Doxorubicin (DOX) is a highly effective chemotherapeutic agent; however, the dose-dependent cardiotoxicity associated with DOX significantly limits its clinical application. In the present study, we investigated whether Rb1 could prevent DOX-induced apoptosis in H9C2 cells
Doxorubicin (DOX), an anthracycline chemotherapeutic agent, has been shown to be effective in treating malignant neoplasias. However, the use of DOX is limited by its dose-dependent cardiotoxicity, which may be either acute or chronic, and could ultimately lead to cardiomyopathy following severe heart failure (Wallace, 2003; Octavia
Cytochrome P450 (CYP) constitutes a major family of phase I xenobiotic metabolizing enzymes (XMEs) that transforms xenobiotics to either active or inactive metabolites (Guengerich, 2004). Although CYPs are expressed predominantly in the liver (Davila and Morris, 1999; Elbekai
Aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor that belongs to the basic-helix-loop-helix (bHLH) family and has the ability to regulate CYP1A. In the absence of ligand, AhR exists primarily in the cytoplasm as an inactive complex with two Hsp90 and p23. Once binding to its ligands such as PAHs, AhR dissociates from the p23 and translocates to the nucleus, where it dissociates from Hsp90 and heterodimerizes with the AhR nuclear translocator (ARNT). The AhR-ARNT complex then binds to the xenobiotic responsive element (XRE), which is located in the promoter region of the receptor regulated genes, such as CYP1A1 and CYP1A2, resulting in transcription and translation of CYP1A genes.
Due to the polycyclic aromatic structure of DOX, it has been demonstrated that DOX can activate AhR and induce the expression of CYP1A1 in ARVM and H9C2 cells (Volkova
Ginseng has been extensively used as a traditional herbal medication in China and other Asian countries for over 2000 years. The therapeutic effects of ginseng have been mainly attributed to the various ginsenoside compounds within the herb. Among these ginsenosides, ginsenoside Rb1 has been demonstrated to possess cardiovascular benefits. Ginsenoside Rb1 has been reported to decrease cardiac contraction in adult rat ventricular myocytes (Scott
Herein, we hypothesized that ginsenoside Rb1 could protect against DOX-induced apoptosis in H9C2 cells and down-regulate the expression of CYP1A genes, which may be a new strategy to reduce the cardiotoxicity of DOX.
We obtained reagents from the following vendors: ginsenoside Rb1 (HPLC≥98%) from Yuanye Biotechnology Corporation (Shanghai, China); doxorubicin (DOX) from Selleck (Boston, MA, USA); dimethyl sulfoxide (DMSO) and TRIzol Reagent from Sigma-Aldrich Chemicals (St Louis, MO, USA); Dulbecco’s modified Eagle’s medium (DMEM) and Phosphate Buffered Saline (PBS) from Gibco (Grand Island, NY, USA); fetal bovine serum (FBS) from HyClone (South Logan, UT, USA); Cell Counting kit-8 from Dojindo (Dojindo Molecular Technologies, Inc., Kumamoto, Japan); PRL-TK plasmid, Dual-Glo® Luciferase Assay System and Caspase-Glo®3/7,8,9 Assay from Promega (Madison, WI, USA); Cell Death Detection ELISA kit from Roche (Roche Diagnostics Corporation, Indianapolis, IN, USA); Hoechst 33258 from Beyotime Institute of Biotecnology (Shanghai, China); Lipofectamine® 2000 Reagent from Invitrogen (Carlsbad, CA, USA); Human pGL4.17-1A1 luciferase reporter plasmid and Human pcDNA3.1 AhR plasmid were pre-constructed by our laboratory; TransScript® First-Strand cDNA Synthesis Super-Mix and TransStart® Green q-PCR SuperMix from TransGen Biotechnology Corporation (Beijing, China); BCA protein Assay Kit from CW Biotechnology (Beijing, China); polyvinylidene diflouride (PVDF) and enhanced chemiluminescence reagent from Millipore Corporation (Billerica, MA, USA); 2-Methyl-2H-pyrazole-3-carboxylic acid-(2-methyl-4-o-tolylazophenyl)-amide (CH-223191), Ah Receptor siRNA(r) and anti-CYP1A1, anti-CYP1A2, anti-AhR from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-caspase-3, anti-Bax, anti-Bcl-2, anti-PARP and anti-cyt
H9C2 cells (Cell Resource Center of Xiehe, Beijing, China) were cultured in DMEM, supplemented with 0.45% glucose, L-glutamine, 0.11% sodium pyruvate, and 10% FBS, at 37°C in a 5% CO2 humidified incubator.
Cells were treated with Rb1 at concentrations of 50, 100, 200, 400 μM or vehicle for 6 h, and then exposed to 1 μM DOX for indicated times. In separate experiments, cells were pre-incubated with CH-223191, an AhR antagonist, 2 h before addition of 200 μM Rb1 and/or 1 μM DOX. The same volumes of corresponding solvents were added to the controls.
To measure cytotoxicity, H9C2 cells and MCF-7 breast cancer cells were seeded with culture medium in 96-well microplates (4000 cells/well) respectively and incubated at 37°C for 24 h before drugs exposures. After treatment with DOX or/and Rb1 for indicated times, cell viability was examined with the Cell Counting kit-8 as the manufacturer’s instructions. The absorbance (OD) values were detected at 450 nm with a plate reader. Data are reported as the percentage of cell viability in comparison to that of its respective non-treated control group (100%).
For apoptosis analysis, DNA fragmentation was determined by a Cell Death Detection ELISA kit. H9C2 cells were seeded with culture medium in 6-well plates and cultured at about 80% confluency prior to the indicated treatments. At 24 h after DOX, Rb1 and/or vehicle treatment, the cytoplasmic monoand oligonucleosomes (histone/DNA fragments) were extracted and assayed according to the manufacturer’s instructions. Absorbance was measured at 405 nm using a microplate reader (reference wavelength approx. 490 nm). The specific enrichment of mono- and oligonucleosomes was expressed as enrichment factor and calculated using the following formula: enrichment factor=the ratio of absorbance of the sample: absorbance of the negative control group.
Hoechst 33258 staining was used to identify the morphological features of apoptosis. Briefly, H9C2 cells (5×104/well) were seeded in 6-well plates and cultured 24 h, then treated with DOX, Rb1 or/and vehicle for 24 h. The cells were fixed with 1mL of 4% paraformaldehyde for 20 min. Then, the cells were incubated in 1mL PBS containing 10 μM Hoechst 33258 at 37°C for 30 min. Cells were washed with PBS twice and dead cells and apoptotic bodies were identified by condensed or fragmented nuclei using fluorescence microscopy (Olympus, Tokyo, Japan) at ×200 magnification.
The present caspase activity assay used a luminogenic substrate containing the DEVD sequence. Activity of caspase-3/7, 8, 9 was determined using a commercial kit according to the manufacturer’s instructions. Briefly, H9c2 cells were seeded in 96-well plates and cultured 24 h, after 12 h treatments with DOX in the presence or absence of Rb1, caspase reagent was added and incubated for 30 min. The production of light was measured with a luminescence spectrometer LS55 (Perkin-Elmer, CA, USA) at an excitation wavelength of 499 nm and an emission wavelength of 521 nm.
Total RNA of H9C2 cells was isolated after various periods of time incubation with the different treatments using TRIzol reagent according to the manufacturer’s instructions to assess the specific induction of CYP1A1, CYP1A2 and AhR expression. The quality of the RNA was confirmed by an A260/ A280 ratio of >1.8. cDNA synthesis was performed by using the TransScript® First-Strand cDNA Synthesis SuperMix and 1.5 μg of total RNA from each sample was used. Quantitative real-time PCR reactions were performed on an ABI StepOne PlusTM (Foster city, CA, USA) real time PCR instrument using SYBR Green PCR SuperMix. The amplification reactions were performed as follows: 30 s at 94°C, and 40 cycles of 94°C for 5 s and 60°C for 30 s. The primers used in the experiments are listed in Table 1. The data were analyzed as described in the instrument manual using relative gene expression. Briefly, the data are presented as the fold change in gene expression normalized to the endogenous reference gene (GAPDH) and relative to the untreated control.
The H9C2 cells were lysed in RIPA buffer at 4–8°C and then the supernatant lysates were collected after centrifugation. The protein concentration was measured by BCA protein Assay Kit. Equal amounts of protein from each sample were loaded on and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and then electrophoretically transferred to immobilon polyvinylidene diflouride membranes. Protein blots were then blocked with 5% skimmed milk powder in TBST at room temperature for 2 h, followed by incubation with primary antibodies overnight at 4°C. The membranes were incubated with HRP-conjugated secondary antibodies for 1 h. The bands were visualized using the enhanced chemiluminescence reagent and quantified relative to GAPDH bands using ImageJ® (National Institute of Health, MD, USA) image processing program.
Human umbilical vein endothelial cells (HUVECs) were cultured in RPMI1640, at 37°C in a 5% CO2 humidified incubator. HUVECs were plated onto 24-well cell culture plates and each well of cells was transfected with pcDNA3.1 AhR plasmid and pGL4.17-1A1 luciferase reporter plasmid plus the Renilla luciferase PRL-TK plasmid, used for normalization. Cells were transfected with 500 ng pcDNA3.1 AhR plasmid and 250 ng pGL4.17-1A1 plasmid plus 50 ng PRL-TK plasmid using Lipofectamine 2000 reagent according to the manufacturer’s instructions. After incubation with test compounds for 24 h, Luciferase assay was performed according to the manufacturer’s instructions (Promega). Briefly, initially firefly luciferase activity was measured with a proper luminometer, and then Renilla luciferase activity was measured. Luciferase activity was reported as light emitted per well, calculating ratio of firefly: Renilla luminescence for each well and normalizing the sample well ratio to the ratio from a control.
The AhR siRNA was a pool of three target-specific 21 nucleotide siRNAs designed to knockdown AhR gene expression. The transfection was performed in 24 wells plates. AhR siRNA and negative control oligonucleotides were transfected using the Lipofectamine 2000 according to the manufacturer’s instructions. The final concentration of the AhR siRNAs was 60 nmol/L. After 6 hours, transfected cells were washed with PBS, and then incubated in new culture media containing 10% FBS. Then cells were incubated for an additional 24 hours before drug treatment.
All data are expressed as the mean ± SD and were analyzed using GraphPad Prism 5.01 software (GraphPad software Inc., CA, USA). Comparisons among groups were made by one-way ANOVA.
To explore the effect of ginsenoside Rb1 against the cardiotoxic effect of DOX, H9C2 cells were treated with DOX for 24 h or Rb1 (50 μM, 100 μM, 200 μM, and 400 μM) pre-treated 6 h before DOX exposure and CCK-8 assay was performed to measure cell viability. The results showed that 5 μM DOX significantly inhibited the growth of H9C2 cells. Additionally, Rb1 pre-treatment significantly increased the cell viability of DOX-induced cell death of H9C2 cells in a dose-dependent manner (Fig. 1A), with the highest effect seen at 200 μM. Rb1 alone at 400 μM was slightly toxic to cells and still could prevent DOX-induced cytotoxicity. These results suggested that Rb1 could reduce DOX-induced H9C2 cell death. In addition, various concentrations of ginsenoside Rb1 had no significant effect on the antitumor activity of DOX in MCF-7 cells (Fig. 1B).
To evaluate the effect of Rb1 on H9C2 cells after DOX treatment, H9C2 cells were pre-treated with 50–400 μM for 6 h and then treated with 1 μM DOX for an additional of 24 h. The positive apoptosis was determined by an ELISA assay to quantify histone-associated DNA fragmentation generated in apoptotic H9C2 cells. We found that 1 μM DOX treatment for 24 h resulted in a noticeable increase in DNA fragmentation and Rb1 treatment reduced DOX-induced DNA fragmentation in H9C2 cells. As shown in Fig. 2, the enrichment factor was significantly higher in the DOX-treated group than in control group and was significantly lower in the Rb1-treated group than in DOX-treated group. These results suggested that Rb1 reduced DOX-induced apoptosis in H9C2 cells.
In order to determine the protective effects of Rb1 on DOX-induced apoptosis, Hoechst 33258 staining was used to further confirm the anti-apoptotic effect of Rb1. In the control group and Rb1 treated group, the nuclei were stained a less bright blue and the color was homogeneous (Fig. 3A, 3D). Compared with the control group, DOX treatment resulted in increased apoptosis with obvious changes in heterogeneous intensity, chromatin condensation, and fragmentation under fluorescent microscopy, which are the classic characteristics of apoptotic cells (Fig. 3B). However, when cells were pre-treated with Rb1 6h before DOX treatment, the morphological changes showed less apoptotic characteristics (Fig. 3C).
Previous study has shown that DOX-induced apoptosis is a multifactorial process (Zhang
DOX is a polyaromatic hydrocarbon. It was previously reported that treatment with DOX induced AhR migration to the nucleus, increased AhR binding with its co-factor, aryl hydrocarbon receptor nuclear translocator-1 (ARNT1), and increased the expression of AhR-regulated phase I (CYP1A1) drug-metabolizing enzymes in H9C2 cells (Volkova
To determine whether the observed effects upon exposure to Rb1 in the presence and absence of DOX on CYP1A mRNA and protein were
To further examine whether AhR was directly involved in the effect of Rb1 on DOX-mediated induction of AhR downstream target genes in H9C2 cells, we knocked down AhR expression with siRNA. H9C2 cells were transfected with a control siRNA (siCON) or a siRNA against AhR (siAhR), and then cultured 24 h before drug treatment. RT-PCR and western blot analysis showed that siAhR dramatically reduced AhR mRNA and protein levels (Fig. 7A), indicating the effectiveness of the siRNA-mediated AhR gene silencing. Knockdown of AhR resulted in a decrease in the expression of CYP1A1 and CYP1A2 under basal conditions. Furthermore, the inhibition of AhR expression blocked DOX-induced up regulation of CYP1A1, CYP1A2 and AhR mRNA, and the effect of Rb1 on DOX-mediated induction of CYP1A1, CYP1A2 and AhR mRNA was significantly blocked compared with siCON group (Fig. 7B). In addition, pretreatment with CH-223191 (10 μM) for 2 h, an AhR antagonist, increased the basal expression of CYP1A1, CYP1A2 and AhR, an effect that has been previously described in ARVM that may be due to ligand-dependent effects of CH-223191 (Volkova
DOX-induced cardiomyopathy is a major obstacle that limits the clinical application of doxorubicin in cancer chemotherapy. To mitigate this hurdle, recent studies have focused on identifying agents that can protect against cardiac dysfunction. In the present study, using H9C2 cells, which is the most popular cell model used to carry out cardiovascular research (Shi
Ginseng, as a natural product, has been shown to exert potential benefits on cardiovascular diseases. Ginsenoside Rb1, as a compound of ginseng, has been reported to reduce isoproterenol-induced cardiomyocytes apoptosis
In the current study, the inhibition of DOX-induced H9C2 cells apoptosis by ginsenoside Rb1 was associated with proportional decrease in the mRNA and protein levels of the CYP1A1 and CYP1A2 genes regulated by AhR (Fig. 5), indicating an AhR-dependent mechanism. These results were consistent with previous studies, which showed that TCDD-induced CYP1A1 expression, an index of dioxin toxicity, was suppressed by flavonoids permeating the human intestinal Caco-2 cell monolayers (Hamada
Benzo(a)pyrene and 3-methylcholanthrene, as an AhR ligand, induced CYP1A1 and CYP1B1 expression and caused cardiac hypertrophy in SD rats (Aboutabl
The activity of CYP1A is an index of dioxin toxicity. There is published evidence supporting the role of CYP1A1 in TCDD toxicities that TCDD-induced weight loss, liver pathology, and wasting syndrome were alleviated in CYP1A1–/– mice (Uno
In order to verify whether the effects of ginsenoside Rb1 were mediated by the AhR, we examined the effects of Rb1 and DOX on CYP1A1 luciferase reporter activity. The results showed that both DOX and Rb1 were capable of enhancing the interaction between AhR and CYP1A1 promoter region and DOX induction was more significant than Rb1; however, different concentrations of Rb1 could attenuate the effects of different levels of DOX (Fig. 6). It further confirms our conjecture that Rb1 is a weaker AhR agonist than DOX, and Rb1 can inhibit AhR agonist activity of DOX. Interestingly, Rb1 at 200 μM was the most efficacious concentration and this was consistent with the results of cytotoxic activity and DNA fragmentation assays. This may be due to Rb1 being a partial agonist; full agonist in combination could produce antagonism by competing for binding and depending on both drug doses and achieving the efficacy of partial agonists, thus Rb1 at 200 μM may be the most appropriate dose. In addition, ginsenoside Rb1 can also induce CYP1A1 expression in a dose-dependent manner and 400 μM Rb1 may have slight toxicity. The direct evidence for the involvement of AhR in the capability of Rb1 decreasing the induction of CYP1A induced by DOX was supported by knockdown of AhR using AhR siRNA or blocking of AhR using CH-223191, a novel AhR antagonist, both of which significantly abolished the previous effects of Rb1 (Fig. 7B, 7C). Importantly, the abolished effects of Rb1 on CYP1A expression were accompanied with the same effect on the protein expression of caspase-3 (Fig. 7D). These results suggested that the mechanism was AhR dependent. Based on data Fig. 7D, DOX-mediated caspase3 activation is independent of AhR because DOX induced caspase3 activation even in siAhR or AhR antagonist treated cells. But in the condition of siAhR or AhR antagonist, the increased caspase3 activation by DOX was not reversed with Rg1 treated. Such results indicated that the toxicity of DOX is only partly related with AhR. Ginsenoside Rb1 inhibited DOX-induced cells apoptosis may through, at least in part, competing with DOX for AhR to decrease the CYP1A induction.
In conclusion, to the best of our knowledge, the present study is the first report that clearly demonstrates that ginsenoside Rb1 inhibits doxorubicin-mediated H9C2 cells apoptosis is associated with the aryl hydrocarbon receptor signaling pathway. However, these findings remain to be confirmed
This study was supported by National Natural Science Foundation of China (grant 81073149), and National Basic Research Program of China (“973 Program”) (grant 2012CB518402).