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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) prevent the doxorubicin (Dox)-induced release of cytochrome c and Bax activation. (A) H9c2 cells were treated with 1 µM Dox for the indicated periods of time. Cells were processed into cytosolic and mitochondrial fractions and subjected to western blot analysis of Bax and cytochrome c (upper panel). The lower panel shows the results of densitometric analysis. *P<0.05 vs. controls. (B) H9c2 cells were 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 processed into cytosolic and mitochondrial fractions and subjected to western blot analysis of Bax and cytochrome c (upper panel). The lower panel shows the results of densitometric analysis. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group (C) H9c2 cells were stimulated 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 double-immunostained for Bax and cytochrome c and the nuclei were visualized by DAPI staining. Cyto. c, cytochrome c.
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f2-ijmm-36-01-0053: L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) prevent the doxorubicin (Dox)-induced release of cytochrome c and Bax activation. (A) H9c2 cells were treated with 1 µM Dox for the indicated periods of time. Cells were processed into cytosolic and mitochondrial fractions and subjected to western blot analysis of Bax and cytochrome c (upper panel). The lower panel shows the results of densitometric analysis. *P<0.05 vs. controls. (B) H9c2 cells were 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 processed into cytosolic and mitochondrial fractions and subjected to western blot analysis of Bax and cytochrome c (upper panel). The lower panel shows the results of densitometric analysis. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group (C) H9c2 cells were stimulated 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 double-immunostained for Bax and cytochrome c and the nuclei were visualized by DAPI staining. Cyto. c, cytochrome c.

Mentions: We then evaluated the effects of L-sulforaphane and D,L-sulforaphane on translocation of Bax to the mitochondria and the subsequent release of cytochrome c following treatment with doxorubicin using cellular fractionation and western blot analysis. Kinetic analysis of the appearance of the main signs of apoptosis in the doxorubicin-treated cells revealed the rapid release of mitochondrial cytochrome c into the cytosol of H9c2 cells within 4 h of treatment (Fig. 2A). The presence of L-sulforaphane and D,L-sulforaphane prevented the release of cytochrome c into the cytosol in comparison to the group treated with doxorubicin alone (Fig. 2B). Similarly, in the cells treated with doxorubicin alone, we observed a time-dependent increase in the translocation of Bax to the mitochondria and a concomitant decrease in cytosolic Bax levels (Fig. 2A). Pre-treatment with L-sulforaphane and D,L-sulforaphane prevented the translocation of Bax into the cytosol compared to the cells treated with doxorubicin alone (Fig. 2B). We also investigated the subcellular distribution of Bax and cytochrome c in the H9c2 cells by dual immunofluorescence staining of Bax and cytochrome c. The control cells displayed a cytosolic distribution pattern of Bax and a punctate pattern of cytochrome c immunostaining (Fig. 2C). During apoptosis induced by doxorubicin, Bax translocated to the mitochondria and displayed a punctate pattern. The Bax-positive cells displayed a diffuse cytosolic pattern of cytochrome c staining, as well as a condensed and shrunken nucleus as assessed by Hoechst 33258 staining (Fig. 1C). Consistent with the results from western blot analysis (Fig. 2B), pre-treatment with L-sulforaphane and D,L-sulforaphane prevented the translocation of Bax to the mitochondria and the release of cytochrome c (Fig. 2C).


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) prevent the doxorubicin (Dox)-induced release of cytochrome c and Bax activation. (A) H9c2 cells were treated with 1 µM Dox for the indicated periods of time. Cells were processed into cytosolic and mitochondrial fractions and subjected to western blot analysis of Bax and cytochrome c (upper panel). The lower panel shows the results of densitometric analysis. *P<0.05 vs. controls. (B) H9c2 cells were 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 processed into cytosolic and mitochondrial fractions and subjected to western blot analysis of Bax and cytochrome c (upper panel). The lower panel shows the results of densitometric analysis. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group (C) H9c2 cells were stimulated 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 double-immunostained for Bax and cytochrome c and the nuclei were visualized by DAPI staining. Cyto. c, cytochrome c.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-ijmm-36-01-0053: L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) prevent the doxorubicin (Dox)-induced release of cytochrome c and Bax activation. (A) H9c2 cells were treated with 1 µM Dox for the indicated periods of time. Cells were processed into cytosolic and mitochondrial fractions and subjected to western blot analysis of Bax and cytochrome c (upper panel). The lower panel shows the results of densitometric analysis. *P<0.05 vs. controls. (B) H9c2 cells were 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 processed into cytosolic and mitochondrial fractions and subjected to western blot analysis of Bax and cytochrome c (upper panel). The lower panel shows the results of densitometric analysis. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group (C) H9c2 cells were stimulated 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 double-immunostained for Bax and cytochrome c and the nuclei were visualized by DAPI staining. Cyto. c, cytochrome c.
Mentions: We then evaluated the effects of L-sulforaphane and D,L-sulforaphane on translocation of Bax to the mitochondria and the subsequent release of cytochrome c following treatment with doxorubicin using cellular fractionation and western blot analysis. Kinetic analysis of the appearance of the main signs of apoptosis in the doxorubicin-treated cells revealed the rapid release of mitochondrial cytochrome c into the cytosol of H9c2 cells within 4 h of treatment (Fig. 2A). The presence of L-sulforaphane and D,L-sulforaphane prevented the release of cytochrome c into the cytosol in comparison to the group treated with doxorubicin alone (Fig. 2B). Similarly, in the cells treated with doxorubicin alone, we observed a time-dependent increase in the translocation of Bax to the mitochondria and a concomitant decrease in cytosolic Bax levels (Fig. 2A). Pre-treatment with L-sulforaphane and D,L-sulforaphane prevented the translocation of Bax into the cytosol compared to the cells treated with doxorubicin alone (Fig. 2B). We also investigated the subcellular distribution of Bax and cytochrome c in the H9c2 cells by dual immunofluorescence staining of Bax and cytochrome c. The control cells displayed a cytosolic distribution pattern of Bax and a punctate pattern of cytochrome c immunostaining (Fig. 2C). During apoptosis induced by doxorubicin, Bax translocated to the mitochondria and displayed a punctate pattern. The Bax-positive cells displayed a diffuse cytosolic pattern of cytochrome c staining, as well as a condensed and shrunken nucleus as assessed by Hoechst 33258 staining (Fig. 1C). Consistent with the results from western blot analysis (Fig. 2B), pre-treatment with L-sulforaphane and D,L-sulforaphane prevented the translocation of Bax to the mitochondria and the release of cytochrome c (Fig. 2C).

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