Transient induction of cytochromes P450 1A1 and 1B1 in MCF-7 human breast cancer cells by indirubin

Abstract

The aryl hydrocarbon receptor (AhR), when activated by exogenous ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), regulates expression of several phase I and phase II enzymes and is also involved in the regulation of cell proliferation. Several studies suggest that endogenous AhR ligand(s) may exist. One putative endogenous ligand is indirubin, which was recently identified in human urine and bovine serum. We determined the effect of indirubin in MCF-7 breast cancer cells on induction of the activities of cytochromes P450 (CYP) 1A1 and 1B1, as measured by estradiol and ethoxyresorufin metabolism, and on induction of the CYP1A1 and CYP1B1 mRNAs. With 4-hr exposure, the effects of indirubin and TCDD at 10 nM on CYP activity were comparable, but the effects of indirubin, unlike those of TCDD, were transitory. Indirubin-induced ethoxyresorufin-O-deethylase activity was maximal by 6–9 hr post-exposure and had disappeared by 24 hr, whereas TCDD-induced activities remained elevated for at least 72 hr. The effects of indirubin on CYP mRNA induction were maximal at 3 hr. Indirubin was metabolized by microsomes containing cDNA-expressed human CYP1A1 or CYP1B1. The potency of indirubin was comparable to that of TCDD in a CYP1B1-promoter-driven luciferase assay, when MCF-7 cells were co-exposed to the AhR ligands together with the CYP inhibitor, ellipticine. Thus, if indirubin is an endogenous AhR ligand, then AhR-mediated signaling by indirubin is likely to be transient and tightly controlled by the ability of indirubin to induce CYP1A1 and CYP1B1, and hence its own metabolism.

Introduction

The AhR was originally identified and characterized as a xenobiotic receptor, and it has been extensively studied in this context. Many of the toxic effects of environmental contaminants, including polycyclic aromatic hydrocarbons and polyhalogenated dioxins, furans, and biphenyls, are mediated by binding to and activation of the AhR. In its unliganded state, the AhR is found in the cytoplasm in a complex with two molecules of heat shock protein 90 and additional cellular chaperones, ARA9 (also known as AIP1 or XAP2) and p23 (reviewed in [1]). Binding of ligand results in dissociation of this complex and translocation of the AhR to the nucleus, where, together with its dimerization partner, the AhR nuclear translocator, it binds to DREs in the 5′-regulatory regions of a number of genes, thereby enhancing the rates of transcription. These genes, which are often referred to as the Ah gene battery, encode the phase I enzymes, CYP1A1, CYP1A2, and CYP1B1, as well as several phase II enzymes that are involved in the metabolism of xenobiotics and endogenous compounds [2]. The regulation of the Ah gene battery, however, appears to represent only a subset of the physiologic roles of the AhR.

When activated by an exogenous ligand such as TCDD, the AhR is known to affect cell proliferation and cell-cycle control in a cell-specific manner [3]. Ligand-activated AhR was recently shown to interact with the retinoblastoma protein, and to delay cell-cycle progression in 5L hepatoma cells [4] and MCF-7 breast cancer cells [5]. Activation of the AhR by TCDD resulted in suppression of cellular proliferation of 5L hepatoma cells by inducing the cell-cycle inhibitor, p27^Kip1[6]. In MCF-7 cells exposed to benzo[a]pyrene, BRCA-1 protein expression was reduced, concomitant with accumulation of p53 and with cell-cycle arrest [7]. Liganded AhR increased the mRNA levels for the immediate-early response genes, fos and jun, in mouse hepatoma cells [8].

A number of studies have reported that AhR-mediated processes occur in the absence of exogenous AhR ligands. Elevated AhR expression led to increased rates of proliferation of mouse Hepa 1c1c7 hepatoma [9] and human A549 lung [10] cells. Embryonic fibroblasts derived from AhR-null mice showed reduced rates of cell proliferation compared with fibroblasts from controls [11], and primary hepatocyte cultures from AhR-null mice had elevated numbers of cells undergoing apoptosis [12]. CYP1A1 was shown to be induced by altered cell culture conditions such as suspension [13], [14] and hydrodynamic shear [15], and to be induced more than 100-fold during early embryogenesis of the mouse [16]. In HeLa cells cultured without exposure to exogenous ligands, approximately 16% of the AhR pool was found to be tightly associated with the nuclear fraction, suggesting that it is in an activated state and is involved in transcriptional regulation [17]. These studies suggest that, in addition to the known xenobiotic ligands, there may exist an endogenous ligand(s) for the AhR that mediates autocrine or paracrine functions.

There have been numerous investigations with the goal of identifying endogenous AhR ligands [18]. Recently, indirubin and indigo, two high-affinity AhR ligands [19], were isolated from human urine and fetal bovine serum and were shown to elicit AhR-mediated responses [20]. The potency of indirubin exceeded that of TCDD by about 50-fold in a yeast AhR-dependent reporter assay. Although indirubin is not known to be a product of metabolism in any animal species, it is formed spontaneously by the dimerization of oxidation products of indoxyl, a product of hepatic CYP-catalyzed metabolism of indole [21], [22]. In this study, we evaluated the efficacy of indirubin in MCF-7 breast cancer cells in the AhR-regulated transcription of two extra-hepatic, phase I metabolic enzymes, CYP1A1 and CYP1B1. We found that indirubin is a potent inducer of CYP1A1 and CYP1B1, but is rapidly metabolized by the enzymes it induces.