The poor prognosis for Chronic Myelomonocytic Leukemia (CMML) patients is a major indication that there is a serious need for a more effective and non-toxic alternative to the conventionally available forms of chemotherapy and surgical procedures 
. With the introduction of natural products, not only as sources of nourishment, but also for their therapeutic benefits, it is therefore necessary to study the vast array of natural products as non-toxic and less expensive alternatives for the treatment of CMML. Apoptosis and autophagy, two necessary modes of programmed cell death, are important mechanisms, which cells utilize for the maintenance of cellular homeostasis 
. Cancer cells, however, have developed mechanisms to evade these programs, so as to enable enhanced proliferation, aggressiveness and resistance 
. In this study, we demonstrate the selective efficacy of dandelion root extract in inducing apoptosis and autophagy in highly aggressive and resistant CMML cell lines.
We observed the induction of apoptosis in three of the CMML cell lines used for this study (). More importantly, this effect was selective, as non-cancerous PBMCs and NHFs remained unsusceptible to DRE-induced apoptosis ().
Although various extracts of dandelions have been used for centuries for the treatment of various diseases, there have been very few scientific studies done to ascertain the mechanism by which these extracts act and although some of the compounds present in dandelion extracts have been isolated and identified, they still are not fully characterized 
. The major aim of the study was therefore to determine the mechanism of DRE-induced cell death in CMML cells.
In this study, we report a rapid activation of caspase-8 (within minutes) and subsequent activation of caspase-3 in human CMML cells, indicative of a speedy activation of the death receptor mediated extrinsic pathway of apoptosis (). This activation has been shown to be necessary for the induction of apoptosis observed after DRE treatment, as the inhibition of caspase activity, using a pan-caspase inhibitor, ZVAD, prevented the induced cell death observed (). These results suggest that caspases, more importantly, caspase-8 and 3, are required for the induction of apoptosis by DRE. The Fas-Associated Death Domain (FADD), as well as other death domains, are highly important for activation of caspase-8 through extrinsic apoptosis 
. The binding of the death ligand to its corresponding death receptor leads to the recruitment of specific receptor death domains for the formation of the death inducing signalling complex (DISC) 
. The absence of FADD should therefore prevent the activation of the extrinsic pathway of apoptosis through inhibition of the activation of caspase-8 
. To further confirm the effect of DRE on the extrinsic pathway of apoptosis, DnFADD cells were treated with DRE and analyzed for apoptosis induction. According to our results, no nuclear condensation and morphological changes were observed with the treatment of DRE. There was no change in the number of cells treated with DRE, compared to the control untreated cells, suggesting that DRE did not have a significant effect on the cells with a truncated FADD protein (). Furthermore, these cells were not susceptible to DRE-induced rapid activation of caspases, specifically caspase-8 (). As we observed late activation of caspase-3, we wanted to observe the connection between the intrinsic and the extrinsic pathway of apoptosis. It is known that the activation of caspase-8 could yield two main results. First, the activated caspase-8 could lead directly to the activation of the effector caspase, caspase-3 or it could lead to mitochondrial changes, through the cleavage of pro-apoptotic protein, Bid. The truncation of Bid causes mitochondrial membrane destabilization, which leads to the release of pro-apoptotic proteins for the activation of caspase-3 
. In this study, we observed the destabilization of the mitochondrial membrane potential 24 hours after treatment with DRE. This destabilization of mitochondrial membrane potential was only observed in MV-4-11 cells, not in DnFADD cells (). These results suggest that DRE indirectly targets the mitochondria after the activation of the extrinsic pathway of apoptosis. It confirms the requirement of the adapter domain for DRE-induced apoptosis, as we do not observe a similar loss of mitochondrial membrane potential in DnFADD cells treated with DRE. As the mitochondria are major players in the production of ROS 
, the next step was to observe the effect of DRE on the production of ROS. Results confirm the production of ROS from isolated mitochondria (), further confirming the effect of DRE on the mitochondria of CMML cells.
The integrity of the mitochondria is essential for the maintenance of cellular homeostasis and defects in the mitochondria caused by treatment with DRE could provide sufficient signals for the induction of programmed cell death type II, also known as Autophagy, a process induced by the presence of defective organelles, proteins and starvation 
. Autophagy has been shown to play a significant role in cancer survival and its role in programmed cell death has been controversial. In response to stressors, cells undergo “self-digestion” as a means of temporary survival, where macromolecules are digested in order to provide an alternate energy source. However, excessive exposure to stressors could lead to excessive autophagy, ultimately resulting in cell death 
. The loss of mitochondrial membrane potential, observed after treatment with DRE, therefore led us to determine if the stress induced by our extract, leading to mitochondrial membrane destabilization was a sufficient signal for the induction of autophagy. Our results indicate that treatment with DRE triggered the induction of a pro-death form of autophagy in MV-4-11 cells (; top panel). Comparing this to treatment with tamoxifen, which is known to induce pro-survival in cancer cells, treatment with DRE not only triggered autophagy, comparable to TAM, the cells were also propidium iodide positive, indicative of cell death. Moreover, ncPBMCs stained with MDC and PI showed no induction of autophagy in these cells after DRE treatment (; bottom panel). These results suggest that DRE is effective in selectively inducing both forms of programmed cell death in human CMML cells.
In conclusion, Dandelion Root Extract has shown selective efficacy in inducing two forms of programmed cell death in highly aggressive and resistant CMML cell lines. The rapid activation of caspase-8 not only activated the extrinsic pathway of apoptosis, but also triggered pro-death autophagy selectively in these cells, suggesting that this extract has components that enhance its selective efficacy in targeting CMML cells. These results indicate that within the vast array of available natural products and compounds, there are non-toxic alternatives to conventional chemotherapy that are safe and effective.