Mechanisms underlying nonsteroidal anti-inflammatory drug-induced p27(Kip1) expression.
We demonstrated previously that nonsteroidal anti-inflammatory drugs (NSAIDs) increased p27(Kip1) by inhibiting protein degradation to suppress the proliferation of human lung cancer cells. In this study, we elucidate the molecular mechanism by which NSAIDs modulate p27(Kip1) proteolysis. Immunoblotting and in vitro ubiquitination assays indicated that the expression of Cul1 and Skp2 and ubiquitination activity toward p27(Kip1) were not regulated by NSAIDs. On the contrary, we found that NSAIDs inhibited proteasome activity to increase p27(Kip1) protein levels. NSAIDs suppressed the expression of chymotrypsin-like catalytic subunits (beta5, LMP7, and LMP2), but did not directly block enzymatic activity, to inhibit proteasome activity. Reverse transcriptase-competitive polymerase chain reaction and promoter activity assays showed that this inhibition occurred at the transcriptional level. In vitro degradation experiments showed that p27(Kip1) degradation was inhibited by NS398, and the addition of purified 26S proteasome reversed this inhibitory effect. Collectively, our results revealed the mechanism by which NSAIDs modulate p27(Kip1) protein degradation and suggest that NSAIDs are a novel class of proteasome inhibitors.
Huang
Yu-Chun
YC
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chuang
Lea-Yea
LY
Hung
Wen-Chun
WC
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D044842:Cullin Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation; D004347:Drug Interactions; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism; D009578:Nitrobenzenes / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D044786:S-Phase Kinase-Associated Proteins; D013223:Statistics as Topic; D013449:Sulfonamides / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
D000894:Anti-Inflammatory Agents, Non-Steroidal; D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D009097:Multienzyme Complexes; D009578:Nitrobenzenes; D044786:S-Phase Kinase-Associated Proteins; D013449:Sulfonamides; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D050760:Cyclin-Dependent Kinase Inhibitor p27; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease