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.

Purinergic receptor‐mediated effects of ATP in high‐grade bladder cancer

“…UTP caused an overall increase in the number of viable cells, which is contrary to the findings of other groups investigating oesophageal and colon cancer [11,12], but concordant with the recognized growthstimulatory effects of P2Y receptor subtypes as previously described [14].…”

“…Extracellular nucleotides, such as ATP, have previously been shown to be involved in the control of cell growth in various malignancies including oesophageal [11], colon [12], ovarian [13] and hormone-refractory prostate cancers [7]. This trophic response has been found to be mediated via P2 receptor subtypes.…”

“…Each sample was further treated with Amplification Grade DNase I (Sigma) to remove any residual DNA present that could generate falsepositive results. Briefly, 1 µ g of total RNA was reverse transcribed using the pd(T) [12][13][14][15][16][17][18] as the first-strand primer at 42 ° C for 30 min and the enzyme was denatured at 95 ° C for 5 min. The sequence specific primers (Life Technologies, NY, USA) for the P2X and P2Y receptors were then added to the reaction mixtures.…”

“…ATP acts via specific P2 receptor subtypes, P2X ionotropic (P2X [1][2][3][4][5][6][7] ) and P2Y metabotropic (P2Y 1,2,4,6,11,12,13,14 ), and can induce trophic responses in various cells that include cell proliferation, cell differentiation, and cell death [6]. It has been shown that purinergic agonists are able to inhibit the growth of poorly differentiated hormone-refractory prostate adenocarcinoma cells in vitro via P2 receptor-mediated apoptosis [7].…”

ATP-induced apoptosis of human granulosa luteal cells cultured in vitro

“…In human endometrial cancer cell lines, ATP was found to be associated with a significant suppression of cell proliferation through a signaling pathway involving mobilization of cytosolic stored Ca 2ϩ (19). In human colorectal carcinoma cells, extracellular ATP induced growth inhibition and apoptosis through [Ca 2ϩ ] i -or cAMP-mediated signaling (20). Of the many biological functions known to be exerted by extracellular ATP, cellular apoptosis or antiproliferation appears to be widespread rather than an exception.…”