Hexamethylene amiloride engages a novel reactive oxygen species- and lysosome-dependent programmed necrotic mechanism to selectively target breast cancer cells

Abstract

Anticancer chemotherapeutics often rely on induction of apoptosis in rapidly dividing cells. While these treatment strategies are generally effective in debulking the primary tumor, post-therapeutic recurrence and metastasis are pervasive concerns with potentially devastating consequences. We demonstrate that the amiloride derivative 5-(N,N-hexamethylene) amiloride (HMA) harbors cytotoxic properties particularly attractive for a novel class of therapeutic agent. HMA is potently and specifically cytotoxic toward breast cancer cells, with remarkable selectivity for transformed cells relative to non-transformed or primary cells. Nonetheless, HMA is similarly cytotoxic to breast cancer cells irrespective of their molecular profile, proliferative status, or species of origin, suggesting that it engages a cell death mechanism common to all breast tumor subtypes. We observed that HMA induces a novel form of caspase- and autophagy-independent programmed necrosis relying on the orchestration of mitochondrial and lysosomal pro-death mechanisms, where its cytotoxicity was attenuated with ROS-scavengers or lysosomal cathepsin inhibition. Overall, our findings suggest HMA may efficiently target the heterogeneous populations of cancer cells known to reside within a single breast tumor by induction of a ROS- and lysosome-mediated form of programmed necrosis.

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

“…When administered together with HMA (30 μM and 40 μM) for 24 h, NEC did not rescue HMA-induced cell death (Fig. 3b), thus suggesting that in HCT-116 cells RIPK1 is not involved in the cellular response to HMA, as already shown in breast cancer cells [9]. To go deeper into the necroptosis issue by addressing the impact of the other key regulator RIPK3, we used the HT-29 cell line, being HCT-116 cells characterised by a low expression of RIPK3 [27].…”

“…We previously detected some autophagy markers in HMA-treated colon carcinoma SW613-B3 cells [10], as also confirmed in breast cancer cells [9]; now, we addressed whether HMA drives a pro-survival or pro-death role of autophagy. To do this, we silenced Atg7 and Beclin 1 autophagy effectors (Fig.…”

“…Is PAR pro-survival function connected to autophagy? To answer this question, we monitored the autophagy marker LC3, generally overexpressed in HMA-treated cells [9, 10]; in samples co-treated with OLAP, a significant reduction of the LC3 signal with respect to the HMA treatment alone was observed (Fig. 5c), suggesting a possible requirement of PAR in the autophagy machinery [25, 30].…”

“…The interest toward HMA is legitimated by the evidence that it is poorly toxic to non-transformed breast cells and mouse mammary cells compared to transformed breast cancer cell lines and mouse mammary cancer cells, suggesting that HMA targets selectively cancer cells [9]. As for identifying the death pathways responsible for HMA cytotoxic effect, we and others [9, 10] previously reported that HMA affects cell survival by triggering caspase-independent paradigms of cell death in human cancer cell lines [9, 10], as it occurred also for other amiloride derivatives [11, 12].…”

“…As for identifying the death pathways responsible for HMA cytotoxic effect, we and others [9, 10] previously reported that HMA affects cell survival by triggering caspase-independent paradigms of cell death in human cancer cell lines [9, 10], as it occurred also for other amiloride derivatives [11, 12]. In the present study, we characterised the molecular events regulating HMA cytotoxicity in colon cancer cells, reporting alterations in mitochondrial structure and function triggering ROS (reactive oxygen species) production, DNA damage and poly(ADP-ribose) synthesis.…”