The human gastric cancer SGC-7901 cell line was obtained from the cell resource center of the Shanghai Biological Sciences Institute (Chinese Academy of Sciences, Shanghai, China). KFX oral liquid was received from Sichuan Good Doctor Pharmaceutical Group (Sichuan, China), comprising 1 g/ml P. americana dried whole body in water.

SGC-7901 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere with 5% CO₂ at 37°C. The cultured cells were passaged with 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc. Waltham, MA, USA) when cell confluence reached ~80%. Cells between passage numbers 3 and 10 were selected for experimentation. Before starting the experimental procedures, the desired final concentrations of KFX (0, 0.25, 0.5, 2.5 mg/ml) were achieved by diluting the stock solution (1 g/ml) in RPMI-1640 culture medium. Then the SGC-7901 cells were placed in RPMI-1640 in the presence or absence of KFX for 12 or 24 h. In some experiments, SGC-7901 cells were exposed to a MEK inhibitor U0126 (0.2 µM, dissolved in RPMI-1640 culture medium) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 12 h. For signaling pathway analysis, SGC-7901 cells were treated with phorbol 12-myristate 13-acetate (PMA) (3 nM, dissolved in DMSO) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), a specific activator of protein kinase C, nuclear factor-κB and ERK, for 12 h in combination with KFX treatment.

The SGC-7901 cells were placed in RPMI-1640 in the presence or absence of KFX (0, 0.25, 0.5, 2.5 mg/ml) for 12 or 24 h. Four µg RNA and oligo dT₁₈ were then incubated at 80°C for 5 min. The cDNA synthesis reaction was performed at 42°C for 1 h with M-MLV reverse transcriptase (cat. no., A5001; Promega Corporation), followed by incubation at 70°C for 15 min to inactivate the reverse transcriptase. Following RT, samples were diluted by adding 60 µl purified water. For the PCR, PCR MasterMix (cat. no., PR1700; BioTeke Corporation, Beijing, China) was used and the reactions were performed in a T100 Thermo Cycler (Bio-Rad Laboratories) with the following profile: Incubation for 3 min at 95°C, followed by 32 cycles of denaturation for 30 sec at 95°C, annealing for 30 sec at 72°C, and extension for 5 min at 72°C. The products were resolved in a 1% agarose gel stained with SYBR Safe (Invitrogen; Thermo Fisher Scientific, Inc.). ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA) was used to quantify the bands (17). Primer sequences of peroxisome proliferator-activated receptor (PPAR)-γ and GAPDH for RT-PCR were as follows: PPAR-γ forward, 5′-TCTGGCCCACCAACTTTGGG-3′ and reverse, 5′-CTTCACAAGCATGAACTCCA-3′; and GAPDH forward, 5′-GCCAAGGTCATCCATGACAACT-3′ and reverse, 5′-GAGGGGCCATCCACAGTCTT-3′.

SGC-7901 cells were lysed in a buffer consisting of 7 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate, 40 mM Trizma base, 40 mM dithiothreitol and 1% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Following centrifugation at 21,885 × g for 15 min at 4°C, the total protein concentration in the supernatant was determined with a Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of protein (50 µg/lane) were subjected to SDS-PAGE (10% gel) and transferred onto polyvinylidene difluoride membranes. Samples were then blocked with 5% skimmed dried milk in Tris-buffered saline containing 0.1% TritonX-100 (TBST) at room temperature for 2 h, and incubated overnight at 4°C with the following primary antibodies: Cleaved-Caspase-3 (cat. no., 9661; dilution, 1:1,000; Cell Signaling Technology Inc., Danvers, MA, USA), Bax (cat. no., ab32503; dilution, 1:1,000; Abcam), Bcl-2 (cat. no., ab59348; dilution, 1:1,000; Abcam), p53 (cat. no., 2524; dilution, 1:1,000; Cell Signaling Technology, Inc.), IL-1β (cat. no., ab106035; dilution, 1:1,000; Abcam;), IL-6 (cat. no., ab6672; dilution, 1:1,000; Abcam), TNF-α (cat. no., ab1793; dilution, 1:5,000; Abcam), p-Erk (cat. no., 9101; dilution, 1:1,000; Cell Signaling Technology, Inc.), Erk (cat. no., 9102; dilution, 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat. no., 4970; dilution, 1:1,000; Cell Signaling Technology, Inc.). The membrane was washed with TBST three times, 5 min each. Subsequently, the membrane was incubated with a horseradish peroxidase-conjugated goat-anti-mouse (cat. no., ab6789; dilution, 1:300; Abcam) or HRP-goat-anti-rabbit (cat. no., ab6721; dilution, 1:300; Abcam) for 2 h at room temperature. Membranes were washed with TBST three times for 5 min each, prior to incubation with enhanced chemiluminescence (cat. no., WBKLS0500; Merck KGaA) for 1 min. The protein levels were normalized against that of internal protein β-actin using ImageJ software (version 1.48; National Institute of Health, Bethesda, MD, USA).

The TUNEL assay was performed using a commercially available In situ Cell Death Detection kit (Roche Diagnostics GmbH; cat. no., 11684817910), according to the manufacturer's protocol, as previously described (18). SGC-7901 cells cultured on 6-mm plates were fixed with 4% paraformaldehyde solution for 30 min at room temperature. Following a PBS wash, cells were treated with permeation solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min at 4°C. Following washing with PBS, samples were incubated with TUNEL reagent containing 10% terminal deoxynucleotidyl transferase and 2% fluorescent isothiocyanate-dUTP for 1 h at 37°C. Subsequently, the cells were stained with 1 µg/ml DAPI for 30 min at room temperature to detect the cellular nuclei. Finally, the cells are mounted on coverslips with antifade mounting medium (Beyotime, P0126). Using an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nm (green), the number of TUNEL-positive SGC-7901 cells and apoptotic bodies were determined. The percentage of apoptotic cells were calculated by dividing the number of TUNEL-positive cells by the total number of cells visualized in ≥6 separate fields using a fluorescence microscope. Three digitized images of similar total cell numbers were selected from each cover slip for counting and averaging, and were considered as one independent experiment. Three independent experiments were then averaged and statistically analyzed.

SGC-7901 cells were plated in 96-well plates at a density of 5,000 cells/well in 120 µl complete medium (RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.; cat. no., 11875093) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.; cat. no., 16000044). To investigate the cytotoxic effect of KFX, SGC-7901 cells were treated with 0.00, 0.25, 0.5, 0.75, 1 and 2.5 mg/ml for 12 and 24 h. Each group was repeated in 9 separate wells. Following treatment, 15 µl MTT reagent (5 mg/ml) was added to each well for 4 h, and then 150 µl DMSO was added to each well. Absorbance was detected at a wavelength of 490 nm using a microplate reader.

Results are expressed as the mean ± standard error of the mean. Statistical differences were assessed with the unpaired 2-tailed Student's t-test for two experimental groups and one-way ANOVA for multiple groups, using SPSS 19.0 software (IBM Corp, Armonk, NY, USA). Bonferroni's post-hoc test was employed following one-way ANOVA for determining significant differences between groups. A two-tailed P-value of <0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using GraphPad Prism (version 5; GraphPad Software).

PPAR-γ, a member of the nuclear receptor superfamily, regulates lipid metabolism, inflammation and cancer progression (19). Usually, PPAR-γ regulates target genes by binding to the PPAR-γ response element in the promoter region of target genes, resulting in either promotion or inhibition. PPAR-γ displays antitumor effects through inhibition of proliferation and induction of differentiation and apoptosis by targeting tumor-associated genes, including tumor protein p63, tumor protein p73, tumor protein p21, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax), caspase-3 and MYC proto-oncogene (17,20–22). It has been demonstrated that PPAR-γ activation inhibits cell growth (23) and promotes differentiation and apoptosis in a variety of types of cancer cell.

To investigate whether KFX treatment may lead to the upregulation of PPAR-γ, RT-qPCR analysis was conducted for SGC-7901 cells treated with KFX at 0, 0.25, 0.5 and 2.5 mg/ml for 12 and 24 h. As presented in Fig. 1A, PPAR-γ mRNA expression level was significantly increased at all concentrations after 24 h and at 2.5 mg/ml after 12 h of KFX treatment, compared with that of the control, in a dose-dependent manner (P<0.05); no significant differences were identified between the control and cells treated with 0.25 or 0.5 mg/ml after 12 h. In addition, cell viability was analyzed by MTT assay. The results demonstrated that exposure to KFX resulted in a dose-dependent decrease in cell viability in the SGC-7901 cells. The half maximal inhibitory concentration values of KFX at 12 and 24 h were 1.19±0.06 and 0.64±0.04 mg/ml (P<0.05), respectively, for the SGC-7901 cells (Fig. 1B).

To investigate the hypothesis that KFX is capable of inducing apoptosis in SGC-7901 cells, caspase-3 activity was detected using western blot analysis following treatment with 0, 0.25 or 0.50 mg/ml KFX for 12 h. Activated caspase-3, denoted by increased expression of cleaved caspase-3, was detected in the SGC-7901 cells following KFX treatment (Fig. 2A); this difference was significant compared with the negative control (P<0.05). Furthermore, to demonstrate the capability of KFX to induce apoptosis in SGC-7901 cells, the pro-survival protein Bcl-2 and the pro-apoptotic protein Bax were evaluated using western blot analysis. A decrease in the expression of Bcl-2 was revealed with KFX treatment; however, Bax expression appeared to remain unaffected. Additionally, the expression levels of p53, an initiator of cellular apoptosis, were upregulated in SGC-7901 cells following KFX treatment (Fig. 2A). These results suggested that the decrease in cell viability observed was due to cell apoptosis induced by KFX. Furthermore, a TUNEL assay was performed to detect the pro-apoptotic effect of KFX on SGC-7901 cells. Compared with the untreated control, the number of apoptosis cells was significantly increased following KFX treatment, in a dose-dependent manner (Fig. 2B).

The magnitude of inflammation is often augmented during aging and age, in turn, is a major risk factor for developing oncological diseases (24). Previous studies have demonstrated that interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α recruit immune cells into the site of a developing tumor or tumor microenvironment, thereby enhancing inflammation (25,26). To investigate the ability of KFX to abate inflammation in the microenvironment of SGC-7901 cells, the expression of IL-1β, IL-6 and TNF-α were detected with western blot analysis in the present study. Fig. 2C shows that IL-1β, IL-6 and TNF-α protein levels significantly decreased following KFX treatment compared with the levels in the untreated cells. These results indicated that KFX may alleviate the production of inflammatory cytokines in SGC-7901 cells.

Mitogen-activated protein kinase kinase (MAPKK) is involved in a number of cellular biological functions, including proliferation, differentiation, motility and death (27–29). ERKs are the main members of the MAPKK signaling pathway, and the activation of ERK1/2 is an anticancer target (30,31). Therefore, to clarify whether the ERK signaling pathway is activated in SGC-7901 cells by KFX, cells were treated with 0, 0.25 and 0.5 mg/ml KFX for 12 h in the present study. The results demonstrated that phosphorylated (p)-ERK1/2 was significantly decreased following KFX treatment, in a dose-depended manner, whereas total ERK expression remained consistent (Fig. 2D).

To further investigate the role of p-ERK1/2 in KFX-mediated SGC-7901 cell apoptosis, PMA, a specific activator of protein kinase C, NF-κB and ERK, was introduced (32). Following cell treatment with PMA, ERK phosphorylation was markedly increased (Fig. 3A). Additionally, pro-survival protein Bcl-2, inflammatory cytokines IL-1β, IL-6 and TNF-α, along with the downregulated p53 and cleaved caspase-3, were significantly increased, whereas the pro-survival protein Bax remained unchanged (Fig. 3B and C). As expected, KFX exhibited an inhibitory effect on PMA-induced anti-apoptosis via the ERK signaling pathway, as demonstrated by the decreased expression following use of KFX and PMA together.

By contrast, U0126, an inhibitor of p-ERK1/2, was used to inhibit p-ERK1/2 expression and to mimic the function of KFX on SGC-7901 cells. Following treatment with U0126, ERK phosphorylation was blocked and cleaved-caspase 3 expression was increased (Fig. 4A and B). Consistent with the results obtained by KFX treatment, U0126 treatment also exhibited a significant pro-apoptotic and anti-inflammatory effect on SGC-7901 cells (Fig. 4C), which suggested that KFX may inhibit phosphorylation in the ERK1/2 signaling pathway, blocking cell proliferation similar to U0126. In addition, the role of ERK-mediated SGC-7901 cell apoptosis and anti-proliferation induced by KFX was further investigated with an MTT assay. Cell viability of SGC-7901 cells incubated with PMA was significantly increased compared with that of the control, but largely abolished by KFX or U0126 (Fig. 5).