Cancer Res Treat.  2009 Mar;41(1):36-44.

Proapoptotic Ginsenosides Compound K and Rh2 Enhance Fas-induced Cell Death of Human Astrocytoma Cells Through Distinct Apoptotic Signaling Pathways

Affiliations
  • 1Laboratory of Computational Cell Biology, Department of Bio and Brain Engineering, KAIST, Daejeon, Korea. cchoi@kaist.ac.kr
  • 2Graduate School of Medical Science and Engineering, KAIST, Daejeon, Korea.
  • 3KI for the BioCentury, KAIST, Daejeon, Korea.

Abstract

PURPOSE: Malignant astrocytomas are among the commonest primary brain tumors and they have a grave prognosis, and so there is an urgent need to develop effective treatment. In this study, we investigated the molecular mechanisms that are responsible for the anti-tumor effect of ginsenosides on human astrocytoma cells.
MATERIALS AND METHODS
We tested 13 different ginsenosides for their anti-tumor effect on human malignant astrocytoma cells in conjunction with Fas stimulation. In addition, the cell signaling pathways were explored by using pharmacological inhibitors and performing immunoblot analysis. DCF-DA staining and antioxidant experiments were performed to investigate the role of reactive oxygen species as one of the apoptosis-inducing mechanisms.
RESULTS
Among the 13 different ginsenoside metabolites, compound K and Rh2 induced apoptotic cell death of the astrocytoma cells in a caspase- and p38 MAPK-dependent manner, yet the same treatment had no cytotoxic effect on the primary cultured human astrocytes. Combined treatment with ginsenosides and Fas ligand showed a synergistic cytotoxic effect, which was mediated by the reduction of intracellular reactive oxygen species.
CONCLUSION
These results suggest that ginsenoside metabolites in combination with Fas ligand may provide a new strategy to treat malignant astrocytomas, which are tumors that are quite resistant to conventional anti-cancer treatment.

Keyword

Apoptosis; Ginsenoside; Fas; Reactive oxygen species; Astrocytoma

MeSH Terms

Apoptosis
Astrocytes
Astrocytoma
Brain Neoplasms
Cell Death
Fas Ligand Protein
Ginsenosides
Humans
Prognosis
Reactive Oxygen Species
Fas Ligand Protein
Ginsenosides
Reactive Oxygen Species

Figure

  • Fig. 1 Caspase-dependent apoptosis by the ginsenosides C-K and Rh2 in human astrocytoma cells. (A) The cells were treated with varying doses (0~50 mg/L) of 13 different ginsenosides, and the cell death was determined by MTT assay. *, significantly different from the control sample without any treatment (p<0.01). (B) Human primary cultured astrocytes and CRT-MG cells were treated with 25 mg/L C-K for 6 h. (C) The cells were incubated in the absence or presence of z-VAD-Fmk (10 mmol/L) for 1 h, and then they were treated with C-K or Rh2 for an additional 24 h, and the cell death was next determined after staining with Annexin-V-FITC and PI by FACS analysis. (D) The cells were incubated in the absence or presence of z-VAD-Fmk (10 mmol/L) for 1 h; they were next treated with C-K or Rh2 for an additional 24 h and then they were stained with TMRE. *, significantly different from the control sample (p<0.001).

  • Fig. 2 The involvement of caspase-3 and cytochrome C release in ginsenosides-induced cytotoxicity. (A) CRT-MG cells were incubated with Rh2 for varying time periods, and the cell lysates were subjected to western blot analysis for caspase-3 and b-actin. (B) CRT-MG cells were incubated with C-K for varying time periods, and the cell lysates were subjected to western blot analysis for caspase-3, PARP and b-actin. (C) The total cell lysates (T) were divided into the cytosolic fraction (C) and the mitochondrial fraction (M), and peroxiredoxin-1 was examined to test the separation efficiency (upper panel). The mitochondrial fraction (M) and the cytosolic fraction (C) were tested for their cytochrome-c levels.

  • Fig. 3 The involvement of p38MAPK in C-K-mediated cell death. (A) CRT-MG cells were incubated in the absence or presence of various MAPK inhibitors for 1 h, and then they were treated with C-K or Rh2 for an additional 24 h, and cell death was measured by MTT assay. (B) The cells were treated with C-K (25 mg/L) for varying time periods, and the total cell lysates were subjected to western blot analysis for p38 MAPK, phospho-p38 MAPK and β-actin.

  • Fig. 4 Ginsenosides augment fas-mediated cell death. (A) The cells were incubated in the absence or presence of varying doses of C-K for 1 h, and then they were treated with CH-11 for an additional 24 h, and the cell death was measured by MTT assay. (B) The cells were incubated in the absence of presence of C-K or Rh2 for 1 h, and then they were treated with CH-11 for an additional 24 h, and the cell death was measured after staining with Annexin-V and PI by FACS analysis. (C) The cells were incubated in the absence or presence of C-K or Rh2 for 1 h, and then they were treated with CH-11 for an additional 24 h, and the Δψm was measured after staining with TMRE.

  • Fig. 5 Ginsenosides Suppress Fas-Induced ROS Generation. (A) The CRT-MG cells were incubated with anti-Fas antibody (CH-11) for varying time periods, and then they were examined for their intracellular ROS levels by staining with DCF-DA. *, significantly different from the control sample (p<0.05). (B) The cells were incubated in the absence or presence of C-K or Rh2 for 1 h, and then they were treated with CH-11 for an additional 30 min, and the intracellular ROS levels were measured. *, significantly different from the control sample treated with CH-11 alone (p<0.01). (C) The cells were incubated in the absence or presence of C-K or Rh2 for 1 h, and then they were treated with CH-11 for an additional 2 h, and the in vitro DEVDase enzymatic activity was measured. *, significantly different from the control sample without any treatment (p<0.01).

  • Fig. 6 The proposed model for the anti-tumor activity of ginsenosides and the therapeutic strategies for treating malignant tumors. Ginsenosides induce apoptotic cell death in a mitochondria-dependent manner, while the sensitizing effect of ginsenosides for Fas treatment occurs by ROS inhibition. Since ginsenosides can induce intrinsic cell death and they augment Fas-induced extrinsic cell death, a combined treatment of Fas and ginsenosides might be employed as a useful therapeutic strategy for treating multi-drug resistant malignant astrocytomas.


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