ET-18-OCH3 INHIBITS NUCLEAR FACTOR-KB ACTIVATION BY 12-O-TETRADECANOYLPHORBOL-13-ACETATE BUT NOT BY TUMOR-NECROSIS-FACTOR-ALPHA OR INTERLEUKIN-1-ALPHA

-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is a s ynthetic diether phospholipid that is competitive with phosphatidylser ine binding to the regulatory domain of protein kinase C (PKC), Our pr evious studies indicate that the selective inhibition of tumor cell gr owth by ET-18-OCH3 may be due to altered signal transduction mechanism s, including the inhibition of PKC. To further define the mechanism of action of ET-18-OCH3, we have used it to study the role of PKC in reg ulation of the transcription factor NK-kappa B, which is activated by diverse stimuli. In the 293.27.2 human kidney cell line, as in hematop oietic cells of all Lineages, NK-kappa B is stimulated by 12-O-tetrade canoylphorbol-13-acetate (TPA), tumor necrosis factor-alpha (TNF-alpha ), and interleukin-1 alpha (IL-1 alpha). The response to either TNF-al pha or IL-1 alpha is synergistically enhanced by TPA. However, the reg ulatory mechanisms and signal transduction systems responsible for NK- kappa B activation in response to these different stimuli have not bee n determined in detail. We have used ET-18-OCH3 and auranofin, which i nhibit PKC by different mechanisms, to assess the role of PKC in NK-ka ppa B activation, ET-18-OCH3 markedly inhibits TPA-induced NK-kappa B activation, as measured by HIV long terminal repeat-directed expressio n of beta-galactosidase. The IC50 for inhibition by ET-18-OCH3 is appr oximately 2 mu M, a noncytotoxic concentration. Inhibition of TPA-indu ced NK-kappa B activation was dependent upon preincubation with ET-18- OCH3, and the drug was active at approximately 2 mol% of total cellula r phospholipid, ET-18-OCH3 did not inhibit NK-kappa B activation by ei ther TNF-alpha or IL-1 alpha, indicating that there are multiple disti nct signal transduction pathways leading to activation of NK-kappa B. We have confirmed these results using auranofin, an antirheumatic drug that is a specific PKC inhibitor interacting with the catalytic domai n. Like ET-18-OCH3, auranofin blocked NF-kappa B activation by TPA but not by TNF-alpha or IL-1 alpha. Also like the ether lipid, auranofin only partially blocked the synergy exhibited by TPA and TNF-alpha. To confirm the role of NK-kappa B in this response, we measured NK-kappa B by electrophoretic mobility shift assay, Both ET-18-OCH3 and auranof in inhibited cellular induction of the active NK-kappa B complex in re sponse to TPA but not in response to TNF-alpha. Together, these data d efine distinct pathways leading to NF-kappa B activation. One pathway, induced by TPA, is PKC dependent; the other, induced by TNF-alpha or IL-1 alpha, is PKC independent. ET-18-OCH3 inhibits only the PKC-depen dent pathway and may be useful as a biological response modifier in co mbination with other chemotherapeutic agents.