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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 activation of caspase-3 and protect against Dox-induced changes in mitochondrial transmembrane potential. (A) H9c2 cells were treated with 1 µM Dox for the indicated periods of time. Cells were lysed and examined for the levels of cleaved caspase-3 by western blot analysis (upper panel). The relative cleaved caspase-3 protein level was normalized to the β-actin level (lower panel). *P<0.05 and **P<0.001 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 lysed and examined for the levels of cleaved caspase-3 by western blot analysis (upper panel). Relative cleaved caspase-3 protein levels were normalized to β-actin level (lower panel). #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. Mitochondrial membrane potential was detected by JC-1 fluorescence staining. Specifically, the cells were stained with JC-1 and examined under a fluorescence microscope for the detection of red JC-1 aggregates (590 nm emission) or green JC-1 monomers (527 nm emission). Typical images are shown at ×600 magnification. (D) Quantification of data in (C). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
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f3-ijmm-36-01-0053: L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) prevent the doxorubicin (Dox)-induced activation of caspase-3 and protect against Dox-induced changes in mitochondrial transmembrane potential. (A) H9c2 cells were treated with 1 µM Dox for the indicated periods of time. Cells were lysed and examined for the levels of cleaved caspase-3 by western blot analysis (upper panel). The relative cleaved caspase-3 protein level was normalized to the β-actin level (lower panel). *P<0.05 and **P<0.001 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 lysed and examined for the levels of cleaved caspase-3 by western blot analysis (upper panel). Relative cleaved caspase-3 protein levels were normalized to β-actin level (lower panel). #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. Mitochondrial membrane potential was detected by JC-1 fluorescence staining. Specifically, the cells were stained with JC-1 and examined under a fluorescence microscope for the detection of red JC-1 aggregates (590 nm emission) or green JC-1 monomers (527 nm emission). Typical images are shown at ×600 magnification. (D) Quantification of data in (C). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.

Mentions: Doxorubicin was found to induce time-dependent cell apoptosis in the H9c2 cells. Treatment with doxorubicin significantly increased the levels of cleaved caspase-3 at 8 h; these levels increased further in a time-dependent manner (Fig. 3A). As shown in Fig. 3B, both L-sulforaphane and D,L-sulforaphane attenuated the doxorubicin-induced increase in the levels of cleaved caspase-3. ΔΨm is one of the key events during apoptosis (38). Mitochondrial permeability transition has been implicated in the collapse of ΔΨm. To monitor ΔΨm, we used the JC-1 dye and measured the emission ratio at 590 to 527 nm. As shown in Fig. 3C, the control cells exhibited mostly brightly stained mitochondria emitting red fluorescence, whereas the H9c2 cells treated with doxorubicin produced green fluorescence indicative of mitochondrial depolarization and the collapse of ΔΨm. We observed an approximately 80% loss in ΔΨm in the cells treated with doxorubicin alone compared with the control cells (Fig. 3D). Conversely, the cells pre-treated with either L-sulforaphane or D,L-sulforaphane exhibited a significant preservation of red fluorescence compared with the cells treated with doxorubicin alone (Fig. 3C and D).


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 activation of caspase-3 and protect against Dox-induced changes in mitochondrial transmembrane potential. (A) H9c2 cells were treated with 1 µM Dox for the indicated periods of time. Cells were lysed and examined for the levels of cleaved caspase-3 by western blot analysis (upper panel). The relative cleaved caspase-3 protein level was normalized to the β-actin level (lower panel). *P<0.05 and **P<0.001 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 lysed and examined for the levels of cleaved caspase-3 by western blot analysis (upper panel). Relative cleaved caspase-3 protein levels were normalized to β-actin level (lower panel). #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. Mitochondrial membrane potential was detected by JC-1 fluorescence staining. Specifically, the cells were stained with JC-1 and examined under a fluorescence microscope for the detection of red JC-1 aggregates (590 nm emission) or green JC-1 monomers (527 nm emission). Typical images are shown at ×600 magnification. (D) Quantification of data in (C). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
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f3-ijmm-36-01-0053: L-sulforaphane (L-Sul) and D,L-sulforaphane (D,L-Sul) prevent the doxorubicin (Dox)-induced activation of caspase-3 and protect against Dox-induced changes in mitochondrial transmembrane potential. (A) H9c2 cells were treated with 1 µM Dox for the indicated periods of time. Cells were lysed and examined for the levels of cleaved caspase-3 by western blot analysis (upper panel). The relative cleaved caspase-3 protein level was normalized to the β-actin level (lower panel). *P<0.05 and **P<0.001 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 lysed and examined for the levels of cleaved caspase-3 by western blot analysis (upper panel). Relative cleaved caspase-3 protein levels were normalized to β-actin level (lower panel). #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. Mitochondrial membrane potential was detected by JC-1 fluorescence staining. Specifically, the cells were stained with JC-1 and examined under a fluorescence microscope for the detection of red JC-1 aggregates (590 nm emission) or green JC-1 monomers (527 nm emission). Typical images are shown at ×600 magnification. (D) Quantification of data in (C). #P<0.05 vs. controls; *P<0.05 vs. Dox-treated group.
Mentions: Doxorubicin was found to induce time-dependent cell apoptosis in the H9c2 cells. Treatment with doxorubicin significantly increased the levels of cleaved caspase-3 at 8 h; these levels increased further in a time-dependent manner (Fig. 3A). As shown in Fig. 3B, both L-sulforaphane and D,L-sulforaphane attenuated the doxorubicin-induced increase in the levels of cleaved caspase-3. ΔΨm is one of the key events during apoptosis (38). Mitochondrial permeability transition has been implicated in the collapse of ΔΨm. To monitor ΔΨm, we used the JC-1 dye and measured the emission ratio at 590 to 527 nm. As shown in Fig. 3C, the control cells exhibited mostly brightly stained mitochondria emitting red fluorescence, whereas the H9c2 cells treated with doxorubicin produced green fluorescence indicative of mitochondrial depolarization and the collapse of ΔΨm. We observed an approximately 80% loss in ΔΨm in the cells treated with doxorubicin alone compared with the control cells (Fig. 3D). Conversely, the cells pre-treated with either L-sulforaphane or D,L-sulforaphane exhibited a significant preservation of red fluorescence compared with the cells treated with doxorubicin alone (Fig. 3C and D).

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