The aerial parts of I. excisoides were collected from Luanchuan County in Henan Province, China in July 2014 and authenticated by Professor Jicheng Li of Zhengzhou University (Zhengzhou, China). A voucher specimen (no. 20140706167LY) was deposited in the herbarium of the College of Pharmacy, Zhengzhou University. The human GBC cell lines GBC-SD (cat. no. CC2502) and NOZ (cat. no. CC2501), which exhibit correct short tandem repeat profiles, were originally purchased from Guangzhou Cellcook Biotech Co., Ltd (Guangzhou, China) and stored in the Henan Key Laboratory for Pharmacology of Liver Diseases (Institute of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China). The reagents used included RPMI-1640 medium (cat. no. SH30809.01) and high-glucose Dulbecco's modified Eagle's medium (DMEM; cat. no. SH30022.01; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), fetal bovine serum (FBS; cat. no. 900-108; Gemini Bio Products, West Sacramento, CA, USA), Cell Counting Kit-8 (CCK-8; cat. no. CK04; Dojindo Molecular Technologies Inc., Shanghai, China), Annexin V-FITC/Propidium iodide (PI) Apoptosis Detection kit (cat. no. 70-AP101-60; Multi Sciences Biotech Co., Ltd., Hangzhou, China), Transwell chambers with polycarbonate filters (8-µm pore size; cat. no. 3422; Corning, Inc., Corning, NY, USA), Matrigel (cat. no. 356234; BD Biosciences, Franklin Lakes, NJ, USA), TRIzol reagent (cat. no. 15596-026; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), PrimeScript™ RT Reagent kit (cat. no. DRR037A; Takara Biotechnology Co., Ltd., Dalian, China), RT² Profiler ‘human apoptosis’ polymerase chain reaction (PCR) arrays (cat. no. PAHS-012Z) and RT² Profiler ‘human extracellular matrix and adhesion molecules’ PCR arrays (cat. no. PAHS-013Z; Qiagen, Inc., Valencia, CA, USA), EvaGreen 2X qPCR MasterMix-No Dye kit (cat. no. MasterMix-S; Applied Biological Materials, Richmond, BC, Canada), Dimethylsulfoxide (DMSO; cat. no. D8371), bovine serum albumin (BSA; cat. no. A8010), RIPA lysis buffer (cat. no. R0020; Solarbio Science and Technology, Beijing, China), phosphorylated (phosphor)-NF-κB p65 (Ser536) antibody (cat. no. 3033), NF-κB p65 antibody (cat. no. 3034), phospho-Akt (Ser473) antibody (cat. no. 9271), Akt antibody (cat. no. 9272; Cell Signaling Technology, Inc., Beverly, MA, USA) and GAPDH monoclonal antibody (cat. no. 60004-1-lg; ProteinTech Group, Inc., Chicago, IL, USA). Other chemicals and reagents were of analytical grade.

The dried and powdered aerial parts of I. excisoides (3 kg) were extracted with anhydrous ether (12 L). The extract was then filtered and evaporated in a rotatory evaporator under reduced pressure. The concentrated residue (89 g) was dissolved in methanol (3.6 L) and activated carbon (108 g) was added. The mixture was heated under reflux and further filtered and evaporated to produce a crude product (71 g). The crude product was then successively separated by silica gel chromatographic column and Sephadex LH-20 column chromatography, giving a compound (56 mg). This compound was identified as UA on the basis of its mass and nuclear magnetic resonance spectra. UA (ursolic acid, C₃₀H₄₈O₃): HR-EIMS m/z 456.3608 (456.3603 calcd. for C₃₀H₄₈O₃); ¹H-NMR (C₅D₅N, 400 MHz): δ 5.52 (1H, t, J=3.5 Hz, H-12), 3.48 (1H, dd, J=9.9, 5.4 Hz, H-3α), 2.68 (1H, d, J=11.4 Hz, H-18), 1.27, 1.05, 0.91, 1.08 and 1.25 (each 3H, s, H-23, H-24, H-25, H-26 and H-27), 0.97 (3H, d, J=6.0 Hz, H-29) and 1.03 (3H, d, J=6.3 Hz, H-30); ¹³C-NMR (C₅D₅N, 100 MHz): δ 39.1 (C-1), 28.1 (C-2), 78.1 (C-3), 39.4 (C-4), 55.8 (C-5), 18.8 (C-6), 33.6 (C-7), 40.0 (C-8), 48.1 (C-9), 37.3 (C-10), 25.0 (C-11), 125.6 (C-12), 139.4 (C-13), 42.5 (C-14), 28.7 (C-15), 23.9 (C-16), 48.1 (C-17), 53.6 (C-18), 39.5 (C-19), 39.5 (C-20), 31.1 (C-21), 37.3 (C-22), 28.8 (C-23), 16.6 (C-24), 15.7 (C-25), 17.6 (C-26), 23.7 (C-27), 180.2 (C-28), 17.5 (C-29) and 21.5 (C-30).

The GBC-SD and NOZ cells were cultured in RPMI-1640 or high-glucose DMEM supplemented with 10% FBS at 37°C in a 5% CO₂ humidified atmosphere, and routinely passaged at 2–3-day intervals. UA was dissolved in DMSO to a 50 mM stock concentration. The final DMSO concentration was accounted for ≤0.1% (v/v). DMSO (0.05%)-treated cells were used as a vehicle control.

A CCK-8 assay was performed to evaluate the effect of UA on cell viability. A total of 1.2×10⁴ cells/well were seeded in 96-well plates overnight and then treated with varying concentrations of UA (0, 10, 20, 30, 40, 45 and 50 µM). The cells were incubated for either 24 or 48 h at 37°C in a humidified incubator, following which 10 µl CCK-8 solution was added into each well and incubated for a further 1 h. The absorbance was measured at 450 nm in each well using a microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). The results are presented as the mean values of three independent experiments performed over multiple days. Cell viability was calculated using the following formula: Cell viability (%)=[optical density (OD) of the experiment samples/OD of the control] × 100%. The half maximal inhibitory concentration (IC₅₀) value of UA against the GBC-SD or NOZ cells was calculated using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA).

Cell apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection kit. In brief, the cells from the UA-treated (10, 30 and 50 µM) and untreated groups were seeded in 6-well plates (1×10⁶ cells/well) and cultured for 24 h. The cells were collected, washed with ice-cold PBS, and resuspended in binding buffer at a cell density of 1×10⁶ cells/ml. The cells were stained with 5 µl Annexin V-FITC and 10 µl PI (20 µg/ml) and then incubated in the dark at 25°C for 15 min. Apoptotic cells were analyzed by flow cytometry (CytoFlex; Beckman Coulter Inc., Brea, CA, USA). The percentage of apoptotic cells was analyzed using FlowJo software (version 9.8.3, FlowJo LLC, Ashland, OR, USA). The experiments were repeated three times.

A cell invasion assay was performed using 8-µm pore size Transwell chambers. The upper side of the Transwell filter inserts was coated with 80 µl diluted (1:8 in serum-free medium) Matrigel in 24-well plates. The GBC-SD cells at a density of 1×10⁵ cells/well were suspended in serum-free RPMI-1640 medium and added to the upper chambers containing various concentrations of UA (10, 30 and 50 µM). The lower chambers were filled with 500 µl RPMI-1640 medium containing 20% FBS. After 24 h, the non-invaded cells were removed, and the invasive cells were fixed with 95% ethanol, stained with 0.1% crystal violet and images were captured (magnification, ×100) with a light microscope (XDS-1B inverted biological microscope, Chongqing Optical & Electrical Instrument Co., Ltd., Chongqing, China). The assay was repeated in three independent experiments.

In brief, cells from the UA-treated (50 µM) and untreated groups were seeded in 6-well plates (1×10⁶ cells/well) and cultured for 24 h. Total RNA was extracted from each experimental group using TRIzol reagent and quantified by spectrophotometry. Subsequently, 1 µg of total RNA was reverse transcribed with the PrimeScript™ RT Reagent kit, according to the manufacturer's protocol. RT² Profiler ‘human apoptosis’ PCR arrays and RT² Profiler ‘human extracellular matrix and adhesion molecules’ PCR arrays were performed in duplicate according to the manufacturer's protocol. The PCR was performed as follows: 10 min at 95°C and 40 cycles (15 sec at 95°C, 1 min at 60°C). The specificity of the SYBR Green assay was confirmed by melting curve analysis. Relative fold changes in mRNA levels were calculated following normalization to housekeeping control gene targets using the comparative Cq method (14).

In order to investigate the signaling pathways that may be involved in the effects of UA on GBC-SD cells, KEGG pathway analysis of differentially expressed genes was performed using the Database for Annotation, Visualization and Integrated Discovery (https://david.ncifcrf.gov/) (15,16). Pathway terms with P<0.05 were considered statistically significant.

Following treatment with UA (0, 10, 30 and 50 µM) for 6 h, the GBC-SD cells were washed with PBS and lysed in RIPA lysis buffer. Based on total protein concentrations calculated from the BCA assays, the total cell lysates (15 µg total protein) were separated via SDS-PAGE (8% gel) and then transferred onto polyvinylidene difluoride membranes in a standard transfer buffer. Following blocking with 1% BSA (blocking solution) for 1.5 h at room temperature, the membranes were incubated with primary antibodies to phospho-NF-κB p65 (Ser536; 1:1,000), NF-κB p65 (1:1,000), phospho-Akt (Ser473; 1:1,000), Akt (1:1,000) or GAPDH (1:5,000) overnight at 4°C. The membranes were washed three times with TBST and then incubated with HRP-conjugated secondary antibodies (1:5,000; cat. no. SA00001-1/SA00001-2; ProteinTech Group, Inc., Chicago, IL, USA) for 2 h at room temperature. Following extensive washing in TBST, the protein signals were visualized using enhanced chemiluminescence (ECL) western blotting substrate (cat. no. B500014; ProteinTech Group, Inc., Chicago, IL, USA) and an ECL system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal protein loading was assessed by normalizing against the expression of GAPDH. Quantification of protein bands densitometry was carried out using ImageJ software (version 1.8.0_112; National Institutes of Health, Bethesda, MD, USA).

All data are expressed as the mean ± standard deviation. Statistical analysis was performed using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Groups were compared using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Consistent with previous results (17,18), UA was isolated from an Isodon species (I. excisoides). UA was identified by comparison of its HR-EIMS, ¹H-NMR and ¹³C-NMR spectra data with those previously reported for UA (19,20), and to an authentic sample (Sigma-Aldrich; Merck KGaA, Darmstadt Germany). The proliferation inhibition effect of UA on GBC-SD and NOZ cells was determined using a CCK-8 assay. Within the dose range and time period measured, UA was able to inhibit cell proliferation in a dose- and time-dependent manner (Fig. 1B and C). Furthermore, the IC₅₀ values of UA at the same exposure time for GBC-SD cells were marginally lower compared with those for NOZ cells. The IC₅₀ values for GBC-SD cells at 24 and 48 h were 57.44 and 39.12 µM, respectively, and the values for NOZ cells were 61.58 and 41.81 µM, respectively. Therefore, GBC-SD cells were selected to further investigate the effect of UA on the invasive capacity of GBC cells. In addition, the IC₅₀ value for GBC-SD cells at 48 h was lower than that reported in the literature (for example, the IC₅₀ value for GBC-SD cells at 48 h was previously reported to be 47.6 µM) (13), which may derive from differences in experimental design and operation.

The UA-induced apoptosis of GBC-SD cells was determined using an Annexin V-FITC/PI assay. As indicated in Fig. 1D and E, UA treatment increased the apoptosis of GBC-SD cells in a dose-dependent manner. The apoptotic rates of the cells were 11.98, 17.37 and 55.50% following treatment with 10, 30 and 50 µM UA, respectively, which were all increased compared with the apoptotic rate of GBC-SD cells cultured under normal conditions (9.96%). The apoptotic rates of GBC-SD cells in the middle (30 µM) and high (50 µM) dose groups were statistically significant (P<0.05). UA (10, 30 or 50 µM) treatment had no significant effect on the distribution of GBC-SD cells in the cell cycle (data not shown). These results indicate that UA inhibits GBC-SD cell proliferation by inducing apoptosis.

Cell invasion is a driving force in the process of tumor metastasis formation. Therefore, the effects of UA on invasion in GBC-SD cells was evaluated (Fig. 2A and B). The in vitro invasion assay indicated that UA at concentrations of 10–50 µM significantly reduced the rate of GBC-SD cell invasion when compared with the control group following cell treatment for 24 h (P<0.01). Furthermore, UA at concentrations of 10 and 30 µM did not significantly reduce the viability of GBC-SD cells following cell treatment for 24 h (Fig. 1B). These results suggested that the inhibition of GBC-SD cell invasion by UA did not result from a reduction of cell viability. These observations suggested that UA was able to regulate the invasive capacity of GBC-SD cells in a dose-dependent manner.

As UA induced apoptosis and inhibited invasion in GBC-SD cells, the expression of 168 key genes involved in these two processes was subsequently evaluated with a PCR array. The cells were incubated with UA (50 µM) for 24 h, and 168 related genes (84 apoptosis-related genes and 84 adhesion/invasion-related genes) were analyzed, compared with untreated GBC-SD cells. Of these 168 genes, there were 24 with log₂ fold change values of either >1.5 or ≥1.5, which were considered differentially expressed (Table I). Of these 24 genes, eight apoptosis-related genes were screened. Of these eight genes, three genes encoding pro-apoptotic members of the tumor necrosis factor (TNF) family, including TNF (log₂ fold change: 3.17), lymphotoxin-α (log₂ fold change: 2.19) and CD27 (log₂ fold change: 3.56) were enhanced in the UA-treated GBC-SD cells compared with the control groups (Table I). Therefore, these results suggested that UA induces apoptosis in GBC-SD cells through activation of the cell extrinsic pathway, which is initiated by members of the TNF superfamily (21,22). TNF is able to trigger either the formation of complex-I, driving the activation of NF-κB and an inflammatory response, or the formation of complex-II, which can trigger apoptosis (23). Of the 24 differentially expressed genes, a further 16 adhesion/invasion-related genes were screened out, including nine upregulated and seven downregulated genes. These upregulated and downregulated genes may have multiple effects on the invasion of GBC-SD cells. KEGG pathway analysis of the 24 differentially expressed genes was performed using the Database for Annotation, Visualization and Integrated Discovery (15,16). According to KEGG pathway enrichment analysis, the differentially expressed genes were significantly associated with NF-κB, TNF, PI3K-Akt and other signaling pathways (Table II). To determine whether UA regulates the NF-κB and Akt signaling pathways, the effect of UA on the expression and activation of proteins in these signaling pathways was detected by western blotting. As shown in Fig. 2C, the GBC-SD cells treated with UA exhibited a significant and dose-dependent reduction in total NF-κB p65 and Akt, and a marginal decrease in total p-NF-κB p65 (Ser536) and p-Akt (Ser473). Therefore, it was demonstrated that the effects of UA on GBC-SD cells were, at least in part, mediated through the NF-κB and Akt signaling pathways.