Cell lines

Four GC cell lines (AGS, NCI-N87, MKN45, and HGC-27) were purchased from ATCC. The human GC cell line SNU-1 was obtained from Boster Biologics (Wuhan, China), and the human esophageal adenocarcinoma cell line SK-GT-4 was purchased from Meisen CTCC (CTCC-007-0167, China). AGS and SNU-1 are gastric adenocarcinoma cell lines with wild-type (WT) TP53. NCI-N87 is a gastric adenocarcinoma cell line harboring R248Q TP53 mutation. HGC-27 is a gastric adenocarcinoma cell line harboring P153fs TP53 mutation. SK-GT4 is an esophageal adenocarcinoma cell line harboring Q100Ter TP53 mutation (lacking NLS). All cell lines were cultured in DMEM or RPMI 1640(SNU-1, SK-GT-4) supplemented with 10% FBS and maintained at 37 °C with 5% CO2.

Immunoblotting assay

Cell and tissue samples were lysed using RIPA lysis buffer supplemented with 1% Protease Inhibitor Cocktail (CWBIO, CW2200S) and 1 mM PMSF (Beyotime, ST506). The samples were then centrifuged for 30 min at 14,000 rpm after ultrasonication. The bicinchoninic acid kit (Yeasen, #20201ES86) was used to measure protein concentration. Total proteins were separated by a 10% precast protein plus gel (Yeasen, #36252-ES10) and transferred to PVDF membranes (Millipore, IPVH00010). The PVDF membranes were blocked with 5% milk for 1 h and incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies (Jackson, #115-035-003 and #111–035-003, 1:10,000) for 2 h. Finally, the bands were visualized using an ECL Kit (Vazyme, #E412-02). The following antibodies were used for Western blotting: anti-LSD1 (CST, #4218, 1:1000), anti-p53 (ABclonal, A19585, 1:1000) and anti-Cyclin A2 (ABclonal, A19036, 1:1000).

Cell proliferation and colony formation assays

Cells were treated separately and plated into 96-well or 6-well plates at a density of 2000 cells per well and treated with GSK690 (MCE, HY-117226A) at different concentrations. For cell proliferation assays, cells in the 96-well plates were incubated in medium containing 10% Cell Counting Kit 8 (Beyotime, CCK-8) at 37 °C for 2 h. Afterward, OD values at 450 nm were measured using a microplate reader. For colony formation assays, cells in the 6-well plates were fixed with 4% paraformaldehyde fix solution (Sangon, E672002) after being treated with GSK690 for 10–15 days; GSK690 was changed every 2 days during treatment. The colonies were stained with 0.1% crystal violet (Beyotime, C0121) and photographed using a scanner. The colonies were counted using ImageJ. The above experiments were repeated at least 3 times.

Histone demethylation drug screening

A total of 3000 cells were plated into 96-well plates and then sequentially treated for 4 days with one of the inhibitors from the epigenetics compound library (MCE, #HY-L005, 5 μM). Afterward, cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. The drugs used in this study are listed in Supplementary Table S1.

Half maximal inhibitory concentration (IC50) assay

Cells were plated in 96-well plates at a density of 3000 cells per well and treated with various concentrations of GSK690 or GSK2879552. After 4 days, cell proliferation was assayed using the CCK-8 method, and the relative inhibition rate and IC50 values were calculated using GraphPad Prism 8.0.

Quantitative reverse transcription PCR (qRT-PCR)

Total RNA was extracted from tumor cells using the RNA Isolater Total RNA Extraction Reagent (Vazyme, R401-01) and then reverse transcribed into cDNA using the HiFiScript cDNA Synthesis Kit (CWBIO, CW2569M). Gene expression levels were measured by qRT-PCR using the 2 × SuperFast Universal SYBR Master Mix (CWBIO, CW3888M). The relative levels of gene expression were normalized to the expression levels of β-actin and calculated using the 2−△△Ct method. The relevant primer sequences are listed in Supplementary Table S2.

Cell cycle assay

Cells treated with GSK690 or DMSO were collected and fixed overnight in 70% ethanol at − 20 °C. The cells were stained using a cell cycle and apoptosis analysis kit (Beyotime, C1052) and analyzed using a NovoCyte flow cytometer (Agilen).

5-Ethynyl-2′-deoxyuridine (EdU) assay

The EdU assay was performed using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, C0078L) according to the manufacturer’s instructions. An Olympus fluorescence microscope was used to capture fluorescent images.

In vivo anti-cancer effect of GSK690

The 5-week-old male nude mice were purchased from SPF (Beijing, China) and maintained under specific pathogen-free conditions. A total of 5 × 106 HGC-27 cells were resuspended in a 1:1 mixture of Matrigel Matrix (Yeasen, #40183ES08) and PBS, which were injected dorsally into the right subcutaneous side of the nude mice. When tumors became visible, the mice were randomly assigned to the control, GSK690 5 mg/kg, or GSK690 10 mg/kg treatment groups and were administered GSK690 or DMSO every other day for 21 days. Every 3 days, the size of the tumors and the body weight of the mice were measured using calipers and electronic scales, respectively. After 21 days, the mice were sacrificed, and the tumors were removed, photographed, and weighed. Tumor volume was calculated using the formula: volume = length × width2 × 0.5.

Immunohistochemistry (IHC)

Xenograft tumors were fixed in 10% neutral formalin fix solution (Sangon, E672001) for 24 h after resection and then embedded in paraffin. Fixed tumors and tissues were processed into 4 mm paraffin sections, which were dried at 72 °C, dewaxed, dehydrated, antigen-repaired, sealed, and incubated overnight with primary antibodies at 4 °C. Secondary antibody incubation, DAB staining, and other related procedures were performed on the 2nd day. Images were captured using an Olympus microscope. The primary antibodies used in the assay included anti-LSD1 (CST, #4218, 1:200) and anti-Cyclin A2 (ABclonal, A19036, 1:200).

Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR)

Initially, cells were fixed with formaldehyde at a final concentration of 1% for 10 min. The cell lysates were sheared by sonication to generate 400–800 bp fragments. ChIP assays were performed using anti-p53 (ABclonal, A19585), anti-H3K9me2 (Abcam, ab1220) and IgG (Beyotime, A0208) via a ChIP Assay Kit (Beyotime, P2078). The immunoprecipitation products were purified and quantified by qPCR. The relevant primer sequences are listed in Supplementary Table S2.

Dual-luciferase reporter assays

The wild-type or mutant promoter regions of LSD1 were cloned into the reporter gene vector PGL3-basic, respectively. The resultant reporter plasmids were co-transfected with the p53 overexpression plasmid into cells, and 48-h post-transfection, the samples were subjected to dual-luciferase reporter assays using the dual-luciferase reporter assay kit (Vazyme, DL101-01) according to the manufacturer’s instructions. The relevant primer sequences are listed in Supplementary Table S2.

Study approval

The animal experiments were approved by the Institutional Animal Care and Use Committee of the affiliated hospital of Jiangnan University (approval number: JN.No2024093-0b0301207 [522]).

Statistical analyses

GraphPad Prism 8.0 was used for all statistical analyses. An unpaired Student’s t-test was used to analyze differences between 2 groups. One-way analysis of variance (ANOVA) was used to analyze differences among multiple groups. P < 0.05 indicated a significant difference.