The MIA PaCa-2, HPAC and Capan-2 cells were transfected with pcDNA3.1 mammalian expression vector containing full-length cDNA encoding human mesothelin, or with the empty pcDNA3.1 vector. After 2 weeks of selection with G418, mesothelin-expressing cells and vector control cells were obtained for each of the three pancreatic cancer cell lines. Mesothelin protein expression were measured by Western blot analysis (Figure 2C). All three mesothelin -expressing cells expressed high levels of mesothelin protein, whereas none of the three vector control cell lines expressed detectably increased levels of mesothelin protein Epacadostat (Figure 2C). Overexpression of mesothelin

increases cell proliferation in pancreatic cancer cells with wt-p53 by p53-dependent pathway To elucidate the role of mesothelin overexpression in pancreatic cancer cell proliferation, we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, comparing the cell growth rate among the mesothelin -overexpressing MIA PaCa-2 stable cell line, the empty vector MIA PaCa-2 stable cell line, and ZVADFMK the unrelated MIA PaCa-2 cell

line. The MTT assay showed that Mesothelin transfected cells proliferated almost 3.1 times faster than the control cells at day 3 (P < 0.05; Figure 3A), and almost 2.6 times faster at day 6 (P < 0.05; Figure 3A). To confirm the role of mesothelin in cell proliferation, we did the above assay with another stably mesothelin -overexpressing pancreatic cancer cell line, Capan-2. The similarity of the results provides further evidence for the role of mesothelin in inducing cell proliferation (Figure 3B). The similarity of the results was also found in HPAC cells (data not shown). Figure 3 Overexpression of mesothelin promotes pancreatic cancer cell survival and proliferation. A, Cell proliferation of MIA PaCa-2 and Capan-2 cells according to MTT assay. Stable mesothelin Thiamine-diphosphate kinase transfected MIA PaCa-2 and Capan-2 cells

and control cells were seeded in 96-well plates (2 × 103 cells/well), serum-starved (0% fetal bovine serum, FBS) for 24 h before changing to 2% FBS growth medium, and cultured for 6 day. Viability was measured with MTT. Relative increase in viability was measured by dividing viability at one time point by viability of the same cell at day 0 (day of addition of growth medium after initial serum starvation) and is plotted along the Y-axis. Points, mean of triplicate wells. B, cells grown in soft agar were counted. bars, SD. *, P < 0.05, relative to control or mock(at 14 days). C and D , Mesothelin increases bcl-2 and decrease Bax via p53-dependent pathway. Whole cell extract from cells were probed for western blot. E , Mesothelin increases bcl-2 and decrease Bax by p53-independent pathway. Whole cell extract from cells were detected for western blot.