Silencing of glyoxalase I defines a role in apoptosis in LNCaP and PC3 human prostate cancer cells.

SILENCING OF GLYOXALASE I DEFINES A ROLE IN APOPTOSIS IN LNCaP AND PC3 HUMAN PROSTATE CANCER CELLS Glyoxalase I (GI) and Glyoxalase II (GII) constitute the glyoxalase system, a ubiquitous detoxification pathway protecting against cellular damage caused by potent cytotoxic metabolites such as methylglyoxal (MG). MG, a highly reactive alpha-oxoaldehyde, generated by oxidation of carbohydrate and glycolysis, binds to proteins and forms advanced glycation end products (AGE). Formation of AGE contributes to the development of pathological conditions such as diabetes and cancer. MG is converted to S-D-lactoylglutathione (SLG) by GI, with reduced glutathione as a cofactor, and GLS in turn is hydrolyzed by GII to D-lactate along with generation of reduced glutathione. MG is a cytotoxic metabolite, potent inhibitor of cellular growth and capable of inducing apoptosis (1). GI inhibitors have been proposed as potential anti-cancer agents in vivo, inducing intracellular increased of cytotoxic MG. Furthermore, most tumor cells displayed increased expression of GI, suggesting its involvement in cellular growth regulation. Furthermore, a possible involvement of GI in apoptosis mediated by MG-derived AGE, has been proposed (2). In order to further elucidate the biological function of GI in the regulation of cell proliferation, and or apoptosis, RNA silencing of GI gene by RNA interference has been performed in human prostate non aggressive and metastatic cell line, LNCaP and PC3, respectively. A comparative study of cell proliferation and apoptosis was performed, analyzing incorporation [3H]thymidine and flow cytometry, 72 h after GI silencing. AGE levels were further assessed by Western blot. The results show that GI silencing has not effect on cell growth of the two cell lines, but induces apoptosis, mediated by MG-derived AGE, in PC3 cells. On the contrary, neither apoptosis nor AGE significant modification were observed in LNCaP cells, after GI silencing. In order to evaluate if the AGE increased, observed in PC3 cell line, was effectively related to MG, the cells were treated for 72 h with a concentration of 0.8 and 1 mM of MG in absence or presence of GI silencing. A comparative evaluation of the possible effect of MG on cell proliferation and apoptosis was performed, assessing the same assay on LNCaP cells. The evaluation of cell proliferation, apoptosis and AGE levels did not show any effect of MG on cell proliferation, but shows an induction of apoptosis in both cell lines, significantly increased in presence of GI silencing. However, the degree of the biological apoptotic effect induced by MG, and the dose related to the activation of the response, different in the two cell lines, could suggest different apoptotic mechanisms. The evaluation of protein levels of caspase3, Bax, BCl2 and BCl-XL, independent from AGE in PC3 and related to them in LNCaP cell lines, could seem to confirm this hypothesis. The apparent paradox in the obtained results could be explained on the basis of the different susceptibility of the two cell lines to MG. Its intracellular concentration, increased after GI knockdown or MG exposure or both, could be different and trigger different AGE levels. The present study pointed out, for the first time, the involvement of GI in the apoptosis process of adenocarcinoma cells and provides further evidence on the different susceptibility of cells to MG (2). 1. de Hemptinne V, Rondas D, Toepoel M, Vancompernolle K. Phosphorylation on Thr-106 and NO-modification of glyoxalase I suppress the TNF-induced transcriptional activity of NF-kappaB. Mol Cell Biochem. 2009,325(1-2):169-178. 2. Van Herreweghe F, Mao J, Chaplen FWR, Grooten J, Gevaert K, Vandekerckhove J, Vancompernolle K: Tumor necrosis factor induced modulation of glyoxalase I activities through phosphorylation by PKA results in cell death and is accompanied by the formation of a specific methylglyoxal-derived AGE. PNAS 99(2): 949–954, 2001.