Apoptosis 1999; 4: 151–162 C  1999 Kluwer Academic Publishers Cyclooxygenase-independent induction of p21 WAF-1/cip1 , apoptosis and differentiation by L-745,337, a selective PGH synthase-2 inhibitor, and salicylate in HT-29 cells G. Santini, M. G. Sciulli, R. Marinacci, O. Fusco, L. Spoletini, A. Pace, A. Ricciardulli, C. Natoli, A. Procopio, J. Maclouf and P. Patrignani Departments of Medicine and Aging, Division of Pharmacology (G. Santini, M. G. Sciulli, A. Pace, A. Ricciardulli, P. Patrignani), Oncology and Neuroscience (O. Fusco, L. Spoletini, C. Natoli, A. Procopio) University of Chieti ‘G. D’Annunzio’ School of Medicine, 66013, Chieti, Italy and IFR Biologie de la Circulation-Lariboisi ` ere (J. Maclouf), Institut National de la Sant` e et de la Recherche M´ edicale, Unit ´ e 348, Hopital Lariboisi ` ere, 75475 Paris, France In order to dissect out cyclooxygenase-dependent from cyclooxygenase-independent mechanisms in the antipro- liferative effects of selective prostaglandin H synthase (PGHS)-2 inhibitors, we compared the effects of L-745,337 (a highly selective PGHS-2 inhibitor) with sodium salicy- late (a weak PGHS inhibitor) on prostanoid production, induction of the cyclin-dependent kinase inhibitor p21 WAF-1/cip1 , mutant p53 (m273-p53) levels, apoptosis and differentiation in human colon adenocarcinoma HT-29 cells. L-745,337 dose-dependently suppressed the cyclo- oxygenase activity of HT-29 cells (IC 50 : 0.24 µM). Four-day treatment with L-745,337 caused a concentration-depe- ndent inhibition of cell growth (IC 50 : 0.9 mM) associated with the induction of p21 WAF-1/cip1 and an increase in the proportion of apoptotic nuclei (EC 50 : 0.1 and 0.34 mM, respectively) while reducing the levels of m273-p53 (IC 50 : 0.2 mM). Sodium salicylate, at the concentration of 10 mM that did not affect prostanoid formation, caused a 60% reduction of cell growth associated with a 3-fold in- duction of p21 WAF-1/cip1 and a 60% increase in the propor- tion of apoptotic nuclei. Ultrastructural analysis showed that L-745,337 (0.5 mM) and sodium salicylate (10 mM) caused the induction of a differentiated phenotype. We conclude that high concentrations of L-745,337 and so- dium salicylate inhibit colon cancer cell growth by a mechanism unrelated to cyclooxygenase inhibition that may involve p53-independent induction of the tumor sup- pressor p21 WAF-1/cip1 . Supported by grants from Consiglio Nazionale delle Ricerche (97.04492.CT04 and 98.03111.CT04) and Associazione Italiana per la Ricerca sul Cancro (to Antonio Procopio). Giovanna Santini holds a Searle doctoral fellowship. Correspondence to: P. Patrignani, Universit ` a di Chieti ‘G. D’Annunzio,’ Cattedra di Farmacologia I, Palazzina delle Scuole di Specializzazione, Via dei Vestini 31, 66013 Chieti, Italy. Tel: +39-0871-3556775; Fax: +39-0871-3556718; email: ppatrignani @unich.it Keywords: Apoptosis; differentiation; HT-29 cells; L-745,337; p21 WAF-1/cip1 ; sodium salicylate. (Received 14 January 1999; accepted 21 January 1999) Introduction Epidemiological studies have shown that regular use of aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) is associated with reduced risk of colorectal cancer. 1,2 Moreover, randomized clinical trials have shown that sulindac reversibly reduces the number and size of col- orectal polyps in familial adenomatous polyposis (FAP). 3 The mechanism(s) of the antiproliferative effect of NSAIDs is not completely understood but most hypothe- ses have focussed on their property to reduce the levels of prostanoids [prostaglandin (PG)s, prostacyclin and thro- mboxane (TX) A 2 ] in gastrointestinal tissues. 4 Several lines of evidence suggest that prostanoids may affect tumor formation in the intestine. Elevated levels of PGE 2 have been demonstrated in human colon cancer samples com- pared to normal mucosa. 5 In the human colon adenocar- cinoma cell line HT-29, exogenous PGE 2 as well as two stable analogues stimulated cell proliferation. 6 Multiple enzymatic steps are involved in the biosyn- thesis of prostanoids. The first step is the release of arachidonic acid (AA) from membrane phospholipids by phospholipases, including phospholipase A 2 . AA is then converted to PGH 2 by PGH synthase (PGHS) which ex- hibits two distinct catalytic activities, cyclooxygenase and peroxidase. 7 Two isoforms of PGHS have been identified 7 : PGHS-1 and PGHS-2 [also referred to as cyclooxygenase- 1 (COX-1) and cyclooxygenase-2 (COX-2)]. PGHS-1 is constitutively expressed at relatively stable levels in most Apoptosis · Vol 4 · No 3 · 1999 151