Identification of carbonyl reductase 1 as a resveratrol-binding protein by affinity chromatography using 4'-amino-3,5-dihydroxy-trans-stilbene.
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.
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University.
Yuta
Y
Ito
Takakazu
T
Mitani
Naoki
N
Harada
Atsushi
A
Isayama
Shinji
S
Tanimori
Shigeo
S
Takenaka
Yoshihisa
Y
Nakano
Hiroshi
H
Inui
Ryoichi
R
Yamaji
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors*; D000903:Antibiotics, Antineoplastic / Q000378:metabolism* / Q000627:therapeutic use; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D004317:Doxorubicin / Q000031:analogs & derivatives / Q000378:metabolism* / Q000627:therapeutic use; D005260:Female; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D061986:MCF-7 Cells; D009249:NADP / Q000378:metabolism; D008517:Phytotherapy; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D011485:Protein Binding / Q000187:drug effects; D000077185:Resveratrol; D013267:Stilbenes / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use
D000903:Antibiotics, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D006873:Hydroquinones; D010936:Plant Extracts; D013267:Stilbenes; D009249:NADP; D004317:Doxorubicin; D000429:Alcohol Oxidoreductases; C531129:CBR1 protein, human; C010013:adriamycinol; D000077185:Resveratrol; C031927:hydroquinone