Figure 4.

Multiple HETE metabolites are produced from arachidonic acid, and 5-HETE specifically induces expression of AKR1C2 and AKR1C3 and results in androgen reduction. (A) Hierarchical clustering heat map (left) and table (right) shows the top intracellular metabolites altered in C4-2 cells treated with DMSO (control) or AA (100 μM) for 24 hours as measured by untargeted metabolomics. Data are representative of 6 biological replicates. *Represents compounds further validated by targeted mass spectrometry. (B) Absolute concentrations of arachidonic acid and the major HETE metabolites in C4-2 cells treated with DMSO (vehicle) or 100 μM arachidonic acid. (C) Protein (top panel) and transcript (bottom panel) levels of AKR1C2 and AKR1C3 in C4-2 cells treated with DMSO or 25 μM of the indicated HETEs. (D) AKR1C2 and AKR1C3 protein and transcript expression in C4-2 cells (left panels) and LNCaP cells (right panels) treated with increasing concentrations of 5-HETE for 24 hours. (E) HPLC analysis of 5α-dione, 5α-diol, and AST in C4-2 (top) and LNCaP (bottom) cells treated with DMSO (blue) or 25 μM 5-HETE (red) along with 100 nM [³H]-AD. Experiments were performed in triplicate and represent at least 3 biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant.