Cytochromes(CYP) P450 are class of heme-containing enzymes involved in Phase I metabolism of a large number of xenobiotics. The CYP family member CYP2E1 is involved in the metabolism of many xenobiotics and procarcinogens including ethanol, acetone, nitrosamine, pyridine, isoniazid and carbon tetrachloride (CCl4), which are also CYP2E1-inducing agents. CYP2E1 comprises approximately 7% of the liver CYP content and it is also expressed in kidney, lung, brain, gastrointestinal tract and breast tissue implying that this enzyme is implicated in other biological processes aside from its role in Phase I metabolism. Several studies converge to the conclusion that CYP2E1 is induced under many pathological conditions including cancer, obesity, and type 2 diabetes. Increased hepatic ketogenesis and insulin resistance are the possible mechanisms mediating CYP2E1 induction in these conditions. Elevated CYP2E1 expression has been reported in the presence of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha). CYP2E1 generates the highest level of reactive oxygen species (ROS) among the CYP450 superfamily and transient overexpression of this CYP family member in COS cells increases the production of mitochondrial ROS, even in the absence of substrate. Oxidative stress induced by CYP2E1 disturbs the folding capacity of the endoplasmic reticulum (ER), with concomitant alterations in the mRNA and protein expression of the ER stress proteins GRP78 and GRP94. Initially the unfolded protein response (UPR) restores ER homeostasis, and cell viability, but at later stages the accumulation of unfolded proteins stimulates pro-apoptotic signals. Cell death induced by ER stress has been reported in several conditions including hypoxia, and diseases such as diabetes and heart disease. To gain better understanding of the factors regulating CYP2E1 gene expression in breast cancer cells we studied CYP2E1 mRNA and protein levels in MCF7 (p53wt) and MDA-MB-231 (p53 mutated) breast cancer cells and identified that CYP2E1 was under p53 and HIF-1alpha transcriptional control in both of these cell lines treated with etoposide or desferrioxamine respectively. In addition, CYP2E1 was differentially expressed in the low metastatic potential MCF7 cell line compared to highly metastatic MDA-MB-231 cells in accord with clinical studies indicating that CYP2E1 isoenzyme is expressed in lower levels in patients at clinical stages II, III, and IV which exhibit higher metastatic potential than in patients at stage I of breast cancer. To further investigate the functional significance of the difference in CYP2E1 levels the generation of ROS in breast cancer cells ectopically overexpressing CYP2E1 or in cells in which CYP2E1 expression had been silenced was assessed. Our results indicated that CYP2E1 overexpression resulted in higher ROS generation, which coincided with increased autophagy biomarker expression, increased endoplasmic reticulum stress, detected by XBP1 mRNA splicing in fluorescent reporter assays, and inhibition of metastatic potential. These studies show that CYP2E1 exerts an important role in breast cancer cells by inducing generation of oxidants the levels of which differentially influence the unfolding protein response and apoptosis in high and low metastatic potential breast cancer cells and suggest that targeting CYP2E1 might be a powerful approach to modulate breast cancer initiation, progression, and metastasis.
|Date of Award||1 Aug 2013|
- The University of Manchester
|Supervisor||Costas Demonacos (Supervisor) & Kaye Williams (Supervisor)|