Cytochrome P450 2E1 (CYP2E1) regulates the response to oxidative stress and migration of breast cancer cells

Travis Leung 0 Ramkumar Rajendran 0 Subir Singh 0 Richa Garva 2 Marija Krstic-Demonacos 1 2 Constantinos Demonacos 0 0 School of Pharmacy and Pharmaceutical Sciences , Stopford Building , University of Manchester , Oxford Road, Manchester M13 9PT , UK 1 School of Environment & Life Sciences, College of Science & Technology , Cockcroft Building , University of Salford , The Crescent, Salford M5 4WT , UK 2 Faculty of Life Sciences , Michael Smith Building , University of Manchester , Oxford Road, Manchester M13 9PT , UK Introduction: The cytochrome P450 (CYP) enzymes are a class of heme-containing enzymes involved in phase I metabolism of a large number of xenobiotics. The CYP family member CYP2E1 metabolises many xenobiotics and pro-carcinogens, it is not just expressed in the liver but also in many other tissues such as the kidney, the lung, the brain, the gastrointestinal tract and the breast tissue. It is induced in several pathological conditions including cancer, obesity, and type II diabetes implying that this enzyme is implicated in other biological processes beyond its role in phase I metabolism. Despite the detailed description of the role of CYP2E1 in the liver, its functions in other tissues have not been extensively studied. In this study, we investigated the functional significance of CYP2E1 in breast carcinogenesis. Methods: Cellular levels of reactive oxygen species (ROS) were measured by H2DCFDA (2 2.9.2 2,7dichlorodihydrofluorescein diacetate) staining and autophagy was assessed by tracing the cellular levels of autophagy markers using western blot assays. The endoplasmic reticulum stress and the unfolded protein response (UPR) were detected by luciferase assays reflecting the splicing of mRNA encoding the X-box binding protein 1 (XBP1) transcription factor and cell migration was evaluated using the scratch wound assay. Gene expression was recorded with standard transcription assays including luciferase reporter and chromatin immunoprecipitation. Results: Ectopic expression of CYP2E1 induced ROS generation, affected autophagy, stimulated endoplasmic reticulum stress and inhibited migration in breast cancer cells with different metastatic potential and p53 status. Furthermore, evidence is presented indicating that CYP2E1 gene expression is under the transcriptional control of the p53 tumor suppressor. Conclusions: These results support the notion that CYP2E1 exerts an important role in mammary carcinogenesis, provide a potential link between ethanol metabolism and breast cancer and suggest that progression, and metastasis, of advanced stages of breast cancer can be modulated by induction of CYP2E1 activity. - Introduction Cytochrome P450 (CYP450) is a superfamily of hemoproteins essential for the biotransformation of drugs [1]. They are mainly localised in the liver, participating in the phase I metabolism of a wide range of exogenous compounds and the biosynthesis and metabolism of endogenous hormones [2]. Apart from the liver, CYPs are also expressed in other tissues such as lung, kidney and hematopoietic tissue [3], and specific isoenzymes of the superfamily have been identified in tumours [4] where they are suggested to affect the response to anticancer therapy [4,5]. CYP450s are highly conserved across species implying that, in addition to their function in the metabolism of xenobiotics, these enzymes possibly exert broader physiological functions [6]. Consistent with this view, the CYP2E1 isoenzyme has been implicated in a variety of pathological conditions such as diabetes, nonalcoholic steatohepatitis (NASH) and cancer, possibly as a result of its capacity to produce high levels of reactive oxygen species (ROS) [7]. CYP2E1 metabolizes several small molecules such as ethanol, acetaminophen and pro-carcinogens like nitrosamines and azo compounds [3]. CYP2E1-mediated metabolism of these compounds generates toxic intermediates and excessive amounts of ROS [7]. High ROS levels, and hence oxidative stress due to increased CYP2E1 protein levels and induced enzymatic activity, are the main causes of various liver diseases associated with chronic alcohol consumption [8] and a variety of other pathophysiological conditions including diabetes type II and obesity [9]. Since CYP2E1 is a key determinant of the cellular redox state generating free radicals in a non-specific manner, even in the absence of a substrate the gene expression of this enzyme is tightly regulated [10]. Indeed, links between CYP2E1 protein levels and cytokines activity have been shown in recent reports [11] as well as variable CYP2E1 gene expression in numerous inflammatory diseases including cancer [12,13]. Autophagy is one of the pathways induced by elevated ROS levels which triggers the accumulation of various autophagyregulated genes (ATGs) including beclin-1 and the light chain 3 (LC3) [14], thereby stimulating the formation of the autophagosome in cancer [15,16]. Furthermore, oxidative stress and other cellular tensions, such as DNA damage and viral infection, impair the protein-folding process resulting in the accumulation of misfolded proteins within the endoplasmic reticulum (ER) lumen [17], stimulating the initiation of the unfolded protein response (UPR) [18]. UPR takes place in the ER lumen and is a major signal transduction pathway aiming to alleviate ER stress by removing accumulated unfolded proteins from this cellular compartment [18]. Clinical studies have indicated that stage I breast tumours express higher CYP2E1 mRNA levels compared to stages II, III and IV [19]. Taken together, the differential expression of CYP2E1 in different tumours and various stages of breast cancer, with its capacity to induce ROS production [7], raises the questions whether CYP2E1 cellular levels could be an indicator of breast cancer progression and which are the factors involved in its differential regulation of gene expression in the various stages of breast cancer. Here we present evidence to suggest that ectopically expressed CYP2E1-mediated oxidative stress regulates autophagy, ER stress and migratory potential and its gene expression is regulated by the p53 tumour suppressor in a cell-type-dependent manner in breast cancer cells. p53) [20,21] and MDA-MB-157 (p53-/-) [20] (obtained from the European Collection of Cell Cultures (ECACC)) were maintained in Dulbeccos modified Eagles medium (Sigma-Aldrich, Gillingham, UK), supplemented with 10% foetal bovine serum (Gibco, Paisley, UK) and 1% penicillin/streptomycin (Lonza, Allendale, NJ, USA) at 37C in a humidified atmosphere containing 5% CO2. Cells were treated with 10 M etoposide (Sigma-Aldrich) for 16 hours, 500 M N-acetylcysteine (NAC) (SigmaAldrich) for 16 hours, 2.5 mM acetaminophen (APAP) (Sigma-Aldrich) for 3 h, 20 M chlormethiazole (CMZ) (Sigma-Aldrich) for 16 h, 1 M bortezomib (Bort) (Selleckchem, Stratech Scientific Ltd., Newmarket, UK) the MCF7 for 24 and the MDA-MB-231 cells for 8 h and 100 mM ethanol for either 24 o (...truncated)