Nordihydroguaiaretic acid (NDGA),
quercetin, eicosatetraynoic
acid (
ETYA),
phenidone, and
esculetin, agents known to inhibit cellular
lipoxygenase (LO) activity, also inhibit human natural killer cell-mediated cytotoxicity (NK-CMC) of K562
tumor target cells (TC) in a dose-dependent fashion. Kinetic analysis demonstrated that LO inhibitors blocked an early event in the activation of the lytic mechanism but did not impair conjugate formation. LO inhibitors also did not affect subsequent
chromium release, indicating that their site of inhibition was the NK cell and not the TC. The
lipoxygenase products 5-hydroperoxyeicosatetraenoic
acid (5-HPETE) and leukotriene-B4 significantly enhanced NK activity, with
5-HPETE being the more effective. Other LO products tested included
15-HPETE and the hydroxy derivatives 15-hydroxyeicosatetraenoic
acid (15-HETE) and
5-HETE. These LO metabolites were either without effect on NK-CMC or inhibitory, depending upon the concentration. Additionally, we examined the ability of
5-HPETE to circumvent the effects of LO inhibitors and found that, in the presence of NDGA,
ETYA or
quercetin,
5-HPETE significantly (p less than 0.001) restored lytic activity. Inhibitors of
LTB4 and
LTC4 synthesis,
diethylcarbamazine and
U-60,257 respectively, produced no inhibition of NK activity. In fact,
U-60,257 significantly (p less than 0.05) enhanced NK-CMC. Previous studies in our laboratory, with a new technique which allows for the separation of NK cells from K562 cells, have shown that K562-treated effector cells are greater than 90% inactivated when retested against fresh K562 in the standard
chromium release assay.
Lipids were extracted from K562-treated,
Percoll-purified LGL and evaluated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). No significant increases were seen in the
arachidonic acid-derived LO products evaluated. Thus, our studies indicate that lipoxygenation may be required in the activation of NK-CMC, possibly as a means to generate
oxygen radicals which have been previously implicated in NK-CMC.