Vasoactive intestinal peptide (VIP) induces c- fos expression in LNCaP prostate cancer cells through a mechanism that involves Ca 2+ signalling. Implications in angiogenesis and neuroendocrine differentiation

Abstract

The effect of vasoactive intestinal peptide (VIP) on intracellular Ca 2+ levels and its relationship with the expression of c- fos and vascular endothelial growth factor (VEGF) as well as with neuroendocrine (NE) differentiation were investigated in human prostate LNCaP cells. VIP induced the expression of c- fos mRNA as studied by reverse transcription polymerase chain reaction (RT-PCR). It was accompanied by VIP stimulation of c- fos protein synthesis, as measured by Western blot analysis. VIP enhanced intracellular Ca 2+ levels as evaluated using the calcium probe fura-2. VIP regulation of c- fos expression depended on [Ca 2+] i concentration since the intracellular calcium chelator BAPTA/AM decreased c- fos expression (both mRNA and protein) to basal levels. As shown by means of real-time RT-PCR, VIP stimulated VEGF mRNA expression: the effect was inhibited by 40% in the presence of curcumin (an inhibitor of AP-1 binding), and it was dependent on Ca 2+ since BAPTA/AM inhibited this VIP action by 43%. Similar observations were made on the effects of BAPTA/AM and curcumin on VIP stimulation of VEGF protein expression. Simultaneous treatment of cells with the protein kinase A inhibitor H89 and BAPTA/AM completely blocked this VIP effect, whereas each agent alone led only to a partial inhibition. In addition, the calcium chelator blocked by 37% the ability of VIP to induce NE cell differentiation as estimated by the observation of neurite development. These features support a VIP signalling pathway that could be mediated through both cAMP and [Ca 2+] i increase in prostate LNCaP cancer cells. Moreover, our data suggest the implication of c-Fos on the induction of the main angiogenic factor VEGF since the promoter region of the VEGF gene possesses AP-1 (i.e., c-Fos/c-Jun heterodimer) response elements. This feature represents a link between the nuclear oncogene c- fos, angiogenesis and NE differentiation by means of an initiating signal upon VIP receptors.