Abstract
Background
Flavonoids are compounds of interest in the search for new anti-cancer therapies. We have previously isolated the methoxyflavones 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone (8-methoxycirsilineol), 5,4′-dihydroxy-6,7,8-trimethoxyflavone (xanthomicrol), and 5,4,'3′-trihydroxy-6,7,8-trimethoxyflavone (sideritoflavone) from Baccharis densiflora. Herein, we investigate the toxicity of these methoxyflavones in human breast-derived cell line. Our main aim was to focus on the cancer stem cell (CSC) sub-population of JIMT-1 breast cancer cells.
Methods (urval)
Initially, dose response experiments yielding inhibitory concentration 50 (IC50) values were performed using MCF-7, HCC1937, and JIMT-1 breast cancer, and the MCF-10A normal-like breast cell lines to get an understanding of toxic ranges. Due to a clear difference in the toxicity of the flavones, only sideritoflavone was selected for further studies using the JIMT-1 cell line. Effects on the CSC sub-population was investigated using flow cytometry-based methods. A wound healing assay and digital holographic microscopy were used to investigate effects on cell movement. A reporter assay was used to study effects on signal transduction pathways and Western blot for protein expression.
Plant material
Aerial parts of B. densiflora Wedd, previously identified as Baccharis pentlandii DC, were collected on February of 2017 in Cota Cota (3422 m.a.s.l.; 16° 32,270′ S 68° 4016′ W), located in the outskirts of La Paz, Bolivia. The plant was identified by Esther Valenzuela at the JBLP (Jardín Botánico del Herbario Nacional de Bolivia) where the voucher specimen is kept (code Beck St G. 2858).
Extraction and isolation of compounds
Compounds and stock solutions
The compounds were dissolved in 100% dimethyl sulfoxide (DMSO) at a concentration of 100 mM and kept at 4 °C. Controls were treated with the same final DMSO concentration as the DMSO concentration in treated cultures maximally at 0.1% DMSO depending on the assay and chosen concentration.
Cell lines and culture conditions
The JIMT-1 human breast carcinoma cell line (ACC589) was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The normal-like breast epithelial MCF-10A cell line (CRL-10317), the cancer cell lines MCF-7 (HTB-22) and HCC1937 (CRL-2336) were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were tested negative for mycoplasma (Eurofins, Konstanz, Germany).
The JIMT-1 cells were routinely cultured at 37 °C in a humidified incubator with 5% CO2 in air. The cells were cultured in DMEM/Ham’s F-12 medium supplemented with 10% fetal bovine serum (FBS), glutamine (2 mM), non-essential amino acids (1 mM), insulin (10 μg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml).
MCF-10A, MCF-7, and HCC1937 cell lines were cultured in RPMI 1640 medium (VWR) supplemented with 10% heat-inactivated FBS (VWR, Lund, Sweden), glutamine (2 mM), 1 mM non-essential amino acids (VWR), 10 μg/ml insulin (Sigma-Aldrich, Stockholm, Sweden), and 100 U/ml penicillin/100 μg/ml streptomycin (VWR). The MCF-10A cells were also supplemented with 20 ng/ml epidermal growth factor (Sigma-Aldrich), 50 ng/ml cholera toxin (Sigma-Aldrich), and 250 ng/ml hydrocortisol (Sigma-Aldrich). Finally, the HCC1937 medium was supplemented with 20 ng/ml epidermal growth factor (Sigma-Aldrich) besides the mentioned supplements.
Phase holographic imaging
Phase holographic imaging was used to monitor cell motility and cell proliferation. The JIMT-1 cells were seeded at a density of 135,000 cells per well in a 6-well plate. Cells were incubated for 24 h to let them attach to the bottom of the plate. Sideritoflavone was added to a concentration of 2 μM or 2.5 μM. The standard lid was replaced by HoloLid™ (71,110 PHI), and the 6-well plate placed on the motorized stage of a HoloMonitor® M4 (Phase Holographic Imaging AB (PHI), Lund, Sweden) placed in a CO2 incubator for cell culturing. Images were acquired with the software App Suite™ 2 (PHI) at three positions per well, every 5 min for 72 h. The experiment was repeated three times with two independent cultures for each treatment. |
| Sideritoflavone inhibits cell proliferation and affects cell cycle phase distribution of JIMT-1 breast cancer cells. Cells were seeded day 0, and the compound was added 1 day after seeding to reach the final the concentrations of 2 μM or 2.5 μM. Control was treated with 0.1% DMSO which was the same DMSO concentration as in treated cultures. A The cell number was determined using a Holomonitor® M4. Data are presented as the mean of 6 independent samples from 3 independent experiments and bars ± SD. B At 72 h of treatment, cells were sampled for analysis of cell cycle phase distribution using flow cytometry of cells labelled with propidium iodide. The numbers show the cell cycle phase distribution in % for n = 3 ± SD |
Cell movement
As the ability to migrate is important for cancer cell metastasis, we investigated the effect of sideritoflavone treatment on the migration of the JIMT-1 cells in a wound healing assay using serum free medium to minimise the influence of cell proliferation on the data (Fig. 4). In this assay, the cells move from an edge into a wound area. Sideritoflavone treatment reduced the directed migration of JIMT-1 cells into the wounded area (Fig. 4).
To obtain further details, cell motility was studied in real time using a phase holographic microscope where images were taken every 5 min. The cells were cultured in their regular medium containing FBS in the absence or presence of 2 or 2.5 μM sideritoflavone. Cell motility was evaluated using App Suite™ (Fig. 5). Treatment with sideritoflavone increased the motility of the cells. Motility is defined as the total accumulated distance a cell has moved over the time of tracking. Motility is not a measure of how far the cells are migrating from a starting point but is a measure of how the cells are moving around. Thus, a cell can have low migratory ability but may be moving around in a small spot resulting in high motility. This is the case with JIMT-1 cells treated with sideritoflavone. The time laps movies are found in supplementary information (Time-lapse movie S1, S2, and S3 for control, and 2 and 2.5 μM sideritoflavone, respectively).
 |
| Treatment of JIMT-1 cells with sideritoflavone increases motility determined from time-lapse movies obtained using a digital holographic microscope. Cells were seeded and 1 day later (time 0 in the figure), sideritoflavone was added to the final concentrations of 2 or 2.5 μM. Images were taken every 5 min. Motility was evaluated using the software App Suite™ 2 (PHI). The data are from three independent experiments with n = 2 in each. The data points are very close, thus forming a line. |
Conclusions
In conclusion, we show here considerable differences in toxicity among the methoxyflavones 8-methoxycirsilineol, xanthomicrol, and sideritoflavone isolated from B. densiflora despite the fact that they have similar chemical structures. Sideritoflavone was markedly more toxic than xanthomicrol while 8-methoxycirsilineol was not toxic up to a 100 μM concentration, showing the importance of a catechol fragment in the phenyl ring of these methoxyflavones. We have not elucidated the exact mechanism by which sideritoflavone inhibits cell proliferation but our data together with reports by others, suggests that DNA intercalation may be the cause for the toxic response. Sideritoflavone treatment inhibited directed cell migration which is a favourable trait for a cancer treatment drug. Sideritoflavone inhibited the non-CSCs and the CSCs to the same extent. Thus, it may be favourable to combine sideritoflavone with CSC targeting compounds.
Acknowledgements
The authors are grateful to the Swedish International Development Agency (SIDA) for the support of this study, which is part of the project “Biomolecules anticancer” developed between the University of San Andres (UMSA La Paz–Bolivia) and Lund University (Sweden). We also wish to thank the Royal Physiographical Society in Lund, Carolina LePrince with the “Kalenderflickorna”, and associated sponsors. We thank Birgit Janicke and Kersti Alm at PHI for constructive help regarding phase holographic microscopy.
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RaderaMicropos Medical ja,det var några år sen jag kollade in företaget.
Sympatisk produkt deras raypilot,borde vara lätt att gilla.
Men det finns en hake med tekniken vill jag minnas och det är precisionsförmågan.Tekniken är lämplig för de med nån enstaka ansamling av cancerceller vid ett och samma ställe på prostatan.Men som i mitt fall hade jag ansamlingar på 6 ställen spridda runt prostatan.Det hade inte raypilot klarat vill jag minnas att jag tänkte.För min del var det bästa alternativet ren kirurgi.Ok,en jobbig strapats men idag 4 år senare har man inte kunnat hitta spår av ev cancerceller kvar. Micropos teknik kan bli en nischprodukt för en begränsad del av de med prostatacancer.Men jag har svårt att tro att de kommer bli nån större aktör.
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