The HeLa cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Resveratrol and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Antibodies against Stat3 and p-Stat3 (phosphorylated at tyrosine 705) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against GRIM-19 were purchased from eBioscience (San Diego, CA, USA). Secondary antibodies were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Penicillin, streptomycin, Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco-BRL (Grand Island, NY, USA). HeLa cells were grown in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 mg/ml streptomycin for 48 h at 37°C/5% CO₂. The HeLa cells were divided into experimental groups and each treatment condition was a single dose of resveratrol at the indicated concentration. The vehicle control was 0.1% dimethyl sulfoxide (DMSO).

Western blotting was used to analyze the expression levels of p-Stat3, Stat3, GRIM-19 and β-actin. The cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA) for 45 min at 4°C. Approximately 30 μg of total protein was loaded into each lane of 10 and 15% polyacrylamide gels and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were then transferred onto nitrocellulose membranes (Novex; Invitrogen Life Technologies, Carlsbad, CA, USA) and the blots were blocked in 5% (w/v) non-fat milk for 1 h at room temperature. The blots were incubated with the appropriate primary antibodies overnight at 4°C (GRIM-19 polyclonal mouse, 1:1,000 dilution; p-Stat3 monoclonal rabbit, 1:2,000; Stat3 monoclonal rabbit, 1:2,000; and β-actin polyclonal mouse, 1:3,000). The blots were washed three times for 5 min per wash and exposed to horseradish peroxidase-conjugated secondary antibodies (Biosynthesis Biotechnology Co., Ltd.) for 2 h. The blots were then examined using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA) and the band intensities were measured and quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).

Total RNA was isolated from HeLa cells using TRIzol^® reagent (Life Technologies, Rockville, MD, USA) following 24 h treatment with resveratrol or 48 h after transfection with GRIM-19 or a GRIM-19 short interfering RNA (siRNA). cDNA was generated from 1 μg of total RNA using a cDNA synthesis kit (Takara Biotechnology Co., Ltd., Dalian, Lianning, China) according to the manufacturer’s instructions. Primer sequences (GeneCore, Shanghai, China) specific for cyclin B1, cyclin D1, B-cell lymphoma 2 (Bcl-2), vascular endothelial growth factor (VEGF), Stat3 and GRIM-19 were used (Table I). β-actin was used for normalization of the cDNA input levels. Following cDNA synthesis, the PCR thermal cycling profile comprised 32 cycles of denaturation (95°C for 30 sec), annealing (56°C for 30 sec) and extension (72°C for 30 sec). The reaction was terminated with a final extension step (72°C for 5 min) following 32 cycles. The amplicons were separated by electrophoresis on 2% (w/v) agarose gel and visualized by staining with ethidium bromide. Three biological replicates were analyzed for each sample point and at least three reactions were used to calculate the expression levels. Relative expression was quantified using densitometry and the Gel Image Version 3.74 System (Tianon, Shanghai, China).

GRIM-19 was knocked down using small siRNAs, with a non-targeting siRNA used in parallel as a negative control (GenePharma Co., Shanghai, China). Primary cultures were transfected with a GRIM-19 siRNA or an irrelevant siRNA (as a control) using the X-tremeGENE HP DNA transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany). After 2 days, the protein expression levels of GRIM-19, p-Stat3, Stat3 and β-actin in HeLa cells were analyzed.

The human GRIM-19 sequence was amplified from HeLa cells using RT-PCR and cloned between the NotI and EcoRV sites of the Pflag-CMV™-4 mammalian expression vector. The pFLAG tag was added to the N-terminus of the GRIM-19 sequences in all the constructs. The transfection of plasmids into the cells was conducted using X-tremeGENE HP DNA transfection reagent according to the manufacturer’s instructions. Following 2 days, the protein expression of GRIM-19, p-Stat3, Stat3 and β-actin in HeLa cells was analyzed.

Cell viability was determined using MTT assays according to the manufacturer’s instructions. HeLa cells were seeded in 96-well culture plates at an optimal density of 1×10⁴ cells/well. Briefly, phosphate-buffered saline containing MTT at a final concentration of 0.5 mg/ml was added to each well following treatment with resveratrol for 24 h, and then incubated at 37°C for 4 h. The medium was gently aspirated and 150 μl DMSO was added to each well. The plates were agitated for 10 min on a shaker to dissolve the formazan product. A well containing DMSO without cells was used as a blank control and the optical density at 490 nm in each well was determined using a spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA).

The data were expressed as the mean ± standard deviation for three or more independent experiments. Statistical significance was estimated using one-way analysis of variance followed by the Student-Newman-Keuls test for comparison of several groups. P<0.05 was considered to indicate a statistically significant difference.

The present study examined the effects of resveratrol on the proliferation and cell viability of HeLa cells. Analyses by MTT assays demonstrated that the treatment of HeLa cells with resveratrol (10 and 100 μM) induced cell shrinkage (Fig. 1A) and decreased cell viability in a dose-dependent manner compared with the control and DMSO groups (Fig. 1B).

The present study investigated the effects of resveratrol on Stat3 activation and GRIM-19 expression in HeLa cells. The cells were treated with resveratrol (10 and 100 μM) for 24 h. HeLa cells treated with resveratrol significantly induced the mRNA and protein expression of GRIM-19 (Fig. 2A–E). At the same time, p-Stat3 protein expression levels were downregulated (Fig. 2F), however, Stat3 mRNA expression levels were unaltered (Fig. 2C).

To understand the function of GRIM-19 on the Stat3 signaling pathway, GRIM-19 with a FLAG tag was overexpressed. The levels of p-Stat3 were decreased by the overexpression of GRIM-19 (Fig. 3A). The relative expression level of p-Stat3 is shown as ratio to β-actin (Fig. 3B). The expression of GRIM-19 is shown in Fig. 3C. Stat3-associated genes (cyclin B1, VEGF and Bcl-2) were downregulated by the overexpression of GRIM-19. However, GRIM-19 had no effect on the transcription levels of cyclin D1 (Fig. 3D).

The cells transfected with GRIM-19 siRNA clearly downregulated the cell cytotoxicity induced by resveratrol (Fig. 4F). Furthermore, the expression of GRIM-19, p-Stat3 and Stat3 was detected (Fig. 4A–C). Resveratrol downregulated p-Stat3 expression, while transfection with GRIM-19 siRNA resulted in the suppression of p-Stat3 downregulation induced by resveratrol (Fig. 4C and D). The results confirmed that GRIM-19 expression, induced by resveratrol, is involved in p-Stat3 suppression induced by resveratrol. GRIM-19 was also involved in the proliferation and cytotoxicity induced by resveratrol in HeLa cells.