br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion In particular, we showed that pancreatic neuroendocrine tumor Loxapine Succinate recognize cAMP as a stimulatory signal for cell proliferation. Conversely, cAMP was found to have an inhibitory effect on cell proliferation in bronchial carcinoids cells. The divergent effects of cAMP on cell proliferation was associated with a corresponding modification in cyclin D1 and p27 expression, thus confirming the cytostatic or proliferative effect of cAMP in these two different neuroendocrine tumor models. Interestingly, we showed that the mechanism involved in the divergent cAMP effects cell type-specific, could be attributed to the different expression of B-Raf and Raf1 in QGP1 and H727 cell lines, in according with previous studies showing that cAMP inhibited MAPK when signaling was through Raf1 and activating MAPK when signaling was through B-Raf [10], [46]. cAMP physiologic effects are mediated by cAMP dependent protein kinase A (PKA) and the more recently discovered two Exchange Protein Activated by cAMP (Epac1/2). In adrenocortical cancer cells Epac2 has been shown to be involved in mediating the effects exerted by cAMP on cytoskeleton integrity and cell migration [1]. Moreover, Epac is required for cAMP-dependent ERK1/2 activation in mouse pituitary AtT20 cells [44] and involved in the release of α−MSH in the pituitary [41]. On the contrary, the involvement of Epac in mediating TSH action is controversial. In dog thyrocytes, Epac activation is not required for none of the known cAMP-mediated TSH effects [9], whereas in PCCL3, a well-differentiated TSH-dependent rat thyroid follicular cell line, Epac acts synergistically with PKA in mediating the stimulatory effects of TSH on DNA synthesis and cell proliferation [17]. The present study demonstrates that both PKA and Epac exert similar effects on tumoral neuroendocrine cell proliferation, mediating the divergent action of cAMP on pancreatic and bronchial neuroendocrine cells. The effects on cell proliferation obtained by measuring incorporation of BrdU in newly synthesized DNA were consistent with the changes in cyclin D1 or p27 expression induced by the exposure to Epac-selective and PKA-selective analogs. Although Rap1 is the main direct downstream effector of Epac [37], [7], other studies reported that also Rap1 activation induced by cAMP requires PKA [10], [15], [32], [39], [40], [43], [46], [47]. In particular, our results are consistent with a previous study in which both PKA and Epac were effective in activating GTP- bound Rap1 [38]. In order to better define the role of Epac in mediating the effects on cell proliferation induced by cAMP, we replicated our experiments after Epac 1 or 2 silencing. In bronchial carcinoids H727 cell line, we demonstrated that both Epac isoforms were required to exert the antiproliferative effect exerted by Epac while in QGP-1 Epac-1 was the main involved isoform, as already reported in human lung fibroblasts [16], [21]. Moreover, we showed that EPAC effects were PKA independent, in fact PKA inhibitor did not affect the response to the selective Epac activator. In addition to cell growth, cell adhesion represents another important process modulated by cAMP and involved in cancer progression. The present study shows that cAMP promoted cell adhesion on collagene type IV coated plates in QGP-1 and H727 cells, and this stimulatory effect was mimicked by both EPAC and PKA effectors, thus suggesting that both Epac and PKA are involved in the adhesion process in neuroendocrine tumor cells. In this respect, several studies demonstrated that Epac-Rap signaling is involved in the control of adhesion-associated processes [14], [25], [29], [48]. It has also been reported that PKA could mediate cAMP- induced adhesion of different cells types [2], [20], [3]. Confirming the involvement of Rap1 in mediating the effects of cAMP on cell adhesion, we demonstrated that Rap1 silencing was associated with the abolishment of the effects exerted by cAMP on both cell growth and adhesion. These data seem to suggest the putative Rap1 involvement in neuroendocrine tumorigenesis as yet suggested in other cancer models [11], [26].