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

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.

Figure 1

Cytotoxic Effect of diosgenin in HepG2 cells. (a) Cells were treated with diosgenin by dose-dependent manner for 24 and 48 h. The ratios of cell viability were measured by MTT assay. Data are presented as mean ± SD of six replicates from three independent experiments. **P < 0.01 compared to control. (b) Nuclear alterations were observed by DAPI staining and fluorescence microscopy (×100). After cells were treated with diosgenin (0–40 μM) for 48 h, marked morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentations were found clearly using DAPI staining.

Figure 2

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.

Figure 3

Effects of diosgenin on caspases (a), Bcl-2 family proteins, and cytochrome c (b). Cells were incubated without or with 20 μM, 30 μM, and 40 μM of diosgenin for 24 h. Total cell lysates were analyzed by immunoblotting with antibody against caspase-3 (pro and cleavage), procaspase-8, procaspase-9, PARP, Bcl-2, Bax, Bid, and cytochrom c.

Figure 4

Diosgenin generated ROS in HepG2 cells. (a) Diosgenin generated ROS in HepG2 cells. Cells were treated with different concentrations of diosgenin (20–40 μM) plus 40 μM of DCFH-DA for 24 h, and ROS productions were determined by FACS analysis. (b) For observation of intracellular ROS by fluorescence microscope, cells were treated with diosgenin (20–40 μM) and then incubated with DCFH-DA.

Figure 5

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.

Figure 6

Effects of diosgenin on MAPK (a) and ASK1 (b). Cells were treated with varying concentrations of diosgenin (20–40 μM) for 24 h. Total cell lysates were analyzed by immunoblotting with antibody against phospho-p38, p38, phospho-JNK, JNK, phosphoASK1, and ASK1.