Macrophage-derived nitric oxide induces apoptosis of rat hepatoma cells invivo

Although nitric oxide (NO) has been postulated to play important roles in h ost defense mechanisms against tumor cells, a direct evidence supporting th is hypothesis is lacking, To obtain molecular insights into the antitumor a ction of NO, its metabolism and effect on ascites hepatoma (AH-130) cells w ere investigated in tumor-bearing rats. Kinetic analysis revealed that subs tantial amounts of nitrite and nitrate, metabolites of NO, appeared in plas ma and ascites of AH-130-inoculated rats. Western blot analysis revealed th at a large number of macrophages that expressed inducible type of NO syntha se (iNOS) appeared in cancerous ascites, particularly during I to 2 weeks a fter inoculation of AH-130 cells. When NO generation by peritoneal macropha ges increased, a significant fraction of AH-130 in ascites fluid underwent apoptosis as judged from the fragmentation of their nuclear DNA. Kinetic an alysis 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 dono r strongly enhanced DNA fragmentation of AH-130 cells. Antimycin A, a speci fic inhibitor for mitochondrial electron transport, also induced DNA fragme ntation of AH-130 cells by a mechanism that was inhibited by adding ascorba te and tetramethyl-p-phenylene diamine (TMPD) as electron donors. These res ults indicate that NO derived from peritoneal macrophages inhibits mitochon drial electron transport and disturbs calcium homeostasis in ascites hepato ma AH-130 cells, thereby inducing their apoptosis in vivo.