Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53 - PubMed (original) (raw)

Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53

Binghui Li et al. Cancer Lett. 2011.

Abstract

Ginsenosides are the main bioactive components in American ginseng, a commonly used herb. In this study, we showed that the ginsenoside Rh2 exhibited significantly more potent cell death activity than the ginsenoside Rg3 in HCT116 and SW480 colorectal cancer cells. Cell death induced by Rh2 is mediated in part by the caspase-dependent apoptosis and in part by the caspase-independent paraptosis, a type of cell death that is characterized by the accumulation of cytoplasmic vacuoles. Treatment of cells with Rh2 activated the p53 pathway and significantly increased the levels of the pro-apoptotic regulator, Bax, while decreasing the levels of anti-apoptosis regulator Bcl-2. Removal of p53 significantly blocked Rh2-induced cell death as well as vacuole formation, suggesting that both types of cell death induced by Rh2 are mediated by p53 activity. Furthermore, we show that Rh2 increased ROS levels and activated the NF-κB survival pathway. Blockage of ROS by NAC or catalase inhibited the activation of NF-κB signaling and enhanced Rh2-induced cell death, suggesting that the anti-cancer effect of Rh2 can be enhanced by antioxidants.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest.

Figures

Figure 1. The effects of ginsenosides on colorectal cancer cell lines

A) The structures of Rg3 and Rh2. B and C) HCT116 cells were treated with different concentrations of Rg3 or Rh2 for 48h, and the cell death was quantified. D) HCT116 cells were treated with 35 μM of Rh2 for 0–72 hours, and the cell death was determined. E) SW480 cells were treated with different concentrations of Rh2 for 48h, and cell death was measured. F) HCT116 and FHC cells were treated with different concentrations of Rh2 for 48h and cell death was determined by staining with Trypan Blue.

Figure 2. Ginsenoside Rh2 induced ROS generation in colorectal cancer cells

(A) HCT116 cells were treated with 35 μM of Rh2, and then ROS level was measured at the indicated times. (B) HCT116 cells were treated with different concentrations of Rh2 for 6h, and then ROS level was determined. (C) SW480 cells were treated with different concentrations of Rh2 for 6h, and then ROS was determined.

Figure 3. Antioxidants increased Rh2-induced cell death of colorectal cancer cells

(A) HCT116 cells were treated with 35 μM of Rh2 in the presence or absence 10 mM of NAC, an antioxidant, for 6h, and then ROS was determined. (B) SW480 cells were treated with 30 or 40 μM of Rh2 in the presence or absence 10 mM of NAC for 6h. (C and D) HCT116 cells or SW480 cells were treated with Rh2 in the presence or absence 10 mM of NAC for 48h, and the cell death was measured. (E and F) HCT116 (E) or SW480 (F) cells with or without Catalase expression were treated with Rh2 for 48h and the levels of cell death were determined.

Figure 4. Gisenosides activated NF-κB pathway via ROS

(A) Twenty four hours after transient transfection of NF-κB reporter plasmids, HCT116 cells were treated with different concentrations of Rh2 for 24 h before the luciferase reporter activity was determined. (B and C) After HCT116 cells or SW480 cells were treated with 30 μM Rh2 for 24h in the presence or absence of NAC (10 mM) or PS1145 (50 μM), NF-κB reporter activity was determined. (D and E) HCT116 or SW480 cells were treated with PS1145 (50 μM) for 48 hours and cell death was measured.

Figure 5. Ginsenoside Rh2 induced apoptosis and paraptosis-like cell death

(A) HCT116 cells were treated with 35 μM of Rh2 with or without the indicated amount of the pan-inhibitor of caspases, Z-VAD, for 48h, and then the cell death was determined. (B) HCT116 cells were treated with 35 μM of Rh2 with or without 10μg/ml of cycloheximide for 6 hours and were imaged (top and middle). HCT116 cells expressing EGFP-LC3 were treated 35 μM of Rh2 for 6 hours and were imaged with a fluorescence microscope (bottom). The serum-free (SF) treatment for 48h induced autophagy, and was taken as the positive control. (C) HCT116 cells were treated with the indicated concentration of Rh2 for 6 hours and the fraction of cells with cytoplasmic vacuoles were determined. (D) HCT116 cells were treated with 35 μM of Rh2 with either vehicle control, 10 μM U0126, or 10μg/ml cycloheximide for 6 hours. The fraction of cells with cytoplasmic vacuoles were determined. (E) HCT116/p53−/− cells were treated with 35 μM of Rh2 for 6h and were imaged (top). HCT116/p53−/− cells expressing EGFP-LC3 were treated 35 μM of Rh2 for 6h and imaged with fluorescence microscope (bottom)

Figure 6. Rh2 treatment resulted in altered levels of apoptosis-associated proteins and activated transcriptional activity of p53

(A) HCT116 cells were treated with 35 μM of Rh2, and then protein samples were collected at different times for Western blot analysis. (B) HCT116 cells expressed Bcl-XL or empty vector were treated with 35 μM of Rh2 for 48h, and the cell death was determined. (C) Twenty four hour after transient transfection of p53 reporter plasmids and treatment with 35 μM of Rh2, the luciferase reporter activity was measured. (D) Cells were treated with 35 μM Rh2 for 48h, and then cell death was quantified.

Figure 7. A working model for the action of ginsenoside Rh2 on colorectal cancer cells

Ginsenoside Rh2 induces both apoptosis and paraptosis in colorectal cancer cells. In addition, Rh2 also activates ROS-NF-κB pathway, which counteracts cell death induction.

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Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53 - PubMed (original) (raw)