The Cationic Amphiphilic Drug Hexamethylene Amiloride Eradicates Bulk Breast Cancer Cells and Therapy-Resistant Subpopulations with Similar Efficiencies

Abstract

The resistance of cancer cell subpopulations, including cancer stem cell (CSC) populations, to apoptosis-inducing chemotherapeutic agents is a key barrier to improved outcomes for cancer patients. The cationic amphiphilic drug hexamethylene amiloride (HMA) has been previously demonstrated to efficiently kill bulk breast cancer cells independent of tumor subtype or species but acts poorly toward non-transformed cells derived from multiple tissues. Here, we demonstrate that HMA is similarly cytotoxic toward breast CSC-related subpopulations that are resistant to conventional chemotherapeutic agents, but poorly cytotoxic toward normal mammary stem cells. HMA inhibits the sphere-forming capacity of FACS-sorted human and mouse mammary CSC-related cells in vitro, specifically kills tumor but not normal mammary organoids ex vivo, and inhibits metastatic outgrowth in vivo, consistent with CSC suppression. Moreover, HMA inhibits viability and sphere formation by lung, colon, pancreatic, brain, liver, prostate, and bladder tumor cell lines, suggesting that its effects may be applicable to multiple malignancies. Our observations expose a key vulnerability intrinsic to cancer stem cells and point to novel strategies for the exploitation of cationic amphiphilic drugs in cancer treatment.

Saponin and Ribosome-Inactivating Protein Synergistically Trigger Lysosome-Dependent Apoptosis by Inhibiting Lysophagy: Potential to Become a New Antitumor Strategy

“…Lysosomes secrete proteases to degrade extracellular matrix components and promote tumor invasion, angiogenesis, and metastasis . Therefore, more and more studies are trying to kill tumor cells by inducing LDCD, developing it as another promising antitumor therapy. − …”

“…However, the pharmacological effects of CADs at low concentrations are insufficient to cause LDCD because cells have a robust set of lysosomal repair mechanisms. For instance, hexamethylene amiloride significantly inhibits mouse breast tumor tissue only at concentrations higher than 40 μM, whereas in an in vivo model of metastatic breast tumor in mice, it needs to be injected at a dose of 30 mg/kg . LLC1, a structurally optimized amiloride derivative, still exhibits a high EC 50 of nearly 20 μM against MCF-7 chemoresistant cells .…”

Structure-Activity Relationship Study Identifies a Novel Lipophilic Amiloride Derivative that Efficiently Kills Chemoresistant Breast Cancer Cells

“…HMA efficiently kills bulk breast tumor cells independent of tumor subtype, proliferation state or species of origin, but does not efficiently kill non-transformed cells derived from a variety of tissues at the same concentrations. Indeed, cell lines derived from diverse tumor types are equally susceptible to HMA, suggesting that its mechanism of cytotoxic action may be dependent on cellular transformation rather than patient-specific genetic alterations [17,18]. Moreover, HMA is cytotoxic toward breast cancer cell populations that are resistant to currently-employed therapies, including the highly refractory cancer stem cell population.…”

“…Moreover, HMA is cytotoxic toward breast cancer cell populations that are resistant to currently-employed therapies, including the highly refractory cancer stem cell population. Finally, HMA induces morphological changes within the lysosomes of tumor cells, and cytotoxicity is rescued by an inhibitor of the lysosomal protease cathepsin [17,18], pointing a central role for the lysosome in its mechanism of action.…”

“…While our previous in vitro studies have revealed many attractive properties of amiloride derivatives, and suggest that derivatization of the C(5) position of its pyrazine ring can markedly enhance the tumor-selective cytotoxic properties of the drug, further translation of HMA is hampered by its relatively modest potency [17,18] and poor pharmacokinetic properties in mice [19]. In this study we aimed to glean further insight into structure-activity relationships (SARs) within the amiloride pharmacophore by comparing the cytotoxic potential of derivatives modified at the C(2) and C(5) positions.…”