Growth inhibition and apoptosis induced by P2Y₂ receptors in human colorectal carcinoma cells: involvement of intracellular calcium and cyclic adenosine monophosphate

Abstract.

Extracellular nucleotides induce apoptosis and inhibit growth of colorectal cancer cells. To understand the underlying signaling pathways, we investigated the role of nucleotide-sensitive P2 receptors and focused on the receptor-mediated signaling of intracellular Ca²⁺ and cyclic adenosine monophosphate (cAMP) in two colorectal carcinoma cell lines (HT29, Colo320 DM). Expression and functionality of P2 receptor subtypes evaluated by RT-PCR and [Ca²⁺]_i imaging revealed that solely metabotropic P2 receptors of the subtype P2Y₂ were expressed on a functional level in both cell lines. Short-term stimulation of P2Y₂ receptors caused Ca²⁺ mobilization from intracellular stores and a subsequent transmembrane Ca²⁺ influx. The receptor-induced [Ca²⁺]_i elevation was shown to increase basal-stimulated [cAMP]_i moderately and to potentiate forskolin-stimulated [cAMP]_i vigorously, since the 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 P2Y₂ receptor-induced increases in [cAMP]_i. Prolonged application of P2Y₂ receptor agonists induced a time-dependent increase in apoptosis (up to 50% above control values) in both cell lines and caused dose-dependent inhibition of cell proliferation of up to 85% (Colo320 DM) or 64% (HT29). Chelating [Ca²⁺]_i with BAPTA almost completely abolished P2Y₂ receptor-induced cell death. Rises in [cAMP]_i elicited by either forskolin or cAMP derivatives inhibited growth in both cell lines, too. In line with the potentiating effect of P2Y₂ receptors on forskolin-stimulated [cAMP]_i increases, costimulation with forskolin and P2Y₂ receptor agonists led to synergistic antiproliferative effects. Moreover, a synergistic growth inhibition was observed when coincubating the cells with the P2Y₂ receptor agonist ATP and the cytostatic drug 5-fluorouracil, which forms the basis for most currently applied chemotherapeutic regimes in colorectal cancer treatment. Our results demonstrate the growth inhibitory potency of P2Y₂ receptors in colorectal carcinoma cells. Receptor-induced [Ca²⁺]_i signaling appears to play a major role in the observed antiproliferative and apoptosis-inducing effects.