Human MGC803 gastric cancer cells (American Type Culture Collection, Manassas, VA, USA) were cultured using 6-well plates (10⁴ per well) in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; both Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 µg/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) at 37°C in 5% CO₂ for 24 h. For stimulation experiments, MGC803 cells were seeded in serum-free medium in 6-well plates (1×10⁵/well) and cultured overnight. Cells were stimulated with 1, 10 and 30 µmol/l casticin (Sigma-Aldrich; Merck Millipore) for indicative time intervals. Equal volumes of DMSO (Sigma-Aldrich; Merck Millipore) were used as the controls.

Following treatment, total RNA was extracted from the cells and purified using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. RNA was reversely transcribed into cDNA by a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.). A total of 2 µg RNA was subject to RT-qPCR using SuperReal PreMix kit (Tiangen Biotech, Co., Ltd., Beijing, China). The primers used were as follows: RECK, forward, 5′-ATCATTCCCGTCGATCACTATC-3′ and reverse, 5′-ATATGTCCAGAGCAAGTGCAAG-3′; DNA methyltransferase 1 (DNMT1) forward, 5′-AACCTTCACCTAGCCCCAG-3′ and reverse, 5′-CTCATCCGATTTGGCTCTTCA-3′; β-actin forward, 5′-CATCCTGCGTCTGGACCTGG-3′ and reverse, 5′-TAATGTCACGCACGATTTCC-3′. PCR amplification was performed on an ABI Prism^® 7700 (Applied Biosystems; Thermo Fisher Scientific, Inc.) as follows: 5 min at 95°C; 40 cycles of 30 sec at 95°C, 30 sec at 60°C and 1 min at 72°C. The 2^−ΔΔCt method (24) was used to calculate the relative levels of target mRNAs and β-actin was used as a reference gene.

Following treatment, cells were centrifuged at 1,000 × g and 4°C (Centrifuge 5418R; Eppendorf, Hamburg, Germany) for 5 min and supernatants were subsequently discarded. Following two washes with ice-cold phosphate-buffered saline, cells were re-suspended using 200 µl cell lysis buffer (P0013; Beyotime Institute of Biotechnology, Shanghai, China) for lysis on ice for 40 min. Following centrifugation at 1,000 × g and 4°C for 15 min, supernatants were collected. Protein samples were mixed with 2X sodium dodecyl sulfate (SDS) loading buffer and boiled for 5 min to induce denaturation. A total of 80 µg protein was resolved using 12% SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Sigma-Aldrich; Merck Millipore) under 30 V at 4°C. Following blocking at 4°C and washing with Tris-buffered saline with Tween-20 (TBST) for 30 min, the membranes were incubated with polyclonal rabbit anti-human RECK (1:400; sc-28918) or DNMT1 antibodies (1:400; sc-20701) or rabbit anti-β-actin antibodies (1:1,000; sc-7210; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C for 8 h. After washing with TBST for 30 min, the membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G antibody (1:5,000; sc-2054; Santa Cruz Biotechnology, Inc.) for 4 h at room temperature. After washing with TBST for 30 min, bands were visualized by enhanced chemiluminescence (sc-2048; Santa Cruz Biotechnology, Inc.). The level of target protein expression in each sample was determined by normalizing protein band intensity to β-actin band intensity using ImageJ analysis software (version 1.46; National Institutes of Health, Bethesda, MD, USA). Tests were performed in triplicate.

Following treatment with casticin, MGC803 cells (1×10⁷) underwent genomic DNA extraction and purification using the DNeasy Blood & Tissue kit (Qiagen GmbH, Hilden, Germany). A total of 200 ng genomic DNA was denatured at 100°C for 3 min and placed on ice for cooling. Afterwards, 1/10 (volume ratio) ammonium acetate (0.1 mol/l, pH 5.3) and 2 units nuclease P1 were added. After 2 h incubation at 45°C, 1/10 (volume ratio) NH₄HCO₃ (1 mol/l) and 0.002 units venom phosphodiesterase I were added prior to incubation at 37°C for 1 h. Subsequently, 0.5 units alkaline phosphatase were added, followed by further incubation at 37°C for 1 h. An EpiQuik DNMT Activity/Inhibition Assay Ultra Kit (EpiGentek, Farmingdale, NY, USA) was used to determine the methylation activity of nucleoprotein in MGC803 cells, following the manufacturer's protocol. For high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) analysis of a 10-µl sample, an Agilent 1100 series HPLC system was used (Agilent Technologies, Inc., Santa Clara, CA, USA). The Atlantis™ dC₁₈ column (inner diameter, 2.1 mm; length, 150 mm; particle size, 3 µm) was purchased from Waters Corporation (Milford, MA, USA) and the mobile phase was 0.1% formic acid-methanol with a flow rate of 0.2 ml/min. The mode of electrospray ionization was positive ions, with the following conditions: Scanning range, 100–2,000 m/z; ion source temperature, 450°C; electrospray voltage, 415 kV; cluster breaking voltage, 55 V; entrance voltage, 6 V; collision energy, 13 V; air curtain pressure, 138 kPa; gas 1 pressure, 221 kPa; gas 2 pressure, 379 kPa; collision gas pressure, 41 kPa (25). Analyst Software version 1.3.1 (AB SCIEX, Framingham, MA, USA) was used for data analysis.

MGC803 cells (100 µl) were seeded onto 96-well plates (~3×10³ cells per well) containing 100 µl RPMI-1640 medium supplemented with 5% fetal bovine serum (both Thermo Fisher Scientific, Inc.). On the second day, the medium was changed to RPMI-1640 supplemented with 5% FBS, 1% penicillin/streptomycin and 1, 10 or 30 µmol/l casticin (98% purity; Sigma-Aldrich; Merck Millipore) prior to incubation for 72 h. Untreated cells served as the control. A total of 4 h before the end of incubation, 20 µl MTT (5 mg/ml; Sigma-Aldrich; Merck Millipore) was added. Supernatants were subsequently discarded and 200 µl dimethylsulfoxide was added to each well before shaking gently for 10 min to dissolve crystals. The absorbance of each well was measured at 570 nm using a Synergy HT microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The following formulae were used to calculate the survival and inhibitory rates of the tumor cells. Survival rate of tumor cells (%) = (absorbance of treatment wells / absorbance of control well) × 100. Inhibitory rate of tumor cells (%) = (absorbance of control well - absorbance of treatment wells) / absorbance of control well × 100. Tests were performed in triplicate.

MGC803 cells were left untreated or treated with 1–30 µmol/l casticin and nuclear protein was extracted using a NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce; Thermo Fisher Scientific, Inc.). Nuclear protein concentrations were determined with the Bradford assay using the Coomassie Plus reagent (Thermo Fisher Scientific, Inc.). A total of 15 µg nuclear protein from each treatment group was analyzed for Sp1 activity using the Transcription Factor ELISA kit (Affymetrix, Inc., Santa Clara, CA, USA). Sp1 antibody was used as the primary antibody and anti-rabbit IgG HRP was used as secondary antibody, which were provided in the kit. The absorbance was measured at a wavelength of 450 nm on a spectrophotometer (BioTek Instruments, Inc.).

The results were analyzed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Two groups of mean values were compared using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of casticin on the mRNA levels and protein expression of RECK, RT-qPCR and western blotting were performed, respectively. Western blot analysis indicated that the RECK protein band in the control group was relatively thin, whereas treatment with 1, 10 or 30 µmol/l casticin significantly increased band thickness (P<0.05; Fig. 1A). Results from RT-qPCR showed that treatment with 1, 10 or 30 µmol/l casticin also significantly increased RECK mRNA levels in a dose-dependent manner (P<0.05; Fig. 1B). These results suggest that casticin increases RECK mRNA levels and protein expression in MGC803 cells.

To detect the effect of casticin on methylation, the EpiQuik DNMT kit was used. The results demonstrated that casticin decreased RECK promoter methylation level, global DNA methylation level and methylation activity in nuclear extracts in a dose-dependent manner. Treatment with 30 µmol/l casticin reduced RECK promoter methylation level by 31%, global DNA methylation level by 39% and nuclear methylation activity by 71.6% (Table I). These results indicate that casticin reduces the RECK promoter methylation level, global DNA methylation level and nuclear methylation activity.

RT-qPCR and western blotting were performed to measure the DNMT1 mRNA and protein expression levels, respectively. Western blot analysis demonstrated that the DNMT1 protein band in the control was relatively thick, whereas treatment with 1, 10 or 30 µmol/l casticin significantly decreased band thickness (P<0.05; Fig. 2A). Quantification of mRNA levels and protein expression indicated that treatment with 1, 10 or 30 µmol/l casticin significantly reduced levels of DNMT1 mRNA and protein in a dose-dependent manner (P<0.05; Fig. 2B). These results suggest that casticin downregulates the levels of DNMT1 mRNA and protein in MGC803 cells.

To investigate the effect of casticin on MGC803 cell proliferation and the DNA-binding activity of Sp1 transcription factor, MTT assay and ELISA were performed, respectively. The MTT assay demonstrated that the proliferation rates of MGC803 cells following treatment with 10 and 30 µmol/l casticin were 44 and 23% of that in the control, respectively (P<0.05; Fig. 3A). Furthermore, the results from ELISA indicated that treatment with casticin (1, 10 and 30 µmol/l) reduced the DNA-binding activity of Sp1 in MGC803 cells (P<0.05; Fig. 3B). These results suggest that casticin inhibits the proliferation and DNA-binding activity of transcription factor Sp1 in MGC803 cells.