Figure 5. Aspirin inhibits tumor growth and invasion in ovo and enhances gemcitabine efficacy.

(A) PANC-1 cells were transplanted into a plastic ring placed on the CAM of 9-day-old chick embryos. Double-distilled water (CO, 40 μL), aspirin (ASA, 30 μL, 15 mM), gemcitabine (GEM, 10 μL, 100 nM) or aspirin and gemcitabine together (A + G) were placed on Whatman filter papers (0.5 cm²) directly adjacent to the tumor xenografts. Aspirin was applied at days 11, 13, 15 and 17 and gemcitabine at days 11 and 15. The tumor xenografts were resected at day 17, and the tumor volumes were measured with calipers. Representative images of each group are shown. The diagram shows single measurements and the means ± SD (**P < 0.01, *P < 0.05). (B) Genomic DNA was isolated from CAM tissue (n = 6) directly adjacent to the tumor xenografts, and a PCR with primers for human Alu sequences was performed. Double-distilled water served as a negative control (Neg CO), and genomic DNA isolated from a tumor xenograft served as a positive control (Pos CO). The DNA marker is shown in the first lane (Marker), and the table on the right summarizes the number of positive bands per group (No. Alu⁺ CAM). (C) Tumor tissue sections from xenografts were evaluated by immunohistochemistry for the expression of c-Met, CD133, Ki67, cleaved active fragment of caspase-3, c-Rel and p65. Representative images at 400× magnification are shown. The bar indicates 50 μm. Positive cells are colored red to dark-red. For evaluation of the expression levels, a semi-quantitative scoring system was used based on the percentage of positive cells: very high (++++), high (+++), medium (++), low (+) and absent (−).