In the mouse leukemic monocyte cell line RAW 264.7, the vacuolar-type (H ϩ )-ATPase (V-ATPase) inhibitors bafilomycin A1 and concanamycin A induced nitric oxide (NO) production through the expression of inducible nitric-oxide synthase mRNA and its protein and decreased cell growth and survival as determined by 3-(4,5-dimethyl(thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Bafilomycin A1 and concanamycin A activated nuclear factor (NF)-B and activator protein-1 and decreased the level of IB-␣ and increased that of phosphorylated c-Jun N-terminal kinase (JNK). NO production induced by these V-ATPase inhibitors was suppressed by the NF-B inhibitor Bay 11-7082 [(E)3-[(4-methylphenyl)sulfonyl])-2-propenenitrile] and the JNK inhibitor SP600125 [anthra[1,9-cd]pyrazol-6(2H)-one] in parallel with the partial alleviation of the V-ATPase inhibitor-induced decrease in MTT response. The Na ϩ ,K ϩ -ATPase inhibitor dibucaine and the F-ATPase inhibitor oligomycin did not induce NO production at which concentrations the MTT response was decreased. The NO donor Snitroso-N-acetyl-DL-penicillamine further lowered the V-ATPase inhibitor-induced decrease in the MTT response, and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (carboxy-PTIO) alleviated it partially. Mitochondrial depolarization, an index of apoptosis, was induced by bafilomycin A1 and concanamycin A. On treatment with the nitric-oxide synthase inhibitor N G -monomethyl-L-arginine acetate, the disruption of mitochondrial membrane potential induced by bafilomycin A1 and concanamycin A was alleviated partially in parallel with the decrease in NO production. Carboxy-PTIO also alleviated it partially. Our findings suggest that the V-ATPase inhibitors bafilomycin A1 and concanamycin A similarly induce NO production and the newly produced NO participates partially in the V-ATPase inhibitorinduced apoptosis in RAW 264.7 cells.

-Treatment of the mouse leukemic cell line RAW 264 with bafilomycin A1 or concanamycin A, inhibitors of vacuolar-type (H + )-ATPases (V-ATPases), significantly increased the production of reactive oxygen species (ROS) and decreased cell viability. These effects were significantly suppressed by the presence of N-acetyl cysteine (NAC), an ROS scavenger. si-RNA mediated knockdown of the gene for the c subunit of the V0 domain of V-ATPase also resulted in an increase in ROS production and a decrease in cell viability. These results suggest that decreased cellular V-ATPase activity decreases cell viability by increasing ROS production in RAW 264 cells.

Apicularen A and the known vacuolar-type (H + )-ATPase (V-ATPase) inhibitor bafilomycin A 1 induced apoptosis of RAW 264.7 cells, while apicularen B, an N-acetyl-glucosamine glycoside of apicularen A, was far less effective. Apicularen A inhibited vital staining with acridine orange of the intracellular organelles of RAW 264.7 cells, inhibited the ATP-dependent proton transport into inside-out microsome vesicles, and inhibited the bafilomycin A 1 -sensitive ATP hydrolysis. The IC 50 values of the proton transport were 0.58 nM for apicularen A, 13 nM for apicularen B, and 0.95 nM for bafilomycin A 1 . Furthermore, apicularen A inhibited the bafilomycin A 1 -sensitive ATP hydrolysis more potently than apicularen B. F-ATPase and P-ATPase were not inhibited by apicularen A. We concluded that apicularen A inhibits V-ATPase, and thus induces 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) 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-B (NF-B) and activator protein-1 (AP-1) and decreased the level of IB-␣ and increased that of phosphorylated c-Jun N-terminal kinase (JNK). Furthermore, the apicularen A-induced nitrite production was suppressed by the NF-B 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-B 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. The cytostatic macrolides apicularens A and B have been isolated from a variety of strains of the myxobacterial genusChondromyces (i.e., C. apiculatus, C. lanuginosus, C. pediculatus, and C. robustus) (Kunze et al., 1998). Structurally, apicularen A features a trans-hydroxypyran with a salicylic acid residue within a 10-membered lactone, which bears a highly unsaturated enamide side chain (Fig. 1). This natural product is usually found with varying amounts of its glycoconjugate with N-acetyl glucose, known as apicularen B (Fig. 1).This study was supported in part by Exploratory Research (11877381) and Scientific Research on Priority Areas (12139202) from the Ministry of Education, Science, Sports and Culture of Japan.Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.077248. ABBREVIATIONS:MAPK, mitogen-activated protein kinase; apicula...