Chloroacridine derivatives as potential anticancer agents which may act as tricarboxylic acid cycle enzyme inhibitors

Article abstract of DOI:10.1016/j.biopha.2020.110515

Purpose: This paper concerns the cytotoxicity of 9-chloro-1-nitroacridine (1a) and 9-chloro-4-methyl-1-nitroacridine (1b) against two biologically different melanoma forms: melanotic and amelanotic. Melanomas are tumors characterized by high heterogeneity

Full text of DOI:10.1016/j.biopha.2020.110515

Biomedicine&Pharmacotherapy130(2020)110515
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Original article
Chloroacridine derivatives as potential anticancer agents which may act as
tricarboxylic acid cycle enzyme inhibitors
Miroslawa Cichoreka,*, Anna Ronowskab, Krystyna Dzierzbickac, Monika Gensicka-Kowalewskac,
Milena Deptulaa, Iwona Pelikant-Maleckad,e
a Department of Embryology, Medical University of Gdansk, Debinki 1 St. PL, 80-210, Gdansk, Poland
b Department of Laboratory Medicine, Medical University of Gdansk, Debinki 7 St. PL, 80-211, Gdansk, Poland
c Department of Organic Chemistry, Gdansk University of Technology, Narutowicza St. 11/12. PL, 80-233, Gdansk, Poland
d Department of Biochemistry, Medical University of Gdansk, Debinki 1 St. PL, 80-210, Gdansk, Poland
e Department of Medical Laboratory Diagnostics, Central Bank of Frozen Tissues and Genetic Specimens, Medical University of Gdansk, Biobanking and Biomolecular
Resources Research Infrastructure Poland, Debinki 7 St. PL, 80-211, Gdansk, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chloroacridine
Purpose: This paper concerns the cytotoxicity of 9-chloro-1-nitroacridine (1a) and 9-chloro-4-methyl-1-ni-
troacridine (1b) against two biologically different melanoma forms: melanotic and amelanotic. Melanomas are
tumors characterized by high heterogeneity and poor susceptibility to chemotherapies. Among new analogs
synthesized by us, compound 1b exhibited the highest anticancer potency. Because of that, in this study, we
analyzed the mechanism of action for 1a and its 4-methylated derivative, 1b, against a pair of biological mel-
anoma forms, with regard to proliferation, cell death mechanism and energetic state.
Methods: Cytotoxicity was evaluated by XTT assay. Cell death was estimated by plasma membrane structure
changes (phosphatidylserine externalization), caspase activation, and ROS presence. The energetic state of cells
was estimated based on NAD and ATP levels, and the activity of tricarboxylic acid cycle enzymes (pyruvate
dehydrogenase complex, aconitase, isocitrate dehydrogenase).
Amelanotic melanoma
Melanotic melanoma
Apoptosis
Tricarboxylic acid cycle enzymes
Cell death
Results: The chloroacridines affect biological forms of melanoma in different ways. Amelanotic (Ab) melanoma
(with inhibited melanogenesis and higher malignancy) was particularly sensitive to the action of the chlor-
oacridines. The Ab melanoma cells died through apoptosis and through death without caspase activation.
Diminished activity of TAC enzymes was noticed among Ab melanoma cells together with ATP/NAD depletion,
especially in the case of 1b.
Conclusion: Our data show that the biological forms of the tumors responded to 1a and its 4-methylated analog
in different ways. 1a and 1b could be inducers of regulated melanoma cell death, especially the amelanotic form.
Although the mechanism of the cell death is not fully understood, 1b may act by interfering with the TAC
enzymes and blocking specific pathways leading to tumor growth. This could encourage further investigation of
its anticancer activity, especially against the amelanotic form of melanoma.
1. Introduction
(skin, eye, mucous membranes) and histological analysis (superficial
spreading, lentigo maligna, nodular, acral lentiginous, desmoplastic,
In the search for a cure for melanoma, new methods of early diag-
nosis, surgical excision and new adjuvant therapies have produced
more positive responses than in earlier years [1–4]. Unfortunately, a
large number of melanoma patients develop drug resistance during
their treatment [5–8]. Therefore, new compounds that would enhance
the effects of melanoma treatment are highly desirable. Melanomas are
molecularly heterogeneous neoplasm. Melanoma has been classified
into subtypes based on the tissue from which the primary tumor arises
amelanotic) [7,9,10]. Despite significant progress in the understanding
of the molecular alterations in melanomas, the degree of melanoma
molecular heterogeneity that is associated with its histologic hetero-
geneity is still unknown [10–12]. The striking features of melanoma as
high genetic instability, cell plasticity in response to perceived changes
in the microenvironment, dedifferentiation to a variety of states under
cellular stress, drives therapy resistance [13–16]. Melanoma is a highly
aggressive solid tumor, showing also an impressive metabolic plasticity
⁎ Corresponding author.
E-mail address: miroslawa.cichorek@gumed.edu.pl (M. Cichorek).
https://doi.org/10.1016/j.biopha.2020.110515
Received 13 March 2020; Received in revised form 3 July 2020; Accepted 7 July 2020
0753-3322/©2020TheAuthor(s).PublishedbyElsevierMassonSAS.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/BY/4.0/).

M. Cichorek, et al.
Biomedicine&Pharmacotherapy130(2020)110515
modulated by oncogenic activation [17,18]. The possible switch be-
tween diverse phenotypic states creates a plethora of opportunities for
melanoma cells to escape the treatment [19–21]. Heterogeneity and
plasticity of melanoma cells are well recognized as causative factors of
chemoresistance [8,15,16,19,22,23].
2. Material and methods
2.1. Transplantable melanomas – Bomirski hamster melanoma model
The original transplantable melanotic melanoma (Ma) was derived
from a spontaneous melanoma of the skin that had appeared in a breed
of golden hamster [50]. The amelanotic melanoma line (Ab) originated
from the Ma form by a spontaneous alteration. The loss of melanin was
accompanied by changes in many biological features of the Ab line –
faster tumor growth rate, shorter animal survival, and changes in cell
ultrastructure [50,51]. Since their discovery, each melanoma line has
been maintained in vivo by consecutive, subcutaneous transplantations
of tumor material every 21 (Ma) or 11 (Ab) days. The hamsters were
injected with a suspension of melanoma tissue obtained by mincing in a
glass homogenizer. The tumor tissue was injected subcutaneously into
the flank region in an amount of 200 mg of Ma per one hamster and 50
mg of Ab melanoma per one hamster. Differences in the quantity of
transplanted tumors and time of getting animals for experiments were
adequate to the known rate of growth of these two melanoma lines
[50].
Pathobiological characteristic of Bomirski hamster melanoma
model is described in the Supplementary material (S1. Pathobiological
BHM model characteristic).
All experiments were conducted in accordance with a Guide for the
Care and Use of Laboratory Animals published by the European
Parliament, Directive 2010/63/EU and were performed with approval
of the Animal Ethics Committee at the Medical University of Gdansk
(24/2015).
Based on cell’s ability to synthesize melanin, melanomas can also be
divided into two biologically distinct forms: melanotic and amelanotic/
hypomelanotic [24–26]. Amelanotic melanoma is very rare, making up
2–4 % of all melanomas [24,27], though some analyses report this
figure to be as high as 20 % [26]. Cells of this melanoma type lack or
show a decreased level of the melanogenesis process, and as a con-
sequence the melanoma becomes nearly white in color [24,26,28].
According to the presence (melanotic melanoma) or absence (amela-
notic melanoma) of melanogenesis, cell sensitivity to cure method may
differ, however the level of melanoma cell pigmentation and the best
method of treatment still remains a controversial subject [28–32].
Acridines are heteroaromatic compounds with a wide range of
biological activities as anticancer, anti-inflammatory, antimicrobial,
antiparasitic, antiviral and fungicidal agents [33,34]. Acridines are
capable to bind nucleic acids by hydrophobic interactions namely in-
tercalation, i.e. auto-insertion between two adjacent base pairs of nu-
cleic acids that influence many cell processes [35–37]. However, the
more recent studies favor an explanation based on direct interaction of
acridines with biologically important proteins [33,38,39]. Thus in re-
ference to these properties, acridines are explored as potential anti-
cancer compounds [38,33,39].
Our previous studies indicated that the Ab amelanotic melanoma of
the Bomirski hamster melanoma model is particularly sensitive to a new
acridine-retro-tuftsin compound, when compared to the melanotic Ma
line of the same model [40,41]. Having considered that, in this paper
we present antimelanoma research with 9-chloroacridine and its me-
thylated derivative 9-chloro-4-methyl-1-nitroacridine (Fig.1), the pre-
paration of which is one of the steps for the synthesis of acridines by the
Ullmann condensation method. According to our knowledge, the an-
ticancer activity of these compounds has not been studied before al-
though the 9-chloroacridines are the common precursors for obtaining
new acridine derivatives at position 9 [33].
2.2. Isolation of melanotic and amelanotic melanoma cells
Melanoma cells were isolated for each experiment from solid tumors
by a non-enzymatic, mechanical dissociation of tissue (taken at the time
of routine passages). The melanoma cells were isolated by means of a
density gradient of Histopaque-1077 (Sigma-Aldrich, USA) [52]. The
suspension consisted of 95–98 % viable cells (estimated by trypan blue
test).
The most commonly used method of acridine synthesis is the cy-
clization of N-phenylanthranilic acid derivatives obtained as a result of
Ullmann condensation. This reaction generates by-products (probably
products of chlorination of other acridine core positions), which were
separated by TLC. The 9-chloroacridine derivatives were further iso-
lated and purified by usual techniques [42–48].
Among new analogs synthesized by us, the compound 9-chloro-4-
methyl-1-nitroacridine (1b, Fig.1) exhibited the highest anticancer
potency. C4-methyl substituents significantly affect thermodynamic
mechanisms associated with complex formation and molecular inter-
actions between the ligand and its DNA binding site and as such could
influence the biological activity [49]. In this study, we analyzed the
mechanism of action of 9-chloro-1-nitroacridine and its 4-methylated
derivative against a pair of biological forms of melanoma (melanotic,
amelanotic), in terms of proliferation, cell death mechanism, and en-
ergetic state.
2.3. Synthesis of acridine compounds 9-chloro-1-nitroacridine (1a) and 9-
chloro-4-methyl-1-nitroacridine (1b) (Fig. 1)
The preparation of 9-chloro-1-nitroacridine was done by an
Ullmann condensation reaction of the potassium salt of o-chlorobenzoic
acid 2 and m-nitroaniline 3a in the presence of copper at 125 °C to give
N-(3′-nitrophenyl)anthranilic acid 4a, which was refluxed in POCl3 to
afford the 9-chloroacridine derivative 1a [45–47,53,54]. 9-Chloro-4-
methyl-1-nitroacridine 1b was synthesized in two steps: Ullmann re-
action of o-chlorobenzoic acid with 3-nitro-6-methylaniline in the
presence of anhydrous potassium carbonate and copper(II), cyclization
of N-(2′-methyl-5′-nitrophenyl)anthranilic acid 4b to derivative 1b
(Fig. 2) [41]. The final products 1a,b was purified with preparative TLC
and its identity was confirmed by high resolution 1H NMR (500 MHz),
13C NMR (125 MHz) and MS mass spectrometry analysis (MALDI-TOF
MS, Biflex III Bruker).
The general preparation of 1-nitro-9-chloroacridine (1a) and 9-
chloro-4-methyl-1-nitrocridine (1b) was described in literature
[43,45,46,48].
o-Chlorobenzoic acid (127.7 mmol) was dissolved in 1 L of water
and added in portions of KOH (about 139 mmol) to pH = 7. The water
was evaporated. A mixture of potassium salt of o-chlorobenzoic acid
(62.5 mmol), m-nitroaniline or 3-nitro-6-methylaniline (187.5 mmol)
and freshly precipitated copper dust (1.2 mmola) was heated at 125 °C
for 1 h. Then, the hot melt was poured into 0.3 ml of intensely stirring
hot 5% potassium carbonate solution. The mixture was heated to reflux
with the addition of activated carbon, and filtered. After cooling, the
precipitated excess of m-nitroaniline or 3-nitro-6-methylaniline was
Fig. 1. Structure of 9-chloro-1-nitroacridine 1a and 9-chloro-4-methyl-1-ni-
troacridine 1b.
2

M. Cichorek, et al.
Biomedicine&Pharmacotherapy130(2020)110515
Fig. 2. Synthesis of 9-chloro-1-nitroacridine 1a and 9-chloro-4-methyl-1-nitroacridine 1b.
filtered off. The filtrate was acidified with conc. HCl up to pH = 5−6.
The precipitate was filtered. The crude product was crystallized from
ethanol, obtaining N-(3′-nitrophenyl)anthranilic acid or N-(2′-methyl-
5′-nitrophenyl)anthranilic acid. Then, a mixture of N-(3′-nitrophenyl)
anthranilic acid derivatives (26.6 mmol) and of POCl3 (266 mmol) was
heated at 120 °C. After 20 min the excess of POCl3 was distilled off. To
the residue was added a small amount of chloroform, then the solution
was poured into a mixture of NH3:CH3Cl (1:3) cooled to −70 °C. The
organic layer was separated and dried with anhydrous MgSO4. After
filtration and distillation of the solvent, pyridine was added and the
precipitate was filtered off. Then, the filtrate was heated at 60 °C for 20
min. After cooling, a 9-chloro-1-nitroacridine precipitate was obtained,
melting point 150−151 °C [54].
for Ab melanoma and 500 × 103 for Ma melanoma, incubated with 1a
and 1b at 100 μM for Ma cells and at 15 μM for Ab cells, for 48 and 72
h. For the analysis in the flow cytometer 1 × 106 cells were stained
with antibodies conjugated with fluorochrome, or the cells were in-
cubated with fluorogenic substrate. After incubation the cells were
analyzed using
a
C6 flow cytometer (Becton Dickinson
Immunocytometry Systems, USA). After gating out small-sized (e.g.
noncellular debris) objects, 10,000 events were collected from each
sample. Results were analyzed off-line using Cyflogic v.1.2.1 software.
2.6. Activated caspases
9-Chloro-1-nitroacridine 1a, Yield 84 %, yellow product; 1H NMR
(500 MHz, DMSO-d6) δ ppm: 6.82 (m, 1H, 7), 7.37 (m, 2H, 6, 3), 8.21
(m, 2H, 2, 5), 8.40 (m, 2H, 4, 8); 13C NMR (500 MHz, CD3OD) δ ppm:
148.94 (C-9), 148.15 (C-1), 146.37 (C-4a), 137.25 (C-5a), 134.51 (C-6),
132.97 (C-2), 129.98 (C-8), 129.87 (C-7), 129.44 (C-4), 125.11 (C-5),
124.94 (C-8a), 124.50 (C-3), 115.16 (C-1a). MS m/z calculated for
We used a FLICA test (fluorochrome-labeled inhibitors of caspases)
to estimate cells containing activated caspases [55]. The rationale for
this method is that fluorochrome-labeled inhibitors of caspases cova-
lently react with the reactive enzymatic center of activated caspases.
We used a FITC (fluorescein) labeled pan-inhibitor of caspases, VAD-
FMK, which detects most active caspases in the cell. After 48 and 72 h
incubation with the acridines the cells were collected and 1 × 106 cells
from each experimental point were incubated with 5 μM FITC-VAD-
FMK (CaspACETM FITC-VAD-FMK, Promega, USA) for 30 min at room
temperature in the dark. The cells were analyzed using a flow cyt-
ometer.
C
13H7ClN2O2 258.66 found 260.31 [M+H]+
.
9-Chloro-4-methyl-1-nitroacridine 1b, Yield 82 %, yellow product; 1
H
NMR (500 MHz, DMSO-d6) δ ppm: 2.52 (s, 3H, Ar−CH3), 6.82 (m, 1H,
7), 7.37 (m, 2H, 6, 3), 8.21 (m, 2H, 2, 5), 8.40 (m, 2H, 4, 8); 13C NMR
(500 MHz, CD3OD) δ ppm: 148.94 (C-9), 148.15 (C-1), 146.37 (C-4a),
137.25 (C-5a), 134.51 (C-6), 132.97 (C-2), 129.98 (C-8), 129.87 (C-7),
129.44 (C-4), 125.11 (C-5), 124.94 (C-8a), 124.50 (C-3), 115.16 (C-1a).
MS m/z calculated for C14H9ClN2O2 272.68; found 272.43 [M+H]+
.
2.7. Phosphatidylserine (PS) externalization assay
An early apoptotic change of the plasma membrane structure, PS
externalization, was determined by Annexin V-FITC and propidium
iodide (PI) staining (BD Pharmingen, USA) according to the manu-
facturer’s instructions. The staining allows populations of cells to be
determined and categorized as: viable An-PI-; early apoptotic An+PI-
[have green fluorescence (PS externalization) but excluded PI (plasma
membrane is not damaged)]; late apoptotic An+PI+ [have green
fluorescence (PS externalization) and stained PI (plasma membrane is
damaged)]; and final apoptosis/necrosis An-PI+ [absence of green
fluorescence (lack of PS externalization) and stained PI (plasma mem-
brane is damaged)]. Fluorescence intensity was measured by a flow
cytometer.
2.4. Cell viability assay (XTT)
Cell viability was determined by XTT assay (Roche Diagnostic, USA)
which measures the cells’ ability to reduce the tetrazolium salt XTT
(2.3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-
anilide) to a water-soluble formazan product. Cells were seeded at a
density of 5 × 103 for Ab melanoma and 50 × 103 for Ma melanoma
cells into 96-well plates with suitable cultivation media. After 24 h the
media were exchanged and cells were stimulated with appropriate
concentrations (0.1–150 μM) of the examined compounds for 48 and 72
h. The orange-colored formazan product was quantified at 450 nm in a
microplate reader (Multiscan FC. Thermoscientific USA). Cell viability
was normalized with respect to an untreated control (100 %) and the
half maximal inhibitory concentration IC50 was estimated. The tested
compounds were primary dissolved in 0.10 mM DMSO then diluted to
the final concentration in a growth medium. The final concentration of
DMSO in the culture did not exceeded 0.001 mM and did not influence
cellular viability.
2.8. Reactive oxygen species
DCFDA (2′.7′-dichlorofluorescin diacetate),
a fluorogenic dye,
measures reactive oxygen species (ROS) activity within a cell. After
diffusion into a cell, DCFDA is deacetylated by cellular esterases to a
non-fluorescent compound, which is later oxidized by ROS into 2′.7′-
dichlorofluorescein (DCF). DCF is a highly fluorescent compound which
can be detected by flow cytometry. DCFDA (Sigma-Aldrich, USA) was
added to cells according to the manufacturer’s protocol, and after 30
min incubation at 37 °C the fluorescence intensity was analyzed in a
flow cytometer. As a positive control, cells were incubated with 3%
H202.(1:30).
2.5. Cytofluorimetric analysis of apoptosis
Flow cytometry analysis was the method used for estimation of
activated caspases, plasma membrane changes (phosphatidylserine ex-
ternalization), reactive oxygen species (ROS) and cell cycle changes.
Cells were seeded in 6-well plates at a concentration of 200 × 103/well
3

M. Cichorek, et al.
Biomedicine&Pharmacotherapy130(2020)110515
2.9. Cell cycle analysis
shown). At a tenfold higher dose, 150 μM, with melanotic Ma mela-
noma, only over 30 % and over 40 % of cells showed mitochondrial
changes for 1a and 1b, respectively (Fig. 3A, B).
The IC50 dose of dacarbazine, a chemotherapeutic against mela-
noma, reached about 70 μM for the Ab melanoma line, only after
prolonged incubation for 72 h – a much more higher dose than for both
examined acridine compounds (Fig. 3B). Among melanotic Ma mela-
noma cells over 60 % were still alive at a dose 150 μM of dacarbazine
(Fig. 3A), similar to the examined acridine compounds.
Cell cycle distribution was determined by the flow cytometry
method, based on the DNA content in cell nuclei, as described earlier
[56]. 1 × 106 ethanol-fixed cells were resuspended in 1 ml of a staining
solution containing 40 μg/ml propidium iodide (Sigma Chemicals,
USA) and 100 μg/ml RNase A (Sigma Chemicals, USA) and incubated
for 30 min at 37 °C. Results were analyzed by flow cytometry.
2.10. Activity of enzymes as a function of the energetic state of cells
4.2. Cell cycle changes
PDHC (pyruvate dehydrogenase complex; EC1.2.4.1), aconitase
(EC4.2.1.3), NADP-isocitrate dehydrogenase (ICDH; EC1.1.1.42), lac-
tate dehydrogenase (LDH EC1.1.1.27) activities were estimated in cell
lysates after 48 h culture with 1a and 1b. Cells were harvested into ice
cold Puck’s solution (140 mmol/L NaCl, 5 mmol/L KCl, 5 mmol/L
glucose, 1.7 mmol/L Na-phosphate buffer, pH 7.4), centrifuged at 200 g
for 7 min, and suspended in 0.320 mmol/L sucrose buffered with
HEPES (pH 7.4) and 0.1 mM EDTA-Na. The protein level was measured
using the Bradford method, with human immunoglobulin as standard.
The protein concentration in suspension was about 2 mg/ml in Ab
melanoma cells. The cells were immediately counted using a Trypan
Blue assay and the tricarboxylic acid cycle (TAC) enzymatic activities
were measured. Before the assay, samples were diluted to the desired
protein concentration in 0.2 % Triton X-100. PDHC activity was assayed
by the citrate synthase coupled method followed by citrate quantisation
using citrate lyase [57]. Aconitase and ICDH activity were examined by
a direct measurement of NAD reduction [58,59]. The influence of 1a
and 1b on enzyme activities is presented as the half maximal inhibitory
concentration (IC50).
After incubation with 1a the content of cells in S/G2/M phases did
not change significantly in comparison to control, for both melanoma
types, and there were 37 % and 21 % of Ab and Ma melanoma cells in
these phases, respectively (Fig. 4B). However, as the result of Ab mel-
anoma cell incubation with 1b, the number of cells in S/G2/M phases
decreased to 28 % after 48 h and, after an additional 24 h, the number
of cells in G0/G1 phases decreased significantly to 29 % (Fig. 4B).
These changes were accompanied by a statistically significant in-
creased number of cells in the sub-G0 area, which comprised 36 % of all
Ab cells after 72 h incubation with 1b (Fig. 4B). Thus, under the in-
fluence of 1b, Ab melanoma cells were broken into fragments with
decreased DNA content (sub G0 area), and dying cells were found to
originate from all cell cycle phases. Under the same conditions, 1b did
not cause significant cell cycle changes among Ma melanoma cells.
4.3. Changes to the plasma membrane structure of dying cells
1a does not induce significant changes in the plasma membrane
structure, such as the externalization of phosphatidylserine (An+; an-
nexin-positive cells) among Ab melanoma cells (Table 1, Fig. 4A). When
affected by its methylated form 1b, about 40 % of Ab cells were An+
after 48 h (p < 0.05). This 40 % figure was made up of 16 %, which
were An+PI- (early apoptotic) cells, and 24 % which were An+PI+
(late apoptotic) cells. With prolonged incubation time to 72 h, the
content of An+ cells increased to 45 % (Table 1, Fig. 4A). Under the
same culture conditions, after 72 h, the number of An+ Ma melanoma
cells slightly decreased to 16 % and 13 % for 1a and 1b respectively
(Table 1, Fig. 4A). 1a and 1b did not increase the number of necrotic
cells (An-PI+) among Ab cells, with necrotic cells being at a level of
4–5 %, but 1a induced a slight increase in necrotic cells among Ma cells
to about 16 % (Table 1). Changes in dying Ab melanoma cell mor-
phology are documented in the Supplementary material (S2. Morpho-
logical changes of Ab melanoma cells).
2.11. Energetic and oxidoreduction potential of cells
The energetic state was assayed by determining the content of ATP
and NAD in cell extracts prepared from freeze-dried cells using 0.4 M
perchloric acid (HClO4300 μl) and centrifuged (14,000 rpm; 10 min, 4
°C). Supernatants were neutralized to pH 6 with 3 M K3PO4 and after 15
min on ice centrifuged again. The concentration of nucleotides in su-
pernatants was measured by high-performance liquid chromatography
(HPLC) with UV-DAD detection as described earlier [60].
3. Statistical analyses
The statistical analyses were performed using the data analysis
software system STATISTICA version 12 from StatSoft Inc. (2016). Data
are expressed as arithmetical means SD (Standard Deviation) or SEM
(Standard Error of the Mean). The results were analyzed by a non-
parametric tests: Jonckheere test (to determine the significance of a
trend in data), Mann-Whitney U test and Steel test (to compare the
differences between the examined groups). p < 0.05 was considered
statistically significant.
4.4. Caspase activation
After 48 h 9% of Ab melanoma cells had activated caspases (C+),
but this group of cells increased significantly with time only under the
influence of 1a (Table 1). Its methylated form 1b did not increase the
number of C+ cells, even after 72 h, although an increased number of
cells without activated caspases but with a damaged plasma membrane
(C-PI+, cells in the final stage of apoptosis) was noticed (Table 1).
Among Ma cells, the examined compounds did not affect caspase acti-
vation (Table 1).
4. Results
4.1. Cell viability (XTT)
There was a statistically significant decreasing trend of melanoma
cells viability with the increasing 1a and 1b concentration (Fig. 3A,
p < 0.001). Nevertheless, it seemed that compounds 1a and 1b caused
a slight viability increase of Ma cells at a dose up to 10 μM and 1 μM
respectively, but then a cytotoxic effect against this melanotic form was
dose-dependent (Fig. 3A). Similar observation in Ab melanoma cells
referred only to compound 1b at a dose up to 1 μM after 72 h (Fig. 3A).
After 48 h incubation, the IC50 for 1a and 1b against Ab melanoma
reached 14.3 μM and 15.4 μM respectively (Fig. 3B). After additional 24
h, these values decreased to 3.4 μM and 7.8 μM respectively (data not
4.5. ROS activation
Both melanoma lines show similar numbers of cells with the ROS
activity, about 40 %. These values did not change in Ma melanoma cells
in the presence of either acridine compound, however in Ab melanoma
cells it increased slightly to 61 % and 58 %, after 48 h incubation, with
1a and 1b respectively (Table 1, statistically insignificant).
It was observed that 1a increased the content of cells with activated
caspases among Ab melanoma cells, but with only slight PS
4

M. Cichorek, et al.
Biomedicine&Pharmacotherapy130(2020)110515
Fig. 3. Cytotoxicity of 1a, 1b and dacarbazine against Ma melanotic melanoma and Ab amelanotic melanoma. A. Cell XTT viability test. B. IC 50 dose of 1a,1b and
dacarbazine for Ab cells. Viability of Ma cells under 150 μM of 1a,1b and dacarbazine. Dac* IC50 after 72 h incubation. For both melanoma lines at least three
experiments were done and values are presented as mean SEM. Statistically significant change (p < 0.05) in comparison to control values. * (black stars for 1a) *
(grey stars for 1b).
externalization (early apoptotic plasma membrane change, statistically
insignificant increase) and did not change the number of cells localized
in the sub-G0 area (e.g. apoptotic bodies). The methylated form 1b did
not influence caspase activation although this compound significantly
increased the number of cells with apoptotic plasma membrane changes
(PS externalization) and the number of cells in sub-G0. Counting cells in
a hemocytometer confirmed that the total number of cells decreased by
about 30 % after incubation with 1b for 48 h (data not shown). Both
compounds slightly increased the number of cells with ROS presence.
Thus 1a and its methylated form 1b act on amelanotic Ab mela-
noma (melanogenesis is inhibited) but in different ways. It seems that
apoptosis and caspase-independent death are the main routes toward
Ab melanoma cell death. Due to the importance of energetic metabo-
lism in the mechanism of cell death, we followed some elements of the
energetic state of Ab cells after 1a and 1b action as the next step of our
experiment.
4.6. Activity of enzymes associated with the energetic state of cells
4.6.1. Pyruvate dehydrogenase complex (PDHC)
The activity of PDHC in Ab cells in control was about 2.01 0.06
nmols/min/mg of protein. There was a statistically significant de-
creasing trend of PDHC activity in Ab melanoma with the increasing 1a
and 1b concentration (Fig. 5A, p < 0.05 and p < 0.001 respectively).
5

M. Cichorek, et al.
Biomedicine&Pharmacotherapy130(2020)110515
Fig. 4. Elements of the potential mechanism of 1a and 1b cytotoxicity. A. Flow cytometry analysis of one representative experiment that documents the apoptotic
change in plasma membrane structure; PS externalization results analysis: An+PI- (early apoptotic), An+PI+ (late apoptotic)), An-PI+ (necrotic) after 48 h
incubation without (control) and with 1a,1b. B. The cell cycle analysis showing percentage of cells in cell cycle phases: G0/G1, S/G2/M, and cells with decreased
DNA content (sub G0). Melanoma lines at least three experiments were done and values are presented as mean SD. * Statistically significant change (p < 0.05) in
comparison to control values.
6

M. Cichorek, et al.
Biomedicine&Pharmacotherapy130(2020)110515
Compound 1a at a dose of 10 μM caused unexpected increase of the
PDHC activity (statistically insignificant) while a dose of 50 μM almost
totally inhibited PDHC activity (Fig. 5A, p < 0.001). On the other hand
the enzyme’s activity was inhibited by 1b at a concentration dependent
manner, IC50 for 1b was 60 μM (Fig. 5A).
4.6.2. Aconitase
The activity of aconitase in control conditions after 48 h was
9.07 0.87 nmol/min/mg of protein. There was a statistically sig-
nificant decreasing trend of aconitase activity in Ab melanoma with the
increasing 1a and 1b concentration (Fig. 5B, p < 0.001). 1a and 1b
strongly inhibited the activity of aconitase, with IC50 values of 20 μM
and 26 μM for 1a and 1b respectively (Fig. 5B).
4.6.3. NADP-isocitrate dehydrogenase (ICDH)
The activity of ICDH in Ab cells after 48 h, under control conditions,
was 26.01 1.6 nmol/min/mg of protein. There was a statistically
significant decreasing trend of ICDH activity in Ab melanoma with the
increasing 1a and 1b concentration (Fig.5C, p < 0.001). Compound 1a
at 5 μM concentration caused 30 % fall of ICDH activity while further
concentration increase up to 100 μM caused 50 % inhibition of ICDH
activity (Fig. 5C). This enzyme was particularly sensitive to 1b with the
IC50 14 μM, while the IC50 for 1a was over 60 μM (Fig. 5C). There was a
slight increase of ICDH activity under 5 μM of 1b treatment but it was
not statistically significant.
4.6.4. Lactate dehydrogenase (LDH)
The activity of LDH in Ab cells after 48 h, under control conditions,
was 1.501 0.02 μmol/min/mg of protein. There was a decreasing
trend of LDH activity in Ab melanoma cells with the increasing 1a
concentration (Fig. 5D, p < 0.05).
There was a strong direct correlation between PDHC and ICDH ac-
tivities and total Ab melanoma cell number, in cells treated with 1b but
not 1a (Fig. 6 A,C). On the other hand there was a direct correlation
between aconitase and the total cell number in cells treated with 1a and
1b (Fig. 6 B).
4.7. NAD and ATP
The NAD level was decreased by 20 % and 35 % after 48 h in-
cubation with 1a and 1b, respectively (Fig. 7A), while the corre-
sponding ATP levels were depleted by 14 % and 46 %, respectively
(Fig. 7B). This illustrates that more significant changes in ATP and NAD
content in Ab melanoma cells appeared under the influence of the
methylated compound 1b.
5. Discussion
Our results indicate that 9-chloro-1-nitroacridine (1a) and its me-
thylated derivative 9-chloro-4-methyl-1-nitroacridine (1b) affect mel-
anotic and amelanotic melanoma in different ways. In comparison to
melanotic Ma melanoma, its amelanotic form – characterized by ab-
sence of melanin production, higher proliferation rate, and higher
malignancy – was particularly sensitive to the action of these chlor-
oacridines.
The observed increase in damaged cells (located in the sub-G0 area),
and decreasing number of cells in the G0/G1 and S/G2/M phases, in-
dicated that 1b induced Ab melanoma cell death independently of the
cell cycle phase. We did not observe cell cycle arrest in any phases
among either type of melanoma, although acridines interacting with
DNA have caused cell cycle arrest in many tumor lines [61–65]. 1b
cytotoxicity also confirmed decreasing number of cells counting with a
hemocytometer. Thus, we looked at elements of cell death machinery
that might be regulated by 1a and 1b action, e.g. plasma membrane
structure changes that could induce other cells to phagocytize dying
apoptotic cells (phosphatidylserine externalization as an “eat me”
7

M. Cichorek, et al.
Biomedicine&Pharmacotherapy130(2020)110515
Fig. 5. Concentration-dependent effect of 1a and 1b incubated with Ab cells for 48 h on the activity of selected enzymes of the TCA cycle: A. PDHC, B. aconitase, C.
ICDH, D. LDH. Data are given as mean SEM from 3-4 independent cellular cultures. Significantly different from respective control: *p < 0.05, ** p < 0.01,***
p < 0.001.
signal), caspase activation, energetic state (ATP, NAD), and activity of
metabolic enzymes, in this case tricarboxylic acid cycle enzymes [66].
At a dose that inhibited 50 % of mitochondria, 1a activated caspases
with only slight PS externalization and without an increase in damaged
cells localized in the sub-G0 phase (e.g. apoptotic bodies), while its
methylated form 1b did not influence caspase activation, yet sig-
nificantly increased the level of cells with apoptotic plasma membrane
changes (PS externalization), and those localized in the sub-G0 phase.
Thus, 1b seemed to destroy Ab melanoma cells more effectively than
the unmethylated 1a, but without involvement of caspases. Apoptosis
which usually leads to caspase activation, is the best understood type of
regulated cell death, but cells may also die by a process called caspase-
independent cell death (CICD). Cells undergoing CICD produce several
cytokines that are important for activation of the immune response
[67–69]. It has been observed that this type of melanoma cell death
does not stimulate cell proliferation, which is opposite to what is ob-
served with the so-called apoptosis-induced proliferation [70]. Thus, it
seems that cell death dependent and independent of caspase activation
could be a mechanism by which these chloroacridine compounds act,
although a detailed mechanism to explain the better effectiveness of 1b
Fig. 6. The correlation between total cell number and PDHC (A), aconitase (B) and ICDH (C) activities in Ab cells after 48 h of incubation with 1b or 1a. Data are
given as mean SEM from 3-6 independent cellular cultures.
8

M. Cichorek, et al.
Biomedicine&Pharmacotherapy130(2020)110515
Fig. 7. The effect of 15 μM of 1a and 1b incubated with Ab cells for 48 h on levels of NAD (A) and ATP (B). Data are given as mean SEM from 3-6 independent
cellular cultures. Significantly different from respective control:*p < 0.05.
against amelanotic melanoma needs further elucidation.
6. Conclusions
The energetic state analysis confirmed that cells dying as the result
of the action of chloroacridines 1a and 1b lost ATP and NAD. Inhibition
of examined metabolic enzymes, such as PDHC, aconitase and ICDH,
was also observed. There is a lot of data showing that interference in the
energy metabolism in cancer cells might be a successful therapeutic
approach [71–74]. This is results from the fact that malignant cells
favor conversion of glucose into lactate, instead of pyruvate, even when
there is high oxygen pressure. This phenomenon was first described by
Warburg, and is known as the Warburg effect [75]. However, activation
of lactic acid fermentation does not supply ATP for all energy needs. In
consequence, the Warburg metabolism is almost 100-fold faster than
oxidative one, thanks to the high glucose influx [76]. Therefore, the
mitochondria of cancer cells exhibit physiological disturbances that
lead to malignancy development [77]. Moreover, there is data showing
that changes in the micro-RNA of human melanocytes lead to disrup-
tion of normal cellular energy metabolism with anaerobic glycolysis
[78]. Consequently, one of the mechanisms that could exert an antic-
ancer effect could be interfering the activity of mitochondrial enzymes
what could be the way of action. The strong correlation between ICDH
activity with a total Ab melanoma cell number and stronger inhibition
of ICDH activity as the result of 1b action, indicated that compound 1b
interrupt the activity of carboxylic acid cycle enzymes. In tumor cells,
the cytosolic form of the enzyme ICDH1 was found to support mi-
tochondrial oxidative metabolism and NADPH synthesis by its mi-
tochondrial isoenzyme ICDH2 [79]. This mechanism plays a role in
decreasing mitochondrial ROS formation in cancer cells [79]. Therefore
compounds that are able to inhibit the activity of ICDH activity can be
considered to be a novel anticancer compound. 1b, inhibiting ICDH
activity more effectively than 1a, was such a compound.
Additionally aconitase inhibition by 1b could be a promising ob-
servation, as Gonzales-Sanchez et al. have noted that inhibition of this
enzyme inhibits tumor growth, including in melanoma cells [80].
The significant ATP decrease in 1b-treated cells was probably due to
the decrease in total NAD level, initiated by the depletion of PDHC
activity (NAD is a cofactor of the E2 subunit of PDHC). On the other
hand, a slight increase of LDH activity observed with low concentration
of 1a may be the result of glycolytic pathway activation, as PDHC ac-
tivity also increased in these conditions.
There are studies showing that, apart from oxygen from ATP pro-
duction, mitochondrial respiration is needed to complement NAD [81].
Thus inhibition of aconitase activity can enhance NAD depletion.
The better effectiveness of 1b, i.e. the methylated form of 1a,
against amelanotic melanoma, needs further examination. However,
there are reports indicating that methylation of acridine (AMSA) im-
proved its binding to DNA and DNA cleavage [82].
Our results indicated that melanotic and amelanotic melanomas
responded to chloroacridine action in different ways. Elements of the
regulated cell death pathways were observed only in amelanotic mel-
anoma cells, although the methylated form of chloroacridine induced
death without caspase activation (caspase-independent cell death); this
needs further studying. The mechanism of the interrelationships be-
tween energy metabolism and cell death is not fully understood, though
the fact that 1b interferes with the activities of tricarboxylic acid cycle
enzymes could be a way towards possible therapeutic use, especially
against the amelanotic form of melanoma. As such it justified further
studies.
Funding
This work was financially supported by the Polish National Science
Center, grant no. 2014/13/B/NZ7/02234.
Authors’ contribution
MC and AR analyzed and interpreted the data. MC performed tests
for means of cell death, AR performed tests for enzyme activities, MD
performed the viability test and IPN estimated ATP and ADP levels,
MGK and KD performed acridine syntheses; MC, AR, and KD were the
major contributors in writing the manuscript. All authors read and
approved the final manuscript.
Ethical approval
All procedures performed in these studies which involved animals
were in accordance with the ethical standards of the institution or
practice at which the studies were conducted.
Declaration of Competing Interest
The authors declare that there are no conflict of interest.
Acknowledgement
We would like to thank professor Ewa Augustin from the
Department of Pharmaceutical Technology and Biochemistry, Chemical
Faculty, Gdansk University of Technology (Poland) for the flow cyto-
metry analysis support.
9

M. Cichorek, et al.
Biomedicine&Pharmacotherapy130(2020)110515
Appendix A. Supplementary data
[22] R. Fisher, L. Pusztai, C. Swanton, Cancer heterogeneity: implications for targeted
therapeutics, Br. J. Cancer 108 (2013) 479–485, https://doi.org/10.1038/bjc.2012.
581.
[23] J. Tsoi, L. Robert, K. Paraiso, C. Galvan, K.M. Sheu, J. Lay, et al., Multi-stage dif-
ferentiation defines melanoma subtypes with differential vulnerability to drug-in-
duced iron-dependent oxidative stress, Cancer Cell 33 (2018) 890–904, https://doi.
org/10.1016/j.ccell.2018.03.017 e5.
[24] E. Wee, R. Wolfe, C. Mclean, J.W. Kelly, Y. Pan, Clinically amelanotic or hypo-
melanotic melanoma: anatomic distribution, risk factors, and survival, J. Am. Acad.
Dermatol. 79 (2018) 645–651, https://doi.org/10.1016/j.jaad.2018.04.045.
[25] A. Slominski, T.K. Kim, A. Brozyna, Z. Janjetovic, D.L.P. Brooks, L.P. Schwab, et al.,
The role of melanogenesis in regulation of melanoma behavior: melanogenesis leads
to stimulation of HIF-1α expression and HIF-dependent attendant pathways, Arch.
Biochem. Biophys. 56 (3) (2014) 79–93, https://doi.org/10.1016/j.abb.2014.06.
030.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2020.110515.
References
[1] S.A. Rosenberg, J.C. Yang, R.M. Sherry, U.S. Kammula, M.S. Hughes, G.Q. Phan,
et al., Durable complete responses in heavily pretreated patients with metastatic
melanoma using T cell transfer immunotherapy, Clin. Cancer Res. 17 (2011)
4550–4557, https://doi.org/10.1158/1078-0432.CCR-11-0116.
[2] A. Ribas, R. Kefford, M.A. Marshall, C.J. Punt, J.B. Haanen, M. Marmol, et al., Phase
III randomized clinical trial comparing tremelimumab with standard-of care che-
motherapy in patients with advanced melanoma, J. Clin. Oncol. 31 (2013)
616–622, https://doi.org/10.1200/JCO.2012.44.6112.
[26] H.Z. Gong, H.Y. Zheng, J. Li, Amelanotic melanoma, Melanoma Res. 29 (2019)
221–230, https://doi.org/10.1097/CMR.0000000000000571.
[27] L. Gualandri, R. Betti, C. Crosti, Clinical features of 36 cases of amelanotic mela-
nomas and considerations about the relationship between histologic subtypes and
diagnostic delay, J. Eur. Acad. Dermatol. Venereol. 23 (2009) 283–287, https://doi.
org/10.1111/j.1468-3083.2008.03041.
[28] N.E. Thomas, A. Kricker, W.T. Waxweiler, P.M. Dillon, K.J. Busman, L. From L,
et al., Comparison of clinicopathologic features and survival of histopathologically
amelanotic and pigmented melanomas: a population-based study, JAMA Dermatol.
150 (2014) 1306–1314, https://doi.org/10.1001/jamadermatol.2014.1348.
[29] T. Wasiewicz, P. Szyszka, M. Cichorek, Z. Janjetovic, R.C. Tuckey, A.T. Slominski,
et al., Antitumor effects of vitamin D analogs on hamster and mouse melanoma cell
lines in relation to melanin pigmentation, Int. J. Mol. Sci. 16 (2015) 6645–6667,
https://doi.org/10.3390/ijms16046645.
[30] K.G. Chen, R.D. Leapman, G. Zhang, B. Lai, J.C. Valencia, C.O. Cardarelli, et al.,
Influence of melanosome dynamics on melanoma drug sensitivity, J. Natl. Cancer
Inst. 101 (2009) 1259–1271, https://doi.org/10.1093/jnci/djp259.
[31] B.S. Larsson, Interaction between chemicals and melanin, Pigment Cell Res. 6
(1993) 127–133, https://doi.org/10.1111/j.1600-0749.1993.tb00591.x.
[32] M. Pawlikowska, J. Piotrowski, T. Jędrzejewski, W. Kozak, A.T. Slominski,
A.A. Brożyna, Coriolus versicolor-derived protein-bound polysaccharides trigger
the caspase-independent cell death pathway in amelanotic but not melanotic mel-
anoma cells, Phytother. Res. 34 (2020) 173–183, https://doi.org/10.1002/ptr.
6513.
[33] M. Gensicka-Kowalewska, G. Cholewiński, K. Dzierzbicka, Recent developments in
the synthesis and biological activity of acridine/acridone analogues, RSC Adv. 7
(2017) 15776–15804, https://doi.org/10.1039/C7RA01026E.
[34] M. Kukowska M, Amino acid or peptide conjugates of acridine/acridone and qui-
noline/quinolone-containing drugs. A critical examination of their clinical effec-
tiveness within a twenty-year timeframe in antitumor chemotherapy and treatment
of infectious diseases, Eur. J. Pharm. Sci. 109 (2017) 587–615, https://doi.org/10.
1016/j.ejps.2017.08.027.
[35] K. Pawlak, J.W. Pawlak, J. Konopa, Cytotoxic and antitumor activity of 1-ni-
troacridines as an after effect of their interstrand DNA cross-linking, Cancer Res. 44
(1984) 4289–4296.
[36] G. Moloney, D. Kelly, P. Mack, Synthesis of acridine-based DNA bis-intercalating
agents, Molecules 6 (2001) 230–243, https://doi.org/10.3390/2F60300230.
[37] Z. Mazerska, J. Dziegielewski, J. Konopa, Enzymatic activation of a new antitumour
drug, 5-diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311, observed
after its intercalation into DNA, Biochem. Pharmacol. 61 (2001) 685–694, https://
doi.org/10.1016/s0006-2952(01)00527-5.
[3] J. Schachter, A. Ribas, G.V. Long, A. Arance, J.J. Grob, L. Mortier, et al.,
Pembrolizumab versus ipilimumab for advanced melanoma: final overall survival
results of a multicentre, randomised, open-label phase 3 study (KEYNOTE-006),
Lancet 390 (2017) 1853–1862, https://doi.org/10.1016/S0140-6736(17)31601–X.
[4] J.L. da Silva, A.L.S. Dos Santos, N.C.C. Nunes, F. de Moraes Lino da Silva,
C.G.M. Ferreira, A.C. de Melo, Cancer immunotherapy: the art of targeting the
tumor immune microenvironment, Cancer Chemother. Pharmacol. 84 (2019)
227–240, https://doi.org/10.1007/s00280-019-03894-3.
[5] M. Polkowska, P. Ekk-Cierniakowski, E. Czepielewska, W. Wysoczański,
W. Matusewicz, M.J. Kozłowska-Wojciechowska MJ, Survival of melanoma patients
treated with novel drugs: retrospective analysis of real-world data, Cancer Res. Clin.
Oncol. Open Access 143 (2017) 2087–2094, https://doi.org/10.1007/s00432-017-
2453-z.
[6] T. Swe, K.B. Kim, Update on systemic therapy for advanced cutaneous melanoma
and recent development of novel drugs, Clin. Exp. Metastasis 35 (2018) 503–520
https://doi.org/0.1007/s10585-018-9913-y.
[7] P.R. Pereira, A.N. Odashiro, L.A. Lim, C. Miyamoto, P.L. BlancoL, M. Odashiro,
et al., Current and emerging treatment options for uveal melanoma, Clin.
Ophthalmol. 7 (2013) 1669–1682, https://doi.org/10.2147/OPTH.S28863.
[8] S. Mourah, B. Louveau, N. Dumaz, Mechanisms of resistance and predictive bio-
markers of response to targeted therapies and immunotherapies in metastatic
melanoma, Curr. Opin. Oncol. 32 (2020) 91–97, https://doi.org/10.1097/CCO.
0000000000000603.
[9] M. Cichorek, M. Wachulska, A. Stasiewicz, A. Tymińska, Skin melanocytes: biology
and development, Postepy Dermatol. Alergol. 30 (2013) 30–41, https://doi.org/10.
5114/pdia.2013.33376.
[10] R. Rabbie, P. Ferguson, C. Molina-Aguilar, D.J. Adams, C.D. Robles-Espinoza,
Melanoma subtypes: genomic profiles, prognostic molecular markers and ther-
apeutic possibilities, J. Pathol. 247 (2019) 539–551, https://doi.org/10.1002/path.
5213.
[11] G. Palmieri, M.N. Ombra, M. Colombino, M. Casula, M.C. Sini, A. Manca, et al.,
Multiple molecular pathways in melanomagenesis: characterization of therapeutic
targets, Front. Oncol. 5 (2015) 183, https://doi.org/10.3389/fonc.2015.00183.
[12] H.A. Mejbel, S.K.C. Arudra, D. Pradhan, C.A. Torres-Cabala, P. Nagarajan,
M.T. Tetzlaff, et al., Immunohistochemical and molecular features of melanomas
exhibiting intratumor and intertumor histomorphologic heterogeneity, Cancers
(Basel) 11 (2019) 1714, https://doi.org/10.3390/cancers11111714.
[13] F. Rambow, J.-Ch. Marine, C.R. Goding, Melanoma plasticity and phenotypic di-
versity: therapeutic barriers and opportunities, Genes Dev. (33) (2019) 1295–1318,
https://doi.org/10.1101/gad.329771.119.
[38] B. Zhang, X. Li, B. Li, C. Gao, Y. Jiang, Acridine and its derivatives: a patent review
(2009 – 2013), Expert Opin. Ther. Patents 24 (2014) 647–664, https://doi.org/10.
1517/13543776.2014.902052.
[14] T. Grzywa, W. Paskal, P. Wlodarski, Intratumor and intertumor heterogeneity in
melanoma, Transl. Oncol. 10 (2017) 956–975, https://doi.org/10.1016/j.tranon.
2017.09.007.
[39] J. Jesek, J. Hlavacek, J. Sebestik, Biomedical applications of acridines, in:
K.D. Rainsford (Ed.), Progress in Drug Research 72, Springer International
Publishing AG, 2017, pp. 1–243, , https://doi.org/10.1007/978-3-319-63953-6_5.
[40] M. Cichorek, A. Ronowska, M. Gensicka-Kowalewska, M. Deptula, I. Pelikant-
Malecka, K. Dzierzbicka, Novel therapeutic compound acridine-retrotuftsin action
on biological forms of melanoma and neuroblastoma, J. Cancer Res. Clin. Oncol.
145 (2019) 165–179, https://doi.org/10.1007/s00432-018-2776-4.
[41] M. Gensicka-Kowalewska, K. Dzierzbicka, M. Cichorek, M. Deptuła, A. Ronowska,
Synthesis new analogs of 4-methyl-1-nitroacridine and its biological evaluation as
potential anticancer drugs, Asian J. Pharmaceut, Anal. Med. Chem. 6 (2018) 42–60.
[42] R.M. Acheson, Acridines, Interscience Publishers, NY, London, 1973.
[43] A. Achari, S. Neidle, 9-Chloroacridine, Acta Cryst B33 (1977) 3269–3270.
[44] G. Rouband, R. Faure, J.P. Galy, H-1 and C-13 chemical shifts for acridines: part
XVIII. 9-chloroacridine and 9-(N-allyl)- and 9-(N-propargyl)acridinamine deriva-
tives, Magn. Reson. Chem. 41 (2003) 549–553, https://doi.org/10.1002/mrc.1170.
[45] A. LedochowskI, Ledacrin - anticancerous medicine 1-nitro-9(3-dimethyloamino-
propylamino)-acridine-2HCl-H2O, Mater. Med. Pol. 8 (1976) 237–251.
[46] K. Dzierzbicka, A.M. Kołodziejczyk, B. Wysocka-Skrzela, A. Myśliwski,
D. Sosnowska, Synthesis and antitumor activity of conjugates of muramyldipeptide,
normuramyl dipeptide, and desmuramylpeptides with acridine/acridone deriva-
tives, J. Med. Chem. 44 (2001) 3606–3615, https://doi.org/10.1021/jm001115g.
[47] K. Dzierzbicka, A.M. Kołodziejczyk, Synthesis and antitumor activity of conjugates
of muramyldipeptide or normuramyldipeptide with hydroxy-acridine/acridone
derivatives, J. Med. Chem. 46 (2003) 183–189, https://doi.org/10.1021/
jm001115g.
[15] L. Boeckmann, A.C. Nickel, C. Kuschal, A. Schaefer, K.M. Thoms, M.P. Schön, et al.,
Temozolomide chemoresistance heterogeneity in melanoma with different treat-
ment regimens: DNA damage accumulation contribution, Melanoma Res. 21 (2011)
206–216, https://doi.org/10.1097/CMR.0b013e328345af95.
[16] S.C. Searles, E.K. Santosa, J.D. Bui, Cell-cell fusion as a mechanism of DNA ex-
change in cancer, Oncotarget 9 (2017) 6156–6173, https://doi.org/10.18632/
oncotarget.23715.
[17] A. Avagliano, G. Fiume, A. Pelagalli, G. Sanità, M.R. Ruocco, S. Montagnani,
A. Arcucci, Metabolic plasticity of melanoma cells and their crosstalk with tumor
microenvironment, Front. Oncol. 10 (2020) 722, https://doi.org/10.3389/fonc.
2020.00722.
[18] R. Avolio, D.S. Matassa, D. Criscuolo, M. Landriscina, F. Esposito, Modulation of
mitochondrial metabolic reprogramming and oxidative stress to overcome che-
moresistance in cancer, Biomolecules 10 (2020) 135, https://doi.org/10.3390/
biom10010135.
[19] M. Saez-Ayala, M.F. Montenegro, L. Sanchez-Del-Campo, M.P. Fernandez-Perez,
S. Chazarra, R. Freter, et al., Directed phenotype switching as an effective anti-
melanoma strategy, Cancer Cell 24 (2013) 105–119, https://doi.org/10.1016/j.ccr.
2013.05.009.
[20] F.G. Cordaro, A.L. De Presbiteris, R. Camerlingo, N. Mozzillo, G. Pirozzi,
E. Cavalcanti, et al., Phenotype characterization of human melanoma cells resistant
to dabrafenib, Oncol. Rep. 38 (2017) 2741–2751, https://doi.org/10.3892/or.
2017.5963.
[21] F. Ahmed, N.K. Haass, Microenvironment-driven dynamic heterogeneity and phe-
notypic plasticity as a mechanism of melanoma therapy resistance, Front. Oncol.
(2018) 173, https://doi.org/10.3389/fonc.2018.00173.
[48] M. Robin, R. Faure, A. Perichaud, J.P. Galy, Synthesis of new thiazolo[5,4-a]acri-
dine derivatives, Heterocycles 53 (2000) 387–395, https://doi.org/10.3987/COM-
10

M. Cichorek, et al.
Biomedicine&Pharmacotherapy130(2020)110515
binding and Topoisomerase inhibition: How can these mechanisms be explored to
design more specific anticancer agents? Biomed. Pharmacother. 96 (2017)
1538–1556, https://doi.org/10.1016/j.biopha.2017.11.054.
99-8794.
[49] B.T. Ashok, K. Tadi, D. Banerjee, J. Konopa, M. Iatropoulos, R.K. Tiwari, Pre-clinical
toxicology and pathology of 9-(2′-hydroxyethylamino)-4-methyl-1-nitroacridine (C-
1748), a novel anti-cancer agent in male Beagle dogs, Life Sci. 79 (2006)
1334–1342, https://doi.org/10.1016/j.lfs.2006.03.043.
[50] A. Bomirski, A. Slomiński, J. Bigda, The natural history of a family of transplantable
melanomas in hamsters, Cancer Metastasis Rev. 7 (1988) 95–118, https://doi.org/
10.1007/bf00046481.
[51] M. Sniegocka, E. Podgórska, P.M. Płonka, M. Elas, B. Romanowska-Dixon,
M. Szczygieł, et al., Transplantable melanomas in hamsters and gerbils as models
for human melanoma. Sensitization in melanoma radiotherapy-from animal models
to clinical trials, Int. J. Mol. Sci. 19 (2018) 1048, https://doi.org/10.3390/
ijms19041048.
[52] K. Zielińska, K. Kozłowska, M. Cichorek, M. Wachulska, Fas and FasL expression on
cells of two transplantable melanoma lines according to their different biological
properties, Folia Histochem. Cytobiol. 46 (2008) 337–343, https://doi.org/10.
2478/v10042-008-0041-4.
[66] L. Galluzzi, I. Vitale, S.A. Aaronson, J.M. Abrams, D. Adam, P. Agostinis, et al.,
Molecular mechanisms of cell death: recommendations of the Nomenclature
Committee on Cell Death 2018, Cell Death Differ. 25 (2018) 486–541, https://doi.
org/10.1038/s41418-017-0012-4.
[67] N. Yatim, H. Jusforgues-Saklani, S. Orozco, O. Schulz, R. Barreira da Silva, C. Reis e
Sousa, et al., RIPK1 and NF-κB signaling in dying cells determines cross-priming of
CD8+ T cells, Science 350 (2015) 328–334, https://doi.org/10.1126/science.
aad0395.
[68] E. Giampazolias, B. Zunino, S. Dhayade, F. Bock, C. Cloix, K. Cao, et al.,
Mitochondrial permeabilization engages NF-κB-dependent anti-tumour activity
under caspase deficiency, Nat. Cell Biol. 19 (2017) 1116–1129, https://doi.org/10.
1038/ncb3596.
[69] E. Giampazolias, S.W.G. Tait, Caspase-independent cell death: an anti-cancer
double whammy, Cell Cycle 17 (2018) 269–270, https://doi.org/10.1080/
15384101.2017.1408229.
[53] M. Gensicka-Kowalewska, M. Cichorek, A. Ronowska, M. Deptula, I. Klejbor,
K. Dzierzbicka, Synthesis and biological evaluation of acridine/acridone analogs as
potential anticancer agents, Med. Chem. (Los Angeles) 15 (2019) 729–737, https://
doi.org/10.2174/1573406414666181015145120.
[70] A. Roumane, K. Berthenet, C. El Fassi, G. Ichim, Caspase-independent cell death
does not elicit a proliferative response in melanoma cancer cells, BMC Cell Biol. 19
(2018) 11, https://doi.org/10.1186/s12860-018-0164-1.
[54] J.K. Konopa, B. Wysocka-Skrzela, R. Tiwari, 9-Alkylamino-1-nitroacridine deriva-
tives. WO 2001/60801A2, patent published 2001-08-23. (2001).
[55] P. Smolewski, J. Grabarek, H. Halicka, Z. Darżynkiewicz, Assay of caspase activa-
tion in situ combined with probing plasma membrane integrity to detect three
distinct stages of apoptosis, J. Immunol. Methods 265 (2002) 111–121, https://doi.
org/10.1016/s0022-1759(02)00074-1.
[56] M. Cichorek, Camptothecin-induced death of amelanotic and melanotic melanoma
cells in different phases of cell cycle, Neoplasma 58 (2011) 227–234, https://doi.
org/10.4149/neo_2011_03_227.
[57] A. Szutowicz, M. Stepien, G. Piec, Determination of pyruvate dehydrogenase and
acetyl-CoA synthetase activities using citrate synthase, Anal. Biochem. 115 (1981)
81–88, https://doi.org/10.1016/0003-2697(81)90527-3.
[58] J. Villafranca, The mechanism of aconitase action: evidence for an enzyme iso-
merization by studies of inhibition by tricarboxylic acids, J. Biol. Chem. 249 (1974)
6149–6155.
[71] L. Xu, L. Wang, L. Zhou, R.G. Dorfman, Y. Pan, D. Tang, et al., The SIRT2/cMYC
pathway inhibits peroxidation-related apoptosis in cholangiocarcinoma through
metabolic reprogramming, Neoplasia 29 (2019) 429–441, https://doi.org/10.
1016/j.neo.2019.03.002.
[72] E. Andreucci, S. Pietrobono, S. Peppicelli, J. Ruzzolini, F. Bianchini, A. Biagioni,
et al., SOX2 as a novel contributor of oxidative metabolism in melanoma cells, Cell
Commun. Signal 16 (2018) 87, https://doi.org/10.1186/s12964-018-0297-z.
[73] G. Pathria, D.A. Scott, Y. Feng, J. Sang Lee, Y. Fujita, G. Zhang, et al., Targeting the
Warburg effect via LDHA inhibition engages ATF4 signaling for cancer cell survival,
EMBO J. 37 (20) (2018), https://doi.org/10.15252/embj.201899735 pii: e99735.
[74] T.A. Fedele, A.C. Galdos-Riveros, H. Jose de Farias e Melo, A. Magalhaes,
D.A. Maria, Prognostic relationship of metabolic profile obtained of melanoma
B16F10, Biomed. Pharmacother. 67 (2013) 146–156, https://doi.org/10.1016/j.
biopha.2012.10.013.
[75] O. Warburg, On respiratory impairment in cancer cells, Science 124 (1956)
[59] G.W. Plaut, T. Aogaichi, Purification and properties of diphosphopyridine nuleo-
tide-linked isocitrate dehydrogenase of mammalian liver, J. Biol. Chem. 243 (1968)
5572–5583.
[60] R. Smolenski, D. Lachno, S. Ledingham, M. Yacoub, Determination of sixteen nu-
cleotides, nucleosides and bases using high-performance liquid chromatography
and its application to the study of purine metabolism in hearts for transplantation,
J. Chromatogr. 527 (1990) 414–420, https://doi.org/10.1016/s0378-4347(00)
82125-8.
[61] T.L. Su, T.C. Chou, J.Y. Kim, J.T. Huang, G. Ciszewska, W.Y. Ren, et al., 9-sub-
stituted acridine derivatives with long half-life and potent antitumor activity:
synthesis and structure-activity relationships, J. Med. Chem. 38 (1995) 3226–3235,
https://doi.org/10.1021/jm00017a006.
[62] I. Antonini, DNA-binding antitumor agents: from pyrimido 5,6,1-de/acridines to
other intriguing classes of acridine derivatives, Curr. Med. Chem. 9 (2002)
1701–1716, https://doi.org/10.2174/092986702336926.
[63] J. Polewska, A. Skwarska, E. Augustin, J. Konopa, DNA-damaging imidazoacridi-
none C-1311 induces autophagy followed by irreversible growth arrest and senes-
cence in human lung cancer cells, J. Pharmacol. Exp. Ther. 346 (2013) 393–405,
https://doi.org/10.1124/jpet.113.203851.
[64] A. Skladanowski, Modulation of G(2) arrest enhances cell death induced by the
antitumor 1-nitroacridine derivative, Nitracrine, Apoptosis 7 (2002) 347–359,
https://doi.org/10.1023/a:1016127513947.
269–270.
[76] A. Maus, G.J. Peters, Glutamate and α-ketoglutarate: key players in glioma meta-
bolism, Amino Acids 49 (2017) 21–32, https://doi.org/10.1007/s00726-016-
2342-9.
[77] S. Vyas, E. Zaganjor, M.C. Haigis, Mitochondria and cancer, Cell 28 (2016)
555–566, https://doi.org/10.1016/j.cell.2016.07.002.
[78] H. Kaushik, D. Malik, D. Parsad, D. Kaul, Mitochondrial respiration is restricted by
miR-2909 within human melanocytes, Pigment Cell Melanoma Res. 32 (2018)
584–587, https://doi.org/10.1111/pcmr.12758.
[79] L. Jiang, A.A. Shestov, P. Swain, C. Yang, S.J. Parker, Q.A. Wang, et al., Reductive
carboxylation supports redox homeostasis during anchorage-independent growth,
Nature 532 (2016) 255–258, https://doi.org/10.1038/nature17393.
[80] L. Gonzalez-Sanchez, M.A. Cobos-Fernandez, P. Lopez-Nieva, M. Villa-Morales,
K. Stamatakis, J.M. Cuezva, et al., Exploiting the passenger ACO1-deficiency arising
from 9p21 deletions to kill T-cell lymphoblastic neoplasia cells, Carcinogenesis
(2019), https://doi.org/10.1093/carcin/bgz185 pii: bgz185.
[81] K. Birsoy, T. Wang, W.W. Chen, E. Freinkman, M. Abu-Remaileh, D.M. Sabatini, An
essential role of the mitochondrial electron transport chain in cell proliferation is to
enable aspartate synthesis, Cell 162 (2015) 540–551, https://doi.org/10.1016/j.
cell.2015.07.016.
[82] A.C. Ketron, W.A. Denny, D.E. Graves, N. Osheroff, Amsacrine as a topoisomerase II
poison: importance of Drug-DNA interactions, Biochemistry 51 (2012) 1730–1739,
https://doi.org/10.1021/bi201159b.
[65] S. Almeida, A. Ribeiro, G. de Lima Silva, J. Ferreira Alves, E. Beltrao, et al., DNA
11