Syringic acid, trypsin-EDTA solution, dichloro-dihydro-fluorescein diacetate (DCFH-DA), 4,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), Nitro Blue Tetrazolium (NBT), Sodium Dodecyl Sulfate (SDS), malondialdehyde bis(dimethyl acetal) and sodium nitrate were procured from Sigma (Steinheim, Germany). Dulbecco's modified eagle medium (DMEM), penicillin, streptomycin, β-actin, Phosphate-Buffered Saline (PBS), 3-(4,5-Di-2-yl)-2,5-ditetrazolium bromide (MTT), fetal bovine serum (FBS), Rhodamine123 (Rh-123), xanthine, catalase, glutathione reductase, Nicotinamide Adenine Dinucleotide Phosphate (NADPH) were acquired from Gibco (Suzhou, China).

Gastric cancer cell lines (AGS) were secured from Peking Union Cell Resource Center (Beijing, China) and cultured in a DMEM medium containing 10% FBS and 1% antibiotic solution. Cells were sustained at 37°C under 5% CO₂, and the nutrient medium was replaced after 2–3 days. Cell passage was performed using the trypsin-EDTA solution to obtain a 75–80% confluency.

Cytotoxicity effects of syringic acid were assessed by MTT assay as previously described (14). In 96-well plates, 1 × 10⁴ cells per each well were first seeded, after 24 h, treated with syringic acid (5, 10, 15, 20, 25, 30, 35, and 40 μg/mL) and incubated for 24 h. The MTT solution (10 μL) was added and incubated for 4 h in a CO₂ incubator, after which absorbance was measured at 540 nm by a microplate reader (BioTek Instruments, Colmar, France). Cell viability was defined as percentage to untreated cells and calculated as follows: Cell viability % = (Abs _treatedcells /Abs _{untreatedcells}) × 100.

Dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining was used to detect ROS in cells. In brief, cells were first seeded, allowed to achieve cell adhesion for 24 h, after which exposed to 25 and 30 μg/mL syringic acid. A 15 μg DCFH-DA stain was introduced to the cells and incubated in the dark at 37°C for 30 min, centrifuged and washed 3 times using ice-cold PBS to remove excess DCFH-DA staining and measured by spectrofluorometer emission filters at 488/530 nm. Ultimately, fluorescence microscopy images of the control and treated cells were measured at 450/490 nm.

Apoptotic morphological changes were determined by acridine orange/ethidium bromide (AO/EB) dual staining as previously described (15). In brief, AGS cells (1 × 10⁴ cells per each well) were first seeded in 96-well plates, after 24 h cells were treated with 25 and 30 μg/mL syringic acid and incubated at 37°C for 2 h. Cells were washed with PBS and stained with AO/EB dual stains (1:1 ratio). The control and syringic acid-treated cells were stained by AO (100 μg/mL) and EB (100 μg/mL) for 5 min, after which, cell morphologies were evaluated using a fluorescent microscope.

The diamidino-2-phenylindole (DAPI) staining was carried out as previously described (16), and Mitochondrial Membrane Potential (MMP) was also measured by Rh-123 (17). Cells were first added to 6-well plates for adherence and incubated at 37°C for 24 h in an incubator containing 5% CO₂. Cells were treated with 25 and 30 μg/mL concentrations of syringic acid for 24 h, after which, washed twice with PBS, stained with DAPI and incubated for 20 min. Subsequently, DAPI-stained cells were treated with Rh-123 stain at 37°C for 30 min, washed twice with PBS to remove the surplus stains, and analyzed for changes in Δψ_m using fluorescent microscopy with 40× magnification.

To analyse DNA fragmentation in apoptosis, the Propidium iodide (PI) staining assay was carried out as previously described (18). After cell incubation, 25 and 30 μg/mL syringic acid were added to the cells for 24 h. Cells were washed twice with PBS and stained by PI (10 μg/mL) for 5 min after which analyzed by fluorescence microscopy.

AGS cells (1 × 10⁴ cells per each well) were first seeded, after 24 h, cells were treated with 25 and 30 μg/mL syringic acid and incubated at 37°C for 2 h. Cells were harvested for superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and thiobarbituric acid reactive substances (TBARS) assays as follows: For SOD assay the xanthine-xanthine oxidase system to reduce Nitro Blue Tetrazolium (NBT) was performed as previously described (19). The reaction mixture contained 1 mM EDTA, 50 mM potassium phosphate, 100 μM xanthine, catalase (100 U/mL), 56 μM NBT, 0.02% Triton X-100, 006% BSA and cellular lysate (0.125 mg protein) at pH 7.8 and 37°C. The absorbance was recorded for 5 min at 560 nm for monitoring NBT reduction. Upon a steady state absorbance change, the reaction was started by adding xanthine oxidase (XO, 8 mU/mL). The highest NBT reduction (100%) was obtained by replacing cellular sample with distilled water in the reaction tube. Total protein concentration was measured by the method of Bradford (20) and bovine serum albumin was used as standard. The amount of protein which inhibited 50% NBT reduction was defined as one unit of SOD activity. Results were expressed as 50% NBT reduction/min/mg protein.

The TBARS assay was carried out as previously described (21). For malondialdehyde standard, a 200 μM MDA bis(dimethyl acetal) solution was prepared fresh every time the TBARS assay was performed. In a glass tube, 200 μL sodium dodecyl sulfate (SDS 8% w/v) was added to 100 μL sample and gently whirled, after which 1.5 mL sodium acetate buffer (3.5 M, pH 4) and 1.5 mL thiobarbituric acid solution (aqueous 0.8%, pH 4) was added. The final volume was brought to 4 mL by adding distilled water. The reaction mixture was vortexed and heated in boiling water for 45 min, after which, cooled and centrifuged at 6000 rpm for 10 min. The absorbance of supernatants was read at 532 nm using a spectrophotometer (Novaspec II VisibleSpectro, Pharmacia Biotech, Uppsala, Sweden). MDA level was reported as nmol MDA per mg protein.

The activity of GSH-Px was determined as previously described (22). In brief, to each sample was added 2.68 mL phosphate buffer (0.05 M, pH 7.0), 0.1 mL ethylenediaminetetraacetic acid (EDTA, 0.005 M), 0.01 mL glutathione reductase, 0.10 mL NADPH (0.0084 M), 0.01 mL sodium nitrate (1.125 M), and 0.1 mL reduced glutathione (GSH, 0.15 M). A 0.1 mL 0.0022 M hydrogen peroxide was added to the mixture to initiate the enzymatic reaction. The conversion of NADPH to oxidized NADP⁺ was recorded by the changes in the absorbance at 340 nm between 2–4 min once the enzymatic reaction was initiated. An equal volume of cell lysis buffer was used as a control. One unit of GSH-Px enzyme activity was defined as 1 mM NADPH as converted to mM NADP⁺ utilized per mg protein per min.

The activity of CAT was determined as previously described (23). Briefly, to 10 μL supernatant was added 50 μL H₂O₂ (0.036% w/w) and incubated at 37°C for 1 min, after which the reaction was ceased by addition of sulfuric acid 5N and potassium permanganate solution 0.005 N. The reaction yielded a pink color and measured at 490 nm. The difference in absorbance per unit indicated catalase activity and data were expressed as μmol H₂O₂ utilized/min/mg protein. All experiments were performed in triplicates and results were shown as mean ± standard deviation.

Total RNA was isolated using the Rneasy Mini Kit (Qiagen, Hilden, German) as per the manufacturer's protocol. cDNAs were synthesized from 1 μg of total RNA using ImProm-II Reverse Transcriptase (Promega, Madison, USA). The set of primers used in the PCR reactions are listed in Table 1. A 20 μL sample was used in the amplification reaction at 95°C for 5 min, followed by 28 cycles at 95°C for 30 s, at 54°C for 45 s, at 72°C for 30 s, and ultimately 10 min at 72°C. To validate the effect of cDNA synthesis from modified cells, β-actin was used as a control. PCR products were investigated on 1.2% Seakem agarose gels.

For Western blotting, syringic acid-treated cells were added to the protein lysis buffer and incubated at 4°C for 30 min, after which centrifuged with 14,000 rpm at 4°C for 30 min. The protein concentration in the supernatant was measured by the Bradford method. From the cell extracts, 25 and 30 μg/mL were subjected to SDS-PAGE based upon mass or volume from 8–15% and relocated to polyvinylidene fluoride (PVDF, Bio-Rad, CA, USA). The 5% skim milk was added in TBST buffer (153 mM NaCl, 10 mM Tris-HCl, 0.05% Tween 20, pH 7.5) at 20°C and incubated for 1 h. The PVDF membranes were incubated with primary antibody (p53, B-cell lymphoma 2 (BCL-2), Bad, caspase-3, caspase-9, Poly (ADP-ribose) polymerase (PARP), phosphorylated Akt (p-Akt), protein kinase B (AKT), phosphorylated mTOR (p-mTOR), mammalian target of rapamycin (mTOR), and β-actin at 4°C overnight. The PVDF membranes were washed thrice by TBST and incubated with secondary antibody (HRP conjugated) at 25°C for 2 h. The PVDF membranes were again washed thrice by TBST buffer. Enhanced chemiluminescence (ECL) detection agent (Thermo Fisher Scientific, Waltham, MA, USA) was employed to develop blots.

Data were expressed as mean ± standard deviation in triplicate equivalent experiments. The statistical analyses were performed using one-way ANOVA by SPSS (Ver. 25.0, SPSS Inc., Chicago, IL, USA) and α at 95% was defined as a statistically significant difference.

Syringic acid cytotoxicity effect at varying concentrations (5–40 μg/mL) against gastric cancer cells is represented in Figure 1A. The inhibition concentration (IC₅₀) of syringic acid was found to be at 30 μg/mL and 50% of cell death occurred in the concentration ranges between 25 and 30 μg/mL. Therefore 25 and 30 μg/mL doses of syringic acid were selected for further investigations. A significant loss of morphological changes was also observed at 25 μg/mL syringic acid treatments, and the increase in syringic acid dose to 30 μg/mL resulted in further morphological alterations (Figure 1B).

Images of DAPI-stained cells with nuclear condensation and fragmentation indicated a significant increase in apoptotic cells upon treatment with 30 μg/mL syringic acid than the control (Figure 2A). The mitochondrial membrane potential (Δψ_m) of cells treated with syringic acid (25 and 30 μg/mL) is also illustrated (Figure 2B). The fluorescence dye red colouration highlights the accumulation of Rh-123 whereas the decrease in Rh-123 accumulation can be observed as yellow greenish (Figures 2B,C). The finding therefore indicates that Δψ_m decreased in syringic acid-treated cells which consequently led to mitochondrial-mediated apoptosis. Instead, substantial apoptosis-mediated morphological changes (i.e., apoptotic body formation and chromatin condensation) were observed. The apoptotic morphological changes demonstrated by dual staining AO/EB indicated red fluorescence stains of EB, which entered nuclei of apoptotic cells (Figure 2D). In contrast, a green dye of AO was generated only by un-damaged cells. Control cells showed a bright green fluorescence nucleus, which indicates live cells (Figure 2D). Cells also showed orange colouration, indicating untimely apoptosis, whereas red-stained cells indicate DNA damaged and delayed apoptosis (Figure 2D). The PI staining also showed apoptotic and necrotic changes suggesting that 30 μg/mL syringic acid induced apoptotic morphological damage in gastric cancer cells compared to the control (Figure 2E).

The ROS status in samples treated with 25 and 30 μg/mL syringic acid significantly (P < 0.05) increased (Figure 3A) and the oxidative stress in the antioxidant enzymes (GSH-Px, CAT, SOD) declined indicating the ROS generation process (Figure 3B). Such observation is because of two methoxy groups attached to the aromatic ring of syringic acid molecule at positions 3 and 5 (11, 24).

The immunoblotting analysis showed syringic acid at 25 and 30 μg/mL notably increased cleaved caspase-3, caspase-9, BAD and PARP whereas p53 and BCL-2 decreased (Figures 4A,B). The regulation of the mTOR/AKT signaling mechanism was also assessed to validate the effect of syringic acid on cells. Therefore, the occurrence of p-AKT and p-mTOR were assessed in syringic acid-treated cells, and results showed 30 μg/mL syringic acid effectively reduced the p-AKT and p-mTOR (Figures 4C,D) in which syringic acid down-regulated p-AKT and p-mTOR expression pattern. Other changes were also identified in the levels of AKT and mTOR (Figures 4C,D), indicating that phosphorylation of AKT and mTOR were downregulated by syringic acid. Findings, therefore, suggested syringic acid promoted apoptosis in cells by suppressing the activity of the mTOR/AKT. Suppressed expression of IL-6, NF-κB, TNF-α, and COX-2 in syringic acid-treated cells showed a more than 2-fold decrease at 30 μg/mL syringic acid concentration (Figure 5).