Tumor growth experiments were per formed 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, JP and Gem, 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 intra peritoneal PDAC tumor model as previously described. Female SCID mice received 0. 75 106 human AsPC 1 cells intraperitoneally. The animals were ran domly grouped and treated intra peritoneally with PBS, JP, Gem and DT, 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. Subse quently, animals were examined for presence and extent of intra abdominal tumor. Statistical analysis In vitro cell proliferation assay results are expressed as mean standard deviation. Statistical significance was analyzed by the two tailed Students t test using Graph Pad Prism 4 Software. For in vivo studies, statistical analysis was per formed by ANOVA for multiple group comparison and Students 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 sig nificant group difference. Results Effect of JP and Gem on PDAC cell proliferation 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 and Gem Entinostat inhibited the AsPC 1 cell prolifera tion by 31% and 58%, respectively. 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. At these concentrations, the inhi bition 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. Effect of JP and Gem on PDAC apoptosis We examined if the inhibition in AsPC 1 cell viability by JP and Gem could in part be due to induction of apop tosis. 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.