Rg3 and Rh2 are the main bioactive components in steamed American ginseng extracts, which exhibited potent ability to kill colorectal cancer cells. In this report, we showed that Rh2 is significantly more potent at inducing cell death in colorectal cancer cells than Rg3 and that Rh2-induced cell death in colorectal cancer cells is mediated partially by caspase-dependent apoptosis and caspase-independent paraptosis-like cell death. Furthermore, we show that both types of cell death induced by Rh2 in colorectal cancer cells are p53-dependent.
Apoptosis and paraptosis are two different types of programmed cell death. Both are accompanied by the phosphatidylserine translocation from the inner (cytoplasmic) leaflet of the plasma membrane to the outer (cell surface) leaflet and can be detected by Annexin V staining[22
]. Besides morphological differences, a key difference between the two types of cell death is that apoptosis is generally caspase-dependent while paraptosis is caspase-independent. Our results that Rh2 significantly alters levels of the Bcl-2 family of pro and anti apoptotic proteins in conjunction with the observation that inhibition of caspases by Z-VAD partially inhibited Rh2-induced cell death in colorectal cancer cells indicate that a mitochondria-dependent apoptosis contributes to Rh2-induced cell death. Consistent with this, overexpression of Bcl-XL significantly suppressed Rh2-induced cell death.
In addition to inducing apoptosis, Rh2 also induced extensive cytosolic vacuolization in HCT116 cells, suggesting the involvement of paraptosis. Rh2 induced vacuolization was significantly inhibited by cycloheximide and MEK1/2 specific inhibitor U0126, indicating the requirements for protein translation and MAP kinase signaling, which are also consistent with parapotosis. In addition, Rh2 induced cytosolic vacuolization is not due to autophagy since Rh2 did not induce autophagosome formation in these colorectal cancer cells. The pan-caspases inhibitor Z-VAD can only partially inhibit Rh2 induced cell death and further increasing the level of Z-VAD did not further decrease Rh2-induced cell death or decrease the level of cytosolic vacuolization suggesting that there is another type of cell death being induced which exhibits vacuolization. Taken together these results suggest that Rh2 also induces a paraptosis-like cell death in colorectal cancer cells. Interestingly, although 48 hour treatment of HCT116 cells with CHX led to 29.6% cell death, similar level of cell death was observed when HCT116 cells were treated with Rh2 alone or Rh2+CHX (52.2±1.8% and 53.0±1.9%, P=0.6). It is likely that the effect of CHX on paraptosis is compensated by the cytotoxic effect of CHX alone. It is worth noting that although the effectiveness of chemotherapeutic drugs generally depend on their ability to induce cell death in cancer cells, there are forms of chemotherapy-induced cell death that cannot readily be classified as apoptosis or necrosis but fit more in the apoptosis-like/necrosis-like programmed cell death model[12
]. It has been suggested that multiple cell death program can be activated in the same cells and the dominant cell death phenotype is determined by the relative speed of those death programs [12
]. As cancer cells often developed some resistance to undergoing apoptosis, it is possible that such alternative forms of programmed cell death play important roles in cancer therapy. In support of this notion, chemotherapeutic agents such as Paclitaxel, Arsenic trioxide, Doxorubicin, etc can all induce caspase-independent cell death[12
Consistent with previous reports that Rh2 can induce ROS in some cell lines [24
], we found that Rh2 increased ROS level in HCT116 cells. ROS has been reported either to induce cell death or to activate some survival pathways to protect cells from death. The exact effect of ROS on a particular cell type is likely depends on the cell type involved and the nature and levels of ROS induced. Similar to our observation with steamed ginseng extracts, we found that ROS scavengers, NAC and catalase, block ROS generation and enhance Rh2-induced cell death in colorectal cancer cells. We showed that this survival effect of ROS in colorectal cancer cells is mediated by the activation of the NF-κB survival pathway.
Given the results presented here we propose a model in which Rh2 treatment activates the NF-κB pathway to protect the cells from cell death while simultaneously activates two different cell death pathways. Rh2 treatment likely activates the p53 pathway, which contributes to the induction of both apoptosis and parapotosis-like cell death.
Since Rg3 can be metabolized to Rh2 and to PPD by human intestinal bacteria, it will be interesting to determine the effect of PPD on colorectal cancer cells in comparison to Rh2 and Rg3. Furthermore our results about the effects of anti oxidants on Rh2-induced cell death in colorectal cancer cells suggest that steamed American ginseng or its ginsenosides in combination with the safe antioxidants can potentially be used as chemoprevention agents to prevent the development of colorectal cancers.