Could the Next Chemotherapy be Derived from a Weed?

As much as I may complain about weeds, one that I enjoy (in moderation and not among my vegetables) is dandelions. The bright yellow flowers herald spring, and the seed puffballs, while not as visually interesting, offer entertainment as I watch birds landing on the shaft, bending it and eating the seeds. When I am pulling out the taproots with my dandelion weeding tool, I like to leave them on my lawn to break down because the roots are known to draw up nutrients. As it turns out, dandelion root is more than a nutrient source for other plants; it has been used for medicinal purposes. And now Ovadje, Hamm and Pandey have published research showing that dandelion root extract is able to induce apoptosis of leukemia cell lines while leaving normal blood cells untouched.

The scientists were interested in using dandelion root extract to treat of chronic myelomonocytic leukemia (CMML), an aggressive form of leukemia that has few treatment options and poor prognosis. Current chemotherapies have significant side effects, and CMML quickly develops resistance to treatment. In other studies by Ovadje and colleagues, dandelion root extract (DRE) induced apoptosis in cultured cells, but the researchers were interested in CMML due to its lack of efficacious chemotherapies. Three human CMML cell lines (MV-4-11, HL-60 and U-937 cells) were treated with 0.25–5mg/ml DRE for 48 hours. The article text indicated the treatments were given over increasing time, but I could not find supporting information in the figure legend or Material and Methods section for a time period other 48 hours. Apoptosis was assessed using two stains: Hoechst dye, which brightly stains cells and nuclei when apoptotic, and annexin V, which binds phosphatidylserine that is translocated to the cell surface after apoptosis induction. Regardless of cell line, increasing concentrations of DRE induced apoptosis in an increasing number of cells.

To assess DNA fragmentation, another indicator of apoptosis, MV-4-11 cells were treated with DRE or etoposide, a positive control for DNA fragmentation, then assessed using a TdT-mediated dUTP nick-end labeling (TUNEL) assay after 1, 3 and 24 hours. A TUNEL assay transfers a fluorescent dye to the ends of DNA to visualize fragmented DNA under a microscope. DNA was stained with increasing intensity over time in the DRE-treated cells while the positive control had bright staining from the start of treatment. Measuring cell viability in MV-4-11 cells mimicked the TUNEL assay results: The cells became less viable over 24–96 hours. However, 40% of the cells seemed unaffected by DRE treatment. To better understand what might be happening with these resistant cells, MV-4-11 cells were again treated with DRE for 48 hours, the medium was replaced with fresh medium without DRE and the cells allowed to grow for 96 hours. These cells began growing again but at a much slower rate than control, untreated cells. This experiment was repeated with a new twist: The MV-4-11 cells were treated with DRE, the medium was changed with fresh medium with DRE, then cells were incubated another 48 hours. This time there was almost no growth, demonstrating the second dose was able to induce apoptosis in the resistant cells.

DRE seemed to induce cell death in CMML cultured cells but by which mechanism? There are two induction pathways for apoptosis: intrinsic (activated by internal cell events and involving caspase-9) and extrinsic (via Death domain receptors like FADD and involving caspase-8). Results with MV-4-11 and HL-60 cell extracts incubated with DRE and caspase-8 or -3 substrates show that caspase-8 was present 15–60 minutes after treatment and caspase-3 was present 6–48 hours after treatment. When MV-4-11 cells were pretreated with Z-VAD-FMK, a pan-caspase inhibitor, then treated with DRE, the cells did not undergo apoptosis as indicated by Hoechst and annexin V staining and the lack of caspase-8 activity in the cell lysate. These results led the authors to hypothesize that DRE was acting through the extrinsic apoptosis pathway. Pursuing this line of inquiry, they used a Jurkat cell line with a dominant-negative Fas-associated Death Domain (FADD) mutant, treated cells with DRE for 96 hours and saw no visual indications of apoptosis. The FADD mutant Jurkat cells also were treated with DRE for 15–1,440 minutes, lysed and incubated with the caspase-8 substrate. Again, there was no difference between treated and untreated cells (i.e., no caspase-8 present). These data seemed to reinforce the hypothesis of extrinsic apoptotic signaling.

The authors also tested what might be happening within the mitochondria. This organelle is involved in apoptotic changes but DRE might ahve other effects on mitochondria. MV-4-11 and FADD mutant Jurkat cells were treated with DRE for 24 hours, and a dye was used to detect loss of mitochondrial membrane potential, a sign of apoptosis. For the MV-4-11 cells, there was a change in membrane potential; none was seen for FADD mutant Jurkat cells. Mitochondria are known produce reactive oxygen species (ROS), which also play a role in cell death. Mitochondria from HL-60 and MV-4-11 cells were treated with DRE or paraquat (a positive control for inducing ROS activity), and samples were removed every 5 minutes over 4 hours. The fluorescence of the substrate used to detect ROS activity increased over the tested interval, most dramatically in MV-4-11-derived mitochondria where the activity was greater for DRE treatment than the positive control inducer.

Finally, we come to the really interesting part of the research: Comparing what happens with CMML cell lines and freshly isolated normal peripheral blood mononuclear cells (PBMCs). MV-4-11 cells and PBMCs were treated with DRE or tamoxifen, an inducer of autophagy, for 48 hours. Autophagy, a self-degradation process, is an important because it is a survival strategy when cells undergo stress. The DRE treatment induced autophagy in the CMML cultured cells but not normal PBMCs. Tamoxifen treatment did induce autophagy in PBMCs but not MV-4-11 cells. Apoptosis in PBMCs was assessed by incubating with 1–5mg/ml DRE for 96 hours and staining with Hoechst dye and annexin V. Using this method, there was no sign of apoptosis. Even when the PBMCs, which normally do not grow in culture, were treated with concanavalin A, which stimulates T cell proliferation, apoptosis did not occur.

This research offered some intriguing hints about the potential of dandelion root extract in selectively inducing apoptosis in CMML cell lines but not freshly isolated PBMCs. However, this is an artificial environment (using cultured cells rather than CMML cells and in a cell culture dish, not an organism). Furthermore, the research has some caveats: most of the work was done with only one of three CMML cell lines, inconsistency in the concentration of DRE used across experiments even for a single figure and the activity of other caspases (e.g., caspase-9) is unknown. It is a long, difficult road to go from interesting results in cultured cells on a plate to a chemotherapy used for treating human beings. Only time and further research will tell us if dandelion root extract could be a useful treatment for leukemia or any other cancer.

Reference
ResearchBlogging.orgOvadje, P., Hamm, C., & Pandey, S. (2012). Efficient Induction of Extrinsic Cell Death by Dandelion Root Extract in Human Chronic Myelomonocytic Leukemia (CMML) Cells. PLoS ONE, 7 (2) PMID: 22363452

The following two tabs change content below.

Sara Klink

Scientific Communication Specialist at Promega Corporation
Sara is a native Wisconsinite who grew up on a fifth-generation dairy farm and decided she wanted to be a scientist at age 12. She was educated at the University of Wisconsin—Parkside, where she earned a B.S. in Biology and a Master’s degree in Molecular Biology before earning her second Master’s degree in Oncology at the University of Wisconsin—Madison. She has worked for Promega Corporation for more than 10 years, first as a Technical Services Scientist, currently as a Scientific Communication Specialist. Sara is camera shy but may succumb to peer pressure and post an image.

Leave a Reply