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. 2012;7(2):e30604.
doi: 10.1371/journal.pone.0030604. Epub 2012 Feb 17.

Efficient Induction of Extrinsic Cell Death by Dandelion Root Extract in Human Chronic Myelomonocytic Leukemia (CMML) Cells

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Free PMC article

Efficient Induction of Extrinsic Cell Death by Dandelion Root Extract in Human Chronic Myelomonocytic Leukemia (CMML) Cells

Pamela Ovadje et al. PLoS One. .
Free PMC article

Abstract

Background: Chronic Myelomonocytic Leukemia (CMML) is a heterogeneous disease that is not only hard to diagnose and classify, but is also highly resistant to treatment. Available forms of therapy for this disease have not shown significant effects and patients rapidly develop resistance early on in therapy. These factors lead to the very poor prognosis observed with CMML patients, with median survival duration between 12 and 24 months after diagnosis. This study is therefore centered around evaluating the selective efficacy of a natural extract from dandelion roots, in inducing programmed cell death in aggressive and resistant CMML cell lines.

Methodology/principal findings: To confirm the induction of programmed cell death in three human CMML cell lines, nuclear condensation and externalization of the phosphatidylserine, two main characteristics of apoptosis, were detected using Hoechst staining and annexin-V binding assay. The induction of another mode of cell death, autophagy, was determined using a monodansylcadaverine (MDC) stain, to detect the formation of autophagy vacuoles. The results from this study indicate that Dandelion Root Extract (DRE) is able to efficiently and selectively induce apoptosis and autophagy in these cell lines in a dose and time dependent manner, with no significant toxicity on non-cancerous peripheral blood mononuclear cells. More importantly, we observed early activation of initiator caspase-8, which led to mitochondrial destabilization and the induction of autophagy, suggesting that DRE acts through the extrinsic pathway of apoptosis. The inability of DRE to induce apoptosis in dominant-negative FADD cells, confirms the mechanism of action of DRE in in vitro models of CMML.

Conclusion: The results from this study indicate that natural products, in particular Dandelion Root Extract, have great potential, as non-toxic and effective alternatives to conventional modes of chemotherapy available today.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. DRE induces apoptosis in CMML cells in a dose dependent manner.
MV-4-11, HL-60 and U-937 cells were treated with increasing doses of DRE and analyzed for the induction of apoptosis. (a) Hoechst and Annexin-V staining of DRE-treated CMML cells 48 hours after treatment. Magnification 400×. (b) Manual quantification of three individual experiments of DRE-treated cells, 48 hours after. 6 pictures were obtained for every sample and live versus dead cells were counted to ascertain the percent apoptotic cell count for every sample. Experiment was performed three times and the mean and standard deviation of the three experiments were obtained.
Figure 2
Figure 2. DRE induces late stage DNA damage in MV-4-11 cells.
MV-4-11 cells were treated with 1.0 mg/ml DRE and 10 µM VP-16 at the indicated time points to determine if DNA damage was the cause or effect of apoptosis observed in CMML cells. Following treatment, cells were fixed and immunostained with anti-BrdU antibody to observe DNA damage. Increase in TUNEL positive staining corresponds with an increase in levels of DNA damage following treatment. More positively stained cells are observed in the final stages of apoptosis caused by DRE, compared to the DNA targeting drug, VP-16. magnification: 200×.
Figure 3
Figure 3. DRE reduces the viability of MV-4-11 cells in a time dependent manner.
(a). MV-4-11 cells were plated in 96-well clear bottom plates and treated with DRE at indicated time points and analyzed for the ability of DRE to reduce the viability of these cells, measured by a decrease in metabolic activity. Following treatment, the WST-1 reagent was added to each well, the absorbance readings were taken at 450 nm, and expressed at a percentage of the control. The absorbance readings were analyzed using GraphPad Prism version5.0 and values are expressed as mean ± SD from quadruplicates of 3 independent experiments. (b). Following treatment with DRE for 48 hours, MV-4-11 cells were removed from media containing DRE and equal number of cells were replated in fresh media, with no extract and allowed to grow for 96 hours, observing growth every 24 hours by trypan blue exclusion assay (left). As a single dose of DRE slowed down growth of MV-4-11 cells but did not completely halt this growth, MV-4-11 cells were treated with DRE for 48 hours, then treated with a second dose of DRE after the first 48 hours. Subsequent to the second treatment, cells were observed for growth ability (right), using trypan blue exclusion assay.
Figure 4
Figure 4. DRE activates the death-receptor-mediated extrinsic pathway of apoptosis.
Following treatment with DRE, at indicated time points and concentrations, MV-4-11 cells were collected, washed and incubated with lysis buffer to obtain cell lysate. The cell lysate was incubated with caspase substrates, specific to each caspase (3,8 and 9) and incubated for an hour. Fluorescence readings were obtained using a spectrofluorometer. An average of 6 readings per well and a minimum of three wells were run per experiment. The results here are reported as activity per µg of protein (in fold) and the average of three experiments are shown (a,b). (c). Prior to DRE treatment, MV-4-11 cells were pre-treated with a pan-caspase inhibitor, Z-VAD-fmk for an hour and then treated with DRE at the indicated concentration for indicated time points. These cells were then incubated with caspase-8 substrate and fluorescence readings were obtained. These cells were also analyzed for the induction of apoptosis by Hoechst and annexin-V staining (d). Magnification: 400×.
Figure 5
Figure 5. DRE required the Fas-Associated Death Domain (FADD) for its activity.
(a). Dominant-negative FADD (DnFADD) cells were treated with DRE at indicated concentrations, for 96 hours and analyzed for the induction of apoptosis by nuclear condensation (Hoechst) and change in morphology (Phase contrast). Magnification: 400×. (b). Following treatment with DRE, DnFADD cells were collected and cell number was obtained using the trypan blue exclusion assay. Live cells were impermeable to the trypan blue dye. (c). DnFADD cells were treated with DRE for the indicated time points and analyzed for the activation of caspase-8, using caspase-8 specific substrate and fluorescence readings were obtained. An average of 6 readings per well and a minimum of three wells were run per experiment. The results here are reported as activity per µg of protein (in fold) and the average of three experiments are shown.
Figure 6
Figure 6. DRE destabilizes the mitochondria membrane potential of MV-4-11 cells.
(a). Following treatment with DRE, MV-4-11 cells were incubated with JC-1 dye to detect the loss of mitochondrial potential (ref to Materials and Methods). Red fluorescence indicates only cells that have healthy mitochondria. The mitochondria of MV-4-11 cells are completely destabilized by DRE treatment. On the other hand, the mitochondria of DnFADD cells remained unaffected by DRE treatment (b). Magnification: 400×.
Figure 7
Figure 7. DRE increases ROS production in isolated mitochondria from CMML cells.
Isolated mitochondria from (a) MV-4-11 and (b) HL-60 cells were treated directly with 1.0 mg/ml DRE and ROS production was measured using Amplex Red substrate in the presence of horseradish peroxidase (HRP). Results were compared to control untreated mitochondria and positive control, paraquat (PQ). Fluorescence readings were taken in 5 min intervals for 4 h at Ex. 560 nm and Em.590 nm and expressed as relative fluorescence units (RFU). Analysis of results were performed using GraphPad Prism version 5.0 and results shown are representative of 3 independent experiments demonstrating similar trends.
Figure 8
Figure 8. DRE triggers pro-death autophagy in MV-4-11 cells but not in non-cancerous Peripheral blood mononuclear cells (ncPBMCs).
MV-4-11 cells were treated with DRE for 48 hours and analyzed for the induction of autophagy; Tamoxifen (TAM) was used as a positive control for the induction of pro-surivival autophagy. Following treatment, the cells were stained with MDC to detect autophagic vacuoles and counterstained with PI to detect cell death. Fluorescence images were captured. Magnification: 400×.
Figure 9
Figure 9. DRE does not target normal non-cancerous peripheral blood mononuclear cells (ncPBMCs).
(a). ncPBMCs were isolated from healthy volunteers and plated in six-well plates. These cells were treated with DRE at increasing concentrations for 96 hours and analyzed for the induction of apoptosis by nuclear condensation (Hoechst) and externalization of the phosphatidylserine (Annexin-V binding). Magnification: 400×. (b). Results from three different experiments were quantified to determine the percentage of apoptosis occurring in ncPBMCs treated with DRE.

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