Our inhibitor studies revealed the presence of a signaling n

Our inhibitor studies revealed the presence of a signaling network triggered by dicarbonyl stress. Nevertheless, p38-MAPK activation was clearly necessary for NADPH-oxidase mediated production of ROS. This is consistent with data from other groups using different cell culture systems [37], [38], [39], [40], [41]. ERK1/2, AKT and NF-κB seem to be activated independently, although further interactions between these pathways were demonstrated. As inhibition of NADPH-oxidases by DPI caused an increase in ERK1/2 and IκBα phosphorylation, ROS, most probably the direct NADPH-oxidase product superoxide O2−, were involved in the regulation of these pathways. ERK1/2 and AKT cross talk was shown by the effect of the UO126 inhibitor. We [22] and others [42] have described earlier that GLO1 expression in MCF-7 Nogo-66 (1-40) was not regulated by aldehyde stress. Also TamR-Md cells did express GLO1 to a similar level as the parental MCF-7 cells, however, the decreased amount of the cofactor glutathione would result in decreased glyoxalase-I activity. We therefore proposed that GLO1 activity is a major determinant of dicarbonyl resistance. Consistently, we here observed that inhibition of GLO1 activity by a specific inhibitor or siRNA experiments resulted in increased sensitivity towards the α-oxo-aldehydes. Thus, GLO1 is indeed important for dicarbonyl resistance although the potential contribution of other aldehyde detoxifying enzymes was not analyzed in this investigation. Our hypothesis that GLO1 expression might be associated with tamoxifen resistance of breast cancer was supported by the Kaplan Meier analysis presented here. High GLO1-mRNA abundance was associated with poor prognosis, showing the highest hazard ratio in tamoxifen treated ER-positive cases (Fig. 8). This is consistent with our hypothesis that tamoxifen resistant cells try to escape increased endogenous aldehyde toxicity by increased expression of glyoxalase as GLO1 is activated by these aldehydes via the transcription factor nrf 2 [43]. Noteworthy, MCF-7 cells were unable to further increase GLO1 expression upon exogenous dicarbonyl stress, which seems contradictory to the observation made for real cancer cases. However, other breast cancer cell lines are able to increase GLO1 expression under stress and therefore, the data on one cell line might not be representative for clinical cancer samples. In line with our data, it has been described that tamoxifen induced oxidative stress in a xenograft model [44]. Here, tamoxifen sensitive and -resistant tumors were analyzed for the expression of antioxidant defense enzymes, such as superoxide dismutase (SOD), catalase, glutathione S-transferase (GST), and lipid peroxidation. Besides an increased expression of these enzymes and decreased lipid peroxidation the authors observed a reduction in glutathione content of the tumors, which resembles the situation in our cell culture model. Additionally, several studies have reported that high glutathione S-transferase (GST) expression is associated with poor response to chemotherapy, as these enzymes are also important for detoxification processes [45], [46], [47], [48], [49], [50]. This underlines the importance of the network of oxidative stress, glutathione synthesis as well as glyoxalase activity and maybe also AGE-formation for endocrine related breast cancer biology.
Conclusions
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Acknowledgments We wish to thank Kerstin Werner and Martina Stoklasek for excellent technical assistance. Parts of this work were supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) to T.K. (KA2663/3-1).
Introduction The cellular glyoxalase system detoxifies cytosolic reactive aldehydes that occur with metabolism. It is composed of the enzymes glyoxalase I (GLO1, homodimer)1, 2 and glyoxalase II (GLO2, monomer) and is responsible for the conversion of the methylglyoxal (MG) to d-lactate via the intermediate S-d-lactoylglutathione using catalytic amounts of glutathione (GSH, Scheme 1). MG is a small dicarbonyl that arises primarily through the non-catalytic breakdown of triosephosphates (i.e., dihydroxyacetone phosphate and glyceraldehyde-3-phosphate)5, 6 and from the spontaneous breakdown of glucose. MG has the ability to form advanced glycation end-products (AGEs) on proteins,8, 9 lipids and DNA, resulting in detrimental effects in aging, as well in diseases such as diabetes13, 14, 15 and Alzheimer’s disease,16, 17 where GLO1 protein expression and activity have been shown to be reduced. GLO1 and MG have also been linked to behavioral conditions such as anxiety18, 19, 20 and depression. Distler et al. identified MG as a GABAA receptor agonist and reported that overexpression of GLO1 increases anxiety by reducing levels of MG and pretreatment with MG or inhibition of GLO1 reduced pharmacologically-induced seizures in mice.