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rostaglandin E-2 increases intestinal epithelialermeability in vitroikael Petrosyan MD, Yigit Guner MD, Catherine Hunter MD,ikunj Chokshi MD, Monica Williams,

effrey Upperman MD, FACS, Anatoly Grishin PhD,enri R Ford MD, FACShildrens Hospital Los Angeles, Los Angeles, CA

NTRODUCTION: Necrotizing enterocolitis (NEC) is character-zed by loss of intestinal epithelial barrier integrity. Sustained upregu-ation of cyclooxygenase 2 (COX-2) and various prostanoids haveeen linked to the pathogenesis of epithelial injury seen in NEC.ne of the most important prostanoids in the gut is prostaglandin-2 (PGE-2). We hypothesize that high levels of PGE-2 contribute to

he development of gut barrier failure in NEC by causing intestinalpithelial injury, and/or by altering epithelial permeability.

ETHODS: Caco-2 cells were grown as polarized monolayers onollagen-coated membranes in bicameral chambers and exposed toarious concentrations (2-50 microg/ml) of exogenous PGE-2.ransepithelial electrical resistance (TEER), an indicator of epithelialonolayer integrity, was measured at various time points (1-5 min)

sing a voltammeter. Apoptosis was assayed with TUNEL stain.

ESULTS: At concentrations normally found in inflamed tissues inivo (1-50 microg/ml), PGE2 caused a dose-dependent and time-ependent decrease in TEER in Caco-2 monolayers ( Table). De-rease in TEER was significantly faster with higher concentrations ofGE-2 (p�0.005). The decrease in TEER was long-lasting, but re-urned to baseline within 24 h after removal of PGE2. PGE-2 alonead no effect on enterocyte apoptosis.

GE-2 �g/ml% TEERat 30 s

% TEER at 1min

% TEER at2 min

% TEER at5 min

1 95 81 65 512 91 79 54 495 88 77 55 530 89 78 53 515 87 45 (p�0.005) 44 440 85 49 (p�0.005) 46 45

ONCLUSIONS: Addition of PGE2 to Caco-2 monolayers in-uces profound and long-lasting disruption of epithelial barrier func-ion, as evidenced by a decrease in TEER. PGE-2 alone does notnduce epithelial apoptosis. These results suggest that PGE-2 maylay a role in gut barrier failure seen in NEC by increasing paracel-

ular permeability through disruption of tight junctions.

ilencing of heat shock factor 1: A potentialherapeutic strategy for pancreatic cancerikas Dudeja MD, Rohit Chugh MD, Steve Skube,umi Yokoyama PhD, Daniel Borja-Cacho MD, Paul Zwolak MD,ara B Antonoff MD, Rajinder Dawara PhD,

elwyn M Vickers MD, FACS, Ashok K Saluja PhDniversity of Minnesota, Minneapolis, MN

NTRODUCTION: Heat Shock Factor 1 (HSF1) is a transcription

actor for Heat Shock Proteins (HSP70, HSP27), multiple drug t

S212008 by the American College of Surgeons

ublished by Elsevier Inc.

esistance genes and other survival proteins. We have previously dem-nstrated that HSP70 is overexpressed in pancreatic cancer cells sug-esting that HSF1 is overactive in pancreatic cancer cells. We hypoth-size that silencing HSF1 expression should induce apoptosis inancreatic cancer cells.

ETHODS: HSF1 expression was reduced in MiaPaCa-2 andanc-1 pancreatic cancer lines by treatment with HSF1 siRNA. Twonique sequences of HSF1 siRNA were used to rule out any off-arget effects of siRNA. Cells treated with non-silencing siRNA se-uence (sequence with no homology to any gene) were used as con-rol. Cell viability (MTT assay) and apoptosis (annexin V staining,aspase 3 and 9) were measured.

ESULTS: Inhibition of HSF1 expression in MiaPaCa-2 andanc-1 reduced the viability at 48h and 72h of treatment (viabilityalues [mean�SEM] expressed as % of control. 48h: Panc17.2�4.3, MiaPaCa-2 61.0�3.9, 72h: Panc1 49.2�3.4, MiaPaCa-21.2�8.8). Similar results were obtained with the other HSF1iRNA sequence used. The cell death was associated with markednnexin positivity and activation of caspase 3 and 9 starting at 24h.

ONCLUSIONS: Silencing of HSF1 expression activates caspaseependent apoptotic cell death in pancreatic cancer cells. Implica-ion: Targeting HSF1 has a therapeutic potential in the treatment ofancreatic cancer and holds a great promise as a potential candidateor the drug development. This approach offers the advantage ofbility to target multiple genes involved in resistance to apoptosis,ell death and conventional chemotherapy.

isplatin retains high-mobility group box 1HMGB1) intranuclearly and protects againstepatic ischemia reperfusion (I/R) injury

on S Cardinal MD, Pinhua Pan MD, Rajeev Dhupar MD,ohn Klune BS, Sung Cho MD, David A Geller MD, FACS,llan Tsung MDniversity of Pittsburgh Medical Center, Pittsburgh, PA

NTRODUCTION: The nuclear protein HMGB1 activates inflam-atory pathways upon release from ischemic cells. We have previ-

usly shown neutralizing antibodies to HMGB1 protect against in-lammation and organ injuryfollowing liver I/R. Platinatinghemotherapeutics induce DNA cross links which effectively seques-er HMGB1 in the nucleus. The purpose of this study was to test theypothesis that cisplatin may protect against hepatic IR by prevent-

ng the release of HMGB1.

ETHODS: In vitro, primary hepatocytes were exposed to hypoxia1% O2) or treatment with hydrogen peroxide (H2O2) in the pres-nce or absence of cisplatin. Immunoblotting and immunofluores-ent staining for HMGB1 were performed. In vivo, partial warmepatic I/R was performed on C57BL/6 mice after 12 hour pretreat-ent with either NSS or cisplatin. Serum and tissue samples were

nalyzed for enzyme, protein and inflammatory mediator levels.

ESULTS: Oxidative stress (hypoxia or H2O2 treatment) inducedhe nucleocytoplasmic shuttling and release of HMGB1 from cul-

ured hepatocytes. Cisplatin abrogated this effect and resulted in the

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