39. Transient Receptor Potential A1 (TRPA1) Mediates Pancreatic Inflammatory Pain

192 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 37. COMBINATION THERAPY WITH LITHIUM CHLORIDE AND VALPROIC ACID: A NOVEL TREATMENT FOR GASTROINTESTINAL NEUROENDOCRINE TUMORS. Joel T. Adler, Muthusamy Kunnimalaiyaan, Herbert Chen; University of Wisconsin, Madison, WI Background: Gastrointestinal (GI) neuroendocrine (NE) tumors frequently present with debilitating symptoms and hepatic metastases. Because conventional cancer treatments are largely ineffective against NE tumors, there is an urgent need for new therapeutic approaches. In the human GI NE cell line BON, valproic acid (VPA) has been shown to inhibit growth, upregulate Notch-1, and decrease NE markers. Moreover, lithium chloride is known to inhibit the growth of NE tumor cells in vitro. We hypothesized that lower-dose combination therapy could achieve similar growth inhibition to that of either drug alone. Methods: BON cells were treated with varying combinations of 0-20 mM lithium chloride and 0-3 mM VPA for up to 4 days. Western analysis was used to measure NE tumor markers achaete-scute complex-like 1 (ASCL-1) and chromogranin A (CgA). Growth was measured by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) cellular proliferation assay. Western analysis was used to determine the mechanism of growth regulation. A standard luciferase reporter assay incorporating the centromere-binding factor 1 (CBF-1) binding site was utilized to measure the activity of Notch-1. Results: A dose-dependent decrease in ASCL-1 and CgA with the combination of 20 mM lithium and 1-3 mM VPA was observed. Furthermore, VPA increased the amount of Notch-1 protein. The combination of 20 mM lithium and 2 mM VPA was as effective at growth inhibition as 3 mM VPA at 6 days. An increase in the levels of both p21 and p27 proteins suggested that the growth inhibition is through cell cycle arrest. Conclusions: Combination therapy in BON cells with VPA and lithium chloride effectively reduces growth through cell cycle arrest, upregulates Notch-1, and suppresses NE tumor markers. Significantly, these effects were achieved with lower doses when used in combination than when each drug was used alone. With the prospect of equal efficacy and decreased toxicity to patients, this approach may prove useful in the treatment of GI NE tumors. 38. AKT-1 INHIBITION IS IMPORTANT FOR SUPPRESSION OF NEUROENDOCRINE HORMONE SECRETION IN CARCINOID TUMOR CELLS. Susan Pitt, Herbert Chen, Muthusamy Kunnimalaiyaan; University of Wisconsin School of Medicine and Public Health, Madison, WI Introduction: The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is important in tumorigensis by promoting tumor cell proliferation and suppressing apoptosis. We have previously shown that inhibition of the PI3K-Akt pathway by LY294002 in human carcinoid cells decreases tumor cell growth and inhibits the neuroendocrine (NE) tumor markers human acheate-scute complex 1 (ASCL1) and chromogranin A (CgA) in a dose dependent manner. However, whether or not Akt inhibition is required for NE hormone reduction in carcinoid tumor cells is still unknown. Additionally, the question of which Akt isoforms is most important, Akt-1, ⫺2, or ⫺3, remains unanswered. We hypothesized that knock out of Akt-1 would result in decreased NE marker expression. Methods: Human metastatic pancreatic carcinoid BON cells were transfected with control (non-specific, NS) and Akt-1 siRNAs with dosages ranging from 25-100nM for a 2 day time period. Western analysis was performed for the level of Akt proteins in these Akt siRNA treated cells. In addition, western analysis was utilized to examine the degree of ASCL1 and CgA production. Results: Treatment of BON cells with Akt-1 siRNA led to progressive silencing of Akt-1 protein expression with no affect on Akt-2 or Akt-3 production. Furthermore, this depletion of Akt-1 caused a reduction in NE tumor markers. Specifically, a dose dependent decrease in ASCL1 and CgA was observed

with decreasing Akt-1 protein. Conclusion: In human pancreatic carcinoid tumor cells, inhibition of Akt-1 appears to be necessary for suppression of NE hormone production. As a result, suppression of the PI3K-Akt pathway or elimination of Akt-1 protein itself may provide a novel approach to control hormone production in carcinoid tumors. 39. TRANSIENT RECEPTOR POTENTIAL A1 (TRPA1) MEDIATES PANCREATIC INFLAMMATORY PAIN. Eugene P. Ceppa1, Natalya Vaksman2, Eileen F. Grady2, Nigel W. Bunnett2, Kimberly S. Kirkwood2; 1Duke University Medical Center, Durham, NC; 2University of California, San Francisco, San Francisco, CA The founding member of the TRP family is TRPV1 which is activated by trypsins in the pancreas leading to inflammation and pain. In a somatic model of pain, the cation channel TRPA1 can be activated by multiple pro-inflammatory mediators acting in concert, including the aldehyde 4-hydroxy-2-nonenal (HNE), a product of oxidative tissue injury, which produce neurogenic inflammation in the affected paw and expression of fos protein in laminae I and II of the dorsal horn of the spinal cord, a validated measure of pain. The role of TRPA1 in visceral pain is unknown. We hypothesized that activation of TRPA1 would promote inflammation and pain in the rodent pancreas. Methods: The TRPA1 selective agonist mustard oil (MO, 1%), the endogenous aldehyde HNE (1x10 ⫺4 M), or vehicle was injected into pancreatic duct of anesthetized rats or mice at constant pressure over 10 min (250␮l rat, 25␮l mice). After 2.5h we measured pancreatic inflammation (serum amylase, histology severity score, and pancreas MPO activity) and pain (spinal cord fos expression in the dorsal horn at T8-10 with T6 and T12 serving as internal controls). Cerulein pancreatitis was induced in ⫹/⫹ and ⫺/⫺ mice (50␮g/kg x 12 hourly injections). Results: Intrapancreatic injection of MO significantly increased all indices of pancreatic inflammation (amylase, histology severity score (not shown), and MPO (11⫹2, Veh; 24⫹5, MO; UMPO/mg protein) and pain (spinal cord fos expression at T9-10 (Fig. 1), p ⬍ 0.05 all figures) as compared to vehicle controls in rats and mice. The effect was due to activation of TRPA1 since it was absent in TRPA1⫺/⫺ mice (Fig. 2). Injection of the endogenous aldehyde HNE produced similar results in wt mice (12⫹2, Veh; 16⫹1, HNE). Deletion of TRPA1 reduced the severity of both inflammation and pain in cerulein pancreatitis (Fig. 3). We conclude that TRPA1 activation promotes inflammation and pain in acute pancreatitis and that products of oxidative tissue injury may be important endogenous mediators of pancreatitis pain.

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 193 was grossly edematous and inflamed, weighing significantly more in IBD compared to control animals (6.3 ⫾ 0.8 vs. 2.2 ⫾ 0.1 grams, p ⫽ 0.001). Microscopic analysis demonstrated a 77% decrease of DiI labeling in the DMNV of IBD animals compared to controls (p ⫽ 0.005). BrdU cell proliferation ELISA assay was performed on cultured DMNV neurons after 24 hours of exposure to cytokine. The cytokines examined were IL-1␤, IL-6, or TNF-␣, each at concentrations of 0.1, 1, 10, and 100 ng/ml. Incubation with IL-1␤ at 0.1, 10, and 100 ng/ml resulted in significantly decreased proliferation (70% ⫾ 7%, 74% ⫾ 6%, 65% ⫾ 8%, and 58% ⫾ 9% vs. control, respectively; p ⬍ 0.05), as did incubation with IL-6 at 1, 10 and 100 ng/ml (57% ⫾ 5%, 58% ⫾ 9%, and 68% ⫾ 8% vs. control, respectively; p ⬍ 0.05) or TNF-␣ at 1, 10 and 100 ng/ml (57% ⫾ 2%, 61% ⫾ 7%, and 36% ⫾ 9% vs. control, respectively; p ⬍ 0.05). Apoptosis was measured after 24 hours incubation with IL-1␤, IL-6, or TNF-␣ each at concentrations of 0.1, 1, 10, and 100 ng/ml. Apoptosis of DMNV neurons was increased after exposure to IL-1␤ at concentrations of 10 and 100 ng/ml (156% ⫾ 21% and 161% ⫾ 24% vs. control, respectively; p ⬍ 0.05), as well as TNF-␣ at 10 and 100 ng/ml (160% ⫾ 25% and 164% ⫾ 25% vs. control, respectively; p ⬍ 0.05). Exposure to IL-6 did not significantly change neuronal apoptosis. Conclusions: DMNV neurons projecting to the stomach are reduced in number after induction of colitis in rats. In vitro, pro-inflammatory cytokines diminish DMNV neuronal proliferation and increase apoptosis. These results indicate that IBD results in systemic inflammation which adversely affects neuronal survival in the DMNV.

ONCOLOGY I: GENETICS

40. INFLAMMATORY BOWEL DISEASE AFFECTS NEURONAL SURVIVAL IN THE DORSAL MOTOR NUCLEUS OF THE VAGUS. John B. Ammori, Wei-Zhen Zhang, Ji-Yao Li, Biaoxin Chai, Michael W. Mulholland; University of Michigan, Ann Arbor, MI Introduction: Inflammatory bowel disease (IBD) is a common clinical problem. Enteric neurons in the intestinal wall have been found to be reduced in number and functionally altered by intestinal inflammation. However, the effects of IBD on the central neurons projecting to the enteric nervous system are largely unknown. The dorsal motor nucleus of the vagus (DMNV) is located in the brainstem and integrates peripheral and central signals, sending efferent signals to the gastrointestinal system. The purpose of this study was to determine the effects of IBD on neuronal survival of the DMNV in an animal model. Methods: At laparotomy, the fluorescent carbocyanine dye, DiI, was injected into the stomach wall of adult Sprague-Dawley rats in order to retrogradely label neurons of the DMNV. One week later, IBD was induced with enemas containing trinitrobenzene sulphonic acid (TNBS) in 50% ethanol while control animals were given 50% ethanol enemas. After 7 days the rats were sacrificed and tissue obtained for study. Fluorescent microscopy was used to measure DiI staining in the DMNV. In vitro studies were performed using primary culture of DMNV neurons which were isolated from neonatal rat brainstem using microdissection and enzymatic digestion. Cell proliferation was measured by BrdU incorporation assay. Apoptosis was measured by an ELISA assay. Data were analyzed using ANOVA. Results are expressed as mean ⫾ SEM. Results: Animals treated with TNBS lost weight (8.8 ⫾ 10.5 grams) compared to control animals which gained 44.6 ⫾ 5.5 grams. Food intake for seven days was significantly decreased compared to control animals (66 ⫾ 1.2 vs. 182 ⫾ 2.2 grams, p ⬍ 0.001). The colon

41. CYCLOOXYGENASE-2 INDUCES GENOMIC INSTABILITY, BCL2 EXPRESSION, DOXORUBICIN RESISTANCE, AND ALTERED CANCER INITIATING CELL PHENOTYPE IN MCF7 BREAST CANCER CELLS. Kendra R. Cook, Balraj Singh, Laura Vincent, Jacob A. Berry, Asha S. Multani, Anthony Lucci, Jr.; The University of Texas MD Anderson Cancer Center, Houston, TX Introduction: COX-2 expression in primary breast cancer correlates with a worse prognosis. We reported previously that COX-2 expression in MCF10A breast epithelial cells of basal subtype induces genomic instability. To understand the role of COX-2 in estrogen receptor- positive (non-basal) breast cancer, we transfected the MCF7 cell line with COX-2 and analyzed its chromosomal profile, BCL2 protein expression, and resistance to doxorubicin. We also analyzed cell cultures grown as mammospheres to determine whether COX-2 expression affects the cancer-initiating (“stem”) cell phenotype in MCF7 cells. Methods: MCF7 Tet On cells (obtained from Clontech) were stably transfected with pTRE2pur-COX-2 or pTRE2pur-COX2-GFP to produce COX-2 or COX-2-GFP protein, respectively. BCL2 protein was detected by western blotting. Sensitivity of cells to drug treatment was analyzed by MTT assay. Groups were compared using Chi-Square test. We analyzed the genomic instability phenotype by chromosome analysis of control and COX-2 transfected metaphase-arrested MCF7 cells after Giemsa staining. We assessed the tumorigenic potential of cells grown as mammospheres with a clonogenic assay. Results: Cytogenetic analysis of early passage COX-2 transfected MCF7 cells demonstrated significant genomic instability as compared to parental MCF7 cells. COX-2 overexpression was associated with a significant increase in chromosomal aberrations (chromatid breaks, chromosome fusions, C anaphase; see Table). COX-2 transfected MCF7 cells produced a significantly higher level of the anti-apoptotic protein BCL2 than the parental cells. In a functional assay, we found that COX-2 expression correlated with increased resistance to doxorubicin. In a complimentary approach to determine tumorigenic potential of cells, we found that COX-2 increased the ability of MCF7 cells to grow as mammospheres in culture, which correlated with an increase in clonogenic