Development of a cell death-based method for the screening of nuclear factor-kappaB inhibitors.
Nuclear factor kappa B (NF-kappaB) plays a significant role in immunity and inflammation and represents a first choice as pharmacological target for anti-inflammatory therapy. However, research in this field has been hampered by the fact that no convenient assay suitable for large-scale screening procedures is available. The present study provides a cell death-based assay method for screening of nuclear factor-kappaB inhibitors. In this study, we observed that four distinct pharmacologic inhibitors of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), N-tosyl-L-lysyl chloromethyl ketone (TPCK), genistein and BAY11-7082, resulted in the cell death of murine macrophages, J774A.1. DNA-binding experiments showed that lethal doses were consistent with those required for NF-kappaB inhibition. DNA fragmentation analysis showed that cell death is apoptotic in nature. Further studies suggested that NF-kappaB inhibitors induced apoptosis is independent of the involvement of other markers of cell death such as caspases and p38 MAP (Mitogen activated protein) kinase. From this study, we conclude that NF-kappaB activation may represent an important survival mechanism in macrophages. This study also provides a new cell-based screening method, as any compound that will inhibit NF-kappaB activity will result in the death of macrophages.
Chopra
Puneet
P
Department of Pharmacology, New Drug Discovery Research, Ranbaxy Research Laboratories, Gurgaon, Haryana, India. p.chopra@ranbaxy.com
Bajpai
Malini
M
Dastidar
Sunanda G
SG
Ray
Abhijit
A
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001681:Biological Assay; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D019833:Genistein / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D009570:Nitriles / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011759:Pyrrolidines / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology; D013859:Thiocarbamates / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D000893:Anti-Inflammatory Agents; D016328:NF-kappa B; D009570:Nitriles; D011759:Pyrrolidines; D013450:Sulfones; D013859:Thiocarbamates; C020972:pyrrolidine dithiocarbamic acid; D014108:Tosylphenylalanyl Chloromethyl Ketone; D019833:Genistein; D051057:Proto-Oncogene Proteins c-akt; D048051:p38 Mitogen-Activated Protein Kinases; D020169:Caspases