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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

FACS analyses of Annexin V and PI staining. HepG2 cells was treated with diosgenin (0–40 μM) for 24 h (a) and 40 μM for 0, 24, 48 h (b). Lower right quadrant, early apoptosis cells, that is, Annexin V-FITC-positive/PI-negative cells; upper right quadrant, necrosis or late-apoptotic cells, that is, Annexin V-FITC-positive/PI-positive cells.
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fig2: FACS analyses of Annexin V and PI staining. HepG2 cells was treated with diosgenin (0–40 μM) for 24 h (a) and 40 μM for 0, 24, 48 h (b). Lower right quadrant, early apoptosis cells, that is, Annexin V-FITC-positive/PI-negative cells; upper right quadrant, necrosis or late-apoptotic cells, that is, Annexin V-FITC-positive/PI-positive cells.

Mentions: A previous study has shown that diosgenin inhibited proliferation and induced apoptosis in HepG2 cells [11]. To confirm whether diosgenin influences the viability of hepatoma cells, HepG2 cells were challenged with diosgenin (0–40 μM). Cytotoxicity is measured by MTT assay following a brief during exposure. Diosgenin markedly induced cell death in HepG2 cells in a dose- and time-dependent manner as compared with vehicle controls (Figure 1). Apoptosis is initially characterized by morphological features, such as chromatin condensation, nuclear fragmentation, and membrane blebbing [24]. In the current study, morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentation were clearly observed by DAPI staining after 24 h of diosgenin (Figure 1). Cell death was also assessed with flow cytometry after double staining with annexin V and PI. We challenged the cells with increasing doses of diosgenin at 24 h of treatment. According to Figure 2(a), the combined early and late apoptotic cells (Annexin V positive) were elevated in a dose-dependent fashion. Consistent with the progression of apoptosis, late apoptotic cells become dominant at later time, because we observed gradual diminution of early apoptotic cells and increment of the late apoptotic cells after 48 h (Figure 2(b)). These findings demonstrate that diosgenin induced the apoptosis of HepG2 cells in both dose- and time-dependent manners.


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)

FACS analyses of Annexin V and PI staining. HepG2 cells was treated with diosgenin (0–40 μM) for 24 h (a) and 40 μM for 0, 24, 48 h (b). Lower right quadrant, early apoptosis cells, that is, Annexin V-FITC-positive/PI-negative cells; upper right quadrant, necrosis or late-apoptotic cells, that is, Annexin V-FITC-positive/PI-positive cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375183&req=5

fig2: FACS analyses of Annexin V and PI staining. HepG2 cells was treated with diosgenin (0–40 μM) for 24 h (a) and 40 μM for 0, 24, 48 h (b). Lower right quadrant, early apoptosis cells, that is, Annexin V-FITC-positive/PI-negative cells; upper right quadrant, necrosis or late-apoptotic cells, that is, Annexin V-FITC-positive/PI-positive cells.
Mentions: A previous study has shown that diosgenin inhibited proliferation and induced apoptosis in HepG2 cells [11]. To confirm whether diosgenin influences the viability of hepatoma cells, HepG2 cells were challenged with diosgenin (0–40 μM). Cytotoxicity is measured by MTT assay following a brief during exposure. Diosgenin markedly induced cell death in HepG2 cells in a dose- and time-dependent manner as compared with vehicle controls (Figure 1). Apoptosis is initially characterized by morphological features, such as chromatin condensation, nuclear fragmentation, and membrane blebbing [24]. In the current study, morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentation were clearly observed by DAPI staining after 24 h of diosgenin (Figure 1). Cell death was also assessed with flow cytometry after double staining with annexin V and PI. We challenged the cells with increasing doses of diosgenin at 24 h of treatment. According to Figure 2(a), the combined early and late apoptotic cells (Annexin V positive) were elevated in a dose-dependent fashion. Consistent with the progression of apoptosis, late apoptotic cells become dominant at later time, because we observed gradual diminution of early apoptotic cells and increment of the late apoptotic cells after 48 h (Figure 2(b)). These findings demonstrate that diosgenin induced the apoptosis of HepG2 cells in both dose- and time-dependent manners.

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