title: 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.
authors.affiliation: Gastroenterology and Infectious Diseases, Medical Clinic I, Universitätsklinikum Benjamin Franklin, Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Höpfner
authors.forename: K
authors.initials: K
authors.lastname: Maaser
authors.forename: B
authors.initials: B
authors.lastname: Barthel
authors.forename: B
authors.initials: B
authors.lastname: von Lampe
authors.forename: C
authors.initials: C
authors.lastname: Hanski
authors.forename: E O
authors.initials: EO
authors.lastname: Riecken
authors.forename: M
authors.initials: M
authors.lastname: Zeitz
authors.forename: H
authors.initials: H
authors.lastname: Scherübl
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology*; D000242:Cyclic AMP / Q000378:metabolism*; D005472:Fluorouracil / Q000627:therapeutic use; D006801:Humans; D018048:Receptors, Purinergic P2 / Q000378:metabolism / Q000502:physiology*; D058473:Receptors, Purinergic P2Y2; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D000964:Antimetabolites, Antineoplastic; C546385:P2RY2 protein, human; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D002118:Calcium; D005472:Fluorouracil; D014544:Uridine Triphosphate