The HeLa human cervical cancer cell line was purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and 2.5 µg/ml amphotericin B (Sangon Biotech Co., Ltd., Shanghai, China) at 37°C in a 5% CO₂ incubator. The medium was replaced every 2 days.

For poly (I:C) transfection, 1×10⁵ HeLa cells were seeded in a 12-well plate and maintained at 37°C in a 5% CO₂ incubator for 15 h. A dose of 2 µl Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was added to 100 µl of serum-free medium, following which the mixture was incubated at room temperature for 5 min. At the same time, 10 µl poly (I:C), purchased from Sigma-Aldrich (St. Louis, MO, USA) was added to the 100 µl of serum-free medium. Subsequently, the Lipofectamine™ 2000 and poly (I:C) were mixed gently and incubated at room temperature for 20 min. Following incubation, the cell culture medium was replaced with serum-free medium, which was added to each well containing the Lipofectamine™ 2000 and poly (I:C) mixture, and incubated at 37°C for 4 h. Finally, the medium in each well was replaced with a fresh serum-containing medium.

Cell apoptosis was measured by flow cytometry using an Annexin V-propidium iodide (PI) kit (cat. no. 556420; BD Pharmingen, San Diego, CA, USA), according to the manufacturer's protocol. Briefly, the cells were harvested and washed three times with phosphate-buffered saline (PBS). Following centrifugation at 300 × g for 10 min at 4°C, the cells were resuspended in 500 µl binding buffer (0.1 M HEPES/NaOH, pH 7.4; 1.4 M NaCl; 25 mM CaCl₂) containing 5 µl fluorescein isothiocyanate-conjugated Annexin V, the mixture was incubated at 25°C in the dark for 10 min, following which 5 µl PI was added. Finally, cell apoptosis was analyzed using flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA) with CellQuest software (BD Biosciences), with the results are expressed as a percentage of the total cells counted.

The proteins were extracted from the cells using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Nantong, China). Western blot analyses were performed, as previously reported (17). Briefly, total protein was quantified using a bicinchoninic acid kit (Beyotime Institute of Biotechnology) and 40 µg protein per lane was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis prior to electroblotting onto a nitrocellulose membrane (GE Healthcare, Munich, Germany). Non-specific binding was blocked by incubating the membrane with 5% non-fat milk in Tris-buffered-saline with Tween (TBST; 10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.05% Tween-20) at room temperature for 1 h. After blocking, the membrane was incubated with various primary antibodies overnight at 4°C. The antibodies used included the following: Mouse monoclonal anti-cytochrome c (1:1,000; cat. no. ab13575; Abcam, Cambridge, UK), rabbit polyclonal anti-cleaved caspase-9 (1:500; cat. no. ab2325; Abcam) and -3 (1:500; cat. no. ab13847; Abcam), rabbit polyclonal anti-IFN-β (1:400; cat. no. sc-83256; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-phosphorylated (p)-H2A.X (cat. no. 07-627; 1:1,500; EMD Millipore, Billerica, MA, USA) and rabbit polyclonal anti-β-actin (1:2,000; cat. no. ab59381; Abcam). The membrane was then incubated at room temperature for 2 h with anti-mouse horseradish peroxidase-conjugated (1:5,000) secondary antibodies (cat. no. sc-2497), obtained from Santa Cruz Biotechnology Inc. The blots were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) and normalized to β-actin.

The total RNA was isolated from the HeLa cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1–2 µg) was reverse transcribed using SuperScript^® IV Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). The RT-qPCR reactions were performed on a Rotor-Gene RG-3000 Real-Time Thermal Cycler (Corbett Research, Sydney, Australia) using a SYBR^® Premix Ex Taq™ II kit (Takara Biotechnology Co., Ltd., Dalian, China). PCR primers specific for IFN-β were designed, as previously reported (20): sense 5′-TTGAATGGGAGGCTTGAATA-3′ and antisense 5′-CTATGGTCCAGGCACAGTGA-3′. These primers were synthesized by Takara Biotechnology Co., Ltd. The PCR procedure was as follows: Polymerase activation for 30 sec at 95°C, 40 cycles of amplification, each consisting of 95°C for 5 sec and 60°C for 20 sec, and 1 cycle of dissociation consisting of 95°C for 15 sec, 60°C for 30 sec and 95°C for 15 sec. All reactions were performed in triplicate. Fluorescence data were analyzed using Rotor-Gene 6 software (version 6.0; Corbett Research). The mRNA expression levels were calculated using the 2^−ΔΔCq method (21), and were normalized to β-actin and reported as arbitrary units.

The generation of intracellular ROS generation in the HeLa cells was evaluated in the homogenate using 5-(and−6)-carboxy-2′, 7′-dichlorohydrofluorescein diacetate (carboxy-H2DCFDA; DCFH), which is a specific ROS-detecting fluorescent dye. DCFH is sensitive to ROS, and can be oxidized to the highly fluorescent dichlorofluorescein (DCF) (22). The protocol was performed, according to a previous report (23). Briefly, 1×10⁶ HeLa cells were incubated with 10 µl DCFH (Sigma-Aldrich) for 30 min at 37°C in the dark. The cells were then washed twice in PBS and analyzed using flow cytometry (Cytomics FC 500; Beckman Coulter, Brea, CA, USA) or observed using a fluorescence microscope (BX53; Olympus Corporation, Tokyo, Japan). The redox state of the samples can be monitored by detecting increases in fluorescence. Accumulation of DCF in the cells is measured by an increase in fluorescence at 530 nm.

The mitochondrial ΔΨm in the cells was determined according to a previous report (24). The HeLa cells were collected following the different treatments in 6-well plates. Following being washed twice with PBS, the cells were incubated with MitoProbe™ 3, 3′-diethyloxacarbicyanine iodide using a Molecular Probes DiOC 2 (3) kit (Thermo Fisher Scientific, Inc.) at a concentration of 8 nM for 30 min at 37°C. These stained cells were examined using a flow cytometer (FACSCalibur; BD Biosciences).

The cell lysates were prepared following the different treatments using cell lysis buffer (Beyotime Institute of Biotechnology). Briefly, 5×10⁶ cells were suspended in 50 µl chilled cell lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM Na₂EDTA; 1 mM EGTA; 1% Triton; 2.5 mM sodium pyrophosphate; 1 mM beta-glycerophosphate; 1 mM Na₃VO₄; 1 µg/ml leupeptin) and incubated on ice for 10 min. Following centrifugation at 14,000 × g and 4°C for 10 min, the supernatant (cytosolic extract) was transferred to a fresh tube and placed on ice, and 300 µg of the protein was diluted in 50 µl cell lysis buffer. The activity of caspase-3 was determined using a Caspase-3 Activity kit (cat. no. C1116; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The assays were performed on 96-well microtitre plates by incubating 10 µl cell lysate protein/sample in 80 µl reaction buffer, containing 1% NP-40, 20 mM Tris-HCl (pH 7.5), 137 mM Nad and 10% glycerol, and 10 µl caspase-3 substrate (acetyl-Asp-Glu-Val-Asp p-nitroanilide; Ac-DEVDpNA; 2 mM; BioVision, Inc., Milpitas, CA, USA). The lysates were incubated at 37°C for 4 h, following which the samples were measured using an ELISA reader (Labsystems, Helsinki, Finland) at an absorbance of 405 nm (25). Caspase-4 activity was measured using a commercially available Caspase-4 Assay kit (cat. no. C1122; Beyotime Institute of Biotechnology). The procedure was performed, according to the manufacturer's protocol. Briefly, ~300 µg protein was diluted in 50 µl cell lysis buffer. Subsequently, 50 µl of 2X reaction buffer, containing 10 mM dithiothreitol, was added to each sample. Finally, 5 µl of the 4 mM LEVD-pNA substrate (final concentration, 200 µM; BioVision, Inc.) was added and incubated at 37°C for 1.5 h. The absorbance was measured in an ELISA reader (Labsystems, Helsinki, Finland) at 405 nm. The caspase-9 activity assay was performed using the caspase-9 Assay kit (cat. no. ab119508; Abcam), according to the manufacture's protocol, and the samples were prepared using the same method used for caspase-3, described above. Subsequently, 85 µl reaction buffer and 5 µl Leu-Glu-His-Asp-p-nitroanilide (LEHD-pNA) were added to each sample, and incubated at 37°C for 2 h. The absorbance was measured in an ELISA reader (Labsystems, Helsinki, Finland) at 405 nm.

Data are presented as the mean ± standard deviation. The results were analyzed using a two-tailed t-test or one-way analysis of variance followed by Duncan's test to evaluate the differences among groups. Statistical analysis was performed using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The effects of poly (I:C) transfected into the HeLa cervical cancer cell line was first examined. Flow cytometry following Annexin V/PI staining revealed that poly (I:C) transfection increased the percentage of apoptosis cells between 4.5 and 65%, compared with those in the control group (Fig. 1A and B). In addition, poly (I:C) transfection markedly increased the protein levels of the pro-apoptotic Bax and Bid, whereas it decreased the protein levels of anti-apoptotic Bcl-2 and Survivin, compared with the control group. However, vector transfection had no marked effect on either cervical cancer cell apoptosis or the protein levels of the apoptosis-associated markers, Bacl-2, Bax, Survivin or Bid (Fig. 1C and D).

IFN-β has been reported to be involved in apoptosis in cancer (26). In order to elucidate whether IFN-β is involved in poly (I:C) transfection-induced cervical cancer cell apoptosis, the present study determined the mRNA and protein levels of IFN-β following poly (I:C) transfection. It was found that vector transfection had no significant effect on the mRNA and protein levels of IFN-β; however, poly (I:C) transfection significantly promoted the mRNA and protein levels of IFN-β, compared with the vector control (Fig. 2A and C). An increase in ROS stress can induce apoptosis in cancer cells (27); in order to investigate whether poly (I:C) induced cancer cell apoptosis through the induction of oxidative stress, the present study determined whether poly (I:C) transfection triggered the generation of ROS. The results demonstrated that the staining intensity of carboxy-H2DCFDA increased significantly in the HeLa cells following poly (I:C) transfection, compared with the vector control, and this increase was inhibited by IFN-β siRNA treatment (Fig. 2B). Excessive ROS production has the potential to damage cellular macromolecules, including DNA, eventually leading to cell death (28). In order to investigate whether poly (I:C) transfection also induced DNA damage, the present study examined DNA damage by analyzing the phosphorylation levels of γH2A.X at Ser 139. The western blot analyses showed that the levels of p-γH2A.X increased in the HeLa cells following poly (I:C) transfection; however, treatment with IFN-β siRNA subsequent to poly (I:C) transfection decreased the levels of p-γH2A.X (Fig. 2D).

MOMP is often required for activation of the caspase proteases, which cause apoptotic cell death (29). To understand the underlying mechanism by which poly (I:C) transfection induces apoptosis, as well as the role of IFN-β in this process, the present study investigated the change in ∆Ψm, which is the cause of MOMP. Following poly (I:C) transfection, the number of cells exhibiting a high ΔΨm decreased, compared with those transfected with the vector (Fig. 3A); and IFN-β siRNA treatment following poly (I:C) transfection markedly increased the number of cells with a high ΔΨm. This data suggested that poly (I:C) transfection may disrupt the ΔΨm, and that IFN-β is involved in this process (Fig. 3A). To further confirm the involvement of the mitochondrial signaling pathway during poly (I:C) transfection-induced apoptosis, the present study measured the release of cytochrome c from the mitochondria into cytosol, a hallmark of mitochondria-mediated apoptosis. As shown in Figs. 3B and C, poly (I:C) transfection increased the content of cytosolic cytochrome c and decreased the content of mitochondrial cytochrome c; however, IFN-β siRNA treatment following poly (I:C) transfection decreased the content of cytosolic cytochrome c and increased the content of mitochondrial cytochrome c. These results suggested that IFN-β attenuated the poly (I:C)-induced release of cytochrome c from mitochondria into cytosol.

The release of cytochrome c from the mitochondria into the cytosol is a key event for caspase activation (6). In order to further confirm whether poly (I:C) induced caspase activation, and whether IFN-β was involved in the process of caspase activation, the present study subsequently examined caspase-9 and caspase-3 activity and processing. As shown in Fig. 4, poly (I:C) transfection significantly increased the activities of caspase-3 and caspase-9, and IFN-β siRNA markedly decreased the poly (I:C)-induced increases in caspase-3 and caspase-9 activity (Fig. 4A and B). The results of the western blot analysis showed that poly (I:C) transfection also promoted the cleavage of caspase-3 and caspase-9.

Caspase-4 activation is required for endoplasmic reticulum (ER) stress-induced apoptosis in human cells (30). In order to investigate whether poly (I:C) transfection also induces cervical cancer cell apoptosis through the ER-mediated pathway, caspase-4 activity and processing were examined following poly (I:C) transfection. The results showed that poly (I:C) transfection marginally increased caspase-4 activity, however, the difference was not significant when compared with the vector-transfected group (Fig. 4C). In addition, IFN-β siRNA had no significant effect on caspase-4 activity (Fig. 4C). These results suggested that poly (I:C) induced the cervical cancer cell apoptosis predominantly through the mitochondrial-mediated pathway.

The results described above suggested that poly (I:C) induced cervical cancer cell apoptosis, and that IFN-β was involved in this progress. In order to confirm the involvement of IFN-β in poly (I:C)-induced cervical cancer cell apoptosis, the present study determined the levels of cervical cancer cell apoptosis following poly (I:C) transfection and IFN-β siRNA treatment. The results showed that poly (I:C) transfection significantly induced cervical cancer cell apoptosis, compared with vector transfection (Fig. 5). However, IFN-β siRNA sharply attenuated the cervical cancer cell apoptosis induced by poly (I:C) transfection (Fig. 5). These results demonstrated that poly (I:C) induced cervical cancer cell apoptosis partly by promoting the expression of IFN-β.