ET-18-OCH3 inhibits nuclear factor-kappa B activation by 12-O-tetradecanoylphorbol-13-acetate but not by tumor necrosis factor-alpha or interleukin 1 alpha.
1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is a synthetic diether phospholipid that is competitive with phosphatidylserine binding to the regulatory domain of protein kinase C (PKC). Our previous studies indicate that the selective inhibition of tumor cell growth by ET-18-OCH3 may be due to altered signal transduction mechanisms, 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 regulation of the transcription factor NF-kappa B, which is activated by diverse stimuli. In the 293.27.2 human kidney cell line, as in hematopoietic cells of all lineages, NF-kappa B is stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 alpha (IL-1 alpha). The response to either TNF-alpha or IL-1 alpha is synergistically enhanced by TPA. However, the regulatory mechanisms and signal transduction systems responsible for NF-kappa B activation in response to these different stimuli have not been determined in detail. We have used ET-18-OCH3 and auranofin, which inhibit PKC by different mechanisms, to assess the role of PKC in NF-kappa B activation. ET-18-OCH3 markedly inhibits TPA-induced NF-kappa B activation, as measured by HIV long terminal repeat-directed expression of beta-galactosidase. The IC50 for inhibition by ET-18-OCH3 is approximately 2 microM, a noncytotoxic concentration. Inhibition of TPA-induced NF-kappa B activation was dependent upon preincubation with ET-18-OCH3, and the drug was active at approximately 2 mol% of total cellular phospholipid. ET-18-OCH3 did not inhibit NF-kappa B activation by either TNF-alpha or IL-1 alpha, indicating that there are multiple distinct signal transduction pathways leading to activation of NF-kappa B. We have confirmed these results using auranofin, an antirheumatic drug that is a specific PKC inhibitor interacting with the catalytic domain. 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 NF-kappa B in this response, we measured NF-kappa B by electrophoretic mobility shift assay. Both ET-18-OCH3 and auranofin inhibited cellular induction of the active NF-kappa B complex in response to TPA but not in response to TNF-alpha.(ABSTRACT TRUNCATED AT 400 WORDS)
Daniel
L W
LW
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157-1016, USA.
Civoli
F
F
Rogers
M A
MA
Smitherman
P K
PK
Raju
P A
PA
Roederer
M
M
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001310:Auranofin / Q000494:pharmacology; D002273:Carcinogens / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006678:HIV / Q000235:genetics; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000187:drug effects*; D010742:Phospholipid Ethers / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D012091:Repetitive Sequences, Nucleic Acid; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000970:Antineoplastic Agents; D002273:Carcinogens; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D016328:NF-kappa B; D010742:Phospholipid Ethers; D014409:Tumor Necrosis Factor-alpha; C026659:edelfosine; D001310:Auranofin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate