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Diosgenin Induces Apoptosis in HepG2 Cells through Generation of Reactive Oxygen Species and Mitochondrial Pathway.

Kim DS, Jeon BK, Lee YE, Woo WH, Mun YJ - Evid Based Complement Alternat Med (2012)

Bottom Line: Diosgenin, a naturally occurring steroid saponin found abundantly in legumes and yams, is a precursor of various synthetic steroidal drugs.In the upstream, diosgenin increased the expression of Bax, decreased the expression of Bid and Bcl-2, and augmented the Bax/Bcl-2 ratio.These results suggest that diosgenin-induced apoptosis in HepG2 cells through Bcl-2 protein family-mediated mitochndria/caspase-3-dependent pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Herbal Resources, Professional Graduate School of Oriental Medicine, Wonkwang University, Republic of Korea.

ABSTRACT
Diosgenin, a naturally occurring steroid saponin found abundantly in legumes and yams, is a precursor of various synthetic steroidal drugs. Diosgenin is studied for the mechanism of its action in apoptotic pathway in human hepatocellular carcinoma cells. Based on DAPI staining, diosgenin-treated cells manifested nuclear shrinkage, condensation, and fragmentation. Treatment of HepG2 cells with 40 μM diosgenin resulted in activation of the caspase-3, -8, -9 and cleavage of poly-ADP-ribose polymerase (PARP) and the release of cytochrome c. In the upstream, diosgenin increased the expression of Bax, decreased the expression of Bid and Bcl-2, and augmented the Bax/Bcl-2 ratio. Diosgenin-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK, p38 MAPK and apoptosis signal-regulating kinase (ASK)-1, as well as generation of the ROS. NAC administration, a scavenger of ROS, reversed diosgene-induced cell death. These results suggest that diosgenin-induced apoptosis in HepG2 cells through Bcl-2 protein family-mediated mitochndria/caspase-3-dependent pathway. Also, diosgenin strongly generated ROS and this oxidative stress might induce apoptosis through activation of ASK1, which are critical upstream signals for JNK/p38 MAPK activation in HepG2 cancer cells.

No MeSH data available.


Related in: MedlinePlus

Diosgenin-generated ROS induced apoptosis in HepG2 cells. (a) The cells were pretreated with/without NAC (10 mM) at least 2 hr before the treatment of 40 μM diosgenin. After 24 h, quantitative assessment of the percentage of cell viability was determined by MTT assay. **P < 0.01 compared to control, ##P < 0.01 compared to diosgenin 40 μM-treated group. (b) Also cells were pretreated with/without NAC (10 mM) at least 2 h before the treatment of diosgenin. ROS production was confirmed by fluorescence microscope.
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fig5: Diosgenin-generated ROS induced apoptosis in HepG2 cells. (a) The cells were pretreated with/without NAC (10 mM) at least 2 hr before the treatment of 40 μM diosgenin. After 24 h, quantitative assessment of the percentage of cell viability was determined by MTT assay. **P < 0.01 compared to control, ##P < 0.01 compared to diosgenin 40 μM-treated group. (b) Also cells were pretreated with/without NAC (10 mM) at least 2 h before the treatment of diosgenin. ROS production was confirmed by fluorescence microscope.

Mentions: As reactive oxygen species (ROS) generation is an important role in apoptosis, we investigated the ability of diosgenin to generate ROS. Cells were exposed to diosgenin (0–40 μM) for 24 hr and analyzed for the present of ROS by flow cytometry The generation of ROS by diosgenin was increased in dose-dependent manner (Figure 4(a)). We also confirmed intracellular ROS production by fluorescence microscope after staining with carboxy-H2DCFDA, ROS were generated by treatment of diosgenin (Figure 4(b)). To examine whether diosgenin-generated ROS induce apoptosis in HepG2 cells, we measured cell death after treatment of diosgenin only or with NAC. NAC is a potent antioxidant that can inhibit oxidative stress by directly scavenging ROS and replenishing GSH [29]. If ROS production mediates diosgenin-induced cell death, we expect that NAC should have the ability to inhibit diosgenin-induced cell death. As shown in Figure 5(a), diosgenin (40 μM) increased cell death, whereas removing diosgenin-generated ROS by NAC led to decreasing cell death. Also, the decrement of intracellular ROS by treating NAC was observed after DCFH-DA staining (Figure 5(b)). These results indicate that diosgenin induced cell death of HepG2 cells by the generation of ROS.


Diosgenin Induces Apoptosis in HepG2 Cells through Generation of Reactive Oxygen Species and Mitochondrial Pathway.

Kim DS, Jeon BK, Lee YE, Woo WH, Mun YJ - Evid Based Complement Alternat Med (2012)

Diosgenin-generated ROS induced apoptosis in HepG2 cells. (a) The cells were pretreated with/without NAC (10 mM) at least 2 hr before the treatment of 40 μM diosgenin. After 24 h, quantitative assessment of the percentage of cell viability was determined by MTT assay. **P < 0.01 compared to control, ##P < 0.01 compared to diosgenin 40 μM-treated group. (b) Also cells were pretreated with/without NAC (10 mM) at least 2 h before the treatment of diosgenin. ROS production was confirmed by fluorescence microscope.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Diosgenin-generated ROS induced apoptosis in HepG2 cells. (a) The cells were pretreated with/without NAC (10 mM) at least 2 hr before the treatment of 40 μM diosgenin. After 24 h, quantitative assessment of the percentage of cell viability was determined by MTT assay. **P < 0.01 compared to control, ##P < 0.01 compared to diosgenin 40 μM-treated group. (b) Also cells were pretreated with/without NAC (10 mM) at least 2 h before the treatment of diosgenin. ROS production was confirmed by fluorescence microscope.
Mentions: As reactive oxygen species (ROS) generation is an important role in apoptosis, we investigated the ability of diosgenin to generate ROS. Cells were exposed to diosgenin (0–40 μM) for 24 hr and analyzed for the present of ROS by flow cytometry The generation of ROS by diosgenin was increased in dose-dependent manner (Figure 4(a)). We also confirmed intracellular ROS production by fluorescence microscope after staining with carboxy-H2DCFDA, ROS were generated by treatment of diosgenin (Figure 4(b)). To examine whether diosgenin-generated ROS induce apoptosis in HepG2 cells, we measured cell death after treatment of diosgenin only or with NAC. NAC is a potent antioxidant that can inhibit oxidative stress by directly scavenging ROS and replenishing GSH [29]. If ROS production mediates diosgenin-induced cell death, we expect that NAC should have the ability to inhibit diosgenin-induced cell death. As shown in Figure 5(a), diosgenin (40 μM) increased cell death, whereas removing diosgenin-generated ROS by NAC led to decreasing cell death. Also, the decrement of intracellular ROS by treating NAC was observed after DCFH-DA staining (Figure 5(b)). These results indicate that diosgenin induced cell death of HepG2 cells by the generation of ROS.

Bottom Line: Diosgenin, a naturally occurring steroid saponin found abundantly in legumes and yams, is a precursor of various synthetic steroidal drugs.In the upstream, diosgenin increased the expression of Bax, decreased the expression of Bid and Bcl-2, and augmented the Bax/Bcl-2 ratio.These results suggest that diosgenin-induced apoptosis in HepG2 cells through Bcl-2 protein family-mediated mitochndria/caspase-3-dependent pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Herbal Resources, Professional Graduate School of Oriental Medicine, Wonkwang University, Republic of Korea.

ABSTRACT
Diosgenin, a naturally occurring steroid saponin found abundantly in legumes and yams, is a precursor of various synthetic steroidal drugs. Diosgenin is studied for the mechanism of its action in apoptotic pathway in human hepatocellular carcinoma cells. Based on DAPI staining, diosgenin-treated cells manifested nuclear shrinkage, condensation, and fragmentation. Treatment of HepG2 cells with 40 μM diosgenin resulted in activation of the caspase-3, -8, -9 and cleavage of poly-ADP-ribose polymerase (PARP) and the release of cytochrome c. In the upstream, diosgenin increased the expression of Bax, decreased the expression of Bid and Bcl-2, and augmented the Bax/Bcl-2 ratio. Diosgenin-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK, p38 MAPK and apoptosis signal-regulating kinase (ASK)-1, as well as generation of the ROS. NAC administration, a scavenger of ROS, reversed diosgene-induced cell death. These results suggest that diosgenin-induced apoptosis in HepG2 cells through Bcl-2 protein family-mediated mitochndria/caspase-3-dependent pathway. Also, diosgenin strongly generated ROS and this oxidative stress might induce apoptosis through activation of ASK1, which are critical upstream signals for JNK/p38 MAPK activation in HepG2 cancer cells.

No MeSH data available.


Related in: MedlinePlus