Induction of inducible nitric oxide synthase is an essential part of tumor necrosis factor-alpha-induced apoptosis in MCF-7 and other epithelial tumor cells.
TNF-alpha-induced cytotoxicity is mediated by the intracellular "death domain" of the 55-kDa TNF-alpha receptor and has been demonstrated to be coupled with induction of inducible nitric oxide synthase (iNOS), leading to generation of nitric oxide radicals (NO*). Because it is still widely unknown, to what extent NO* participates in the execution of TNF-alpha-induced apoptosis, NO* production, iNOS expression, and enzyme activity in relation to TNF-alpha-induced apoptotic cell death were investigated in the human breast cancer cell line MCF-7 and various other malignant cell lines. Incubation with TNF-alpha led to induction of iNOS mRNA and protein as well as enhancement of NOS activity. Augmented synthesis of NO2, the stable end product of NO* generation, was significantly correlated with augmented rates of cell death. Measurement of TNF-alpha-triggered production of reactive oxygen species (ROS) suggested a major role for NO* within the generated oxygen radicals. Dying cells showed characteristic features of apoptosis. Addition of cycloheximide (CX) enhanced apoptotic cell death by increasing iNOS activity. L-Nitro-arginine-methylester (L-NAME), a competitive NOS-inhibitor, and iNOS antisense oligonucleotides effectively prevented NO2 generation and apoptosis. Evaluation of iNOS expression during TNF-alpha-induced cell death in various malignant cell lines demonstrated iNOS positivity for all TNF-alpha-sensitive cells. The only primarily resistant line was iNOS negative. In a resistant variant of MCF-7, iNOS mRNA was still detectable; however, treatment with TNF-alpha did not enhance NOS activity. TNF-alpha sensitivity and NO2 production were completely restored by the addition of CX. Taken together, iNOS induction plays an essential role in TNF-alpha-induced apoptosis of the investigated cell lines. Further studies are necessary to define the impact of NO* in relation to other specific effectors of apoptosis such as the caspases.
Binder
C
C
Department of Haematology/Oncology, Georg-August-University, Göttingen, Germany. cbinder@med.uni-goettingen.de
Schulz
M
M
Hiddemann
W
W
Oellerich
M
M
D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009336:Necrosis; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II