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Protective effect of guggulsterone against cardiomyocyte injury induced by doxorubicin in vitro
- Wen-Ching Wang†1,
- Yih-Huei Uen†1, 2,
- Ming-Long Chang3,
- Khoot-Peng Cheah3,
- Joe-Sharg Li3,
- Wen-Yu Yu3,
- Kock-Chee Lee3,
- Cheuk-Sing Choy4, 5 and
- Chien-Ming Hu3, 5Email author
© Wang et al.; licensee BioMed Central Ltd. 2012
Received: 16 March 2012
Accepted: 17 August 2012
Published: 27 August 2012
Doxorubicin (DOX) is an effective antineoplastic drug; however, clinical use of DOX is limited by its dose-dependent cardiotoxicity. It is well known that reactive oxygen species (ROS) play a vital role in the pathological process of DOX-induced cardiotoxicity. For this study, we evaluated the protective effects of guggulsterone (GS), a steroid obtained from myrrh, to determine its preliminary mechanisms in defending against DOX-induced cytotoxicity in H9C2 cells.
In this study, we used a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release measurements, and Hoechst 33258 staining to evaluate the protective effect of GS against DOX-induced cytotoxicity in H9C2 cells. In addition, we observed the immunofluorescence of intracellular ROS and measured lipid peroxidation, caspase-3 activity, and apoptosis-related proteins by using Western blotting.
The MTT assay and LDH release showed that treatment using GS (1–30 μM) did not cause cytotoxicity. Furthermore, GS inhibited DOX (1 μM)-induced cytotoxicity in a concentration-dependent manner. Hoechst 33258 staining showed that GS significantly reduced DOX-induced apoptosis and cell death. Using GS at a dose of 10–30 μM significantly reduced intracellular ROS and the formation of MDA in the supernatant of DOX-treated H9C2 cells and suppressed caspase-3 activity to reference levels. In immunoblot analysis, pretreatment using GS significantly reversed DOX-induced decrease of PARP, caspase-3 and bcl-2, and increase of bax, cytochrome C release, cleaved-PARP and cleaved-caspase-3. In addition, the properties of DOX-induced cancer cell (DLD-1 cells) death did not interfere when combined GS and DOX.
These data provide considerable evidence that GS could serve as a novel cardioprotective agent against DOX-induced cardiotoxicity.
Doxorubicin (DOX), a member of the anthracycline class of chemotherapeutic drugs, is a potent antineoplastic drug that has exhibited a wide spectrum of antitumor activity including in leukemia, lymphomas, soft tissue sarcomas, and breast cancer for several decades. Despite the efficacy of DOX, its use has been limited by the dose-dependent cardiotoxicity associated with acute and chronic treatments in anticancer therapy[1–3]. Both acute and chronic DOX-induced cardiotoxicity may cause cardiac dysfunction or cardiomyopathy, and may ultimately lead to rigorous heart failure and death[3, 4]. Previous studies have suggested that one mechanism responsible for DOX cardiotoxicity is the formation of reactive oxygen species (ROS)[5, 6], which can harm membrane lipids and other cellular components, leading to cardiomyocyte apoptosis and death. Certain antioxidants have been examined to reduce ROS formation, but many have demonstrated only a limited cardioprotective effect or have other side effects[8, 9].
Dulbecco’s Modified Eagle’s Medium, an RPMI 1640 medium, fetal bovine serum, penicillin/streptomycin, and medium supplements were purchased from Life Technologies (Gibco, Grand Island, NY). Monoclonal antibodies and a peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). DOX, GS, and other agents were obtained from Sigma Chemical (St. Louis, MO).
All rat cardiac H9C2 myocardial cells, spontaneously immortalized ventricular rat embryo myoblasts, and DLD-1 cells (human colon adenocarcinoma) were purchased from the Food Industry Research and Development Institute, Taiwan (BCRC). The H9C2 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in 5% CO2, and DLD-1 cells were cultured in the RPMI 1640 medium. The media were changed every 2–3 d.
Sub-confluent cells were trypsinized and seeded onto 96-well plates at a density of 1.5 × 105 cells/ml and incubated for 24 h before treatment. Thereafter, the cells were exposed to DOX 1 μM for 24 h and then incubated in a fresh medium with Z-GS at various concentrations for an additional 24 h. The effects of GS on DOX-induced cytotoxicity were assessed using the MTT assay, as previously described. The unwashed dye was eluted and quantified spectrophotometrically at 550 nm using a microplate reader. Cell viability was determined as the percentage of surviving cells compared with that of the DOX-treated control.
Lactate dehydrogenase (LDH) release assay for cytotoxicity
GS-induced cytotoxicity leading to plasma membrane damage was measured using the LDH Cytotoxicity Detection Kit (Boehringer Mannheim, Mannheim, Germany). The LDH release assay has been widely used in cytotoxicity studies. The detailed assay was performed as previously described.
Intracellular ROS induced by DOX was measured using 2’7’-dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescent probe. H9C2 cells were loaded with DCFH-DA (20 μ) for 10 min, followed by 2 washes with HBSS. Dichlorodihydrofluorescein (DCF) fluorescence was detected using a fluorescence spectrophotometer with an excitation of 485 nm and an emission of 520 nm, and the fluorescence image was visualized using a fluorescence microscope.
Lipid peroxidation was assayed using the thiobarbituric acid (TBA) reaction. The amount of thiobarbituric acid reactive substance (TBARS) was determined using a standard curve of 1,1,3,3-tetramethoxypropane. The detailed assay procedure was performed as previously described.
Preparation of total cell lysates and nuclear and cytosolic extracts
H9C2 cells (5 × 105 cells/well) or DLD-1 cells in 6-well plates were incubated with or without concentrations of GS and DOX (1 μM) for 24 h. The total cell lysates were obtained using a lysis buffer (250 mM Tris–HCl (pH 6.8), 1% Triton-100, 0.1% SDS, 1 mM Na3VO4, 1 mM EDTA, 5 mM sodium fluoride, 1 mM PMSF, and 1 mg/ml leupeptin), and cell debris was removed using a centrifuge at 10 000 × g for 10 min at 4°C and stored at −80°C until required. The protein content of the cell lysates was determined using the Bradford assay.
Western blot analysis
Equal amounts of cell lysates (30 μg) were electroblotted onto a nitrocellulose membrane (Millipore, MA), following separation using 8%-12% SDS-polyacrylamide gel electrophoresis. The blot was probed using a primary antibody against poly (ADP-ribose) polymerase (PARP), caspase-3, bcl-2, bax, cytochrome C, and β-actin (Santa Cruz Biochemicals, Santa Cruz, CA). The intensity of each band was quantified using density analysis software (MetaMorph Imaging System, Meta Imaging Series 4.5), and the density ratio represented the relative intensity of each band against controls in each experiment.
Hoechst 33258 staining
Cells were rinsed twice in 4°C PBS and fixed in 4% formaldehyde at 4°C for 10 min. After washing, the cells were incubated using Hoechst 33258 (5 μg/ml) staining at room temperature for 10 min in the dark. The cells were then observed and imaged using a laser scanning confocal microscope (Bio-Rad MRC-1000, American Laboratory, USA) with an excitation of 350 nm and an emission of 460 nm.
Caspase-3 activity assay
Caspase-3 activity was determined using the ApoAlert Caspase Colorimetric Assay kit (Clontech Laboratories, Inc. USA), according to manufacturer’s protocol.
Data and statistical analysis
The results of all the experiments were expressed as the mean ± standard error (SE) obtained from the number of replicate treatments. Data were analyzed using analysis of variance (ANOVA), followed by Dunn’s post hoc test for comparison, and P values of < 0.05 were considered statistically significant.
Effects of guggulsterone (GS) on cell viability and cytotoxicity
GS protected H9C2 cells from DOX-induced cell death
GS relieved oxidative stress induced by DOX in H9C2 cells
GS protected against apoptosis of H9C2 cells treated with DOX
GS did not interfere with DOX-induced cell death in DLD-1 cells
DOX, as an anthracycline antibiotic, has been used in cancer therapy for nearly three decades. However, the clinical use of DOX is restricted considerably by an increased risk of cardiotoxicity associated with DOX-induced cardiomyocyte apoptosis[27–29]. Adult cardiomyocytes of the heart are well known to finally differentiated muscle cells that do not progress proliferation. In addition, DOX seemed to affect specific enzymes (NADH and FADH2), transporters (Ca2+-ATPase and Na+, and K+-ATPase), and metabolic pathways (AMP-activated protein kinase) to varying extents in the cardiac muscle. The accumulation of these defects may ultimately result in irreversible cardiac failure. This study elucidated the protection of GS from DOX-induced apoptosis and cell death by conducting the following experiments: MTT assay, LDH release, DHE-staining, fluorescence intensity of DCFH, lipid peroxidation, Hoechst 33258 staining, caspase-3 activity measurement and immunoblot analysis. The production of ROS as a derivative of the DOX metabolism has been suggested to be the main mechanism of DOX-induced cardiotoxicity[32–34]. However, superoxide radicals are involved in other ROS and generate hydroxyl radical and hydrogen peroxide. According to current reports, generating these ROS causes mitochondrial damage, which may lead to cardiomyocyte apoptosis, necrosis, or death. In addition, these DOX-induced mitochondrial injuries in cardiomyocytes were prevented by some natural substances, such as curcumin, naringenin-7-O-glucoside, and plantainoside D[20, 36, 37]. In our study, we used DCFH-DA as an intracellular method of ROS detection. The results demonstrated that GS can significantly reduce the intracellular ROS produced by DOX in H9C2 cells. We also measured lipid peroxidation (MDA formation) in H9C2 cells. The reduction of MDA content suggested that GS can attenuate the oxidative stress induced by DOX in H9C2 cells.
According to the identified apoptosis of cell signaling, PARP is a nuclear enzyme activated by strand breaks in DNA and implicated in DNA repair, apoptosis, organ dysfunction, or necrosis[39, 40]. In addition, it is known that members of the bcl-2 protein family are known to be major regulators of cytochrome C release and downstream caspases activation. Thus, bcl-2 plays a vital role in regulating cardiomyocyte apoptosis[29, 41]. Using Western blot analysis, we found that GS treatment attenuated DOX-induced apoptotic proteins (cleaved-PARP, cleaved-caspase-3, and bax). In contrast, treatment with GS also enhanced anti-apoptotic protein (bcl-2) expression in cardiomyocytes. Finally, another type of cancer cell line, DLD-1, was used to exclude the possible interference of GS with DOX in cancer therapy. The data showed that the efficacy of DOX did not diminish co-treatment of GS and DOX.
Numerous studies have shown that myocardial impairment caused by DOX may be due to cardiomyocyte apoptosis, and DOX may also cause injury to endothelial cells. This study shows that GS is a novel potent protector against DOX-induced cardiotoxicity, providing protection using its antioxidative activity.
This work was supported by the Chi Mei Medical Center, Taiwan, R.O.C. (99CM-TMU-11), and the National Science Council, Taiwan, R.O.C. (NSC99-2320-B-038-004-MY2).
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