Modulation of CYP1A1 by PKC Inhibitors and TPA Pre-Treatments in MH1C1 Rat Hepatoma Cells Exposed to 3 -Methylcholanthrene

Clara De Pascale,Roberta Sanguineti,Sabina Ledda,Barbara Marengo,Mariafrancesca Catalano,Chiara Scanarotti,Susanna Penco,Mariapaola Nitti,Maura Siri,Anna M Bassi,Cinzia Domenicotti

doi:10.2174/1874340400903010047

Abstract

Cytochrome P4501A1 (CYP1A1), an enzyme known to metabolize polycyclic aromatic hydrocarbons, is regulated by the aryl hydrocarbon receptor (AhR). The involvement of protein kinase C (PKC) in the regulation of AhR signal transduction pathway, has been widely studied but the role of specific PKC isoform(s) involved in this process it is not well clarified. To study which PKC isoform(s) is implicated in the regulation of CYP1A1, in the poorly tumorigenic MH1C1 rat hepatoma cells, we examined the effects of some PKC pharmacological inhibitors, Calphostin C (CAL), Staurosporine (STA) and H7, and of 12-0-tetradecanoyl phorbol 13-acetate (TPA), a PKC activator, on basal and 3- methylcholanthrene (MC)-induced CYP1A1 protein expression and mediated ethoxyresorufin O-deethylation (EROD) activity. In parallel, the activities of PKC-α, -βI, -δ and -e isoforms, the most expressed in MH1C1 cells, were monitored. After pre-treatment with CAL, STA and H7, the MC-induced CYP1A1 protein and EROD activity were rapidly reduced with temporal profile similar to the profile of the activity of α and β1 PKC isoforms. Moreover, TPA pre-treatment induced a biphasic effect on EROD activity, and a decline of PKC -βI and -α, in first instance, and -δ and -e activities later on. These findings clearly show that, in MH1C1 cells, PKC is involved in CYP1A1 regulation and that α and βI classic PKC isoforms play an active role in modulating this process.

Highlights

Among cytochrome P450s (CYPs) one of the most widely studied is the Cytochrome P4501A1 (CYP1A1) isoenzyme, that despite its low expression level in many tissues, is highly inducible by polycyclic aromatic hydrocarbons (PAHs) and is able of bioactivating a large number of xenobiotics to toxic derivatives [1,2,3,4].Mechanisms of CYP1A1 induction by the products of the aryl hydrocarbon (Ah)-inducible gene battery, have been widely studied, as reviewed by Whitlock in 1999 [5]
The AhR/ AhR nuclear translocator (Arnt) heterodimer binds to a specific DNA element termed the xenobiotic response element (XRE), in the transcriptional regulatory region to regulate the transcription of AhR target genes including CYP1A1, CYP1A2, CYP1B1, and AhR repressor genes [11,12,13]
We demonstrated that the well differentiated, poorly tumorigenic MH1C1 rat hepatoma cell line is highly responsive to classical CYP1A1 inducers [3739], such as 3-methylcholanthrene (MC); it was reported that MH1C1 cells express protein kinase C (PKC), - and - isoforms, and that their proliferation rate is PKC- and not PKC--dependent [40, 41]

Summary

Introduction

Among cytochrome P450s (CYPs) one of the most widely studied is the CYP1A1 isoenzyme, that despite its low expression level in many tissues, is highly inducible by polycyclic aromatic hydrocarbons (PAHs) and is able of bioactivating a large number of xenobiotics to toxic derivatives [1,2,3,4]. Mechanisms of CYP1A1 induction by the products of the aryl hydrocarbon (Ah)-inducible gene battery, have been widely studied, as reviewed by Whitlock in 1999 [5]. Unliganded AhR exists in a complex with heat shock protein 90 (HSP90), the HSP90 co-chaperone p23, and a hepatitis B virus X-associated protein 2 (XAP2) in the cytoplasm [7,8,9,10]. I.e. PAH, activated AhR is translocated from the cytoplasm into the nucleus where dimerizes with AhR nuclear translocator (Arnt). The AhR/ Arnt heterodimer binds to a specific DNA element termed the xenobiotic response element (XRE), in the transcriptional regulatory region to regulate the transcription of AhR target genes including CYP1A1, CYP1A2, CYP1B1, and AhR repressor genes [11,12,13]

Results

Discussion

Conclusion