Cell culture
The human umbilical cords were received from healthy women at full-term delivery time, and the hWJSCs were derived according to our previous study [8]. In addition, the GC cell line AGS was purchased from Cell Bank of Pasteur Institute, Tehran, Iran. Both hWJSCs and AGS cells culture was performed using Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin (100 units/mL) and streptomycin (100 µg/mL). The cells incubation was performed at standard condition (37oC with 95% humidity and 5% CO2).
Preparation of hWJSC-CM and hWJSC-CL
The conditioned medium and cell lysates were prepared from cultured hWJSCs with 70% confluence, according to our previous study [8]. Briley, the culture medium was substituted with antibiotic and FBS free medium, and the hWJSCs conditioned medium (hWJSC-CM) was collected after 72 hours. Next, the cultured hWJSCs were washed once with calcium and magnesium free phosphate buffered saline (PBS(-)), dissociated using trypsin-EDTA, and centrifuged. The obtained cells pellet mixed with the cell lysis buffer with protease inhibitor. Next, cells suspension was centrifuged and supernatant was collected as hWJSCs cell lysate (hWJSC-CL). The both hWJSC-CM and hWJSC-CL were filtersterilized, and the pH were determined, and stored at -20°C.
Cancer cells viability assay
The GC cell line AGS were seeded in a 96‑well plates at density of 1.5×104 cells/well in 200 μl culture medium, and then incubated for 24 hours. Next, the cancer cells were treated with various concentrations of hWJSC-CM (38, 40, 42, 44, and 46%) and hWJSC-CL (11, 12, 13, 14, and 15%) for 24-72 hours. The treated cancer cells were washed once with PBS, and then 200 µL MTT solution (5 mg/mL MTT in culture medium) was added to the each well and incubated for 4 hours at standard condition. Next, 50 µL dimethyl sulfoxide (DMSO) was added and incubated further for 30 minutes. The optical densities (OD) at 570 nm wavelength were detected using a plate reader for each well. The relative cancer cell viability was calculated as following: cell viability=(OD570 of treated samples / OD570 of untreated samples)×100.
Wound‐healing migration assay
The cancer cells were seeded in a 6‑well plates at density of 1.5×105 cells/well in 2 ml complete culture medium until cells reached 85% confluence. Next, a wound was produced by scraping with a sterile pipette tip in middle of each well, and detached cells were washed with PBS. The cancer cells were treated with hWJSC-CM (44%) and hWJSC-CL (14%), and incubated in standard condition for 48 hours. Finally, the migrated cancer cells were counted by inverted phase‐contrast microscope at 0-20 hours.
Transwell invasion assay
The cancer cells were seeded in upper chamber of a transwell chamber at density of 2×104 cells/well in FBS serum-free culture medium and treated with hWJSC-CM (44%) and hWJSC-CL (14%). Moreover, complete culture medium supplemented with 10% FBS was added to lower chamber. After incubation for 24 hours at standard condition, the migrated cancer cells to lower chamber were fixed in 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 10 minutes. Finally, cancer cells in lower chamber were counted in 5 random fields by inverted phase‐contrast microscope.
Cancer cells apoptosis assay
The cancer cells were seeded in a 6‑well plates at density of 1.5×105 cells/well in 2 ml complete culture medium for 24 hours. The cancer cells were treated with hWJSC-CM (44%) and hWJSC-CL (14%), and incubated in standard condition for 48 hours. Next, the cancer cells were dissociated using trypsin-EDTA and resuspended in binding buffer (400 μL). Next, the FITC‑conjugated Annexin‑V (5 μl) and propidium iodide (PI) (10 μl) were added and incubated at room temperature in dark for 5 minutes. Finally, apoptosis of cancer cells was determined by flow cytometry instrument (Becton Dickinson Bioscience).
Gene expression assay by Real-Time PCR
The cancer cells were seeded in a 6‑well plates at density of 1.5×105 cells/well in 2 ml complete culture medium for 24 hours. The cancer cells were treated with hWJSC-CM (44%) and hWJSC-CL (14%), and incubated in standard condition for 48 hours. Next, the cancer cells were dissociated using trypsin-EDTA and resuspended in TRIzol reagent for extraction of total RNA. The complementary DNA (cDNA) was synthesized from 2 µg total RNA using reverse transcription reagents. The quantitative Real‐Time polymerase chain reaction (qRT‐PCR) was performed with SYBR green master mix and specific primers of BAX, BCL2, SMAC, and SURVIVIN genes (Table 1). The ACTIN (β‐actin) gene was used as endogenous control. The calculations were performed by 2-ΔΔCt (Livak) formula.
Proteins expression assay by western blotting
The cancer cells were seeded in a 6‑well plates at density of 1.5×105 cells/well in 2 ml complete culture medium for 24 hours. The cancer cells were treated with hWJSC-CM (44%) and hWJSC-CL (14%), and incubated in standard condition for 48 hours. Next, the cancer cells were dissociated using trypsin-EDTA, and lysed in ice-cold RIPA lysis buffer containing protease inhibitor. The cell lysates centrifuged and supernatant collected. The protein levels of p-JNK, JNK, p-p38, p38, p-p65, and p65 were evaluated using western blot analysis. The protein content of cells (~100 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to polyvinylidene difluoride (PVDF) membrane. The membrane was blocked using non-fat dry milk (5%) and then incubated with primary antibodies at 4°C for 24 hours. The horseradish peroxidase (HRP) conjugated antibody was used as secondary antibody for 30 minutes at room temperature. The protein detection was performed using enhanced chemiluminescent (ECL) detection system.
Statistical analysis
The all performed experiments were repeated for three times, and obtained data were presented as mean ± standard deviation (SD). The Student's t‑test, Tukey (post‑hoc), and one‑way analysis of variance (ANOVA) were used to statistical analysis using the Graph Pad Prism software. The p-value<0.05 was considered as significant.