Human PDAC cell lines, AsPC-1, Panc-1, BxPC-3 and MIA PaCa-2 were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cell lines were cultured in RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin solution (Sigma) at 37°C in a humidified 5% CO₂ atmosphere. JP was obtained from Joyant Pharmaceuticals (Dallas, TX), Gem was purchased from Eli Lilly Corporations (Indianapolis, IN), doxorubicin (Dox) was purchased from Ben Venue Laboratories (Bedford, OH) and docetaxel (DT) was purchased from Sanofi-aventis (Bridgewater, NJ).

In vitro cell viability of PDAC cell lines was evaluated by using the colorimetric WST-1 reagent (Roche Diagnostics, Indianapolis, IN) as described earlier 24 . The assay is based on the ability of viable cells to cleave the sulfonated tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) by mitochondrial dehydrogenases. Briefly, PDAC cells (4,000 cells per well) were plated in a 96-well plate in regular growth medium and after 16 hours the medium was replaced with 2% FBS containing medium. After 5 hours incubation the cells were treated with JP, Gem, Dox or DT, either alone or in combination. After additional incubation of 72 hours, 10 μl WST-1 reagent was added in each well followed by incubation for 2 hours. The absorbance at 450 nm was measured using a microplate reader.

A monolayer of cells at 75 to 80% confluence was placed in 2% FBS containing medium for at least 5 hour before treatment with JP (10 μM), gemcitabine (10 μM) for 24 hours. Cells were lysed and equal amounts of total protein were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA). The membranes were blocked for 1 hour at room temperature with gentle shaking in TBS-T (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.05% Tween 20). The membranes were incubated overnight at 4°C with the following antibodies: phospho-JNK, (Santa Cruz Biotechnologies, Santa Cruz, CA), poly (ADP-ribose) polymerase-1 (PARP-1) (Cell Signaling Technology, Beverly, MA), α-tubulin or GAPDH (Sigma). The membranes were then incubated with corresponding HRP-conjugated secondary antibodies (Pierce Biotechnologies, Rockford, IL) for 1 hour at room temperature. Specific bands were detected using the enhanced chemiluminescence reagent (ECL, Perkin Elmer Life Sciences, Boston, MA) on autoradiographic film and quantitated by densitometry.

Effect of JP and Gem on AsPC-1 cell apoptosis was detected by using the Annexin V-FITC apoptosis detection kit (BioVision, CA) as per manufacturer's protocol. Briefly, AsPC-1 cells were grown in 10% serum containing medium up to 80% confluence, medium was changed to 2% serum containing medium and incubated for 5 hours and then treated with JP (10 μM) and Gem (10 μM) for 12 hours. After incubation, cells were trypsinized and suspended in 1 ml of low serum medium. From each cell suspension 2 × 10⁵ cell were centrifuged and re-suspended in 500 μl 1X binding buffer. In each sample 5 μl of Annexin V-FITC and 5 μl of PI was added, incubated for 5 minutes in dark and immediately analyzed by flow cytometry (FACS Calibur System, BD Biosciences, Franklin Lakes, NJ). The annexin V - FITC positive and PI-negative cells were considered to be early apoptotic cells.

All animal studies were performed in accordance with the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center (Dallas, TX). In vivo animal studies were performed using 4-6 weeks old SCID mice purchased from an on campus facility. Tumor growth experiments were performed by orthotopically injecting 1 × 10⁶ Panc-1 cells. Animals were examined by ultrasound and randomized into treatment groups when tumor size averaged 500 mm³ at approximately 8 weeks after tumor cell injection. Animals were injected intraperitoneally with PBS (control), JP (6 mg/kg in 100 μl PBS, three times weekly) and Gem (25 mg/kg in 100 μl PBS, three times weekly), either alone or in combination. Three mice from each treatment group were sacrificed at 24 hours of therapy while the remaining animals in each group received 2 weeks of therapy prior to sacrifice.

Animal survival studies were conducted in an intraperitoneal PDAC tumor model as previously described 25 . Female SCID mice received 0.75 × 10⁶ human AsPC-1 cells intraperitoneally. The animals were randomly grouped (n = 6 to 8 per group) and treated intraperitoneally with PBS (control), JP (6 mg/kg in 100 μl PBS, twice weekly), Gem (100 mg/kg in 100 μl PBS, twice weekly) and DT (3 mg/kg in 100 μl PBS, twice weekly), either alone or in combination for 14 days or as maintenance therapy. Animal weight was measured twice weekly and all animals were examined daily for signs of distress or development of jaundice. Moribund mice at risk for distress were euthanized in accordance with the local animal care committee protocol. Subsequently, animals were examined for presence and extent of intra-abdominal tumor.

In vitro cell proliferation assay results are expressed as mean ± standard deviation. Statistical significance was analyzed by the two-tailed Student's t-test using GraphPad Prism 4 Software (GraphPad Software, San Diego, CA). For in vivo studies, statistical analysis was performed by ANOVA for multiple group comparison and Student's t-test for the individual group comparison. In survival studies, statistical differences were analyzed by nonparametric survival statistics and logrank test. Values of p < 0.05 were considered to represent statistically significant group difference.

In vitro WST-1 assay was performed to examine the effect of JP and Gem on PDAC cell proliferation. In AsPC-1 cells, JP and Gem significantly inhibited the cell proliferation. After 72 hours of incubation, JP (1 μM) and Gem (500 nM) inhibited the AsPC-1 cell proliferation by 31% and 58%, respectively (Figure 1). The combination of JP and Gem had an additive effect on inhibition of AsPC-1 cell proliferation, with an inhibition in cell proliferation of 70% after 72 hours of incubation (Figure 1). At these concentrations, the inhibition in cell proliferation of other PDAC lines in JP, Gem and JP + Gem groups were 54%, 10% and 79% for Panc-1; 42%, 56% and 77% for BxPC-3; and 9%, 43% and 77% for MIA PaCa-2, respectively.

We examined if the inhibition in AsPC-1 cell viability by JP and Gem could in part be due to induction of apoptosis. Annexin V/PI staining assay revealed an increase in early apoptotic cells by JP and Gem treatment that was further increased by combination of these agents (Figure 2A). At 10 μM concentration of either agent, the percentage of early apoptotic cells was 17% in controls, 26% in JP or Gem, and 38% in the JP + Gem group. (Figure 2A).

We also measured cleavage of PARP-1 protein, a caspase-dependent apoptosis-marker protein, after JP and Gem treatment. A significant increase in the expression of cleaved PARP-1 protein was observed after treatment with JP, but not after exposure to Gem (Figure 2B).

We next evaluated if JP can sensitize other chemotherapeutic agents. In vitro WST-1 assay revealed that JP, Dox and DT inhibited the proliferation of all four PDAC cell lines tested in a dose-dependent manner (Figure 3). Interestingly, the combinations of JP with either doxorubicin or docetaxel had additive effects on inhibition of proliferation of PDAC cell lines (Figure 3). At intermediate concentrations of these agents, inhibition in cell proliferation in AsPC-1 cells in JP (5 μM), Dox (1 μM), DT (1 μM), JP + Dox and JP + DT groups were 40%, 39%, 48%, 73% and 73%, respectively. Similar additive combination effects of JP with Dox or DT were observed regarding Panc-1, MIA PaCa-2 and BxPC-3 proliferation (Figure 3).

Evaluation of PARP-1 cleavage after JP, Dox and DT treatment by Western blot analysis revealed that Dox and DT had no effect on PARP-1 cleavage, while JP exposure led to a significant increase in PARP-1 cleavage (Figure 4).

The antitumor impact of JP, Gem and DT, either alone or in combination, was evaluated in murine PDAC xenografts. In an orthotopic Panc-1 xenograft model, tumor weights were measured at completion of therapy and compared to tumors harvested at 24 hours of therapy (Figure 5). The increase in tumor weight was 87% in controls, while Gem and JP treatment alone trended towards decreased tumor growth, albeit not significant. JP + Gem combination treatment resulted in decreased tumor weight compared to that at treatment start, with a mean reduction of approximately 30% (p < 0.05 verses control) (Figure 5). In survival studies with maintenance therapy, a statistically significant improvement in animal survival was observed in mice treated with the combination of JP + Gem (median: 38 days, p = 0.01) compared to controls (22 days) or single agent treatment with JP (28 days) or Gem (32 days, p = 0.02) (Figure 6A). Animal survival was also significantly improved in a 14-day combination treatment with JP + DT (32 days) as compared to controls (16 days) or single agent treatment with JP (21 days) or DT (27 days) (Figure 6B). In addition to survival impact, we also evaluated the treatment effects of JP and DT on inhibition of local tumor growth in subcutaneous AsPC-1 pancreatic cancer xenografts. JP enhanced the DT-mediated local antitumor effects: compared with controls, addition of JP enhanced the inhibition in net tumor growth by DT of 57% to 91% in combination (p = NS), respectively (data not shown).