AGA Abstracts W1929 Interplay Between GSK3 and JNK Pathways Regulates Human Pancreatic Cancer Cell Survival Benoit Marchand, Marie-Josee Boucher The glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase involved in glycogen metabolism but also in other important cellular functions such as proliferation and survival. We have previously demonstrated that prolonged inhibition of GSK3 activity induced apoptosis in human pancreatic cancer cells. However, the mechanisms involved remain unresolved. METHODS. Experiments were done in human pancreatic normal ductal cells (HPDE) and human pancreatic cancer cells (BxPC-3, PANC-1, MIA PaCa-2). GSK3 activity was inhibited using a specific GSK3 inhibitor (SB216763, 20μM) and specific GSK3α/β siRNA and shRNA. Apoptosis was evaluated by Annexin-V staining assays, labeling of active mitochondria and PARP cleavage. RESULTS. 1-Prolonged inhibition (48,72h) of GSK3 activity induced apoptosis in pancreatic cancer cells but not in normal pancreatic cells. 2- Increased Bim and decreased Bcl-2 protein expression levels were observed in SB216763- treated cells (24,48,72h). No modulation in their mRNA expression levels (RT-PCR) was noted. Furthermore, protein expression levels of Mcl-1, Bcl-Xl, Bad, Bax and PUMA remained unaffected following inhibition of GSK3 activity. 3-Treatment of pancreatic cancer cells with SB216763 (48,72h) induced caspase-7 activation. More importantly, we observed caspase- 7 activation and PARP cleavage in cells treated for 24h with SB216763 followed by a 48h- recovery period in complete medium suggesting that inhibition of GSK3 activity for 24h is sufficient to induce irreversible pancreatic cancer cell death. 4-Inhibition of GSK3 activity neither influence Akt nor Erk activities, two signaling pathways reported to mediate survival signals in pancreatic cancer cells. However, increased c-jun expression was observed in SB216763-treated cells (24,48,72h) suggesting that inhibition of GSK3 activity up-regulated the activity of the JNK pathway. 5-Interestingly, combined treatment with SB216763 and the specific JNK inhibitor SP600125 reduced the SB216763-induced c-jun expression as well as attenuated the SB216763-induced apoptosis. CONCLUSION. Taking together, our data demonstrated that prolonged inhibition of GSK3 activity initiates apoptosis of pancreatic cancer cells which is associated with increased Bim and decreased Bcl-2 protein expression levels and caspase-7 activation. Moreover, we uncovered a crosstalk between the GSK3 and JNK pathways which seem to play an important role in determining between cell survival and cell death. More interestingly, the critical survival signals mediated by GSK3 activity are specific to pancreatic cancer cells since cell survival of normal pancreatic cells remain unaffected by GSK3 activity inhibition. W1930 Nuclear Galectin-3 Confers Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Resistance to Colon Cancer Cells By Inhibiting Caspase-8 Activation Nachman Mazurek, James C. Byrd, Yun Jie Sun, Robert Bresalier Background: Galectin-3 exhibits pro- and anti-apoptotic effects depending on its subcellular localization, nature of stimulus and cell type. TRAIL is a potential cancer therapeutic agent currently in clinical trials. However, drug-resistance may limit its application. Objective: To elucidate mechanisms responsible for TRAIL-resistance in colon cancer. Methods: Cell viabil- ity was assessed by the MTS assay and apoptosis was determined by PARP degradation and flow cytometry. Western blots were used to study the underlying mechanism of cell death. Forced nuclear expression of galectin-3 was achieved by either mutating the nuclear export signal or adding 3 nuclear localization signals to its C-terminus. Results: Exposure of LS- LiM6 colon cancer cells to TRAIL resulted in a dose- and time-dependant apoptotic cell death which plateaued within 6 hrs at 65% cell death. The residual cells were propagated with periodic exposure to TRAIL to yield a TRAIL-resistant cell line, LiM6-TR. Exposure of LS-LiM6 but not LiM6-TR cells to TRAIL lead to the recruitment of the initiator caspase-8 followed by Bid cleavage, cytochrome C release, activation of caspase-9, processing of caspase- 3 and PARP degradation. TRAIL also induced a caspase-3-dependent degradation of beta- catenin and nuclear PTEN in LS-LiM6 but not in LiM6-TR. Analysis of components of the Death-Inducing Signaling Complex (DISC) and of endogenous caspase-8 inhibitors revealed no significant differences, indicating that the TRAIL resistance of LiM6-TR results from inhibition of caspase-8 activation. Since TRAIL decreased p-Akt in both LiM6 and LiM6- TR, apoptosis triggered by TRAIL was independent of the PI3K/Akt signaling pathway. Although TRAIL induced p-JNK only in LS-LiM6, the specific JNK inhibitor SP600125 did not rescue LS-LiM6, and the JNK activator parthenolide failed to sensitize LiM6-TR to TRAIL, indicating that TRAIL-induced apoptosis is also independent of JNK signaling. Subcellular fractionation revealed a 6-fold higher level of nuclear galectin-3 in LiM6-TR than in LS- LiM6, with no difference in cytoplasmic galectin-3. Forced nuclear expression of galectin- 3 in LS-LiM6 inhibited caspase-8 activation, conferred TRAIL-resistance and prevented the degradation of nuclear PTEN and beta-catenin. Conclusion: Accumulation of nuclear galectin- 3 causes TRAIL-resistance in colon cancer cells by inhibiting the activation of caspase-8. The TRAIL-induced apoptotic pathway involves nuclear PTEN and blocking the Wnt pathway, but is independent of the PI3K/Akt and JNK signaling pathways. Elevated levels of nuclear galectin-3 may predict unfavorable outcome to TRAIL-based therapy of colon cancer patients. W1931 Helicobacter pylori Remodeling of the Gastric Epithelium: Pathogen Targeting of Mitochondria to Induce Cellular Dysfunction Prashant Jain, Akshra Verma, Steven Blanke Chronic infection with the gastric pathogen Helicobacter pylori is a risk factor for the develop- ment of gastric ulcer disease or gastric adenocarcinoma in humans. However, the molecular basis underlying H. pylori persistence within the harsh environment of the gastric mucosa remains poorly understood. We established an In Vitro model for evaluating the changes to gastric epithelial cells induced by the vacuolating cytotoxin (VacA), the only known exotoxin released by H. pylori. When incubated with the human-derived AZ-521 gastric epithelial A-756 AGA Abstracts cell line, VacA induced a dose- and time-dependent fragmentation of mitochondria. VacA- dependent mitochondrial damage was accompanied by a gradual and cumulative loss of mitochondrial membrane potential (Dpsi) and ATP production, indicating progressive loss of mitochondrial function. VacA entered cells and localized to mitochondria in a manner correlated to the cumulative Dpsi loss, suggesting that VacA may exert its biological activity directly at this organelle. Progressive Dpsi dissipation progressed over 4-6 h until a rapid and irreversible release of mitochondrial-associated cytochrome c into the cytosol, consistent with increased permeability at the mitochondrial outer membrane. Over the next 12-16 h, there was a sequential release of mitochondrial-associated inter-membrane effectors that promote caspase-dependent (Smac and HtrA2), and caspase-independent (AIF and endonu- clease G) cellular death mechanisms. Permeabilization of the mitochondrial outer membrane required VacA-dependent recruitment of the endogenous mitochondrial targeted cellular stress sensor Bid to the mitochondrial outer membrane. Collectively, these data suggest that VacA intoxication of AZ-521 cells induces changes in at least three stages. Early intoxication results in progressive mitochondrial dysfunction, followed by activation of the pro-apoptotic stress sensor protein Bid, followed finally by permeabilization of the mitochondrial outer membrane and an ordered release of caspase-dependent and -independent pro-death effectors. We propose that at low VacA concentrations that occur distal to the site of infection, toxin-dependent mitochondrial modulation results in dysfunctional cells contributing to an epithelial barrier with impaired functions including decreased acid production or attenuation in the capacity of the innate immune system to sense and respond appropriately to the presence of H. pylori. Moreover, our data suggest that at higher VacA concentrations that occur immediately at the site of infection, mitochondrial dysfunction occurs to a greater extent, resulting in apoptotic cell death and the eventual release of nutrients. W1932 The BCL-2 Family of Proteins As a New Therapeutic Target for the Treatment of Colorectal Cancer Mary D. Cannon, Caoimhin Concannon, Frank E. Murray, Jochen H. Prehn BACKGROUND Colorectal cancer is a leading cause of cancer related death. In addition to surgery, radiochemotherapy improves prognosis and survival in a subgroup of patients. Current treatment paradigms involve 5-fluorouracil and oxaliplatin based regimens. These compounds inhibit cell proliferation and reduce tumour size by activating apoptosis. Apoptosis is regulated by the Bcl-2 family of proteins which contains both pro- and anti- apoptotic members. P53 initiates apoptosis and PUMA, a member of the pro-apoptotic BH3-only subfamily of Bcl-2 proteins, mediates most of the pro-apoptotic activity of p53. Dysregulation of the expression levels of Bcl-2 family members is observed in many cancer types. BH3 mimetics, which function in a manner similar to BH3 only proteins, have recently evolved as novel chemotherapeutic agents. AIM This study aimed to assess the role of the Bcl-2 family of proteins on the induction of apoptosis by 5FU and oxaliplatin treatment, and whether the addition of BH3 mimetics would enhance the therapeutic efficacy of standard treatment regimens. METHODS A qualitative and quantitative analysis of the expression levels of the major pro- and anti-apoptotic Bcl-2 proteins as well as p53 status was performed in both a panel of colon cancer cell lines (DLD-1, LoVo, Colo205, HT29, HCT116, HCT116 p53-/- & HCT116 PUMA-/-) and patient tumour samples using western blotting techniques. In parallel, the sensitivity of these cell lines following treatment with 5FU and oxaliplatin in the presence and absence of BH3 mimetics was assessed by Annexin V/PI staining and flow cytometry to measure levels of apoptosis induced by this treatment. Long term cell survival was assessed using clonogenic survival assays. RESULTS Western blotting demon- strated that expression levels of Bcl-2 family proteins varied strongly between individual patients and cell lines, with LoVo cell line expressing high levels of Bak, Bcl-2 and Bcl-XL and no detectable Bax. P53 appears central to the cell death process with cell lines containing wild type p53 (LoVo, Colo205, HCT116) having greater sensitivity to apoptosis induced by 5FU and oxaliplatin. Additionally, HCT116 WT cells demonstrated greater sensitivity to 5FU and oxaliplatin treatment than HCT116 p53-/- or HCT116 PUMA-/- cells as assessed by clonogenic survival assays. Utilization of BH3 mimetics enhanced the cytotoxicity of 5FU and oxaliplatin treatment. CONCLUSION Our data suggest that the Bcl-2 family of proteins are an important new therapeutic target for the treatment of colorectal cancer, and that individual protein expression patterns may dictate the ability of BH3 mimetics to induce or reactivate apoptosis. W1933 Increased Level of Cell Surface Annexin II Promotes Pancreatic and Colon Cancer Cells Growth In Vitro Agata Ptak-Belowska, Agata Gawad, Tomasz Brzozowski, Wieslaw W. Pawlik Background: Gastrin exerts growth stimulatory and co-carcinogenic effects in gastrointestinal (GI) tract. Gastrin peptide mediates its growth effects by binding to CCK 1 and CCK 2 receptors. However, recently annexin II was identified as a high affinity binding protein for gastrin and its precursors. It's known that annexin II binds gastrin and mediates growth factor effects on GI cancer cells. Proliferator-activated receptor γ (PPARγ) is a member of nuclear hormone receptor family of transcription factors. The effects of thiazolidinedione PPARγ agonists on GI carcinogenesis remain controversial. Recently we have shown that the PPARγ agonist, ciglitazone, increases anti-apoptotic potential indirectly through up regulation of the gastrin gene in pancreatic cancer cell lines. The link between PPARγ and cell surface annexin II has not been evaluated yet. Aims: We assessed the role and possible interactions between annexin II, gastrin and PPARγ in the development of colon and pancre- atic cancer. Moreover, a possible role for gastrin, annexin II and PPARγ in circumvention of apoptosis using human pancreatic and colon cancer cell lines as In Vitro models was investigated. Materials & methods: The human pancreatic (PANC1), colon (HCT116, HT29) cancer cell lines and control cells were used. Cell lines were treated with the PPARγ agonist ciglitazone and gastrin to investigate apoptotic potential in relation to PPARγ, gastrin and annexin II. PPARγ, gastrin and annexin II genes expression by means of Real-Time PCR and confirmatory Western Blot methods. Additionally, PPARγ level in cells was downregulated by specific small interfering RNA and the growth effects of gastrin and annexin II were measured using MTT assays. Results: Annexin II mRNA and cell surface annexin II protein