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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

Activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2) pathway by L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) in H9c2 cells. (A) 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. Cells were double immunostained for Nrf2 and Keap1 and nuclei were visualized by DAPI staining. (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. In parallel, cells were also treated with 10 µM L-Sul or D,L-Sul alone for 24 h. Nuclear and cytosolic fractions of H9c2 cells were obtained and subjected to western blot analysis using Nrf2 and Keap1 antibodies (top panel). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a cytosolic marker, while histone H3 was used to identify nuclear fractions. Densitometric analysis is shown on the lower panel. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
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f6-ijmm-36-01-0053: Activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2) pathway by L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) in H9c2 cells. (A) 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. Cells were double immunostained for Nrf2 and Keap1 and nuclei were visualized by DAPI staining. (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. In parallel, cells were also treated with 10 µM L-Sul or D,L-Sul alone for 24 h. Nuclear and cytosolic fractions of H9c2 cells were obtained and subjected to western blot analysis using Nrf2 and Keap1 antibodies (top panel). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a cytosolic marker, while histone H3 was used to identify nuclear fractions. Densitometric analysis is shown on the lower panel. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.

Mentions: We then investigated the activation status of Nrf2 in the H9c2 cells by assessing the nuclear translocation of Nrf2 by western blot analysis of the cytosolic and nuclear fractions and by immunofluorescence staining of Nrf2 and Keap1 (Fig. 6). Immunofluorescence staining and confocal microscopy revealed that Nrf2 and Keap1 were predominantly localized in the cytoplasm under basal conditions. In the doxorubicin-treated positive cells, Nrf2 was almost completely absent in the nuclear fraction. Conversely, the nuclear Nrf2 content was increased in the presence of L-sulforaphane and D,L-sulforaphane, whereas Keap1 remained localized in the cytoplasm (Fig. 6A). Similarly, western blot analysis revealed that Nrf2 expression induced by L-sulforaphane and D,L-sulforaphane was present at much higher levels in the nucleus than in the cytoplasmic fraction (Fig. 6B). Taken together, these results demonstrate that L-sulforaphane and D,L-sulforaphane activate the Keap1/Nrf2 pathway in H9c2 cells.


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)

Activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2) pathway by L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) in H9c2 cells. (A) 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. Cells were double immunostained for Nrf2 and Keap1 and nuclei were visualized by DAPI staining. (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. In parallel, cells were also treated with 10 µM L-Sul or D,L-Sul alone for 24 h. Nuclear and cytosolic fractions of H9c2 cells were obtained and subjected to western blot analysis using Nrf2 and Keap1 antibodies (top panel). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a cytosolic marker, while histone H3 was used to identify nuclear fractions. Densitometric analysis is shown on the lower panel. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
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f6-ijmm-36-01-0053: Activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2) pathway by L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) in H9c2 cells. (A) 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. Cells were double immunostained for Nrf2 and Keap1 and nuclei were visualized by DAPI staining. (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. In parallel, cells were also treated with 10 µM L-Sul or D,L-Sul alone for 24 h. Nuclear and cytosolic fractions of H9c2 cells were obtained and subjected to western blot analysis using Nrf2 and Keap1 antibodies (top panel). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a cytosolic marker, while histone H3 was used to identify nuclear fractions. Densitometric analysis is shown on the lower panel. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
Mentions: We then investigated the activation status of Nrf2 in the H9c2 cells by assessing the nuclear translocation of Nrf2 by western blot analysis of the cytosolic and nuclear fractions and by immunofluorescence staining of Nrf2 and Keap1 (Fig. 6). Immunofluorescence staining and confocal microscopy revealed that Nrf2 and Keap1 were predominantly localized in the cytoplasm under basal conditions. In the doxorubicin-treated positive cells, Nrf2 was almost completely absent in the nuclear fraction. Conversely, the nuclear Nrf2 content was increased in the presence of L-sulforaphane and D,L-sulforaphane, whereas Keap1 remained localized in the cytoplasm (Fig. 6A). Similarly, western blot analysis revealed that Nrf2 expression induced by L-sulforaphane and D,L-sulforaphane was present at much higher levels in the nucleus than in the cytoplasmic fraction (Fig. 6B). Taken together, these results demonstrate that L-sulforaphane and D,L-sulforaphane activate the Keap1/Nrf2 pathway in H9c2 cells.

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