Flax contains large amounts of hormone-like compounds, especially lignans. These socalled phytoestrogens are thought to inhibit the cell growth of hormone-sensitive cancers. Hence, we analysed the influence of flax root extracts at various stages of maturity on the proliferation and cytotoxicity in oestrogen-receptor-positive breast cancer cells (MCF7) in vitro. Flax root extracts were prepared by using lignan extraction. The extracted compounds were analysed by Pyrolysis-Field Ionisation Mass Spectrometry. Various extract concentrations were applied to the cells to test for proliferation (BrdU test) and cytotoxicity (LDH test). A significantly higher inhibition of cell proliferation was observed with an extract made from 9-week-old flax roots in comparison with that of 3- and 6-week-old roots. Older roots contained more lignans and other phenolic substances than younger roots. The maturity grade of plants or their various parts is thus important for the production and concentration of secondary metabolites and leads to different biological effects on breast cancer cell growth.
Cases of mammary gland carcinoma have been increasing continuously for years. In addition to traditional approaches in oncological pharmacology and surgery, great interest has been shown in the development of new avenues in the prevention and therapy of breast cancer.
Cases of hormone-dependent carcinoma are significantly lower in regions with a traditional diet high in phytoestrogens than in regions with minor intake. The outstanding constituents in the group of phytoestrogens are isoflavones and lignans, which are structurally similar to endogen oestrogens and have the ability to bind to the human oestrogen-receptors alpha and beta [
Prior to this study, we have been able to demonstrate a difference in cancer cells with regard to the effects of extracts from the leaves, stems and roots of Linum usitatissimum. Our results have indicated that root extract is more effective than leaf and stem extracts [
In this study, we wished to determine any influences on the proliferation and cytotoxicity of breast cancer cells depending on the stage of maturity of the flax roots and the differences in the chemical composition of their extracts.
The plants were sown under field conditions and harvested at intervals of three weeks. Based on these condi- tions, three different stages of maturity could be used for the study: roots at 9 weeks (9 w), 6 weeks (6 w) or 3 weeks (3 w). The extracts were prepared according to Luyengi et al. [
By using Py-FIMS, we were able to analyse the components of the root extracts [
The human breast cancer cell line MCF7 was obtained from the Department of Human and Animal Cell Culture, Braunschweig, Germany. Cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Al- drich-Chemie, Germany). This medium was supplemented with 4.5% glucose; L-glutamine; 2.6 g/l HEPES pH 7.4; 3.0 g/l NaHCO3; 10% foetal calf serum; 1% penicillin; 1% streptomycin and 0.5% amphotericin B. The MCF7 cell line was cultured in an incubator under physiological conditions (37˚C; 5% CO2, humidified atmos- phere).
For proliferation and cytotoxicity tests, a cell concentration of 5 × 105 cells/ml was used. The breast cancer cells (100 µl/well) were plated onto 96-well micro-plates. After 24 hours, the flax root extracts at different concentrations (0.01 µg/ml - 1000 µg/ml) and all controls were added. 17β-oestradiol and tamoxifen (anti-oestrogen) were used as positive controls (final concentrations: 50 µg/ml; 1 µg/ml; 0.1 µg/ml). Both controls were commercially acquired from Sigma (Germany). One of the negative controls was the solvent ethanol (Roth; Germany), the other one being the untreated MCF7 cell suspension.
For measuring cytotoxicity, the Cytotoxicity Detection Kit LDH (Roche Diagnostics, Germany) and, for cell proliferation, the BrdU assay (colorimetric, Roche Diagnostics) were used according the manufacturer’s protocol.
Cell culture tests were performed in quadruplicate and repeated at least four times. Statistical analysis was exe- cuted by using the Student’s t-test for a comparison of the means. Data are presented as means ± standard devia- tion. All values with P ≤ 0.01 differ significantly from the negative control (ethanol) and are denoted with an as- terisk (*). Significance between different stages of maturity (P ≤ 0.05) is symbolised with a hatch sign (#).
Py-FIMS allowed the characterisation of substance classes in the extracts being tested. The analysis showed a high fraction of free saturated and unsaturated fatty acids. Sterols and triterpenes were detected in all three stag- es, but their total ion intensity (TII) was higher in 3 w than in 6 w and 9 w extracts (
The lignan fraction (0.3%) in 3 w extracts was striking. Phenols and lignin/lignan monomers were verified, as were lignin dimers. In the 6-week-old flax root, we detected lignans (2.2%), lignin dimers, phenols and lignin/
lignan monomers. The 9 w root showed the highest TII of lignans (3%) and lignin dimers but the proportion of fatty acids was the lowest compared with the other two stages. Flavonoids were also found in all three flax roots, increasing with the maturity of the flax (0.4%, 0.8% and 1.3%). The percentage of groups of phenolic compounds, potentially containing phytoestrogens, was much higher in the extract of the 9 w (23%) root than in 6 w (16.3%) and 3 w (6.8%) roots.
A significant cytotoxic effect was detected for all three stages of maturity at the highest extract concentration (1000 µg/ml). The extracts of 3 w and 9 w roots caused a cytotoxicity of more than 50%. For the concentrations of 500 µg/ml, a cytotoxicity of about 35% was determined. At other concentrations, no cytotoxic effect was measured (
In addition to cytotoxicity, the influence of extracts on cell proliferation was of interest (
In previous investigations, a number of phytoestrogens have been found in the flax root. We have identified, for example, the lignans secoisolariciresinol, lariciresinol, pinoresinol and matairesinol and the isoflavones geniste- in, daidzein and biochanin A [
The present results of Py-FIMS show differences in the composition between the various states of maturity of flax roots. Although all three extracts were prepared by the same method of lignan extraction, the extract of the 3-week-old flax root contained a low lignan fraction compared with the older roots. We suggest that the lignans develop during the growing period and are influenced by abiotic environmental factors. Booth et al. have de- scribed a seasonal variation of red clover isoflavones and oestrogenic activity [
The high contingent of lignin dimers is notable; they are the third highest fraction at 9 w. In some plants, the contents of cellulose and lignin increase within the growing season [
Concentration-dependent biphasic effects have been described for genistein [
Flax root extracts show a cytotoxic effect on oestrogen-receptor-positive breast cancer cells at high concentra- tion levels and from all three stages of maturity. This, however, is reduced at lower concentrations and might in- dicate a dose-dependent interaction between the flax root extract and MCF7 breast cancer cells. The correlation between the dose of lignans and the effect dependent on the status of oestrogen-receptor has been described in many in vitro studies [
The maturity grade of plants or their various parts is thus important for the development, quality and quantity of secondary metabolites and results in different biological effects on breast cancer cell growth. These results are promising and make further research into new methods for the prevention and adjuvant therapy of breast cancer with products from endemic plants worthwhile. In order to determine the extent of the potential of flax roots in cancer therapy, however, more in vitro and in vivo studies are necessary. Further analyses will also be needed to classify the substance classes into their chemical compounds.
The authors thank Mrs. E. Greschkowitz and Mrs. C. Bauer for technical assistance.
This work was supported by The Deutsche Krebshilfe, Project No.: 107820.