Growth inhibition and apoptosis induced by P2Y(2) receptors in human colorectal carcinoma cells: involvement of intracellular calcium and cyclic adenosine monophosphate

Extracellular nucleotides induce apoptosis and inhibit growth of colorectal cancer cells. To understand the underlying signaling pathways, we investig ated the role of nucleotide-sensitive P2 receptors and focused on the recep tor-mediated signaling of intracellular Ca2+ and cyclic adenosine monophosp hate (cAMP) in two colorectal carcinoma cell lines (HT29, Colo320 DM). Expr ession and functionality of P2 receptor subtypes evaluated by RT-PCR and [C a2+](i) imaging revealed that solely metabotropic P2 receptors of the subty pe P2Y(2) were expressed on a functional level in both cell lines. Short-te rm stimulation of P2Y(2) receptors caused Ca2+ mobilization from intracellu lar stores and a subsequent transmembrane Ca2+ influx. The receptor-induced [Ca2+](i) elevation was shown to increase basal-stimulated [cAMP](i) moder ately and to potentiate forskolin-stimulated [cAMP](i) vigorously, since th e effects were dose-dependently inhibited by preloading the cells with the [Ca2+](i) chelator BAPTA. In contrast, activation of protein kinase C (PKC) did not contribute to a receptor-mediated rise in [cAMP](i), since the PKC inhibitor staurosporine completely failed to reduce P2Y(2) receptor-induce d increases in [cAMP](1). Prolonged application of P2Y(2) receptor agonists induced a time-dependent increase in apoptosis (up to 50% above control va lues) in both cell lines and caused dose-dependent inhibition of cell proli feration of up to 85% (Colo320 DM) or 64% (HT29). Chelating [Ca2+](i) with BAPTA almost completely abolished P2Y(2) receptor-induced cell death. Rises in [cAMP](i) elicited by either forskolin or cAMP derivatives inhibited gr owth in both cell lines, too. In line with the potentiating effect of P2Y(2 ) receptors on forskolin-stimulated [cAMP](i) increases, costimulation with forskolin and P2Y(2) receptor agonists led to synergistic antiproliferativ e effects. Moreover, a synergistic growth inhibition was observed when coin cubating the cells with the P2Y(2) receptor agonist ATP and the cytostatic drug 5-fluorouracil, which forms the basis for most currently applied chemo therapeutic regimes in colorectal cancer treatment. Our results demonstrate the growth inhibitory potency of P2Y(2) receptors in colorectal carcinoma cells. Receptor-induced [Ca2+](i) signaling appears to play a major role in the observed antiproliferative and apoptosis-inducing. effects.