Pharmacodynamics of Ginsenosides: Antioxidant Activities, Activation of Nrf2 and Potential Synergistic Effects of Combinations

Materials

All compounds were purchased from Sigma Aldrich Chemical Company (St Louis, USA): ginsenoside Rb1 (Rb1, catalog #00170580, 98.31%, Fig. 1A), ginsenoside Rg1 (Rg1, catalog #68317, 98.73%, Fig. 1B) and ginsenoside 20S-protopanaxatriol (20S, catalog #P0034, 99.4%, Fig. 1C). The ginsenosides were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO in the media was less than 0.1% DMSO. The vehicle control of 0.1% DMSO was used for all tests.

Cell culture

The Nrf2-mediated ARE in human hepatoma HepG2-C8-ARE luciferase cells, previously established in our laboratory using the pARE-TI-luciferase reporter gene was utilized in this experiment.¹³ The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1.17 g/L sodium bicarbonate, and 100 unit/mL penicillin, 100 μg/ml streptomycin at 37 °C in a humidified incubator with 5% CO₂.

Cell proliferation MTS assay

The cytotoxicity of the ginsenosides was tested in HepG2-C8 cells using the CellTiter 96 aqueous non-radioactive cell proliferation assay MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] (Promega, Madison, WI). The cells were first cultured in 96-well plates for 24 h and then were treated with ginsenosides at various concentrations for 24 h using 1% FBS medium. The cells were then treated with MTS for 1 h at 37°C. Absorbance of the formazan product was read at 490 nm with μQuant Biomolecular Spectrophotometer from Bio-Tek Instruments Inc. (Winooski, VT). At least three independent studies were conducted and four samples were performed for each treatment each time.

Total antioxidant activity test

The antioxidant potential of ginsenosides in HepG2-C8 cells were tested using the Total Antioxidant Power kit (Oxford Biomedical Research, MI, USA; catalog # TA02) according to the manufacturer' protocol. Briefly, the cells were first cultured in 12-well plates for 24 h and then were treated with ginsenosides at various concentrations for 24 h using 1% FBS medium. After 24 h, the experiment was terminated by washing the cells with ice-cold phosphate buffered-saline (PBS, pH 7.4), the cells were homogenized with lysis buffer (Promega, WI, USA). The lysate was then subjected to centrifugation at 604 × g for 12 min at 4°C, supernatant was collected for the assays and protein concentrations were determined by BCA protein assay (Pierce, Rockford, USA). For normalization of the total antioxidant activity of the treatment, the activity is expressed as percentage compared with the control 0.1% DMSO.

The reduction potential of the samples or the standards which effectively converts Cu²⁺ to Cu¹⁺, thus changing the ion's absorption characteristics. This reduced form of Cu¹⁺ would selectively form a stable 2:1 complex with the chromogenic reagent with an absorption maximum at 450 nm. A known concentration of Trolox was used to create a calibration curve, and the results were expressed as mM Trolox equivalents or in μM copper reducing equivalents and the activity is expressed as percentage compared with 0.1% DMSO. At least three independent studies were conducted in triplicates. All samples were diluted to fall within the standard curve.

ARE-luciferase assay

HepG2-C8 cells were cultured in 12-well plates and each well contained 100,000 cells in 1 ml of 1% FBS medium. The cells were treated with compounds for 24 h. The luciferase activity was determined as described previously^14-21 using a luciferase kit from Promega (Madison, USA) according to the manufacturer's instructions. Briefly, after treatments for 24 h, the cells were washed twice with ice-cold PBS and harvested in 1 × reporter lysis buffer and kept at -20°C overnight. After centrifugation at 4°C, 9,660 × g for 5 min, a 10 μl aliquot of the supernatant was assayed for luciferase activity with a SIRIUS luminometer (Berthold Detection System GmbH, Pforzheim, Germany). The luciferase activity was normalized against protein concentration determined by BCA protein assay (Pierce, Rockford, USA). The luciferase activity was expressed as percentage of induction of the samples over that of control vehicle-treated cells. At least three independent studies were conducted in triplicates.

Combination index calculation

To determine the synergistic effect between the combination of two different compounds, the combination index (CI) can be calculated with the following formula: CI= d1/Dx,₁ + d2/Dx,₂ where d1 and d2 are doses of drugs 1 and 2 in combination, which produces an effect x. Dx,₁ and Dx,₂ are the doses of drug 1 and drug 2 that produce the same effect x when given alone.^{21, 22} When the CI is equal to, less than or greater than 1, the combination will be additive, synergistic or antagonistic, respectively, as we have described previously.^{21, 22} This approach is based on the Loewe additivity model and although the exact mechanism of interaction may be unknown, this model is one of the most commonly used reference models for evaluating potential drug-drug interactions²³ and was first introduced by Chou TC el al.²⁴ Using this CI calculation for the ARE-luciferase activity and total antioxidant activity induced by the combinations of ginsenosides, it is possible to identify whether the combination of these ginsenosides at some concentrations would be synergistic, antagonistic or additive.

Western blotting

The cells were treated similarly as the MTS, and ARE-luciferase assays described above using 1% FBS medium. HepG2-C8 cells were treated with the compound for 48 h. Cells were harvested in 100 μl of Cell Culture Lysis Reagent (Promega E153A, Madison, WI) after washed with ice-cold PBS (pH 7.4) and kept at -20°C overnight. The next day, the frozen samples were thawed and collected as the homogenate. Then, the homogenate was centrifuged at 4°C, 9,660 × g for 5 min. The supernatants were collected and 15 μg of total protein were mixed with 5 μl Laemmli's SDS-Sample Buffer (Boston Bioproducts, Ashland, MA, USA) and denatured at 95°C for 5 minutes. The samples and the protein standard (Bio-Rad, Hercules, CA, USA) were then loaded onto a polyacrylamide gel (Criterion Tris–HCl gel, Bio-Rad Lab, Hercules, CA, USA) and gel electrophoresis was ran at 130 mA for 60 min. Proteins were transferred onto a polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA, USA) over 2 h using a semi-dry transfer system (BioRad, Hercules, CA, USA). The membranes were blocked with 5% BSA solution for 1 h at room temperature and incubated with the primary antibody (1:1000, in 3% BSA in Tris-buffered-saline and Tween 20, TBST) overnight at 4°C. Antibody against Actin (catalog no. sc-1616) was purchased from Santa Cruz (Santa Cruz Biotechnology, Inc., CA, USA). Antibody against Nrf2 (catalog no. 2178-1) was purchased from Epitomics (Burlingame, CA, USA). After hybridization with primary antibody, membranes were washed with TBST four times. The immunoreactions were continued with the respective secondary antibodies (1:5000, in 3% BSA in TBST) purchased from Santa Cruz Biotechnology, Inc., CA, USA, for 1 h at room temperature. After washing four times with TBST, the immunocomplexes were determined using the enhanced chemiluminescent system to detect the horseradish peroxidase on the immunoblots (Thermo Scientific, Rockford, IL, USA) and the bands were visualized and captured by BioRad ChemiDoc XRS system (Hercules, CA, USA). The protein bands were quantified by densitometry using Image J (Version 1.44, National Institute of Health, USA).

RNA isolation and quantitative real-time polymerase chain reaction (qPCR) in HepG2-C8 and murine prostate cancer TRAMP C1 cell lines

The HepG2-C8 cells were treated similarly as the MTS and ARE-luciferase assays described above using 1% FBS medium. The incubation of the compounds with the cells was terminated at 6 h later. On the other hand, TRAMP C1 cells at a density of 1 × 10⁴ cells/dish were cultured in 3 cm dishes, and the cells were treated with 20S (12.5 μM) after 24 h. The treatment medium was changed after 2 days, and total RNA was extracted from the cells using Trizol reagent (Invitrogen, Carlsbad, CA) after 3 days treatment. SuperScript III First-Strand Synthesis System from Invitrogen (Grand Island, NY) was further used to synthesize first-strand cDNA from total RNA according to the manufacturer's instructions. The first-strand cDNA was used as the template for the determination of mRNA expression levels by quantitative real time-PCR (qPCR) using Power SYBR Green PCR Master Mix (Applied Biosystem, Carlsbads, CA) in ABI7900HT system. The primer pairs were designed using Primer Quest Oligo Design and Analysis Tool by Integrated DNA Technologies Inc. (Coralville, IA, USA) and the sequences are listed in Table 1. At least 3 independent treatments were performed and duplicate samples were carried out for each treatment.

Statistical analysis

The results are presented as means ± standard error of the mean (SEM). The data were analyzed using one-way ANOVA with a post hoc multiple comparison analysis by Tukey's (PASW Statistics, version 18, SPSS Inc., Chicago, Illinois) or Student's t-test. P value of less than 0.05 was considered to be statistically significance. At least three independent experiments of each assay with three samples were conducted for each experiment.

Ginsenosides and their combinations are relatively non-toxic

MTS assay was performed to determine the effects of Rb1, Rg1 and 20S and their combinations of 12.5 μM of 20S with 12.5 μM of Rb1 or Rg1 (designated as 20S Rb1 12.5 and 20S Rg1 12.5 respectively). Rb1 and Rg1 are relatively non-toxic as compared to the control (0.1% DMSO) even up to 100 μM (Figure 2), while 20S decreased cell proliferation in a dose-dependent manner at 50 μM and above 50 μM caused significant cell death (ANOVA, p < 0.01 for 50 and 100 μM compared with 0.1 % DMSO; Figure 2). All subsequent experiments were conducted below 50 μM for compound 20S. Interestingly, the combinations of 20S Rb1 12.5 and 20S Rg1 12.5 did not increased the toxicities.

Induction of total antioxidant activity by ginsenosides and combination of ginsenosides show potential synergistic effect

To determine the antioxidant activity of Rb1, Rg1 and 20S and the combinations 20S Rb1 12.5 and 20S Rg1 12.5, the antioxidant activity was examined. The induction of the total antioxidant activity was increased by treatment of Rb1, Rg1 and 20S alone dose-dependently (Figure 3). Figure 3 shows the combinations 20S Rb1 12.5 and 20S Rg1 12.5 significantly induced total antioxidant activity (ANOVA, p < 0.01 compared with 0.1 % DMSO), significantly greater than the individual 12.5 μM Rb1 or Rg1 alone (both p < 0.05, data not shown). Table 2 shows the CI of the combinations resulted in CI < 1 significantly (Table 2), indicating synergistic effects when combining 12.5 μM 20S with 12.5 μM of Rb1 or Rg1.

Induction of ARE-luciferase activity by ginsenosides and their combinations show potential synergistic induction of ARE-luciferase activity

To test the transcriptional activation of ARE by ginsenosides, ARE-luciferase reporter assay was performed.^14-21 Figure 4 shows that the three ginsenosides alone or in combinations induced ARE-luciferase activity in HepG2-C8 cells with different potency. 20S at 25 μM strongly induced ARE-luciferase activity as compared to any other treatments (p<0.01). Combination of 12.5 μM 20S with 12.5 μM Rb1 or Rg1 synergistically induced the ARE luciferase activity with CI < 1 (Table 2), similar to the total antioxidant activity above (Figure 3).

Ginseonisdes induce Nrf2 protein expression

Figure 5 shows the protein expression of Nrf2 after the cells were treated with the ginsenosides individually or in combinations. The Nrf2 protein was not significantly induced at low doses of 12.5 μM by the three ginsenosides. However, the combinations of low doses of ginsenosides were able to significantly induce higher Nrf2 protein level as compared to the individual agent (Figures 5A & 5B). This result correlates with the synergistic effects (CI < 1) shown above in the total antioxidant activity (Figure 3) and ARE-luciferase assay (Figure 4).

Ginseonisdes induce mRNA expression of Nrf2 and Nrf2-target genes

To confirm that ginseonisdes can also induce endogenous Nrf2 and Nrf2 downstream target genes in cells, we conducted qPCR to quantify the mRNA expression (Figure 6). Values higher than one were considered positive in comparison to cells treated with control 0.1% DMSO. Figure 6A shows the results for the induction of Nrf2 and HO-1, Nrf2-dependent ARE-driven endogenous gene in HepG2-C8 cells (Figure 6A). When ginsenosides were tested alone at 12.5 μM, they showed two-fold induction of Nrf2 mRNA. Similar trend of mRNA induction of HO-1 was also shown (Figure 6A). Additionally, the mRNA induction of endogenous Nrf2 and Nrf2-mediated antioxidant and detoxifying enzymes such as HO-1, NQO1 and UGT1A1 in murine prostate cancer TRAMP C1 cells were also quantified using qPCR after treatments with 20S for 3 days (Figure 6B). The results show that 20S (12.5 μM) significantly increased Nrf2 and UGT1A1 mRNA expressions compared to the controls (p < 0.05). 20S (12.5 μM) also slightly induced HO-1 and NQO1 mRNA expressions compared to the controls although not statistically significance. These results suggest that ginsenosides, especially 20S, have the potential of increasing endogenous mRNA expression of Nrf2 and Nrf2-mediated antioxidant and detoxifying enzymes in cells.