12(S)-HETE ENHANCEMENT OF PROSTATE TUMOR-CELL INVASION - SELECTIVE ROLE OF PKC-ALPHA

Background: Prostate carcinoma has become the second most fatal cancer in American men. In Dunning R3327 rat prostate adenocarcinoma cells, elevated invasiveness positively correlates with metastatic potential. However, the mechanism(s) responsible for regulation of tumor cell motility and invasion is poorly understood. We have reported that a lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], augments tumor cell metastatic potential through activation of protein kinase C (PKC). Purpose: We proposed to determine the effect of 12(S)-HETE on the motility and invasion of low-metastatic rat prostate AT2.1 tumor cells and the effect of 12(S)-HETE activation of specific PKC isoform(s) in these processes. Methods: The motility of AT2.1 cells was determined by the colloidal gold phagokinetic track assay and the invasiveness measured as their ability to invade through basement membrane Matrigel-coated filters. Expression of PKC isoforms was determined by Western blotting of the whole cell lysate with isoform-specific anti-PKC antibodies. Cytosol and membrane fractions were prepared and the subcellular distribution of PKC was analyzed by Western blotting and activity assay. The effect of 12(S)-HETE on cell proliferation was examined. Data were analyzed for significance of difference with the two-sampled, two-sided Student's t test. Results: 12(S)-HETE increased the motility and invasion of AT2.1 cells, and this 12(S)HETE-increased motility and invasion were inhibited by a selective PKC inhibitor, calphostin C, as well as a Ca-2 chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/tetra(acetoxy-methyl)ester, AT2.1 cells expressed the PKC isoforms alpha and delta, and 12(S)-HETE increased the membrane association of PKC alpha but not delta. Further, the motility and invasion of AT2.1 cells were increased by thymelea toxin, a selective activator of PKC alpha over PKC delta. Conclusion: 12(S)-HETE augments the invasiveness of AT2.1 cells via selective activation of PKC alpha. Implications: 12(S)-HETE modulation of PKC alpha invasiveness may be an important mechanism of action for the regulation of the invasive potential of rat prostate carcinoma cells, and the 12-lipoxygenase enzyme and/or PKC alpha may serve as key targets for the development of anti-invasive agents useful for combating the spread of prostate cancer.