Abstract
Plants are an infinite source of bioactive compounds. We screened the Israeli flora for compounds that interfere with the organization of the actin cytoskeleton. We found an activity in lipidic extract from Iris germanica that was able to increase HeLa cell area and adhesion and augment the formation of actin stress fibers. This effect was not observed when Ref52 fibroblasts were tested and was not the result of disruption of microtubules. Further, the increase in cell area was Rac1-dependent, and the iris extract led to slight Rac activation. Inhibitor of RhoA kinase did not interfere with the ability of the iris extract to increase HeLa cell area. The increase in HeLa cell area in the presence of iris extract was accompanied by impairment of cell migration and arrest of the cell cycle at G1 although the involvement of Rac1 in these processes is not clear. Biochemical verification of the extract based on activity-mediated fractionation and nuclear magnetic resonance analysis revealed that the active compounds belong to the group of iridals, a known group of triterpenoid. Purified iripallidal was able to increase cell area of both HeLa and SW480 cells.
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Abbreviations
- DIC:
-
Differential interference contrast
- FACS:
-
Fluorescence-activated cell sorting
- PKC:
-
Protein kinase C
- GAP:
-
GTPase-activating protein
- ECM:
-
Extracellular matrix
- TLC:
-
Thin layer chromatography
- DN:
-
Dominant negative
- CA:
-
Constitutive active
- PI:
-
Propidium iodide
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Acknowledgment
We thank Dr. Zohar Kerem (Institute of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem, Rehovot) for his help in the isolation of the iris active compounds. This research was financed by the foundation of the Chief Scientist of the Ministry of Agriculture and Rural Development, Israel.
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The authors declare that they have no conflict of interest.
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Supplementary Fig. 1
Increase focal contacts in HeLa cells treated with the iris extract. HeLa cells were treated with the iris extract, fixed, and stained for actin (red) and for vinculin (green). Arrows mark increased focal contact stained for vinculin in the treated cells. Scale bars = 50 μm (GIF 47 kb)
Supplementary Fig. 2
Hela cells in the presence of purified iridals. Crude extract was fractionated by TLC and activity was checked. According to cell area measurements (shown in the graph) and Scheffe statistical analysis (p < 0.05), fractions 1c, 2a, and 2b were active. Fraction 1c included 90% iripallidal and 10% iriflorental, 2a contained 60% 17-hydroxyiridal, 30% 16-hydroxyiridal, and 10% iripallidal. 2b was a mixture of several unidentified materials. Scale bars = 20 μm (GIF 102 kb)
Supplementary Fig. 3
The iridals isolated (GIF 117 kb)
Supplementary Table 1
NMR data of iridals. Iripallidal and iriflorental (DOC 48 kb)
Supplementary Table 2
NMR data of iridals. Three hydroxyiridals (DOC 67 kb)
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Halpert, M., Abu-Abied, M., Avisar, D. et al. Rac-dependent doubling of HeLa cell area and impairment of cell migration and cell cycle by compounds from Iris germanica . Protoplasma 248, 785–797 (2011). https://doi.org/10.1007/s00709-010-0254-1
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DOI: https://doi.org/10.1007/s00709-010-0254-1