The MNK28 human gastric cancer cell line was obtained from the American Type Culture Collection (Manassas, VA, USA.) and maintained in RPMI-1640 supplemented with 10% (v/v) heat inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO₂.

Maslinic acid was obtained from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany) and stock solution was prepared in dimethyl sulfoxide at 1 mM. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). The bicinchoninic acid assay (BCA) kit (71285–3) was purchased from Beyotime Institute of Biotechnology (Haimen, China). Antibodies against STAT3 (cat. no. ab119352), p-STAT3 (Tyr705; cat. no. ab76315), JAK2 (cat. no. ab108596) and p-JAK2 (cat. no. ab32101) were purchased from Abcam (Cambridge, UK). B-cell lymphoma 2 (Bcl-2; cat. no. 2870), Bcl-2 associated agonist of cell death (Bad; cat. no. 9292), Bcl-2 associated X protein (Bax; cat. no. 2772) and β-actin (cat. no. 3700) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Enhanced chemiluminescence (ECL) reagent was purchased from EMD Millipore (Billerica, MA, USA). A Human IL-6 Quantikine ELISA kit (D6050), recombinant human IL-6 protein (cat. no. 206-IL-050/CF) and human IL-6 antibody (cat. no. MAB206-100) were obtained from R&D Systems, Inc. (Minneapolis, MN, USA).

MKN28 cells were seeded into 24-well plates at a density of 1×10⁴ cells/well and treated with various concentrations of maslinic acid (0, 0.1, 1 or 10 µM) at 37°C for 24 h; subsequently CCK-8 reagent was added for a further 2-h incubation at 37°C. Optical density was evaluated at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell number was determined using the trypan blue dye exclusion method (17).

The apoptotic rate of MNK28 cells was evaluated by flow cytometry using the Annexin V-fluorescein isothiocyanate (FITC)/PI double staining method. MNK28 cells were seeded in 6-well plates and treated with maslinic acid, as described above. Cells were trypsinized with 0.25% EDTA-free trypsin, then washed with PBS and centrifuged at 300 × g for 3 min, prior to incubation with 1 µg/ml Annexin V-FITC and 10 µg/ml PI for 15 min at room temperature in the dark. Samples were analyzed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and presented as two parameter dot-plots.

For each treatment group, ~1×10² cells were seeded into each well of a 6-well plate. Following incubation with 0, 0.1, 1 and 10 µM of maslinic acid at 37°C for 12 days, the cells were washed with PBS and images of each clone were captured under a light microscope.

Following treatment with maslinic acid for 24 h, MKN28 cells were collected and lysed in radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF and a protease inhibitor cocktail). The extracts were incubated on ice for 30 min and supernatants were collected by centrifugation at 13,400 × g for 10 min at 4°C. Subsequently, protein concentrations were determined by BCA assay, 30 µg protein were separated by electrophoresis on 10% SDS-PAGE gel and electro-transferred onto a polyvinylidine fluoride membrane with transfer buffer (25 mM Tris, 250 mM glycine and 20% methanol) at 100 V for 2 h. The membrane was blocked in 5% nonfat skimmed milk and probed with the corresponding primary antibodies at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated goat-anti mouse (1:5,000; cat. no., sc-2005) and mouse-anti rabbit (1:5,000; cat. no. sc-2357) secondary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Primary antibodies included anti-β-actin (1:5,000), rabbit anti-human anti-STAT3 (1:1,000), rabbit anti-human anti-p-STAT3 (1:1,000), rabbit anti-human anti-Bad (1:1,000), rabbit anti-human anti-Bcl-2 (1:1,000), rabbit anti-human anti-Bax (1:1,000), rabbit anti-human anti-JAK2 (1:1,000) and rabbit anti-human anti-p-JAK2 (1:1,000). Protein expression was detected using an ECL system (GE Healthcare Biosciences, Pittsburgh, PA, USA). For western blotting, band density was determined with Quantity One 1-D software (Bio-Rad Laboratories, Inc.). Western blotting was performed in triplicate.

The concentration of IL-6 in the cell culture supernatants was evaluated using the ELISA method. Briefly, MKN28 cells were plated in 24-well plates. Following treatment with maslinic acid for 24 h, the supernatant was harvested at 13,400 × g for 10 min at 4°C for analysis. Supernatants were analyzed using an IL-6 ELISA protein assay kit, according to the manufacturer's instructions. Color development was determined using a microplate reader (MK3; Thermo Fisher Scientific, Inc.) set to 450 nm. A standard curve was plotted using data generated by evaluation of the absorbance of recombinant-IL-6 serial dilutions.

Following serum starvation for 4 h, 10 µg/ml maslinic acid, 10 ng/ml recombinant IL-6 protein or 10 µg/ml anti-IL-6 antibody were added to the medium for 24 h of treatment at 37°C. Cell viability was determined using CCK-8 and the phosphorylation status of STAT3 was evaluated by western blot analysis as previously described.

Data are expressed as the mean ± standard deviation of the mean of separate experiments (n≥3). Student's t-test was performed for comparison of the means between the two groups, and one-way analysis of variance was used for analyzing the means of multiple groups. P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the inhibitory effects of maslinic acid on MKN28 cell viability and proliferation, the present study evaluated the cell number using trypan blue dye exclusion, and determined cell viability using the CCK-8. As presented in Fig. 1A and B, maslinic acid significantly reduced cell viability and proliferation in a dose-dependent manner with a half-maximal inhibitory concentration of 8.45 µM, P=0.032, 0.00092 and 0.000036, respectively. Colony formation assays also demonstrated a significant reduction in the size and number of colonies that received treatment with maslinic acid, P=0.047, 0.00083 and 0.00006, respectively (Fig. 1C). The results revealed that maslinic acid has an inhibitory effect on the viability and proliferation of MKN28 cells.

Subsequently, the present study examined the apoptosis rate of MKN28 cells following treatment with maslinic acid for 24 h (Fig. 1D). A significantly dose-dependent increase in the percentage of early apoptotic (Annexin V⁺/PI⁻) and late apoptotic/necrotic (Annexin V⁺/PI⁺) cells was observed following culturing of MKN28 cells with maslinic acid for 24 h, P=0.048, 0.0082 and 0.00074, respectively. Totals of 5.83% for early apoptotic and 2.64% for late apoptotic/necrotic in the control group, 12.94% for early apoptotic and 4.90% for late apoptotic/necrotic in the 0.1 µM maslinic acid treatment group, 49.81% for early apoptotic and 12.64% for late apoptotic/necrotic in the 1 µM maslinic acid treatment group and 39.38% for early apoptotic and 38.15% for late apoptotic/necrotic in the 10 µM maslinic acid treatment group, were observed. These results indicate that maslinic acid inhibits MKN28 cell viability by inducing apoptosis.

JAK/STAT signaling is involved in oncogenesis and cancer progression by upregulation of anti-apoptotic genes (18). To explore the mechanisms underlying maslinic acid induced apoptosis, the Bcl-2 protein family expression was analyzed by western blotting. Maslinic acid treatment significantly increased the expression level of Bad compared with DMSO group, P=0.0089, 0.00035 and 0.00001, respectively. Bcl-2/Bax expression level was significantly decreased in MKN28 cells treated with maslinic acid for 24 h compared with the DMSO group, P=0.0049, 0.00088 and 0.000053, respectively (Fig. 2A). These results indicated that the inhibition of proliferation due to maslinic acid treatment may result from significantly attenuated expression levels of Bcl-2 protein family products, including Bcl-2, Bax and Bad.

JAK/STAT3 signaling regulates gene products involved in various cellular processes including survival, proliferation and cell cycle progression (19). Western blot analysis revealed that maslinic acid treatment resulted in a marked downregulation of STAT3 phosphorylation levels without observable significant effects on the total STAT3 protein level in MKN28 cells. Inhibition of JAK2 phosphorylation at a concentration of 1 and 10 µM maslinic acid was observed, with negligible effects on total JAK2 protein levels, suggesting that the inhibition of p-JAK2 may contribute to the inhibition of STAT3 activity induced by maslinic acid (Fig. 2B).

IL-6 is one of the most prevalent cytokines that mediates its effects via the phosphorylation of STAT3 (20). As JAK/STAT3 activation is known to be involved in hematologic malignancies (21), it was imperative to determine whether maslinic acid had deleterious effects on the production of IL-6. The present study investigated the production of IL-6 in MKN28 cells using ELISA prior to and following maslinic acid treatment. A decrease of IL-6 protein in the medium following maslinic acid treatment was revealed (Fig. 3A). Therefore, it is likely that suppression of the JAK/STAT3 signaling pathway following maslinic acid treatment may be induced by the upstream inhibition of IL-6 expression.

Pretreatment with the anti-IL-6 antibody for 24 h also resulted in the inhibition of cell viability and phosphorylation of STAT3. The addition of recombinant IL-6 to the medium resulted in an increase in cell viability and the reactivation of STAT3 phosphorylation, suggesting that IL-6 is responsible for STAT3 activation (Fig. 3B and C). Taken together, these findings suggest that maslinic acid serves an important role in the inhibition of IL-6-mediated STAT3 activation in the MKN28 gastric cancer cell line.