Diverse roles of 2-arachidonoylglycerol in invasion of prostate carcinoma cells: Location, hydrolysis and 12-lipoxygenase metabolism - PubMed (original) (raw)

Diverse roles of 2-arachidonoylglycerol in invasion of prostate carcinoma cells: Location, hydrolysis and 12-lipoxygenase metabolism

Michael P Endsley et al. Int J Cancer. 2007.

Abstract

Endogenous 2-arachidonoylglycerol (2-AG) is antiinvasive in androgen-independent prostate carcinoma (PC-3) cells. Invasion of PC-3 cells is also inhibited by exogenously added noladin ether, a non-hydrolyzable analog of 2-AG. In contrast, exogenous 2-AG has the opposite effect. Cell invasion significantly increased with high concentrations of exogenous 2-AG. In PC-3 cells, arachidonic acid (AA) and 12-hydroxyeicosatetraenoic acid (12-HETE) concentrations increased along with exogenously added 2-AG, and 12-HETE concentrations increased with exogenously added AA. Invasion of PC-3 cells also increased with exogenously added AA and 12(S)-HETE but not 12(R)-HETE. The exogenous 2-AG-induced invasion of PC-3 cells was inhibited by 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP, an inhibitor of 2-AG hydrolysis) and baicalein (a 12-LO inhibitor). Western blot and RT-PCR analyses indicated expression of 12-HETE producing lipoxygenases (LOs), platelet-type 12-LO (P-12-LO) and leukocyte-type 12-LO (L-12-LO), in PC-3 cells. These results suggest that exogenous 2-AG induced, rather inhibited, cell invasion because of its rapid hydrolysis to free AA, and further metabolism by 12-LO of AA to 12(S)-HETE, a promoter of PC cell invasion. The results also suggest that PC-3 cells and human prostate stromal (WPMY-1) cells released free AA, 2-AG, and 12-HETE. In the microenvironment of the PC cells, this may contribute to the cell invasion. The 2-AG hydrolysis and concentration of 2-AG in microenvironment are critical for PC cell's fate. Therefore, inhibitors of 2-AG hydrolysis could potentially serve as therapeutic agents for the treatment of prostate cancer. (c) 2007 Wiley-Liss, Inc.

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Figures

Figure 1

Metabolism of 2-AG in PC-3 cells. (a) Diagram depicting the hydrolysis of 2-AG to AA and the 12-LO metabolism of AA to 12-HETE. (b) Chromatogram of exogenous [14C]2-AG incubated with PC-3 cells for 30 min indicating the formation of AA and more polar products including a radiolabeled peak that comigrates with the 12-HETE standard. (c) Effects of OTFP (1 μM) and exogenous 2-AG (1 μM) on the concentrations of free AA in PC-3 cells. (d) Effects of OTFP (1 μM), baicalein (10 μM) and exogenous 2-AG (1 μM) on the concentrations of 12-HETE in PC-3 cells. Values are mean ± S.E.M. (n = 4–6). *, significantly lower than control, p < 0.05; #, significantly higher than control, p < 0.005; $, significantly lower than 2-AG treatment, p < 0.005.

Figure 2

Stereoisomers of 12-HETE. (a) Chromatogram of 12(R)-HETE and 12(S)-HETE standards separated on a chiral column and detected by UV detection. (b) Chromatogram of 15-, 11-, 12- and 5-HETE separated on a reverse phase C18 column and detected by ESI-MS. Note: The LC-ESI-MS technique used in this study cannot differentiate the 12-HETE stereoisomers. Chromatograms of (c) 12(S)-HETE and (d) 12(R)-HETE in PC-3 cells treated with exogenous 2-AG (1 μM). 12-HETE mixtures were first separated on an HPLC chiral column as in (a) and subsequently analyzed by LC-ESI-MS.

Figure 3

Expression of L-12-LO and P-12-LO in PC-3 cells. (a) Western blots depicting immunoreactive bands corresponding to L-12-LO (at 75 kDa of molecular weight markers). Lane 1 = L-12-LO protein standard and Lane 2 = PC-3 cell lysate. The upper blot was probed by a primary antibody (Cayman) that cross reacts with human L-12-LO and the lower blot was probed with the antibody raised in our laboratory against the NH2 terminus peptide sequence for the L-12-LO. (b) RT-PCR blots for P-12-LO mRNA in PC-3 cells. Left blot indicates the markers (Lane 1), blank negative control (Lane 2), and GAPDH (Lane 3) corresponding to marker of 284 bp. Right blot indicates the markers (Lane 1), negative control without reverse transcriptase (Lane 2), and the radioactive band of P-12-LO (Lane 3) corresponding to marker of 159 bp.

Figure 4

Invasion of PC-3 cells treated with exogenous 2-AG, AA and 12-HETE. (a) Invasion of PC-3 cells treated with exogenous 2-AG at various concentrations (0.1–10.0 μM) and OTFP (1 μM). (b) Effect of exogenous AA at various concentrations (0.1–10.0 μM) and baicalein (10 μM) on invasion of PC-3 cells. (c) Effect of exogenous 12(S)-HETE at various concentrations (0.1–10.0 μM) on invasion of PC-3 cells. (d) Effect of exogenous 12(R)-HETE at various concentrations (0.1–10.0 μM) on invasion of PC-3 cells. Invasion was normalized to the control cells. Values are mean ± SEM (n = 12–18). *, significantly lower than control with p < 0.01; #, significantly higher than control with p < 0.005.

Figure 5

Release of 2-AG, AA, and 12-HETE by PC-3 cells and WPMY-1 cells and effects of conditioned media on invasion of PC-3 cells. (a) LC-MS chromatograms of 12-HETE, 2-AG and AA standards (upper panel), in conditioned media of PC-3 cells (middle panel), and in conditioned media of WPMY-1 cells (lower panel), respectively. (b) Effects of PC-3 cell conditioned media at 25, 50 and 100% on invasion of PC-3 cells. (c) Effects of WPMY-1 cell conditioned media at 25, 50 and 100% on invasion of PC-3 cells. Invasion was normalized to the control cells. Values are mean ± SEM (n = 6–12). #, significantly higher than control with p < 0.005.

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Diverse roles of 2-arachidonoylglycerol in invasion of prostate carcinoma cells: Location, hydrolysis and 12-lipoxygenase metabolism - PubMed (original) (raw)