Molecular features of the cytotoxicity of an NHE inhibitor: Evidence of mitochondrial alterations, ROS overproduction and DNA damage

Abstract

BackgroundNH exchangers (NHEs) play a crucial role in regulating intra/extracellular pH, which is altered in cancer cells, and are therefore suitable targets to alter cancer cell metabolism in order to inhibit cell survival and proliferation. Among NHE inhibitors, amiloride family members are commonly used in clinical practice as diuretics; we focused on the amiloride HMA, reporting a net cytotoxic effect on a panel of human cancer cell lines; now we aim to provide new insights into the molecular events leading to cell death by HMA.MethodsColon cancer cell lines were treated with HMA and analysed with: morphological and cellular assays for cell viability and death, and autophagy; biochemical approaches to evaluate mitochondrial function and ROS production; in situ detection of DNA damage; molecular tools to silence crucial autophagy/necroptosis factors.ResultsHMA affects cellular morphology, alters mitochondrial structure and function, causes an increase in ROS, which is detrimental to DNA integrity, stimulates poly(ADP-ribose) synthesis, activates RIPK3-dependent death and triggers autophagy, which is unable to rescue cell survival. These features are hot points of an intricate network of processes, including necroptosis and autophagy, regulating the homeostasis between survival and death.ConclusionOur results allow the identification of multiple events leading to cell death in cancer cells treated with HMA. The here-defined intricate network activated by HMA could be instrumental to selectively target the key players of each pathway in the attempt to improve the global response to HMA. Our data could be the starting point for developing a newly designed targeted therapy.

Quinalizarin exerts an anti-tumour effect on lung cancer A549 cells by modulating the Akt, MAPK, STAT3 and p53 signalling pathways

“…Intracellular ROS levels have been reported to activate the MAPK signalling pathways (40). Imbalances due to either increased ROS production or decreased ROS degradation may cause excessive ROS accumulation, thus damaging to cell structures and compromising cellular functions (41). Our study showed that NAC completely restored the apoptotic mechanism of A549 cells induced by quinalizarin.…”

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

“…Small molecule NHE1 inhibitors have therefore been explored as an anticancer approach, alone or in combination treatment schemes 6,31,32,45 . However, evidence from 2D systems has shown that at high concentrations (20-40 µM, and up to 500 µM for amiloride, compare to IC 50 values for NHE1 inhibition, Table 1), some pyrazinoylguanidine-type NHE1 inhibitors exert NHE1-independent cytotoxicity 24,25,28,46 . Furthermore, pyrazinoylguanidine-type inhibitors inhibit several other NHE isoforms than NHE1 and some additionally target the epithelial Na + channel (ENaC) and Na + /Ca 2+ exchangers [13][14][15] .…”

“…In cancer cells, the long-term effects of pyrazinoylguanidine-type NHE1 inhibitors, which have generally been assumed to be downstream of NHE1 inhibition, include decreased proliferation, reduced metastatic potential and -viability, and sensitization to chemotherapy 32,45,47,48 . There are, however, reports of NHE1-unrelated effects of these compounds, including dysregulation of ER Ca 2+ homeostasis 24 , reactive oxygen species production 25 , urokinase plasminogen activator inhibition 26 , and cell death 27,28 . The present study is the first to use NHE1 CRISPR/Cas9 KO in conjunction with extensive pharmacological analyses, to unequivocally establish the NHE1-independence of these effects.…”

“…Despite this, essentially all studies of NHE1 inhibitors in cancer cells were conducted under two-dimensional (2D) growth conditions which poorly reflect in vivo conditions 20 . Furthermore, several studies point to NHE1-independent effects of NHE1 inhibitors [24][25][26][27][28][29][30] , yet mechanistic insight into these effects is lacking.…”

The Antifungal Activity of HMA, an Amiloride Analog and Inhibitor of Na+/H+ Exchangers

“…Alternatively, off-target effects of HMA in fungal cells could be possible and would be consistent with other reports that demonstrated HMA activity toward G-protein-coupled receptors, such as the adenosine 2A receptor ( Garritsen et al, 1991 ; Gao and Ijzerman, 2000 ; Soudijn et al, 2004 ). The reported anticancer activity of HMA and its analogs stems from their antitumor/metastasis effects due in part to the inhibition of the human urokinase plasminogen protease that functions as a major driver of cell invasiveness ( Kleyman and Cragoe, 1988 ; Aredia et al, 2016 ; Buckley et al, 2018 ). These various biological activities of HMA may be indicative of other targets in fungal cells, possibly in addition to Nhx1 or Nhe1.…”