The p53 Inhibitor Pifithrin- Is a Potent Agonist of the Aryl Hydrocarbon Receptor

The tumor suppressor protein p53 is currently a target of emerging drug therapies directed toward neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, and side effects associated with cancer treatments. Of this group of drugs, the best characterized is pifithrin, a small molecule that inhibits p53-dependent apoptosis through an undetermined mechanism. In this study, we have used a number of molecular approaches to test the hypothesis that pifithrinacts as an aryl hydrocarbon receptor (AhR) agonist and, in this manner, inhibits the actions of p53. Toward this end, we have found that pifithrinis a potent AhR agonist as determined by its ability to bind the AhR, induce formation of its DNA binding complex, activate reporter activity, and up-regulate the classic AhR target gene CYP1A1. However, examination of its ability to inhibit p53-mediated gene activation and apoptosis revealed that these actions occurred via an AhR-independent manner. The significance of this study is based on the fact that activation of the AhR is typically associated with an increase in phase I and phase II metabolizing enzymes and adverse biological events such as tumor promotion that may contribute to untoward effects of pifithrin. Hence, this work will aid in the future design of more specific members of this important class of p53 inhibitors for use in a clinical setting. The tumor suppressor protein p53 is a transcription factor that functions as a cellular gatekeeper and is often deregulated in human tumors (Hofseth et al., 2004). Although the lack of functional p53 expression is associated with the development of cancers, its up-regulation of the intrinsic apoptotic pathway is implicated in the cell death that occurs during the progression of a number of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease (Waldmeier, 2003) and during chemoand radiotherapies in the normal tissue surrounding the tumors (Gudkov and Komarova, 2003). Given the therapeutic potential of p53 inhibitors, a chemical screen was used to identify pifithrin([2-(2-imino-4,5,6,7-tetrahydrobenzothiazol-3-yl)-1-p-tolyethanone] hydrobromide) as an effective inhibitor of p53-mediated gene activation and apoptosis that was capable of protecting mice from lethal genotoxic stress elicited by gamma irradiation (Komarov et al., 1999). Further developments in the design of p53 inhibitors have identified a series of pifithrinanalogs that display potent neuroprotective effects and show promise in their potential as therapeutic agents to be used to reduce or prevent neurodegeneration and protect the cancer patient from the dehabilitating effects that occur during current chemoand radiotherapies (Zhu et al., 2002). In addition to its promise as a clinical tool, pifithrinhas also proven to be effective in the laboratory using a variety of cell types and apoptotic-inducing agents to characterize p53-mediated events (Lorenzo et al., 2000; Zhu et al., 2002; Kaji et al., 2003; Schafer et al., 2003; Chramostova et al., 2004; Wang et al., 2004). An important consideration of all clinical and laboratory tools is the specificity with which the therapeutic agent interacts with its intended target. With this in mind, we noted the structural similarities between pifithrinand ligands of the aryl hydrocarbon receptor (AhR; Fig. 1), as well as recent observations that ligand activation of the AhR can inhibit apoptosis (Schrenk et al., 2004) and senescence (Ray and Swanson, 2003, 2004), two p53-mediated events, and questioned whether pifithrinmay act as an AhR agonist. The AhR is best characterized as a transcriptional activator of phase I and phase II metabolizing enzymes (Rushmore This work is supported by the National Institutes of Environmental Health Sciences Grant ES 008088. This work in part was presented at the 95th Annual Meeting of the American Association for Cancer Research, 2004 March 27–31; Orlando, FL; and the 43rd Annual Meeting of the Society of Toxicology, 2004 March 21–25; Baltimore, MD. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.084186. ABBREVIATIONS: AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; DRE, dioxin response element; NFB, nuclear factor B; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDF, 2,3,7,8-tetrachlorodibenzo-p-furan; MNF, 3 -methoxy-4 -nitroflavone; DMSO, dimethyl sulfoxide; conDRE, consensus DRE; PCR, polymerase chain reaction; EMSA, electromobility shift assay; MOPS, 3-(Nmorpholino)propanesulfonic acid. 0022-3565/05/3142-603–610$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 314, No. 2 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 84186/3039842 JPET 314:603–610, 2005 Printed in U.S.A. 603 at A PE T Jornals on Jne 1, 2017 jpet.asjournals.org D ow nladed from