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 were 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.

Zyflamend, a polyherbal mixture, inhibits lipogenesis and mTORC1 signalling via activation of AMPK

“…Our data pertaining to Zyflamend and several studies from other laboratories (Bemis et al, 2005;Burke et al, 2015;Capodice et al, 2009;Ekmekcioglu et al, 2011;Huang, Chen, et al, 2011;Huang et al, 2014;Kim et al, 2012;Kunnumakkara et al, 2012;Mohebati et al, 2012;Rafailov et al, 2007;Sandur et al, 2007;Yan et al, 2012;Yang et al, 2007Yang et al, , 2008 reveal diverse mechanisms of action, possibly explaining its robust effects using in vivo models of tumorigenesis at oral doses that mimic human intakes (Huang, Chen, et al, 2011;Kunnumakkara et al, 2012). Because herbal extracts are composed of multiple compounds, it is difficult to directly extrapolate a standard concentration.…”

“…Its original formulation almost 2 decades ago was based on those herbs that had antioxidant and antiinflammatory properties. Its subsequent use in experimental investigations in an assortment of cancer models has drawn attention by a variety of laboratories interested in the role of natural products in health and disease (Bemis et al, 2005;Burke et al, 2015;Capodice et al, 2009;Ekmekcioglu et al, 2011;Huang, Chen, et al, 2011;Huang et al, 2014;Kim et al, 2012;Kunnumakkara et al, 2012;Mohebati et al, 2012;Rafailov et al, 2007;Sandur et al, 2007;Yan et al, 2012;Yang et al, 2007Yang et al, , 2008. There are always concerns regarding reproducibility and quality control of commercial products.…”

“…Group comparisons, AICAR or Zyflamend versus control,* p < 0.05. Huang et al, 2014;Kim et al, 2012;Kunnumakkara et al, 2012;Mohebati et al, 2012;Rafailov et al, 2007;Sandur et al, 2007;Yan et al, 2012;Yang et al, 2007Yang et al, , 2008. The best combinations of extracts within this formulation have not been systematically determined because it would be infeasible to reproduce the results from 1028 possible combinations.…”

“…Zyflamend® (New Chapter, Brattleboro, VT, USA) is a blend of 10 herbal extracts (ginger, rosemary, turmeric, Chinese goldthread, holy basil, hu zhang, barberry, oregano, green tea and baikal skullcap) formulated based on their antioxidant and potentially anti-inflammatory properties. To date, there have been 14 research articles investigating the anti-cancer effects of this blend, covering in vitro, preclinical and clinical studies involving the prostate, bone, skin, kidney, breast and pancreas (Bemis, Capodice, Anastasiadis, Katz, & Buttyan, 2005;Burke et al, 2015;Capodice et al, 2009;Ekmekcioglu et al, 2011;Huang, Chen, et al, 2011;Huang et al, 2014;Kim et al, 2012;Kunnumakkara et al, 2012;Mohebati et al, 2012;Rafailov, Cammack, Stone, & Katz, 2007;Sandur et al, 2007;Yan, Xie, Capodice, & Katz, 2012;Yang et al, 2007Yang et al, , 2008, with prostate being the predominant focus. It has been reported that a number of its constituents act synergistically such that physiological doses are possible (Zhao, Collier, Huang, & Whelan, 2014) and multiple mechanisms of action have been linked in a variety of cancer cell lines by down regulating the androgen receptor (Huang, Chen, et al, 2011;Huang et al, 2014;Yan et al, 2012), insulin-like growth factor-1R (Huang, Chen, et al, 2011;, class I and II HDACs (Huang, Chen, et al, 2011;Huang et al, 2014), COX-1 and COX-2 activities (Bemis et al, 2005;Ekmekcioglu et al, 2011;Kunnumakkara et al, 2012), 12-LOX and Rb protein phosphorylation (Yang et al, 2007), and the up regulation of tumour suppressor genes such as p21 and p27 (Bemis et al, 2005;Huang et al, 2014).…”

“…It has been reported that a number of its constituents act synergistically such that physiological doses are possible (Zhao, Collier, Huang, & Whelan, 2014) and multiple mechanisms of action have been linked in a variety of cancer cell lines by down regulating the androgen receptor (Huang, Chen, et al, 2011;Huang et al, 2014;Yan et al, 2012), insulin-like growth factor-1R (Huang, Chen, et al, 2011;, class I and II HDACs (Huang, Chen, et al, 2011;Huang et al, 2014), COX-1 and COX-2 activities (Bemis et al, 2005;Ekmekcioglu et al, 2011;Kunnumakkara et al, 2012), 12-LOX and Rb protein phosphorylation (Yang et al, 2007), and the up regulation of tumour suppressor genes such as p21 and p27 (Bemis et al, 2005;Huang et al, 2014). Zyflamend has also been shown to modulate NF-κB activation (Burke et al, 2015;Kunnumakkara et al, 2012;Sandur et al, 2007), iNOS (Ekmekcioglu et al, 2011), apoptotic pathways (Ekmekcioglu et al, 2011;Kim et al, 2012;Kunnumakkara et al, 2012), and aryl hydrocarbon receptor expression (Mohebati et al, 2012). It is believed that it is the combination of bioactives that accounts for modulation of multiple pathways at relatively low doses (as described in Huang, Chen, et al, 2011;Zhao et al, 2014).…”