Pharmacological interactions between phenylbenzothiazoles and aryl hydrocarbon receptor (AhR)

Bazzi, Rana (2008) Pharmacological interactions between phenylbenzothiazoles and aryl hydrocarbon receptor (AhR). PhD thesis, University of Nottingham.

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Abstract

The aryl hydrocarbon receptor is a ligand-dependent transcription factor that induces expression of a number of genes encoding drug metabolizing enzymes, such as CYP1A1, CYP1A2 and CYP1B1. Recently, it was suggested that the AhR signaling pathway may be involved in mediating the anticancer activity of novel 2-(4-aminophenyl) benzothiazole drugs in MCF-7 breast cancer cells. There is no direct proof of direct binding between these drugs and AhR, and it is also unclear how AhR signaling per se plays a role in the activity of these drugs. This study investigates the role of AhR in the mechanism of action of the benzothiazole drugs by determining the ability of these drugs to bind to the rat hepatic AhR, to induce CYP1A1 mRNA and to inhibit cell growth in rat hepatoma H4-II-E cells.

The apparent binding kinetics of [3H]-TCDD to AhR in rat liver cytosol were, KD= 0.37nM and Bmax = 40 fmol/mg cytosolic protein. Using the standard assay conditions, 18 compounds competitively displaced [3H]-TCDD from specific sites, and are ligands for AhR. Induction of CYP1A1 mRNA by 5 compounds was determined in H4-II-E cells. The highest affinity ligand, IH445, was the most potent with an EC50 ~ 80-fold lower than that of TCDD (60 pM) with no detectable antagonistic activity in H4-II-E cells. The other high-affinity benzothiazoles tested were (30-100) x 103-fold less potent for inducing CYP1A1 mRNA than TCDD. The binding affinities of these compounds were 200-1000-fold higher than induction potency. For example, 5F 203 has a Ki value of 2.8 nM, induced CYP1A1 mRNA to similar maximal levels as seen with TCDD, and has an EC50 of 3 μM. The 1000-fold difference for 5F 203 between binding and CYP1A1 RNA induction was suggested to be a result of metabolism or that 5F 203 exhibits partial AhR antagonist activity. The time course effect on the CYP1A1/β-actin mRNA ratios by 5F 203 revealed that the response was increasing linearly in response to 5F 203 at 4 h treatment, indicating that the former possibilty is less likely to be a major factor. To address the second possibility, the antagonistic activity of 5F 203 on TCDD-induced CYP1A1 mRNA was investigated. H4-II-E cells were treated with increasing concentrations of TCDD ± 1μM 5F 203. The results demonstrated that 5F 203 shifted the EC50 of TCDD 100-fold to the right. Schild analysis on the antagonism of TCDD-induced CYP1A1 mRNA by different concentrations of 5F 203 provided a quantitative explanation for the 1000-fold difference between binding and induction for 5F 203. In contrast, the EC50 of 5F 203 in human MCF-7 cells was 2 nM, which is ~ 10-fold less potent than TCDD. Moreover, 5F 203 had no detectable antagonistic activity on TCDDinduced CYP1A1 mRNA. When 5F 203 was assessed for cell growth inhibition by MTT assay, it was found active in MCF-7 cells with a GI50 of 18 nM, but failed to elicit the same effect in H4-II-E cells.

These results prove that 5F 203 is a potent agonist in MCF-7 cells, but a partial agonist in H4-II-E cells. The partial agonism observed with 5F 203 is a compound-specific property given that another analogue, IH 445, was found potent inducer of CYP1A1 mRNA with no antagonistic activity. The results of this study reveal species-specific partial agonism of the AhR. The potency of the cytostatic effect of 5F 203 parallel potency for inducing CYP1A1 mRNA in both cells. Moreover, both, the cytostatic effect of 5F 203 and partial agonism of AhR for inducing CYP1A1 mRNA is species-specific. Whether agonism/antagonism for the induction of CYP1A1 mRNA is related to the anticancer activity of 5F 203 remains to be elucidated.

Item Type:Thesis (PhD)
Supervisors:Bell, D.R.
Faculties/Schools:UK Campuses > Faculty of Medicine and Health Sciences > School of Biology
ID Code:999
Deposited By:Dr David R Bell
Deposited On:04 Dec 2009 16:16
Last Modified:04 Dec 2009 16:16

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