All the solvents used in the study are of analytical quality and were procured from Sigma Aldrich (Castle Hill, NSW, Australia). Magnesium acetate (MgA), sodium propionate (NaP), sodium butyrate (NaB), dexamethasone (Dex) and doxorubicin (Dox) were also purchased from Sigma Aldrich (Castle Hill, NSW, Australia). Furthermore, all reagents were prepared according to the standard methods and protocols provided with the assay kits.

AGS (CRL-1739, ATCC) and HS738.St/Int cell lines (CRL-7869, ATCC) were purchased from the American Type Culture Collection (ATCC, United States). AGS cells were grown in the ATCC-formulated F-12 K medium (Kaighn’s Modification of Ham’s F-12 Medium) containing 2 mM L-glutamine and 1500 mg/L sodium bicarbonate with 10% fetal bovine serum (Bio-Strategy PTY, Campbellfield, VIC, Australia), and supplemented with 1% penicillin and streptomycin (Sigma-Aldrich, Castle Hill, NSW, Australia). The HS738.St/Int cells were grown in the ATCC-formulated DMEM (Dulbecco’s Modified Eagle Medium) comprised of 4.5 g/L glucose, L-glutamine, and sodium pyruvate supplemented with 10% fetal bovine serum (Bio-Strategy PTY Campbellfield, VIC, Australia), and supplemented with 1% penicillin and streptomycin (Sigma Aldrich, Castle Hill, NSW, Australia). These cells were maintained at 37°C in a 5% controlled CO₂ atmosphere and cell maintenance was performed every 48–72 h, the time necessary for cells to achieve confluent monolayers.

The murine RAW264.7 macrophage cells were cultured in DMEM (Lonza Australia, Mount Waverley, VIC, Australia) supplemented with 5% FBS (Bio-Strategy PTY Campbellfield, VIC, Australia), and supplemented with 1% penicillin and streptomycin (Sigma-Aldrich, Castle Hill, NSW, Australia) at 37°C in a humidified incubator with 5% CO₂.

The cell viability of the AGS cells after treatment with different concentrations of the three postbiotics (MgA, NaP, NaB) and Dex was determined using the Alamar blue assay as per the method described earlier (28, 29). Briefly, 100 μL of cells were cultured in 96 well-plates at a seeding density of 10⁵ cells/mL. After 24 h, the cells were treated with the compounds and incubated for 72 h. A positive control using Dox at a concentration of 1 μM was included, and a negative control with 0.1% DMSO was added to every plate. At the end of the incubation period, the culture media were removed, and 100 μL of a 0.1 mg/mL Alamar blue solution (resazurin, prepared as a stock solution at 1 mg/mL in freshly made PBS followed by a 1:10 dilution with serum-free media) was added to each well. The fluorescence levels were assessed with a microplate spectrophotometer (BMG CLARIOstar, Mornington, VIC, Australia) using an excitation wavelength of 555 nm and measuring emission at 595 nm. The compounds were tested in triplicate, with the negative control taken as 100% cell viability.

Dex was combined with the most active postbiotic in nine different ratios (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, v/v) for combination index (CI) analyses. The potential interactions between Dex and the most active postbiotic were analyzed using the CI model and the CompuSyn version 2.0 (Biosoft, CA, United States) was used for the CI calculations based on the median-effect equation, which was derived from mass action law (28). In the current study, the nine pairwise combinations of Dex and the postbiotic were studied with a six-point dose–response curve using the CI model.

The effect of the most active gut metabolites and their most synergistic combinations on the oxidative stress of the cancer cells was studied as per our recently reported protocol using the H2DCFDA (2′,7′-dichlorofluorescein diacetate) cellular reactive oxygen species (ROS) Detection Assay Kit (#ab113851; Abcam, Melbourne, VIC, Australia) (19, 29). Briefly, AGS cells (2.5 × 10⁵ cells/mL) were cultured in a 96-well plate, adhered overnight, and treated with 20 μM H2DCFDA for 45 min to assess ROS levels. The dye solution was removed, and cells were washed with 1x buffer. Next, the cells were treated with 750, 1,500 and 3,000 μg/mL of SCFAs, 50, 100 and 200 μg/mL of Dex, 0.54 μg/mL (1 μM) of Dox, and 250 μM of tert-Butyl hydroperoxide (TBHP) and incubated at 37°C for 4 h. Finally, the plate was immediately read at Ex/Em = 485/535 nm using a microplate spectrophotometer (BMG CLARIOstar, VIC, Australia). The fold increase in ROS production was determined relative to the untreated control (cells treated with the supplement buffer according to the manufacturer’s protocol).

Nitric oxide (NO) production in the RAW264.7 macrophage cells, stimulated with lipopolysaccharides (LPS), was assessed by measuring total nitrite content using Griess reagents (a mixture of an equal amount of 1% sulphanilamide in 5% phosphoric acid and 0.1% N-1-(naphthyl)ethylenediamine dihydrochloride). The cells were seeded at a density of 0.85 × 10⁶/mL in a 96-well culture plate, incubated for 45 h, treated with different concentrations of Dex, the most active postbiotic and their combination and then stimulated with 50 ng/mL of LPS for an additional 16 h. Cell supernatant was collected, mixed with Griess reagents, and absorbance was measured at 540 nm using a microplate spectrophotometer (BMG CLARIOstar, Mornington, VIC, Australia).

The impact of the most potent postbiotic and its most synergistic combination with Dex on the apoptosis profiles of the AGS adenocarcinoma cells was studied using an annexin V and 7-AAD-based kit (#ab214663, Abcam, Melbourne, VIC, Australia) (19, 29). The AGS cells were cultured in T75 cell culture flasks with an initial density of 1 × 10⁶ cells per 10 mL at 37°C in the presence of 5% CO₂ for 24 h. The following day, the cell culture media was removed from each flask and replaced with fresh FBS-containing media, and the cultured flasks were then treated with the highest concentration of the most active postbiotic (3,000 μg/mL), 200 μg/mL of Dex, 0.54 μg/mL (1 μM) of Dox (used as a positive control). Serum-containing medium was used as the untreated control. The flasks were then incubated at 37°C with 5% CO₂ for an additional 24 h.

The apoptotic profiles of the AGS cells after 24 h were studied using the Abcam apoptosis detection kit following the manufacturer’s instructions. Briefly, the cell culture media from each flask was collected. Subsequently, trypsin (0.25% w/v) was applied to the flasks for 3 min at 37°C. The trypsin reaction was neutralized with an equal volume of 10% FBS serum-containing media, and the cells were combined with the previously collected media. The cell pellets were obtained by centrifuging at 500 × g for 5 min at room temperature (RT). This procedure was repeated by suspending the cell pellets in 1 mL of PBS each time. The collected cell pellets from each treatment were immediately suspended in 500 μL of 1x binding buffer and gently mixed by pipetting. Annexin V-CF blue (5 μL) and 7- AAD (5 μL) staining solutions were added to 100 μL of cell suspension. The stained cells were incubated for 15 min in the dark at RT, after which 400 μL of a 1x assay buffer was added to each cell suspension. Subsequently, the cells were examined using a flow cytometer (Novocyte 3,000, ACEA Biosciences Inc., CA, United States), and data analysis and processing were performed using NovoExpress software (version 1.5.0, ACEA Biosciences Inc., CA, United States). In the initial step, the cells were gated on forward and side scatter modes to exclude cell aggregates and debris near the origin. Following that, the cells were gated on dot plots, where Annexin V-CF in Pacific Blue was plotted against 7-AAD fluorescence in PerCP. Quadrants were positioned relative to the untreated control, indicating live cells (+Annexin V and − 7-AAD) appearing in the lower-left quadrant, early apoptotic cells (+Annexin V and − 7-AAD) in the lower-right quadrant, late apoptotic cells (+Annexin V and + 7-AAD) in the upper-right quadrant, and necrotic cells (−Annexin V and + 7-AAD) in the upper-left quadrant. For statistical analyses and visualization, the percentage data of cells in each quadrant after different treatments (n = 3) were exported to GraphPad Prism software (version 9.0, San Diego, CA, United States).

Data were collected and managed using MS Office - Excel and GraphPad Prism for both statistical analyses and visualization. Data collection and analyses were carried out in triplicate, and the outcomes were presented as the mean ± standard deviation. Statistical significance between the mean values was determined at p < 0.05 employing a two-way ANOVA. To perform multiple comparisons, Tukey and Dunnett’s tests were utilized within the GraphPad Prism software. Furthermore, the IC₅₀ value (representing the concentration of a drug required to achieve a 50% cell growth inhibition) was computed using GraphPad Prism software. Nonlinear regression and determination of IC₅₀ values were carried out using GraphPad Prism 9.0.

Three SCFA salts, namely MgA, NaP and NaB at different concentrations, were investigated for their anti-proliferative activity against the AGS adenocarcinoma cells following a 72-h treatment using the Alamar Blue assay. All three SCFA salts exhibited significant anti-proliferative activity against the AGS cells, with NaB showing the most significant dose-dependent activity (93.75–3,000 μg/mL) followed by NaP and MgA (Table 1). The standard anti-inflammatory and chemotherapeutic drug Dex also displayed dose-dependent activity (6.25–200 μg/mL) against the AGS cells (Table 1).

The antiproliferative effect of postbiotics has been extensively investigated in different types of cancer including breast and colon cancers; however, only a few studies have explored the antiproliferative action of postbiotics on GC (8, 35–37). In a previous study, NaB at 4.0 mM was found to inhibit the growth of the AGS cells by 81.54% using the colorimetric MTS assay (26). Other studies found that the inhibitory rate of NaB on AGS cells could reach 59.19% at an intervention concentration of 10 mmol/L using the MTT assay (38). Another in vitro study from our research group showed that 3,000 μg/mL (27.25 mM) NaB treatment showed 81.73% growth inhibition of the MCF7 cells using Alamar Blue assay (19). In the current study, 3,000 μg/mL (27.25 mM) and 1,500 μg/mL (18.17 mM) of NaB exhibited AGS anti-proliferative activity of 100.36 ± 1.23% and 99.62 ± 0.84%, respectively using Alamar blue assay with the IC₅₀ value of 193.97 ± 5.55 μg/mL (1.76 mM; Table 1).

NaP displayed a growth inhibition value of 95.15 ± 1.99% at 3000 μg/mL, with an IC₅₀ value of 736.87 ± 157.56 μg/mL (7.67 mM) against the AGS cells (Table 1). While the cell inhibition rates were not as high as those observed with NaB, this investigation stands as one of the first reports of the antiproliferative effects of NaP against the AGS gastric adenocarcinoma cells. A previous investigation examined the impact of NaP on the MCF7 and MDA-MB-231 breast cancer cells, revealing a significant decrease in the viability of both cell lines when exposed to 0.5–20 mM NaP over 48 h in a dose-dependent manner (39). In the present study, NaP emerged as the second most effective postbiotic in inhibiting the growth of AGS cells in a dose-dependent manner. Specifically, at concentrations of 3,000 and 1,500 μg/mL, NaP induced mean cell growth inhibition of 95.15 and 81.51%, respectively.

MgA demonstrated growth inhibition value of 81.47 ± 20.28% at 3000 μg/mL (21.07 mM) and 54.12% at 1500 μg/mL (10.53 mM) against the AGS cells, with an IC₅₀ value of 1033.03 ± 694.18 μg/mL (7.25 mM; Table 1). Few in vitro studies have previously investigated the cytotoxicity and cell proliferation inhibition against the AGS cells demonstrating 50% inhibition of AGS cells at 12.5 mM (40) using Quick cell proliferation assay. Differences in outcomes between the current and the previous studies may arise from the distinct nature of acetate salts, which could exhibit variations in pharmacokinetic and pharmacodynamic activities as well as the differences in the assays implemented.

The highest concentration of Dex (200 μg/mL) resulted in substantial growth inhibition of 80.46% in the AGS cells in a dose-dependent manner (Table 1). At 100 μg/mL, 39.38% cell inhibition was observed with lower concentrations displaying no activity. The IC₅₀ of Dex in AGS cells was determined to be 86.60 ± 11.85 μg/mL. The current study, to the best of our knowledge, is one of the first reports to demonstrate the antiproliferative effects of Dex against GC cells.

Testing anticancer drugs on normal cell lines is crucial to assess their selectivity and potential side effects, providing critical insights into the therapeutic index and safety profile of these drugs in order to minimize harm to healthy tissues during treatment (41). The most active SCFA NaB, and the chemotherapeutic drug Dex were tested against the Hs 738.St/Int human normal Intestine cells using the Alamar blue assay (Table 1). NaB at the highest tested concentrations of 3,000 μg/mL, and Dex at 200 μg/mL displayed cell viability values of 106.91 ± 9.25% and 106.86 ± 2.58%, respectively in the Hs 738.St/Int human normal intestine cells. This is presumably because SCFAs are the primary energy source for intestinal epithelial cells in the gut (42), allowing NaB to induce the proliferation of normal Hs 738.St/Int human normal intestine cells in our study. These results demonstrate the favorable safety profile of NaB and Dex against the healthy cells, indicating their potential for use in future therapies.

The potential synergistic interactions between Dex with NaB were evaluated using the CI model as per our previous study (28). The CI model quantifies the potential interactions between drug–drug combinations into three categories: (a) synergistic effect: CI value <1, (b) additive effect: CI = 1, and (c) antagonistic effect: CI value >1. Nine different ratios of Dex and NaB (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1; Table 2) were evaluated against the AGS cells and strong synergistic interactions were observed between Dex and NaB at a 2:8 (40 μg/mL Dex + 2,400 μg/mL NaB) ratio with CI values <1 compared to the mono treatments of Dex and NaB. The synergistic ratio was also evaluated for its impact on the cell viability (%) of the Hs 738.St/Int normal intestine cell line (Table 2).

Following the synergy study, cell growth inhibition and cell viability of the most active combination (2:8) were evaluated in the AGS cells and the Hs 738.St/Int normal intestine cells, respectively. The synergistic combination at 305–2440 μg/mL demonstrated 80.32–103.78% cell growth inhibition of the AGS gastric adenocarcinoma cells (Table 3). All tested concentrations of the 2:8 combination improved the cell viability of the normal Hs 738.St/Int intestine cells, indicating a good safety profile (Table 3).

Oxidative stress is a hallmark of carcinogenesis. Reactive oxygen species (ROS) function as a significant biomarker in cancer and play a crucial role not only in the initiation of cancer but also in its progression and metastasis (43). Consequently, inhibiting the production of ROS by cancer cells is often regarded as a valuable approach for the development of novel anticancer drugs. Our investigation is focused on examining the impact of the most potent postbiotics and their most synergistic combination on the oxidative stress levels within AGS cancer cells.

MgA and NaP reduced ROS production in the AGS cells at all tested concentrations (3,000 μg/mL, 1,500 μg/mL, and 750 μg/mL; Figure 1). This is in contrast with previous studies which showed that MgA and NaP induced ROS production in Caco-2 colorectal cancer cells and mouse glomerular mesangialn cells (43, 44). At all tested concentrations (3,000 μg/mL, 1,500 μg/mL, and 750 μg/mL), NaB significantly enhanced ROS production compared to the negative control (p < 0.0001) suggesting that NaB may have pro-oxidative properties in the AGS cells (Figure 1). Previously, it was reported NaB increased levels of ROS after 48 h at 1, 5, and 10 mM, triggering the onset of apoptosis in the MCF7 breast cancer cells (19, 45).

Treatment with Dex, (200 μg/mL, 100 μg/mL, and 50 μg/mL), displayed a significant reduction in ROS production compared to the untreated control (p ≤ 0.01). The lower fold change values indicated the potential antioxidative or ROS-suppressing effects of Dex in the AGS cells (Figure 1). The combination treatment showed a unique pattern of ROS modulation, representing a potential interaction between NaB and Dex. The combination demonstrated a substantial fold increase in ROS production compared to Dex alone (p < 0.05), but a decrease compared to NaB alone. This suggested that Dex in the synergistic combination potentially counteracted some of the pro-oxidative effects of NaB observed in isolation. The observed combination effect indicates that Dex, known for its anti-inflammatory properties, might counterbalance or mitigate the pro-oxidative impact of NaB. The ability of the combination to moderate ROS production could have implications for oxidative stress regulation, potentially highlighting a synergistic or protective interaction between NaB and Dex.

The anti-inflammatory activity of Dex, NaB, and their most synergistic combination (2:8) was measured by their ability to inhibit the production of NO in RAW264.7 macrophage cells stimulated with LPS. NaB, Dex, and their combination dose-dependently inhibited NO production, as shown in Figure 2. The standard anti-inflammatory drug Dex showed the greatest inhibition of NO production (IC₅₀ = 81.8 μg/mL) in this study, as predicted. Interestingly, both NaB alone and its synergistic combination with Dex exhibited potent anti-inflammatory effects with IC₅₀ values of 306.7 μg/mL 296.6 μg/mL, respectively. In the Alamar Blue assay, Dex was found to be more cytotoxic to RAW264.7 macrophage cells (IC₅₀ = 24.2 μg/mL), followed by the combination Dex:NaB (IC₅₀ = 603.4 μg/mL) and NaB (IC₅₀ = 1204.0 μg/mL; Figure 2). In a previous study, butyrate significantly reduced NO production in LPS-stimulated RAW cells, and the inhibition was reversed by the removal of butyrate (46). Another study reported from the same group showed that butyrate increased NO production in the murine vascular endothelial cell line (END-D) in response to LPS or IFN-γ (47). Recently, NaB was reported to markedly suppress macrophage-driven inflammation in a non-alcoholic steatohepatitis model. It suppressed LPS-induced proinflammatory gene expression (TNF-α, IL-6) and their secretion (48). This may indicate that the anti-inflammatory effect of NaB and the combination may play a role in their antiproliferative effects against the AGS cells.

In this assay, Annexin V and 7-AAD were used to detect apoptosis and necrosis, respectively using flow cytometry. Annexin V binds phosphatidylserine (at the outer surface of cells during apoptosis) and is used to detect apoptosis, while 7-AAD (with a high affinity for guanine-cytosine residues) intercalates the double-stranded DNA indicating necrosis (49).

After 24 h of treatment, significant distinctions in the populations of viable, early apoptotic, and late apoptotic cells were evident in both the individual and combined treatments compared to the control group (Figure 3). Dex (200 μg/mL) treatment resulted in a notable proportion of living cells (38.95%), with a moderate percentage of early apoptotic cells (31.86%) and late apoptotic cells (28.84%), and a very low proportion of necrotic cells (0.35%). NaB treatment (3,000 μg/mL) demonstrated a substantial increase in early apoptotic (45.20%) and late apoptotic cells (33.83%) and a moderate proportion of living cells (20.03%). The synergistic combination of Dex and NaB (Dex: NaB at 40: 2400 μg/mL, respectively) displayed a significant reduction in living cells (21.75%; p < 0.0001) and an increase in early apoptotic cells (48.97%; p < 0.0001) and comparable levels in late apoptotic cells (28.41%; p < 0.0001) to the vehicle control and the mono treatments. Necrotic cells remained at a relatively low level in the Dex-NaB combination group (Figure 3). The positive control Dox (0.54 μg/mL) showed a very low percentage of living cells (0.23%), a moderate increase in early apoptotic cells (5.38%), a substantial percentage of late apoptotic cells (55.75%), and a significant proportion of necrotic cells (38.66%; Figure 3). The synergistic Dex-NaB combination demonstrated a notable increase in early apoptotic cells compared to the monotreatments, suggesting a potential synergy in inducing apoptosis. These findings highlighted the potential of the Dex:NaB combination in promoting apoptotic pathways and warrant further investigation into its therapeutic implications.

The bottom-up label-free quantification proteomics analysis using NanoUPLC-qTOF-MS was performed as per the protocol from a recent study from our group to understand the primary difference in the expression of key biomarkers (related to apoptotic and cancer pathogenesis) after treatment with the most potent postbiotic- NaB, its most synergistic combination with Dex (2:8), Dex alone and the vehicle control (19). The expressed proteins in NaB only, Dex only and 2:8 Dex-NaB combination treated AGS cells were analyzed in pairwise comparisons to the control group to trace the difference in the proteome-wide dysregulated expressions that may be associated with the cytotoxic effects of the combination. The proteins that exhibited differential expression in the treated AGS cells, in comparison to the control group, were chosen based on significance levels, with an adjusted value of p of ≤0.01 alongside an absolute FC ≥ 2 (absolute log2 FC ≥ 1) cutoffs.

The proteins that demonstrated statistical significance as a result of the analysis were subsequently classified according to whether they were upregulated or downregulated in response to the treatments. In order to elucidate the molecular pathways that might be associated with the quantified proteins, different online tools were used to identify the potential pathways activated by the dysregulated proteins in pairwise comparisons, including Reactome (version 83), UniProt (release 2022_04), g:Profiler (Ensembl version 107, Ensembl Genomes version 54, Wormbase ParaSite version 17), and STRING (version 12.0).