 6A). To verify the impact of COE on actin cytoskeleton remodelling in GC cells, we utilised cytoskeleton staining and TEM to observe the cytoskeleton after inducing significant actin cytoskeleton remodelling with TGF-β (Fig. 6B). The cytoskeleton staining results revealed that, compared to the control group, the fluorescence intensity of cytoskeleton staining in the COE group was reduced, indicating a decrease in the number of filamentous pseudopods surrounding the cells. TEM further confirmed this observation, showing a reduction in the number of microfilaments in COE-treated cells compared to that in the control group, where microfilaments were abundant and clearly visible.Fig. 6 Celastrus orbiculatus stem extract (COE) inhibits the malignant biological behavior of gastric cancer (GC) cells. (A) The left panel shows the cell viability histograms of the four cell lines treated with COE for 24 h. The right panel shows the line plot of the cell viability rate of the four cell lines after 24 h of COE treatment. (B) Transforming growth factor-β (TGF-β) (15 ng/mL) was added to induce actin skeleton remodelling, and the drug group was treated with COE 100 μg/mL. Fluorescence microscopy was used to observe the cytoskeleton staining. Transmission electron microscopy (TEM) was used to observe the structural changes of microfilaments in cells. (C–E) The mean square displacement (C), number (D), and current speed (E) of tracked cells of GC cell after COE treatment were detected using a high-content imaging system. (F) Images of the adhesion experiment results were captured using an inverted microscope. The cloned cells were stained with crystal violet and photographs were taken. The panels on the right show the statistical graphs of the adhesion experiment and the cloning formation experiment, respectively. (G) The protein expression of polypyrimidine tract binding protein 1 (PTBP1), alpha actinin-4 (ACTN4), phospho-ezrin, phospho-radixin, phospho-moesin, and phospho-vasodilator-stimulated phosphoprotein (VASP) decreased with increasing COE concentration. Data are representative of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ns: no statistical significance. IF: immunofluorescence; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Fig. 6

We employed a high-content imaging system to track cell movement dynamically. The mean-squared displacement of cells in the COE group was smaller than that in the control group (Fig. 6C). During the dynamic tracking, the number of cells identified in the COE group was lower than that in the control group (Fig. 6D). Moreover, the average speed of cell movement was significantly inhibited by COE treatment (Fig. 6E). The results of the clone formation and cell adhesion experiments indicated that the proliferation and adhesion abilities of GC cells were significantly inhibited after the addition of COE (Fig. 6F). Western blotting was used to investigate the impact of COE on the signalling pathways associated with actin cytoskeleton remodelling in GC cells. Our findings revealed that as the concentration of COE increased (25, 50, and 100 μg/mL), the protein expression levels of both PTBP1 and ACTN4 decreased. Additionally, there was a reduction in the protein expression of signalling pathways related to actin cytoskeleton remodelling (Fig. 6G). These results suggest that COE inhibits the proliferation and motility of GC cells by regulating actin cytoskeleton remodelling.

3.7 COE regulates the protein interaction between PTBP1 and ACTN4

To explore whether COE directly targets PTBP1 or ACTN4, thereby affecting the interaction between these two proteins, we conducted a CETSA assay. The results showed that in the ACTN4 group, as the temperature increased, the protein expression level in the COE group increased and was higher than that in the control group (Figs. 7A–C). However, this phenomenon was not observed in the PTBP1 group. Proteins undergo degradation with increasing temperature. Furthermore, when comparing the IC50 values of COE in AGS cells with ACTN4 knockdown (shACTN4) and the control group (shNC), we found that the IC50 value in the shACTN4 group was significantly higher than that in the control group. This suggests that COE inhibited GC cell proliferation by targeting ACTN4 (Fig. 7D). Next, we used the Duolink assay to detect the effect impact of COE on the binding between ACTN4 and PTBP1. Upon confirming the protein-protein interaction between ACTN4 and PTBP1, we also discovered that COE inhibited the protein interaction between ACTN4 and PTBP1. Additionally, the interaction between PTBP1 and ACTN4 occurred primarily in the cytoplasm, with a small portion occurring in the nucleus (Fig. 7E). Finally, IF staining indicated the co-expression of PTBP1 and ACTN4 in GC cells, with reduced fluorescence of PTBP1 and ACTN4 in the COE group compared with that into the control group (Fig. 7F). Our findings indicate that the binding of COE to ACTN4 stabilises the protein, suggesting that COE directly targets ACTN4 rather than PTBP1, thereby influencing the interaction between these two proteins and exerting its anti-GC effects. Therefore, ACTN4 may be a direct target of COE.Fig. 7 Celastrus orbiculatus stem extract (COE) regulates the protein interaction between polypyrimidine tract binding protein 1 (PTBP1) and alpha actinin-4 (ACTN4). (A) Western blotting was used to detect the expression of ACTN4 and PTBP1 in the COE and control proteins; protein degradation degree is expressed according to the band colour shade. (B, C) A hot melting curve drawn according to the protein expression levels. If the hot melting curve shifted to the right, the amount of undegraded protein in the COE group was increased compared with the control group. The melting curve of PTBP1 shows no rightward shift (B), whereas the melting curve of ACTN4 exhibits a rightward shift (C). (D) The graph shows the cell survival rate, with the Y-axis representing the cell viability (%) and the X-axis representing the concentration of COE. AGS short hairpin negative control (shNC) half maximal inhibitory concentration (IC50) is 109.9 μg/mL and AGS shACTN4 IC50 is >200 μg/mL. (E) The positive proximity ligation assay (PLA) signal is generated when the spatial distance between two target antigens (epitopes) is within 40 nm. Each PLA signal is composed of approximately 1000 bound fluorescent probes, which together form a clear red dot. (F) Immunofluorescence (IF) staining was performed to observe the expression of PTBP1 and ACTN4 in gastric cancer (GC) cells. Data are representative of three independent experiments. DAPI: 4′,6-diamidino-2-phenylindole.

Fig. 7

3.8 COE inhibits the proliferation of subcutaneous xenograft tumours by regulating the expression of PTBP1 and ACTN4 in vivo

Finally, we validated the anti-tumor activity of COE in vivo. In vivo imaging of subcutaneous xenografts was performed on days 7 and 21 to observe tumour growth. Additionally, the volume of the subcutaneous xenografts was measured every 3 days using a Vernier calliper (Fig. 8A), and the body weights of the mice were recorded. In vivo imaging results showed that the fluorescence intensity of the subcutaneous xenografts in the COE group was lower than that in the control group (Figs. 8B and C). The volume and weight of subcutaneous xenografts in the COE group were lower than those in the control group (Figs. 8D and E). Furthermore, there was no difference in body weight between the COE and control groups, indicating that COE had no adverse effects on the body weight of nude mice (Fig. 8F). IHC experiments were performed on dissected subcutaneous xenografts to detect the proliferative marker Ki67, the highly expressed protein PTBP1 in GC, and the cytoskeleton-related protein ACTN4. Positive staining results for all three markers were significantly lower in the COE group than in the control group. Additionally, there was a high degree of overlap between the positively stained areas for PTBP1 and ACTN4 (Fig. 8G). The qRT-PCR results from the subcutaneous xenografts also showed that the expression of EMT-related markers was lower in the COE group than in the control group (Fig. 8H). Taken together, these results indicate that COE inhibits the proliferation of subcutaneous xenografts in vivo by regulating the expression of PTBP1 and ACTN4.Fig. 8 Celastrus orbiculatus stem extract (COE) inhibits the proliferation of subcutaneous xenograft tumours by regulating the expression of polypyrimidine tract binding protein 1 (PTBP1) and alpha actinin-4 (ACTN4) in vivo. (A) The long and short diameters of subcutaneous transplanted tumours were measured, and the volume was calculated every three days using a vernier calliper. (B) Fluorescence intensity pictures of subcutaneous xenografts tumours in nude mice were taken by in-vivo imaging system (IVIS) living-image technology on days 7 and 21. The fluorescence intensity scale ranged from 1.2 × 109 to 1 × 1010. (C) Average radiant efficiency was analysed using IVIS living-image software. (D, E) On day 21, the nude mice were sacrificed and the xenografted tumours were photographed (D) and weighed (E). (F) Nude mice in the control and COE groups were weighed every three days (n = 6). (G) Immunohistochemical (IHC) analysis was performed on the dissected tumours. (H) Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of epithelial-mesenchymal transition (EMT)-related indicators in the tumours. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ns: no statistical significance. mRNA: messenger RNA.

Fig. 8

4 Discussion

Currently, the primary curative approach for GC involves radical surgical resection. Throughout this process, TCM plays a significant supporting role both before and after surgery. Preoperatively, the application of TCM can effectively enhance patient tolerance and create more favourable surgical conditions. Postoperatively, it promotes treatment effectiveness, reduces the incidence of complications, and accelerates patient recovery rates [44]. Although chemotherapy and targeted drug therapies are highly effective in controlling tumour growth and reducing tumour burden, the side effects of these treatments cannot be ignored [45]. Studies have shown that the combined use of TCM and chemotherapy can effectively alleviate the clinical symptoms of patients [46]. Furthermore, an increasing number of TCM extracts have been proven to exhibit anti-GC activity [47,48], highlighting the unique advantages of TCM in the clinical treatment of GC.

In this study, we explored the specific mechanism by which PTBP1 regulates actin cytoskeleton remodelling in GC cells based on previous research [[22], [23], [24], [25]]. Interestingly, as an RNA-binding protein rather than a cytoskeleton-associated protein, PTBP1 cannot directly regulate actin cytoskeleton remodelling. The specific molecular mechanism by which PTBP1 in regulating actin cytoskeleton remodelling requires further investigation. We used Co-IP-MS to identify the cytoskeleton-associated proteins that may interact with PTBP1, and screened ACTN4 for further study. ACTN4 belongs to the spectrin family of cytoskeletal proteins and is located on human chromosome 19q13.2, spanning from 38647649 to 38731589. It maintains the integrity of the cytoskeleton and regulates cell movement by interacting with actin filaments. ACTN4 interacts with many proteins involved in cytoskeletal signal transduction, including those involved in cell adhesion, metastasis, and immune cell targeting [35]. Some studies have suggested that ACTN4 can promote the metastasis of GC cells and has a carcinogenic effect [49], but the mechanism remains unclear.

Experimental results revealed that ACTN4 expression was higher in GC cells than in human gastric epithelial cells. Furthermore, ACTN4 knockdown in two GC cell lines inhibited their proliferation, motility, and EMT. As a cytoskeleton-associated protein, ACTN4 regulates actin cytoskeleton remodelling in GC cells. To validate the correlation between PTBP1 and ACTN4, we performed IF staining in tissue microarrays of a ptbp1 Cas9-KO mouse model and patient with GC. The results showed co-expression and a correlation between PTBP1 and ACTN4, confirming our hypothesis. Additionally, the high expression of ACTN4 in patients with GC suggests that it may play a crucial role in its diagnosis and treatment.

ACTN4 is an actin-binding protein that belongs to the cell cytoskeleton protein family. It interacts with numerous proteins involved in cell cytoskeleton signalling, regulating cell actin cytoskeleton remodelling. In this study, PTBP1 interacted with ACTN4 in GC cells, regulating the protein expression of ACTN4, and participated in regulating GC cell actin cytoskeleton remodelling. Therefore, PTBP1 and ACTN4 may become attractive targets for developing new therapies for GC in the future.

Next, we functionally demonstrated that COE can suppresse the malignant biological behaviour of GC cells and regulate actin cytoskeleton remodelling in GC cells, which is consistent with our previous findings. In this study, we discovered that COE affected the protein expression of both PTBP1 and ACTN4. To identify the direct target binding proteins of COE, we used CETSA and CCK-8 assays to validate that COE directly targets and binds to ACTN4. Through PLA, we found that the binding capacity between PTBP1 and ACTN4 was weakened after the addition of COE. This indicates that besides regulating PTBP1 and ACTN4 expression, COE can also reduce the protein interaction between PTBP1 and ACTN4, thereby exerting anti-GC effects. Finally, through IF and IHC stainings and in vivo imaging of subcutaneously xenograft tumours in nude mice, we verified that COE inhibited tumour proliferation by regulating the expression of PTBP1 and ACTN4 proteins in vitro and in vivo.

5 Conclusions

COE may directly target and bind to ACTN4, affecting the protein interaction between PTBP1 and ACTN4 and regulating the remodelling of the cellular actin cytoskeleton to exert its anti-GC effect. In this study, we found that PTBP1 regulates alternative splicing (AS), which can selectively splice pre-mRNA, resulting in diverse mRNAs that transcriptionally regulate various proteins and mediate processes such as tumour cell survival, proliferation, and metastasis. We hypothesised that PTBP1 mediates AS of ACTN4 and other cytoskeleton-related proteins in GC cells. In future, we will continue to delve into the relationship between AS events and the development of GC, focusing on the biological characteristics of PTBP1. This will provide a solid theoretical and experimental foundation for the further development of COE into a new anti-tumour TCM.

CRediT authorship contribution statement

Zewen Chu: Formal analysis, Investigation, Visualization, Writing – original draft. Miao Zhu: Formal analysis, Investigation, Validation. Yuanyuan Luo: Formal analysis, Investigation. Yaqi Hu: Investigation. Xinyi Feng: Investigation. Jiacheng Shen: Investigation. Haibo Wang: Conceptualization, Project administration, Writing – review & editing. Masataka Sunagawa: Conceptualization, Methodology, Resources. Yanqing Liu: Conceptualization, Project administration, Resources.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Appendix A Supplementary data

The following are the Supplementary data to this article.Multimedia component 1

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Grant Nos.: 82274603 and 82104946), the Natural Science Foundation of Jiangsu Province, China (Grant No.: BK20210817), the Traditional Chinese Medicine Science and Technology Development Project of Jiangsu Province, China (Project code: QN202008), the Young Scientific and Technological Talents Uplift Project of Jiangsu Association of Integrated Traditional Chinese and Western Medicine, China (Grant No.: JSZXTJ-2024-A05), the Postgraduate Research & Practice Innovation Program of Jiangsu Province, China (Grant No.: KYCX21_3295), and the Yangzhou University Graduate Student International Academic Exchange Special Fund Project, China.

Thanks for the Graphical abstract drawn by using Figdraw. We thank Wuhan Genecreate Biological Engineering Co., Ltd., China for the assistance with the Co-IP-MS analysis. We would like to express our gratitude to Shanghai Outdo Biotech Co., Ltd., China for supplying the tissue microarray of patients with GC and technical assistance for multiple IF. We also extend our gratitude to GemPharmatech Co., Ltd., China for the assistance in developing the PTBP1 Cas9-KO mouse model.

Peer review under responsibility of Xi'an Jiaotong University.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.jpha.2024.101021.
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