TUMOR CELL-DERIVED 12(S)-HYDROXYEICOSATETRAENOIC ACID INDUCES MICROVASCULAR ENDOTHELIAL-CELL RETRACTION

Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] indu ced a nondestructive and reversible retraction of cultured endothelial cells. In the current study we tested the hypothesis that tumor cells produce 12(S)-HETE during their interactions with endothelial cells w hich in turn induces endothelial cell retraction. Coincubation of Lewi s lung carcinoma cells or elutriated B16 amelanotic melanoma (B16a) ce lls but not 3T3 fibroblasts with microvascular endothelial cells (CD3) resulted in a time- and concentration-dependent retraction of the CD3 monolayers as revealed by quantitative binding assays and phase contr ast microscopy. Lewis lung carcinoma cell-induced endothelial cell ret raction was blocked by specific lipoxygenase inhibitors but not by cyc looxygenase inhibitors, suggesting the involvement of a lipoxygenase m etabolite(s). Radioimmunoassay and high-performance liquid chromatogra phy analysis of tumor cell extracts identified 12(S)-HETE as the major lipoxygenase metabolite of arachidonic acid and tumor cell generation of 12(S)-HETE was specifically blocked by a select 12-lipoxygenase in hibitor N-benzyl-N-hydroxy-5-phenylpentamide. The identity and stereoc hemistry of tumor cell-derived 12-HETE was substantiated by gas chroma tography-mass spectrometry analysis and chiral phase high-performance liquid chromatography, respectively. Lewis lung carcinoma cell adhesio n to CD3 monolayers was accompanied by an enhanced 12(S)-HETE biosynth esis by tumor cells, which paralleled the tumor cell-induced endotheli al cell retraction in a cell number-dependent manner. Pretreatment of tumor cells with N-benzyl-N-hydroxy-5-phenylpentamide inhibited both i ncreased 12-(S)-HETE biosynthesis and tumor cell-induced endothelial c ell retraction. Highly metastatic variants of elutriated B16a cells wh ich had been shown to produce large quantities of 12(S)-HETE induced s ignificant CD3 cell retraction, while low metastatic subpopulations of B16a cells which synthesized no or little 12(S)-HETE did not induce e ndothelial cell retraction. These results suggest that 12(S)-HETE synt hesis during tumor cell-endothelial cell interactions may represent a key contributory factor in cancer metastasis.