1. *corresponding author: karasd@seznam.cz
DEHYDROSILYBIN AND ITS GALLOYLDERIVATIVES AS MODULATORS OF ANGIOGENESIS
Daniel Karas*1, Radek Gažák2, Vladimír Křen2, Ivana Oborná3, Jitka Ulrichová1, Kateřina Valentová1
1 Department of Medical Chemistry and Biochemistry, Palacký University, Hněvotínská 3, CZ Olomouc, Czech Republic,
2 Institute of Microbiology AS CR, Vídeňská 1083, Prague 4, CZ , Czech Republic,
3 Department of Obstetrics and Gynecology, University Hospital in Olomouc, I.P. Pavlova 6, Olomouc, Czech Republic
*corresponding author:
Introduction
Angiogenesis, the formation of new vessels from pre-existing ones, is important for growth and development, as well as in wound healing and tissue granulation. But it is also prominent in growth and metastases of solid malignancies. Plants are rich in biologically active natural compounds and to date many of them, e.g. flavonolignan silybin and its derivatives, were tested for various biological activities including anticancer and antiangiogenic properties.
In the present study, 2,3-dehydrosilybin (DHS) and its galloylderivatives were tested for antiangiogenic properties in a variety of in vitro tests with human umbilical vein endothelial cells (HUVEC). Primary screening was performed using wound healing migration and MTT cytotoxicity tests. Subsequently, capillary-like tube formation of HUVEC on matrigel and cell proliferation were evaluated.
Figure 3. Inhibition of HUVEC tube formation by 2,3-dehydrosilybin (DHS) and its galloyl derivatives. The extent of tube formation was quantified using an angiogenic score* in a representative optical field containing around 100 cells and the results were expressed as a % of the non-treated control; * - p < 0.05 compared with non-treated control; 2-ME (2-methoxyestradiol, 10 µM, positive control)
2,3 - dehydrosilybin
Nos = No of sprouting cells, Noc = No of connected cells, Nop = No of polygons, and Not = Total number of cells. Complex mesh 2-3 cells thick (+ 1), > 4 cells thick (+2)
Results
Table 1. The effect of DHS and its derivatives on viability of HUVEC.
Table 2. Inhibitory effects of the studied
compounds on HUVEC proliferation
In HUVEC cell migration test, minimal inhibitory concentrations of 40 µM for 3-galloyl-DHS, 2 µM for 7-galloyl-DHS, 10 µM for 20-galloyl-DHS, 6 µM for 23-galloyl-DHS, and 5 µM for DHS were observed (Figure 1). Similar results were obtained using Matrigel tube formation inhibition test with significant inhibition by DHS already at 5 uM (89,61 ± 2,34 of control), 25 uM (87,77 ± 1,82 of control) by 3-galloyl-DHS, 1 uM (97 ± 1,93 of control) by 7-galloyl-DHS, 10 uM 92,27± 6,71 of control) by 20-galloyl-DHS and 10 uM (96,55 ± 1,16 of control) by 23-galloyl-DHS (Figures 2 and 3). On the basis of these two tests is seen the trend, that 3-galloyl-DHS has a weaker inhibitory effect than DHS and 7-, 20- and 23-galloyl-DHS conversely stronger.. Strongest of the three last mentioned is 7-galloyl-DHS. The same trend were obtained in HUVEC proliferation and cytotoxicity test (table 1 and 2, figure 4), where all the compounds had HUVEC inhibitory effect.
Compound
IC50 (μM)
DHS
13.7 ± 1.58
3-galloyl-DHS
20.6 ± 0.16
7-galloyl-DHS
2.8 ± 0.75
20-galloyl-DHS
10.7 ± 1.73
23-galloyl-DHS
5.1 ± 1.88
Compound
IC50 (μM)
DHS
4.5 ± 1.76
3-galloyl-DHS
12.1 ± 2.28
7-galloyl-DHS
1.2 ± 0.18
20-galloyl-DHS
11.5 ± 1.33
23-galloyl-DHS
2.2 ± 0.93
The cells were grown in 96-well plates to confluency, then incubated with the tested compounds for 16 h. MTT-reducing ability was expressed as IC50 (mean ± SD).
HUVECs (5 × 103) in a total volume of 100 µl were incubated with serial dilutions of the tested compounds for three days and MTT-reducing ability was expressed as IC50 (mean ± SD).
Figure 1. Effect of tested compounds on endothelial cell migration afterovernight (16-20 h) incubation in 24-well plates.
A: T=0, B: control, C: 3-galloyl-DHS (40 μM), D: 7-galloyl-DHS (2 μM), E: 20-galloyl-DHS (10 μM), F: 23-galloyl-DHS (6 μM), G: DHS (5 μM)
Figure 4. Efect of DHS derivatives on HUVEC proliferation. Cell proliferation is presented as a percentage of control cell growth. Each point represents the mean of three independent experiments performed in triplicates, SD values were always below 10 %.
Figure 2. Inhibition of HUVEC tube formation by selected compounds. HUVECs were incubated overnight on Matrigel. A = control, panel B = 20-galloyl-DHS, 25 µM, C = 50 µM, D = 75 µM, E: 2-methoxyestradiol (2-ME, 10 µM, positive control).
References
Aranda; Owen. A semi-quantitative assay to screen for angiogenic compounds and compounds with angiogenic potential using the EA.hy926 endothelial cell line. Biol. Res. 2009, 42,
Financial Support
Supporting by grants LF_2013_008, P301/11/0767 and NT11083