The genus Achillea is a member of the family Asteraceae (Compositae) and is distributed in different geographical regions of the world, being mainly present in Asia, Europe and America. There is a wide range of Achillea and 50 species belong to this genus in Turkey. One of them is Achillea millefolium (1, 2). A. millefolium, known as yarrow, is a perennial herbaceous plant that blooms between June and September.
A. millefolium is an aromatic and medicinal plant. Yarrow is applied in traditional medicine, mixed with tea or given in different types of consumption. Applications of yarrow in traditional medicine can be exemplified in kidney diseases, wound treatment, urinary tract infections, cancer and gynecological diseases. Yarrow essential oil is important supplementary material in the treatment of oncogenic diseases and antimicrobial or antifungal infections (3). In the essential oil of the yarrow, there are more than one hundred bioactive compounds such as camazulene, α pinene, β pinene, casticin, 1,8-cineole, cosmosiin and luteolin (1). Different compounds of yarrow’s essential oil were applied on cancer cell lines; Casticin flavonoid obtained from A. millefolium have anti-tumor activity since it arrests cell cycle in G2 / M phase (4). Furthermore, 1.8 cineole and essential oil of Artemisia lavandulifolia were found to have some biological effects on KB oral epidermoid carcinoma cells, and Kim et al. (2010) indicated that this substance induces endonucleolytic DNA cleavage depending on the essential oil dose applied to the cells (5). There are similar studies conducted on different cancer cell lines investigated the anticancer effect of the yarrow essential oil (6, 7, 8). It has been reported that different concentrations of hydroethanolic extract of A. millefolium were affective on cell cycle, apoptosis and inhibit the growth of NCI-H460 and HCT-15 cancer cell lines (6). Additionally, another member of genus Achillea, A. falcata, extract had an antiproliferative effect, and this extract induced cellular apoptosis and formation of apoptotic bodies in HeLa cell line (7). For instance, Lamia et al. have demonstrated that yarrow’s essential oil plays a role as a complementary adjuvant in pancreatic cancer (8).
Although there are some studies using the essential oil of A. millefolium on cancer cell lines, the link between the compound and the molecules which play a role in cancer related networks, is scarce. The aims of this study were i) to investigate the effects of different concentrations of essential oil obtained from the yarrow plant (A. millefolium) on HeLa cells, and ii) to show the biological pathways of the components of the yarrow plant in network analysis.
Material and Methods
Preparation of the Achillea millefolium (ACH) essential oil for the treatment
Yarrow was collected from its habitats near Kayseri Ali mountain (a registered example is CV4851, Erciyes University Herbarium). Collected plants were dried at room temperature. After weighing of the dried plant, about 10 times more water was added (i.e. 1 liter per 100 grams). Distillation method was applied as described in the procedure of Raal et al (2006) to obtain essential oils (9). The essential oils extracted were stored at 4 oC for cell culture analysis. 500 μL of the essential oil of A. millefolium was prepared for GC-MS analysis. In order to apply to cell culture, essential oil was dissolved in DMSO (1:10 v/v).
500 μL of the essential oil was prepared for GC-MS analysis (Shimadzu-QP2010 ULTRA). The analysis were performed at Bozok University Science and Technology Application and Research Center. GC analysis was performed on SLB-5m colum (Spelco, Milan, Italy). Oven temperature was from 50 to 280 oC with an increments of 4 oC per min, as the carrier gas Helium being used in flowrate of 2 mL per min. C5-C24 n-alkane library library was used to identify the spectras. In order to establish a link between cancer cells and the molecules, the function of the GC-MS identified compounds was investigated by Ingenuity Pathway Analysis (IPA, QIAGEN) software.
In this study, we used ATCC HeLa (CCL-2) cell line to investigate the anticancerogenic effect of essential oil. HeLa cells were cultured in RPMI-1640 (Biological Industries) medium containing 10% FBS (Biological Industries), 1% L-glutamine (Biological Industries) and 1% penicillin-streptomycin (Biological Industries). A. millefolium essential oils were dissolved in DMSO following at 1:10 (v/v) dilution. 0.05%, 0.1%, 0.5% (v/v) essential oil in DMSO were added on culture media and 0.5%, 1% and 5% (v/v) DMSO blank groups were used as control. Since the essential oil is poorly soluble, it was dissolved in high percentage of DMSO. Keeping in mind that high concentration of DMSO is also toxic to the cells, therefore keeping the DMSO level below 5% was the main purpose. After 48 hours incubation, biological tests were performed.
Biological effects of Achillea millefolium (ACH) essential oil application
For the apoptosis assay, MUSE Annexin V / Dead Cell kit was used. After the essential oil application, HeLa cells were liberalized by trypsinization and dissolved in 100 μL of DPBS with 2% FBS. 100 μL of Annexin V and Dead Cell Reagent were added on suspension and incubated for 20 minutes. The analyses were performed by using MUSE TM Cell Analyzer (Merck, Germany).
Cell Cycle assay
HeLa cells were dissolved in 100 μL of 2% FBS containing DPBS and test groups were fixed with 70% ethanol in and incubated at -20 oC overnight. In the following step, ethanol was removed by centrifugation and 100 μL of PI (Propodeum iodide)-containing dye was added. After 30 minutes incubation, cells were analyzed by using MUSETM Cell Analyzer.
In order to complete Ki-67 proliferation assay, HeLa cells were dissolved in 100 μL DPBS containing 2% FBS and washed by centrifugation. Cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes. In order to get rid of the fixative solution, cells were washed with assay buffer. Fixed cells were incubated with permeabilization buffer and washed with assay buffer once. Phycoerythrin (PE)-conjugated Ki-67 antibody was added to cells and incubated for 30 mins. PE-conjugated Hu-IgG were used as isotype control during threshold determination. Analyses was completed by using the MUSE TM Cell Analyzer.
In statistical calculations, the changes of the experimental groups according to the control groups were compared by using One-Way Anova Test. Post-Hoc Tukey test was applied to determine the general differences, multiple comparisons were made. The results were determined according to mean ± standard deviation (X ± SD) and showed a statistical significance of p <0.05 SPSS (16.0-2010) package program was used in all calculations.
Component analysis of essential oil by GC-MS
In GC-MS analysis, 10 different major molecules were identified from yarrow essential oil. Retention time and peak area percentage were provided at Peak report total ion chromatography table (Table 1).
GC-MS analysis of essential oil components from A. millefolium. According to Peak report 10 different molecule were identified in essential oil. Four of these molecules are significantly higher than other molecules.
|Peak Report Total Ion Chromatogram|
|Peak #||Molecule||R. Time||Area||Area % ↓|
|5||Bicyclo[2.2.1]heptan-2-ol. 1.7.7-trimethyl-, (1S-endo)-||9.832||16313677||5.35|
|2||Bicyclo[3.1.1]heptan. 6.6-dimethyl-2-methylene-. (1S)-||7.424||9919490||3.25|
|6||3-Cyclohexen-1-ol. 4-methyl-1-(1-methylethyl)-. (R)-||9.880||8528233||2.80|
The three compounds were more dominant than rest of other compounds in essential oil. These dominant yarrows essential oil content was determined as 1.8 cineole (27.4%), Camphor (24.28%) and β-Eudesmol (18.74%). Additionally with the chemically similar structure carrying compound, Camphene (3.9%), medically noticeable, were also been detected. Although we analyzed all identified compounds of essential oil, only four of the all compunds were recognized by IPA databases. Therefore the biological effects of these four molecules (1,8-Cineole, Camphor, β-Eudesmol and Camphene) were analyzed by using Ingenuity Pathway Analysis software for the network analysis. Data obtained from these four biomolecules were evaluated in terms of biological significance and related canonical pathways were determined.
Our results indicated that the major 4 molecules in essential oil have potential to influence various pathway and network which were crucial for cellular metabolism and viability. Most important of these pathways were cAMP, Protein Kinase A (PKA), AMPK, MAPK, ATM and Apoptosis Signaling Pathways. IPA results also showed that the components of essential oil can influence Type II Diabetes signaling, Superoxide radical degradation process and estrogen biosynthesis (Fig. 1).
Following IPA analysis, it was determined that cineole molecule blocks the Cng channel and increases the expression of Superoxide Dismutase 1. The Cng channel is associated with cAMP signaling pathway and Protein Kinase A pathway. (B.) it was found that the camphor molecule increased the expression of some hepatic enzymes associated with the Pyruvate fermentation and increased expression of the CYP2B6 molecule related to estrogen biosynthesis. (C.) Camphene molecule increased the expression of the ADIPOQ molecule associated with PPARα/RXRα Activation and Glutathione Redox Reactions. Adiponectin also is closely associated with AMPK signaling pathway and Type II Diabetes signaling pathway. (D.) β-Eudesmol molecule influences the cAMP, ATM, Apoptosis and GPCR signaling pathways through MAPK, inositol phosphate, Calcium and CREB1.
Biological analysis of cervical cancer (HeLa cell line) cells treated with essential oil
It was observed that A. millefolium essential oil application induced apoptosis in HeLa cells. While the rate of apoptosis was 20% in the DMSO control group, the ratio was increased to 34% in the ACH treatment group (Fig. 2 and Fig. 3).
This ratio of apoptotic cells was found statistically significant (p <0.05) between the application of 0.5% essential oil and DMSO control. It was observed that 0.5% essential oil application was successful in inducing apoptosis in HeLa cells compared to other doses.
When applied, the A. millefolium essential oil decreased the viability of HeLa cells (Fig. 4). The 80% cell viability in the DMSO control group decreased to 60%. This difference was found statistically significant (P<0.05). The differences between the other doses and DMSO controls were not significant.
Cell Cycle analysis
According to the results obtained from the cell cycle test, the high amount of DMSO blocks the cells in the G0/G1 phase. Difference between ACH and DMSO control groups were found to be significant (P<0.05). The current results indicated that 0.5% (v/v) A. millefolium essential oil concentration was more effective when compared with control groups (Fig. 5). According to cell cycle data number of cycling cell was decreased after essential oil application.
Cell proliferation analysis
The results of Ki-67 test showed that the application of essential oil decreased the proliferation of HeLa cells when compared to the control groups (Fig. 6 and Fig. 7). The data was statistically significant (P<0.05). This showed that the application of essential oil to HeLa cells might prevent the proliferation of cancer cells.
Our study indicated that the components present in yarrow essential oil were associated with various molecular mechanisms. The mixture of these compounds induced apoptosis and inhibited cell proliferation by reducing proliferation in HeLa cells. It has been shown that one or more of the components in yarrow essential oil might have anticancer properties (10).
Various plants have been used medical purpose and applied for different treatments in traditional medicine. The components of the essential oils of plants are shown to be effective in complementary and alternative therapy (1). Some of the well-known components in the essential oils of medicinal plants are monoterpenes and derivatives having a property of anticancer agent that are used in chemotherapy. There are examples of the medical uses of yarrow for complementary and alternative therapies that are related to cancer, diabetes, atherosclerosis and metabolism (11).
In this study, GC-MS analysis detected the presence of monoterpene, sesquiterpene and terpenes in the essential oil of A. millefolium. In order to establish a link between the biological effect of A. millefolium essential oil and medical purposes, IPA analysis were performed. The compounds of essential oil (1,8-cineole, camphor, camphene and beta-eudesmol) were in relation with some molecular pathways, which have important role in cellular functions and metabolism. When considering IPA analysis, it was concluded that the 1,8-cineole molecule directly related to the Superoxide dismutase (SOD) level, which means that A. millefolium has an important antioxidant activity, especially it helps to break down the superoxide radical caused by mitochondrial oxidation. A recent study provided that Achillea alexandri-regis plant extract has antioxidant effects on the HeLa cell line (12). As shown in the study of Kundakovic et al. (2005), other members of the genus Achillea could have similar antioxidant activity like A. alexandri-regis. In addition, 1,8-cineole has been in relation with the Protein Kinase A (PKA) signaling pathway and cAMP secondary messenger signaling pathways. Previous studies indicated that the cineole molecule triggers the production of cAMP and inositol 3-phosphate (13), we also determined similar activity with our IPA analysis (Fig. 1A).
In our GC-MS analysis, camphor molecule was found to be the second most abundant ones. According to IPA analysis, it was detected that camphor was related to the expression of CYP2B6 molecule (Fig. 1B). The CYP2B6 molecule is associated with estrogen biosynthesis and is similar to estrogenic activity of that in the MCF-7 cell line. Extract of the above-ground portions of A. millefolium was demonstrated to have estrogenic activity on the MCF-7 cell line (14). The ability to increasing the level of estrogen related molecule CYP2B6, it could be considered a significant finding especially observed effect on cervix cancer cell line HeLa and it is suggesting that it should be investigated by further studies.
Considering IPA analysis, β-Eudesmol was determined to be affecting G-protein mediated receptor pathways, calcium and inositol 3-phosphate secondary messenger pathways and MAPK pathway (Fig. 1D). Some studies in the literature have shown that β-Eudesmol induces apoptosis via c-Jun N-terminal Kinases (JNK), which are associated with the MAPK signalling pathway (15). Additionally, β-Eudesmol molecules can induce apoptosis in hematopoietic cancer cell lines and hepatocellular carcinoma cell lines (16). Since β-Eudesmol is an effective molecule on ATM, ATR kinases and apoptosis signal, essential oil applied cells must be evaluated in terms of cell cycle and apoptotic properties as already done with our study. Furthermore, the relation of β-Eudesmol with the cAMP mediated signalling pathway and the GPCR signalling pathway were enhancing the value to this molecule in terms of various cell signalling mechanism like glucagon metabolism, inflammatory response, epinephrine prostaglandin etc.
Another molecule identified by GC-MS analysis was the camphene. A recent study showed the antidiabetic effect of A. millefolium extracts (17). In our work, camphene molecule effects the increasing in expression of ADIPOQ (Adiponectin), a protein with a regulatory role in glucose metabolism and fatty acid oxidation (Fig. 1C). In this case, camphene might have an important role in diabetes and obesity mediated diseases. If we consider the obesity as the reason of various disease leading to cancer, it can be mentioned that essential oil has also indirect anticancerogenic effects (18).
As a second part of study, we investigated the effects of essential oil on cervix cancer cell line HeLa. When apoptosis test performed on HeLa cells, ACH treatment at different concentrations increased the number of total apoptotic cells. The treatment of 0.5% (v/v) ACH showed an approximately 15% increase in the percentage of apoptotic cells when compared to the control group, which containing the same amount of DMSO. Similarly, our results, the study by Bali et al. showed that the phenolic compounds containing ACH extracts increased the activation of apoptosis genes such as Bax and Cas3 in PC-3 cells (19). The viability of The number of HeLa cells was reduced about 10-15% with the treatment of 0.5% (v/v) ACH essential oil. The aforementioned study done by Bali et al. (19) also tested membrane integrity that shares common grounds with the dead cell assay we conducted. In order to deeply investigate the essential oil effects on apoptosis pathway, it needs to be investigated the expression of certain genes such as Bax, Bad, Bcl2, and Cas3/7 at transcriptomic level in an extensive study.
We also observed that the application of essential oil blocked cell cycle of HeLa cells on G0/G1 phase of cycle. In accordance with cell cycle data, essential oil application reduced the expression of Ki-67, a marker for cell proliferation. Anti-proliferative properties of A. millefolium essential oil is well known since flavonoids and sesquiterpenes of A. millefolium have shown an inhibitory effect on the proliferation of HeLa and MCF-7 cancer cell lines (20). Both the literature and in this study, experimental results showed that essential oil of A. millefolium anti-proliferative effect on HeLa cancer cell line. For instance, investigation of cell cycle checkpoint genes and proteins with molecular approaches such as q-PCR, Western Blot or Mass Spectrometry studies could give crucial information about mechanism of action of essential oil.
Furthermore, individual analysis of identified molecules by GC-MS could lead valuable data that could be used for anticancerogenic drug discovery studies.
As a consequence, essential oil of A. millefolium have anti-proliferative and apoptotic effects on cervix cancer cell line HeLa. According to experimental and literature originated data it seems possible to use these compounds as therapeutic agents directly or cooperatively with chemotherapeutics for several disease including cervix cancer.
Therapeutic effects of the yarrow essential oils were investigated on cervical cancer cell line, which is important for women’s health (21). We reported that essential oil components may interact with molecular pathways that are closely related to cancers. The yarrow’s essential oil has potential to induce apoptosis in HeLa cells and inhibit cell proliferation. It was found that at least one of the components of the essential oil have anticancer properties. Concurrently, components of the yarrow’s essential oils should be subject to more detailed research. Examination of 1,8-Cineole, Camphor, Beta-eudesmol and Camphene molecules may provide valuable data to lead anti-cancer drug discoveries. Explaining the relationship of these components to molecules, which involved in disease-related mechanisms, may help complementary and alternative therapies.
We are grateful to Bozok University Science and Technology Application and Research Center for GC-MS analysis.
This study was partially supported by Erciyes University Scientific Research Project Department (ERU BAP- FBY-12-3935).
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