Dandelion Root Extract Affects the Proliferation, Survival and Migration of Cervical Cancer Cell Lines


 Background

Taraxacum officinale has been shown to exert diverse biological activities, in someones cancer cell lines. Also in traditional medicine have been its used fresh latex to treat warts (non-malignant HPV infection); however, its effect over cervical cancer cell lines is yet to be explored. The aim was to evaluate the effects of a root extract of Taraxacum officinale on the proliferation, survival and migration of cervical cancer cell lines.
Methods

Ethanolic extract was obtained from T. officinale roots (R-EtOH). Caski (HPV16), HeLa (HPV18), C33A (HPV-) cervical cancer cell lines (CC) and keratinocytes HaCaT (HPV-) were used. Viability and inhibitory concentration values were determined by the MTT assay. Clonogenic assay was used to evaluate the survival. Effect on cell migration was evaluated by wound healing assay. Apoptotic response was observed using the TUNEL and Annexin V/7AAD assay.
Results

R-EtOH extract showed a dose-dependent cytotoxic effect on all cell lines. The number and size of the colonies, mainly in CC, decrease with higher concentrations of extract. R-EtOH effectively inhibits cell migration, and causes a higher percentage of apoptotic cells.
Conclusion

This study reports, for the first time, the antitumor potential of dandelion root extract (R-EtOH) on CC. Our results showed that R-EtOH affect proliferation, survival and cell migration. In addition, R-EtOH leads to apoptosis in all CC cells; thus, it is a promising extract for future studies which will allow to evaluate its usefulness in cervical cancer treatment as in other cancer types.


Conclusion
This study reports, for the rst time, the antitumor potential of dandelion root extract (R-EtOH) on CC. Our results showed that R-EtOH affect proliferation, survival and cell migration. In addition, R-EtOH leads to apoptosis in all CC cells; thus, it is a promising extract for future studies which will allow to evaluate its usefulness in cervical cancer treatment as in other cancer types.

Background
Cervical cancer, one of the most common gynecological tumors, is the third leading cause of cancerassociated deaths among women worldwide with an estimated 530,000 new cases annually and 270,000 deaths. Approximately 85% of worldwide deaths from cervical cancer occur in underdeveloped or developing countries, and the death rate is 18 times higher in low-income and middle-income countries when compared with wealthier countries. [1] In Argentina, 5000 women per year are still diagnosed with cervical cancer and 2,000 die from this tumor [2].
Human papillomavirus (HPV) infection is predominantly responsible, nearly 100%, for the incidence of cervical cancer [3]. According to statistical analyzes, among the 15 high-risk (HR) HPV genotypes, HPV16 and HPV18 are responsible for 75% of cervical cancer cases [4]. While low-risk (LR) HPV genotypes, such as HPV-6 and HPV-11, are associated with non-malignant manifestations of infection, for example, lowgrade squamous intraepithelial lesions, anogenital warts, condyloma acuminata and others [5] There are different treatment methods with their advantages and disadvantages. Therefore, choosing a treatment option is never easy and the choice will be in uenced by multiple factors [6][7][8]. One of the options is chemotherapy; however, the main problems concerning chemotherapeutic agents are severe adverse effects and multiresistance formations [9].
In that sense, natural therapeutic agents have become important for the development of new treatment strategies that could be deployed in cancer therapy. Phytochemical compounds obtained from extracts of plant roots, bulbs, barks, leaves, stems and other plant parts have shown promising potential as anticancer drugs, and as lead compounds in the synthesis of new drugs [10].
In traditional medicine, fresh "dandelion" latex (Taraxacum o cinale G. Weber ex F.H. Wigg) is sometimes used to treat warts (manifestations of HPV infection), although without proven e cacy [11,12]. T. o cinale is a widespread owering herbaceous perennial plant which belongs to the Asteraceae family with wide global distribution. Prominent constituents in dandelion include sesquiterpene lactones, triterpenes, several phenolic acids, avonoids and coumarin [13,14]. It is regarded as a nontoxic herb that can be potentially exploited for its choleretic, diuretic, antirheumatic, and anti-in ammatory properties [15]. Recent studies showed a strong anti-cancer activity of a dandelion root extract. Some authors found that this extract is able to induce a rapid activation of the death-receptor mediated extrinsic pathway of apoptosis in human leukemia and pancreatic cancer cells. In addition, this extract would be cancer cell selective, as the same treatment is not detrimental to non-cancer cells [16][17][18][19]. Moreover, new studies have reported antitumor activity of T. o cinale in cell lines of pediatric cancer, colon cancer and gastric cancer [20][21][22].
It is known that cervical cancer is initially asymptomatic, later transforming into high-grade squamous intraepithelial lesions and invasive cancer. Thus, the ndings of the anti-tumor role of T. o cinale mentioned above, allow as to thinking that bioactive components of this species could have activity in the treatment of lesions of different severity, caused by the HPV.
The aim of this study was to evaluate the effects of a T. o cinale root extract on the proliferation, survival and migration of cervical cancer cell lines infected with human papillomavirus.

Collection of T. o cinale
The vegetal material was collected in Córdoba city, Argentina (between 31º22'47.75 "S, 64º8'46.34" W and 31º22'43.84 "S, 64º 8'24.49" W), in accordance with the national guidelines of Argentina. The species was identi ed by PhD Gloria Barboza, in the Museum of Botany of Facultad de Ciencias Exactas Físicas y Naturales-Universidad Nacional de Córdoba, where a sample of herbarium was deposited (CORD00027086).
Obtaining root extract of T. o cinale (R-EtOH) Individuals of dandelion were transported to the laboratory, washed with water in order to remove all traces of dust, and then dried in an oven stove at 28ºC, limiting its exposure to the light. Once the material was dried, roots were separated from aerial parts and subsequently ground. An extraction with ethanol was carried out by means of a Soxhlet apparatus. Extractive liquids were separated from the plant material which was concentrated to dryness by using a rotary evaporator under reduced pressure and at moderate temperatures. A stock solution in dimethylsulfoxide [100 mg/mL] was prepared from which subsequent tests were performed. dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay. This assay allows to assess the cytotoxic potential of R-EtOH extract, since only mitochondrial dehydrogenases formed in viable cells are able to reduce tetrazolium salt into an insoluble formazan product [23]. Brie y, 1x10 5 cells/mL were seeded in 96-well plates at 37˚C with 5% CO 2 for overnight incubation and treated with different concentrations of R-EtOH (10-1000 µg/mL) for 48 hours in their respective media, supplemented with 2% FBS. The cells were then incubated in a serum-free medium that contained MTT at a nal concentration of 0.5 mg/mL for 1 hour [24]. The crystals formed in intact cells were solubilized in 100 µL of MTT solution (10% of triton X-100, 10% of 0.1N HCl, 80% isopropanol), and the absorbance was measured at 570 nm by using a microplate reader (BioTek ELx800, USA). All samples were assayed in triplicate, and values were normalized to untreated controls. Analysis of results was done with software Origin Pro 8.6.

Clonogenic Survival Assay
Clonogenic assay allows us to estimate cell survival, based on the cell's regenerative capacity after having been exposed to an extract. Cells (200 cells/well) were placed in 12-well plates and incubated for 48 hours in RPMI 1640 or DMEM with 10% FBS at 37°C and 5% CO 2 in a humidi ed incubator. Then, the aforementioned medium was discarded and the cells were treated with [IC 20 ] and [IC 50 ] R-EtOH in culture medium with 2% FBS for 2 hours. Culture medium with 2% FBS was used for the untreated control. Subsequently, the medium with R-EtOH was also discarded, washed with phosphate buffered saline (PBS), and then medium supplemented with 10% FBS in 3% methylcellulose was added. After 7 days, it was xed with methanol and stained with 0.1% violet crystal. The number of visible colonies was counted using an optical microscope with a 40x magni cation. It was interpreted as a colony whose clone comprises 50 cells or more. The results were expressed as surviving fraction (SF) [25].

Migration assay
Cell migration capacity was calculated by wound healing assay. Cells were seeded at a density of 2 x10 5 cells/mL, placed into 12-well plates and incubated for 48 hours reaching 100% con uence, then they were treated with [IC 20 ] R-EtOH in culture medium with 2% FBS for 48 hours. Culture medium with 2% FBS was used for the untreated control. The next day, arti cial wounds in each one of the cell lines were made with 10µL tips, they were washed three times with PBS and incubated in a serum-free medium. Wounds were observed at 0, 6, 12, 18 and 24 hrs, with the OLYMPUS IX81 microscope to 20X. Average extension of the wound closure was evaluated by measuring the area of the wound by the ImageJ/Fiji software, version 1.46 (http://imagej.nih.gov/ij/) [26].

Identi cation of cell apoptosis by means of Hoechst staining and TUNEL assay
Cells were seeded at a density of approximate 1 x10 5 cells/mL, in 96 well plates. The next day, they were treated with [IC 20 ] and [IC 50 ] R-EtOH in culture medium with 2% FBS, in triplicate and for 48 hours. Culture medium with 2% FBS was used for the untreated control. For terminal deoxynucleotidyl transferase dUTP nick-end labeling, TUNEL assay was performed using the In Situ Cell Death Detection Kit, TMR red TUNEL (Roche Diagnostics, Sigma-Aldrich). Cells were xed in 4% paraformaldehyde and then they were washed with PBS. Subsequently, cells were permeabilized with 0.1% Triton X-100, in 0.1% sodium citrate for 2 min on ice and then they were washed with PBS. The cells were incubated with 50 µL TUNEL reaction mixture at 37°C for 60 min in a dark humidi ed atmosphere. Images were captured with an OLYMPUS IX81 uorescence microscope, and subsequently they were evaluated with ImageJ/Fiji software, version 1.46 (http://imagej.nih.gov/ij/). Hoechst 33258 staining (Sigma-Aldrich) was used to visualize the nucleus.
Cells were seeded at a density of 2 x10 5 cells/mL placed into 12-well plates and incubated reaching 100% con uence. In uence of R-EtOH on cell growth using a clonogenic assay. Since the clonogenic assay is useful for testing the proliferative capability of cells by virtue of the cell's ability to undergo su cient proliferation to form a colony, it is especially suitable for assessing long-term effects of cell treatments (e.g., a few weeks after treatment). In particular, as the cell metabolic state and clonogenic potential are not necessarily parallel events, the clonogenic assay can add valuable and distinct information to that provided by the MTT assay and may be seen as complementary [27]. We observed that the reduction in the ability to form colonies in cervical cancer cells was signi cantly greater than in HaCaT cells (Fig. 1).

The inhibitory effect of R-EtOH on cells migration capacity
Besides cell proliferation, migration is an important characteristic of tumor cells [20]. Thus, we performed wound healing assays to assess the impact of R-EtOH on cancer cell migration. It was observed that treatment with R-EtOH inhibited the ability to migrate into the wound in all cell lines, this affects mainly cervical cancer cells, unlike the untreated control group (Fig. 2 a. and b). At the end of the assay HeLa cells had a lower migration capacity than CaSki cells, this could be because the latter are cells of CC metastasis, so they would have greater migration capacity than HeLa.

R-EtOH induces apoptosis in cervical cancer cells
In order to determine whether the cell death induced by R-EtOH was apoptotic, we used the speci c TUNEL assay (Fig 3a). Signi cant differences were observed in the percentage of apoptotic cells between CC and HaCaT for each concentration. HaCaT cells showed a slight apoptotic effect after treatment with R-EtOH, but this was not statistically signi cant (Fig. 3b). It was observed that with higher doses of R-EtOH the percentage of apoptotic cells in cervical cancer cells signi cantly increased (Fig 3b). In [IC 20 ] there were signi cant differences between CaSki-HeLa and CaSki-C33A, but there were not differences between HeLa and C33A. However, for [IC 50 ] there were signi cant differences between all CC lines, mainly affecting CaSki then C33A and nally HeLa (Fig. 3b). This result suggests that R-EtOH contains bioactive components which were effective to induce apoptosis in cervical cancer cells.
Kinetic of apoptotic cell death via Annexin VFITC/7AAD assay Stages of apoptosis were quanti ed using Annexin V as a marker for apoptosis and 7-amino-actinomycinD (7AAD) as a marker of necrosis. In this assay, cells in the early stages of apoptosis stain As shown in Fig. 4a, the majority of untreated cells were viable (lower left quadrant, Q4). There were signi cant differences between the percentages of apoptosis of all the CC lines with respect to HaCaT. After 24 hours of R-EtOH [IC 20 ] treatment, we observed an increase in the percentage of apoptotic cells, mainly in early apoptosis (Q1). This increase was signi cantly different for all CC cells when compared to untreated cells (Fig 4b). After 48 hours of treatment, there was a drastic reduction in viability in CC cells (Q4) and a signi cant increase in the percentage of apoptosis for CaSki and C33A cells when compared to the 24 hour treatment. Although after 24 hours there were not signi cant differences in the percentages of apoptosis between CC cells, after 48 hours signi cant differences between all CC lines were observed, being the highest percentages in CaSki, then C33A and nally HeLa cells (Fig 4b). Although after 48 hours an increase in the percentage of necrotic cells is observed, the fact that cells progress through early apoptosis in a time-dependent manner before staining positive for 7AAD suggests that positive 7AAD staining is indicative of cells dying as a result of apoptosis and not necrosis.

Discussion
Cervical cancer is responsible for 10-15% of cancer-related deaths in women worldwide, where HPV is the causative agent in over 99% of the cases. Although vaccines for HPVs and improvements in early screening have successfully reduced the mortality rate, cervical cancer is still considered a major public health problem in developing countries [28]. Although there is a diversity of treatments for lesions caused by HPV, recent studies report that about one out of six treated women (16%) manifested at least one persistent High Risk HPV type that was associated with recurrent or residual High Squamous Intraepithelial Lesion disease [29]. Therefore, the search for new treatments is of great importance. In recent years, antitumor properties of dandelion extracts have been reported; however, there is little evidence of its effects on cervical cancer.
In MTT assay, we revealed a decrease in dose-dependent cell viability and greater cytotoxicity in CaSki and C33A cells with respect to HaCaT. However, we did not nd signi cant differences between HeLa and HaCaT cells in this assay. R-EtOH showed a great cytotoxic effect, since [IC 50 ] values found were lower than reported in other studies [20][21][22]. This may be due to the nature of different cancer cell lines or to the fact that R-EtOH might contain a higher concentration of bioactive compounds, since the other reports refer to aqueous extracts.
Our results con rmed that exposure to higher concentrations of R-EtOH signi cantly inhibits the proliferation and colony formation of cervical cancer cells. It is known that any substance that inhibits the clonogenic progression of tumor cells could be considered a potential anticancer therapeutic agent [30]. Analysis of wound healing showed the ability of R-EtOH to inhibit cell migration, showing greater effect in cancer cells. This effect has also been reported by other authors in different cancer cell lines treated with dandelion extracts [20,22,31]. HaCaT cells treated with R-EtOH were able to migrate more freely to the wound area than cancer cell lines, showing selectivity of R-EtOH to cancer cells. This inhibition of migration, caused by R-ETOH, could reduce the ability of cancer cells to metastasize to secondary sites.
The loss of balance between cell proliferation and apoptosis is a hallmark that increases the failure of tumoral cells to be eliminated through apoptosis. One essential strategy for cancer therapy is to activate apoptotic pathways in the tumor cells [32]. The Annexin-V/7AAD and TUNEL assays con rmed that the decrease in cell viability is a result of apoptotic processes induced by R-EtOH treatment.

Conclusion
This study reports, for the rst time, the antitumor potential of dandelion root extract (R-EtOH) against cervical cancer cell lines. Our results showed that dandelion root extract (R-EtOH) contains bioactive components that affect proliferation, survival and cell migration. In addition to this, it leads cervical cancer cells to the programmed cell death. Therefore, this is a promising extract for future studies which will allow to evaluate the usefulness of its compounds in the treatment of cervical cancer as well as in other cancer types.

Declarations
Financial support   and HaCaT cells. Asterisks on gray lines correspond to differences between IC20 and IC50 concentrations in each cell line. Asterisks on black lines correspond to different concentrations of IC20 between CC cells.