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Compound K, a metabolite of ginseng saponin, induces mitochondria-dependent and caspase-dependent apoptosis via the generation of reactive oxygen species in human colon cancer cells.

Lee IK, Kang KA, Lim CM, Kim KC, Kim HS, Kim DH, Kim BJ, Chang WY, Choi JH, Hyun JW - Int J Mol Sci (2010)

Bottom Line: Compound K produced intracellular ROS in a time dependent fashion; however, N-acetylcysteine (NAC) pretreatment resulted in the inhibition of this effect and the recovery of cell viability.Compound K induced a mitochondria-dependent apoptotic pathway via the modulation of Bax and Bcl-2 expressions, resulting in the disruption of the mitochondrial membrane potential (Δψ(m)).The apoptotic effect of Compound K, exerted via the activation of c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), was abrogated by specific MAPK inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-799, Korea; E-Mails: inkyeong@korea.ac.kr (I.K.L.); kbumjoon@snu.ac.kr (B.J.K.).

ABSTRACT
The objective of this study was to elucidate the cytotoxic mechanism of Compound K, with respect to the involvement of reactive oxygen species (ROS) and the mitochondrial involved apoptosis, in HT-29 human colon cancer cells. Compound K exhibited a concentration of 50% growth inhibition (IC(50)) at 20 μg/mL and cytotoxicity in a time dependent manner. Compound K produced intracellular ROS in a time dependent fashion; however, N-acetylcysteine (NAC) pretreatment resulted in the inhibition of this effect and the recovery of cell viability. Compound K induced a mitochondria-dependent apoptotic pathway via the modulation of Bax and Bcl-2 expressions, resulting in the disruption of the mitochondrial membrane potential (Δψ(m)). Loss of the Δψ(m) was followed by cytochrome c release from the mitochondria, resulting in the activation of caspase-9, -3, and concomitant poly ADP-ribosyl polymerase (PARP) cleavage, which are the indicators of caspase-dependent apoptosis. The apoptotic effect of Compound K, exerted via the activation of c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), was abrogated by specific MAPK inhibitors. This study demonstrated that Compound K-mediated generation of ROS led to apoptosis through the modulation of a mitochondria-dependent apoptotic pathway and MAPK pathway.

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Induction of mitochondria-dependent and caspase-dependent apoptosis by Compound K treatment. (A) Apoptotic sub-G1 cells were detected by flow cytometry after PI staining. (B) Δψm was analyzed by flow cytometry and (C) confocal microscopy after staining cells with JC-1 dye. (D) Cell lysates were electrophoresed and Bax, Bcl-2, cytochrome c, active caspase-9, active caspase-3, and cleaved PARP proteins were detected using their corresponding antibodies.
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f4-ijms-11-04916: Induction of mitochondria-dependent and caspase-dependent apoptosis by Compound K treatment. (A) Apoptotic sub-G1 cells were detected by flow cytometry after PI staining. (B) Δψm was analyzed by flow cytometry and (C) confocal microscopy after staining cells with JC-1 dye. (D) Cell lysates were electrophoresed and Bax, Bcl-2, cytochrome c, active caspase-9, active caspase-3, and cleaved PARP proteins were detected using their corresponding antibodies.

Mentions: Such increases in intracellular ROS cause loss of mitochondria membrane permeability, resulting in the induction of apoptosis [40,41]. We investigated whether the cytotoxicity of Compound K was associated with the induction of apoptosis. Sub G1-hypodiploid cells, which are an indicator of apoptosis, increased in Compound K-treated cells as compared to control cells (Figure 4A). Apoptotic pathway requires the alteration of the mitochondrial membrane potential (Δψm), which leads to mitochondrial membrane permeabilization and is followed by a release of cytochrome c and caspases activation [4,42]. Compound K-treated cells exhibited a loss of Δψm, as substantiated by an increase of fluorescence intensity in fluorescence (FL-1) using the JC-1 dye (Figure 4B). In non-apoptotic cells, the monomer form of JC-1 accumulates as aggregates in the mitochondria, which then emits red fluorescence; whereas in apoptotic cells, JC-1 does not accumulate and remains a monomer, emitting green fluorescence. The control cells exhibited strong red fluorescence in the mitochondria; however, Compound K-treated cells resulted in a decreased red fluorescence in the mitochondria and increased green fluorescence, suggesting that Compound K treatment disrupted the mitochondrial Δψ (Figure 4C). During the apoptotic process, Bcl-2, an anti-apoptotic regulator, prevents the opening of the mitochondrial membrane pores, whereas Bax, an apoptotic regulator, induces it [43]. Compound K was shown to increase Bax expression while decreasing that of Bcl-2 expression. Therefore, Compound K-induced loss of the Δψm may have been a result of an up-regulation of Bcl-2 and a down-regulation of Bax. The pore opening induces the loss of the Δψm, which in turn induces the release of cytochrome c from the mitochondria [44,45]. Compound K induced the release of cytochrome c from mitochondria into cytosol (Figure 4D). The mitochondrial membrane disruption by Compound K activated caspase-9 (37 and 39 kDa) and caspase-3 (19 and 17 kDa), a target of caspase-9, which was further demonstrated by PARP cleavage (89 kDa) (Figure 4D). These results suggest that Compound K induced apoptosis via a caspase--dependent pathway in the mitochondria. Further, Compound K-induced decreases in the Bcl-2 protein and corresponding increases in the Bax protein, results in the opening of mitochondrial membrane pores, facilitating the release of cytochrome c from mitochondria into cytosol. Cytochrome c is bound to the outer surface of the inner membrane phospholipids. An early process in the release of cytochrome c is its dissociation from the inner membrane. Mitochondrial ROS have been shown to promote cytochrome c release to the cytosol by dissociation from these membrane phospholipids [46,47]. Therefore, Compound K-mediated ROS production may target membrane phospholipids resulting in dissociation of cytochrome c.


Compound K, a metabolite of ginseng saponin, induces mitochondria-dependent and caspase-dependent apoptosis via the generation of reactive oxygen species in human colon cancer cells.

Lee IK, Kang KA, Lim CM, Kim KC, Kim HS, Kim DH, Kim BJ, Chang WY, Choi JH, Hyun JW - Int J Mol Sci (2010)

Induction of mitochondria-dependent and caspase-dependent apoptosis by Compound K treatment. (A) Apoptotic sub-G1 cells were detected by flow cytometry after PI staining. (B) Δψm was analyzed by flow cytometry and (C) confocal microscopy after staining cells with JC-1 dye. (D) Cell lysates were electrophoresed and Bax, Bcl-2, cytochrome c, active caspase-9, active caspase-3, and cleaved PARP proteins were detected using their corresponding antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100836&req=5

f4-ijms-11-04916: Induction of mitochondria-dependent and caspase-dependent apoptosis by Compound K treatment. (A) Apoptotic sub-G1 cells were detected by flow cytometry after PI staining. (B) Δψm was analyzed by flow cytometry and (C) confocal microscopy after staining cells with JC-1 dye. (D) Cell lysates were electrophoresed and Bax, Bcl-2, cytochrome c, active caspase-9, active caspase-3, and cleaved PARP proteins were detected using their corresponding antibodies.
Mentions: Such increases in intracellular ROS cause loss of mitochondria membrane permeability, resulting in the induction of apoptosis [40,41]. We investigated whether the cytotoxicity of Compound K was associated with the induction of apoptosis. Sub G1-hypodiploid cells, which are an indicator of apoptosis, increased in Compound K-treated cells as compared to control cells (Figure 4A). Apoptotic pathway requires the alteration of the mitochondrial membrane potential (Δψm), which leads to mitochondrial membrane permeabilization and is followed by a release of cytochrome c and caspases activation [4,42]. Compound K-treated cells exhibited a loss of Δψm, as substantiated by an increase of fluorescence intensity in fluorescence (FL-1) using the JC-1 dye (Figure 4B). In non-apoptotic cells, the monomer form of JC-1 accumulates as aggregates in the mitochondria, which then emits red fluorescence; whereas in apoptotic cells, JC-1 does not accumulate and remains a monomer, emitting green fluorescence. The control cells exhibited strong red fluorescence in the mitochondria; however, Compound K-treated cells resulted in a decreased red fluorescence in the mitochondria and increased green fluorescence, suggesting that Compound K treatment disrupted the mitochondrial Δψ (Figure 4C). During the apoptotic process, Bcl-2, an anti-apoptotic regulator, prevents the opening of the mitochondrial membrane pores, whereas Bax, an apoptotic regulator, induces it [43]. Compound K was shown to increase Bax expression while decreasing that of Bcl-2 expression. Therefore, Compound K-induced loss of the Δψm may have been a result of an up-regulation of Bcl-2 and a down-regulation of Bax. The pore opening induces the loss of the Δψm, which in turn induces the release of cytochrome c from the mitochondria [44,45]. Compound K induced the release of cytochrome c from mitochondria into cytosol (Figure 4D). The mitochondrial membrane disruption by Compound K activated caspase-9 (37 and 39 kDa) and caspase-3 (19 and 17 kDa), a target of caspase-9, which was further demonstrated by PARP cleavage (89 kDa) (Figure 4D). These results suggest that Compound K induced apoptosis via a caspase--dependent pathway in the mitochondria. Further, Compound K-induced decreases in the Bcl-2 protein and corresponding increases in the Bax protein, results in the opening of mitochondrial membrane pores, facilitating the release of cytochrome c from mitochondria into cytosol. Cytochrome c is bound to the outer surface of the inner membrane phospholipids. An early process in the release of cytochrome c is its dissociation from the inner membrane. Mitochondrial ROS have been shown to promote cytochrome c release to the cytosol by dissociation from these membrane phospholipids [46,47]. Therefore, Compound K-mediated ROS production may target membrane phospholipids resulting in dissociation of cytochrome c.

Bottom Line: Compound K produced intracellular ROS in a time dependent fashion; however, N-acetylcysteine (NAC) pretreatment resulted in the inhibition of this effect and the recovery of cell viability.Compound K induced a mitochondria-dependent apoptotic pathway via the modulation of Bax and Bcl-2 expressions, resulting in the disruption of the mitochondrial membrane potential (Δψ(m)).The apoptotic effect of Compound K, exerted via the activation of c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), was abrogated by specific MAPK inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-799, Korea; E-Mails: inkyeong@korea.ac.kr (I.K.L.); kbumjoon@snu.ac.kr (B.J.K.).

ABSTRACT
The objective of this study was to elucidate the cytotoxic mechanism of Compound K, with respect to the involvement of reactive oxygen species (ROS) and the mitochondrial involved apoptosis, in HT-29 human colon cancer cells. Compound K exhibited a concentration of 50% growth inhibition (IC(50)) at 20 μg/mL and cytotoxicity in a time dependent manner. Compound K produced intracellular ROS in a time dependent fashion; however, N-acetylcysteine (NAC) pretreatment resulted in the inhibition of this effect and the recovery of cell viability. Compound K induced a mitochondria-dependent apoptotic pathway via the modulation of Bax and Bcl-2 expressions, resulting in the disruption of the mitochondrial membrane potential (Δψ(m)). Loss of the Δψ(m) was followed by cytochrome c release from the mitochondria, resulting in the activation of caspase-9, -3, and concomitant poly ADP-ribosyl polymerase (PARP) cleavage, which are the indicators of caspase-dependent apoptosis. The apoptotic effect of Compound K, exerted via the activation of c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), was abrogated by specific MAPK inhibitors. This study demonstrated that Compound K-mediated generation of ROS led to apoptosis through the modulation of a mitochondria-dependent apoptotic pathway and MAPK pathway.

Show MeSH
Related in: MedlinePlus