BMC Cancer 2004, 4:49 http://www.biomedcentral.com/1471-2407/4/49 kELtFieYfnifgte1oiucn1-tr7Pteo0hGf1e3F8tpamorepnosrex6oni-dfkceuenct,toiLo-fYPnTG1Pa1FAn7d01(pB8r)oodtrhuercatelioffxneicfietnndoeufcoteanmd(obAyx)ilfbaeacnstaoclry6s-- 1α 1α Effect of tamoxifen, LY117018 or raloxifene on (A) basal 6- keto-PGF1α production and (B) the effect of tamoxifen or LY117018 on 6-keto-PGF1α production induced by lactacys- tin in the presence of TPA. Rat liver cells were incubated for 6-h. The analyses were performed with triplicate or quadru- plicate dishes. Each bar gives the mean value and the brackets the SEM. * = Statistically significant vs control stimulation. ** = Statistically significant vs lactacystin plus TPA control. The data are representative of several independent experiments with the same results. from the relative effectiveness of inhibition of PGI2 pro- duction by celecoxib and piroxicam. Celecoxib is 7.5 times more effective than piroxicam as an inhibitor of COX-2, but 30,000 times less effective as an inhibitor of COX-1. Piroxicam is a selective inhibitor of COX-1 [17]. Celecoxib, the nonsteroidal anti-inflammatory drug (NSAID) that is a selective inhibitor of COX-2, inhibits both induced and basal COX activity 20–80 times more effectively than piroxicam (Fig. 4-A and 4B). Thus, based on inhibition by these two NSAIDs, COX-2 is the most likely isoform expressed both constitutively and after induction by lactacystin in the presence of TPA in rat liver cells. This conclusion was confirmed by western blot anal- yses (Levine L, Tashjian A, unpublished data). FItcnaaihmcmgyiubos(irtxtieinof)e4nin-ostfhti(emApu)rltaeatsmeednocixneidfeounfcTe(1dP6A6µ)-kMbey)t-ocs-etPilmeGcuFolaxtiebpdr(o*bd)auascantldiaopnnidr(ol(axBci)- 1α Inhibition of (A) tamoxifen (16 µM)-stimulated basal and (B) tamoxifen-stimulated induced 6-keto-PGF1α production (lac- tacystin in the presence of TPA) by celecoxib (*) and piroxi- cam ( ). Rat liver cells were incubated with either (A) tamoxifen (16 µM) or (B) lactacystin plus TPA in the pres- ence of tamoxifen, for 6-h. The analyses were performed with triplicate dishes. Is COX-2 production non-genomic and ER-independent? The time-course of stimulation by tamoxifen (15 µM) of basal activity is shown in Fig. 5. Even after a 5-minute incubation (not shown in Fig. 5), stimulation by tamoxifen is significant statistically [0.08 ± 0.011 (n = 5) vs 0.13 ± 0.011 (n = 5) ng 6-keto-PGF1α per ml in the absence and presence of tamoxifen, respectively]. Similar to AA release [4], stimulation of basal PGI2 production by tamoxifen is not affected by preincubation of the cells for 2-h with 1 µM actinomycin D. The induction obtained by incubation with lactacystin in the presence of TPA is inhibited (Fig. 6-A). Stimulation of basal PGI2 production by tamoxifen is unaffected by ICI-182,780 (Fig. 6-B). This estrogen antagonist, which binds to the ER with high affinity [18] did not affect the release of AA by tamoxifen [4] but does partially inhibit release of AA stimulated by 17β-estradiol, 22 (R)-cholesterol, indomethacin, trans- Page 4 of 8 (page number not for citation purposes)