Background: We have previously demonstrated that the metastatic potential of tumor cells can be increased by treatment with exogenous 12(S)hydroxyeicosatetraenoic acid [12(S)-HETE], a lipoxygenase metabolite of arachidonic acid. However, the biosynthesis of the authentic lipid mediator by tumor cells, and especially the correlation of its biosynthesis to tumor cell metastatic capacity have not been characterized. In addition, a role for other mono HETEs in influencing tumor cell metastatic behavior has been suggested, but conclusive evidence is lacking. In this study, we analyzed the biosynthesis of mono HETEs from arachidonic acid in tumor cells of different metastatic ability and correlated biosynthesis to metastatic potential.
Experimental design: The biosynthesis of mono HETEs by low and high metastatic subpopulations of B16 amelanotic melanoma (B16a) cells was analyzed by high performance liquid chromatography (HPLC). The identity of biosynthetic 12-HETE was confirmed by gas chromatography/mass spectrometry (GC/MS) and its stereochemical structure assigned by chiral phase HPLC. The effect of a lipoxygenase inhibitor on the biosynthesis of mono HETEs and its effect on metastatic behavior was examined.
Results: HPLC analysis revealed that low (LM180) and high (HM340) metastatic B16a cells exhibited different profiles and efficiencies for conversion of arachidonic acid to mono HETEs. LM180 cells produced equal quantities of 12-HETE and 5-HETE. In contrast, HM340 cells synthesized predominantly 12-HETE and small amounts of 15-, 11- and 5-HETEs. At equal concentrations of substrate, four times more 12-HETE was synthesized by HM340 cells than by LM180 cells. The identity of biosynthetic 12-HETE was confirmed by gas chromatography/mass spectrometry and chiral phase HPLC demonstrated that it was the S enantiomer. The biosynthesis of 12(S)-HETE, but not other HETEs, was significantly inhibited by a lipoxygenase inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide. N-benzyl-N-hydroxy-5-phenylpentanamide, in a dose-dependent manner, decreased the adhesion of HM340 cells to murine pulmonary microvessel endothelium in vitro and lung colony formation in vivo. Furthermore, re-introduction of 12(S)-HETE, but not other mono HETEs, to HM340 cells pretreated with N-benzyl-N-hydroxy-5-phenylpentanamide, increased their adhesion to endothelium.
Conclusions: Biosynthesis of 12(S)-HETE by tumor cells is a determinant of their metastatic potential and inhibition of 12(S)-HETE biosynthesis in tumor cells may be a crucial target for intervening in metastasis.