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

  • Ion channels, receptors and transporters
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

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 IC50s of 5–13 μM and Emaxs 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 (IC50 = 2.3 ± 0.3 μM; Emax = 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.

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Abbreviations

VRACs:

volume-regulated anion channels

HEK:

human embryonic kidney

DCPIB:

4-(2-butyl-6, 7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid

HUVECs:

human umbilical vein endothelial cells

EGTA:

ethylene glycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid

HEPES:

N-(hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)

IC50 :

half-maximal concentrations

E max :

maximum effect

References

  1. Bhat TA, Nambiar D, Pal A, Agarwal R, Singh RP (2012) Fisetin inhibits various attributes of angiogenesis in vitro and in vivo--implications for angioprevention. Carcinogenesis 33:385–393

    Article  PubMed  CAS  Google Scholar 

  2. Cho H, Lee HY, Ahn DR, Kim SY, Kim S, Lee KB, Lee YM, Park H, Yang EG (2008) Baicalein induces functional hypoxia-inducible factor-1alpha and angiogenesis. Mol Pharmacol 74:70–81

    Article  PubMed  CAS  Google Scholar 

  3. Decher N, Lang HJ, Nilius B, Bruggemann A, Busch AE, Steinmeyer K (2001) DCPIB is a novel selective blocker of I(Cl,swell) and prevents swelling-induced shortening of Guinea-pig atrial action potential duration. Br J Pharmacol 134:1467–1479

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Dick GM, Kong ID, Sanders KM (1999) Effects of anion channel antagonists in canine colonic myocytes: comparative pharmacology of Cl-, Ca2+ and K+ currents. Br J Pharmacol 127:1819–1831

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Doleckova I, Rarova L, Gruz J, Vondrusova M, Strnad M, Krystof V (2012) Antiproliferative and antiangiogenic effects of flavone eupatorin, an active constituent of chloroform extract of Orthosiphon stamineus leaves. Fitoterapia 83:1000–1007

    Article  PubMed  CAS  Google Scholar 

  6. Graefe EU, Derendorf H, Veit M (1999) Pharmacokinetics and bioavailability of the flavonol quercetin in humans. Int J Clin Pharmacol Ther 37:219–233

    PubMed  CAS  Google Scholar 

  7. Guarneri L, Ibba M, Angelico P, Colombo D, Fredella B, Testa R (1993) Effects of drugs used in the therapy of detrusor hyperactivity on the volume-induced contractions of the rat urinary bladder. Pharmacol Res 27:173–187

    Article  PubMed  CAS  Google Scholar 

  8. Helix N, Strobaek D, Dahl BH, Christophersen P (2003) Inhibition of the endogenous volume-regulated anion channel (VRAC) in HEK293 cells by acidic di-aryl-ureas. J Membr Biol 196:83–94

    Article  PubMed  CAS  Google Scholar 

  9. Hoffmann EK, Holm NB, Lambert IH (2014) Functions of volume-sensitive and calcium-activated chloride channels. IUBMB Life 66:257–267

    Article  PubMed  CAS  Google Scholar 

  10. Huang H, Chen AY, Rojanasakul Y, Ye X, Rankin GO, Chen YC (2015) Dietary compounds galangin and myricetin suppress ovarian cancer cell angiogenesis. J Funct Foods 15:464–475

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Kim JD, Liu L, Guo W, Meydani M (2006) Chemical structure of flavonols in relation to modulation of angiogenesis and immune-endothelial cell adhesion. J Nutr Biochem 17:165–176

    Article  PubMed  CAS  Google Scholar 

  12. Klausen TK, Bergdahl A, Hougaard C, Christophersen P, Pedersen SF, Hoffmann EK (2007) Cell cycle-dependent activity of the volume- and Ca2+-activated anion currents in Ehrlich lettre ascites cells. J Cell Physiol 210:831–842

    Article  PubMed  CAS  Google Scholar 

  13. Liantonio A, Pusch M, Picollo A, Guida P, De Luca A, Pierno S, Fracchiolla G, Loiodice F, Tortorella P, Conte Camerino D (2004) Investigations of pharmacologic properties of the renal CLC-K1 chloride channel co-expressed with barttin by the use of 2-(p-chlorophenoxy)propionic acid derivatives and other structurally unrelated chloride channels blockers. J Am Soc Nephrol 15:13–20

    Article  PubMed  CAS  Google Scholar 

  14. Liu Y, Zhang H, Huang D, Qi J, Xu J, Gao H, Du X, Gamper N (2014) Characterization of the effects of Cl(−) channel modulators on TMEM16A and bestrophin-1 Ca(2)(+) activated Cl(−) channels. Pflugers Arch 467:1417–1430

    Article  PubMed  CAS  Google Scholar 

  15. Maertens C, Droogmans G, Chakraborty P, Nilius B (2001) Inhibition of volume-regulated anion channels in cultured endothelial cells by the anti-oestrogens clomiphene and nafoxidine. Br J Pharmacol 132:135–142

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Manolopoulos VG, Liekens S, Koolwijk P, Voets T, Peters E, Droogmans G, Lelkes PI, De Clercq E, Nilius B (2000) Inhibition of angiogenesis by blockers of volume-regulated anion channels. Gen Pharmacol 34:107–116

    Article  PubMed  CAS  Google Scholar 

  17. Pedersen SF, Okada Y, Nilius B (2016) Biophysics and physiology of the volume-regulated anion channel (VRAC)/volume-sensitive outwardly rectifying anion channel (VSOR). Pflugers Arch 468:371–383

    Article  PubMed  CAS  Google Scholar 

  18. Picollo A, Liantonio A, Didonna MP, Elia L, Camerino DC, Pusch M (2004) Molecular determinants of differential pore blocking of kidney CLC-K chloride channels. EMBO Rep 5:584–589

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Planells-Cases R, Lutter D, Guyader C, Gerhards NM, Ullrich F, Elger DA, Kucukosmanoglu A, Xu G, Voss FK, Reincke SM, Stauber T, Blomen VA, Vis DJ, Wessels LF, Brummelkamp TR, Borst P, Rottenberg S, Jentsch TJ (2015) Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. EMBO J 34:2993–3008

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Pratheeshkumar P, Budhraja A, Son YO, Wang X, Zhang Z, Ding S, Wang L, Hitron A, Lee JC, Xu M, Chen G, Luo J, Shi X (2012) Quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting VEGFR- 2 regulated AKT/mTOR/P70S6K signaling pathways. PLoS One 7:e47516

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Pratheeshkumar P, Son YO, Budhraja A, Wang X, Ding S, Wang L, Hitron A, Lee JC, Kim D, Divya SP, Chen G, Zhang Z, Luo J, Shi X (2012) Luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis. PLoS One 7:e52279

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Qiu Z, Dubin AE, Mathur J, Tu B, Reddy K, Miraglia LJ, Reinhardt J, Orth AP, Patapoutian A (2014) SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel. Cell 157:447–458

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Tomoda T, Aishima M, Takano N, Nakano T, Seki N, Yonemitsu Y, Sueishi K, Naito S, Ito Y, Teramoto N (2005) The effects of flavoxate hydrochloride on voltage-dependent L-type Ca2+ currents in human urinary bladder. Br J Pharmacol 146:25–32

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Tomoda T, Zhu HL, Iwasa K, Aishima M, Shibata A, Seki N, Naito S, Teramoto N (2007) Effects of flavoxate hydrochloride on voltage-dependent Ba2+ currents in human detrusor myocytes at different experimental temperatures. Naunyn Schmiedeberg's Arch Pharmacol 376:195–203

    Article  CAS  Google Scholar 

  25. Voets T, Szucs G, Droogmans G, Nilius B (1995) Blockers of volume-activated Cl- currents inhibit endothelial cell proliferation. Pflugers Arch 431:132–134

    Article  PubMed  CAS  Google Scholar 

  26. Voss FK, Ullrich F, Munch J, Lazarow K, Lutter D, Mah N, Andrade-Navarro MA, von Kries JP, Stauber T, Jentsch TJ (2014) Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. Science 344:634–638

    Article  PubMed  CAS  Google Scholar 

  27. Zhang X, Li H, Zhang H, Liu Y, Huo L, Jia Z, Xue Y, Sun X, Zhang W (2017) Inhibition of transmembrane member 16A calcium-activated chloride channels by natural flavonoids contributes to flavonoid anticancer effects. Br J Pharmacol 174:2334–2345

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Zhao K, Li X, Lin B, Yang D, Zhou Y, Li Z, Guo Q, Lu N (2017) Oroxyloside inhibits angiogenesis through suppressing internalization of VEGFR2/Flk-1 in endothelial cells. J Cell Physiol 233:3454–3464

  29. Zhao K, Song X, Huang Y, Yao J, Zhou M, Li Z, You Q, Guo Q, Lu N (2014) Wogonin inhibits LPS-induced tumor angiogenesis via suppressing PI3K/Akt/NF-kappaB signaling. Eur J Pharmacol 737:57–69

    Article  PubMed  CAS  Google Scholar 

  30. Zhou M, Song X, Huang Y, Wei L, Li Z, You Q, Guo Q, Lu N (2014) Wogonin inhibits H2O2-induced angiogenesis via suppressing PI3K/Akt/NF-kappaB signaling pathway. Vasc Pharmacol 60:110–119

    Article  CAS  Google Scholar 

  31. Ziegelhoeffer T, Scholz D, Friedrich C, Helisch A, Wagner S, Fernandez B, Schaper W (2003) Inhibition of collateral artery growth by mibefradil: possible role of volume-regulated chloride channels. Endothelium 10:237–246

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Research Foundation of Education Bureau of Hebei Province (BJ2018030 to XZ), the Natural Science Foundation of Hebei Province (H2015423014 to XZ), the National Natural Science Foundation of China (31401003 to XZ, 81500057 to HZ), the project funded by China Postdoctoral Science Foundation (2015M581313 to XZ), the Research Foundation of Education Bureau of Hebei Province (QN2017113 to PC, QN2016166 to WL), and the Research Foundation of Administration of Traditional Chinese Medicine of Hebei Province (2017007 to YL).

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Authors and Affiliations

  1. Department of Pharmacology, Hebei University of Chinese Medicine, No. 326 South Xinshi Road, Shijiazhuang, 050091, Hebei, China

    Yucong Xue, Xue Han, Gaohua Zhang, Yue Lin, Pingping Chen, Xiaorun Sun, Yalei Liu, Li Chu, Jianping Zhang & Xuan Zhang

  2. Department of Respiratory, The Second Hospital of Hebei Medical University, Shijiazhuang, China

    Honglin Li & Huiran Zhang

  3. Department of Medicinal Chemistry, Hebei University of Chinese Medicine, Shijiazhuang, China

    Yuanyuan Zhang & Li Chu

  4. Department of Physiology, Hebei University of Chinese Medicine, Shijiazhuang, China

    Wenya Li

  5. Department of Radiotherapy, Cangzhou Central Hospital, No. 16 West Xinhua Road, Cangzhou, China

    Mingyun Zhang

Authors
  1. Yucong Xue
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  2. Honglin Li
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  3. Yuanyuan Zhang
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  4. Xue Han
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  5. Gaohua Zhang
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  6. Wenya Li
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  8. Yue Lin
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  10. Xiaorun Sun
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  11. Yalei Liu
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  12. Li Chu
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  13. Jianping Zhang
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  14. Mingyun Zhang
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  15. Xuan Zhang
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Contributions

Participated in research design: X. Zhang, M. Zhang, J. Zhang, Xue, and Chu

Conducted experiments: Xue, H. Li, Y. Zhang, Han, G. Zhang, W. Li, Lin, H. Zhang, Chen, and Sun

Performed data analysis: Xue, G. Zhang, and Liu

Wrote or contributed to the writing of the manuscript: X. Zhang, M. Zhang, J. Zhang, and Xue

Corresponding authors

Correspondence to Jianping Zhang, Mingyun Zhang or Xuan Zhang.

Additional information

This article is part of the Topical Collection on Ion channels, receptors and transporters

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Figure S1

The effects of five natural flavonoids on VRAC currents in HUVEC cells. (PDF 309 kb)

(A) Representative current traces for the effects of five natural flavonoids (100 μM) on VRAC currents tested at −100 mV in HUVEC cells.

(B) The mean inhibition of VRAC current tested at −100 mV by five natural flavonoids (100 μM) in HUVEC cells. *p < 0.05 compared with the current amplitudes in the absence of drugs.

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Xue, Y., Li, H., Zhang, Y. et al. Natural and synthetic flavonoids, novel blockers of the volume-regulated anion channels, inhibit endothelial cell proliferation. Pflugers Arch - Eur J Physiol 470, 1473–1483 (2018). https://doi.org/10.1007/s00424-018-2170-8

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  • DOI: https://doi.org/10.1007/s00424-018-2170-8

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