Cell Lines and Culture Conditions
Human GC cell line MKN45 cells, AGS cells, and normal gastric epithelial cells (GES-1 cells) were purchased from Procell (Wuhan, China). All cell lines were cultured in RPMI-1640 medium (Hyclone, Beijing, China) supplemented with 10% fetal bovine serum (Procell, Wuhan, China), 1% penicillin/streptomycin (Gibco, Shanghai, China) and 1% glutamine (Beyotime, Shanghai, China). All cells were grown in a 37°C cell incubator maintained at 5% CO2.
Reagents
The following antibodies were used in the experiment: LC3, Beclin1, SQSTM1/p62, phosphorylation-GSK-3β (ser9), GSK-3β, and β-catenin were bought from ABclonal, Wuhan, China. β-Actin, GAPDH, HRP-conjugated anti-mouse and rabbit secondary antibodies from Cell Signaling Technology, Shanghai, China. PCNA from Beyotime, Shanghai, China. ARHGAP29 from Solarbio, Beijing, China. Rhodamine (TRITC) AffiniPure Goat anti-Rabbit IgG and Fluorescein isothiocyanate (FITC) labeled Goat Anti-mouse IgG from ZSGB-BIO, Beijing, China. γ-Tocotrienol (Solarbio, Beijing, China) was dissolved using ethanol absolute and stored at -20 ℃ away from light; Oxaliplatin (Sigma, Shanghai, China) was configured as a 10 mmol solution using DMSO solvent.
Animal treatment
Animal treatment Four-week-old female BALB/c mice were purchased from the Viton Lihua Laboratory Animal Technology, Beijing, China. MKN45 cells (1.0×106) were injected for initiating location at the lower part of the head at the back of the neck. The mice were randomized into the control group (30% ethanol), γ-T3 treatment group (20mg/kg b.w.), OXA (2.0 mg/kg b.w), and γ-T3 + OXA treatment group, and received intraperitoneal injections every two days until the nude mice were sacrificed under anesthesia (phenobarbital 50mg/kg b.w. IP) on the 28th day. The xenograft from each mouse was collected, measured, and weighed. The half of xenograft was fixed in 4% polyformaldehyde for immunohistochemistry or immunofluorescence. The rest of the xenograft was frozen at -80 ºC for subsequent Western blot. The tumor volume was calculated as: W2 × (L/2), L: length; W: width. IRB: The experimental procedures were approved by the Ethics Committee of the Fourth Hospital of Harbin Medical University, China (2022-WZYSLLSC-12).
Cell viability
Cell viability was detected by MTT and MB assays according to our previous article(26, 27). MKN45 cells and AGS cells were seeded in 96-well plates overnight before treatment with different doses of γ-T3 and/or OXA for 24h, 48h, or 72h. MTT (2.0mg/ml) or Methylene blue (MB) solution was added before the end of incubation. DMSO or Elusion solution were used to lysis the cellular crystals and the cells were shaken for 30 min at room temperature. The optical density (OD) values were measured at 492nm or 620nm by an enzyme marker, and the cell viability curve was drawn by GraphPad Prism software 5.0 (GraphPad Software, LLC). All experiments were repeated three different times.
Docking of molecules
Using AutoDock Vina software (http://vina.scripps.edu/), PDB database (https://www.rcsb.org/), ligand structure library (https://pubchem.ncbi.nlm.nih.gov/), pyrx software (https://pyrx.sourceforge.io/) to calculate its Affinity (kcal/mol) value which represented the binding activity. It is generally accepted that a docking energy value of less than − 4. 25 kcal /mol indicates some binding activity, less than − 5. 0 kcal /mol indicates good binding activity, and less than − 7. 0 kcal /mol indicates strong binding activity.
Tumor sphere formation assay
The tumorigenic properties of MKN45 cells were determined by tumor sphere formation assay and using standard techniques as described previously(29). 20 ng/mL human bFGF and 50 ng/mL rhEGF were purchased from Cell Signaling Technology (CST, Shanghai, China).
Flow cytometry
MKN45 cells or AGS cells (4×105) were seeded in the bottom of a 6-well plate overnight before being treated with γ-T3 or/and OXA for 72h. The cells were washed with pre-cooled PBS, collected, and centrifuged at 2850 rpm, and blocked overnight using 75% ethanol. On the next day, Repeated washing and centrifugation were followed by resuspending using 500µl PI-FITC (Beyotime, Shanghai, China), and stained for 30 away from light. Finally, centrifugation and resuspension in 500µl PBS were analyzed by flow cytometer (BD FACSCalibur).
Western blot
MKN45 cells (4×105) were seeded in the bottom of a 6-well plate overnight before being treated with γ-T3 or/and OXA for different time points. the cells were washed with pre-cooled PBS and added the lysis buffer, including RIPA (Beyotime, Shanghai, China), Complete™ ULTRA (5892792001, Roche, Shanghai, China), and phosphatase inhibitor cocktail 2 and 3 (P5726 and P0044, Sigma, Shanghai, China). Cell lysates were centrifuged at 14,000 rpm for 25 minutes and denatured at 95°C. The protein concentrations were detected by a BCA protein assay kit (Sigma-Aldrich). The same amount of total protein in each sample was loaded in SDS-PAGE gel and transferred onto a 0.2mm PVDF membrane. The membrane was blocked with 5% nonfat milk for 1h and incubated the primary antibodies at 4°C overnight, such as ARHGAP29, pGSK-3β (ser9), GSK-3β, β-Catenin, LC3, Beclin1, p62 or GAPDH. Then, the membrane was incubated with the secondary antibody (horseradish peroxidase-coupled goat anti-rabbit antibody) for 2h at room temperature. Protein bands were viewed in the ECL Chemiluminescent System (ChemiScope S6, ClinX, Shanghai, China).
RNA extraction and RT-qPCR
The total RNA from MKN45 cells or xenografts was extracted by TRIzol reagent (HaiGene, Harbin, China) and RNA extraction reagent (G-clone, Beijing, China) according to the manufacturer’s instructions. 2µg of Total RNA was reverse transcribed using the PrimeScriptTMRT reagent Kit with gDNA Eraser (Takara, Shanghai, China), and 50ng cDNA was used for the following PCR reaction. Real-time PCR was performed using TB Green® Premix Ex Taq™ II (TII RNaseH Plus) (Takara, Shanghai, China). The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative expression of the target gene was normalized against the level of GAPDH expression. Relative mRNA expression was determined using the comparative CT (2-ΔΔCt) method. Primer obtained from the NIH Primer-BLAST was as follows: ARHGAP29:
GAPDH:
IHC and immunofluorescence
The sections of xenograft were first deparaffinized, and antigen recovery was performed using 10mmol/L sodium citrate buffer (pH = 6.0); endogenous peroxidase was removed using 3% H2O2 for 10 min, followed by blocking of nonspecific protein-binds; the primary antibody (1:50, PCNA ) and secondary antibody (1:50, goat anti-mouse IgG) were used; the staining was performed using DAB solution (Beyotime, Shanghai, China) for 2-3min, followed by hematoxylin re-staining, gradient dehydration, and neutral resin sealing before observation under the microscope. Under a 400× microscope, the number of positive cells in each field was counted in three visual fields in each section and the results were averaged(30). The steps of immunofluorescence were similar to those of immunohistochemistry, but the corresponding primary antibodies (1:50, LC3, Beclin1, or p62) were conjugated to secondary antibodies (1:50, FITC-goat anti-mouse IgG or TRITC-goat anti-rabbit IgG) with different luciferase labels; DAB staining and hematoxylin re-staining were not required; DAPI (Beyotime, Shanghai, China) was used directly to make the nuclei fluoresce blue.
Over-expression of ARHGAP29 by lentiviral mediation
ARHGAP29 plasmid was purchased from VectorBuilder (Guangzhou, China). The PlV[Exp]-Puro-CMV>Harhgap29[NM_001328664.2](ns)/FLAG:T2A: EGFP was transfected into MKN45 cells seeded in 6-well plates when reaching 30% confluence. Lipofectamine™ 3000 Reagent was purchased from Invitrogen (ThermoFisher SCIENTIFIC, Shanghai, China), and the entire transfection process was followed by Lipofectamine™ 3000 Reagent Protocol (Thermofisher.com/support). After 3 days, the infectious efficiency was evaluated by observing the FLAG expression with an inverted phase contrast microscope (Leica, Germany).
Statistical analysis
All statistical analysis was performed using GraphPad InStat software (GraphPad Software, Inc.). The data is shown as the mean ± standard deviation (SD). p < 0.05 was considered to indicate a statistically significant difference. Number: All results were repeated three times independently.
Results