The SGC-7901 and MGC-803 human GC cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% streptomycin and penicillin in a humidified incubator at 5% CO₂ and 37°C. Rabbit primary antibodies against S-phase kinase-associated protein 2 (Skp2; cat. no. ab183039), mechanistic target of rapamycin kinase (mTOR; cat. no. ab32028), microtubule-associated protein 1 light chain 3β (LC3)-II (cat. no. ab51520), caspase-3 (cat. no. ab13847), cleaved-caspase-3 (cat. no. ab2302), poly(ADP ribose) polymerase (PARP; cat. no. ab32138) and cleaved-PARP (cat. no. ab32064) were purchased from Abcam (Cambridge, UK). Rabbit primary antibodies against β-actin (cat. no. 4970) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibody (goat anti-rabbit; cat. no. sc2004) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rottlerin and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rottlerin was dissolved in DMSO to generate a 10 mM stock solution. Cells cultured with only 0.1% DMSO served as the control group.

Cell proliferation was measured with a Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). SGC-7901 and MGC-803 cells were seeded in 96-well plates at a density of 2,000 cells/well and incubated in a humid environment at 5% CO₂ and 37°C for 4 h. Subsequently, the cells were exposed to 0, 1, 2, 4, 8 and 16 µM rottlerin for 12, 24, 48 and 72 h. CCK-8 reagent was then added and incubated for 30 min at 37°C. Absorbance of the colored formazan product, formed by mitochondrial dehydrogenases, was measured at a wavelength of 450 nm.

SGC-7901 and MGC-803 cells were cultured in a 6-well plate at a density of 500 cells/well with 0, 2, 4 and 8 µM rottlerin at 37°C for 2 weeks. Cells treated with rottlerin-free medium served as the control group. After 2 weeks, the cells were fixed in 4% methanol for 15 min at room temperature. Cells were then stained with 0.1% crystal violet for 5 min at room temperature and imaged using a light microscope (Olympus Corporation, Tokyo, Japan) at ×40 magnification.

SGC-7901 and MGC-803 cells were seeded at a density of 1×10⁶/ml, and then harvested following treatment with 0, 2 4 and 8 µM rottlerin at 37°C for 24 h. The cells were fixed in 70% ethanol at 4°C overnight. The fixed cells were centrifuged at 1,000 × g for 15 min at room temperature and washed with cold PBS three times. The cells were incubated with 50 µg/ml RNase A at 37°C for 30 min. Then cells were incubated with 100 µg/ml propidium iodide (PI) in the dark at 4°C for 30 min. The DNA content was quantified by FCM (BD CellQuest Pro; BD Biosciences, Franklin Lakes, NJ, USA). The percentages of cells in the G₀-G₁, S and G₂-M phases were compared with the control group.

SGC-7901 and MGC-803 cells were cultured at 1×10⁶/ml in 6-well plates following treatment with 0, 2 4 and 8 µM rottlerin at 37°C for 24 h. Cells were collected, washed twice with cold PBS and resuspended in 100 µl binding buffer containing 5 µl fluorescein isothiocyanate-conjugated anti-Annexin V antibody and 5 µl PI using a FITC-Annexin V Apoptosis Detection kit (BD Biosciences). Apoptosis was assessed using a FACS Calibur flow cytometer (BD CellQuest Pro; BD Biosciences). The percentages of apoptotic cells were compared with the control group.

SGC-7901 and MGC-803 cells were cultured at 1×10⁶/ml in 6-well plates. Migration was assessed using a wound healing assay that was performed following treatment with 0, 2 4 and 8 µM rottlerin at 37°C for 0 and 24 h. A scratch was created in a culture plate using the tip of a pipette (Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C and images were captured after 0 and 24 h. For the cell invasion assay, GC cells were incubated with 0, 2, 4 and 8 µM rottlerin at 37°C for 24 h and then harvested. 5×10⁴ cells were added to the upper chambers of a Transwell assay plate, which were coated with Matrigel, containing 200 µl serum-free medium. The lower chambers contained complete medium with 0, 2, 4 and 8 µM rottlerin. After 48 h, the cells in the lower chamber were fixed in cold methanol, stained with 0.1% crystal violet and images were captured.

SGC-7901 and MGC-803 cells were incubated with 0, 2, 4 and 8 µM rottlerin at 37°C for 24 h. Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was synthesized using a PrimeScript™ RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) at 37°C for 15 min, followed by 85°C for 5 sec, determined at 4°C and qPCR was conducted using a SYBR^® Premix Ex Taq™ kit (Takara Biotechnology Co., Ltd.) on a Bio-Rad iQ5 Real-Time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The following thermocycling conditions were used for the PCR: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The primer sequences for amplification were as follows: mTOR, 5′-AGGCCGCATTGTCTCTATCAA-3′ (forward) and 5′-GCAGTAAATGCAGGTAGTCATCCA-3′ (reverse); Skp2, 5′-GCTGCTAAAGGTCTCTGGGT-3′ (forward) and 5′-AGGCTTAGATTCTGCACTTG-3′ (reverse); and GAPDH, 5′-ACCCAGAAGACTGTGGATGG-3′ (forward) and 5′-CAGTGAGCTTCCCGTTCAG-3′ (reverse). The relative expression was calculated using the 2^−ΔΔCq method, with GAPDH used as the internal control (23).

SGC-7901 and MGC-803 cells were seeded in 96-well plates at a density of 2,000 cells/well in rottlerin-containing medium (0, 2, 4 and 8 µM rottlerin) at 37°C for 24 h, followed by incubation with the Autophagosome Detection kit (Sigma-Aldrich; Merck KGaA). Medium was removed from the cells and 100 µl of the autophagosome detection reagent working solution was added to each well and cells were incubated at 37°C with 5% CO₂ for 30 min. Subsequently cells were washed with Wash Buffer 3 times by gently adding 100 µl of Wash Buffer to each well and cells were immediately imaged using a fluorescence microscope (Olympus Corporation, Tokyo, Japan). Autophagy was visualized as the bright blue staining of autophagic vacuoles.

Following treatment with 0, 2, 4 and 8 µM rottlerin at 37°C for 24 h, radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) was added for the extraction of proteins from GC cells for 40 min on ice. The supernatants incubated in 4°C for 30 min and centrifuged at 13,000 × g at 4°C for 15 min, the protein concentrations were measured by Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Protein (20 µg) was loaded and separated via 12.5% SDS-PAGE, with a volume of 20 µl per well. The proteins were transferred onto 0.22-µm polyvinylidene fluoride membranes. The membranes were subsequently incubated with primary antibodies at a dilution of 1:1,000 at 4°C overnight, followed by incubation with secondary antibodies diluted in TBS-Tween-20 (0.1% Tween-20) at room temperature for 1.5 h. Enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.) was used to visualize the bound antibodies using the ChemiDoc imaging system (Abcam). Protein band intensities were semi-quantitated with ImageJ software (version 1.46r; National Institutes of Health, Bethesda, MD, USA) and normalized to β-actin.

Data are presented as the mean ± standard error. Each experiment was performed three times independently. Data analysis was performed by ANOVA using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Bonferroni's test was used as a post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate whether rottlerin suppressed the proliferation of GC cells, a CCK-8 assay was performed using SGC-7901 and MGC-803 cells following rottlerin treatment. The results demonstrated that rottlerin inhibited GC cell proliferation in a dose-dependent manner (Fig. 1A and B). Relative cell viability is presented in Fig. 1C. Rottlerin at 1 µM exerted no obvious suppressive effect on GC cells, 16 µM rottlerin caused cell death, and 2, 4 and 8 µM rottlerin was used as the experimental group.

The colony formation assay revealed that rottlerin suppressed the clonogenic capacity of GC cells compared with the control group (Fig. 1D).

It was previously reported that rottlerin promoted cell cycle arrest and apoptosis in breast cancer cells (11). To further elucidate the effects of rottlerin on cell cycle distribution and apoptosis in GC cells, the percentage of GC cells in each cell cycle phase following rottlerin treatment was determined through PI staining and flow cytometry. As presented in Fig. 2A and B, rottlerin led to evident G₀/G₁ phase arrest in GC cells. Rottlerin at 2, 4 and 8 µM led to a G₁ cell population increase from 45.13±2.21% to 53.43±1.81, 60.45±3.07 and 66.79±1.94%, respectively, in SGC-7901 cells. Similarly, 2, 4 and 8 µM rottlerin caused a G₁ cell population increase from 42.21±1.86% to 50.41±1.87, 56.42±2.38 and 65.82±2.22%, respectively, in MGC-803 cells. Furthermore, flow cytometry following annexin V-FITC and PI staining revealed that, following treatment with rottlerin at 2, 4 and 8 µM, the apoptosis rate of MGC-803 cells were increased from 2.68±1.00% to 8.57±0.80, 12.27±1.06 and 15.19±0.92%, the SGC-7901 cell apoptosis rate from 3.20±1.60% to 16.01±1.15, 23.83±1.67 and 29.99±1.64%, respectively (Fig. 2C and D). Taken together, these results indicate that rottlerin promoted cell cycle arrest and apoptosis in GC cells.

To determine the effects of rottlerin on the migratory activity of GC cells, a wound-healing assay was conducted. The results demonstrated that rottlerin inhibited migration in both types of GC cells in a dose-dependent manner (Fig. 3A and B). To confirm the effect of rottlerin on the invasion ability of GC cells, a Transwell assay was conducted using Matrigel (Fig. 3C and D). Rottlerin reduced the number of GC cells invading through the Matrigel, compared with the control group (Fig. 3C and D). These results indicate that rottlerin inhibited the migration and invasion ability of GC cells.

GC cells treated with 2, 4 and 8 µM rottlerin were examined under a light microscope (Fig. 4A). Cells treated with rottlerin were fewer compared with the control group, as well as smaller and more spindle-shaped, with less cytoplasm. Membrane ruffling was reduced, with a contractive cell appearance. Therefore, the morphology of GC cells treated with rottlerin was clearly affected. The observations were consistent with those in rat C6 glioblastoma cells, as reported by Parmer et al (24).

GC cells treated with 2, 4 and 8 µM rottlerin were used to investigate the association between rottlerin and autophagy. LC3-II expression levels were detected by western blotting and fluorescence microscopy was used to evaluate autophagosome formation. As demonstrated by examination with a fluorescence microscope, the number of autophagosomes was increased in the rottlerin-treated GC cells compared with the control group, as demonstrated by increased light blue fluorescence, whereas the light green cells were those without autophagosomes. (Fig. 4B). During autophagosome formation, the microtubule-associated LC3-I is converted to the membrane-bound form LC3-II (25). The LC3-II expression level was increased in GC cells following treatment with rottlerin and the quantitative results revealed significant differences compared with the control cells (Fig. 4C and D). Therefore, the results indicated that rottlerin may promote autophagy in GC cells.

Rottlerin-induced apoptosis has been reported in several cancer cell lines (11,26). The caspase cascade functions in apoptosis induction and completion (27,28). Western blotting was conducted to evaluate caspase-3 and PARP expression, which exhibited no obvious increase in GC cells following treatment with rottlerin (Fig. 5). Cleaved-caspase-3 and PARP were also evaluated, and no cleaved-caspase-3 expression was detected in GC cells treated with rottlerin (Fig. 5). Cleaved-PARP was detected in GC cells, but the difference between rottlerin-treated GC cells and control cells was not statistically significant (Fig. 5). Similar observations were reported by Torricelli et al (29). Therefore, rottlerin may promote apoptosis independently of caspase in GC cells.

Several studies have reported that rottlerin inhibits Skp2 protein expression in pancreatic and breast cancer (11,13). Wu et al (30) observed that Skp2 promoted autophagy through inhibition of mTOR complex 1 (mTORC1). The expression levels of mTOR and Skp2 were evaluated via RT-qPCR (Fig. 6A and B) and western blot assays (Fig. 6C-E) in GC cells treated with rottlerin. The mRNA levels of mTOR and Skp2 were downregulated following treatment with rottlerin for 24 h (Fig. 6A and B). The protein levels of mTOR and Skp2 following rottlerin treatment were consistent with the results for mRNA expression levels in GC cells (Fig. 6C-E). Consistent with previous studies, these results demonstrate that rottlerin may reduce the expression of Skp2 via the inhibition of mTOR expression in order to promote autophagy in GC cells (11,13).