Macrophage-derived nitric oxide induces apoptosis of rat hepatoma cells in vivo.
Although nitric oxide (NO) has been postulated to play important roles in host defense mechanisms against tumor cells, a direct evidence supporting this hypothesis is lacking. To obtain molecular insights into the antitumor action of NO, its metabolism and effect on ascites hepatoma (AH-130) cells were investigated in tumor-bearing rats. Kinetic analysis revealed that substantial amounts of nitrite and nitrate, metabolites of NO, appeared in plasma and ascites of AH-130-inoculated rats. Western blot analysis revealed that a large number of macrophages that expressed inducible type of NO synthase (iNOS) appeared in cancerous ascites, particularly during 1 to 2 weeks after inoculation of AH-130 cells. When NO generation by peritoneal macrophages increased, a significant fraction of AH-130 in ascites fluid underwent apoptosis as judged from the fragmentation of their nuclear DNA. Kinetic analysis revealed that NO strongly inhibited mitochondrial electron transport and changed calcium status in AH-130 cells, particularly under low oxygen tensions such as in cancerous ascites. Intraperitoneal injection of NO donor strongly enhanced DNA fragmentation of AH-130 cells. Antimycin A, a specific inhibitor for mitochondrial electron transport, also induced DNA fragmentation of AH-130 cells by a mechanism that was inhibited by adding ascorbate and tetramethyl-p-phenylene diamine (TMPD) as electron donors. These results indicate that NO derived from peritoneal macrophages inhibits mitochondrial electron transport and disturbs calcium homeostasis in ascites hepatoma AH-130 cells, thereby inducing their apoptosis in vivo.
Nishikawa
M
M
Department of Biochemistry, Osaka City University Medical School, Abeno-ku, Osaka, Japan.
Sato
E F
EF
Kuroki
T
T
Utsumi
K
K
Inoue
M
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001202:Ascitic Fluid / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000503:physiopathology*; D053938:DNA Fragmentation / Q000187:drug effects; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis / Q000097:blood / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D019335:Nitroarginine / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; C093285:NOC 18; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D019335:Nitroarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat