Flavonoid-Induced Morphological Modifications of Endothelial Cells Through Microtubule Stabilization

Abstract

Flavonoids are common components of the human diet and appear to be of interest in cancer prevention or therapy, but their structure-activity relationships (SAR) remain poorly defined. In this study, were compared 24 flavonoids for their cytotoxicity on cancer cells (B16 and Lewis lung) and their morphological effect on endothelial cells (EC) that could predict antiangiogenic activity. Ten flavonoids presented inhibitory concentrations for 50% of cancer cells (IC ₅₀ , 48 h) below 50 μM: rhamnetin, 3 ^′ ,4 ^′ -dihydroxyflavone, luteolin, 3-hydroxyflavone, acacetin, apigenin, quercetin, baicalein, fisetin, and galangin. Important SAR for cytotoxicity included the C2-C3 double bond and 3 ^′ ,4 ^′ -dihydroxylation. Concerning the morphological effects on EC, only fisetin, quercetin, kaempferol, apigenin, and morin could induce the formation of cell extensions and filopodias at noncytotoxic concentrations. The SAR for morphologic activity differed from cytotoxicity and involved hydroxylation at C-7 and C-4 ^′ . Fisetin, the most active agent, presented cell morphology that was distinct compared to colchicine, combretastatin A-4, docetaxel, and cytochalasin D. Resistance to cold depolymerization and a 2.4-fold increase in acetylated α -tubulin demonstrated that fisetin was a microtubule stabilizer. In conclusion, this study disclosed several SAR that could guide the choice or the rational synthesis of improved flavonoids for cancer prevention or therapy.

ACKNOWLEDGMENTS

This study was supported by the Institut National de la Recherche et de la Santé Médicale (Inserm), by the Centre National de la Recherche Scientifique (CNRS), and by the French Institut National du Cancer (INCa, 92513 Boulogne-Billancourt, France). We thank Johanne Seguin, Sébastien David, Else Hotin, and Aurélie Chiche for their expert technical assistance in cellular experiments. We also wish to thank Dr. Virginie Escriou for her assistance with confocal microscopy. This study was presented in part at the XXVIIth Forum de cancérologie, Paris, June 26–27, 2007: Touil YS, Fellous A, Scherman D, Chabot GG: Effets des flavonoïdes sur la morphologie des cellules endothéliales par stabilisation des microtubules. Bull Cancer 94, 551, 2007 (Abstract No. 84).

Notes

^a B16 melanoma cells or Lewis lung carcinoma cells were exposed to the flavonoid for 48 h and viability was assessed by the MTT test. Results are expressed as the inhibitory concentration for 50% (IC₅₀) of the cells compared to controls (1% dimethylsulfoxide). Mean ± SEM of 3 to 6 independent experiments each performed in triplicate.

^b The EC₅₀ is the effective concentration to obtain a change in the morphology for 50% of EA hy 926 cells after a 2 h exposure time at noncytotoxic concentrations as described in Materials and Methods.

^c IC₅₀ of cells relative to controls for a 48 h exposure time.

^d The therapeutic window for the EA hy 926 endothelial cells represents the ratio of the IC₅₀ value at 48 h (cytotoxicity) divided by the EC₅₀ value at 2 h (morphology).

^e No effect: indicates no cytotoxicity observed at 400 μM, the maximum concentration tested.