Ethanol-induced oxidative stress is associated with EGF receptor phosphorylation in MCF-10A cells overexpressing CYP2E1
Introduction
Previous studies by our laboratories have demonstrated that reactive oxygen species (ROS) activate epidermal growth factor receptor (EGFR) signaling pathways in human mammary epithelial cells by superoxide and hydrogen peroxide-dependent mechanisms (Burdick et al., 2003). Activation of EGFR signaling is associated with tumor promotion and progression in epithelial cells (Mill et al., 2009). We have previously worked with redox-cycling quinones derived from benzo(a)pyrene (BaP), which included the 1,6-benzo(a)pyrene quinone and 3,6-benzo(a)pyrene quinone (1,6-BPQ and 3,6-BPQ). These quinones have been found to generate ROS, increase mammary cell proliferation and replace the need for normal growth factors such as EGF, in long term cultures. BPQs increase EGFR tyrosine phosphorylation on several phosphosites leading to downstream cell signaling pathways including phospholipase Cγ and several STAT pathways (Rodríguez-Fragoso et al., 2009).
Recent epidemiological studies have provided convincing evidence that ethanol (EtOH) consumption is associated with an increased risk of breast cancer in women (Chen et al., 2011). The mechanism(s) whereby EtOH increases breast cancer risk is still unclear. Because EtOH can be metabolized by several different pathways, one of which is associated with a microsomal enzyme oxidizing system (MEOS) and CYP2E1 in liver resulting in the formation of superoxide anion (Cederbaum et al., 2009), we were interested in determining whether CYP2E1 metabolism of EtOH can induce ROS formation in human mammary epithelial cells. Therefore, we examined the possibility that CYP2E1-related metabolism of ethanol may form sufficient amounts of superoxide to activate the EGFR in human mammary epithelial cells. The results of these studies show that ethanol increases oxidative stress and EGFR tyrosine phosphorylation in MCF-10A overexpressing CYP2E1.
Section snippets
Chemicals
All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated. 1,6-BPQ and 3,6-BPQ were purchased from the Midwest Research Institute (Kansas City, MO) at >99% purity and maintained as stock solutions in anhydrous tissue culture grade dimethyl sulfoxide (DMSO). The final concentration of DMSO in all experiments was 0.1%. 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester, CM-H2DCFDA, *mixed isomers* (DCF) was purchased from Invitrogen (Carlsbad,
Expression of CYP2E1 protein in MCF-10A cells
Fig. 1 shows the protein expression of CYP2E1 protein in ethanol-treated MCF-10A cells. MCF-10A cells were found to express low levels of CYP2E1, and these levels were not changed following an 18 h exposure to 10, 30 and 100 mM ethanol (Fig. 1). Because the levels of CYP2E1 protein was found to be quite low in MCF-10A as compared to normal human mammary epithelial cells (data not shown), we decided to develop a stable transfectant of MCF-10A cells overexpressing CYP2E1.
Stable transfection of CYP2E1 into MCF-10A cells results in an increase of EtOH-induced oxidative stress
ROS levels from CYP2E1
Discussion
Breast cancer is the most common cancer and the second leading cause of cancer-related mortality among American women (Draper, 2006). The etiology of breast cancer is very diverse and EtOH consumption is a well-established risk factor for breast cancer in women (Rohan et al., 2000, Boffeta and Hashibe, 2006, Smith-Warner et al., 1998, Hamajima et al., 2002, Singletary and Gapstur, 2011). However the mechanisms through which this agent is involved in the development of breast cancer is not fully
Conflict of interest statement
The authors declare that they have no financial or other conflicts of interest to declare for the studies conducted in this manuscript, nor for the conclusions that have been reached.
Acknowledgments
These studies were supported in part by NIH RO1 ES-07259 and by a Mexico CONACYT Fellowship to Angel León-Buitimea number 21416.
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