Vol. 62, Issue 6, 1515-1521, December 2002

Mechanisms Underlying Nonsteroidal Anti-Inflammatory Drug-Induced p27^Kip1 Expression

Yu-Chun Huang, Lea-Yea Chuang, and Wen-Chun Hung

Graduate Institute of Medicine (Y.-C.H.), Department of Biochemistry, (L.-Y.C.) and School of Technology for Medical Sciences (W.-C.H.), Kaohsiung Medical University, Kaohsiung, Taiwan

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 (5, 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.