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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) protect against doxorubicin (Dox)-induced cell death. (A) 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. The effects of L-Sul or D,L-Sul on Dox-induced cell death and morphological alterations in the H9c2 cells were observed under a light microscope. (B) Determination of H9c2 cell viability by trypan blue exclusion assay. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (C) Morphological apoptosis was determined by Hoechst 33258 staining under a fluorescence microscope (left panel). Bar graph showing the quantification of apoptotic cells as a percentage of total cells (right panel). White sqaure boxes indicate apoptotic cells. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
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f1-ijmm-36-01-0053: L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) protect against doxorubicin (Dox)-induced cell death. (A) 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. The effects of L-Sul or D,L-Sul on Dox-induced cell death and morphological alterations in the H9c2 cells were observed under a light microscope. (B) Determination of H9c2 cell viability by trypan blue exclusion assay. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (C) Morphological apoptosis was determined by Hoechst 33258 staining under a fluorescence microscope (left panel). Bar graph showing the quantification of apoptotic cells as a percentage of total cells (right panel). White sqaure boxes indicate apoptotic cells. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.

Mentions: The H9c2 cells were exposed to doxorubicin and cell viability was examined after 24 h under a light microscope and by trypan blue exclusion assay. Based on the results obtained by light microscopy, treatment of the H9c2 cells with doxorubicin induced morphological changes, including rounding up and detachment. Treatment with L-sulforaphane and D,L-sulforaphane clearly protected the H9c2 cells against doxorubicin-induced cell death (Fig. 1A). In addition, treatment with either L-sulforaphane or D,L-sulforaphane alone was not toxic to the H9c2 cells. Analysis of trypan blue dye uptake also revealed that both L-sulforaphane and D,L-sulforaphane increased cell viability in a dose-dependent manner (Fig. 1B). The results of the analysis of apoptotic cells using Hoechst 33258 staining are shown on the left panel of Fig. 1C. Following treatment with doxorubicin, the H9c2 cells exhibited numerous brightly condensed and broken fluorescent nuclei. Conversely, the number of apoptotic cells treated with doxorubicin was significantly decreased in the cells pre-treated with L-sulforaphane or D,L-sulforaphane. The results of the quantification of apoptotic cells are shown on the right panel of Fig. 1C.


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) protect against doxorubicin (Dox)-induced cell death. (A) 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. The effects of L-Sul or D,L-Sul on Dox-induced cell death and morphological alterations in the H9c2 cells were observed under a light microscope. (B) Determination of H9c2 cell viability by trypan blue exclusion assay. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (C) Morphological apoptosis was determined by Hoechst 33258 staining under a fluorescence microscope (left panel). Bar graph showing the quantification of apoptotic cells as a percentage of total cells (right panel). White sqaure boxes indicate apoptotic cells. #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

f1-ijmm-36-01-0053: L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) protect against doxorubicin (Dox)-induced cell death. (A) 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. The effects of L-Sul or D,L-Sul on Dox-induced cell death and morphological alterations in the H9c2 cells were observed under a light microscope. (B) Determination of H9c2 cell viability by trypan blue exclusion assay. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group. (C) Morphological apoptosis was determined by Hoechst 33258 staining under a fluorescence microscope (left panel). Bar graph showing the quantification of apoptotic cells as a percentage of total cells (right panel). White sqaure boxes indicate apoptotic cells. #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
Mentions: The H9c2 cells were exposed to doxorubicin and cell viability was examined after 24 h under a light microscope and by trypan blue exclusion assay. Based on the results obtained by light microscopy, treatment of the H9c2 cells with doxorubicin induced morphological changes, including rounding up and detachment. Treatment with L-sulforaphane and D,L-sulforaphane clearly protected the H9c2 cells against doxorubicin-induced cell death (Fig. 1A). In addition, treatment with either L-sulforaphane or D,L-sulforaphane alone was not toxic to the H9c2 cells. Analysis of trypan blue dye uptake also revealed that both L-sulforaphane and D,L-sulforaphane increased cell viability in a dose-dependent manner (Fig. 1B). The results of the analysis of apoptotic cells using Hoechst 33258 staining are shown on the left panel of Fig. 1C. Following treatment with doxorubicin, the H9c2 cells exhibited numerous brightly condensed and broken fluorescent nuclei. Conversely, the number of apoptotic cells treated with doxorubicin was significantly decreased in the cells pre-treated with L-sulforaphane or D,L-sulforaphane. The results of the quantification of apoptotic cells are shown on the right panel of Fig. 1C.

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