Dandelion root
Oncotarget. 2016 Nov 8; 7(45): 73080–73100.
Published online 2016 Aug 22. doi: 10.18632/oncotarget.11485
PMCID: PMC5341965
PMID: 27564258
Dandelion root extract affects colorectal cancer proliferation and survival through the activation of multiple death signaling pathways
Pamela Ovadje,1 Saleem Ammar,2 Jose-Antonio Guerrero,2,3 John Thor Arnason,2 and Siyaram Pandey1
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Abstract
Dandelion extracts have been studied extensively in recent years for its anti-depressant and anti-inflammatory activity. Recent work from our lab, with in-vitro systems, shows the anti-cancer potential of an aqueous dandelion root extract (DRE) in several cancer cell models, with no toxicity to non-cancer cells. In this study, we examined the cancer cell-killing effectiveness of an aqueous DRE in colon cancer cell models. Aqueous DRE induced programmed cell death (PCD) selectively in > 95% of colon cancer cells, irrespective of their p53 status, by 48 hours of treatment. The anti-cancer efficacy of this extract was confirmed in in-vivo studies, as the oral administration of DRE retarded the growth of human colon xenograft models by more than 90%. We found the activation of multiple death pathways in cancer cells by DRE treatment, as revealed by gene expression analyses showing the expression of genes implicated in programmed cell death. Phytochemical analyses of the extract showed complex multi-component composition of the DRE, including some known bioactive phytochemicals such as α-amyrin, β-amyrin, lupeol and taraxasterol. This suggested that this natural extract could engage and effectively target multiple vulnerabilities of cancer cells. Therefore, DRE could be a non-toxic and effective anti-cancer alternative, instrumental for reducing the occurrence of cancer cells drug-resistance.
Keywords: cancer, oxidative stress, phytochemical composition, gene expression, natural health product
INTRODUCTION
The increases in the world's aging population, as well as the adoption of cancer-causing behaviors, are the major contributors to a global escalation of different forms of cancers. There are over 12 million new cancer cases arising annually and over 7 million cancer-related deaths worldwide, and even with the introduction of many chemotherapy and chemopreventive approaches, cancer is still one of the leading causes of deaths in the world today, with a statistic of one in four deaths being attributed to cancer alone [1, 2].
Despite the progress made in the development and introduction of many chemotherapy agents, the accompanying toxicities and side effects [1], indicate that further research is required to reduce the incidence of cancer rates, and the amount of cancer-related deaths, as well as to improve the quality of life of patients already diagnosed with the disease.
Natural health products (NHPs) and natural products (NPs) have been essential in the development of many drugs, with over 75% of the currently available chemotherapies having been derived from natural sources (plants, microbes and marine sources), with a common example being paclitaxel [3]. These NHPs have been used in various traditional medicines and recent studies on the use of the NHPs for specific diseases yield some scientific validation for their application [4, 5]. Even with all the incoming evidence, herbal drugs and other NHPs and NPs are usually shunned during systemic chemotherapy because of a possible herb-drug interaction that might enhance chemotherapy-related toxicity [6, 7].
Dandelions (Taraxacum spp) have been used for centuries for the treatment of various ailments; surprisingly enough, they have received little research attention. Some scientific studies report anti-inflammatory, anti-oxidative and diuretic activities of various parts of this plant [8]. Recent studies from our lab show a strong anti-cancer activity of an aqueous dandelion root extract (DRE) [9, 10, 11, 12]. We find that DRE is able to induce a rapid activation of the death-receptor mediated extrinsic pathway of apoptosis in human leukemia and pancreatic cancer cells in a dose and time dependent manner. Furthermore, the induction of apoptosis is dependent on caspase-8 activation. Our data shows that this action of DRE is cancer cell selective, as the same treatment is not detrimental to non-cancer cells. However, the detailed analyses of the efficacy and toxicity of this extract in in-vivo and ex-vivo models, as well as, its mechanism(s) of action still remain unexplored. Furthermore, the pharmacologically active anti-cancer components of this extract are at present unknown.
We report the anti-cancer activity of the DRE obtained with in-vitro (colon cancer cell lines) and in-vivo (mouse xenograft model of colon cancer) models. We hypothesized that due to its compositional complexity (mixture of bioactives), DRE might be able to activate different signaling events and more efficiently induce program cell death (PCD) processes by targeting different metabolic vulnerabilities of cancer cells. Accordingly, we have shown that, although DRE treatment triggered cell death in all cell models examined and led to the activation and localization of active caspase-8 to the mitochondria and the peri-nuclear space, this caspase-8 activation was not essential for the induction of cell death in colon cancer cells as an inhibition of caspase-8 activation did not alter the cytotoxicity of DRE. Therefore, in colorectal cancer cells the DRE treatment must have utilized caspase-8 independent cell death pathway. We have been able to identify four pharmacologically active components, α-amyrin, β-amyrin, lupeol and taraxasterol, in two out of the six bioactive fractions, but the anti-cancer activities of the individual compounds were not as strong as that of the unfractionated DRE indicating, clearly, the benefits of using the whole extract. Taken together our results scientifically validate the use of NHPs, especially dandelion root extracts, as potential anti-cancer agents, which might represent a novel non-toxic alternative to conventional cancer therapy available today.
RESULTS
Dandelion root extract (DRE) induces apoptosis in aggressive colorectal cancer cells
This apoptosis-inducing activity of DRE, as previously reported [9, 11] prompted further studies into its efficacy in highly aggressive colorectal cancer cells, HT-29 (p53−/−) and HCT116 (p53 WT). For comparison, normal colon mucosal epithelial cells (NCM460) were also used to assess the selectivity of DRE to colorectal cancer cells. Furthermore, we compared the efficacy of DRE to the currently utilized colon cancer chemotherapy, FOLFOX (5-fluorouracil, Folinic Acid and Oxaliplatin). The results are summarized in Figure ​Figure1.1. We observed a significant decrease in the viability of both HT-29 and HCT116 colorectal cancer cells following the DRE treatment. This effect was both time and dose dependent and it was similar in both cell lines, irrespective of their p53 status. Employing the WST-1 cell viability assay, we determined the EC50 of DRE in both colon cancer cell lines; 2.0 mg/ml in HCT116 cells and 3.5 mg/ml in HT-29 cells. The selectivity of DRE to cancer cells was once again confirmed, as normal NCM460 cells were DRE refractive and did not lose metabolic activity and cell viability when exposed to the same doses and time points as the colon cancer cells. Furthermore, the efficacy and selectivity of DRE to colorectal cancer cells was compared to that of FOLFOX. It was observed that the FOLFOX combination did not have a selective effect to colorectal cancer cells, as the normal colon mucosal epithelial cells were also affected at the same doses (Figure ​(Figure1A1A).
This reduction in metabolic viability corresponded to an increase in apoptosis induction, as DRE treatment triggered apoptosis selectively in colon cancer cells, but not in normal mucosal cells, which was subsequently confirmed by fluorescence microscopy following Hoechst 33342, propidium iodide and Annexin V staining, to observe the nuclear morphology, cell membrane integrity and externalization of phosphatidylserine respectively. In the DRE-treated colon cancer cells we observed significant increases in propidium iodide and Annexin V positive staining, indicative of apoptosis, while NCM460 cells, again, remained unaffected (Figure ​(Figure1B).1B). Image-based cytometry was used to quantify the apoptotic response and the data showed an approximately 40% increase in Annexin V positive cells and a corresponding 97% increase in propidium iodide staining in DRE responsive cells (Figure ​(Figure1C).1C). These results confirmed the anti-cancer potential of DRE and demonstrated its efficacy in aggressive colorectal cancer cells regardless of their p53 status.
Dandelion root extract selectively impairs the migration of colon cancer cells
To determine if DRE can prevent invasive and metastatic behaviours in colorectal cancer cells, the scratch wound healing assay was employed. HT-29, HCT116 and NCM460 cells were pre-treated with thymidine for 18 hours to halt further proliferation, following which, the cells were treated with DRE at the indicated concentrations. Cells were monitored at 0 hours (at the time of treatment) and at 3, 6, 24 and 48 hours following treatment (Figure ​(Figure2).2). It was observed that treatment with DRE inhibited the ability of colorectal cancer cells, HT-29 and HCT116, to migrate into the wound, unlike the control untreated samples that freely migrated into the wound area. As anticipated, the normal NCM460 cells treated with DRE were able to migrate into the scratch wound area (Figure ​(Figure2),2), confirming the selectivity of DRE to cancer cells. These results clearly indicate that dandelion root extract can inhibit the ability of colorectal cancer cells to migrate and invade, and therefore metastasize to secondary locations.
CONCLUSIONS
Our results showed that aqueous dandelion root extract (DRE) efficiently and selectively triggers programmed cell death pathways in in-vitro and in-vivo colorectal cancer models. The results confirmed our hypothesis that the molecular complexity of the DRE extract is responsible for its anti-cancer activity, as it allows the engaging of multiple signaling pathways in cancer cells, including the mitochondria. Therefore, we can conclude that DRE, as a complex mixture might provide a complementary alternative to currently available chemotherapies. With these results, DRE is approved by Health Canada for Phase I clinical trials in hematological cancers. Their use might prove not only efficacious, but also associated with fewer and less severe side-effects and, thus, improve the quality of life and possibly increase the lifespan of cancer patients.