Huamin Wang1, Gary E. Gallick1; 1UT MD Anderson CancerCenter, Houston, TX; 2Baylor College of Medicine, Houston,TX

Introduction: Previous studies report the non-receptor protein ty-rosine kinase, Src, to be overexpressed in at least 70% of pancreaticadenocarcinomas. Using the ARIOL imaging system, a computerprogram that assigns a numerical value to protein staining, we wereable to objectively confirm Src overexpression in pancreatic adeno-carcinoma compared to normal tissue. However, the levels of Srcexpression are lower than previously reported. In addition, wepresent the novel finding that Src is overexpressed in pancreatitistissue. Methods: Tissue Arrays were created from samples of 81patients with pancreatic cancer and pancreatitis. Tissue cores werestained with phosphorylated Src 416 (1:100 dilution). To analyze thelevels of Src staining, we employed the Ariol imaging system (Ap-plied Imaging, Corp.). In this system, various cells are assignedcolors based on optical density and cell shapes. Positively stainedcells appear brown on microscopy due to its chromogen (diaminoben-zidene). In Ariol, the positively stained cells (tumor or pancreatitis)are assigned the color red. Normal cells (which may nonspecificallystain positive on microscopy), and background tissues are assignedgreen and yellow, respectively. The percentage of positivity is thearea of the red (positive staining) divided by the sum of all areascombined. The values assigned reflect the percentage of the normal,tumor, or pancreatic tissue that stains positive for phosphorylatedSrc. Results: Our data indicates that phosphorylated Src activity isalmost 2-fold (23% vs. 42%) overexpressed in pancreatitis and 2.3-fold (23% vs. 52%) overexpressed in pancreatic adenocarcinoma com-pared to normal pancreatic tissue. Conclusion: With ongoing Srcinhibitor clinical trials, accurate assessment of Src activity may helpgauge its value as an effective target. In addition, the increasedactivity of Src in diseased pancreatic tissue may serve as a valuabletissue marker.

ONCOLOGY IX: APOPTOSIS, IMMUNOLOGY,AND ANGIOGENESIS

QS301. APIGENIN POTENTIATES GEMCITABINE-INDUCEDGROWTH ARREST OF PANCREATIC CANCER CELLSTHROUGH BLOCKING OF GEMCITABINE-INDUCEDUPREGULATION OF CELL CYCLE PROTEINS. Mat-thew J. Strouch, Benjamin M. Milam, Laleh G. Melstrom,Mohammad R. Salabat, Daniel M. Heifferman, Michael J.Heifferman, Xianzhong Ding, Thomas A. Adrian, David J.Bentrem; Northwestern Memorial Hospital and Lurie Com-prehensive Cancer Center, Chicago, IL

Introduction: Despite attempts of curative resection and adjuvanttherapy, patients diagnosed with pancreatic cancer continue to havea poor prognosis. Novel therapeutics are currently under investiga-tion. The flavonoid apigenin exhibits growth inhibition in severalcancer cell lines. We have previously shown that apigenin inhibitspancreatic cancer cell proliferation through the induction of G2/Mcell cycle arrest. In the present study we evaluated the effects ofcombination therapy with apigenin and gemcitabine on cell prolifer-ation, the cell cycle and the pro-survival factor pAkt. Further, weevaluate the effect of Apiginen in Gemcitabine-resistant pancreaticcancer cells. Methods: Cell counting and thymidine incorporationwere utilized to assess the combination effects of apigenin and gem-citabine on AsPC-1 and CD18 human pancreatic cancer cell prolif-eration. Flow cytometry was used to evaluate the effects of apigeninand gemcitabine treatment on the cell cycle. Western blot analysiswas used to evaluate pAkt, cyclin A, cyclin B, cyclin E and CDK2expression as a function of apigenin alone, gemcitabine alone or incombination in CD 18, AsPC-1 and AsPC-1 Gemcitabine-resistantcells. Results: Combination treatment with apigenin (25 �M) andgemcitabine (10 �M) significantly inhibited cell number and cell

proliferation compared to controls and gemcitabine alone in bothCD18 and AsPC-1 human pancreatic cancer cells (P�0.05). Apigenindown-regulated pAkt expression and prevented gemcitabine medi-ated pAkt induction in both CD18 and AsPC-1 cell lines. As deter-mined by flow cytometry, combination treatment induced cells toaccumulate in the S phase. Gemcitabine induced the expression ofcyclin A, cyclin B, cyclin E and CDK2. In the combination treatment,apigenin blocked gemcitabine-induced upregulation of the aforemen-tioned cell cycle proteins. Furthermore, apigenin treatment (0-100�M) inhibited cell proliferation of AsPC-1 cells resistant to gemcit-abine (P�0.01). Conclusions: Combination treatment with apige-nin and gemcitabine inhibited cell growth in human pancreatic cells,causing cell cycle arrest in the S phase. Apigenin blocked gemcitab-ine’s induction of cell cycle regulatory proteins as well as pro-survivalfactor p-Akt. These results indicate combination therapy may proveuseful for the treatment of pancreatic cancer.

QS302. GINKGO BILOBA EXTRACT KAEMPFEROL INHIB-ITS CELL PROLIFERATION AND INDUCES APO-PTOSIS IN PANCERATIC CANCER CELLS. YuqingZhang, Aaron Y. Chen, Min Li, Qizhi Yao, Changyi J. Chen;Baylor College of Medicine, Houston, TX

Background: Kaempferol is one of the most important constituentsin ginkgo flavonoids. Recent studies indicate kaempferol may haveanti-tumor activities. However, it is not clear whether kaempferolhas any inhibitory effects on pancreatic cancer cells. The mechanismof kaempferol action is also unknown. The objective in this study wasto determine the effect and mechanism of kaempferol on pancreaticcancer cell proliferation and apoptosis. Methods: Pancreatic cancercell lines MIA PaCa-2 and Panc-1 were treated with Kampferol at17.5, 35, and 70 �M for 4 days, and the inhibitory effects ofkaempferol on pancreatic cancer cell proliferation were examined bydirect cell counting, 3H-thymidine incorporation, and colorimetricproliferation MTS assay. Apoptosis was analyzed by TUNNELassay. Results: MIA PaCa-2 cell proliferation was significantly in-hibited by 79% and 45.7% as determined by direct cell counting andMTS assay, respectively, upon the treatment with 70 �M kaempferolfor 4 days compared with control cells (p�0.05). Similarly, the treat-ment with kaempferol (70 �M) for 4 days inhibited Panc-1 cellproliferation by 47.5% in direct cell counting and 40.4% in MTSassay compared with control cells (p�0.05). Kaempferol treatmentalso significantly reduced 3H-thymidine incorporation in both MIAPaCa-2 and Panc-1 cells. In addition, kaempferol was found to in-duce apoptosis in pancreatic cancer cells in a time- andconcentration-dependent manner. In MIA PaCa-2 cells, apoptoticcell population was increased by 3.1, 5.4 and 8 fold when treated withkaempferol at concentrations of 17.5, 35.0, and 70.0 �M, respec-tively, compared with DMSO controls (p�0.01). Panc-1 cells treatedwith same amounts of kaempferol significantly increased apoptoticcell population by 1.4, 2.1 and 4 fold, respectively, compared withcontrol cells (p�0.01). Conclusions: Ginkgo biloba extractkaempferol effectively inhibits pancreatic cancer cell proliferationand induces cancer cell apoptosis, which may sensitize pancreatictumor cells to chemotherapy. These findings indicate thatkaempferol, a natural compound, may have clinical applications inthe treatment of pancreatic cancer.

QS303. ERLOTINIB REDUCES FLIP MEDIATED RESIS-TANCE TO TRA-8 AGONISTIC TRAIL-R2 ANTIBODYIN PANCREATIC CANCER CELLS. Joshua W. Long1,Donald J. Buchsbaum1, Jeffery Sellers1, Zhi Huang1, SelwynM. Vickers2, Pablo Arnoletti1; 1UAB, Birmingham, AL; 2Uni-versity of Minnesota, Minneapolis, MN

Background: TRA-8 is an agonistic TRAIL-R2 antibody which iseffective against pancreatic cancer cells and xenografts. While manypancreatic cancer cell lines are sensitive to TRA-8, resistance does

387ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

occur through unknown mechanisms. Erlotinib is a small moleculeepidermal growth factor (EGFR) tyrosine kinase inhibitor (TKI)which by analogy with similar TKI’s may increase sensitivity toTRAIL signaling through modulation of FLIP. The purpose of thisstudy was to determine whether erlotinib could help overcome pan-creatic cancer cell resistance to TRA-8 therapy via FLIP-mediatedmechanisms. Methods: The TRA-8 resistant S2VP10 and Aspc-1pancreatic cancer cell lines were treated with 1-5 �M concentrationsof Erlotinib and 5-125 ng/ml of TRA-8. An ATP-lite cytotoxicity assaywas employed. Changes in cFLIP, pI�B (a surrogate marker ofNF�B-induced cell proliferation), caspase 3 and XIAP were detectedby immunoblot. Results: S2VP10 cells experienced a 16.8% reduc-tion in cell viability in response to TRA-8/Erlotinib combinationtreatment when compared to either agent alone. Similarly Aspc-1cells demonstrated a 40.6 % decrease in cell viability with combina-tion treatment versus dose matched individual treatments. The ad-dition of erlotinib reduced anti-apoptotic signaling as evidenced by areduction in cFLIP-S, pI�B and XIAP protein levels. Increased apo-ptosis signal transduction was further corroborated by an increase inCaspase-3 cleavage among the erlotinib/TRA-8 treated cells. Con-clusions: Erlotinib overcomes pancreatic cancer cell resistance toTRA-8 by modulation of FLIP. Erlotinib may successfully enhancethe therapeutic efficacy of TRA-8.

QS304. NOVEL p53-DERIVED PEPTIDE INDUCES RAPIDHUMAN PANCREATIC CANCER CELL DEATH.Kelley A. Sookraj; State University of New York, HealthScience Center of Brooklyn and Brooklyn Harbor View VAMedical Center, Brooklyn, NY

Introduction: PNC-28 is a peptide from the mdm-2-binding domainof the p53 protein which contains a membrane crossing-penetratinsequence. We have found that this peptide can selectively inducenecrosis of ras-transformed rat pancreatic carcinoma cells (BM-RPA1. TUC-3) in vitro and in vivo without affecting normal cells. Wenow investigate for the ability of PNC-28 to block growth of a lethalhuman pancreatic cancer cell line. Secondly, we studied the mecha-nism of human pancreatic cancer cell death. Methods: Growth In-hibition Studies: Groups of 2 � 10 4 MiaPaCa-2 human pancreaticcarcinoma cells were treated daily with 0.1, 0.3, and 0.5 mgs/ml ofPNC-28 peptide. Control groups received unrelated PNC-29 peptide.A standard group was not treated. Morphologic Studies: All groupswere observed for changes in cell morphology and growth character-istics. Cytotoxicity Assay: Lysis mediated by peptide was determinedmeasuring lactate dehydrogenase (LDH) release at different timepoints. Caspace-3, 7 Assay: Detection of Caspace �3, 7 activities wasemployed to characterize cell death by apoptosis. Results: GrowthInhibition Studies: Dose dependent induction of tumor cell death wasseen starting at 0.1 mg/ml of PNC-28. Remarkably, 100% cell deathwas induced at 0.5 mg/ml following 4 d of drug treatment. There wasno significant difference between the control and standard group.Morphologic Studies: In a dose dependent manner PNC-28 treatedcells withdrew into small clumps with large areas devoid of cells.Cytotoxicity Assay: PNC-28 dependent tumor necrosis was demon-strated as early as 4 hrs with continuous (12-24 hr) LDH releaseotherwise absent in control and untreated groups. In contrast, nocaspace activity was detected during drug treatment. Conclusions:These results suggest that PNC-28 may be effective in treatinghuman pancreatic cancer. This novel peptide also appears to inducenecrosis as the mechanism for cell death. Apoptosis does not appearto play a role in cell death following treatment with PNC-28.

QS305. SOPHOROLIPIDS AND THEIR DERIVATIVES ARELETHAL AGAINST HUMAN PANCREATIC CANCERCELLS. Sophia L. Fu1, Irene Garnett1, Sabine R. Wallner2,Michael E. Zenilman1, Richard Gross2, Martin H. Bluth1;1SUNY Downstate Medical Center, Brooklyn, NY; 2Poly-technic University, Brooklyn, NY

Introduction: We have previously demonstrated that sophorolip-ids, a class of easily chomoenzymatically modifiable glycolipids, pos-sess anti-inflammatory effects in vitro and in vivo. Since glycolipidshave been shown to have anti-cancer activity we investigated theeffects of sophorolipids and their derivatives against pancreaticcancer. Methods: Human pancreatic carcinoma (HPAC) cells(1�104/ml) were treated with increasing concentrations (0.5, 1.0, 1.5,and 2.0 mg/ml) of sophorolipid natural mixture (NM) or six selectderivatives (ethyl ester [EE], ethyl ester monoacetate [EEM], ethylester diacetate [EED], acidic sophorolipid [AS], lactonic sophorolipiddiacetate [LSD]) for 24hrs and assessed for cell necrosis (cytotoxicity- LDH release) and apoptosis (annexin). Controls consisted of cellstreated with media or vehicle alone and sophorolipid treatment ofperipheral blood mononuclear cells (PBMC). Results: Sophorolipidsdemonstrated anti-cancer activity against HPAC cells. NM mediatedconsistent cytotoxicity at all doses tested (20�4%). However MEderivative mediated much greater levels of cytotoxicity (63�5%)compared with other derivatives (EED � 36�6, EEM � 18�7;p�0.05). In contrast, LSD and AS mediated toxicity was inverselyproportional with dose (LSD � 40.3% at 0.5mg/ml; 3.4% at 2.0mg/ml,AS � 49% at 0.5mg/ml; 0% at 2.0mg/ml ). Dose dependent apoptosiswas most notably observed with the AS derivative. Sophorolipidtreatment did not affect PBMC at all doses tested Conclusions:These results suggest that sophorolipids and select derivatives (i.e.methyl ester, acidic sophorolipid) may be effective in treating humanpancreatic cancer. Furthermore select derivatives may utilize differ-ent mechanisms (necrosis, apoptosis) toward this end. The ability tochemoenzymatically modify sophorolipids can provide effective leadcompounds toward the treatment of pancreatic cancer.

QS306. A PANCREAS CANCER TARGETED AKT INHIBITORINDUCES APOPTOSIS. Peter O. Simon, Jr., JonathanMcDunn, Hiroyuki Kashiwagi, Fabian Johnston, NicholasHamilton, David Linehan, Richard Hotchkiss, Robert Mach,William Hawkins; Washington University School of Medi-cine, St. Louis, MO

Background: Pancreatic adenocarcinoma is the fourth leadingcause of cancer death in the US and carries an ominous prognosis.Previous reports have demonstrated that Sigma-2 ligands bind spe-cifically to pancreas adenocarcinoma, are internalized into the cell,and induce cell death in a small subset of cells. Furthermore, it hasbeen demonstrated that carboxyl terminal modulator protein(CTMP) induces apoptosis by inhibiting the Akt/protein kinase Bpathway. Methods: We generated a dual domain compound, termedS2-CTMP4, containing a Sigma-2 ligand (targeting and internaliza-tion domain) and a pro-apoptotic peptide derived from CTMP(CTMP4, cell death domain). An inactive point mutant of CTMP4was also identified and used as a negative control (S2-CTMP4 Inac-tive). Human (AsPC-1, BxPC-3, CFPAC-1, Panc-1) and murine(Panc-02) pancreatic adenocarcinoma cell lines were treated withescalating doses of S2-CTMP4 and S2-CTMP4 Inactive and apoptosiswas measured utilizing flow cytometry to determine percent-activecaspase-3 and percent-TUNEL positive cells. Human donor periph-eral blood mononuclear cells (PBMC) were also treated with S2-CTMP4 and S2-CTMP4 Inactive to determine if this therapeuticagent produces an apoptotic effect in non-malignant cells. Results:In all pancreas cancer cell lines tested, S2-CTMP4 induced a dose-dependent increase in apoptosis as determined by percent-TUNELpositive cells and percent-active caspase-3 (percent-TUNEL positivecells ranged from 43.6 to 52.8 percent at the highest dose tested(10�M)). Specifically, Panc-1 exhibited 48.1 percent-TUNEL positiv-ity compared to the 13.6 percent-TUNEL positivity of the S2-CTMP4Inactive-treated group (negative control, P�0.0002). These resultswere superior to that of either pro-apoptotic agent alone (S2 � 25.6percent-TUNEL positivity and CTMP4 � 42.0 percent-TUNEL pos-itivity). This pro-apoptotic activity was not observed in PBMC of

388 ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS