Metabolism of arachidonic acid by 12-lipoxygenase results in the formation of 12(S)-hydroxy eicosatetraenoic acid, which exhibits profound biological activity and plays an important role in tumor cell proliferation, motility, invasiveness, angiogenesis, and inhibition of apoptosis, all properties essential for the growth and metastasis of cancer cells. Arachidonate 12-lipoxygenase exists in three isoforms, viz., leukocyte-type, platelet-type, and the epidermis-type. Of these isoforms the platelet-type enzyme is highly expressed in many types of cancers and plays an important role in cancer pathophysiology. In a clinical study involving 122 patients, it was shown that elevation of platelet-type 12-LOX mRNA expression correlates with advanced stage and poor differentiation of human prostate cancer. Given the established role of platelet-type 12-lipoxygenase, hence 12(S)-HETE, in cancer metastasis, inhibition of 12-lipoxygenase is attractive from a therapeutic standpoint and isoform-specific 12-lipoxygenase inhibitors would provide a major breakthrough in chemopreventive therapy. In addition to the 12-lipoxygenase, there are two other major lipoxygenases, viz., 5- and 15-lipoxygenases, of mammalian origin. All lipoxygenases catalyze the oxidation of polyunsaturated fatty acids by an essentially identical mechanism. Based on the knowledge from the X-ray crystallographic studies of plant lipoxygenases and human reticulocyte 15-lipoxygenase, it is evident that subtle differences in the three-dimensional structures of these enzymes are responsible for the regiospecificity of the oxidation along the fatty acid chain. While inhibitors for lipoxygenases have been available for some time, not many isozyme-specific inhibitors are available and efforts to develop such inhibitors have been severely hampered by the paucity of structural information on these enzymes. Our long-term objective is to bridge this information gap by deducing the three-dimensional structures of leukocyte and platelet-type 12-lipoxygenases in our attempts to develop platelet-type 12-lipoxygenase-specific inhibitors as potential anticancer agents. In this proposal, we aim to purify multi milligram quantities of both enzymes from bacterial expression clones to electrophoretic homogeneity and screen several methods to obtain crystals suitable for X-ray crystallographic studies. ? ?
