4.1 Chemicals and Reagents
Gypenoside (purity > 99%) was procured from Selleck (Houston, TX, USA), and the working solution was prepared by dissolving it in DMSO to achieve a 100 mM concentration. XMU-MP-1 was procured from MCE (Shanghai, China), and the working solution was prepared by dissolving it in DMSO to achieve a 10 mM concentration. Lactate Content Assay Kit was acquired from Zybio (Chongqing, China), while the Glucose reagent was sourced from Maccura (Chengdu, China).
4.2 Cell Culture
The human gastric cancer cell lines AGS and HGC-27 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco, NY, USA) supplemented with 10% FBS (Biological Industries, KBH, IL) and 1% penicillin-streptomycin (Gibco, NY, USA). The cultures were sustained in cell culture dishes within a controlled humidity environment with 5% CO2 at 37℃.
4.3 CCK8 Assay
Adjust the density of HGC-27 and AGS cells to 3×103/well. Following a 48h treatment with Gyp, 10µL of CCK8 reagent (MCE, Shanghai, China) was added into each well. Subsequently, the absorbance of the samples at 450nm was measured using an enzyme-labeled instrument (Thermo Fisher, MA, USA) following a 2h incubation.
4.4 Colony Formation Assay
HGC-27 and AGS cells were seeded in 6-well plates at a density of 5×102 cells. After 48h of incubation, Gyp was added to wells at concentrations of 75 µM and 100 µM, respectively. The medium with the respective Gyp concentration was refreshed every 3 days. Following a 12-day incubation period, cells were fixed using 4% paraformaldehyde, stained with 0.1% crystal violet, and the number of clonal spots was quantified using Image J software.
4.5 Wound Healing Assay
Following 48h of Gyp treatment, HGC-27 and AGS cells were plated in 6-well plates and incubated 12h, then cell scratches were created using a 200 µL pipette tip. After replacing the 10% FBS medium with 1% FBS medium, incubation was continued and photographs were taken. The extent of the scratched area was analyzed using Image J software.
4.6 Transwell Migration and Invasion Assays
Gyp-treated cells were diluted to a cell density of approximately 5×104 cells per well. For the invasion assay, the upper chamber was exposed to Matrigel (Corning, NY, USA) and incubated at 37℃ for 12h. The cells, suspended in 200 µL of serum-free medium, were introduced into the upper chamber, with an additional 650 µL of medium containing 20% FBS being added to the lower chamber. For the migration assay, following resuspension in 200 µL of serum-free medium, the cells were seeded into the upper chamber. Simultaneously, 650 µL of medium containing 10% FBS was added to the lower chamber. After 48h of incubation, cells were fixed using 4% paraformaldehyde and subjected to 0.1% crystal violet staining. Cells undergoing migration or invasion were subsequently observed and quantified using a microscope.
4.7 Quantitative Reverse Transcription PCR
GC cells treated with Gyp for 48h were harvested, and total RNA was extracted from each cell group using the TRIzol method (Invitrogen, CA, USA). The extracted RNA was reverse transcribed into cDNA using the PrimeScriptTM RT reagent Kit (TaKaRa, Shiga, Japan). The reaction system was prepared according to the ChamQ SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). Relative expression of the target genes was calculated using the 2−△△Ct method with HPRT as the reference gene. The primer sequences for RT-qPCR are presented in Supplementary Table S1.
4.8 Network Pharmacology Analysis
Gyp-related targets were sourced from the PharmMapper database (http://www.lilab-ecust.cn/pharmmapper/), while GC-related genes were acquired through OMIM (https://www.omim.org/), Drugbank (https://www.drugbank.ca/), and Genecards (https://www.genecards.org/) databases. Metascape (https://metascape.org/) and David (https://david.ncifcrf.gov/) databases were utilized to conduct GO and KEGG enrichment analysis. The data screening principle adhered to the standard of p value < 0.05.
4.9 RNA-seq
HGC-27 cells were exposed to 100 µM Gyp for 48h. Cell lysis was carried out for total RNA extraction using Trizol reagent. The transcriptomic data was generated using Illumina Novaseq technology and further analyzed utilizing the DAVID database. The data screening principle adhered to the standard of p value < 0.05.
4.10 Metabolomics Analysis
1×107 HGC-27 cells were harvested for non-targeted metabolomics analysis. Then the cells were gently washed with PBS at 37°C. To eliminate proteins and extract the metabolites, cold methanol/acetonitrile (1:1, v/v) was added. The mixture was then moved to a fresh centrifuge tube and centrifuged at 14,000 g for 5 minutes at 4°C to obtain the supernatant. The obtained supernatant was taken for LC-MS analysis. Analysis was conducted on an ACQUITY UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 µm). The flow rate was set at 0.4 mL/min, with the column temperature maintained at 25°C, and the autosampler temperature set to 5°C. An injection volume of 2 µL was utilized, and the mass spectrometer functioned in both positive and negative ion modes.
4.11 Concentration Measurement of Lactate and Glucose
8×105 HGC-27 cells were plated in 6-cm dishes. After 12h, the experimental group received treatment with either Gyp or XMU-MP-1, while a DMSO control group was established. Following 48h of incubation, cell supernatants were collected for the determination of glucose and lactate using a fully automated biochemical analyzer (Hitachi, TKY, Japan). Protein concentration was determined by BCA assay, and the samples were subsequently normalized.
4.12 Western Blot Analysis
Cells from each group treated with Gyp for 48h were harvested and lysed using RIPA cell lysate (Solarbio, Beijing, China) and the total proteins concentration in the supernatant was determined using a BCA kit (Solarbio, Beijing, China). The proteins were separated via SDS-PAGE and transferred onto a PVDF membrane (Millipore, MA, USA). Then the membrane was blocked with 5% skimmed milk at room temperature for 2h. The primary antibody was incubated overnight at 4°C, followed by the addition of the corresponding secondary antibody and a 2h incubation. Finally, the droplet was supplemented with Super-sensitive ECL luminescence reagent (MeilunBio, Dalian, China), and images were captured using a gel imaging system (Bio-Rad, CA, USA). Details about the primary antibodies employed can be found in Supplementary Table S2.
4.13 Xenograft Model
Six-week-old female immunodeficient BALB/c nude mice were acclimated for one week under controlled conditions (18–25°C with a 12-hour light/dark cycle), with ad libitum access to chow and water. After being randomly grouped according to their body weights, mice were subcutaneously inoculated with 1×107 HGC-27 cells on the right side (4/group). After 7 days of inoculation, the mice received a daily intraperitoneal injection of Gyp or vehicle control at a dosage of 30 mg/kg for 14 consecutive days. Subsequently, the mice were euthanized via cervical dislocation while under isoflurane inhalation anesthesia and the tissues including tumors, hearts, livers, kidneys, and blood were collected for subsequent experiments. Body weight and tumor volume of the mice were measured every two or three days. Alanine Transaminase (ALT), Aspartate Transferase (AST), Blood Urea Nitrogen (BUN), and Creatinine (CREA) were analyzed using a fully automated biochemical analyzer (Hitachi, TKY, Japan). All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, while strictly adhering to the ARRIVE guidelines 2.0, and received approval from the Animal Research Ethics Committee of Guizhou Medical University (Approval No.2201515).
4.14 Immunohistochemical Staining
The tissue sections were baked at 60°C for 2h, followed by deparaffinization and rehydration. After high-pressure antigen retrieval, the slides were treated with 3% H2O2 for 15 minutes, blocked with 10% BSA for 1h, and then incubated overnight at 4°C with the primary antibody. Subsequently, the corresponding secondary antibody was applied, followed by the addition of freshly prepared DAB color development solution and hematoxylin staining. Finally, the slides were dehydrated and sealed with neutral plastic.
Statistical Analysis
All statistical analyses were conducted using GraphPad Prism 8.0 software and SPSS 25.0 software. Data are presented as mean ± standard deviation (SD). Student’s t-test analyzed group comparisons, while one-way ANOVA assessed differences among multiple groups. For comparisons involving multiple groups with independent variables, two-way ANOVA was employed. p value < 0.05 was considered statistically significant.