Degradation of NF-κB, p53 and other regulatory redox-sensitive proteins by thiol-conjugating and -nitrosylating drugs in human tumor cells.
The ionized cysteines present on the surfaces of many redox-sensitive proteins play functionally essential roles and are readily targeted by the reactive oxygen and reactive nitrogen species. Using disulfiram (DSF) and nitroaspirin (NCX4016) as the model compounds that mediate thiol-conjugating and nitrosylating reactions, respectively, we investigated the fate of p53, nuclear factor-kappaB (NF-κB) and other redox-responsive proteins following the exposure of human cancer cell lines to the drugs. Both drugs induced glutathionylation of bulk proteins in tumor cells and cell-free extracts. A prominent finding of this study was a time- and dose-dependent degradation of the redox-regulated proteins after brief treatments of tumor cells with DSF or NCX4016. DSF and copper-chelated DSF at concentrations of 50-200 µM induced the disappearance of wild-type p53, mutant p53, NF-κB subunit p50 and the ubiquitin-activating enzyme E1 (UBE1) in tumor cell lines. DSF also induced the glutathionylation of p53. The recombinant p53 protein modified by DSF was preferentially degraded by rabbit reticulocyte lysates. The proteasome inhibitor PS341 curtailed the DSF-induced degradation of p53 in HCT116 cells. Further, the NCX4016 induced a dose-dependent disappearance of the UBE1 and NF-κB p50 proteins in cell lines, besides a time-dependent degradation of aldehyde dehydrogenase in mouse liver after a single injection of 150 mg/kg. The loss of p53 and NF-kB proteins correlated with decreases in their specific binding to DNA. Our results demonstrate the hitherto unrecognized ability of the non-toxic thiolating and nitrosylating agents to degrade regulatory proteins and highlight the exploitable therapeutic benefits.
Department of Biomedical Sciences, Cancer Biology Research Centre, School of Pharmacy, Texas Tech University Health Sciences Center, 1406 S. Coulter Drive, Amarillo, TX 79106, USA.
Ameya
A
Paranjpe
Kalkunte S
KS
Srivenugopal
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001241:Aspirin / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004221:Disulfiram / Q000494:pharmacology; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D016159:Tumor Suppressor Protein p53; C471538:UBA1 protein, human; D046988:Proteasome Endopeptidase Complex; D044764:Ubiquitin-Activating Enzymes; C102148:nitroaspirin; D001241:Aspirin; D004221:Disulfiram