Carnosol, a constituent of Zyflamend, inhibits aryl hydrocarbon receptor-mediated activation of CYP1A1 and CYP1B1 transcription and mutagenesis

Abstract

The aryl hydrocarbon receptor (AhR), a ligand-activated member of the basic helix-loop-helix family of transcription factors, plays a significant role in polycyclic aromatic hydrocarbon (PAH)-induced carcinogenesis. In the upper aerodigestive tract of humans, tobacco smoke, a source of PAHs, activates the AhR leading to increased expression of CYP1A1 and CYP1B1, which encode proteins that convert PAHs to genotoxic metabolites. Inhibitors of Hsp90 ATPase cause a rapid decrease in levels of AhR, an Hsp90 client protein, and thereby block PAH-mediated induction of CYP1A1 and CYP1B1. The main objective of this study was to determine whether Zyflamend, a polyherbal preparation, suppressed PAH-mediated induction of CYP1A1 and CYP1B1 and inhibited DNA adduct formation and mutagenesis. We also investigated whether carnosol, one of multiple phenolic antioxidants in Zyflamend, had similar inhibitory effects. Treatment of cell lines derived from oral leukoplakia (MSK-Leuk1) and skin (HaCaT) with benzo[a]pyrene (B[a]P), a prototypic PAH, induced CYP1A1 and CYP1B1 transcription, resulting in enhanced levels of message and protein. Both Zyflamend and carnosol suppressed these effects of B[a]P. Notably, both Zyflamend and carnosol inhibited Hsp90 ATPase activity and caused a rapid reduction in AhR levels. The formation of B[a]P-induced DNA adducts and mutagenesis was also inhibited by Zyflamend and carnosol. Collectively, these results show that Zyflamend and carnosol inhibit Hsp90 ATPase leading to reduced levels of AhR, suppression of B[a]P-mediated induction of CYP1A1 and CYP1B1, and inhibition of mutagenesis. Carnosol-mediated inhibition of Hsp90 ATPase activity can help explain the chemopreventive activity of herbs such as Rosemary, which contain this phenolic antioxidant.

Fig. 1

Zyflamend suppresses B[a]P-mediated induction of CYP1A1 and CYP1B1 proteins. MSK-Leuk1 (A) and HaCaT (B) cells were treated with vehicle or the indicated concentrations of Zyflamend for 2 hours. Cells were then treated with vehicle or 1 µmol/L B[a]P. Five hours later, cells were harvested for western blot analysis. Cellular lysate protein (100µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Immunoblots were probed with antibodies specific for CYP1A1, CYP1B1 and β-actin.

Fig. 2

Zyflamend suppresses B[a]P-mediated induction of CYP1A1 and CYP1B1 transcription. MSK-Leuk1 (A) and HaCaT (B) cells were treated with vehicle or the indicated concentration of Zyflamend for 2 hours. Subsequently, cells received vehicle or 1 µmol/L B[a]P for 3 hours. Total RNA was then isolated and quantitative real-time PCR was performed. Values for CYP1A1 and CYP1B1 were normalized to values obtained for β-actin. Columns, mean (n=3); bars, SD. *P < 0.05, **P < 0.01, ***P < 0.001. C, MSK-Leuk1 and HaCaT cells were transfected with 1.8 µg of pGudLuc6.1 (XRE-luciferase) and 0.2 µg of pSVβgal. After transfection, cells were treated with vehicle or the indicated concentration of Zyflamend for 2 hours, followed by treatment with vehicle or 1 µmol/L B[a]P. Reporter activities were measured in cellular extract 10 hours after treatment. Luciferase activity represents data that have been normalized to β-galactosidase activity. Columns, mean (n=6); bars, SD. *P < 0.05, ***P < 0.001.

Fig. 3

Zyflamend and carnosol down-regulate levels of AhR protein in epithelial cells. MSK-Leuk1 (A) and HaCaT (B) cells were treated with vehicle or Zyflamend (0.033 µl/mL) as indicated for 0–2.0 hours. Cells were then harvested for western blot analysis. C, MSK-Leuk1 cells were treated with vehicle or 10 µmol/L carnosol for 0–2.0 hours and were then harvested for western blot analysis. In A-C, cellular lysate protein was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Immunoblots were probed with antibodies specific for AhR, Hsp90, p23, XAP2, and β-actin. Results are representative of a minimum of three independent experiments.

Fig. 4

Zyflamend and carnosol inhibit Hsp90 ATPase activity. MSK-Leuk1 cells were treated with indicated concentrations of Zyflamend (A) or carnosol (B) for 30 minutes. Hsp90 was immunoprecipitated and Hsp90 ATPase activity was measured as described in Materials and Methods. The activity is presented as pmol/min/mg protein. The input indicates the amount of Hsp90 that was immunoprecipitated. Columns, mean (n = 3); bars, SD. ***P < 0.001.

Fig. 5

Carnosol, a component of Rosemary, suppresses B[a]P-mediated induction of CYP1A1 and CYP1B1. A and B, MSK-Leuk1 cells were treated with vehicle, the indicated concentrations of Rosemary extract (A) or carnosol (B) for 2 hours. Rosemary concentrations are presented as ×10⁻³ µl/mL of media. Subsequently, cells received vehicle or 1 µmol/L B[a]P for 5 hours before being harvested for western blot analysis. C, HaCaT cells were treated with vehicle or the indicated concentrations of carnosol for 2 hours. Cells then received vehicle or 1 µmol/L B[a]P for 5 hours before being harvested for western blot analysis. In A-C, immunoblots were probed with antibodies specific for CYP1A1, CYP1B1 and β-actin. D, MSK-Leuk1 cells were treated with vehicle or carnosol for 2 hours. Subsequently, cells received vehicle or 1 µmol/L B[a]P for 3 hours. Total RNA was isolated and quantitative real-time PCR was carried out. Values for CYP1A1 and CYP1B1 were normalized to values obtained for β-actin. Columns, means (n=3); bars, SD. **, P < 0.01; ***, P < 0.001. E, MSK-Leuk1 cells were transfected with 1.8 µg of pGudLuc6.1 (XRE-luciferase), and 0.2 µg of pSVβgal. After transfection, cells were treated with vehicle or the indicated concentration of carnosol for 2 hours, followed by treatment with vehicle or 1 µmol/L B[a]P. Reported activities were measured in cellular extract 10 hours after treatment. Luciferase activity represents data that have been normalized to β-galactosidase activity. Columns, mean (n=6); bars, SD., ***P < 0.001.

Fig. 6

Zyflamend and carnosol inhibit B[a]P induced DNA adduct formation and mutagenesis. In A, B, D and E, MSK-Leuk1 cells were pretreated with vehicle, Zyflamend (1 µL/10 mL), carnosol (10 µmol/L) or 1 µmol/L α-napthoflavone (αNF) as indicated for 2 hours. Cells were then treated with vehicle or 1 µmol/L B[a]P for 8 hours. DNA was isolated for quantification of DNA adducts (A, D); B[a]P-tetrol formation was measured in the media (B, E). C and F, BB® cells were pretreated with the indicated concentrations of Zyflamend or carnosol overnight and subsequently 2 µmol/L B[a]P was added. Cells were then grown to confluence and DNA was isolated. Mutagenesis assays were then carried out using the isolated DNA. Columns, mean (n=6); bars, SD. *P < 0.05, **P < 0.01, ***P < 0.001.