Uptake of resveratrol and role of resveratrol-targeting protein, quinone reductase 2, in normally cultured human prostate cells

Abstract

AbtractResveratrol is a dietary polyphenol espoused to have chemopreventive activity against a variety of human cancer types. We first reported that resveratrol significantly decreases the proliferation of both androgen-dependent and hormone-refractory prostate cancer cells. However, the effects of resveratrol in normal prostate epithelial and stromal cells, particularly with regard to its uptake, subcellular distribution and intracellular targets, have not been investigated. To advance the knowledge on accessibility and cellular disposition of resveratrol in prostate cells, [ 3 H] resveratrol, fractionation of cell extracts into subcellular compartments, Western blot analysis, resveratrol affinity column chromatography and flow cytometry were used to study the uptake and intracellular distribution of resveratrol in normally cultured prostate stromal (PrSCs) and epithelial cells (PrECs). Pretreatment of both PrSCs and PrECs for 2 days with resveratrol modulated its uptake and selectively increased its distribution to the membrane and organelle compartments. Resveratrol affinity column chromatography studies showed differential expression of a previously identified resveratrol-targeting protein, quinone reductase 2 (QR2), in PrSCs and PrECs. Flow cytometric analysis comparing resveratrol-treated and untreated PrSCs showed a large decrease in G 1 -phase and a concomitant increase in S and G 2 /M-phases of the cell cycle. These results suggest that resveratrol suppresses PrSC proliferation by affecting cell cycle phase distribution, which may involve the participation by QR2.

Regulation of p53 and cell proliferation by resveratrol and its derivatives in breast cancer cells: An in silico and biochemical approach targeting integrin αvβ3

“…To analyze the expression of specific proteins in control and treated cells, cell extracts containing 10–20 μg of proteins were boiled for 5 min and separated on 10% SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) 23, 24. The gels were transferred to nitrocellulose membranes by a semidry transfer method.…”

Resveratrol: a cardioprotective substance

“…A specific protein migrating with a molecular weight of 22 kDa, denoted RTP‐22 was found in the resveratrol‐eluted fraction, based on our studies using extracts prepared from human prostate 22,25 and melanoma cells, 23 and from cultured human aortic smooth muscle cells 20 . In all cell extracts tested, the retention of RTP‐22 on the resveratrol affinity column was effectively and almost completely competed when extracts were first incubated with excess resveratrol prior to fractionation 20,22,23,25 . The RTP‐22 was subsequently identified as quinone reductase NQO2 by combining resveratrol‐directed affinity chromatography purification strategy with MALDI‐TOF mass spectrometry and cloning 26 .…”

“…To experimentally test and validate the presence of RTPs, resveratrol was immobilized on epoxyactivated agarose, generating a biospecific affinity platform for the rapid and specific capture of RTPs. 20,22,24,25 Extracts prepared from various sources were fractionated on resveratrol affinity agarose columns by sequential elution with 0.35 M and 1 M NaCl, followed by 1 mM ATP and, finally, 2 mM resveratrol. The variously eluted fractions were concentrated and resolved by 10% SDS-PAGE, followed by silver staining.…”

“…The variously eluted fractions were concentrated and resolved by 10% SDS-PAGE, followed by silver staining. A specific protein migrating with a molecular weight of 22 kDa, denoted RTP-22 was found in the resveratrol-eluted fraction, based on our studies using extracts prepared from human prostate 22,25 and melanoma cells, 23 and from cultured human aortic smooth muscle cells. 20 In all cell extracts tested, the retention of RTP-22 on the resveratrol affinity column was effectively and almost completely competed when extracts were first incubated with excess resveratrol prior to fractionation.…”

“…20 In all cell extracts tested, the retention of RTP-22 on the resveratrol affinity column was effectively and almost completely competed when extracts were first incubated with excess resveratrol prior to fractionation. 20,22,23,25 The RTP-22 was subsequently identified as quinone reductase NQO2 by combining resveratrol-directed affinity chromatography purification strategy with MALDI-TOF mass spectrometry and cloning. 26 Notably, purified recombinant NQO2 showed a high affinity for resveratrol, with a dissociation constant K D ≤ 50 nM.…”