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

Michala G Rolver,Anne P Andersen,Laurent Counillon,Stine F Pedersen,Line O Elingaard-Larsen

doi:10.1038/s41598-020-62430-z

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-N-isopropyl)-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.

Highlights

With more than 2 million estimated new cases in 2018, breast cancer remains the leading cause of cancer death in women globally[1]
Pharmacological inhibition of Na+/H+ exchanger-1 (NHE1) using EIPA or cariporide sensitizes p95HER2-expressing MCF-7 human breast cancer cells grown in 2D culture to cisplatin chemotherapy[31,32]
Spheroid growth was monitored by brightfield imaging (Fig. 1A), and a cell viability assay was performed on day 9 (Fig. 1B)

Summary

Introduction

With more than 2 million estimated new cases in 2018, breast cancer remains the leading cause of cancer death in women globally[1]. Upregulation of acid extrusion is a ubiquitous characteristic of aggressive tumour cells, and we and others have shown that knockdown (KD) or genetic ablation of net acid-extruding transporters reduces tumour growth in several cancer models[5,6,7,8,9,10,11] This renders inhibition of such transporters, alone or as combination therapy, a promising therapeutic approach, as suggested already decades ago[12]. Inhibitors (cariporide (10 μM) or EIPA (10 μM)), breast cancer subtype specific anti-cancer therapy (tamoxifen (Tam, 2 μM) or chemotherapy (Cisplatin (18.75 nM), Doxorubicin (18.75 nM) and 5-FU (0.0625 nM)) or a combination thereof as indicated, on day 2 and 4 (MDA-MB-231) or 2, 4 and 7 (MCF-7). Except for the pyrazinoylguanidine parent compound amiloride, inhibitors in both compound classes generally exhibit Ki values for NHE1 in the nanomolar range[14,15,16,18]

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Conclusion