Nitric oxide suppresses apoptosis in human colon cancer cells by scavenging mitochondrial superoxide anions.
In cells lacking a functional p53 tumor suppressor protein, the endogenous free radical nitric oxide (NO) appears to inhibit apoptosis, and thereby promotes growth of cancer cells. In order to elucidate the underlying mechanisms on a molecular basis, we used HT-29 human colon carcinoma cells, carrying a p53 loss-of-function mutation, and examined the effects of NO on apoptosis when induced either by the flavonoid flavone or by the chemotherapeutic drug camptothecin (CPT), which is able to scavenge NO by superoxide anion production. Caspase-3 activation as well as nuclear fragmentation, both indicative of apoptosis, were dose dependently inhibited by the NO-liberating agents sodium nitroprusside (SNP) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP) when apoptosis was initiated by flavone with only minor effects on apoptosis when initiated by camptothecin. The transcript levels of 9 apoptosis-related genes were assessed and NO liberation was shown to completely and specifically prevent the flavone-induced but not camptothecin-induced decrease in bcl-X(L) mRNA levels. These results were also confirmed at the protein level. The effects of NO on the mitochondrial apoptosis pathway were further evidenced by the scavenging of superoxide anions as produced in mitochondria of cells undergoing apoptosis. Scavenging of mitochondrial superoxide anions by NO prevents the downregulation of bcl-X(L), the depolarization of the mitochondrial membrane potential, the cytochrome c release and finally the activation of caspase-3. In conclusion, NO effectively inhibits apoptosis by scavenging superoxide anions generated in the mitochondria of p53 mutant cells and thereby prevents the downregulation of the antiapoptotic factor bcl-X(L), which controls the mitochondrial apoptosis pathway.
Wenzel
Uwe
U
Molecular Nutrition Unit, Department of Food and Nutrition, Technical University of Munich, Freising, Germany. uwenzel@wzw.tum.de
Kuntz
Sabine
S
De Sousa
Ulrike Jambor
UJ
Daniel
Hannelore
H
D017209:Apoptosis / Q000187:drug effects*; D002166:Camptothecin / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D003574:Cytochrome c Group / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D015536:Down-Regulation; D004789:Enzyme Activation; D016166:Free Radical Scavengers / Q000494:pharmacology*; D019073:HT29 Cells; D006801:Humans; D015151:Immunoblotting; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013481:Superoxides / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D003574:Cytochrome c Group; D017931:DNA Primers; D016166:Free Radical Scavengers; D020030:Nitric Oxide Donors; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D017382:Reactive Oxygen Species; C110881:S-nitro-N-acetylpenicillamine; D016159:Tumor Suppressor Protein p53; D051020:bcl-X Protein; D013481:Superoxides; D009599:Nitroprusside; D009569:Nitric Oxide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D010396:Penicillamine; D002166:Camptothecin