The role of 15-LOX-1 in cancer is complex because the biological activity is highly tissue specific with opposing activity observed in different tissues. In human prostate cancer, the expression of 15-LOX-1 is associated with an increase in prostate cancer (22
). In contrast, 15-LOX-1 appears to act as a “tumor suppressor” in human colorectal cancer (3
). Restoring 15-LOX-1 expression induces apoptosis (26
) and inhibits the growth of human colorectal cells (3
). Investigations from several laboratories (29
) suggest that an increase in the expression of 15-LOX-1 by a number of NSAIDs or COX inhibitors may contribute to the cancer prevention by these drugs. The mechanism by which 15-LOX-1 alters colorectal cancer is not fully understood and several possible mechanisms have been examined. Recently, we reported that the expression of 15-LOX-1 increases the activity of the tumor suppressor p53 by increasing the phosphorylation at Ser15, an important phosphorylation site in the activation of p53 by kinases. One interesting, but unexplained, observation was the inactivity of 13(S)-HODE or 15(S)-HETE, which are the primary metabolites of 15-LOX-1, to increase p53 phosphorylation in HCT-116 cells (4
). Incubation of 15-LOX-1 expressing HCT-116 cells with the substrates arachidonic acid or linoleic acid did increase the formation of metabolites, but did not result in an increase in the phosphorylation of p53, suggesting that metabolism is not required for the p53 activation (data not shown). This finding rules out the metabolites of 15-LOX-1 pathway including secondary metabolites, such as 4-oxo-2-nonenal, as responsible for the increase in p53 phosphorylation. 4-oxo-2-nonenal (4-ONE), one of secondary metabolites from hydroperoxide metabolites of 15-LOX-1 (32
), can induce phosphorylation of p53 in human neuroblastoma SH-SY5Y cells (34
) and we observed a similar response in HCT-116 cells (data not shown).
These data suggest the possibility for the 15-LOX-1 protein to act directly, independent of its enzymatic activity, in the activation of the p53 pathway via an increase in p53 phosphorylation. To test this hypothesis, we prepared a mutant 15-LOX-1 devoid of enzymatic activity. With stably transfected HCT-116 cells, we observed an increase in p53 phosphorylation that correlated with the expression levels of the native and mutant 15-LOX-1 expression. The ratio of p53 phosphorylation to 15-LOX-1 expression in mutant 15-LOX-1 expression clones was approximately the same in wild type 15-LOX-1 expression clones. Secondly, transient transfection of wild type and mutant 15-LOX-1 plasmid into HCT-116 cells increased the phosphorylation of p53 dependent on the level of 15-LOX-1 protein expressions. In addition the expression of 15-LOX-1 increased the expression of p21, NAG-1 and MDM2 all targets of p53. In addition, we observed similar effects in the human lung cells, A-549 which are wild-type for p53. Treatment of these cells with IL-4 induces 15-LOX-1 expression and induces apoptosis (21
). The presence of 15-LOX-1 in A-549 cells treated with IL-4 was confirmed and we observed an increase in the expressions of p21, phosphorylated p53 at Ser15, and MDM2. Thus the activation of p53 by 15-LOX-1 was observed in cells not genetically modified that physiologically express 15-LOX-1 and in cells engineered to overexpress 15-LOX-1. These data support the hypothesis that the expression of 15-LOX-1 protein induces p53 phosphorylation independent of its enzymatic activity.
Our previous data suggested that DNA-PK was required for p53 phosphorylation at Ser15 and is responsible for the increase in p53 phosphorylation observed in 15-LOX-1 expressing cells. In this report, DNA-PK siRNA and the DNA-PK inhibitor Wortmannin, confirmed the involvement of DNA-PK in p53 phosphorylation through overexpression of 15-LOX-1. The inhibition of DNA-PK resulted in a reduction in phosphorylation of p53. Incubation of the cells with the siRNA also inhibited the 15-LOX-1-induced increase in p53 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrated a physical association between DNA-PK and 15-LOX-1, with the binding of 15-LOX-1 to DNA-PK cs resulting in an increase in the kinase activity. Confocal microscopy showed DNA-PK cs and 15-LOX-1 co-localized in 15-LOX-1 expressing HCT-116 cells (). The co-localization of 15-LOX-1 and DNA-PK cs and the enhanced kinase activity observed in vitro suggest that 15-LOX-1 directly activates the kinase. The nuclear localization of 15-LOX-1 () is particularly interesting for several reasons. Nonsteroidal anti-inflammatory drugs (NSAIDs) induced the expression of 15-LOX-1 and stimulated apoptosis. These findings were observed only at high concentrations of metabolites (27
). Therefore, nuclear 15-LOX-1 might interact directly with nuclear protein DNA-PK to achieve activation of p53. Although 15-LOX-1 is a cytosolic enzyme, it can associate with the cell membrane (35
). For example, the rabbit reticulocyte-type 15-LOX (15-LOX-1) associates with biomembranes in a calcium-dependent manner (36
). 15-LOX-1 also has been found both in the cytosol and the plasma membrane in non-stimulated human dendritic cells in the absence of calcium (37
). In our laboratory, early studies using human colorectal carcinoma cells demonstrated that 15-LOX-1 was detected in both nuclear and microsomal fractions, with very little in the cytosol in the presence of calcium (10
). The mechanism of translocation of 15-LOX-1 is not clear, although the N-terminal C2-like domain in the enzyme is thought to be responsible for membrane binding in a calcium dependent manner (38
). In this study, the findings that 15-LOX-1 and DNA-PK co-localized in nuclei in 15-LOX-1 expressing cells raises the question of whether DNA-PK may act as a transporter for the 15-LOX-1. We suspect that the translocation of 15-LOX-1 might be regulated by DNA-PK interaction. However, we could not find any reports in the literature on the binding of 15-LOX-1 with other proteins. Our results clearly demonstrate that 15-LOX-1 directly interacts and binds with the kinase DNA-PK.
The binding of lipoxygenases with other proteins is not without precedent. 5-lipoxygenase is reported to interact with coactosin-like proteins, and this interaction appears to play a modulatory role in actin dynamics (39
). Platelet type 12-LOX is distributed in the membrane fraction (40
) and may interact with some cytoskeletal proteins such as keractin and lamin (41
). However, we could not find any reports in the literature on the binding of 15-LOX-1 with other proteins. Our results clearly demonstrate that 15-LOX-1 interacts and binds with the kinase DNA-PK. The binding of 15-LOX-1 to DNA-PK increases the kinase activity and leads to increased phosphorylation, and presumably, activation of p53. This represents a new and novel mechanism for the activation of a tumor suppressor, p53, by a lipid metabolizing enzyme.