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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) activate heme oxygenase-1 (HO-1) through antioxidant-responsive elements (AREs) in H9c2 cells. (A. panel a) H9c2 cells were treated with 10 µM L-Sul or D,L-Sul for 24 h. (Panel b) H9c2 cells were treated with 1 µM doxorubicin (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. HO-1 mRNA levels were measured by RT-qPCR. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (B) Protein expression of HO-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured by western blot analysis. Densitometric analysis is shown on the lower panel. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated. (C, panel a) H9c2 cells were treated with 10 µM L-Sul or D,L-Sul for 24 h. (Panel 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. ARE luciferase activity was measured. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
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f5-ijmm-36-01-0053: L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) activate heme oxygenase-1 (HO-1) through antioxidant-responsive elements (AREs) in H9c2 cells. (A. panel a) H9c2 cells were treated with 10 µM L-Sul or D,L-Sul for 24 h. (Panel b) H9c2 cells were treated with 1 µM doxorubicin (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. HO-1 mRNA levels were measured by RT-qPCR. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (B) Protein expression of HO-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured by western blot analysis. Densitometric analysis is shown on the lower panel. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated. (C, panel a) H9c2 cells were treated with 10 µM L-Sul or D,L-Sul for 24 h. (Panel 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. ARE luciferase activity was measured. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.

Mentions: L-sulforaphane and D,L-sulforaphane protected the H9c2 cells from doxorubicin-induced oxidative insults (Fig. 4). This protective action of sulforaphane may be related to the induction of HO-1, which, along with other phase II enzymes, serves as a defense system against oxidative stress (44,45). Using RT-qPCR, we found that pre-treatment with L-sulforaphane and D,L-sulforaphane induced HO-1 mRNA expression in a dose-dependent manner (Fig. 5A panel a). In addition, pre-treatment with L-sulforaphane and D,L-sulforaphane reversed the decrease in HO-1 mRNA expression observed in the cells treated with doxorubicin alone (Fig. 5A panel b). We then measured HO-1 protein expression levels by western blot analysis. Consistent with our mRNA data, pre-treatment with L-sulforaphane and D,L-sulforaphane induced a significant increase in HO-1 protein expression (Fig. 5B). Furthermore, HO-1 protein expression was higher in the cells pre-treated with L-sulforaphane and D,L-sulforaphane before the addition of doxorubicin compared with cells treated only with doxorubicin. We also found that L-sulforaphane and D,L-sulforaphane stimulated ARE-dependent transcriptional activity in a dose-dependent manner (Fig. 5C panel a). Similarly, pretreatment with L-sulforaphane and D,L-sulforaphane increased ARE-dependent transcriptional activity compared to the cells treated with doxorubicin alone (Fig. 5C panel b). Taken together, these results demonstrate that sulforaphane induces HO-1 mRNA and protein expression by activating AREs.


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) activate heme oxygenase-1 (HO-1) through antioxidant-responsive elements (AREs) in H9c2 cells. (A. panel a) H9c2 cells were treated with 10 µM L-Sul or D,L-Sul for 24 h. (Panel b) H9c2 cells were treated with 1 µM doxorubicin (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. HO-1 mRNA levels were measured by RT-qPCR. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (B) Protein expression of HO-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured by western blot analysis. Densitometric analysis is shown on the lower panel. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated. (C, panel a) H9c2 cells were treated with 10 µM L-Sul or D,L-Sul for 24 h. (Panel 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. ARE luciferase activity was measured. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494600&req=5

f5-ijmm-36-01-0053: L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) activate heme oxygenase-1 (HO-1) through antioxidant-responsive elements (AREs) in H9c2 cells. (A. panel a) H9c2 cells were treated with 10 µM L-Sul or D,L-Sul for 24 h. (Panel b) H9c2 cells were treated with 1 µM doxorubicin (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. HO-1 mRNA levels were measured by RT-qPCR. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (B) Protein expression of HO-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was measured by western blot analysis. Densitometric analysis is shown on the lower panel. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated. (C, panel a) H9c2 cells were treated with 10 µM L-Sul or D,L-Sul for 24 h. (Panel 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. ARE luciferase activity was measured. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
Mentions: L-sulforaphane and D,L-sulforaphane protected the H9c2 cells from doxorubicin-induced oxidative insults (Fig. 4). This protective action of sulforaphane may be related to the induction of HO-1, which, along with other phase II enzymes, serves as a defense system against oxidative stress (44,45). Using RT-qPCR, we found that pre-treatment with L-sulforaphane and D,L-sulforaphane induced HO-1 mRNA expression in a dose-dependent manner (Fig. 5A panel a). In addition, pre-treatment with L-sulforaphane and D,L-sulforaphane reversed the decrease in HO-1 mRNA expression observed in the cells treated with doxorubicin alone (Fig. 5A panel b). We then measured HO-1 protein expression levels by western blot analysis. Consistent with our mRNA data, pre-treatment with L-sulforaphane and D,L-sulforaphane induced a significant increase in HO-1 protein expression (Fig. 5B). Furthermore, HO-1 protein expression was higher in the cells pre-treated with L-sulforaphane and D,L-sulforaphane before the addition of doxorubicin compared with cells treated only with doxorubicin. We also found that L-sulforaphane and D,L-sulforaphane stimulated ARE-dependent transcriptional activity in a dose-dependent manner (Fig. 5C panel a). Similarly, pretreatment with L-sulforaphane and D,L-sulforaphane increased ARE-dependent transcriptional activity compared to the cells treated with doxorubicin alone (Fig. 5C panel b). Taken together, these results demonstrate that sulforaphane induces HO-1 mRNA and protein expression by activating AREs.

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