Protective effect of guggulsterone against cardiomyocyte injury induced by doxorubicin in vitro

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: These data provide considerable evidence that GS could serve as a novel cardioprotective agent against DOX-induced cardiotoxicity.

Figure 1

Structure ofZ-guggulsterone [trans-4,17(20)-pregnadiene-3,16-dione][12].

Figure 2

Effects of guggulsterone (GS) on the H9C2 cell line. (A) Cells were treated with various concentrations of GS for 24 h. Cell viability was determined using an MTT assay, as described in the Materials and Methods section. (B) GS cytotoxicity was analyzed using LDH release. The results are expressed as mean ± S.E. from 3 independent experiments. Asterisks indicate a significant difference for the control (GS 0 μM) (**P < 0.01).

Figure 3

GS prevention on DOX-induced H9C2 cell death. Cells were incubated with DOX (1 μM) in the presence and absence of GS for 24 h. The percentages of viable cells were determined using an MTT assay, as described in the Materials and Methods section. The results are expressed as mean±S.E. from 3 independent experiments. ^*P < 0.05 and ^**P < 0.01 indicate a significant difference from DOX treatment. ^#P < 0.01 indicates a significant difference from the control (GS 0 μM and DOX 0 μM).

Figure 4

Morphology of DOX-induced cell death in H9C2 cells in the presence or absence of GS for 24 h. Cells were viewed using a video camera (Nikon) attached to a microscope, and the image was projected onto a monitor. Images were obtained with a 200× objective.

Figure 5

GS relieved the oxidative stress induced by DOX in H9C2 cells. Staining of intracellular ROS by dihydroethidium (DHE) staining in H9C2 cells (original magnification × 200).

Figure 6

Prevention of GS on DOX-induced oxidative stress. Subconfluent H9C2 cells were left untreated or exposed to DOX and co-treated with GS for 24 h and oxidative stress was evaluated by measuring DCFH oxidation (A) and the amount of TBARS formation (malondialdehyde, MAD) (B). The results are expressed as mean±S.E. from 3 independent experiments. ^**P < 0.01 indicates a significant difference from DOX treatment. ^##P < 0.01 indicates a significant difference from the control (GS 0 μM and DOX 0 μM).

Figure 7

Effects of GS on DOX-induced apoptosis-related protein in the cytoplasm of H9C2 cells. (A) Cells (5 × 10⁵ cells/ well) were pretreated with the indicated concentrations of GS for 2 h before incubation with DOX (1 μM) for 24 h. The proteins (30 μg) of total cell lysates were analyzed at the expression levels of anti-PARP, caspase-3, bcl-2, bax, and cytosolic lysates that were analyzed for anti-cytochrome C by Western blotting at 10% SDS-PAGE. β-Actin is the internal standard to confirm equal loading. (B) Quantification of PARP, bcl-2, and caspase-3 expression, and cytochrome C release. (C) Quantification of the cleaved form of PARP and caspase-3, as well as the bax expression. Data are expressed as mean ± S.E. from 3 independent experiments. ^*P < 0.05, ^**P < 0.01, and PARP (both the precursor and cleaved form) expression compared to the DOX treatment alone; ^#P < 0.05 and ^##P < 0.01, and caspase-3 (both the precursor and cleaved form) compared to the DOX treatment alone; ^ςP < 0.05 and ^ςςP < 0.01, and bcl-2 and bax compared to the DOX treatment alone. ^ξP < 0.05 and ^ξξP < 0.01, and cytochrome C compared to the DOX treatment alone.

Figure 8

The protective effects of GS on H9C2 cells against DOX-induced apoptosis by Hoechst 33258 staining. Fluorescence images of Hoechst 33258 stained H9C2 cells after a 2 h of exposure to DOX in the absence or presence of GS. Images were obtained using a 200× objective.

Figure 9

Effects of GS on DOX-induced apoptosis using caspase-3 activity assay in H9C2 cells. Subconfluent H9C2 cells were treated with DOX and co-treated with different concentrations of GS for 24 h, and caspase-3 activity was measured using colorimetry. The results are expressed as mean±S.E. from 3 independent experiments. ^**P < 0.01 indicates a significant difference from DOX treatment. ^##P < 0.01 indicates a significant difference from the control (GS 0 μM and DOX 0 μM).

Figure 10

Effects of combining GS and DOX in cancer cells. (A) Morphology of DOX-induced cell death. (B) Cell viability of the MTT assay. (C) Western blotting of PARP, caspase-3 and bax protein expression in DLD-1 cells in the presence or absence of GS for 24 h. Images were obtained with a 200× objective of the microscope. The assay methods were performed in the same manner as that described for the H9C2 cells.