RRC ID 68724
Author Ito Y, Mitani T, Harada N, Isayama A, Tanimori S, Takenaka S, Nakano Y, Inui H, Yamaji R.
Title Identification of carbonyl reductase 1 as a resveratrol-binding protein by affinity chromatography using 4'-amino-3,5-dihydroxy-trans-stilbene.
Journal J Nutr Sci Vitaminol (Tokyo)
Abstract The mechanisms by which resveratrol (3,4',5-trihydroxy-trans-stilbene) elicits diverse health benefits remain unclear because the intracellular target molecules of resveratrol are poorly defined. We screened resveratrol-binding proteins from lysates of MCF-7 breast cancer cells using resveratrol-affinity resin, which was constructed by immobilizing 4'-amino-3,5-dihydroxy-trans-stilbene on activated CH-Sepharose. On SDS-PAGE, two bands were detected as proteins that specifically bound to the resveratrol-affinity resin. One of these, a 30-kDa protein, was identified as human carbonyl reductase 1 (CBR1) by hybrid linear ion trap/time-of-flight mass spectrometry. Similarly, recombinant CBR1 bound to the resveratrol-affinity resin in the absence of resveratrol, but not in the presence of resveratrol. Among its activities, CBR1 catalyzes a NADPH-dependent reduction of the anticancer drug doxorubicin to the cardiotoxin doxorubicinol. The effects of doxorubicin on viability of MCF-7 cells were enhanced by resveratrol, 3,5-dihydroxy-4'-methoxy-trans-stilbene, 3,4'-dihydroxy-5-methoxy-trans-stilbene, and 4'-amino-3,5-dihydroxy-trans-stilbene at concentrations of 1 and 10 μM. Resveratrol and these derivatives inhibited CBR1 activities to a similar degree at concentrations of 100 and 200 μM. However, 3,5-dimethoxy-4'-hydroxy-trans-stilbene and m-hydroquinone had no influence on doxorubicin cytotoxicity or CBR1 activity. Resveratrol inhibited CBR1 activity through an apparent mix of competitive (Ki=55.8 μM) and noncompetitive (αKi=164 μM; α=2.98) inhibition kinetics. These results indicate that (i) resveratrol enhances the cytotoxic effects of doxorubicin on MCF-7 cells; (ii) the moiety that contains the 3,5-dihydroxyl groups of resveratrol, but not the m-hydroquinone structure alone, is required to bind CBR1; and (iii) resveratrol acts as a mixed-type inhibitor of CBR1 activity on doxorubicin.
Volume 59(4)
Pages 358-64
Published 2013-1-1
DOI 10.3177/jnsv.59.358
PII DN/JST.JSTAGE/jnsv/59.358
PMID 24064738
MeSH Alcohol Oxidoreductases / antagonists & inhibitors* Antibiotics, Antineoplastic / metabolism* Antibiotics, Antineoplastic / therapeutic use Antineoplastic Agents, Phytogenic / chemistry Antineoplastic Agents, Phytogenic / pharmacology* Antineoplastic Agents, Phytogenic / therapeutic use Binding, Competitive Breast Neoplasms / drug therapy Breast Neoplasms / metabolism* Chromatography, Affinity / methods Doxorubicin / analogs & derivatives Doxorubicin / metabolism* Doxorubicin / therapeutic use Female Humans Hydroquinones / pharmacology MCF-7 Cells NADP / metabolism Phytotherapy Plant Extracts / chemistry Plant Extracts / pharmacology* Plant Extracts / therapeutic use Protein Binding / drug effects Resveratrol Stilbenes / chemistry Stilbenes / pharmacology* Stilbenes / therapeutic use
IF 1.424
Human and Animal Cells MCF7(RCB1904)