Inhibitory effect of a callophycin A derivative on iNOS expression via inhibition of Akt in lipopolysaccharide-stimulated RAW 264.7 cells.

In previous studies, (R)-2-isobutyl 3-methyl 3,4-dihydro-1H-pyrido[3,4-b]indole-2,3(9H)-dicarboxylate (1), a callophycin A derivative, was found to strongly inhibit nitrite production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, while (R)- or (S)-callophycin A showed only weak inhibition. We currently report additional studies to define the mechanisms underlying the inhibitory action of 1. Expression of inducible nitric oxide synthase (iNOS) was reduced at both protein and mRNA levels. Major upstream signaling molecules and transcription factors regulating iNOS expression were examined, but it was found that 1 did not affect the phosphorylated and total protein levels of p38 mitogen-activated protein kinase (p38 MAPK), Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and signal transducer and activator of transcription 1 (STAT1), nor did it mediate the degradation of the inhibitor of nuclear factor-κB α-isoform (IκBα). However, starting at early time points, 1 consistently inhibited the phosphorylation of protein kinase B/Akt at serine 473. In addition, 1 suppressed the protein expression of octamer-binding transcription factor-2 (Oct-2) and the expression of microRNA 155 (miR-155). In sum, compound 1 inhibits LPS-induced nitrite production by a unique and complex mechanism. Reduction of iNOS expression is accompanied by inhibition of Akt activation, Oct-2 protein expression, and miR-155 expression.