Resveratrol inhibits dioxin-induced expression of human CYP1A1 and CYP1B1 by inhibiting recruitment of the aryl hydrocarbon receptor complex and RNA polymerase II to the regulatory regions of the corresponding genes

Abstract

The CYP1A family of cytochrome P450s (CYPs), comprising CYP1A1, CYP1A2, and CYP1B1, plays a role in bioactivation of several procarcinogens to carcinogenic derivatives, and also in detoxification of several xenobiotic compounds. Resveratrol (3,4,5-trihydroxystelbine) is a naturally occurring compound that has been shown in a number of studies to inhibit the induction of CYP1A1 and CYP1B1 by dioxin (2,3,7,8-tetrachloro-dibenzo-p-dioxin), but the mechanism(s) of resveratrol inhibition is controversial. In the current study, 100nM dioxin treatment for 24, 48, and 72 h induced CYP1A1, CYP1A2, and CYP1B1 mRNA levels in the human breast cancer cell line MCF-7, and CYP1A1 and CYP1A2 mRNA levels in the human hepatocellular carcinoma cell line, HepG2. Simultaneous treatment with 10 microM resveratrol significantly inhibited dioxin-induced mRNA expression levels of these genes in both cell lines. Our studies are novel in that we used the chromatin immunoprecipitation assay to assay dioxin-induced recruitment of the aryl hydrocarbon receptor (AHR), and aryl hydrocarbon nuclear translocator (ARNT) to the enhancer regions and recruitment of RNA polymerase II to the promoter regions, of the CYP1A1 and CYP1B1 genes in their natural chromosomal settings. These recruitments were significantly inhibited in cells cotreated with resveratrol. Our studies thus indicate that resveratrol inhibits dioxin induction of the CYP1 family members either by directly or indirectly inhibiting the recruitment of the transcription factors AHR and ARNT to the xenobiotic response elements of the corresponding genes. The reduced transcriptional factor binding at their enhancers then results in reduced pol II recruitment at the promoters of these genes.

FIG. 1.

Dioxin-induction of the CYP1A1, CYP1B1, CYP1A2, AHR and ARNT genes expression. MCF-7 and HepG2 cells were treated with 100nM dioxin for 0, 24, 48, and 72 h. The relative amounts of the CYP1A1 (A), CYP1A2 (B), CYP1B1 (C), AHR (D), and ARNT (E) mRNAs were measured using SYBR green real-time PCR, and were normalized to the constitutively expressed ribosomal subunit 36B4 gene. The mRNA levels of the MCF-7 cells are indicated with black bars and HepG2 cells are indicated with gray bars. In this and subsequent figures, the relative expression levels of all genes were reported using standard curves generated from MCF-7 cDNA treated for 72 h with dioxin, thus allowing us to report HepG2 mRNA expression levels relative to MCF-7 expression levels. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control DMSO or 0-h treatment.

FIG. 2.

Resveratrol inhibits dioxin induction of CYP1A1, CYP1A2, and CYP1B1 mRNAs in MCF-7 and HepG2 cells. MCF-7 and HepG2 cells were treated with either 10 or 20μM resveratrol (R) or the vehicle, DMSO, for 48 h. The cells were also treated with 100 nM dioxin (D) or the vehicle, DMSO during this time. The cells were either treated with resveratrol alone or cotreated with dioxin and resveratrol for 48 h. DMSO was used as vehicle for resveratrol and dioxin and was therefore included in the negative controls. The mRNA levels of the MCF-7 cells are indicated with black bars (A) and HepG2 (B) cells are indicated with gray bars. *p < 0.05, **p < 0.01, ***p < 0.001 compared with dioxin alone.

FIG. 3.

Resveratrol inhibits dioxin-induced expression of the CYP1A1 and CYP1B1 genes in a dose dependent manner. MCF-7 cells were treated with 1, 5, 10, or 20μM resveratrol or with DMSO (control) for 48 h. The cells were also treated with 100nM dioxin or DMSO (control) for 48 h. Resveratrol inhibited mRNA levels of the CYP1A1 (A) and CYP1B1 (B) genes were measured by real-time PCR. *p < 0.05, **p < 0.01, ***p < 0.001 compared with dioxin alone.

FIG. 4.

Resveratrol inhibits dioxin-induced recruitment of AHR and polII to the enhancer and promoter regions of the CYP1A1 and CYP1B1 genes. MCF-7 (A) and HepG2 (B) cells were treated with DMSO or with100nM dioxin, with or without 20μM resveratrol for 60 min and subjected to ChIP analysis. Sonicated whole cell lysates were probed with 2 μg of anti-human AHR, ARNT, or pol II antibodies. The recruitments of AHR and ARNT were measured using primers targeted to the enhancer regions, and pol II recruitment was performed using primers targeted to the promoter regions of the CYP1A1 and CYP1B1 genes. The results were plotted relative to those of the total input controls (untreated chromatin). The levels of recruitment in the MCF-7 cells are indicated with black bars and those for HepG2 cells are indicated with gray bars. *p < 0.05, **p < 0.01, ***p < 0.001 compared with dioxin alone.