Induction of nitric oxide production by the cytostatic macrolide apicularen A [2,4-heptadienamide, N-[(1E)-3-[(3S,5R,7R,9S)-3,4,5,6,7,8,9,10-octahydro-7,14 dihydroxy-1-oxo-5,9-epoxy-1H-2-benzoxacyclododecin-3-yl]-1 propenyl]-, (2Z,4Z)-(9CI)] and possible role of nitric oxide in apicularen A-induced apoptosis in RAW 264.7 cells.

We previously reported that apicularen A [2,4-heptadienamide, N-[(1E)-3-[(3S,5R,7R,9S)-3,4,5,6,7,8,9,10-octahydro-7,14 dihydroxy-1-oxo-5,9-epoxy-1H-2-benzoxacyclododecin-3-yl]-1 propenyl]-, (2Z,4Z)-(9CI)], a highly cytostatic macrolide isolated from the myxobacterial genus Chondromyces, induces apoptosis in the mouse leukemic monocyte cell line RAW 264.7. To analyze the action mechanism of apicularen A for the induction of apoptosis, effects of apicularen A on nitric oxide (NO) production in RAW 264.7 cells were examined. It was demonstrated that apicularen A at 10 and 100 nM induced nitrite production, whereas apicularen B [2,4-heptadienamide, N-[(1E)-3-[(3S,5R,7R,9S)-7-[[2-(acetylamino)-2-deoxy-beta-d-glucopyranosyl]oxy]-3,4,5,6,7,8,9,10-octahydro-14-hydroxy-1-oxo-5,9-epoxy-1H-2-benzoxacyclododecin-3-yl]-1 propenyl]-, (2Z,4Z)-(9CI)], an N-acetyl-glucosamine glycoside of apicularen A, had no effect at 100 nM. The apicularen A-induced nitrite production was accompanied by an increase in the level of inducible nitric-oxide synthase (iNOS) and its mRNA and was suppressed by the NOS inhibitor N(G)-monomethyl-l-arginine acetate (l-NMMA). In addition, apicularen A activated nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) and decreased the level of IkappaB-alpha and increased that of phosphorylated c-Jun N-terminal kinase (JNK). Furthermore, the apicularen A-induced nitrite production was suppressed by the NF-kappaB inhibitor Bay 11-7082 [(E)-3-(4-methylphenylsulfonyl)-2-propenenitrile] and the JNK inhibitor SP600125 [anthra[1,9-cd]pyrazol-6(2H)-one]. These findings suggested that apicularen A activates NF-kappaB and AP-1, thus triggering the expression of iNOS mRNA and iNOS protein and induces NO production. Finally, apicularen A decreased cell growth and survival and cell viability and disrupted the mitochondrial membrane potential. The addition of l-NMMA partially recovered the apicularen A-induced decrease in cell growth and survival and cell viability and the disruption of mitochondrial membrane potential. These findings suggested that NO produced by apicularen A treatment participate partially in the apicularen A-induced apoptosis in RAW 264.7 cells.