levels were significantly higher in pancreatic and colon cancer cells than in control. Ciglita-zone, a specific PPARγ agonist, increased gastrin gene expression and Bcl-2 and PKB/Aktprotein levels in each of cancer cell lines examined. Furthermore, transfection of the PANC1,HT29 and HCT116 cells with a PPARγ siRNA reversed ciglitazone-induced PKB/Akt phos-phorylation and Bcl-2 protein up-regulation. Conclusions: 1) Gastrin peptide promotescancer cells growth via an increase in annexin II cell surface concentration in these cells;2) gastrin cooperates with the tumour-growth promoting activity of PPARγ agonists.

W1934

Combined Inhibition of mTOR and MAPK Signaling Inhibits Growth, InducesApoptosis, and Prevents Cell Cycle Progression in Colorectal CarcinomaPat Gulhati, Jing Li, Jianli Dong, B. M. Evers

The PI3K-Akt-mTOR and Ras-Raf-ERK (MAPK) signaling pathways are constitutively activ-ated and serve critical functions in the growth and progression of colorectal cancer (CRC).The mTOR pathway, comprised of two distinct complexes mTORC1 and mTORC2, isinvolved in cell proliferation, survival and invasion. Rapamycin inhibits the kinase activityof mTORC1; prolonged rapamycin treatment also inhibits mTORC2 assembly and Aktactivation in certain cells. The MAPK pathway plays an essential role in cell proliferationand differentiation. Sorafenib is a kinase inhibitor that inhibits MAPK signaling at the levelof the Raf kinase. Recent studies suggest that inhibition of mTORC1 with rapamycin leads toMAPK pathway activation through a S6K-PI3K-Ras feedback loop. Therefore, we investigatedwhether combined targeting of mTOR and MAPK signaling pathways, using rapamycin andsorafenib respectively, has therapeutic benefits in CRC. METHODS. HCT116 human coloncancer cells possessing mutant K-Ras and wild-type B-Raf were utilized due to their relativelyhigh activation of Akt-mTOR signaling. Cells were treated with either rapamycin (20 nM)or sorafenib (10 μM) or combinatorial treatment. Effects on cell proliferation (measured byCoulter counter cell counts), apoptosis (measured by detecting the level of histone-associatedDNA fragments in mono- and oligonucleosomes) and cell cycle progression (measured byfluorescence-activated cell sorting) were analyzed. RESULTS. (i) Rapamycin or sorafenibsignificantly decreased cell proliferation compared to the control group; combinatorial treat-ment significantly potentiated growth inhibition compared to single agent treatment alone.(ii) Sorafenib, but not rapamycin, increased apoptosis compared to the control group;combination treatment was more effective than either agent alone. (iii) Single agent treatmentwith rapamycin or sorafenib inhibited G1-S phase cell cycle progression; combinatorialtreatment increased growth arrest compared to rapamycin, but not sorafenib monotherapy.CONCLUSIONS. We show that combinatorial inhibition of the mTOR and MAPK signalingpathways, using rapamycin and sorafenib respectively, significantly enhances growth inhibi-tion compared to single-agent treatment. CRC is a heterogeneous disease with multipleaberrant signaling pathways driving the oncogenic behavior of tumors; simultaneous inhibi-tion of different signaling pathways may be more effective than single pathway inhibitionin CRC therapies.

W1935

The Intracellular Tyrosine Kinase PTK6 Is Induced in Select Crypt Cells inthe Colon After Azoxymethane TreatmentJessica Gierut, Wenjun Bie, Yu Zheng, Angela L. Tyner

Protein Tyrosine Kinase 6 (PTK6) is an intracellular tyrosine kinase that is expressed athighest levels in epithelial cells that are exiting the cell cycle and undergoing terminaldifferentiation in the small intestine and colon. PTK6 is also expressed in other regeneratingepithelial linings such as the skin and oral epithelia, and a decrease in PTK6 expression hasbeen correlated with the development of skin and oral squamous epithelial cancers. Incontrast PTK6 is not expressed in normal breast or ovarian tissue, but it is highly expressedin many breast and ovarian carcinoma cell lines and primary breast and ovarian tumors. Innormal prostate tissue PTK6 is localized in the nucleus, but in prostate tumors PTK6 istranslocated to the cytoplasm. These data indicate that expression and localization of PTK6are tissue-specific and regulate its function. The aim of our study was to determine the roleof PTK6 in the initiation and progression of colon carcinogenesis. Previously we reportedthat although PTK6 expression is correlated with differentiation in the normal intestine,PTK6 can enhance development of ACF and colorectal tumors In Vivo. To address the rolesof PTK6 in tumor initiation, wild type and PTK6 knockout mice were subjected to a singleinjection of the colon carcinogen azoxymethane (AOM) and sacrificed 6 or 72 hours post-injection. Using immunohistochemistry we found that PTK6 expression was induced in thecrypt compartment of the colon where PTK6 is not normally expressed. Induction of PTK6was seen at the base of the crypt where stem cells reside. Following AOM injection wefound that both apoptosis and proliferation were induced in wild type mice, but not inPTK6 null mice treated with AOM. Deregulated apoptosis and proliferation contribute tothe development of cancers. Our lab is currently investigating whether the induction ofPTK6 in select cells of the crypt after AOM treatment is altering the signaling cascade withinthe stem cell niche. Such an alteration may give rise to increased proliferation that contributesto tumor initiation. Although PTK6 expression is correlated with growth suppression anddifferentiation in the normal colon, our data suggest that PTK6 can enhance developmentof colorectal cancers In Vivo, possibly by having an effect on stem cell signaling.

W1936

Celecoxib-Induced Apoptosis Is Enhanced By Inhibition of Autophagy inHuman Colon Cancer CellsShengbing Huang, Frank A. Sinicrope

Background: Celecoxib is a selective inhibitor of the cyclooxygenase-2 (COX-2) enzyme thatinduces cell death In Vitro and In Vivo and has been shown to prevent colorectal adenomarecurrence in humans. We determined the contributions of apoptosis and autophagy tocelecoxib-induced cell death in human colon cancer cells. Autophagy is protein degradationprocess whereby cellular proteins and organelles are delivered to lysosomes for digestion.

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Many anti-neoplastic drugs induce autophagy and we tested the hypothesis that inhibitionof autophagy may enhance apoptosis induction. Materials & Methods: Human colon cancercell lines (HT-29, HCT116) were incubated with celecoxib (0-120 μM) alone and in combina-tion with 3-methyladenine (3-MA; 10 mM). Caspase cleavage, Bid truncation, and LC3I-IIexpression were determined using highly specific antibodies by Western blotting. A GFP-LC3B chimeric plasmid was introduced into HT-29 cells and its distribution pattern wasanalyzed by fluorescence microscopy. Beclin 1 knockdown HT-29 cells were generated usinglentiviral short hairpin RNA (shRNA). Results: Celecoxib treatment induced a dose-dependentapoptosis characterized by caspase cleavage and Annexin V staining. Celecoxib also causeda conversion of autophagosome-associated protein light chain 3 (LC3) from cytosolic (LC3I)to membrane-bound (LC3II) forms. Furthermore, celecoxib-induced autophagic cells weredetected by GFP-LC3 that produced a characteristic punctuate pattern at fluorescence micro-scopy, compared to untreated cells. Addition of 3-methyladenine (3-MA), an inhibitor ofautophagosome formation and a class III PI3 kinase inhibitor, blocked LC-3II induction bycelecoxib and also enhanced celecoxib-induced apoptosis as shown by enhanced cleavageof caspases-8,-9, -3 and truncated Bid expression. To confirm the role of autophagy incelecoxib-induced cell death, knockdown of Beclin 1 expression, a Bcl-2-binding proteinthat is essential for autophagy, using shRNA enhanced celecoxib-induced apoptosis shownby caspase-3 cleavage. Conclusion: Celecoxib induces both apoptosis and autophagy incolon cancer cells. Moreover, suppression of autophagy can enhance apoptotic signaling,suggesting that strategies to inhibit autophagy may increase the anti-tumor efficacy of cele-coxib for the prevention and treatment of colorectal cancer.

W1937

In-Vitro Cytotoxicity of Bile Acids and Fecal Water to Human ColonicEpithelial CellsAnneke Rijnierse, Nicole de Wit, Noortje IJssennagger, Michael Müller, Roelof Van derMeer

Background&Aims: Unconjugated secondary bile acids present in colon can induce epithelialcell hyperproliferation and therefore promote colon cancer. Another nutritional stressor,heme (typical of red meat) is also known to be a high risk factor for the development ofcolorectal cancer by injuring surface mucosa and consequently inducing compensatoryhyperproliferation of crypt cells. The mechanism of action of these cytotoxic compounds ishypothesized to be a disturbed signaling from the injured surface epithelial cells to theproliferative crypt compartment. Therefore we aim to develop an in-vitro model system forthe identification of basolaterally secreted molecules that signal epithelial surface injury dueto nutritional stressors. Methods: Monolayers of human epithelial Caco-2 cells were grownon permeable transwell filters to create a model with an apical and basolateral compartment.Cells were grown for 14-21 days to allow them to differentiate into surface epithelial cellswith distinct apical and basolateral membranes. Cells were exposed for 30 min or 3h to 2mM cholate, 2 mM deoxycholate, and fecal water of rodents fed with heme-enriched dietor control diet. Cytotoxicity was determined by lactate dehydrogenase (LDH) release in theapical and basolateral supernatant and trans-epithelial resistance (TER) measurements. Geneexpression profiles were studied by PCR techniques. Results: Apical exposure of Caco-2cells to deoxycholate led to increased apical LDH release compared to the apical exposureof the non-toxic bile acid cholate. LDH release to the basolateral compartment was negligible.Basolateral stimulation with deoxycholate showed increased cytotoxicity compared to apicalstimulation reflecting the well described differences in lipid composition of the apical andbasolateral membranes of the Caco-2 cells. Furthermore, TER dramatically dropped afterapical stimulation with deoxycholate whereas TER remained at basal levels after stimulationwith cholate. Apical stimulation of Caco-2 cells with fecal water from heme-fed rats showedincreased cytotoxicity compared to fecal water from control-fed animals determined by LDHrelease and TERmeasurements. Gene expression profiles demonstrated an increased epressionof Ki67 (cell proliferation marker) and stress-induced heme oxygenase 1 after 3h of stimula-tion with fecal water from heme-fed rats. Conclusion: This experimental set-up can be usedto study mild cytotoxicity to epithelial cells due to nutrional stressors and to identify putativemolecules that signal surface injury to the proliferative crypt compartment using genomicsapproaches and secretome analysis.

W1938

Methylation Chip Assay for Determination of Gastric Cancer Associated CpGIsland Methylated GeneNayoung Kim, Younmu Jung

Background and aim: Aberrant DNA methylation with Helicobacter pylori (Hp) infection hasbeen recognized as an important factor for gastro-carcinogenesis. This study was performedto evaluate the methylation status of gastric cancer by Illumina GoldenGate methylationassay. Methods: The methylation level of 1506 CpG site in 807 genes were measured byIllumina GoldenGate methylation assay (Illumina, San Diego, CA, USA) in the normal antraltissue from 28 gastric cancer patients and 20 controls To find the target genes whichmay contribute to carcinogenesis by methylation, we used both protein-protein interactionprediction with end gene products and pathway analysis based on KEGG data base. Results:Hp-positive group showed significant hypermethylation in 337 genes and hypomethylationin 170 genes than Hp-negative group. Cancer group showed significant hypermethylationin 118 genes and hypomethylation in 93 genes than control group. To find the target genes,we focused on 83 genes that were significantly hypermethylated both in Hp infection andcancer group. DCC and MOS genes were found to be target genes, according to biologicfunction and previous reports. 40 genes were hypomethylated both in HP positive andcancer group. The target genes were found to be VAV1, PDGFb, and LTA that are knownas oncogenes related to highly ranked significant pathways. In addition, JAK3 and STAT5Awere found to be target genes. Conclusion: Illumina GoldenGate methylation assay was auseful tool in selection of target genes in the aberrant methylation induced by H. pyloriinfection, which may contribute to the H. pylori infection-associated gastric carcinogenesis.Confirmation study using real-time methylation specific PCR is undergoing.

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