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Introduction

Resveratrol (3,5,4′-trihydroxy-trans-stilbene; Fig. 1) is a polyphenolic phytoalexin that is naturally present in food products, including grapes, mulberries and peanuts (1), with demonstrated cardioprotective (2), neuroprotective (3) and anti-inflammatory effects (4). Although these effects are partly attributed to its antioxidant properties, resveratrol has also been demonstrated to produce anti-carcinogenic effects (5). A number of previous studies have revealed the anti-proliferative and apoptotic effects of resveratrol in various cancer cell lines including breast (6–9), lung and prostate cancer (10–12).

Figure 1. Chemical structure of resveratrol.

Human cervical carcinoma is the most common type of cancer in females, accounting for ~8% of all newly diagnosed cancer cases globally (13). However, the effects of resveratrol on the regulation of human cervical carcinoma, and the mechanisms underlying such effects, remain to be established. In the present study, resveratrol treatment induced apoptosis in the HeLa human cervical cancer cell line. Furthermore, resveratrol activated the mitochondrial apoptotic signaling pathway and upregulated the expression of caspase-3 and −9. In addition, resveratrol was able to induce cell cycle arrest at the G2 phase and the expression of p53 was upregulated in resveratrol-treated HeLa cells. These data indicate that resveratrol is able to induce cell death in HeLa human cervical cancer cells through various potential underlying mechanisms.

Materials and methods

Reagents and cell lines

Resveratrol was purchased from The National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and dissolved in sterile dimethyl sulfoxide (DMSO) to prepare a stock solution, which was further diluted in fresh Dulbecco's Modified Eagle's Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the final concentrations of resveratrol used were 0, 5, 10, 20 or 40 µM. The final concentration of DMSO in all cell cultures was <0.01%. P53 antibody (cat. no. 38007) was purchased from Signalway Antibody (College Park, MA, USA) and B-cell lymphoma 2 (Bcl-2) (cat. no. sc-7382), Bcl-2-associated X protein (Bax) (cat. no. sc-7480), Bcl-extra large (XL; cat. no. sc-8392), caspase-3 (cat. no. sc-7272), β-actin (cat. no. sc-8432) and were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Goat anti-mouse horseradish peroxidase (HRP)-conjugated antibody (cat. no. G-21040; dilution, 1:1,000) and protein ladder (cat. no. SM0671) were purchased from Thermo Fisher Scientific, Inc. The P53 antibody was diluted at a ratio of 1:1,000 when the antibody incubated with the target protein in the NC membrane, while the primary antibodies were diluted at a ratio of 1:500. The experimental protocol was approved by the Research Ethics Committee of Nanjing University of Chinese Medicine Medical University (Nanjing, China).

Cell culture

The HeLa human cervical cancer line was purchased from the American Type Culture Collection (Manassas, VA, USA) and grown in DMEM, supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mmol/L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.). The HeLa cells were incubated at 37°C in a humidified atmosphere with 5% carbon dioxide. The HeLa cells were subject to MTT assay upon reaching 70–80% confluency.

Assessment of cell viability using an MTT assay

Upon reaching ~80% confluency, the HeLa cells were treated with resveratrol in complete medium for dose-dependent and time-dependent studies. In the control group, the HeLa cells were treated with DMSO. The cytotoxicity-based MTT assay was performed according to previous reference (14). Briefly, 200 µl of complete culture medium containing 2×104 cells was added to 96-well microtiter plates and incubated with different concentrations (0, 5, 10, 20 or 40 µM) resveratrol at 37°C in a humidified incubator. After 12, 24, 36 and 48 h of incubation with resveratrol, MTT was added and the cell samples were monitored at a wavelength of 595 nm on a scanning multiwell spectrophotometer. The effect of resveratrol on growth inhibition was assessed as the percentage of cell viability, and control cells treated with DMSO were considered 100% viable. Each assay was replicated 4 times and each experiment was repeated ≥3 times.

Hoechst 33342 staining

The HeLa cells were seeded onto coverslips in 6-well plates at 5×104 cells per well and incubated at 37°C. When the cells grown about 70–80% confluency, they were treated with 20 µM resveratrol. After an incubation at 37°C 48 h, the medium was aspirated and the coverslips were washed with PBS and treated with 20 µg/ml Hoechst 33342 at 37°C for 15 min. Subsequently, the cells were washed with PBS and observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Apoptosis detection

Cell apoptosis was evaluated using flow cytometry to quantify the levels of detectable phosphatidylserine on the outer membranes of apoptotic cells as previously reported (15). Briefly, the cells were grown to a density of 2×105 cells in 100 mm culture dishes and treated with 20 µM resveratrol for 48 h. The HeLa cells were trypsinized and washed twice with PBS, and then floating and adherent cells were collected and suspended in binding buffer (cat. no. 556547; BD Pharmingen; BD Biosciences, Franlink Lakes, NJ, USA) at 2×105 cells per 100 µl binding buffer. Subsequently, the HeLa cells were stained using 5 µl Annexin V-fluorescein isothiocyanate (FITC; cat. no. 556547; BD Pharmingen; BD Biosciences) in the dark at room temperature for 15 min. Next, the cells were resuspended in 300 µl binding buffer and stained with 10 µl propidium iodide (PI; 50 µmol/l). Annexin V/PI fluorescence was analyzed immediately with a flow cytometer. Per sample, ≥10,000 events were counted, and the data presented as the proportion of early apoptotic cells (FITC+/PI-) and late apoptotic cells (FITC+/PI+). The percentages of cells in the upper-right phase (late apoptotic cells), upper-left phase (necrotic cells), lower-right phase (early-apoptotic cells) and lower-left phase (viable cells) panels of the resulting histogram were calculated for comparison among these groups (Fig. 4).

Figure 4. Flow cytometry analysis of HeLa cells treated with 20 µmol/l resveratrol for (A) 0 h (B) 24 h and (C) 48 h, with cell death rates of 3.89, 12.23 and 34.16%, respectively. PI, propidium iodide.

Caspase-3 and −9 activity assay

Caspase-3 or −9 activity was determined using Caspase Activity Assay kits (Biolife Co., Ltd., Beijing, China). Briefly, the cells were treated with 0, 5, 10, 20 or 40 µM resveratrol for 48 h, washed twice with PBS, and digested with 0.25% trypsin-EDTA at 37°C for 1 min. The cells were collected at a density of 3×106 by centrifugation at 100 × g at 4°C for 3 min and suspended in 100 µl of ice-cold lysis buffer (cat. no. 89900; Thermo Fisher Scientific, Inc.). The cell lysates were incubated on ice for 10 min and centrifuged at 900 × g for 10 min. The cell supernatants were collected and the protein concentration was determined using the Bradford method, based on standard reference BSA protein concentrations. The supernatant proteins (50 µg) were added to 96-well plates and incubated with caspase-3 and −9 colorimetric substrate (Biolife Co., Ltd.) for 2 h at 37°C. Subsequently, the absorbance was measured at a wavelength of 405 nm using a microplate reader (Titertek Multiskan Plus, Labsystems, Finland). All experiments were performed in triplicate.

Western blot analysis

Protein samples were dissolved in a sample buffer containing 0.5 M Tris hydrochloride (pH 6.8), 20% glycerol, 2% SDS, 0.5% bromophenol blue and 10 mM dithiothreitol. The protein concentrations of cell lysates were evaluated according to the Bradford method using bovine serum albumin as a standard. The proteins (40–100 µg) were separated by 12% SDS-polyacrylamide gel electrophoresis, blotted on nitrocellulose (NC) membranes, and blocked with a solution of 5% (w/v) skimmed milk powder and 0.1% (w/v) Tween-20 in PBS (pH 7.5) for 1 h at room temperature. The NC membranes were incubated with the primary antibodies for 2 h at room temperature or overnight at 4°C. Subsequently, the membranes were washed with PBS and incubated with the secondary antibody for 2 h at 4°C (16). Next, HRP substrate (dilution, 1:1,000) was added and the NC membranes were imaged (Kodak, Japan). The intensities of the bands were quantitated using Image Lab™ Image Analysis software on a Gel Doc™ system (version no. 170-8195; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Molecular weight-markers were electroblotted and analyzed simultaneously.

Statistical analysis

All data were representative of ≥3 independent experiments and are presented as the mean ± standard deviation. The data were analyzed using SPSS version 11.5 for Windows (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance or the Student's t-test was used to compare between the treatment and control groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Resveratrol inhibits the proliferation of HeLa cells

To identify the effects of resveratrol on HeLa cell survival, the cells were cultured in medium containing various concentrations of resveratrol (0–40 µmol/l) for 48 h. Resveratrol treatment induced a concentration-dependent reduction in the viability of the HeLa cells (Fig. 2A). The survival rate of the HeLa cells treated with 20 µmol/l resveratrol was reduced after 24 h (Fig. 2B). On the basis of the MTT assay results, resveratrol treatment at concentrations of 20, 40 and 60 µmol/l, and a 48 h incubation period, was selected for further mechanistic studies.

Figure 2. Cell cytotoxicity effects upon resveratrol-treatment in HeLa cells. (A) HeLa cells were incubated with resveratrol at concentrations of 0, 5, 10, 20 and 40 µmol/l for 48 h and cell viability was evaluated. (B) Cells were incubated with 20 µmol/l resveratrol for 0, 12, 24, 36 and 48 h. Following incubation, cell viability was determined by an MTT assay. Results are presented as the mean ± standard deviation of three separate experiments. bP<0.05 vs. control.

Resveratrol induces HeLa cell apoptosis

After 48 h of incubation with resveratrol (20 µmol/l), Hoechst 33342 staining revealed increased apoptotic bodies and nuclear condensations in the HeLa cells (Fig. 3). Apoptosis was quantified by flow-cytometric analysis using Annexin V FITC/PI double staining in the HeLa cells treated with 20 µmol/l resveratrol at various time points. Resveratrol (0–40 µmol/l) produced a concentration-dependent increase in the apoptotic cell population after 48 h of incubation. After 24 h of treatment with resveratrol, the counts of early (lower-right quadrant) and late (upper-right quadrant) apoptotic cells were increased in a concentration-dependent manner (Fig. 4). Almost 35% of the HeLa cells had advanced to late apoptotic stages upon treatment with 60 µmol/l resveratrol, compared with the vehicle-treated controls (Fig. 4).

Figure 3. Resveratrol treatment induces apoptosis in HeLa cells. Hoechst 33342 fluorescence staining was used to detect apoptosis in HeLa cells treated with (A) vehicle or (B) 20 µM resveratrol for 48 h. Red arrows indicate the fragmentation of condensed nuclei occurring during apoptosis. Scale bar, 10 µm. Light, light microscopy; merged, merged fluorescence and light microscopy images.

Resveratrol upregulates the expression and activity of caspase-3 and −9 in HeLa cells

Following treatment with varying concentrations of resveratrol for 48 h, the expression levels of caspase-3 and −9 were increased in the HeLa cells (Fig. 5A). The expression levels of caspase-3 and −9 were upregulated in the resveratrol-treated (20 µmol/l) HeLa cells (Fig. 5B). Activation of caspase cascades was observed in the process of apoptosis. Subsequently, the HeLa cells were incubated with various concentrations of resveratrol for 48 h to examine caspase-3 and −9 activity. The results revealed that caspase-3-like and caspase-9-like cysteine protease activity increased during the process of resveratrol-induced apoptosis (Fig. 6).

Figure 5. Western blot analysis of caspase-3 and −9 protein expression levels in resveratrol-treated HeLa cells. (A) Cells were exposed to 0, 5, 10, 20 and 40 µmol/l of resveratrol for 48 h. (B) Cells were treated with 20 µmol/l resveratrol for 0, 12, 24, 36 and 48 h. Cells were lysed, and equal quantities of protein were separated using 12% SDS-PAGE prior to protein expression being detected by western blotting.

Figure 6. Resveratrol-induced caspase-3 and −9 activation in HeLa cells. HeLa cells were treated with 0, 5, 10, 20 and 40 µmol/l of resveratrol for 48 h. Cell extracts (50 µg) were then incubated in the presence of fluorescent (A) caspase-3 substrate, Ac-DEVDMCA and (B) caspase-9 substrate, Ac-LEHD-MCA for 4 h at 37°C. Absorbance was measured at a wavelength of 405 nm using a microplate reader. Results are presented as the mean ± standard deviation of three separate experiments. cP<0.05 vs. Control.

Resveratrol alters the expression levels of Bcl-2 family proteins in the HeLa cells

The effect of resveratrol on the expression levels of the pro-apoptotic protein Bax and the anti-apoptotic proteins Bcl-2 and Bcl-XL was examined. Following treatment with resveratrol, the expression levels of Bax were increased, whereas the expression levels of Bcl-2 and Bcl-XL were decreased in a dose-dependent and time-dependent manner in the HeLa cells (Fig. 7).

Figure 7. Western blot analysis of Bax, Bcl-2 and Bcl-XL protein expression levels in resveratrol-treated HeLa cells. (A) Cells were exposed to 0, 5, 10, 20 and 40 µmol/l of resveratrol for 48 h. (B) Cells were treated with 20 µmol/l resveratrol for 0, 12, 24, 36 and 48 h. All cells were lysed, equal quantities of protein were separated using 12% SDS-PAGE and protein expression was detected by western blotting. Bcl-2, B-cell lymphoma 2; Bax, B-cell lymphoma 2-associated X protein; Bcl-2 XL, B-cell lymphoma 2-extra large.

Resveratrol upregulates p53 expression and downregulates cyclin B1 expression in HeLa cells

Western blot analysis revealed that p53 expression levels were gradually increased and Cyclin B1 expression levels were decreased (P<0.05) in a time-dependent manner in the resveratrol-treated (20 µmol/l) HeLa cells, as compared with in the vehicle control cells (Fig. 8).

Figure 8. Western blot analysis of p53 and cyclin B1 protein expression levels in resveratrol-treated HeLa cells. Cells were treated with 20 µmol/l of resveratrol for 0, 12, 24, 36 and 48 h All cells were lysed, equal quantities of protein were separated using 12% SDS-PAGE and protein expression was detected by western blotting.

Discussion

Chemotherapy using non-toxic natural components is a promising therapeutic strategy for the treatment of carcinoma. Recently, numerous studies have attempted to identify novel anticancer treatments obtained from natural products (17). Resveratrol, a natural polyphenolic phytoalexin, has been demonstrated to possess antioxidant, anti-atherosclerotic, anti-inflammatory (18) and anticancer properties (19–21). The chemopreventive efficacy of resveratrol has been identified in hepatocellular, lung, skin and prostate cancer, functioning through a number of underlying regulatory mechanisms (22–24). However, the precise mechanism of action of resveratrol in human cervical carcinoma remains to be investigated, limiting its therapeutic applications. In the present study, it was demonstrated that resveratrol significantly inhibited HeLa cell growth and induced apoptosis in a dose- and time-dependent manner, and the cells exhibited hallmarks of apoptosis, including cell shrinkage, DNA fragmentation and formation of apoptotic bodies.

Numerous external and internal stimuli may trigger cell apoptosis via the activation of caspases. In the process of caspase-9 activation, cytochrome c binds to apoptosis activating factor 1 and procaspase-9 to form an apoptosome complex, which further activates the downstream effector caspase-3 (25). Caspase-8 and −9 are regarded as initiator caspases, and activate additional effector caspases, including caspase-6 and −7 (26). The activation of caspases leads to the cleavage of a set of proteins, including poly (ADP-ribose) polymerase, and the disassembly of cell components, including the fragmentation of DNA (27). The overexpression of Bcl-2 or Bcl-XL results in the inhibition of cytochrome c release and termination of the apoptotic response, whereas the overexpression of Bax or its Bcl-2 homologous domain 3 promotes cytochrome c release (28,29). The present study revealed that resveratrol-treatment was able to significantly increase the activation of caspase-3 and −9, decrease Bcl-2 and Bcl-XL protein levels and increase Bax protein levels (P<0.05). These findings suggest that Bcl-2 family proteins, as well as caspase-3 and −9, are involved in the process of resveratrol-induced apoptosis.

The cell cycle includes four phases progressing from quiescence (G0 phase) to proliferation (G1, S, G2 and M phases), which are driven by the sequential activation of cyclin-dependent kinase (CDK) and its cofactor cyclins. CDK-cyclin B1 complexes are essential for the phosphorylation of a variety of proteins involved in mitotic events, including nuclear envelope breakdown, chromosomal condensation, spindle formation and the attachment of chromosomes to spindle fibers (30). Therefore, cell cycle proteins, including cyclin B1 and CDK1, are associated with the G2/M phase of the cell cycle (31). p53, a tumor suppressor gene, is activated during cellular stresses, including hypoxia, carcinogenesis and oxidative stress, functioning by inhibiting cell cycle progression and activating the DNA repair machinery to promote cell survival and maintain genome integrity. A p53-dependent arrest occurring at the G2 phase of the cell cycle is associated with a proteasome-dependent decrease in cyclin B1 protein levels (32,33). In addition, a p53-dependent increase in p21 protein levels is associated with a decrease in cyclin B1 protein levels (34,35). The results of the present study revealed that resveratrol was able to induce G2/M phase arrest in HeLa cells. To investigate the association between G2/M phase arrest and cyclin B1 expression levels, the effect of resveratrol on cyclin B1 proteins was examined. The results revealed that resveratrol treatment significantly decreased (P<0.05) the expression levels of cyclin B1 protein in HeLa cells, leading to a significant reduction (P<0.05) in the formation of CDK1-cyclin B complexes and G2/M phase cell cycle arrest. In summary, the present study demonstrated that resveratrol is able to increase the expression levels of p53 in HeLa cells in order to inhibit cell cycle progression and activate DNA repair machinery to promote cell survival and maintain genome integrity.

In conclusion, the results of the present study support the hypothesis that resveratrol downregulates the expression levels of the essential signaling proteins Bcl-2 and Bcl-XL, which are involved in the proliferation and survival of HeLa cells. Furthermore, resveratrol treatment promotes apoptosis by increasing the levels of caspase-3 and −9 and p53 protein expression in HeLa cells. In summary, resveratrol may induce cell cycle arrest and apoptosis in HeLa cells through activation of the mitochondrial apoptosis signaling pathway, accompanied by the upregulation of p53 expression and downregulation of cyclin B1 expression. Therefore, resveratrol may be a promising novel inhibitor of human cervical cancer.

Acknowledgements

The authors would like to thank Professor Li Zhang for her guidance, and Professor Li-Yun Shi and laboratory members for discussion and insightful comments.

Funding

This study was supported by the National Natural Science Foundation of China (grant nos. 30371727, 30973940, 30772766 and 81001599).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

LL contributed to study design, performed experiments, data analysis and writing of the manuscript. RLQ made substantial contributions to the conception and design of the study, performed experiments and writing of the manuscript. YL contributed to study design, performed experiments and critically revised the manuscript. YC performed experiments, and data analysis and interpretation. YB analyzed data and contributed to writing of the manuscript. YF and XJG performed experiments and data analysis. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interest.

References