Interestingly, more endogenous mesothelin introduced

caused lower expression of the pro-apoptotic protein Bax. These results indicate that endogenous mesothelin not only enhanced the expression of the anti-apoptotic proteins Bcl-2 and Mcl-1, but also reduced the expression of the pro-apoptotic selleckchem protein Bax [10]. In the present study,we also observed increased bcl-2 expression and decreased bax expression followed by mesothelin overexpression,and vice verse. Furthermore,the expression of bcl-2/bax was p53-dependent. This data shown mesothelin promoted cell survival and proliferation by p53-dependent pathway in pancreatic cancer cells with wt-p53. However, mesothelin did not affect proliferation in HPAC cells in vivo, which suggests that the tumor microenvironment may play an important role. In MIA PaCa-2 cells with mutant p53 which expressed less endogenous mesothelin,we found that mesothelin overexpression is also associated with increased cell proliferation followed by decreased bax and increased bcl-2. In contrast, in AsPC-1 cells with p53-null and Capan-1 cells with mt-p53 that expressed more endogenous mesothelin, reduction in expression of mesothelin by shRNA stable silencing resulted in decreased cell proliferation and increased bax and decreased bcl-2. When

mesothelin was re-expressed in stable mesothelin sliencing cells, cell proliferation and bax expression was increased and bax was decreased(data not shown). However mesothelin did not affect wt-p53 level. Those results indicate that in pancreatic cancer cells VX-680 manufacturer with mt-p53 or null-p53, DCLK1 mesothelin regulates proliferation through p53-independent bcl-2/bax pathway. p53 functions to regulate several pathways, including cell cycle LXH254 nmr arrest, DNA repair and apoptosis through transcriptional upregulation of proapoptotic Bcl-2 genes, in particular Puma/Bbc3 [30, 31]. Loss of p53 protects cells from p53-dependent apoptotic stimuli due to limited PUMA transcriptional upregulation.

The induction of apoptosis is a key tumor suppressor function of p53, particularly in those cells which acquire other oncogenic lesions [32]. p53-dependent Puma upregulation has a central role in this response, inducing apoptosis in the transformed cells [20]. In the present study, silencing endogenous mesothelin by shRNA in Capan-2 (wt-p53) cells increased significant apoptosis followed by increased wt-p53, PUMA and caspase-3 activity. When the p53 or PUMA was blocked by transient p53 siRNA or PUMA siRNA transfection in stable mesothelin shRNA transfected Capan-2 cells,the significant reduction of apoptosis was found. In vivo, mesothelin shRNA also promoted apoptosis, followed by increased p53, PUMA expression and caspase-3 activity. Those results indicate that mesothelin silencing promoted apoptosis through p53-dependent PUMA pathway in cells with wt-p53.