Ester derivatives of salinomycin efficiently eliminate breast cancer cells via ER-stress-induced apoptosis

Article abstract of DOI:10.1016/j.ejphar.2020.173824

The polyether ionophore salinomycin (SAL) has been found to selectively target breast cancer cells, including those with stem-like phenotype. On the other hand, SAL amides and esters obtained through derivatisation of the C1 carboxyl of the ionophore were

Full text of DOI:10.1016/j.ejphar.2020.173824

Journal Pre-proof
Ester derivatives of salinomycin efficiently eliminate breast cancer cells via ER-stress-
induced apoptosis
Dominika Kuran, Sylwia Flis, Michał Antoszczak, Marlena Piskorek, Adam Huczyński
PII:
S0014-2999(20)30929-8
DOI:
https://doi.org/10.1016/j.ejphar.2020.173824
EJP 173824
Reference:
To appear in: European Journal of Pharmacology
Received Date: 21 August 2020
Revised Date: 13 December 2020
Accepted Date: 16 December 2020
Please cite this article as: Kuran, D., Flis, S., Antoszczak, M., Piskorek, M., Huczyński, A., Ester
derivatives of salinomycin efficiently eliminate breast cancer cells via ER-stress-induced apoptosis ,
European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2020.173824.
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CRediT authorship contribution statement
Dominika Kuran: Experiments performing, data analysis, writing – part of the original draft.
Sylwia Flis: Design and performing of the experiments, data analysis, visualization, writing – original
draft, writing – review and editing, funding acquisition.
Marlena Piskorek: Generation of the part of western blot results.
Michał Antoszczak: Investigation, writing – original draft, writing – review and editing, visualization,
funding acquisition.
Adam Huczyński: Resources, writing – review and editing, conceptualization, funding acquisition,
formal analysis, supervision.

Full-length paper
European Journal of Pharmacology
Ester derivatives of salinomycin efficiently eliminate breast cancer cells
via ER-stress-induced apoptosis
Dominika Kuran1, Sylwia Flis1*, Michał Antoszczak2, Marlena Piskorek1,
Adam Huczyński2*
1Department of Pharmacology, National Medicines Institute, Chełmska 30/34, 00‒725
Warsaw, Poland
2Department of Medical Chemistry, Faculty of Chemistry, Adam Mickiewicz University,
Uniwersytetu Poznańskiego 8, 61‒614 Poznań, Poland
Abstract: The polyether ionophore salinomycin (SAL) has been found to selectively target
breast cancer cells, including those with stem-like phenotype. On the other hand, SAL
amides and esters obtained through derivatisation of the C1 carboxyl of the ionophore were
found to exhibit anticancer properties, whilst reducing potential toxicity issues which often
occur during standard chemotherapy. However, the studies on the activity and especially on
the mechanisms of action of this class of semi-synthetic products against breast cancer cells
are very limited. Therefore, in this work, we confirmed the anti-breast cancer activity of SAL,
and further investigated the potential of its selected C1 amide and ester analogs to destroy
breast cancer cells, including the highly aggressive triple-negative MDA-MB-231 cells.
Importantly, SAL esters were found to be more potent than the native structure and their
amide counterparts. Our data revealed that SAL ester derivatives, particularly compounds 5
and 7 (2,2,2-trifluoroethyl and benzotriazole ester of SAL, respectively), increase the level of
p-eIF2α (Ser51) and IRE1α proteins. Additionally, an increased level of DNA damage
indicators such as γH2AX protein and modified guanine (8-oxoG) was observed. These
findings suggest that the apoptosis of MCF-7 and MDA-MB-231 cells induced by the most
promising esters derived from SAL may result from the interaction between ER stress and
DNA damage response mechanisms.
Key words: Breast adenocarcinoma; salinomycin; endoplasmic reticulum stress; DNA
damage; apoptosis
*Correspondence: s.flis@nil.gov.pl (S.F.); adhucz@amu.edu.pl (A.H.)
1

1. Introduction
Breast cancer is the most frequent type of female cancer (Ataollahi et al., 2015),
which is contracted by more than two million women each year throughout the world
(World Health Organization – Cancer, 2020). Moreover, it causes the greatest number of
cancer-related deaths among women (World Health Organization – Cancer, 2020; Shah et
al., 2014), especially in the group of post-menopausal individuals (Akram et al., 2017). In the
last decade, significant progress has been made to develop preventive methods and to
understand breast cancer, which has resulted in more efficient, and less toxic treatment
options for the disease (Sharma et al., 2010); the current ones involve a combination of
various therapies, such as surgery, radiation, hormonal therapy, and/or chemotherapy.
Within the large group of diverse breast cancers, various types could be identified on the
basis of their invasiveness relative to that of the primary tumor sites (Feng et al., 2018),
including triple-negative breast cancer (TNBC) with the poorest prognosis from among all
breast cancer subtypes (Mehanna et al., 2019). TNBC makes up about 10–15% of all
diagnosed breast cancers, and is characterized by a unique molecular profile, very aggressive
nature, significant drug resistance, distinct metastatic patterns, and thus limited options of
effective treatment (Aysola et al., 2013; Dawson et al., 2009). Therefore, although the
mortality rate of breast cancer decreases due to widespread early screenings and advanced
medical therapies (Sun et al., 2017), novel alternative methods with less adverse effects and
a favorable risk-benefit ratio, particularly against TNBC variants, are much in demand.
Salinomycin (SAL, 1, Table 1) is a naturally-occurring polyether ionophore first
isolated from Streptomyces albus, which exhibits a very broad spectrum of bioactivities,
including antibacterial, antiparasitic, antiviral, and also anticancer activity (Antoszczak,
2019a; Antoszczak and Huczyński, 2019; Miyazaki et al., 1974). The significant anticancer
2

properties of SAL were originally identified in the treatment of breast cancer in 2009, when
it showed greater efficacy than the cytostatic drug taxol (paclitaxel) and nearly 16,000 other
compounds used in the screening study, i.a. by selectively targeting a sub-population of
breast cancer stem cells (CSCs) (Gupta et al., 2009). Of note, some preliminary clinical
reports indicated that SAL is well-tolerated by cancer patients when used in appropriate
doses (Naujokat and Steinhart, 2012). Several strategies have been developed to target
specific biomarkers of TNBC or basal-like subtypes (Collignon et al., 2016; Penault-Llorca and
Viale, 2012), and some of the promising therapeutic targets include the Wnt/β-catenin,
Hedgehog and/or NOTCH signaling pathways (O'Toole et al., 2013). Thus, as the effects of
several signal transduction pathways have been invoked to explain the change in phenotype
composition after SAL treatment (Huang et al., 2018), extensive efforts have been made to
explore the anticancer effects of SAL and its derivatives on breast cancer.
Indeed, as far as breast cancer is concerned, SAL was found to be able to induce
growth inhibition, permanent cell cycle arrest, apoptosis and senescence of different human
breast cancer cells, including TNBC variants. SAL could be also applied at a relatively low
concentration for a longer time to overcome drug-resistance of breast cancer cells (Huang et
al., 2018; Al Dhaheri et al., 2013). With respect to its semi-synthetic analogs, selected SAL
amides and esters obtained by the chemical modification of the C1 carboxyl were more
potent than the native structure in cell viability assays against primary acute lymphoblastic
leukemia (ALL) cells (Antoszczak et al., 2018; Urbaniak et al., 2018), as well as identified as
agents that may selectively overcome the resistance to platinum-based drugs in ovarian
cancer (Michalak et al., 2020), which makes them promising candidates for the development
of anticancer drug leads. However, although tertiary amides of SAL have been recently
found to be more potent toward the MDA-MB-231 cell line than the parent structure
3

(Czerwonka et al., 2020), there are only limited studies on the effects of SAL secondary
amides and its ester analogs against breast cancer cells, particularly toward highly aggressive
variants. Furthermore, as the precise anticancer mechanism of action of SAL derivatives is
not fully understood, intensive investigations in this direction are strongly recommended.
We therefore decided to characterize thoroughly the anticancer effects of SAL
analogs obtained via derivatisation of the C1 carboxyl of the ionophore against breast cancer
cells. Given the favorable anticancer potential of SAL, we examined the anti-breast cancer
activity of its semi-synthetic derivatives selected from the group of the most promising C1
amides and esters (Table 1) (Antoszczak, 2019b). Along with the above studies, our aim was
to unravel the mechanism of their anticancer action, with the hope of increasing the efficacy
and reducing potential toxicity issues of the parent biomolecule.
2. Materials and methods
2.1. General procedures
All commercially available reagents and solvents were purchased from Merck
(Germany) or Trimen Chemicals S.A. (Poland), and used in the experiments without further
purification. Detailed description of the general procedures, equipment (NMR and mass
spectrometers), measurement parameters, and software can be found in the Supplementary
material.
2.2. Synthesis of salinomycin derivatives
Salinomycin (SAL) was prepared conveniently by isolation of its sodium salt from the
commercially available veterinary premix SACOX®, followed by the acidic extraction, using
the procedure described by us previously (Huczyński et al., 2012b). The synthesis of the six
4

SAL derivatives investigated in this study, i.e. three C1 amides 2‒4 and three C1 esters 5‒7
(Table 1), is also described elsewhere (Antoszczak et al., 2014a; Antoszczak et al., 2014b;
Huczyński et al., 2012a; Huczyński et al., 2012b). The spectroscopic and spectrometric data
of all these compounds were in good agreement with those found in the reference
literature.
2.2.1. General procedure for preparation of salinomycin amides 2‒4
According to the reference procedures (Antoszczak et al., 2014a; Huczyński et al.,
2012b), to a stirred solution of SAL (1.0 equiv.) in anhydrous CH2Cl2 at 0 °C, the following
reagents were added: DCC (1.5 equiv.), HOBt hydrate (1.1 equiv., dissolved in anhydrous
THF), and a respective amine (1.8 equiv.). The temperature of the reaction mixture was
raised to room temperature, and stirring was continued for 24 h. Then, the solvents were
evaporated under reduced pressure to dryness. The oily residue was suspended in CH2Cl2
and filtered off to remove the formed by-product (DCU). The filtrate was concentrated in
vacuo and purified chromatographically on silica gel using the CombiFlash system to give the
pure products 2‒4 (59‒88% yield) as clear oils. After thrice evaporation to dryness with n-
pentane, the oily products were completely converted into white amorphous solids. The 1H
and 13C NMR spectra of amides 2‒4 can be found in the Supplementary material (Figs. S1–
S6).
Propargyl amide of salinomycin 2: Yield: 138 mg, 88%. Isolated as a white amorphous
solid, >95% pure by NMR and a single spot by TLC. Strains green with PMA; 1H NMR (403
MHz, CD2Cl2) δ 7.07–6.98 (m, 1H), 6.11 (dd, J = 10.7, 2.2 Hz, 1H), 5.96 (dd, J = 10.7, 1.6 Hz,
1H), 4.36 (ddd, J = 17.6, 5.9, 2.5 Hz, 1H), 4.22 (ddd, J = 17.6, 5.3, 2.5 Hz, 1H), 4.13–4.05 (m,
1H), 4.01 (dt, J = 7.8, 1.9 Hz, 1H), 3.90 (ddd, J = 9.6, 5.9, 1.3 Hz, 1H), 3.87–3.76 (m, 2H), 3.73
5

(dd, J = 10.2, 2.6 Hz, 1H), 3.65 (ddd, J = 8.3, 5.3, 1.8 Hz, 2H), 2.97 (dq, J = 9.4, 7.4 Hz, 1H),
2.76–2.61 (m, 2H), 2.61–2.48 (m, 2H), 2.33 (dt, J = 12.6, 10.0 Hz, 1H), 2.22–2.10 (m, 2H),
2.05‒0.50 (m, 52H) ppm; 13C NMR (101 MHz, CD2Cl2) δ 213.0, 175.2, 133.8, 120.7, 106.7,
99.0, 89.1, 82.0, 80.0, 77.3, 75.6, 74.8, 71.3, 71.0, 69.7, 69.3, 67.3, 53.9, 48.0, 46.6, 40.7,
38.7, 37.1, 36.7, 33.0, 31.0, 30.6, 29.5, 28.9, 28.6, 26.9, 26.1, 22.5, 22.3, 20.5, 17.7, 17.3,
15.8, 15.0, 14.7, 14.2, 12.0, 11.4, 8.3, 6.5 ppm; ESI MS (m/z): [M+Na]+ Calcd for
C45H73NNaO10+ 810.5; Found 810.
4-fluorophenethyl amide of salinomycin 3: Yield: 143 mg, 82%. Isolated as a white
amorphous solid, >95% pure by NMR and a single spot by TLC. UV-active and strains green
with PMA; 1H NMR (403 MHz, CD2Cl2) δ 7.36–7.27 (m, 2H), 7.01–6.92 (m, 2H), 6.52 (t, J = 5.8
Hz, 1H), 6.07 (dd, J = 10.7, 2.2 Hz, 1H), 5.95 (dd, J = 10.7, 1.5 Hz, 1H), 4.14 (ddd, J = 9.1, 6.7,
1.3 Hz, 1H), 4.01–3.96 (m, 1H), 3.89–3.74 (m, 3H), 3.74–3.50 (m, 5H), 3.00 (dq, J = 9.5, 7.3 Hz,
1H), 2.93 (d, J = 6.7 Hz, 1H), 2.87 (t, J = 7.6 Hz, 2H), 2.68–2.57 (m, 2H), 2.44 (s, 1H), 2.32 (dt, J
= 12.6, 9.7 Hz, 1H), 2.22–2.13 (m, 1H), 2.05‒0.50 (m, 52H) ppm; 13C NMR (101 MHz, CD2Cl2) δ
213.9, 175.3, 163.0, 160.6, 136.00, 135.97, 133.8, 130.8, 130.7, 120.8, 115.3, 115.1, 106.8,
99.2, 88.7, 79.9, 77.4, 75.8, 74.6, 71.5, 71.0, 69.8, 67.6, 54.8, 49.0, 46.8, 41.2, 40.8, 38.9,
37.1, 36.7, 35.5, 33.3, 31.0, 30.6, 29.5, 28.7, 27.0, 26.0, 22.5, 21.0, 18.5, 17.5, 15.8, 14.9,
14.7, 13.9, 12.1, 11.8, 8.3, 6.5 ppm; ESI MS (m/z): [M+Na]+ Calcd for C50H78FNNaO10+ 894.6;
Found 894.9.
N-benzyl amide of salinomycin 4: Yield: 99 mg, 59%. Isolated as a white amorphous
solid, >95% pure by NMR and a single spot by TLC. UV-active and strains green with PMA; 1H
NMR (403 MHz, CD2Cl2) δ 7.40–7.31 (m, 2H), 7.31–7.23 (m, 2H), 7.23–7.17 (m, 1H), 6.97–
6.91 (m, 1H), 6.08 (dd, J = 10.7, 2.2 Hz, 1H), 5.93 (dd, J = 10.7, 1.5 Hz, 1H), 4.90 (dd, J = 15.3,
7.0 Hz, 1H), 4.60 (dd, J = 15.3, 4.9 Hz, 1H), 4.23–4.17 (m, 1H), 3.99–3.91 (m, 2H), 3.78 (d, J =
6

8.0 Hz, 1H), 3.72 (ddd, J = 10.0, 8.3, 4.5 Hz, 3H), 3.46 (dd, J = 12.1, 2.3 Hz, 1H), 2.99 (ddd, J =
14.7, 9.2, 7.3 Hz, 1H), 2.78–2.69 (m, 2H), 2.58 (dt, J = 8.4, 2.7 Hz, 1H), 2.44 (s, 1H), 2.27 (dt, J
= 12.6, 9.9 Hz, 1H), 2.07 (ddd, J = 12.6, 9.8, 3.0 Hz, 1H), 2.00‒0.50 (m, 52H) ppm; 13C NMR
(101 MHz, CD2Cl2) δ 213.4, 175.6, 140.6, 133.9, 128.5 (2C), 127.8 (2C), 127.0, 120.7, 106.7,
99.1, 88.9, 79.9, 77.2, 75.9, 74.6, 71.4, 70.9, 69.6, 67.4, 54.1, 48.6, 46.6, 43.0, 40.7, 38.8,
37.1, 36.8, 33.1, 31.0, 30.4, 29.6, 28.6, 27.0, 26.2, 22.4, 22.0, 20.7, 18.1, 17.5, 15.8, 14.9,
14.8, 14.3, 12.4, 11.5, 8.4, 6.5 ppm; ESI MS (m/z): [M+Na]+ Calcd for C49H77NNaO10+ 862.5;
Found 862.8.
2.2.2. Synthesis of 2,2,2-trifluoroethyl ester of salinomycin 5
According to the reference procedure (Antoszczak et al., 2014b), to a stirred solution
of SAL (500 mg, 0.66 mmol, 1.0 equiv.) in anhydrous CH2Cl2 the following reagents were
added: DCC (206 mg, 1.00 mmol, 1.5 equiv.), PPy (50 mg, 0.34 mmol, 0.5 equiv.), 2,2,2-
trifluoroethanol (500 mg, 5.00 mmol, 7.5 equiv.) and catalytic PTSA. The reaction mixture
was first stirred at 0 °C for 6 h, and then for further 18 h at room temperature. The organic
solvent was subsequently evaporated under reduced pressure to dryness. The residue was
suspended in CH2Cl2 and filtered off to remove the formed by-product (DCU). Then, the
filtrate was concentrated in vacuo and purified chromatographically on silica gel using the
CombiFlash system to give the pure product 5 (71% yield) as a clear oil. After thrice
evaporation to dryness with n-pentane, the oily product was completely converted into
white amorphous solid. The 1H and 13C NMR spectra of ester 5 can be found in the
Supplementary material (Figs. S7–S8).
Yield: 394 mg, 71%. Isolated as a white amorphous solid, >95% pure by NMR and a
single spot by TLC. Strains green with PMA; 1H NMR (400 MHz, C6D6) δ 6.12 (dd, J = 10.7, 1.5
7

Hz, 1H), 5.78 (dd, J = 10.7, 2.3 Hz, 1H), 5.25 (dq, J = 13.1, 8.9 Hz, 1H), 4.96 (dq, J = 13.1, 8.8
Hz, 1H), 4.31 (dd, J = 10.0, 5.7 Hz, 1H), 4.21 (dt, J = 9.1, 1.9 Hz, 1H), 4.14 (d, J = 9.1 Hz, 1H),
4.06 (dd, J = 10.9, 5.9 Hz, 1H), 3.85–3.72 (m, 2H), 3.69 (dd, J = 9.7, 2.3 Hz, 1H), 3.58 (dd, J =
10.3, 2.4 Hz, 1H), 3.12 (dq, J = 10.0, 7.2 Hz, 1H), 2.94 (td, J = 11.1, 4.0 Hz, 1H), 2.75 (d, J = 5.9
Hz, 1H), 2.62 (dt, J = 9.8, 2.6 Hz, 1H), 2.48 (dt, J = 13.4, 9.8 Hz, 1H), 2.35 (s, 1H), 2.24 (ddq, J =
14.4, 9.7, 7.2 Hz, 1H), 2.11–1.95 (m, 2H), 1.89 (ddd, J = 13.1, 6.7, 4.0 Hz, 1H), 1.80‒0.50 (m,
49H) ppm; 13C NMR (101 MHz, C6D6) δ 212.9, 172.9, 133.5, 120.5, 106.6, 98.9, 88.1, 79.3,
77.0, 74.05, 74.01, 71.8, 70.3, 69.1, 67.5, 60.8, 60.5, 55.3, 48.6, 47.0, 40.4, 38.4, 36.8, 36.6,
34.1, 32.8, 30.9, 30.3, 29.1, 28.1, 26.1, 25.8, 22.5, 22.4, 22.1, 19.6, 18.2, 17.2, 15.6, 14.3,
14.1, 13.9, 13.4, 11.4, 10.9, 7.7, 6.3 ppm, one signal overlapped; ESI MS (m/z): [M+Na]+ Calcd
for C44H71F3NaO11+ 855.5; Found 855.
2.2.3. Synthesis of propargyl ester of salinomycin 6
According to the reference procedure (Antoszczak et al., 2014b), a mixture of SAL
(500 mg, 0.66 mmol, 1.0 equiv.), propargyl bromide (~80% in toluene; 171 mg, 1.45 mmol,
2.2 equiv.), DBU (173 mg, 1.15 mmol, 1.7 equiv.) and anhydrous toluene was heated at 90 °C
for 5 h. After cooling, the mixture was concentrated in vacuo and purified
chromatographically on silica gel using the CombiFlash system to give the pure product 6
(88% yield) as a clear oil. After thrice evaporation to dryness with n-pentane, the oily
product was completely converted into a white amorphous solid. The 1H and 13C NMR
spectra of ester 6 can be found in the Supplementary material (Figs. S9–S10).
Yield: 462 mg, 88%. Isolated as a white amorphous solid, >95% pure by NMR and a
single spot by TLC. Strains green with PMA; 1H NMR (400 MHz, C6D6) δ 6.13 (dd, J = 10.7, 1.3
Hz, 1H), 5.83 (dd, J = 10.8, 2.2 Hz, 1H), 5.36–5.20 (m, 2H), 4.41–4.32 (m, 1H), 4.26–4.12 (m,
8

2H), 4.08 (dd, J = 11.1, 6.0 Hz, 1H), 3.84–3.75 (m, 2H), 3.72 (dd, J = 9.7, 2.3 Hz, 1H), 3.64 (dd, J
= 10.4, 2.4 Hz, 1H), 3.14 (dq, J = 10.1, 7.2 Hz, 1H), 3.00 (d, J = 5.4 Hz, 1H), 2.94 (td, J = 11.2,
4.1 Hz, 1H), 2.66 (dt, J = 9.7, 2.7 Hz, 1H), 2.55–2.43 (m, 1H), 2.28 (ddq, J = 14.4, 9.8, 7.2 Hz,
1H), 2.08 (d, J = 3.1 Hz, 1H), 2.05 (t, J = 2.5 Hz, 1H), 1.90 (dtd, J = 13.5, 6.7, 4.0 Hz, 1H), 1.80‒
0.50 (m, 51H) ppm; 13C NMR (101 MHz, C6D6) δ 212.7, 173.9, 133.3, 120.7, 106.5, 99.0, 87.9,
79.0, 78.5, 77.0, 74.7, 74.3, 74.0, 71.7, 70.3, 69.2, 67.7, 55.9, 52.5, 48.5, 47.3, 40.5, 38.6,
36.8, 36.7, 33.1, 30.9, 30.3, 29.3, 28.1, 26.1, 25.8, 22.7, 22.1, 19.6, 18.4, 17.3, 15.6, 14.3,
14.1, 13.3, 11.6, 10.9, 7.6, 6.3 ppm; ESI MS (m/z): [M+Na]+ Calcd for C45H72NaO11+ 811.5;
Found 811.
2.2.4. Synthesis of benzotriazole ester of salinomycin 7
According to the reference procedure (Huczyński et al., 2012a), a solution of SAL
(1000 mg, 1.33 mmol, 1.0 equiv.) and DCC (418 mg, 2.03 mmol, 1.5 equiv.) in anhydrous
CH2Cl2 as well as HOBt hydrate (197 mg, 1.46 mmol, 1.1 equiv.) dissolved in anhydrous THF
were mixed, and stirred at 0 °C for 1 h. After this time, the reaction mixture was stirred at
room temperature for 48 h. Then, the solvents were evaporated under reduced pressure to
dryness. The oily residue was suspended in CH2Cl2 and filtered off to remove the formed by-
product (DCU). The filtrate was concentrated in vacuo and purified chromatographically on
silica gel using the CombiFlash system to give product 7 (45% yield) as a clear oil. After thrice
evaporation to dryness with n-pentane, the oily product was completely converted into a
white amorphous solid. Pure ester 7 was dissolved in warm ACN, and the solution was
allowed to evaporate at room temperature. After several days, the crystals were formed in
30% yield. The 1H and 13C NMR spectra of ester 7 can be found in the Supplementary
material (Figs. S11–S12).
9

Yield: 520 mg, 45%. Isolated as a white amorphous solid, >95% pure by NMR and a
single spot by TLC. UV-active and strains green with PMA; 1H NMR (600 MHz, CD2Cl2) δ 8.06–
8.00 (m, 1H), 7.66–7.60 (m, 1H), 7.58–7.53 (m, 1H), 7.44–7.38 (m, 1H), 5.99 (dd, J = 10.7, 2.2
Hz, 1H), 5.88 (dd, J = 10.7, 1.2 Hz, 1H), 4.39–4.33 (m, 1H), 4.11 (ddd, J = 9.6, 4.8, 0.9 Hz, 1H),
3.95–3.90 (m, 1H), 3.87 (dd, J = 9.5, 2.3 Hz, 1H), 3.79–3.72 (m, 2H), 3.58–3.48 (m, 3H), 3.03
(dq, J = 9.3, 7.3 Hz, 1H), 2.77 (d, J = 4.4 Hz, 1H), 2.58 (dt, J = 10.2, 2.1 Hz, 1H), 2.46 (s, 1H),
2.27 (dt, J = 12.7, 9.7 Hz, 1H), 2.12 (ddd, J = 13.2, 9.9, 3.7 Hz, 1H), 2.07–1.98 (m, 1H), 1.97‒
0.50 (m, 51H) ppm; 13C NMR (151 MHz, CD2Cl2) δ 213.0, 171.1, 143.8, 133.4, 129.2, 129.1,
125.0, 121.0, 120.4, 109.6, 106.7, 99.2, 88.4, 80.3, 77.5, 74.4 (2C), 72.9, 71.0, 69.8, 67.7,
55.5, 47.9, 46.8, 40.8, 39.0, 36.9, 36.5, 33.2, 31.1, 30.7, 29.4, 29.1, 26.8, 25.9, 23.0, 22.3,
20.9, 18.8, 17.4, 15.7, 14.8, 14.2, 13.5, 12.4, 11.8, 8.3, 6.5 ppm; ESI MS (m/z): [M+Na]+ Calcd
for C48H73N3NaO11+ 890.5; Found 890.8.
2.3. Cell culture and agents treatment
Two human breast cancer cell lines, i.e. MCF-7 (human estrogen-dependent breast
adenocarcinoma) and MDA-MB-231 (human estrogen-independent breast adenocarcinoma),
were used in our studies, together with a normal human HMF cell line (normal breast
fibroblasts). The cells were cultured in appropriate media such as RPMI 1640, Iscove’s
DMEM and MEME, respectively, containing additionally 10% fetal bovine serum (FBS,
BioWest, Cytogen, Poland), 2 mM stabilized L-glutamine (BioWest) and antibiotics: 100 U/ml
sodium penicillin, 100 µg/ml streptomycin and 250 ng/ml amphotericin B (Biowest). All cell
types were incubated at 37 °C in a humidified atmosphere with 5% CO2.
10

Tested compounds were dissolved in 100% dimethylsulfoxide (DMSO, Sigma-Aldrich,
Poland), and then diluted in the media for experiments. The final concentration of DMSO,
without affecting cell survival, was maintained at 0.2%.
2.4. MTT assay
The cells were seeded into 96-well plates at a density of 5 × 103 cells/well. After
treatment with SAL and its six derivatives, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromine (MTT) at the concentration of 5 mg/ml was added at 24, 48,
and 72 h. Then, the cells were incubated for additional 4 h at 37 °C, and next, 180 µl of a
solubilization solution (10% SDS) were added. The mixture was incubated at room
temperature overnight. The product of solubilized formazan was spectrophotometrically
quantified using a microtiter plate reader, Power Wave XS (Bio-Tek, Winooski, VT, USA), at
570 nm wavelength.
2.5. Oxidative DNA damage and cell cycle analysis
The cells (~1.5 × 105) were fixed with 1% paraformaldehyde on ice for 15 min and
washed in phosphate buffered saline (PBS). Then, cells were permeabilinized in 0.1%
PBS/Triton X-100 solution (PBST) for 30 min at room temperature and washed again in
0.01% PBST (230 × g, 5 min). Next, 100 μL of the diluted DNA/RNA damage antibody-FITC
(StresMarq Biosciences Inc., Canada) were added to the cell pellet, and incubated for 1 h at
room temperature in the dark. After cells washing (230 × g, 10 min), the pellet was re-
suspended in propidium iodide (PI)/RNase staining solution (50 µg/ml PI, 100 µg/ml RNase in
0.1% PBST solution). The samples were characterized using a CyFlow Cube 8 (Sysmex,
11

Norderstedt, Germany) flow cytometer. The DNA histograms were analyzed using
FCSExpress 5Flow software (De Novo Software, Glendale, CA, USA).
2.6. Analysis of apoptosis
Apoptosis was measured using annexin-V/fluorescein isothiocyanate (FITC) (BD
Pharmingen, San Jose, California, USA). The staining procedure was conducted according to
the manufacturer’s instructions. The cells were harvested after treatment, washed twice
with PBS, and then centrifuged. The cell pellet was resuspended in ice-cold binding buffer
and annexin-V/FITC was added to the cell suspension. After 15 min of incubation in the dark
at room temperature, the samples were analyzed using a flow cytometer.
Additionally, the cells were seeded into 12-well plates at a density of 7 × 104.
Following exposure to the tested compounds, the cells were double-stained with fluorescein
diacetate (FDA) and PI for 30 min at 37°C. Following two washing procedures in PBS, the
culture medium without phenol red was added. The cell viability was observed under the
ZOE Fluorescent Cell Imager microscope (Bio-Rad, Hercules, CA, USA).
2.7. Clonogenic assay
The cells (500/well) were seeded in 6-well plates and treated with SAL and its analogs
(25 μM) for 24 h. Then, they were washed and allowed to grow for next 8‒10 days in the
growth media. The colonies were fixed with chilled methanol and stained with 0.5% crystal
violet solution for 30 min. The visible colonies were manually calculated in triplicate, and
images of the colonies were scanned.
2.8. Measurement of mitochondrial membrane potential (ΔΨm)
12

The mitochondrial membrane potential (ΔΨm) was measured by flow cytometry using
MitoStatus Red (BD Pharmingen), a cationic lipophilic dye which accumulates within the
mitochondria of healthy cells. The cells were stained according to the manufacturer’s
instructions and examined by flow cytometer. Additionally, the cells seeded into 12-well
plates at a density of 7 × 104 were stained with MitoStatus Red according to the
manufacturer’s instructions, and examined by ZOE Fluorescent Cell Imager (Bio-Rad,
Hercules, CA, USA).
2.9. Reactive oxygen species level analysis
The cells were incubated with redox-sensitive fluorochrome
2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) (Sigma-Aldrich) and analyzed using the
CyFlow Cube 8 (Sysmex, Poland) to detect H2O2 and •OH (Flis et al., 2019).
2.10. Immunoblotting
The cells were washed with cold PBS buffer and total proteins were extracted from
the collected cells using RIPA buffer (25ꢀmM Tris-HCl (pH 7.4), 150ꢀmM NaCl, 0.1% SDS, 0.5%
sodium deoxycholate, 1ꢀmM EDTA, 1% NP-40) and protease inhibitors (Flis et al., 2019). The
samples were fractionated on a 4%–20% TGX™ Precast Protein Gels (Bio-Rad, Poland). Next,
the proteins were transferred onto a nitrocellulose membrane and probed with anti-human
antibodies specific to PARP (#9542), caspase-3 (#9668), IRE1α (#3294), p-eIF2α (Ser51)
(#3398), BiP (#3177), Ero1-Lα (#3264), p-p53 (Ser15) (#9286), p-Rb (Ser807/811) (#8516),
γH2AX (#9718) and β-actin (#8457) (purchased from Cell Signaling Technologies, the
Netherlands). The signals were detected using the enhanced chemiluminescence detection
system (MicroChemi Bio-Imaging Systems, Israel).
13

2.11. Statistical analysis
The data are presented as mean values ± standard deviation (S.D.). Statistical
comparisons between groups were performed by Student’s t-test or one-way analysis of
variance (ANOVA), followed by Student-Newman-Keuls post-hoc test. Significance was
assumed at P<0.05 (marked with asterisks or crosses on graphs).
3. Results
3.1. Salinomycin ester derivatives inhibited cell viability
The first stage of the study was to assess the survival of MCF-7 and MDA-MB-231
breast cancer cells treated with salinomycin (SAL, 1) and its derivatives (2‒7) (Table 1) at
three time intervals of 24, 48, and 72 h (Fig. 1). The tested compounds reduced the
percentage of surviving cancer cells depending on the incubation time and dose used.
Interestingly, both cancer cell lines showed a better response to ester derivatives (5‒7) than
to SAL, which was used as the reference compound. At a concentration of ≥25 µM and after
24 h of incubation, a decrease in cell survival by more than 70% was observed, and the
changes in cancer cells survival induced by SAL derivatives, together with those of the
unmodified ionophore, were statistically significant. Following 48 h and 72 h, the maximal
inhibition of cell growth (90%, P<0.01) was observed for both cell types treated with
products 5‒7 at the concentration of ≥25 µM, whereas for SAL the same effect was observed
at the concentration of 50 µM for MCF-7 and MDA-MB-231 after 48 h and 72 h, respectively.
Our results clearly showed that MDA-MB-231 cells were less sensitive to SAL than MCF-7
cells. As to the amide analogs of SAL, only MDA-MB-231 cells were slightly more sensitive to
the action of compounds 2 and 3 at the concentration of ≥50 µM and ≥25 µM, respectively,
14

and after 24 h treatment, in comparison to SAL. The viability of normal breast fibroblast cells
treated with SAL and its C1 amide and ester analogs was also assessed under the same
experimental conditions as for cancer cells (data not shown).
On the basis of the results obtained from MTT assay for all cell lines, the selectivity
index (SI) indicating the selectivity of the tested compounds towards the cancer cells, was
calculated according to the following formula: SI = IC50(normal cells)/IC50(cancer cells). The values of
the SI parameter depending on the time of incubation are collected in Table 2. The higher
the SI ratio, the less specific a given compound is towards normal cells. In general, SI values
>1.0 indicate some selectivity of the tested compounds, while values of SI >2.0 indicate
highly selective compounds which can be supposed to be potentially safer therapeutic
agents (Segun et al., 2019). However, according to the selectivity criteria, only SAL and three
ester derivatives 5, 6 and 7 could be considered as selective compounds against both MCF-7
and MDA-MB-231 breast cancer cells. Although for amides 2 and 4 also SI >1.0 when applied
to MCF-7 cells, they precipitated in aqueous solution, therefore, only C1 ester derivatives
were tested in the following studies. For further analysis, the tested compounds were used
in the same concentration towards both cancer cell lines, and the established dose was that
at which the lowest IC50 value was obtained for SAL against normal cells, i.e. 25 µM.
3.2. Salinomycin ester derivatives inhibited breast cancer cell proliferation and induced cell
apoptosis
One of the main indicators of cellular stress resulting from the activities of the tested
compounds are changes in cell cycle progression. In this context, Fig. 2A shows changes in
the percentage of cancer cells in particular phases of the cell cycle induced by the presence
of the tested compound, depending on the type of the compound. SAL slightly induced the
15

accumulation of MCF-7 cells in G1 phase, whereas all ester derivatives (5‒7) caused a
significant increase in the number of cells in G1 phase, at the expense of S and G2/M phases.
The observed effects were stronger for MDA-MB-231 cells; both SAL as well as its esters 5‒7
caused the accumulation of cancer cells in G2/M phase with a simultaneous decrease in the
number of cells in G1 and S phases. Moreover, pretreatment of MCF-7 and MDA-MB-231
cells with SAL and 5‒7 gave not only a reduction in cell growth (in terms of cell division
intensity), but also reduced reproductive ability of both breast cancer cell lines. While SAL
induced the loss of colony-forming ability, its C1 ester derivatives, particularly compounds 5
and 7, almost completely diminished the reproductive ability of MCF-7 and MDA-MB-231
cells (Fig. 2B).
As SAL was reported to mediate apoptosis in many human cancer cells, including
breast cancer cells (Antoszczak, 2019a), we were interested to find whether the observed
growth and cell viability inhibition upon the treatment with C1 ester derivatives were also
associated with the induction of apoptosis. For this purpose, Annexin V/PI staining was
performed, followed by flow cytometry. As shown in Fig. 3A, all ester derivatives effectively
increased the percentage of apoptotic cells, and this process was time-dependent. After 48 h
of incubation, the percentage of apoptotic cells was in the range of 45‒90%. Surprisingly, the
response of MDA-MB-231 cells to SAL treatment was even twice higher than that observed
for the corresponding MCF-7 cells.
Microscopic examination also revealed significant changes in the cell morphology
(Fig. 3B). The number of shrinking cells or cells with blebbing membranes was significantly
increased in the SAL ester derivatives-treated group. The advanced level of apoptosis was
also evidenced by a decrease in caspase-3 level, as well as an increase in γH2AX and
poly(ADP-ribose)polymerase (PARP) protein cleavage. Interestingly, treatment of
16

MDA-MB-231 cells with the C1 ester derivatives caused a significant reduction of Rb
phosphorylated at Ser807/811, whereas the changes in the level of p53 phosphorylated at
Ser15 were the opposite in the tested cell lines (Fig. 3C). The latter observation is likely
connected with the genetic status of the TP53 gene, which is mutated in MDA-MB-231 cells.
3.3. Salinomycin ester derivatives induced changes in the mitochondrial membrane potential
(ΔΨm)
To characterize the mechanism of apoptosis induced by SAL esters 5‒7, the
mitochondrial membrane potential (ΔΨm) was measured by MitoStatus Red staining (Fig. 4).
MCF-7 and MDA-MB-231 breast cancer cells revealed morphological changes after
treatment with the tested compounds. The cells shrank and became loosely arranged, and
additionally their adhesion was weakened. The intensity of fluorescence, measured with the
use of a flow cytometer, decreased in MCF-7 cells especially after incubation with analogs 5‒
7 (Figs. 4A and 4B). The observed effects were weaker for MDA-MB-231 cancer cells. A
slight, but statistically significant, decrease in red fluorescence was detected after 48 h of
incubation with compounds 5 and 7. These results suggest that the evaluated C1 ester
derivatives of SAL induce mitochondria-dependent apoptosis and the loss of ΔΨm in MCF-7
cells is more potent than in the corresponding MDA-MB-231 cells.
3.4. Salinomycin ester derivatives induced changes in reactive oxygen species level, DNA
damage and endoplasmic reticulum (ER)-stress
An important consequence of ΔΨm disruption is the enhancement of oxidative stress.
We found that changes in the reactive oxygen species level strongly depend on the type of
compounds tested and the cancer cell lines used. A significantly elevated level of reactive
17

oxygen species was observed in MDA-MB-231 breast cancer cells after 24 h treatment with
esters 5‒7, then the reactive oxygen species level was constant or slightly decreased after
the next 24 h (Fig. 5A). Similar results were observed during the assessment of 8-oxoguanine
(8-oxoG), an indicator of DNA damage caused by reactive oxygen species (Fig. 5B).
Surprisingly, a reversed trend was found for MCF-7 cells, for which a decrease in reactive
oxygen species levels was found to be compound-dependent. Even a four-time higher
decrease in reactive oxygen species was observed for the MCF-7 cells treated with ester 7,
but only marginal changes in the level of 8-oxoG were detected for all compounds.
Nevertheless, in both breast cancer cell lines, changes in the levels of proteins such as eIF2α
phosphorylated at Ser51, BiP and Ero1-Lα were found (Fig. 5C). Additionally, IRE1α level,
which reflects the activation of the IRE1α pathway (Deldicque et al., 2010), increased after
the treatment with C1 ester derivatives when compared with control and SAL (Fig. 5C).
4. Discussion
Although many forms of treatment are available to patients affected by cancer,
chemotherapy is still the most popular and effective option, however, very often it is
burdened with unpleasant and serious side effects. Therefore, the search for new, highly
bioactive compounds that could make the basis of better, more effective therapeutic
strategies with fewer side effects is a top current interest. In this context, various studies
have demonstrated that salinomycin (SAL) shows significant anticancer activity; its semi-
synthetic analogs are thus the objects of intense research interest.
Following this lead, in our work, the anti-breast cancer effects of SAL and its analogs
obtained via derivatisation of the C1 carboxyl of the ionophore were compared; the selected
C1 ester derivatives of SAL were found to be the most promising candidates in this context.
18

Generally, both SAL and its esters reduced the population of breast cancer cells, but the
effectiveness of the semi-synthetic products was higher than that of unmodified SAL on
MCF-7 and MDA-MB-231 cells. The evaluated mechanism of action appeared to be not the
same for all compounds tested as well as for all cell lines used. Nevertheless, SAL ester
derivatives were found to inhibit the growth and viability of human breast cancer cells.
Structural alterations, such as shrinkage and membrane fragmentation, were also observed
after treatment with SAL analogs.
It is known that SAL as an ionophore can disrupt ionic homeostasis in cells, leading to
an increase in intracellular levels of Ca2+ cations. The cells are most sensitive to depletion of
extracellular Ca2+, especially in G1 and at the G1/S and G2/M transitions (Roderick and Cook,
2008). We observed cell cycle arrest in G1 and G2/M phases in MCF-7 and MDA-MB-231
cells, respectively. The Rb and p53 proteins, which act as tumor suppressors, are key
regulators of cell proliferation by controlling progression through the restriction points of
the cell cycle. Treatment of the cells with the evaluated compounds resulted in the
abrogation of Rb phosphorylation at Ser807/811, and as a consequence, the cell cycle arrest.
Additionally, an increased level of p53 phosphorylation at Ser15 was observed in MCF-7
cells. Phosphorylation of p53 at this site is crucial for its stabilization and is essential for the
induction of cell cycle arrest. However, in response to severe DNA damage and/or ER stress,
p53 induces apoptosis (Lin et al., 2012; Zhao et al., 2008). With respect to the MDA-MB-231
cells, inhibition of cell cycle progression in the G2/M phase is p53-independent, and may be
due to alterations in other components in the pathway controlling this checkpoint.
Generally, apoptosis may be induced either by sustained Ca2+ influx via activated
channels or by the release of calcium by a stressed endoplasmic reticulum (ER) (Kaushik et
al., 2018). The latter phenomenon seems to be responsible for the induction of apoptosis in
19

our study. As a response to prolonged ER stress conditions, the unfolded protein response
(UPR) signal transduction cascade is directly activated to counteract the occurring damage,
but when ER stress is not reduced/softened, the UPR triggers apoptosis (Siwecka et al.,
2019). We observed an increased level of IRE1α protein, the one from three main sensor
proteins that recognize ER stress, in breast cancer cells after treatment with C1 ester analogs
of SAL. Upon accumulation of misfolded proteins in ER, BiP protein, the main ER stress
marker, gets released and allows oligomerization of IRE1α and protein kinase-like ER kinase
(PERK). The western blot analysis of UPR-associated proteins revealed that the levels of BiP
and Ero1-Lα (especially in MCF-7 cells) were increased after treatment with C1 esters of SAL.
On the other hand, the level of pospho-eIF2α (Ser51) protein, the main downstream effector
of the PERK-dependent UPR signaling pathway (the second sensor protein), was also
increased after treatment with C1 ester analogs in MDA-MB-231 cells, and to a much lesser
degree in MCF-7 cells. However, the level of phosho-eIF2α was strongly elevated in both
breast cancer cell lines treated with SAL. Similar results have been obtained recently by
Zhang et al. (2019) who showed that SAL treatment triggered ER stress by the PERK sensor in
PC-3 prostate cancer cells.
The final result of the treatment of evaluated cell lines with SAL ester derivatives,
especially compounds 5 and 7, was the induction of cell death confirmed by flow cytometry
analysis using annexin V/PI staining. The advanced level of apoptosis was also demonstrated
by a decrease in the level of caspase-3, a cysteine protease belonging to the group of
executive caspases degrading cellular proteins, indirectly contributing to the degradation of
genetic material as well as PARP protein, which plays an important role in the DNA repair
processes; its proteolytic degradation was also confirmed.
20

Of note is that the SAL C1 ester derivatives induced mitochondrial dysfunction,
especially in MCF-7 breast cancer cells. Often, an important consequence of disruption of
the ΔΨm is an increment in oxidative stress. With respect to the MCF-7 cells, the decrease in
mitochondrial potential was accompanied by a decrease in the level of reactive oxygen
species and oxidative DNA damage (measured by 8-oxoG, the indicator of oxidative DNA
damage). The changes in the mitochondrial potential were not highlighted very well in the
corresponding MDA-MB-231 cells (except for unmodified SAL), although the concomitant
increase in the reactive oxygen species and 8-oxoG levels were observed. These
observations agree with the concept that higher (more polarized) ΔΨm is associated with
greater reactive oxygen species production by mitochondria (Li et al., 2013). Nevertheless,
both mechanisms can be beneficial in the treatment strategy of cancer patients.
A characteristic feature of cancer cells is the generation of high levels of reactive
oxygen species and the accumulation of genomic mutations under increased oxidative
stress. Therefore, lowering the reactive oxygen species level in cancer cells may be
associated with the inhibition of genetic instability and disease progress. On the other hand,
pro-oxidizing agents, such as SAL (Managò et al., 2015) and its derivatives, may in some
circumstances raise the level of reactive oxygen species in cancer cells above the tolerated
threshold. A consequence is the generation of DNA lesions because of an 8-oxoG production
(as a result of guanine oxidation). Insertion of 8-oxoG during replication generates DNA
double-strand breaks (DSBs), which affects genome integrity and can activate cell death
processes. The results that have been obtained by Tyagi and Patro (2019) showed an
approximately 1.5-fold increase in γH2AX in MCF-7DDP cells (cisplatin-resistant variant) after
24 h of treatment with SAL. We also observed an elevated level of γH2AX after treatment
with SAL, but importantly, this effect was more enhanced in the cells treated with the C1
21

ester derivatives of the ionophore. Often, γH2AX is considered only as a DSBs marker, but it
should be noticed that it can also play a pivotal role during apoptosis, as one of the factors
involved in apoptotic DNA fragmentation.
5. Conclusions
To sum up, our studies clearly confirmed the findings of other authors demonstrating
significant anti-breast cancer effects of salinomycin (SAL). Furthermore, we also identified a
cohort of promising SAL derivatives with enhanced activity against breast cancer cells. Of
note is that the breast cancer cells demonstrated increased sensitivity to the action of
compounds derived from SAL when compared to normal mammary fibroblasts. Moreover,
our results showed that the ester analogs obtained through derivatisation of the C1 carboxyl
of SAL, especially compounds 5 and 7 (2,2,2-trifluoroethyl and benzotriazole ester of SAL,
respectively), exhibit a more efficient ability to eliminate triple-negative breast cancer
(TNBC) cells (MDA-MB-231 cell line) than both the unmodified SAL and its C1 amide
counterparts. This observation is essential, because TNBC is an aggressive form of breast
cancer with limited treatment options. On the other hand, there was no denial regarding the
different response (signaling) across the two breast cancer cell lines, and it was independent
from the rate of death; it may be explained by the mutational background of the cell lines. In
this context, SAL derivatives, like ester 7, may be beneficial if treatment is given based on
genetic status (targeted approach).
The chemical modification of SAL biomolecule seems to be thus an extremely
interesting direction of research, as it may lead to the discovery of candidates for new
anticancer agents. Our results showed that SAL esters 5 and 7 should be recognized as the
lead structures for further investigation, including in vivo studies.
22

Acknowledgements
The research was financed from the statutory activity of the National Medicines
Institute. M.A. wishes to acknowledge the Polish Science Centre (NCN) for financial support
by a grant SONATA (2016/23/D/ST5/00242). M.A. wishes also to acknowledge the NCN and
the Polish National Agency for Academic Exchange (NAWA) for the scholarships under the
UWERTURA (2019/32/U/ST4/00092) and the BEKKER programme (PPN/BEK/2019/1/00034),
respectively, and the Polish Ministry of Science and Higher Education (MNiSW) for the
scholarship for outstanding young scientists in the years 2020‒2023 (STYP/15/1665/E-
336/2020).
CRediT authorship contribution statement
Dominika Kuran: Experiments performing, data analysis, writing – part of the original draft.
Sylwia Flis: Design and performing of the experiments, data analysis, visualization, writing –
original draft, writing – review and editing, funding acquisition.
Michał Antoszczak: Investigation, writing – original draft, writing – review and editing,
visualization, funding acquisition.
Marlena Piskorek: Generation of the part of western blot results.
Adam Huczyński: Resources, writing – review and editing, conceptualization, funding
acquisition, formal analysis, supervision.
Declaration of competing interests
23

The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
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