Identification of carbonyl reductase 1 as a resveratrol-binding protein by affinity chromatography using 4'-amino-3,5-dihydroxy-trans-stilbene.

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Ito Y, Mitani T, Harada N, Isayama A, Tanimori S, Takenaka S, Nakano Y, Inui H, Yamaji R

Identification of carbonyl reductase 1 as a resveratrol-binding protein by affinity chromatography using 4'-amino-3,5-dihydroxy-trans-stilbene.

J Nutr Sci Vitaminol (Tokyo). 2013;59(4):358-64.

PubMed ID

24064738 [ View in PubMed

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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 muM. Resveratrol and these derivatives inhibited CBR1 activities to a similar degree at concentrations of 100 and 200 muM. 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 muM) and noncompetitive (alphaKi=164 muM; alpha=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.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Resveratrol	Carbonyl reductase [NADPH] 1	Protein	Humans	Unknown	Inhibitor	Details