Amiloride potentiates TRAIL-induced tumor cell apoptosis by intracellular acidification-dependent Akt inactivation Young-Lai Cho a,b , Kwang-Soon Lee a,b , Seon-Jin Lee a,b , Seung Namkoong a,b , Young-Mi Kim a , Hansoo Lee c , Kwon-Soo Ha a,b , Jeong-A Han b , Young-Guen Kwon d , Young-Myeong Kim a,b, * a Vascular System Research Center, Kangwon National University, Chunchon, Kangwon-do, Republic of Korea b Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, Kangwon-do, Republic of Korea c Division of Life Science, College of Natural Sciences, Kangwon National University, Chunchon, Kangwon-do, Republic of Korea d Department of Biochemistry, College of Sciences, Yonsei University, Seoul, Republic of Korea Received 17 November 2004 Available online 7 December 2004 Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor gene family, is con- sidered as one of the most promising cancer therapeutic agents due to its ability to selectively induce tumor cell apoptosis. In this study, we investigated whether the Na + /H + exchanger inhibitor, amiloride, promotes TRAIL-induced apoptotic death both in sen- sitive and resistant tumor cells, HeLa and LNCaP cells, respectively, and its underlying molecular mechanism. Amiloride enhanced TRAIL-induced apoptosis and activation of caspase-3 and -8 in both cells. This compound increased TRAIL-induced mitochon- drial cytochrome c release and poly(ADP-ribose) polymerase cleavage. Moreover, amiloride-induced intracellular acidification, and inhibited the phosphorylated activation of the serine/threonine kinase Akt, which is known to promote cell survival, in both tumor cells. These data suggest that amiloride sensitizes both tumor cells to TRAIL-induced apoptosis by promoting Akt dephos- phorylation and caspase-8 activation via the intracellular acidification and that Na + /H + exchanger inhibitors may play an impor- tant role in the anti-cancer activity of TRAIL, especially, in TRAIL-resistant tumors with highly active and expressed Akt. Ó 2004 Elsevier Inc. All rights reserved. Keywords: TRAIL; Apoptosis; Amiloride; Na + /H + exchanger; Intracellular acidification; Caspases Tumor necrosis factor-related apoptosis-inducing-li- gand (TRAIL) [1] is a member of the tumor necrosis fac- tor family of cytokines and is capable of inducing apoptosis in a wide variety of cancer cells in culture, but does not cause toxicity in the majority of normal cells. Thus, TRAIL may be suitable for novel anti-can- cer therapies. The apoptosis-inducing receptors for TRAIL include the transmembrane type-I receptors, Trail-R1 (DR4) and Trail-R2 (DR5). These receptors are expressed on the surface of many types of tumor cells and transmit apoptotic signals via cytoplasmic death domains. However, TRAIL also binds to the non-apoptosis-inducing decoy receptors, DcR1 and DcR2, which possess deletions in the functional cyto- plasmic regions containing death domains [2]. It has been shown that ligation of TRAIL to its cyto- toxic receptors recruits FADD and procaspase-8 to the cytoplasmic death domain of the receptors and forms the death-inducing signaling complex (DISC), which subsequently activates apical caspase-8. Active caspase-8 can directly activate downstream effector caspases, including caspase-3, -6, and -7 [3]. In addition, caspase-8 cleaves Bid (p22) to truncate Bid (p15), which 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.109 * Corresponding author. Fax: +82 22 244 3286. E-mail address: ymkim@kangwon.ac.kr (Y.-M. Kim). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 326 (2005) 752–758 BBRC