期刊名称：Journal of Nutritional Science and Vitaminology
印刷版ISSN：0301-4800
电子版ISSN：1881-7742
出版年度：2013
卷号：59
期号：4
页码：358-364
DOI：10.3177/jnsv.59.358
出版社：Center for Academic Publications Japan
摘要：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 μ M . Resveratrol and these derivatives inhibited CBR1 activities to a similar degree at concentrations of 100 and 200 μ M . 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 μ M ) and noncompetitive (αKi=164 μ M ; 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.
关键词：resveratrol;carbonyl reductase 1;affinity chromatography;doxorubicin;breast cancer cells
