The induction of cyclooxygenase-2 in IL-1beta-treated endothelial cells is inhibited by prostaglandin E2 through cAMP.

Prostaglandins (PGs) have numerous cardiovascular and inflammatory effects. Cyclooxygenase (COX), which exists as COX-1 and COX-2 isoforms, is the first enzyme in the pathway in which arachidonic acid is converted to PGs. Prostaglandin E2 (PGE2) exerts a variety of biological activities for the maintenance of local homeostasis in the body. Elucidation of PGE2 involvement in the signalling molecules such as COX could lead to potential therapeutic interventions. Here, we have investigated the effects of PGE2 on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with interleukin-1beta (IL-1beta 1 ng/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and thromboxane B2 (TXB2) in the presence of exogenous arachidonic acids (10 microM for 10 min) using enzyme immunoassay (EIA). COX-1 and COX-2 protein was measured by immunoblotting using specific antibody. Untreated HUVEC contained only COX-1 protein while IL-1beta treated HUVEC contained COX-1 and COX-2 protein. PGE2 (3 microM for 24h) did not affect on COX activity and protein in untreated HUVEC. Interestingly, PGE2 (3 microM for 24h) can inhibit COX-2 protein, but not COX-1 protein, expressed in HUVEC treated with IL-1beta. This inhibition was reversed by coincubation with forskolin (100 microM). The increased COX activity in HUVEC treated with IL-1beta was also inhibited by PGE2 (0.03, 0.3 and 3 microM for 24h) in a dose-dependent manner. Similarly, forskolin (10, 50 or 100 microM) can also reverse the inhibition of PGE2 on increased COX activity in IL-1beta treated HUVEC. The results suggested that (i) PGE2 can initiate negative feedback regulation in the induction of COX-2 elicited by IL-1beta in endothelial cells, (ii) the inhibition of PGE2 on COX-2 protein and activity in IL-1beta treated HUVEC is mediated by cAMP and (iii) the therapeutic use of PGE2 in the condition which COX-2 has been involved may have different roles

The induction of cyclooxygenase-2 in IL-1b -treated endothelial cells is inhibited by prostaglandin E 2 through cAMP

PROSTAGLANDINS (PGS) h ave n um er ous car dio vas cular an d in flam m ator y effects. Cycloox yge nas e (COX), w h ic h e x is ts as COX-1 an d COX-2 is ofo r m s , is th e firs t en zym e in th e p ath w ay in w h ic h arach idon ic acid is con verted to PGs . Pr os taglan din E 2 (PGE Introduction Prostaglandins (PGs) have numerous cardiovasc ular and inflammatory e ffec ts. 1 Cycloox yge nase (COX) is the first enzyme in the pathw ay in w hich arachidonic acid is conve rted to PGs. 2 ,3 COX ex ists in at least tw o isoforms. One is the constitutiv e e nzyme , COX-1, producing re gulatory prostanoids under physiologic al conditions, 4 w hereas the other, COX-2, is induc ed by mitogens, The main PGs produc ed in the body are prostacyclin (PGI 2 ), PGE 2 , PGF 2 a , Thrombox ane A 2 (TXA 2 ) and PGD 2 . Each PGs has different charac te rs and functions. Among the PGs, PGE 2 is a potent lipid molecule w ith c omplex proinflammatory and immunoregulatory p rope rties. 9 PGE 2 is considered a major contributor to the production and maintenanc e of immunosuppression after overw he lming injury. 1 0 PGE 2 is believed to modulate biochemic al and immunologic al e ve nts le ading to p arturition. 11 PGE 2 also ex e rts a variety of biologic al ac tivities for the mainte nance of local homeostasis in the body. 1 2 Inte restingly, w e have show n in p re vious studie s that the induc tion of COX-2 e lic ite d by e ndotox in (lipopolysaccharide , LPS) in e ndothelial c ells is inhibite d by PGE 1 and 13,14-dihydro PGE 1 . 1 3 Eluc idation of the effects of PGE 2 on the signalling molec ule such as COX could lead to potential therape utic interve ntions and unde rstanding of the fee dback re gulation of COX in e ndothelial c ells. Here , w e have inve stigated the effects of PGE 2 on the induc tion of COX-2 in human umbilical ve in endothe lial ce lls (HUVEC) treate d w ith inte rleukin-1b (IL-1b ) (1 ng /ml). 287 Research Paper Mediators of Inflammation, 8, 287-294 (1999) Material and methods Cell culture Human umbilic al ve in e ndothelial ce lls (HUVEC) w ere obtaine d from babies born to normal pregnant w ome n as pre viously de sc ribe d 1 4 and c ulture d in 96-w ell plates w ith Human Endothelial-SFM Basal Grow th Medium (Gibco) containing 10% fetal calf se rum (Gibco), 100 units /ml pe nicillin G sodium and 100 m g /ml streptomycin. Ce lls w ere inc ubate d at 37°C in a humidified inc ubator and grow n to confluenc e before use. Measurement of COX activity Conflue nt HUVEC w e re gently w ashe d tw o times w ith phosp hate-buffe re d saline (PBS) and re plac ed w ith fresh medium (200 m l/w e ll) before use. Ce lls w ere tre ate d w ith no addition, IL-1b (1 ng /ml), IL-1b (1 ng / ml) plus PGE 2 (0.03, 0.3 or 3 m M) or PGE 2 (3 m M) alone for 24 h, afte r w hich time the medium w as remove d and w ashed tw ice w ith PBS. COX ac tivity w as me asured by the production of four COX me tabolites, e.g. 6-keto-PGF 1 a (a stable metabolite of PGI 2 ), PGE 2 , Prostaglandin F 2 a (PGF 2 a ) and thromobox ane B 2 (TXB 2 ; a stable metabolite of TXA 2 ) in the replace d fresh me dium c ontaining ex ogenous arachidonic ac id (10 m M for 10 min) using enzyme immunoassay (EIA). Briefly, 50 m l of standard PGs or samp le s w e re added to pre-c oate d mouse anti-rabbit IgG microtitre plates (96-w e ll). The n, PGs ac etylcholineste rase trac er (Clayman; 50 m l) and rabbit antiserum of PGs w e re adde d. The plate w as c overe d w ith plastic film and inc ubate d for 18 h at 4°C, after w hich time the w ells w ere emptied and rinse d five time s w ith w ash buffe r (PBS containing 0.05% Tw ee n). Ellman's reagent (Cayman; 200 m l) w as adde d to e ach w ell and the plates w ere shaken on a mic rotitre plate shaker. The duration of the reaction w as about 90 min. A yellow c olour develops w hich can be read using a mic roplate reader (BIORAD; OD 415 nm). Immunoblot (Western blot) analysis HUVEC w hich w e re untre ate d, treated w ith IL-1b (1 ng /ml), IL-1b (1 ng /ml) plus PGE 2 (0.03, 0.3 and 3 m M) or PGE 2 (3 m M) alone w ere cultured in six -w e ll culture plates (37°C; for 24 h). After 24 h inc ubation, ce lls w ere ex trac te d and analyse d by immunoblotting using spe cific antibodies for COX-1 and COX-2 prote in (a gene rous gift from Dr Gary O'Ne ill, Merck Frosst, Canada) as pre viously described. 15 The othe r ex periment w as performed to study the signalling mole cule in the e ffec ts of PGE 2 on COX ex pre ssion by using forskolin (cAMP activator). HUVEC w ere treated w ith no addition, IL-1b (1 ng / ml), IL-1b (1 ng /ml) plus PGE 2 (3 m M), IL-1b (1 ng /ml) plus PGE 2 (3 m M) w ith forskolin (10, 50 and 100 m M), IL-1b (1 ng /ml) plus forskolin (100 m M), PGE 2 (3 m M) plus forskolin (100 m M), forskolin (100 m M) alone or PGE 2 (3 m M) alone for 24 h, after w hich time, the me dium w as removed and re place d w ith fre sh me dium c ontaining ex oge nous arachidonic ac id (10 m M for 10 min). The me dium w as then removed to me asure COX activity by 6-ke to-PGF 1 a production. The re mained c ells w ere ex tracted and analyse d by immunoblotting using spe cific antibodies for COX-1 and COX-2 prote in. Measurement of cell viability Ce ll respiration, an indicator of c ell viability, w as asse sse d by the mitochondrial-depe ndent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. Statistical analysis The re sults are show n as mean standard e rror of the me an (SEM) of triplicate de te rminations (w e lls) from at least four separate ex perimental days (n =12). Student's paire d or unpaired t-te sts, as ap propriate, w ere use d for the de te rmination of significanc e of differences betw ee n me ans and a P value of less than 0.05 w as taken as statistically significant. Materials DMSO, phosphate buffe red saline (PBS; pH 7.4), Trizma base , EDTA, triton X-100, phenylme thylsulphonyl fluoride (PMSF), pe pstatin A, le upeptin, glycerol, bromphenol blue, 2-mercaptoethanol, sodium dodec yl sulphate (SDS), forskolin, anti-rabbit IgG antibody, goat IgG, premix e d BCIP/NBT solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe nyltetrazolium bromide (MTT), pe nicillin G sodium and stre ptomycin w ere supplied by Sigma Che mical Comp any (St Louis, MO, USA). PGs (6-keto-PGF 1 a , PGE 2 , PGF 2 a and TXB 2 ) and the ir respective ac etylcholineste rase trac er and rabbit antiserum, pre-coated mouse anti-rabbit IgG microtitre plates (96-w ell) and Ellman's reage nt w ere purchased from Cayman (Sapphire Biosc ie nc e, Australia). Human Endothelial-SFM Basal Grow th Me dium and fetal calf serum w as obtained from GibThai (Thailand). Rec ombinant human IL-1b , w ere purchased from Ge nzyme (USA). Pure nitroc ellulose me mbrane (0.45 mic ron) and filte r pape r w ere purchased from BIO-RAD (USA). P. Ak a ra s e re e n o n t et al. 288 Mediators of Inflammation · Vol 8 · 1999 Results The effect of PGE 2 on COX activity as measured by the production of 6-keto-PGF 1 a , PGE 2 , PGF 2 a and TXB 2 in HUVEC treated with IL-1b (1 ng/ml) Untreate d HUVEC in the presenc e of arachidonic acid (10 m M for 10 min) re le ase low er amounts of 6-ke to-PGF 1 a (3.36 ± 0.1 ng /ml), PGE 2 (0.4 ± 0.04 ng /ml), PGF 2 a (0.78 ± 0.01 ng /ml) and TXB 2 (0.04 ± 0.01 ng /ml). In IL-1b (0.01, 0.1 and 1 ng /ml) tre ate d HUVEC; the produc tion of 6-keto-PGF 1 a , PGE 2 and PGF 2 a w as increase d but not TXB 2 IL-1b alone , PGE 2 alone and IL-1b plus PGE 2 did not affec t on cells viability (97 ± 2, 98 ± 1 and 98 ± 1%, respective ly) w hen compare to the control untreate d ce lls over a 24-h incubation pe riod. The stability of PGE 2 (3 m M) in c ulture d me dium upto 24 h w as also te ste d and has not change d significantly be tw ee n 3 (2.97 ± 0.2), 6 ( 2.98 ± 0.1), 12 (2.95 ± 0.2) and 24 (2.97 ± 0.2) hours inc ubation of PGE 2 . The effect of PGE 2 on COX isoform expressed in HUVEC treated with IL-1b Untre ated HUVEC contained no COX-2 prote in The effect of forskolin on 6-keto-PGF 1 a production in HUVEC treated with IL-1b plus PGE 2 The COX ac tivity (as measured by 6-keto-PGF 1 a production) in HUVEC tre ate d w ith forskolin (100 m M) plus PGE 2 (3 m M) or forskolin (100 m M) alone w as not changed in comparison w ith untre ate d HUVEC ( PGE 2 in hibit COX-2 indu ctio n th ro u g h cAMP Mediators of P. Ak a ra s e re e n o n t et al. 290 Discussion He re, w e show ed that the induction of COX-2 e licite d by IL-1b in HUVEC c an be inhibited by PGE 2 in a dosedepende nt manne r. Moreover, PGE 2 had no affe ct on either COX-1 protein or ac tivity. Inte restingly, forskolin (cAMP activator) can re ve rse this inhibition of PGE 2 on COX-2 protein and activity in IL-1b tre ate d HUVEC. The results sugge ste d that (i) PGE 2 is a ne gative fe edback regulator through c AMP in the induc tion of COX-2 elic ited by IL-1b in e ndothelial ce lls and (ii) the use s of PGE 2 in the c ondition in w hich COX-2 has be en involved may be therapeutic. PGs induc e a w ide range of biological actions that are mediate d by spe cific membrane-bound rec eptors. Among the PGs, PGE 2 is considered to ex ert a varie ty of biologic al activities such as the maintenanc e of loc al homeostasis in the body, 12 it is a major contributor to the production and maintenanc e of immunosuppression afte r ove rw helming injury 10 and an imp ortant factor for implantation and dec idualization. 1 7 Therefore , PGE 2 is a lipid molec ule w ith complex inflammatory modulation and immunoregulatory prop erties. Our results have be en supported that PGE 2 c an ac t as anti-inflammation and immunosuppression in the induction of COX-2 in endothelial ce lls by IL-1b . The ex act mechanisms by w hich PGE 2 inhibite d COX-2 induction in e ndothelial c ells ac tivated w ith IL1b are not know n. These may involve binding to spe cific c ell surface rece ptors and influe nc ing sec ond me sse nger syste ms through G-prote ins. Inde ed, the se should be c omplex because the e ffec ts of PGE 2 are ex e rted by a varie ty of PGE re ceptors w hich are different in their signal transduc tion p rope rties. There are at least four subtypes of PGE rece ptors. The EP1 and EP3 re ce ptors are couple d to Ca 2 + mobiliz ation and the inhibition of ade nylate cyclase, re spe ctively, and the EP2 and EP4 re cep tors are coupled to the same signal transduc tion pathw ay, stimulation of ade nylate cyclase. 1 9 How e ver, our studies show e d that forskolin (cAMP activator) can re verse the inhibiton of PGE 2 on COX-2 induce d in IL-1b tre ate d HUVEC suggesting PGE 2 may inhibit COX-2 ex p re sse d in IL-1b treated HUVEC through c AMP inhibition via EP3 re ceptors. PGE 2 is one of the PGs or COX me tabolites, such as PGI 2 , PGE 2 , PGD 2 , PGF 2 a and TXA 2 , synthe size d by COX-1 and COX-2 w hich are involved in physiology and pathology, 4 -8 respe ctive ly. Each COX is oform c an produc e diffe rent COX me tabolites in different cell type s such as PGI 2 is a major COX-1 and COX-2 metabolite in endothelial c ells w hile PGE 2 is a major COX-2 metabolite in mac rophages. 2 0 These differences in COX metabolite produc tion in different c ell type s may be re sulted from the feedback regulation of each COX metabolite produce d. Our results show e d that PGE 2 (0.03 m M) inhibite d PGE 2 produc tion (30% inhibition; 13 PGE se rie s have bee n use d in clinical disorders such as peripheral vascular oc clusive dise ase s, 2 1 NSAIDsinduc ed gastric ulcer, 2 2 abortion 23 and impote nc e. Thus, w e propose d that uses of PGE 2 in the c ondition in w hich COX-2 has bee n involve d may be therapeutic and the e ffec ts of othe r COX me tabolites such as PGI 2 or PGF 2 a on COX-2 ex pressed in different cells should be e luc idate d. ACKNOWLEDGMENTS: This w ork w as supported by a grant from Siriraj China Medical Board to P. Akarase reenont

The Notch inhibitor cowanin accelerates nicastrin degradation

Abstract Aberrant activation of Notch signaling contributes to the pathogenesis of several different types of cancer, and Notch pathway inhibitors may have significant therapeutic potential. Using a unique cell-based assay system, we isolated twelve compounds, including one new natural product from Garcinia speciosa, that inhibit the Notch signaling pathway. HES1 and HES5 are target genes of the Notch cascade, and compound 2, referred to as cowanin, decreased the protein levels of HES1 and HES5 in assay cells. Furthermore, cowanin (2) showed potent cytotoxicity against human leukemic HPB-ALL cells. The Notch signaling inhibitory activity of cowanin (2) is linked to the increased degradation of nicastrin, which is one of the components of the γ-secretase complex. To the best of our knowledge, this is the first example of a compound with Notch pathway inhibitory activity mediated by nicastrin degradation

PRISMA flow diagram [8] for the systematic review.

<p>PRISMA flow diagram <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116018#pone.0116018-Moher1" target="_blank">[8]</a> for the systematic review.</p

The Notch Inhibitors Isolated from <i>Nerium indicum</i>

Notch signaling plays a crucial role in differentiation and cell maintenance, but once aberrantly activated, it contributes to cancer progression. Notch inhibitors were isolated from plant extracts and tested using an originally constructed cell-based assay system. We isolated eight compounds from <i>Nerium indicum</i> that showed inhibition of the Notch signaling pathway. <i>HES1</i> and <i>HES5</i> are target genes of the Notch signaling pathway, and oleandrin (<b>1</b>) decreased the protein levels of HES1 and HES5 in assay cells. Oleandrin (<b>1</b>) showed potent cytotoxicity against HPB-ALL cells and decreased HES1 and the Notch intracellular domain in these cells. The main mechanism of action of <b>1</b> appears to be inhibition of Notch signaling by acceleration of Notch intracellular domain degradation

Prenylated Flavonoids and Resveratrol Derivatives Isolated from <i>Artocarpus communis</i> with the Ability to Overcome TRAIL Resistance

In a screening program on natural products that can abrogate tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resistance, four new prenylated flavonoid and resveratrol derivatives (<b>1</b>–<b>4</b>) were isolated from <i>Artocarpus communis</i>, together with eight known prenylflavonoids (<b>5</b>–<b>12</b>). The structures of <b>1</b>–<b>4</b> were elucidated spectroscopically. Pannokin E (<b>1</b>) (2 μM) and artonin E (<b>5</b>) (3 μM) potently exhibited the ability to overcome TRAIL resistance. Artonin E (<b>5</b>) induced caspase-dependent apoptosis in combination with TRAIL, increased caspase 3/7 activity, and enhanced the protein levels of p53 and DR5. Moreover, this substance decreased cell viability in combination with TRAIL and enhanced the protein levels of DR5, and these effects were mediated by increases in the production of ROS (reactive oxygen species). Thus, artonin E (<b>5</b>) was found to induce extrinsic apoptotic cell death by the ROS- and p53-mediated up-regulation of DR5 expression in AGS cells

Bar chart showing data extracted by the systematic review relating to the presentation of individual study results.

<p>Bar chart showing data extracted by the systematic review relating to the presentation of individual study results.</p