Pyrazine ring-based Na+/H+ exchanger (NHE) inhibitors potently inhibit cancer cell growth in 3D culture, independent of NHE1

Abstract

the na + /H + exchanger-1 (NHE1) supports tumour growth, making NHE1 inhibitors of interest in anticancer therapy, yet their molecular effects are incompletely characterized. Here, we demonstrate that widely used pyrazinoylguanidine-type NHE1 inhibitors potently inhibit growth and survival of cancer cell spheroids, in a manner unrelated to NHE1 inhibition. Cancer and non-cancer cells were grown as 3-dimensional (3D) spheroids and treated with pyrazinoylguanidine-type (amiloride, 5-(N-ethyl-Nisopropyl)-amiloride (EIPA), 5-(N,N-dimethyl)-amiloride (DMA), and 5-(N,N-hexamethylene)-amiloride (HMA)) or benzoylguanidine-type (eniporide, cariporide) NHE1 inhibitors for 2-7 days, followed by analyses of viability, compound accumulation, and stress-and death-associated signalling. EIPA, DMA and HMA dose-dependently reduced breast cancer spheroid viability while cariporide and eniporide had no effect. Although both compound types inhibited NHE1, the toxic effects were NHE1-independent, as inhibitor-induced viability loss was unaffected by NHE1 CRISPR/Cas9 knockout. EIPA and HMA accumulated extensively in spheroids, and this was associated with marked vacuolization, apparent autophagic arrest, ER stress, mitochondrial-and DNA damage and poly-ADP-ribose-polymerase (PARP) cleavage, indicative of severe stress and paraptosis-like cell death. Pyrazinoylguanidine-induced cell death was partially additive to that induced by conventional anticancer therapies and strongly additive to extracellular-signal-regulated-kinase (ERK) pathway inhibition. Thus, in addition to inhibiting NHE1, pyrazinoylguanidines exert potent, NHE1-independent cancer cell death, pointing to a novel relevance for these compounds in anticancer therapy.

The Interplay between Dysregulated Ion Transport and Mitochondrial Architecture as a Dangerous Liaison in Cancer

“…One such pathway is paraptosis, in which ER and mitochondria dilate in absence of classical features of apoptosis and necrosis [ 187 ]. The process is induced by a number of natural compounds and anticancer drugs [ 188 ], including compounds previously considered specific NHE1 inhibitors [ 189 ] and is of interest in the treatment of cancer cells, which have downregulated apoptotic machinery. Notably, while the mechanisms involved in paraptosis are still incompletely understood, substantial evidence links it to Ca 2+ dysregulation, and also BK channel dysfunction and disturbed K + homeostasis has been suggested [ 188 ].…”

An optogenetic tool to raise intracellular pH in single cells and drive localized membrane dynamics

“…11 Furthermore, the use of ion transport inhibitors is very common 12,13 but recent studies have reported significant off-target effects of various pHi-lowering drugs. 14,15 Micropipette pHi manipulation techniques are highly specific and adaptable to single-cell analysis. 16 However, micropipette pHi manipulation is technically challenging, has poor temporal control, is damaging to the cell, and is low-throughput.…”

Cancer and pH Dynamics: Transcriptional Regulation, Proteostasis, and the Need for New Molecular Tools

“…The work described above suggests that the cell biological and biochemical effects of increased pHi in cancer correlate with and perhaps reinforce transcriptional and proteostatic changes associated with cancer. However, several recent papers that note off-target effects of key pHi manipulating drugs [ 130 , 131 ] have begun to call into question the previous causative role of cytosolic pH in driving tumorigenic phenotypes at the population level. Indeed, the work in this field so far has been crippled by the lack of tools to spatiotemporally manipulate and measure pHi across multiple cancer model systems (cell lines, organoids, and in vivo models) to determine the roles of pHi dynamics across biological scales.…”