GA, PDTC and H₂O₂ were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Huh7 hepatocarcinoma cells (donated by Dr. Koromilas; McGill University, Montreal, Canada) were cultured in advanced Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Grand Island, NY, USA) supplemented with 1% nonessential amino acids, 2% heat-inactivated fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 1% antibiotics (100 U penicillin G and 100 µg/ml streptomycin; Thermo Fisher Scientific, Inc.) and 1% glutamine, in a humidified atmosphere with 5% CO₂ at 37°C. In addition, a genotype 1b HCV subgenomic replicon cell culture system was established (24), which was maintained at the same conditions, but in the presence of 500 µg/ml G418 (Geneticin; Thermo Fisher Scientific, Inc.). Cells grown to 80–85% confluence were trypsinized with 2.5 ml trypsin diluted with fresh medium and counted using a hematocytometer (Marienfield-Superior, Lauda-Königshofen, Germany) with trypan blue (Gibco).

Huh7 parental and HCV replicon cells were seeded onto 96-well plates (2×10⁴ cells/well) and cultured for 24 h. Next, the medium was changed and the cells were treated with different concentrations of GA (100, 300 and 600 µM) dissolved in sterile phosphate buffered saline (PBS) and incubated for 0, 24, 48 and 72 h (25). Following incubation, cell viability was evaluated using an MTT [also known as 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, according to the standard experimental protocol (26,27).

Huh7 HCV replicon cells (~6×10⁵ cells/well) were seeded onto 6-well plates, cultured for 24 h and then treated with 300 µM GA for a duration between 0 and 72 h. After each time point, the cells were harvested and total protein extraction was performed. Briefly, the cells were washed twice with ice-cold 1X PBS/0.5 M EDTA, and proteins were extracted with 1X lysis buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 2 mM MgCl₂, 1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 3 µg/ml aprotinin, 1 µg/ml leupeptin and 1 µg/ml pepstatin. Upon incubation, the cell lysates were centrifuged at 16,438 × g for 5 min at 4°C (28). The supernatant was recovered, the protein concentration was measured using the Bradford method with a Bio-Rad Protein Assay kit (500-0006; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and a standard curve was obtained using bovine serum albumin (Amresco LLC, Solon, OH, USA).

Total cellular protein extracts were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, the samples were transferred to Hybond-P polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Little Chalfont, UK), activated with methanol, washed with pure water and equilibrated with 1X transfer buffer. The electrotransfer was performed at 100 V for 1 h at 4°C, and then the membrane was blocked with mouse anti-HCV NS5A monoclonal antibody (MAb; dilution, 1:1,000; AB20342; Abcam, Cambridge, UK) and anti-actin MAb (β-actin; dilution, 1:1,000; MAB1501; EMD Millipore, Billerica, MA, USA). Immunocomplexes on the membranes were detected using an enhanced chemiluminescence assay (Luminol, ImmunoCruz; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) (10). The expression of NS5A relative to β-actin protein was quantified using ImageJ software, version 1.46r (http://imagej.nih.gov/ij/docs/guide/146.html).

Huh7 HCV replicon cells (~2×10⁵ cells/well) were seeded onto 24-well plates, cultured for 24 h and then treated with 300 µM GA for a duration between 0 and 72 h. Cells were harvested after each time point and total RNA was extracted using TRIzol reagent (Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's specifications. RNA precipitates were washed with 75% alcohol and resuspended in 12 µl RNase-free water, and then the samples were stored at −80°C (29).

The total RNA extracted was subjected to RT to obtain complementary DNA (cDNA) using a SuperScript III RT kit according to the manufacturer's specifications (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA). Subsequently, 200 ng cDNA was amplified by qPCR to quantify the levels of HCV and GAPDH mRNA using an ABI-7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers used were as follows: HCV forward (75–93 nt), 5′-GCGTCTAGCCATGGCGTTA-3′, and reverse (138–157 nt), 5′-GGTTCCGCAGACCACTATGG-3′; GAPDH forward, 5′-GTGTTCCTACCCCCAATGTGT-3′, and reverse, 5′-ATTGTCATACCAGGAAATGAGCTT-3′; and the TaqMan probe (94–110 nt), 5′-FAM-CTGCACGACACTCATAC-NFQ-3′. For each PCR reaction, the following were used: 1 µl assay mix, 9 µl cDNA diluted in RNase-free water and 10 µl TaqMan PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermal cycling conditions were as follows: Initial setup at 50°C for 2 min, then 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. GAPDH-RNA expression was used to normalize the cDNA concentration and the amplification plots were analyzed using the ABI-7500 Real-Time PCR System software, version 2.0.1, according to the manufacturer's specifications (Applied Biosystems; Thermo Fisher Scientific, Inc.) (30). The 2^−ΔΔCq method was used to calculate relative changes in gene expression determined from RT-qPCR experiments.

To evaluate the effect of GA on the oxidative stress level, ROS production was measured. Briefly, Huh7 HCV replicon cells (~2×10⁴ cells/well) were seeded onto 96-well plates, cultured for 24 h and then treated with 300 µM GA, 2 µM H₂O₂ as a damage control or 5 µM PDTC as an antioxidant control, for 0.5, 1, 3, 6, 12 and 24 h at 37°C. Next, 2 µl of 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Invitrogen Molecular Probes, Eugene, OR, USA) were added 30 min before the end of the treatment. The fluorescence of cells was measured at room temperature, using 485 nm and 528 nm as the excitation and emission wavelengths, respectively (BioTek Synergy H5; BioTek Instruments, Inc., Winooski, VT, USA) (31,32).

All variables were tested in triplicate, and experiments were repeated at least three times. Values were presented as the mean ± standard deviation. Statistically significant differences between control and treated groups were determined by Student's t-test. Differences were considered to be statistically significant for values of P<0.05.

Initially, the study investigated whether GA induces a cytotoxic effect on treated cells. Huh7 parental and Huh7 HCV replicon cells were exposed to three different concentrations of GA (100, 300 and 600 µM) and incubated for a time between 24 and 72 h. Next, total cell count and viability determinations were performed using an MTT assay. Fig. 1 shows that after 72 h of treatment no statistically significant differences in cell number and viability were detectable between the untreated (cell viability, 100%) and treated cell lines (Fig. 1A, parental cells; Fig. 1B, HCV replicon cells) when using the 100 and 300 µM GA concentrations (cell viability, ~98 and 95%, respectively). By contrast, cells treated with 600 µM GA showed a lower cell survival rate in the two cell lines at all times of exposure. Based on this finding, we selected the concentration of 300 µM GA in all subsequent experiments.

To evaluate the effect of GA on HCV-RNA expression in HCV replicon-containing cells, these cells were incubated with 300 µM GA for three different durations (24, 48 and 72 h). Subsequently, the total cellular RNA was extracted and subjected to RT-qPCR for HCV-RNA quantification, as described in the Materials and methods. GA was found to inhibit HCV-RNA expression in a time-dependent manner compared with the untreated cells, showing a higher effect at 72 h post-treatment, at which the lowest HCV-RNA expression was observed (22% inhibition at 48 h and 44% inhibition at 72 h, P<0.01; Fig. 2). Collectively, these results reveal that 300 µM GA is able to decrease HCV expression at the transcriptional level in the HCV replicon cell culture system.

To investigate whether GA can influence the synthesis of HCV viral proteins, NS5A and actin protein levels were examined by western blot analysis in untreated HCV replicon cells or cells treated with 300 µM GA, and incubated for 24, 48 and 72 h (Fig. 3A). Cells treated with 300 µM GA expressed lower levels of NS5A-HCV proteins, as shown by the lower ratio of NS5A-HCV protein at the three time points after exposure compared with the untreated cells (0.33, 0.45 and 0.49 for the time points 24, 48 and 72 h, respectively, compared with the control value; P<0.05; Fig. 3B). These data suggest that GA treatment may diminish the translational rate of viral proteins or decrease viral protein stability, in addition to the negative effect on HCV-RNA levels.

It has been reported that HCV promotes ROS production in infected hepatocytes, which further promote lipid and protein oxidation, leading to cell death (33). To determine whether GA treatment has an antioxidant activity in HCV replicon cells, ROS levels were evaluated using the H₂DCF-DA assay. HCV replicon cells were incubated in the presence or absence of 300 µM GA and incubated for 24, 48 and 72 h, following which the ROS levels were quantified. GA was found to reduce ROS levels in a time-dependent manner (Fig. 4), with a greater effect at earlier time points, including 0.5, 1 and 3 h after treatment (~50, 30 and 20% reduction, respectively; P<0.05). In addition, at 6, 12 and 24 h post-treatment, ROS production was maintained <20% compared with the control cells. As a positive control of antioxidant activity, HCV replicon cells were treated with 5 µM PDTC. Fig. 4 shows that PDTC decreased ROS levels starting at 0.5 h and showing a similar effect as that observed at 3 h after GA treatment (P<0.05). In addition, the present study investigated whether GA is able to decrease the levels of ROS generated by a strong oxidizing agent, such as H₂O₂. Huh7 replicon cells were treated simultaneously with 2 µM H₂O₂ and GA, and incubated between 0.5 and 48 h. Subsequently, ROS levels were measured at the end of each time point. It was observed that GA was able to abate the increased ROS levels induced by H₂O₂, reaching levels similar to those of cells without the oxidizing agent (Fig. 4). This suggested that GA acid was able to mitigate the cellular oxidative stress induced by an oxidizing agent. Therefore, these results confirm that GA was able to decrease oxidative stress markers in the same way the antioxidant agent, PDTC (5 µM).