Page 85 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

SDIRSACR Oncology Insights

L25

Targeting Multidrug Resistance and Cancer Stem Cells with Ionophores

Marijeta Kralj , Marija Mioč , Marko Marjanović , Ana-Matea Mikecin Dražić , Mladen Paradžik 1
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1 Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička c. 54, 10000 Zagreb, Croatia
Keywords: Cancer stem cells, epithelial-to-mesenchymal transition, Golgi apparatus, Ionophores, multidrug resistance,
salinomycin

Background: Cancer stem cell (CSC) populations are considered among the most therapy-resistant tumor cell populations
and are believed to be key drivers of cancer relapse. Their resistance to chemo- and radiotherapy arises from several
mechanisms. One such mechanism is epithelial-to-mesenchymal transition (EMT), a cell reprogramming process
during which epithelial cells lose their polarity and adhesion properties and acquire mesenchymal traits, including
motility. Epithelial carcinoma cells that have undergone EMT display CSC-like characteristics—such as invasiveness,
drug resistance, and the ability to initiate metastases—thereby contributing to cancer progression and relapse [1].
Targeting the EMT program to eliminate CSCs selectively is thus a promising strategy to enhance cancer treatment
efficacy.
It has been demonstrated that induction of EMT in immortalized human mammary epithelial cells (HMLEs) leads to
the expression of stem cell markers and acquisition of the breast CSC phenotype. As such, HMLEs represent a robust
experimental model of CSCs. High-throughput screening using this model identified several compounds with selective
activity toward EMT/CSCs. The most potent was salinomycin (Sal), a naturally occurring K⁺/H⁺ ionophore widely used
as a coccidiostat, followed by nigericin (Nig), another ionophore with similar selectivity [2]. Salinomycin has since been
shown to be highly effective and selective against CSCs across multiple cancer types [3]. Despite numerous proposed
mechanisms of action, the basis of its selectivity remains incompletely understood.
Ionophores are characterized by their ability to transport ions across lipid membranes, thereby disturbing membrane
potential and ion homeostasis, which leads to physiological and osmotic stress. Consequently, natural ionophores (e.g.,
Sal, Nig, valinomycin) have been used as antibiotics and are being increasingly investigated in cancer treatment [4].
The EMT/CSC phenotype is often associated with overexpression of ATP-binding cassette (ABC) transporters, including
ABCG2 and P-glycoprotein (P-gp), which contribute to multidrug resistance (MDR) by preventing the intracellular
accumulation of chemotherapeutic agents. Therefore, modulation of ABC transporter activity or expression is of great
clinical importance for overcoming MDR and improving the efficacy of anticancer therapies [5] Previous studies have
shown that some well-known K⁺/H⁺ ionophores inhibit P-gp and sensitize resistant leukemia cells to chemotherapeutics
such as adriamycin, docetaxel, and vinblastine [6]. However, their effects on ABCG2 activity have not been systematically
addressed [4].
Our group has extensively studied the biological and potential antitumor activity of various compounds, particularly
focusing on CSC selectivity. These include ion transport-disrupting agents such as crown ether compounds and natural
ionophores (e.g., Sal, monensin (Mon), and Nig). In our recent work, we investigated the mechanisms underlying the
CSC/EMT-selective activity of ionophores.
Materials and Methods: Human myeloid leukemia cell line PLB-985/ABCG2 and Madin-Darby canine kidney cells type
II (MDCKII/ABCG2), both overexpressing human ABCG2, as well as their parental counterparts (PLB-985 and MDCKII,
respectively), were used as model systems. A range of functional assays was employed to study the interaction of the
ionophores salinomycin, nigericin, and monensin with the ABCG2 transporter. Inhibitory activity on ABCG2 function
was assessed using the Pheophorbide A (PhA) efflux assay or the Hoechst 33342 accumulation assay in MDCKII/ABCG2
and PLB-985/ABCG2 cell lines.[7]
Additionally, a mammary CSC model generated by EMT induction in immortalized human mammary epithelial (HMLE)
cells was used. These included HMLE cells expressing Twist or an empty vector (HMLE-Twist and HMLE-pBp), as well
as HMLE cells with shRNA targeting E-cadherin or GFP (HMLE-shEcad and HMLE-shGFP). Other breast cancer cell lines,
such as MCF-7 and SUM159, were also included [2],[8].
Cell proliferation was assessed using the MTT assay and flow cytometry. Protein expression was evaluated by Western
blotting and flow cytometry. Cell morphology and specific protein localization were analyzed using confocal microscopy.
Global gene expression profiling (RNA-Seq) was performed using the NextSeq500 platform (Illumina). Sequence reads
were uploaded to the BaseSpace Sequence Hub (Illumina) and aligned to the human reference genome (hg19) using
the RNA-Seq Alignment app. Differential gene expression after treatment, for each cell line and between EMT and non-
EMT cells, was analyzed using the DESeq2 app. The N-glycome of secreted proteins was analyzed by mass spectrometry.
Results: We found that Sal, Mon, and Nig do not inhibit ABCG2 activity, suggesting that their CSC selectivity arises

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