Activation of p90RSK and growth stimulation of multicellular tumor spheroids are dependent on reactive oxygen species generated after purinergic receptor stimulation by ATP.

Abstract

Mitogenic stimulation by growth factors may be mediated through intracellular reactive oxygen species (ROS) acting as signaling molecules. Incubation of multicellular prostate tumor spheroids with adenosine 5' triphosphate (ATP) dose-dependently stimulated tumor growth. ATP, uridine 5'-triphosphate (UTP), adenosine 5'-diphosphate (ADP), and 2-methylthio-ATP (2-MeS-ATP) increased intracellular ROS levels significantly. ROS generation by ATP was inhibited by the P2 receptor antagonist suramin, by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors diphenylene iodonium chloride (DPI) and 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), as well as by the Ca2+-dependent phospholipase A2 (PLA2) inhibitors indomethacin and methyl arachidonyl fluorophosphonate (MAFP). The generation of ROS was dependent on the intracellular Ca2+ response evoked by ATP. Exogenous ATP activated the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) pathway, which was blunted by the MAPK/ERK kinase 1/2 (MEK1/2) antagonist PD98059. The radical scavengers vitamin E, dimethyl thiourea (DMTU), and N-acetyl cysteine (NAC) failed to inhibit ERK1/2 activation but abolished p90 ribosomal S6 kinase (p90RSK) activation downstream of ERK1/2, as well as the growth stimulation of tumor spheroids. Our data indicate that p90RSK downstream of ERK1/2 is the molecular target for ROS generated through stimulation of purinergic receptors by ATP.