title: Amiloride augments TRAIL-induced apoptotic death by inhibiting phosphorylation of kinases and phosphatases associated with the P13K-Akt pathway.
abstract: We have previously shown that low extracellular pH (pHe) promotes cell killing by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we examined whether amiloride, an inhibitor of the Na(+)/H(+) antiporter capable of lowering the intracellular pH (pHi), can potentiate TRAIL-induced apoptotic death. Human prostate adenocarcinoma DU-145 cells were treated with various concentrations of TRAIL (10-200 ng/ml) and/or amiloride (0.1-1 mM) for 4 h. Amiloride, which caused little or no cytotoxicity by itself, enhanced TRAIL-induced apoptosis. The TRAIL-mediated activation of caspase, and PARP (poly (ADP-ribose) polymerase) cleavage were both promoted by amiloride. Western blot analysis showed that combined treatment with TRAIL and amiloride did not change the levels of TRAIL receptors (death receptor (DR)4, DR5, and DcR2 (decoy recepter 2) or antiapoptotic proteins (FLICE-inhibitory protein (FLIP), inhibitor of apoptosis (IAP), and Bcl-2). However, unlike pHe, amiloride promoted the dephosphorylation of Akt. Interestingly, amiloride also induced the dephosphorylation of P13K (phosphatidylinositol 3-kinase) and PDK-1 (phosphoinositide-dependent kinase-1) kinases along with PTEN (phosphatase and tensin homolog deleted on chromosome 10) and PP1alpha phosphatases. In vitro kinase assays revealed that amiloride inhibited phosphorylation of kinases and phosphatases by competing with ATP. Taken together, the present studies suggest that amiloride enhances TRAIL-induced cytotoxicity by inhibiting phosphorylation of the PI3K-Akt pathway-associated kinases and phosphatases.
authors.affiliation: Department of Surgery and Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: Ki M
authors.initials: KM
authors.lastname: Kim
authors.forename: Yong J
authors.initials: YJ
authors.lastname: Lee
mesh_terms: D000230:Adenocarcinoma; D000584:Amiloride / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D002460:Cell Line; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D004847:Epithelial Cells; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011467:Prostate / Q000166:cytology; D011471:Prostatic Neoplasms; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D051017:Apoptosis Regulatory Proteins; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D000584:Amiloride; D011494:Protein Kinases; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D010749:Phosphoprotein Phosphatases