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Sulforaphane prevents doxorubicin-induced oxidative stress and cell death in rat H9c2 cells.

Li B, Kim do S, Yadav RK, Kim HR, Chae HJ - Int. J. Mol. Med. (2015)

Bottom Line: Sulforaphane, a natural isothiocyanate compound found in cruciferous vegetables, has been shown to exert cardioprotective effects during ischemic heart injury.Furthermore, sulforaphane increased the mRNA and protein expression of heme oxygenase-1 (HO-1, measured by RT-qPCR), which consequently reduced the levels of reactive oxygen species (ROS, measured using MitoSOX Red reagent) in the mitochondria which were induced by doxorubicin.The cardioprotective effects of sulforaphane were found to be mediated by the activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2)/antioxidant-responsive element (ARE) pathway, which in turn mediates the induction of HO-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Institute of Cardiovascular Research, School of Medicine, Chonbuk National University, Jeonju, Chonbuk 561-180, Republic of Korea.

ABSTRACT
Sulforaphane, a natural isothiocyanate compound found in cruciferous vegetables, has been shown to exert cardioprotective effects during ischemic heart injury. However, the effects of sulforaphane on cardiotoxicity induced by doxorubicin are unknown. Thus, in the present study, H9c2 rat myoblasts were pre-treated with sulforaphane and its effects on cardiotoxicity were then examined. The results revealed that the pre-treatment of H9c2 rat myoblasts with sulforaphane decreased the apoptotic cell number (as shown by trypan blue exclusion assay) and the expression of pro-apoptotic proteins (Bax, caspase-3 and cytochrome c; as shown by western blot analysis and immunostaining), as well as the doxorubicin-induced increase in mitochondrial membrane potential (measured by JC-1 assay). Furthermore, sulforaphane increased the mRNA and protein expression of heme oxygenase-1 (HO-1, measured by RT-qPCR), which consequently reduced the levels of reactive oxygen species (ROS, measured using MitoSOX Red reagent) in the mitochondria which were induced by doxorubicin. The cardioprotective effects of sulforaphane were found to be mediated by the activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2)/antioxidant-responsive element (ARE) pathway, which in turn mediates the induction of HO-1. Taken together, the findings of this study demonstrate that sulforaphane prevents doxorubicin-induced oxidative stress and cell death in H9c2 cells through the induction of HO-1 expression.

No MeSH data available.


Related in: MedlinePlus

L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) reduce the doxorubicin (Dox)-induced generation of mitochondrial reactive oxygen species (ROS) in H9c2 cells. (A) H9c2 cells were treated with 1 µM doxorubicin or pre-treated with 10 µM L-Sul or D,L-Sul for 2 h, and then treated with 1 µM Dox for 24 h. Cells were then analyzed for mitochondrial superoxide generation by fluorescence microscopy using MitoSOX Red. Representative micrographs are shown on the left panel, and quantification plots are shown on the right panel. Results were calculated as a percentage of control in fluorescence intensity compared with the control group. Plots are the means ± SE (n=3). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (B) H9c2 cells were treated with 1 µM Dox or pre-treated with 10 µM L-Sul or D,L-Sul for 2 h, and then treated with 1 µM Dox for 24 h. Cells were then analyzed for mitochondrial peroxynitrite generation by fluorescence microscopy using DHR123. Representative micrographs are shown on the left panel, and quantification plots are shown on the right panel. Results are calculated as a percentage of control in fluorescence intensity compared with the control group. Plots are the means ± SE (n=3). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
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f4-ijmm-36-01-0053: L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) reduce the doxorubicin (Dox)-induced generation of mitochondrial reactive oxygen species (ROS) in H9c2 cells. (A) H9c2 cells were treated with 1 µM doxorubicin or pre-treated with 10 µM L-Sul or D,L-Sul for 2 h, and then treated with 1 µM Dox for 24 h. Cells were then analyzed for mitochondrial superoxide generation by fluorescence microscopy using MitoSOX Red. Representative micrographs are shown on the left panel, and quantification plots are shown on the right panel. Results were calculated as a percentage of control in fluorescence intensity compared with the control group. Plots are the means ± SE (n=3). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (B) H9c2 cells were treated with 1 µM Dox or pre-treated with 10 µM L-Sul or D,L-Sul for 2 h, and then treated with 1 µM Dox for 24 h. Cells were then analyzed for mitochondrial peroxynitrite generation by fluorescence microscopy using DHR123. Representative micrographs are shown on the left panel, and quantification plots are shown on the right panel. Results are calculated as a percentage of control in fluorescence intensity compared with the control group. Plots are the means ± SE (n=3). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.

Mentions: ROS are involved in doxorubicin-induced cell apoptosis (39–41). Previous studies have suggested that cardiomyocyte mitochondria are important intracellular targets of ROS during doxorubicin-induced cardiotoxicity. Superoxide and peroxynitrite are the major ROS induced by doxorubicin (42,43). Thus, in the present study, we evaluated whether L-sulforaphane and D,L-sulforaphane influence the generation of doxorubicin-induced ROS. The cells were analyzed for mitochondrial superoxide generation by fluorescence microscopy using the mitochondria-targeted dye, dihydroethidium (MitoSOX Red), as a probe (Fig. 4A). Our results indicated that doxorubicin increased mitochondrial superoxide generation compared to the untreated control cells. Conversely, the doxorubicin-induced MitoSOX fluorescence intensity was attenuated by pre-treatment with L-sulforaphane and D,L-sulforaphane (Fig. 4A). We also investigated the generation of mitochondrial peroxynitrite using the dye, DHR123, as a probe. As shown in Fig. 4B, the doxorubicin-induced DHR123 fluorescence was attenuated by pre-treatment wtih L-sulforaphane and D,L-sulforaphane. Taken together, these results suggest that both L-sulforaphane and D,L-sulforaphane protect H9c2 cells against doxorubicin-induced apoptosis by preventing doxorubicin-induced mitochondrial ROS generation.


Sulforaphane prevents doxorubicin-induced oxidative stress and cell death in rat H9c2 cells.

Li B, Kim do S, Yadav RK, Kim HR, Chae HJ - Int. J. Mol. Med. (2015)

L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) reduce the doxorubicin (Dox)-induced generation of mitochondrial reactive oxygen species (ROS) in H9c2 cells. (A) H9c2 cells were treated with 1 µM doxorubicin or pre-treated with 10 µM L-Sul or D,L-Sul for 2 h, and then treated with 1 µM Dox for 24 h. Cells were then analyzed for mitochondrial superoxide generation by fluorescence microscopy using MitoSOX Red. Representative micrographs are shown on the left panel, and quantification plots are shown on the right panel. Results were calculated as a percentage of control in fluorescence intensity compared with the control group. Plots are the means ± SE (n=3). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (B) H9c2 cells were treated with 1 µM Dox or pre-treated with 10 µM L-Sul or D,L-Sul for 2 h, and then treated with 1 µM Dox for 24 h. Cells were then analyzed for mitochondrial peroxynitrite generation by fluorescence microscopy using DHR123. Representative micrographs are shown on the left panel, and quantification plots are shown on the right panel. Results are calculated as a percentage of control in fluorescence intensity compared with the control group. Plots are the means ± SE (n=3). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494600&req=5

f4-ijmm-36-01-0053: L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) reduce the doxorubicin (Dox)-induced generation of mitochondrial reactive oxygen species (ROS) in H9c2 cells. (A) H9c2 cells were treated with 1 µM doxorubicin or pre-treated with 10 µM L-Sul or D,L-Sul for 2 h, and then treated with 1 µM Dox for 24 h. Cells were then analyzed for mitochondrial superoxide generation by fluorescence microscopy using MitoSOX Red. Representative micrographs are shown on the left panel, and quantification plots are shown on the right panel. Results were calculated as a percentage of control in fluorescence intensity compared with the control group. Plots are the means ± SE (n=3). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (B) H9c2 cells were treated with 1 µM Dox or pre-treated with 10 µM L-Sul or D,L-Sul for 2 h, and then treated with 1 µM Dox for 24 h. Cells were then analyzed for mitochondrial peroxynitrite generation by fluorescence microscopy using DHR123. Representative micrographs are shown on the left panel, and quantification plots are shown on the right panel. Results are calculated as a percentage of control in fluorescence intensity compared with the control group. Plots are the means ± SE (n=3). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
Mentions: ROS are involved in doxorubicin-induced cell apoptosis (39–41). Previous studies have suggested that cardiomyocyte mitochondria are important intracellular targets of ROS during doxorubicin-induced cardiotoxicity. Superoxide and peroxynitrite are the major ROS induced by doxorubicin (42,43). Thus, in the present study, we evaluated whether L-sulforaphane and D,L-sulforaphane influence the generation of doxorubicin-induced ROS. The cells were analyzed for mitochondrial superoxide generation by fluorescence microscopy using the mitochondria-targeted dye, dihydroethidium (MitoSOX Red), as a probe (Fig. 4A). Our results indicated that doxorubicin increased mitochondrial superoxide generation compared to the untreated control cells. Conversely, the doxorubicin-induced MitoSOX fluorescence intensity was attenuated by pre-treatment with L-sulforaphane and D,L-sulforaphane (Fig. 4A). We also investigated the generation of mitochondrial peroxynitrite using the dye, DHR123, as a probe. As shown in Fig. 4B, the doxorubicin-induced DHR123 fluorescence was attenuated by pre-treatment wtih L-sulforaphane and D,L-sulforaphane. Taken together, these results suggest that both L-sulforaphane and D,L-sulforaphane protect H9c2 cells against doxorubicin-induced apoptosis by preventing doxorubicin-induced mitochondrial ROS generation.

Bottom Line: Sulforaphane, a natural isothiocyanate compound found in cruciferous vegetables, has been shown to exert cardioprotective effects during ischemic heart injury.Furthermore, sulforaphane increased the mRNA and protein expression of heme oxygenase-1 (HO-1, measured by RT-qPCR), which consequently reduced the levels of reactive oxygen species (ROS, measured using MitoSOX Red reagent) in the mitochondria which were induced by doxorubicin.The cardioprotective effects of sulforaphane were found to be mediated by the activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2)/antioxidant-responsive element (ARE) pathway, which in turn mediates the induction of HO-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Institute of Cardiovascular Research, School of Medicine, Chonbuk National University, Jeonju, Chonbuk 561-180, Republic of Korea.

ABSTRACT
Sulforaphane, a natural isothiocyanate compound found in cruciferous vegetables, has been shown to exert cardioprotective effects during ischemic heart injury. However, the effects of sulforaphane on cardiotoxicity induced by doxorubicin are unknown. Thus, in the present study, H9c2 rat myoblasts were pre-treated with sulforaphane and its effects on cardiotoxicity were then examined. The results revealed that the pre-treatment of H9c2 rat myoblasts with sulforaphane decreased the apoptotic cell number (as shown by trypan blue exclusion assay) and the expression of pro-apoptotic proteins (Bax, caspase-3 and cytochrome c; as shown by western blot analysis and immunostaining), as well as the doxorubicin-induced increase in mitochondrial membrane potential (measured by JC-1 assay). Furthermore, sulforaphane increased the mRNA and protein expression of heme oxygenase-1 (HO-1, measured by RT-qPCR), which consequently reduced the levels of reactive oxygen species (ROS, measured using MitoSOX Red reagent) in the mitochondria which were induced by doxorubicin. The cardioprotective effects of sulforaphane were found to be mediated by the activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2)/antioxidant-responsive element (ARE) pathway, which in turn mediates the induction of HO-1. Taken together, the findings of this study demonstrate that sulforaphane prevents doxorubicin-induced oxidative stress and cell death in H9c2 cells through the induction of HO-1 expression.

No MeSH data available.


Related in: MedlinePlus