Natural and synthetic flavonoids, novel blockers of the volume-regulated anion channels, inhibit endothelial cell proliferation

Abstract

Natural flavonoids are ubiquitous in dietary plants and vegetables and have been proposed to have antiviral, antioxidant, cardiovascular protective, and anticancer effects. Volume-regulated anion channels (VRACs), which are essential for cell volume regulation, have been proposed to play a key role in cell proliferation and migration, apoptosis, transepithelial transport, and cancer development. In this study, we screened a group of 53 structurally related natural flavonoids and three synthetic flavonoids for their inhibitory activities on VRAC currents. A whole-cell patch technique was used to record VRAC currents in the human embryonic kidney (HEK) 293 and human umbilical vein endothelial (HUVEC) cells. The 5'-bromo-2-deoxyuridine (BrdU) assay technique was used to investigate cell proliferation. At 100 μM, 34 of 53 compounds significantly inhibited hypotonic extrasolution-induced VRAC currents by > 50% in HEK293 cells. Among these compounds, luteolin, baicalein, eupatorin, galangin, quercetin, fisetin, karanjin, Dh-morin, genistein, irisolidone, and prunetin exhibited the highest efficacy for VRAC blockade (the mean inhibition > 80%) with ICs of 5-13 μM and Es of about 87-99%. We also studied the effects of three synthetic flavonoids on VRAC currents in HEK293 cells. Flavoxate showed high inhibition efficacy toward VRAC currents (IC = 2.3 ± 0.3 μM; E = 91.8% ± 2.7%). Finally, these flavonoids inhibited endogenous VRAC currents and cell proliferation in endothelial cells. This study demonstrates that natural and synthetic flavonoids are potent VRAC current inhibitors, and VRAC inhibition by flavonoids might be responsible for their anti-angiogenic effects.

The Volume-Regulated Anion Channel LRRC8/VRAC Is Dispensable for Cell Proliferation and Migration

“…Other studies corroborate the notion that VRAC is dispensable for cell proliferation. The flavonoid Dh-morin suppressed VRAC currents but did not reduce proliferation of HUVEC cells [43], and siRNA against LRRC8A did not affect the proliferation rate in HeLa cells [42]. The DCPIB treatment even inhibited the antiproliferative effect of cardiac glycosides that correlated with an increase in VRAC activity in HT-29 cells [44].…”

“…The proliferation of HeLa cells was reported to be unaffected by the siRNA-mediated knockdown of LRRC8A [42]. Recently, the flavonoid Dh-morin was shown to effectively inhibit endogenous VRAC currents in endothelial cells, without impairing the proliferation of human umbilical vein endothelial (HUVEC) cells [43], arguing against a crucial role for the VRAC in the cell cycle progression of this cell type. The anti-proliferative effect of submicromolar concentrations of cardiac glycosides was even linked, albeit not necessarily directly, to an increase of VRAC activity in HT-29 colorectal cancer cells and could be blocked by the VRAC inhibitor DCPIB [44].…”

A 30-year journey from volume-regulated anion currents to molecular structure of the LRRC8 channel

“…VRAC current amplitude varies during the cell cycle (Shen et al, 2000; Chen et al, 2002), suggesting that the channel may play a role in cell proliferation. Pharmacological inhibition of VRAC slows proliferation of endothelial cells (Maertens et al, 2001; Xue et al, 2018), hepatocytes (Wondergem et al, 2001), glioblastoma cells (Rouzaire-Dubois et al, 2000), and vascular smooth muscle cells (Liang et al, 2014). Knockdown of the essential VRAC subunit LRRC8A was shown recently to inhibit proliferation of glioblastoma cells (Rubino et al, 2018).…”

Tannins, novel inhibitors of the volume regulation and the volume-sensitive anion channel

“…However, pharmacology of this biologically important ion channel remains poorly explored. Thus far, a number of structurally divers compounds including stilbene derivatives, etacrynic acid analogs and flavonoids have been shown to suppress the activity of VSOR in a voltage-dependent and independent manner (Okada et al, 2019;Xue et al, 2018). Tannins are structurally heterogenous polyphenols, which bind to proteins and can trigger their precipitation (Mavlyanov et al, 2001).…”

“…Tannins are secondary metabolites and constitute a part of the plants' defense system against pathogens and insect's invasion. In addition, these substances exhibit a wide spectrum of biological activities such as antimicrobial (Scalbert, 1991); antioxidant, (Rice-Evans et al, 1995, 1996; anti-inflammatory (Terra et al, 2007;Xue et al, 2018); neuroprotective (Behravan et al, 2014). Tannins, along with other polyphenolic compounds, have been considered to be responsible for the health benefits of red wine and green tea, possibly by inhibiting the activity of the Caactivated chloride channels (CaCCs) (Namkung et al, 2010).…”