A Structurally Optimized Celecoxib Derivative Inhibits Human Pancreatic Cancer Cell Growth Junan Li, Ph.D., Jiuxiang Zhu, Ph.D., W. Scott Melvin, M.D., Tanios S. Bekaii-Saab, M.D., Ching-Shih Chen, Ph.D., Peter Muscarella, M.D.
Deregulation of the phosphatidylinositol 3-kinase (PI-3K)/PDK-l/Akt signaling cascade is associated with pancreatic cancer tumor invasion, angiogenesis, and tumor progression. As such, it has been postulated that PDK-1/Akt signaling inhibitors may hold promise as novel therapeutic agents for pancreatic cancer. Disadvantages of currently available Akt inhibitors include tumor resistance, poor specificity, potential toxicity, and poor bioavailability. Previous studies have demonstrated that OSU-03012, a celecoxib derivative, specifically inhibits PDK-1 mediated phosphorylation of Akt with IC50 values in the low mM range. Human pancreatic cancer cell lines AsPC-1, BxPC-3, Mia-PaCa 2, and PANC-1 were cultured in media containing varying concentrations of OSU-03012, 5-fluorouracil (5-FU), and gemcitabine, and changes in Akt phosphorylation and cell viability were evaluated using western blotting and a 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, respectively. Treatment with OSU-03012 resulted in decreased PDK-1-mediated Akt phosphorylation and cell growth inhibition for all cell lines with IC50 values ranging between 1.0 and 2.5 mM. Resistance to 5-FU and gemcitabine was observed in cell lines AsPC-1 and BxPC-3. Further analyses indicate that OSU-03012 induces both proapoptotic and antiproliferative effects in these cells. Taken together, these data suggest that OSU-03012 has potential value as a novel therapy for pancreatic cancer. ( J GASTROINTEST SURG 2006;10:207–214) Ó 2006 The Society for Surgery of the Alimentary Tract KEY
Akt signaling, PDK-1 specific inhibitor, celecoxib, pancreatic cancer
Pancreatic cancer is the fifth most common cancer-causing death in the United States1 and prognosis is almost universally dismal with currently reported five-year survival rates of only 4%.2 Surgical resection remains the only hope for cure, but most patients present with locally advanced or metastatic disease, factors that preclude attempts at resection. Chemotherapy and radiation offer only modest improvements in survival.3,4 Novel therapeutic strategies are desperately needed, and this topic has been the subject of a recent progress review group sponsored by the National Institutes of Health.5 Recent advances in pancreatic cancer research have identified a number of tumorigenesis-associated genetic events including Akt activation.6–8 As a key component in the PI3K/Akt signaling pathway, activated Akt is involved in the regulation of several important cellular events, including apoptosis, cellular proliferation, and the response to hypoxic stress
(Fig. 1). Constitutive activation of Akt has been reported in 78% of pancreatic adenocarcinoma cell lines,9 and mutations in PTEN, a negative regulator of Akt activation, have also been identified in pancreatic tumor specimens.10–12 Moreover, PI3K/Aktinhibiting agents have been shown to induce apoptosis in several pancreatic cancer cell lines exhibiting Akt activation.13–17 Therefore, Akt activation appears to represent a viable target for therapeutic intervention in pancreatic cancer. We have recently identified a series of Akt signaling inhibitors with high antiproliferative and proapoptotic activities in human prostate cancer cell lines and mouse xenografts.18 One of these, OSU03012, has been approved by National Institutes of Health–Rapid Access to Interventional Development (NCI RAID) for further bioavailability and preInvestigational New Drug Application (IND) toxicology studies. As a structurally optimized celecoxib
Presented at the 2005 American Hepato-Pancreato-Biliary Association Congress, Hollywood Florida, April 14–17, 2005. From the Departments of Surgery (J.L., W.S.M., P.M.) and Internal Medicine (T.S. B.-S.), College of Medicine; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy (J.Z., C.-S.C); Comprehensive Cancer Center (W.S.M., T.S.B.-S., C.-S.C., P.M.), The Ohio State University, Columbus, Ohio. Reprint requests: Peter Muscarella, M.D., Department of Surgery, College of Medicine, The Ohio State University, N711 Doan Hall, 410 West 10th Avenue, Columbus, OH 43210. e-mail: [email protected] Ó 2006 The Society for Surgery of the Alimentary Tract Published by Elsevier Inc.
1091-255X/06/$dsee front matter doi:10.1016/j.gassur.2005.07.025
Journal of Gastrointestinal Surgery
Li et al.
Fig. 1. The PI3K/Akt signaling pathway, modified from reference 7. Arrows represent positive effects, and stop bars indicate negative regulation. P-phosphorylation.
derivative18–21 (Fig. 2), OSU-03012 specifically inhibits PDK-1 in PC-3 cells and blocks Akt and p70S6K activation, resulting in apoptotic cell death and cell growth inhibition at low mM concentrations. We hypothesize that treatment with OSU-03012 will result in enhanced apoptosis and an antiproliferative effect through modulation of Akt signaling in pancreatic cancer cells. The purpose of the present study was to evaluate the effects of OSU-03012 treatment on pancreatic cancer cell growth, Akt phosporylation, cellular proliferation, and apoptosis. MATERIALS AND METHODS Cell Lines Human pancreatic cancer cell lines AsPC-1, BxPC-3, MiaPaCa-2, and Panc-1 were purchased
from American Type Culture Collection (Manassas, VA). AsPC-1 and BxPC-3 cells were cultured in RPMI1640 (Invitrogen Corp., Carlsbad, CA) containing 10% fetal bovine serum (FBS; Invitrogen Corp., Carlsbad, CA) and 2 mM glutamine in a humidified 37 C incubator supplied by 5% CO2. Dulbecco’s modified Eagle medium (DMEM, Invitrogen Corp., Carlsbad, CA) was used for MiaPaCa-2 and PANC-1 cells. Reagents and Antibodies 5-Fluorouracil, 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT), trypsin, and Tween 20 were purchased from Sigma-Aldrich (St. Louis, MO), and gemcitabine was from Eli Lilly and Company (Indianapolis, IN). M-PER mammalian
Fig. 2. The molecular structures of celecoxib (A) and OSU-03012 (B).
Vol. 10, No. 2 2006
New Agent Inhibits Pancreatic Cancer Cell Growth
protein extraction reagent and BCA protein concentration determination kits were purchased from Pierce (Rockford, IL), and ECL western blotting detection reagents (RPN 2209) were purchased from Amersham Biosciences (Piscataway, NJ). Nonfat dry milk was purchased from Bio-Rad (Hercules, CA). Rabbit antihuman Akt, rabbit antihuman phosphorylated Akt (Ser473), and rabbit antihuman poly (ADP-ribose) polymerase (PARP) antibodies were purchased from Cell Signaling Technology, Inc. (Bevery, MA). Immuno monoclonal mouse antihuman actin antibody Clone C4 was purchased from MP Biomedicals, Inc. (Livermore, CA). Peroxidaseconjugated AffiniPure rabbit antimouse IgG C IgM (H C L) and peroxidase-conjugated AffinitiPure goat antirabbit IgG (H C L) was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Cell Viability Assay Five thousand cells per well were seeded into a 96-well plate in media containing 10% FBS. After incubation at 37 C, 5% CO2 for 24 hours, cells were treated with 1% FBS media containing various concentrations of compounds and incubated at 37 C for another 48 hours.18 Cell viability was evaluated using an MTT assay. Briefly, 200 uL of 10% FBSsupplemented medium containing MTT at a concentration of 75 mg/ml was added to each well, and cells were incubated in the presence of MTT for 2 hours. Mitochondrial dehydrogenate activity reduced the yellow MTT dye to a purple formazan, which was then solubilized with 200 uL of DMSO, and absorbance at 570 nm was read on an enzyme-linked immunosorbent assay (ELISA) plate reader; 0.1% of DMSO was used as a control. For AsPC-1 and BxPC-3, the medium was RPMI1640, while DMEM was used for MiaPaCa-2 and PANC-1. The value of IC50 was defined as the concentration at which cell viability decreased to 50%. The experiments were performed in six replicates. Western Blotting 5
Pancreatic cancer cells (5 3 10 ) were cultured in a T-25 flask and incubated for 24 hours in the presence of 10% FBS. Cells were treated with media containing different concentrations of agents under investigation, or 0.1% DMSO (negative control) for 4–24 hours. Cells were collected by centrifugation at 6-hr intervals. Following PBS washing, the cells were resuspended in 200–400 mL of M-PER lysate buffer. After being gently vortexed at room temperature for 10 minutes, the cells were centrifuged at 14,000 rpm at room temperature for 10 minutes, and the supernatants were transferred into new
Eppendorf tubes and stored at 220 C. After protein concentration determination using a BCA kit, the supernatants were mixed with 5 3 SDS-PAGE loading buffer and boiled at 100 C for 5 minutes. Equal amounts (50 mg) of the supernatants were loaded onto 10% SDS-PAGE gels and separated by electrophoresis. Proteins were then transferred to nitrocellulose membranes using a BioRad Mini-II system. The transblotted nitrocellulose membranes were washed three times with TBS buffer (15 mM Tris-Borate0.15 M Sodium Chloride, pH 7.5) containing 0.05% Tween 20 (TBST), and then blocked with TBST containing 5% nonfat dry milk at room temperature for 2 hours. After incubation with the primary antibody at a 1:1000 dilution in TBST-5% nonfat dry milk at 4 C overnight, the membranes were washed three times with TBST and probed with peroxidase-conjugated secondary antibody at a 1:10,000 dilution in TBST-5% nonfat dry milk for 1.5 hours at room temperature. Immunoblots were visualized by enhanced chemiluminescence using an ECL kit after washing with TBST. For all western blot assays, actin was used as an internal control. Cell Growth Inhibition Analysis Pancreatic cancer cells were seeded into six-well plates at 50,000-75,000 cells per well in 10% FBScontaining RPMI1640 or DMEM media.18 After incubation at 37 C in 5% CO2 for 24 hours, cells were treated in duplicate with 1 mM or 5 mM of OSU-03012 in 10% FBS-containing media, and the control groups were treated with 0.1% DMSO. Cells were harvested by trypsin digestion at various time intervals and numerated using a Coulter counter (model Zl D/T) (Beckman Coulter, Fullerton, CA). The relative proliferation index was defined as the ratio of OSU-03012-treated cells to DMSO-treated cells for any given time point. RESULTS Expression and Phosphorylation of Akt in Pancreatic Cancer Cell Lines Because OSU-03012 acts as a specific PDK-1 inhibitor, we first investigated the expression and phosphorylation of Akt in these cell lines by Western blotting. While high concentrations of Akt were detected in the cell lysates of AsPC-1, MiaPaCa-2, and PANC-1, only basal levels were detected in the cell lysate of BxPC-3 (Fig. 3). Furthermore, increased concentrations of phosphorylated Akt (Ser473) were detected only in the cell lysate of AsPC-1, and phosphorylation of Akt in MiaPaCa-2 and PANC-1
Journal of Gastrointestinal Surgery
Li et al.
phosphorylation after treatment with varying concentrations of OSU-03012. Treatment with OSU03012 resulted in a dose-dependent inhibition of Akt phosphorylation in all cell lines tested (Fig. 5). These findings indicate that OSU-03012 specifically and efficiently inhibits Akt phosphorylation in pancreatic cancer cells regardless of basal Akt phosphorylation status.18
Fig. 3. Phosphorylation status of Akt in pancreatic cancer cell lines.
approximated that of BxPC-3. In regards to the expression and phosphorylation of Akt, these four cell lines can be divided into three groups: (1) AsPC-1 with high expression and phosphorylation of Akt, (2) MiaPaCa-2 and PANC-1 with high expression but low phosphorylation Akt, and (3) BxPC3 with low expression and phosphorylation of Akt. OSU-03012 Inhibits the Growth of All Tested Pancreatic Cancer Cell Lines Treatment with OSU-03012 resulted in a dosedependent inhibition of cell viability for all pancreatic cancer cell lines tested (Fig. 4, A). In the presence of 5 mM of OSU-03012, only trace cell viability was observed. Although the four cell lines varied widely in regards to expression and phosphorylation of Akt (Fig. 3), effects on cell viability were comparable (Table 1). While the IC50 values of AsPC-1, BxPC-3, and MiaPaCa-2 are nearly identical (1.5 mM, 1.8 mM, and 1.3 mM, respectively), there was a moderate increase in the IC50 value of PANC-1 (2.3 mM). OSU-03012 appears to be an order of magnitude more potent than celecoxib (IC50 20–50 mM).22 As controls, two drugs commonly used for human pancreatic cancer therapy, 5-fluorouracil (5-FU) and gemcitabine, were also evaluated. Results varied among the four cell lines (Fig. 4, B, C ). While MiaPaCa-2 and PANC-1 are sensitive to both 5-FU and gemcitabine, AsPC-1 is resistant to both 5-FU and gemcitabine. BxPC-3 is sensitive to 5-FU but resistant to gemcitabine. OSU-03012 demonstrated more potent inhibition of cell proliferation than either 5-FU or gemcitabine for all cell lines tested (Table 1).
OSU-03012 Induces Antiproliferative and Proapoptotic Effects in All Tested Pancreatic Cancer Cell Lines To investigate the biological consequences resulting from OSU-03012-mediated inhibition of Akt phosphorylation, we examined the effects of OSU03012 treatment on pancreatic cancer cell proliferation (Fig. 6). In general, OSU-03012 treatment resulted in decreased cell proliferation in a time and dose-dependent manner. The antiproliferative effect of OSU-03012 treatment was most pronounced in the MiaPaCa-2 cell line. Of note, the effects of OSU-03012 on cell growth are strikingly different in the presence of varying concentrations of FBS. As demonstrated in the cell viability assay (Fig. 4, A), the cell growth of all four cell lines was completely inhibited after incubation with media containing 1% FBS and 5 mM of OSU-03012 for 48 hours (2 days), while 40–60% of proliferation activity was retained after incubation with media containing 10% FBS and 5 uM of OSU-03012 for 3 days. This apparent inconsistency was also observed in previous studies using PC-3 cells and may be attributed to serum-induced growth factor/receptor tyrosine kinase-mediated activation of PI3K/Akt signaling or altered cellular uptake of OSU-03012. Finally, OSU-03012-mediated changes in apoptosis were evaluated using a poly (ADP-ribose) polymerase (PARP) cleavage assay18 (Fig. 7). Treatment of all pancreatic cancer cell lines with OSU-03012 resulted in a dose-dependent induction of apoptosis as measured by PARP cleavage. The proapoptotic effect of OSU-03012 did vary among cell lines and was most pronounced in the AsPC-1 and BxPC-3 cell lines. The findings indicate that OSU-03012 treatment results in both antiproliferative and proapoptotic effects in all tested pancreatic cancer cells. DISCUSSION
OSU-03012 Inhibits Akt Phosphorylation in All Tested Pancreatic Cancer Cell Lines To explore the molecular mechanisms underlying OSU-03012-induced growth inhibition in these pancreatic cancer cell lines, we analyzed the status of Akt
The K-ras oncogene and multiple receptor tyrosine kinases affect intracellular signaling in malignant cells through the activation of downstream oncogenic pathways such as PI3K/Akt, MEK/Erk, and IkB/NF-kB. Downstream components result in
Vol. 10, No. 2 2006
New Agent Inhibits Pancreatic Cancer Cell Growth
Fig. 4. Cell viability of pancreatic cancer cells in the presence of (A) OSU-03012, (B) 5-FU, and (C) gemcitabine.
uncontrolled cell proliferation, angiogenesis, and inhibition of apoptosis.23 After binding of the regulatory p85 subunit of PI3K to an activated receptor tyrosine kinase, or through binding of the p110 catalytic subunit of PI3K to activated ras, PI3K is activated and subsequently leads to the translocation of Akt to the plasma membrane.7,8,24 The p110 subunit
of PI3K catalyzes the conversion of PIP2 to PIP3. PIP3 promotes phosphorylation of Akt by PDK1 and PDK2, at Thr308 and Ser473, respectively, resulting in full activation. Activated Akt (protein kinase B, PKB) is involved in several important cellular events including apoptosis, cellular proliferation, and the response to hypoxic stress.
Journal of Gastrointestinal Surgery
Li et al.
Table 1. IC50 values of tested agents Cell line
AsPC-1 BxPC-3 MiaPaCa-2 PANC-1
1.5 1.8 1.3 2.3
6 6 6 6
0.4 0.3 0.4 0.8
O250* 71.6 6 14.2 23.4 6 5.3 100.0 6 22.7
O250* O250* 14.5 6 2.5 7.2 6 3.1
*IC50 was out of the tested concentration range of the agent. This indicates minimal or no growth inhibition in this cell line.
Antiapoptotic activity is partially mediated through Bad, IkB kinase, forkhead transcription factor, and caspase-9. Downstream regulators of cell cycle progression mediated by PI3K/Akt include p21, GSK3b, and p27. Phosphorylation of GSK3b inhibits its kinase activity and allows cyclin Dl to accumulate.24 In addition, activated Akt enhances expression of hypoxia-inducible factor-1, resulting in increased cellular hypoxia tolerance.25,26 A number of recent investigations support the evalution of PI3K/Akt pathway inhibitors as potential targets for therapeutic intervention in pancreatic cancer.7,8 The 5-lipoxygenase metabolite, 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE), is known to significantly stimulate the proliferation of pancreatic cancer cells lines and markedly increases Akt phosphorylation.13 Treatment of these cells with the PI3K inhibitor, wortmannin, results in the blockade of 5(S)-HETE-induced Akt phosphorylation and DNA synthesis. Inhibition of the antiapoptotic regulator, NF-kB, has been reported to result in potentiation of gemcitabine-induced apoptosis in a group of gemcitabine-resistant pancreatic cancer cell lines.14 In this study, inhibition of the PI3K/ Akt pathway by LY294002 did not result in an attenuation of NF-kB mediated apoptosis. Furthermore, basal Akt activity did not correspond to gemcitabine resistance. These findings suggest that gemcitabineinduced apoptosis may not be modulated by the PI3K/ Akt pathway in certain pancreatic cancers. Others have reported, however, that PI3K/Akt pathway inhibitors may induce apoptosis in a dose-dependent manner in gemcitabine-resistant pancreatic cancer cell lines.15–17 Possible downstream mediators of apoptosis following PI3K/Akt inhibition in gemcitabine-treated pancreatic cancer cells lines include NF-kB, Bcl-2, and Bax. PI3K/Akt inhibition augments the proapoptotic and antiproliferative effects of TNF-a induced NF-kB inhibition in several pancreatic cancer cell lines. This suggests that the PI3K/Akt pathway may, in part, modulate NF-kB mediated chemoresistance in pancreatic cancer. Finally, treatment of pancreatic cancer cells with LY294002 and the cyclooxygenase
Fig. 5. OSU-03012 inhibits Akt phosphorylation in pancreatic cancer cell lines.
inhibitor, sulindac, resulted in enhanced growth inhibition, alteration of cell cycle distribution, and decreased apoptotic threshold as compared to treatment with sulindac alone.27 Despite these potentially encouraging findings, early generation PI3K/Akt inhibitors exhibit a number of clinically significant disadvantages, including poor specificity, potential toxicity, and poor bioavailability.7,8 Wortmannin is known to have unfavorable pharmacokinetic properties. Both wortmannin and LY294002 are likely to have broad inhibitory activities and may have nonspecific effects on other regulatory cellular molecules. For example, there are different isoforms of p110, the PI3K catalytic subunit. Wortmannin and LY294002 exhibit inhibitory activities against all of these isoforms, as well as distant PI3K-like kinases such as ATM and ATR. Finally, delivery issues have precluded the study of PI3K/Akt inhibitors in available animal models of pancreatic cancer. In this study, all tested pancreatic cancer cell lines demonstrated sensitivity to OSU-03012. The IC50 values (1.5–2.5 mM) are an order of magnitude lower than those previously reported values for other PI3K/Akt inhibitors.7,8,27,28 The high potency of OSU-03012 appears to be mediated through both antiproliferative and proapoptotic mechanisms.18 It has been previously reported that inhibition of PDK-1/Akt signaling represents the underlying antitumor mechanism for OSU-03012 in PC-3 cells.18 Considering the conserved role of PDK-1/Akt signaling in cell proliferation and survival, we believe that a similar molecular mechanism underlies the high antitumor activity of OSU-03012 in different pancreatic cancer cell lines. Our data are further
Vol. 10, No. 2 2006
New Agent Inhibits Pancreatic Cancer Cell Growth
Fig. 6. The effect of OSU-03012 treatment on pancreatic cancer cell line proliferation. (A) 1 mM OSU03012; (B) 5 mM OSU-03012.
supported by antitumor screening studies of OSU03012 in the Developmental Therapeutic Program at the National Cancer Institute.18 OSU-03012 demonstrated potent growth inhibition in 60 cell lines from human lung, colon, brain, ovary, breast, prostate, kidney, leukemia, and melanoma cancers with a mean IC50 value of 1.2 mM. While it is still unclear whether antiproliferative or proapoptotic effects are predominant in specific cancer cell lines, the bifunctionality of OSU-03012 appears to counteract cell resistance. The data presented here indicate that OSU-03012 uniformly inhibits pancreatic cancer cell growth and suggest that this effect is mediated by inhibition of
Fig. 7. The effect of OSU-03012 treatment on apoptosis.
Journal of Gastrointestinal Surgery
Li et al.
Akt phosphorylation. The widely varying effects of 5-FU and gemcitabine on pancreatic cancer cell growth undoubtedly are a result of heterogeneity among various cell lines with regards to their mechanisms of action.27,28 This may explain why these agents have demonstrated only limited clinical activity for the treatment of pancreatic cancer. These results support further investigation of this novel therapeutic agent for pancreatic cancer therapy in preclinical and clinical studies.
18. REFERENCES 1. Cancer Facts and Figures-2003. New York: American Cancer Society, 2003, pp 12–24. 2. Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ. Cancer statistics 2003. CA Cancer J Clin 2003; 53:5–26. 3. Rosenberg L. Treatment of pancreatic cancer. Int J Pancreatol 1997;22:81–93. 4. Kollmannsberger C, Peters HD, Fink U. Chemotherapy in advanced pancreatic adenocarcinoma. Cancer Treat Rev 1998;24:133–156. 5. Pancreatic Cancer: An Agenda for Action. Report of the Pancreatic Cancer Progress Review Group. NIH Publication Number 01-4940. Bethesda, MD: National Cancer Institute, 2001. 6. Cowgill SM, Muscarella P. The gentics of pancreatic cancer. Am J Surg 2003;186:279–286. 7. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinaseAkt pathway in human cancer. Nat Rev Cancer 2002;2: 489–501. 8. Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002;296:1655–1657. 9. Schlieman MG, Fahy BN, Ramsamooj R, Beckett L, Bold RJ. Incidence, mechanism and prognostic value of activated AKT in pancreas cancer. Br J Cancer 2003;89:2110–2115. 10. Altomare DA, Tanno S, De Rienzo A, et al. Frequent activation of Akt2 kinase in human pancreatic carcinomas. J Cell Biochem 2003;88:470–476. 11. Perren A, Komminoth P, Saremaslani P, et al. Mutation and expression analyses reveal differential subcellular compartmentation of PTEN in endocrine pancreatic tumors compared to normal islet cells. Am J Pathol 2000;157:1097–1103. 12. Stambolic V, Suzuki A, Delapompa JL, et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998;95:29–39. 13. Ding XZ, Tong WG, Adrian TE. Multiple signal pathways are involved in the mitogenic effect of 5(S)-HETE in human pancreatic cancer. Oncology 2003;65:285–294. 14. Arlt A, Gehrz A, Muerkoster S, et al. Role of NF-kappaB Akt/PI3K in the resistance of pancreatic carcinoma cell lines against gemcitabine-induxed cell death. Oncogene 2003;22: 3243–3251. 15. Ng SS, Tsao M-S, Nicklee T, Hedley DW. Effects of the epidermal growth factor receptor inhibitor OSI-774,
Tarceva, on downstream signaling pathways and apoptosis in human pancreatic adenocarcinoma. Mol Cancer Ther 2002;1:777–783. Bondar VM, Sweeney-Gotsch B, Andreeff M, Mills GB, McConkey DJ. Inhibition of the phosphatidylinositol 3’kinase-Akt pathway induces apoptosis in pancreatic carcinoma cells in vitro in vivo. Mol Cancer Ther 2002;1: 989–997. Ng SSW, Tsao M-S, Chow S, Hedley DW. Inhibition of phosphatidylinositide 3-kinase enhances gemcitabine-induced apoptosis in human pancreatic cancer cells. Cancer Res 2000; 60:5451–5455. Zhu J, Huang JW, Fowble J, et al. From the cyclooxygenase2 inhibitor Celecoxib to a novel class of 3-phosphoinositidedependent protein kinase-1 inhibitors. Cancer Res 2004;64: 4309–4318. Hsu AL, Ching TT, Wang DS, Song X, Rangnekar VM, Chen C-S. The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem 2000;275: 11397–11403. Johnson AJ, Song X, Hsu A, Chen C-S. Apoptosis signaling pathways mediated by cyclooxygenase-2 inhibitors in prostate cancer cells. Adv Enzyme Regul 2001;41:221–235. Kulp SK, Yang YT, Huang CC, et al. 3-Phosphoinositidedependent protein kinase-1/Akt signaling represents a major cyclooxygenase-2-independent target for Celecoxib in prostate cancer cells. Cancer Res 2004;64:1444–1451. Levitt RJ, Pollak M. Insulin-like growth factor-I antagonizes the antiproliferative effects of cyclooxygenase-2 inhibitors on BxPC-3 pancreatic cancer cells. Cancer Res 2002;62:7372– 7376. Yokoi K, Fidler IJ. Hypoxia increases resistance of human pancreatic cancer cells to apoptosis induced by gemicitabine. Clin Cancer Res 2004;10:2299–2306. Chang F, Lee JT, Navolanic PM, et al. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: A target for cancer chemotherapy. Leukemia 2003;17:590–603. Matsumoto J, Kaneda M, Tada M, et al. Differential mechanisms of constitutive Akt/PKB activation its influence on gene expression in pancreatic cancer cells. Jpn J Cancer Res 2002;93:1317–1326. Zhong H, Chiles K, Feldser D, et al. Modulztion of hypoxia-inducible factor 1 alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/ FRAP pathway in human prostate cancer cells: Implications for tumor anhiogenesis and therapeutics. Cancer Res 2000; 60:1541–1545. Yip-Schneider MT, Wiesenauer CA, Schmidt CM. Inhibition of the phophatidylinositol 3’-kinase signaling pathway increases the responsiveness of pancreatic carcinoma cells to sulindac. J GASTROINTEST SURG 2003;7:354–363. Halloran CM, Ghaneh P, Shore S, et al. 5-Fluorouracil or gemcitabine combined with adenoviral-mediated reintroduction of p16INK4A greatly enhanced cytotoxicity in Panc-1 pancreatic adenocarcinoma cells. J Gene Med 2004;6:514– 525.