phosphoinositide 3-kinase ( pi3k ) is a family of lipid kinases that phosphorylate the 3-hydroxyl group of phosphoinositides and phosphatidylinositol . the lipid products of pi3k reactions create binding sites for specific , lipid - binding domains on many intracellular signaling proteins . therefore , it contributes to a wide range of cellular responses to extracellular signals and in various signal transduction pathways [ 3 , 4 ] . pi3k is revealed to play an essential role in the regulation of intestinal epithelial proliferation , survival , and wound healing . its pathways have been shown to be associated with many cancers such as breast , colorectal , and esophageal cancer [ 6 , 7 ] . they are also involved in the development of inflammatory diseases such as arthritis and atherosclerosis [ 8 , 9 ] . pi3k is an important mediator in the signaling cascade leading to the initiation of the inflammatory response including an enhanced release of proinflammatory cytokines [ 10 , 11 ] . pi3k is on the basis of structural and functional aspects divided into three classes ( i , ii , and iii ) . while the involvement of class ii and class iii pi3k in inflammation has not yet been defined , class i enzymes are considered as an important element in many steps of the inflammatory reaction [ 12 , 13 ] . class i pi3k is consisting of a complex formed by catalytic subunits ( c ) and regulatory subunits ( r ) . three r ( p85 , p85 , and p55 ) and four c ( p110 , p110 , p110 , and p110 ) isoforms are known [ 4 , 1417 ] . it is well established that pi3k isoforms could be distinctively expressed according to the cell type [ 4 , 18 ] . targeting pi3k is compelling therapeutic approach and some inhibitors have been clinically tested to be developed as potent drugs [ 19 , 20 ] . nonselective pi3k inhibitors , wortmannin , and ly294002 are unsuitable for a therapeutic approach because its lack of selectivity may produce toxicity issues . many studies have established that an increased production of proinflammatory cytokines is critical for the pathogenesis of inflammatory disorders . various inflammation inducers such as tnf- enhance the secretion of il-1 , il-6 , and il-8 [ 23 , 24 ] . the levels of il-8 have significantly increased in the gastroesophageal reflux disease ( gerd ) [ 25 , 26 ] . also the esophageal epithelium is believed to be the major source of il-8 production . increased ros in mucosa have been implicated as a trigger of gastrointestinal diseases , including inflammatory disorders and acid - related peptic diseases . hydrogen peroxide is a well - known inducer of esophageal epithelial inflammation and is elevated in gerd [ 30 , 31 ] . in the present study , we investigated the expression of pi3k isoforms and the anti - inflammatory potential effect of pik-75 as a selective p110 inhibitor   in feline eecs . hydrogen peroxide , 4-(2-hydroxyethyl)-1-piperazine - n-2-ethane sulfonic acid ( hepes ) , leupeptin , aprotinin , bovine serum albumin ( bsa ) , -mercaptoethanol , ethylene glycol - bis-(-aminoethylether)-n , n , n,n-tetraacetic acid ( egta ) , phenylmethylsulfonyl fluoride ( pmsf ) , ethylenediamine tetra acetic acid ( edta ) , thiazolyl blue tetrazolium bromide , and hank 's balanced salt solution - modified ( hbss ) were purchased from sigma chemical co. ( st . louis , mo , usa ) . fetal bovine serum ( fbs ) , antibiotic - antimycotic ( penicillin , streptomycin , and amphotericin b ) , and trypsin - edta were purchased from invitrogen ( grand island , ny , usa ) tris - buffered saline ( tbs ) , dulbecco 's modified eagle 's medium ( dmem ) , and phosphate - buffered saline ( pbs ) were purchased from welgene inc . p - akt and t - akt antibodies were purchased from cell signaling technology ( beverly , ma , usa ) . il-1 antibody was purchased from santa cruz biotechnology ( santa cruz , ca , usa ) . il-8 antibody was purchased from bioworld ( dublin , oh , usa ) . goat anti - rabbit igg - hrp , rabbit anti - goat igg - hrp , and goat anti - mouse igg - hrp were purchased from bethyl ( montgomery , tx , usa ) . rainbow prestained molecular weight marker was purchased from amersham ( arlington heights , il , usa ) . n , n,n-tetramethylethylenediamine ( temed ) , and enhanced chemiluminescence ( ecl ) agents were purchased from perkinelmer life sciences ( boston , ma , usa ) . nitrocellulose ( nc ) membrane , tris / glycine / sds buffer , tris / glycine buffer , and restore tm western blot stripping buffer were purchased from thermo ( rockford , il , usa ) ; il-6 elisa kit was purchased from enzo ( farmingdale , ny , usa ) and the 30% acrylamide / bis solution was purchased from bio - rad ( richmond , ca , usa ) . all animal experiments were performed in accordance with the guidelines of the institutional animal care and use committee of the institute for molecules - based new drug development . adult cats of either sex weighing between 2.5 and 3.5  kg were anesthetized with zoletil 50 ( 12.5  mg/0.25  ml / kg ) before the abdomen was opened with a midline incision . the esophagus was excised , washed , and maintained in krebs buffer composed of 116.6  mm nacl , 1.2  mm nah2po4 , 21.9  mm nahco3 , 3.4  mm kcl , 5.4  mm glucose , 2.5  mm cacl2 , and 1.2  mm mgcl2 . the mucosa from esophagus was sliced into 0.5  mm thick sections with a stadie riggs tissue slicer ( thomas scientific apparatus , philadelphia , pa , usa ) . the last slices were cut into 2  mm  2  mm tissue squares with scissors . the sliced tissue was placed into dmem supplemented with 10% fbs containing 100  u / ml penicillin , 0.1  mg / ml streptomycin , and 0.25  g / ml amphotericin b and incubated in a humidified atmosphere of 5% co2 and 95% air at 37c . after 10 days , the medium was exchanged with fresh dmem containing 10% fbs . after reaching confluence , the cells were then counted , seeded at 2  10  cells / ml on 100  mm culture dishes , and maintained in dmem containing 10% fbs . the cell viability was determined by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) reduction assay . in this assay , viable cells convert mtt to insoluble blue formazan crystals via the mitochondrial respiratory chain enzyme succinate dehydrogenase . the cells were plated at a density of 1  10/well on 6-well plates and maintained in dmem containing 10% fbs . when the cells were made quiescent at confluence by incubation , the media was changed with serum - free dmem for 24 hours to arrest cell growth and silence gene activity , followed by treatment with each indicated agent for the indicated time periods . after incubation , the cells were rapidly washed twice with ice - cold pbs and incubated with mtt solution ( final concentration , 5  mg / ml ) for 4 hours at 37c . absorbance was monitored at 570  nm with a microplate reader ( molecular devices , sunnyvale , ca , usa ) . when the cells reached confluence , they were serum starved by incubation in serum - free dmem for 24 hours . the cells were then stimulated with each compound for the indicated time periods or at the indicated concentrations . after incubation , the cells were rapidly washed twice with ice - cold pbs and lysed with ice - cold lysis buffer ( 20  mm tris - hcl ( ph 7.4 ) , 0.5  mm edta , 0.5  mm egta , 1% ( w / v ) triton x-100 , 0.01% ( w / v ) sds , 10  g / ml leupeptin , 10  g / ml aprotinin , 1  mm pmsf , and 0.7  g / ml -mercaptoethanol ) for 5 minutes . they were then sonicated ( 6 seconds , 3x ) and centrifuged to remove cellular debris for 10 minutes with 13,000  rpm at 4c ; the supernatants were collected and stored at 70c for protein assay and western blot analysis . the protein concentration of the supernatant in each reaction vial was measured spectrophotometrically using the bio - rad assay ( bio - rad chemical division , richmond , ca , usa ) . equal amounts of protein from each sample were subjected to electrophoresis on a 10% sds - polyacrylamide gel and transferred to a nc membrane using the power pac 1000 ( bio - rad , melville , ny , usa ) power supply . to block any nonspecific binding , the nc membrane was incubated in 5% nonfat dry milk or 3% bsa in tbs for 60 minutes followed by three rinses in milk - free tbs . the membranes were with shaking with primary antibodies raised against p - akt , il-1 , and il-8 incubated overnight at 4c , followed by three washes with tbs containing 0.1% tween 20 . the same blots were subsequently stripped with western blot stripping buffer and reprobed with actin and t - akt antibodies to confirm the uniformity of protein loading . the results were analyzed by quantity one analysis software ( bio - rad chemical division , richmond , ca , usa ) . the percentage of akt activation or il-1 and il-8 expression was calculated as the ratio of phosphorylated akt to total akt or il-1 and il-8 to actin . the levels of il-6 released into the culture medium were quantified using an il-6 elisa kit . differences among the groups were analyzed using one - way anova and student 's t - test . mtt assays were performed in cultured eecs to investigate the cytotoxic effect of hydrogen peroxide . the cells were incubated with hydrogen peroxide at the indicated concentration for 24 hours and then cell viability was measured using the mtt assay ( figure 1 ) . the cell viability was decreased by 300  m hydrogen peroxide in a dose - dependent manner and its decrease was apparent at a higher concentration of h2o2 . the viability of cells exposed to 600  m hydrogen peroxide was reduced to 40% when compared to control . serum - starved cells were exposed to 300  m hydrogen peroxide at the indicated time periods to examine whether hydrogen peroxide induces il-1 and il-8 expression in cultured eecs . 300  m of hydrogen peroxide which made the maximal expression of 5-lox was used for inflammation induction . hydrogen peroxide induced the increased expression of il-1 and il-8 with a maximal reach at 6 hours . a longer stimulation with hydrogen peroxide reduced the il-1 and il-8 expression only slightly . the expression profile of class i pi3k r and c isoforms in feline eecs was established ( figure 3 ) . the verification of protein expression by western blot confirmed that p110 , p85 , p85 , and p55 are indeed predominantly expressed and that p110 , p110 , and p110 are weakly expressed when the cells were untreated . after the treatment with 300m hydrogen peroxide for 6 hours the expression of p110 was significantly increased while the expression of p110 , p110 , p85 , p85 , and p55 was little changed only and slightly increased after the treatment with hydrogen peroxide . mtt assay had been performed and the morphology of eecs was observed to identify the cell viability and the morphologic changes after the treatment of pik-75 ( figure 4 ) . feline eecs were pretreated with pik-75 at the indicated concentrations ( 0.1 , 0.5 , 1 , and 5  m ) for 1 hour and treated with 300  m hydrogen peroxide for 6 hours . treatment with pik-75 for 1 hour did not cause strong changes of cell viability and morphology in eecs . akt as a major downstream effector of pi3k was examined to determine the effect of pik-75-mediated pi3k inhibition on downstream signaling events ( figure 5 ) . the cells were treated with 300  m h2o2 for 30 minutes to investigate the phosphorylation of akt . the western blot analysis of the lysates from h2o2-stimulated eecs showed a prominent upregulation in the phosphorylation status of akt compared with lysates from nave cells . more importantly , the pretreatment of eecs with pik-75 inhibited the h2o2-induced phosphorylation of akt . the cells were pretreated with pik-75 at the indicated concentrations and exposed to 300  m hydrogen peroxide and then il-1 expression was measured by western blot to examine whether pik-75 downregulates il-1 expression in cultured eecs ( figure 6 ) . the expression of il-1 was upregulated when treated with 300  m hydrogen peroxide for 6 hours . the cells were pretreated with different doses of pik-75 ( 0.1 , 0.5 , 1 , and 5  m ) for 1 hour to examine the effect of pik-75 on h2o2-induced il-1 expression . the cells were pretreated with pik-75 at the indicated concentrations and exposed to 300  m hydrogen peroxide , and then il-8 expression was measured by western blot to examine whether pik-75 downregulates il-8 expression in cultured eecs ( figure 7 ) . the expression of il-8 was upregulated when treated with 300  m hydrogen peroxide for 6 hours . the cells were pretreated with different doses of pik-75 ( 0.1 , 0.5 , 1 , and 5  m ) for 1 hour to examine the effect of pik-75 on h2o2-induced il-8 expression.pik-75 inhibited the il-8 expression in a dose - dependent manner . the inhibitory effect of pik-75 in the hydrogen peroxide - induced il-6 release was determined using il-6 elisa kit ( figure 8) . the data showed that the treatment of cultured eecs with hydrogen peroxide caused a significant increase in the il-6 production . however , the il-6 production level was reduced in a dose - dependent manner when the eecs were treated with pik-75 . pi3k is an important regulator of inflammations and its isoforms have been studied as therapeutic targets . it seems to be related with esophageal diseases such as barrett 's esophagus and esophageal cancer [ 7 , 35 ] . in this study , we investigated the expression of pi3k isoforms in feline eecs and their role in the mediation of esophageal inflammations . in the present study , hydrogen peroxide , as one type of ros that is elevated in gerd , exhibited significant cytotoxicity and decreased the cell viability in eecs this cytotoxicity of hydrogen peroxide seemed to be related with its ability to induce il-1 , il-8 , and il-6 , suggesting a role for classic stress signaling pathways during esophagitis and gerd [ 3638 ] . it is consistent with the effect of hydrogen peroxide in the esophagus , inducing il-1 and il-6 . in this study , p110 isoform was the mostly predominant pi3k isoform in eecs when it came to the inflammatory responses induced by hydrogen peroxide . although the expression of p85 , p85 , and p55 was at high level in normal condition , it was not significantly changed after the treatment with hydrogen peroxide . for those reasons , we assume p110 may play an essential role in esophageal inflammation . this result is consistent in the distribution of human intestinal epithelial crypt ( hiec ) cells meaning that p110 is a predominant pi3k isoform . in this study , pik-75 was used as a preferential p110 pi3k inhibitor   to confirm the role of p110 in esophageal epithelial inflammation . for instance , it regulates the secretion of intestinal peptide neurotensin in the small bowel . furthermore , the delivery system of pik-75 was investigated due to an increasing interest in pik-75 [ 43 , 44 ] . the inhibition of p110 was confirmed due to an expression examination of p - akt because p110-positive cases were also positive for p - akt . the expression of p - akt was decreased depending on the concentration of pik-75 if p - akt was treated with hydrogen peroxide . moreover , pik-75 downregulated the production of il-1 , il-6 , and il-8 as proinflammatory cytokines when the cells were cotreated with hydrogen peroxide , while the production of il-1 , il-6 , and il-8 was increased when it was only treated with hydrogen peroxide . the il-8 expression was increased as an esophagitis inducer in human eecs if it was treated with bile acids . patients with reflux esophagitis have high levels of il-8 and il-1 and patients with adenocarcinoma show markedly elevated levels of il-8 and il-1 . the expression of il-6 and il-8 is elevated in the esophageal mucosa of patients with gerd . for those reasons , it is important that pik-75 decreases the production of il-1 , il-6 , and il-8 in feline eecs . furthermore , the requirement for pi3k / akt in the induction of il-6 genes in response to il-1 is revealed [ 49 , 50 ] . the present study data indicate an engagement of p110 in the inflammation suppression in eecs as well as a role as an important mediator in the signaling cascade that leads to an initiation of the inflammatory response . although pi3k is a good target for the development of anti - inflammation drugs , pi3k inhibitors have some limitations . pi3k inhibitors are associated with toxicities resulting from off - target effects that can not not be fully explained yet . targeting a single isoform would have fewer side effects and avoid the toxicity in immune system . however , it remains a challenge to identify the optimal concentration and exposure time for therapeutic uses and a more specific signaling in eecs . furthermore , full investigation of the expression and the roles of pi3k isoforms in many different cells could prevent side effects when inhibitors are used as drugs . as a result of this study we found that hydrogen peroxide , which is highly expressed in esophagitis , activates pi3k signaling and that p110 plays an important role in esophageal inflammation . by hydrogen peroxide therefore , these data suggest that pik-75 has an anti - inflammatory effect in feline eecs .
isoform - selective inhibitors of phosphoinositide 3-kinase ( pi3k ) activation have an anti - inflammatory effect by reducing proinflammatory cytokines . 
 cultured feline esophageal epithelial cells ( eec ) of passages 3~4 were treated with hydrogen peroxide and pik-75 . 
 the cell viability was measured by a mtt incorporation assay . the distribution of pi3k isoforms , p - akt , il-1 , and il-8 was inferred from western blots . 
 the release of il-6 was determined by elisa . 
 the cell morphology was not considerably different from nontreated cells if the cells were pretreated with pik-75 and treated with 300  m hydrogen peroxide . 
 the density of p110 of pi3k was increased , but that of other types was not affected after the treatment with hydrogen peroxide . the density of p - akt , when the cells were exposed to pik-75 and hydrogen peroxide , was diminished dose dependently more than that of hydrogen peroxide treatment only . 
 the decrease of p - akt showed an inhibition of pi3k by pik-75 . pik-75 
 dose dependently reduced the expression of il-1 , il-8 , and the level of il-6 compared with hydrogen peroxide treatment only . 
 these results suggest evidence that p110 mediates esophageal inflammation and that pik-75 has an anti - inflammatory effect by reducing proinflammatory cytokines on feline esophageal epithelial cultured cells .