Natural Polyphenols Inhibit Lysine-Specific Demethylase-1 in vitro

Abstract

Natural polyphenols, such as resveratrol, have beneficial functions on major human diseases such as cancer, diabetes, and cardiovascular disease. Besides acting as antioxidants, some of these polyphenols can also target proteins to modulate specific biological pathways. The lysine-specific histone demethylase LSD1 plays important roles in cell growth, differentiation and nutrient metabolism. Here, we studied the effect of natural polyphenols resveratrol, curcumin, quercetin and analogs on LSD1. Using in vitro LSD1 enzymatic assays, we show that resveratrol, curcumin and quercetin displayed a potent inhibitory effect on the LSD1 activity and were more potent than the known LSD1 inhibitor trans-2-phenylcyclopropylamine (TCP). The new function of resveratrol, curcumin and quercetin is independent of their antioxidant properties, as other antioxidants had no effect on LSD1 under the similar conditions. In C2C12 fibroblasts, resveratrol and curcumin can efficiently inhibit myogenic expression and differentiation, for which LSD1 is required. Thus, our study has identified LSD1 as a novel target of bioactive natural compounds, such as resveratrol, curcumin and quercetin, and such finding suggests that LSD1 inhibition can at least partially contribute to some of the previously observed beneficial effects of these compounds.

Keywords: LSD1; curcumin; demethylation; myogenesis; polyphenol; quercetin; resveratrol.

Fig. 1

A. Chemical structure of TCP. B. Effects of the indicated doses of TCP on SIRT1 protein levels in HEK293 cells by immunoblotting. Cells were treated with TCP for 18 hours. The amount of β-tubulin was used as the invariant control for total proteins. C. Relative mRNA levels of SIRT1, as detected by quantitative RT-PCR, in HEK293 cells treated with 50 µM of TCP for 18 hours. Cyclophilin B was used as the invariant control for total mRNA. D. Effects of the indicated doses of TCP on SIRT1 protein levels in primary rat hepatocytes by immunoblotting. Treatment was similar to that in B.

Fig. 2

A. Chemical structure of resveratrol. B. Dose-dependent effect of resveratrol on the demethylase activity of recombinant LSD1 in vitro as examined using an LSD1 assay kit. Data represent Mean ± S.D. of triplicates of a representative experiment, which was independently repeated three times. C. Effects of resveratrol and TCP on the demethylase activity of recombinant LSD1 using in vitro demethylase reactions. The substrates were bulky histones. The amount of di-methyl H3K4 histones was examined by immunoblotting using specific antibody. Total amount of histone H3 was used as the invariant control.

Fig. 3

Chemical structure of curcumin (A), (−)-Epigallocatechin gallate (B), and N-acetyl-L-cysteine (C). D. Effects of the indicated compounds (50 µM) on the demethylase activity of recombinant LSD1 in vitro as examined using a LSD1 assay kit. RES: resveratrol. Data represent Mean ± S.D. of triplicates of a representative experiment (n=3), * p<0.01 vs. control (DMSO).

Fig. 4

Chemical structure of quercetin (A), myricetin (B), luteolin (C), apigenin (D), and genistein (E). F. Effects of the indicated compounds (50 µM) on the demethylase activity of recombinant LSD1 in vitro as examined using a LSD1 assay kit. Data represent Mean ± S.D. of triplicates of a representative experiment (n=3), * p<0.01 vs. control (DMSO).

Fig. 5

A. Effects of the indicated compounds on C2C12 cell differentiation into myotubes using the protocol as detailed in Material and Method section. Microscopic photos (10×) were taken at day 1 and day 4 after incubation in the differentiation medium. B. Effect of the indicated compounds on the expression of myogenin and myosin heavy chain (MHC) in C2C12 cells at 4 days of incubation in the differentiation medium. Relative mRNA levels of myogenin and MHC were detected by quantitative RT-PCR, and cyclophilin B was used as the invariant control. Data represent Mean ± S.D. (n=3), * p<0.01 vs. control (DMSO). TCP: trans-2-phenylcyclopropylamine; RES: resveratrol; CUR: curcumin.