Chalcone methoxy derivatives exhibit antiproliferative and proapoptotic activity on canine lymphoma and leukemia cells

Article abstract of DOI:10.3390/molecules25194362

Chalcones are interesting candidates for anti-cancer drugs due to the ease of their synthesis and their extensive biological activity. The study presents antitumor activity of newly synthesized chalcone analogues with a methoxy group on a panel of canine lymphoma and leukemia cell lines. The antiproliferative effect of the 2⁰-hydroxychalcone and its methoxylated derivatives was evaluated in MTT assay after 48 h of treatment in different concentrations. The proapoptotic activity was studied by cytometric analysis of cells stained with Annexin V/FITC and propidium iodide and by measure caspases 3/7 and 8 activation. The DNA damage was evaluated by Western blot analysis of phosphorylated histone H2AX. The new compounds had selective antiproliferative activity against the studied cell lines, the most effective were the 2⁰-hydroxy-2⁰⁰,5⁰⁰-dimethoxychalcone and 2⁰-hydroxy-4⁰,6⁰-dimethoxychalcone. 2⁰-Hydroxychalcone and the two most active derivatives induced apoptosis and caspases participation, but some percentage of necrotic cells was also observed. Comparing phosphatidylserine externalization after treatment with the different compounds it was noted that the addition of two methoxy groups increased the proapoptotic potential. The most active compounds triggered DNA damage even in the cell lines resistant to chalcone-induced apoptosis. The results confirmed that the analogues could have anticancer potential in the treatment of canine lymphoma or leukemia.

Full text of DOI:10.3390/molecules25194362

molecules
Article
Chalcone Methoxy Derivatives Exhibit
Antiproliferative and Proapoptotic Activity
on Canine Lymphoma and Leukemia Cells
1
,
1
, Beatriz Hernández Suárez 1
,
Aleksandra Pawlak * , Marta Henklewska
Mateusz Łu z˙ ny 2 , Ewa Kozłowska 2 , Bo z˙ ena Obmi n´ ska-Mrukowicz 1 and
Tomasz Janeczko 2
1
Department of Pharmacology and Toxicology, Wrocław University of Environmental and Life Sciences,
C.K. Norwida 31, 50-375 Wrocław, Poland; marta.henklewska@upwr.edu.pl (M.H.);
beatriz.hernandez-suarez@upwr.edu.pl (B.H.S.); b.mrukowicz@gmail.com (B.O.-M.)
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25,
2
5
0-375 Wrocław, Poland; mateusz.luzny@upwr.edu.pl (M.Ł.); ewa.kozlowska1@upwr.edu.pl (E.K.);
tomasz.janeczko@upwr.edu.pl (T.J.)
*
Correspondence: aleksandra.pawlak@upwr.edu.pl
Academic Editor: H.P. Vasantha Rupasinghe
Received: 4 September 2020; Accepted: 21 September 2020; Published: 23 September 2020
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Chalcones are interesting candidates for anti-cancer drugs due to the ease of their synthesis
and their extensive biological activity. The study presents antitumor activity of newly synthesized
chalcone analogues with a methoxy group on a panel of canine lymphoma and leukemia cell
0
lines. The antiproliferative effect of the 2 -hydroxychalcone and its methoxylated derivatives was
evaluated in MTT assay after 48 h of treatment in different concentrations. The proapoptotic activity
was studied by cytometric analysis of cells stained with Annexin V/FITC and propidium iodide
and by measure caspases 3/7 and 8 activation. The DNA damage was evaluated by Western blot
analysis of phosphorylated histone H2AX. The new compounds had selective antiproliferative activity
0
00 00
against the studied cell lines, the most effective were the 2 -hydroxy-2 ,5 -dimethoxychalcone
0
0
0
0
and 2 -hydroxy-4 ,6 -dimethoxychalcone. 2 -Hydroxychalcone and the two most active derivatives
induced apoptosis and caspases participation, but some percentage of necrotic cells was also observed.
Comparing phosphatidylserine externalization after treatment with the different compounds it was
noted that the addition of two methoxy groups increased the proapoptotic potential. The most active
compounds triggered DNA damage even in the cell lines resistant to chalcone-induced apoptosis.
The results confirmed that the analogues could have anticancer potential in the treatment of canine
lymphoma or leukemia.
Keywords: chalcones; apoptosis; DNA damage; anticancer activity; canine cancer cell lines
1
. Introduction
The search for novel natural or synthetic compounds with potential antitumor activity is one
of the major goals of contemporary oncology. The research involves both known compounds with
proven biological activity as well as newly synthesized molecules that are structural analogues
of biologically active substances already used in the treatment of cancer. Interest is aroused by
compounds that may constitute the basis of chemotherapy in the future, as well as compounds that
may support therapy or sensitize cancer cells to classic cytostatic drugs. Because the high toxicity of
many anticancer substances (particularly bone marrow toxicity) significantly reduces the effectiveness
of the treatment, to maximize the efficacy and reduce adverse effects, chemotherapy is usually based
Molecules 2020, 25, 4362; doi:10.3390/molecules25194362
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4362
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on a combination of drugs with various molecular mechanisms of action and different side effects.
With regard to this principle, research focusing on the antitumor activity of new natural compounds
or their derivatives is highly justified. Such a potentially interesting group of compounds may be
chalcones, an essential group of natural compounds classified as flavonoids [1]. Their typical structural
feature is an open, -unsaturated three-carbon fragment connecting two aromatic rings [2
α
,
β
]. They are
yellow [
3
] and commonly occur in the world of plants: in citrus fruits, vegetables and spices [
4
,
5
].
0
Chalcones (1-(2 -hydroxyphenyl)-3-phenylprop-2-en-1-ones) and their derivatives are often obtained
from natural sources or as a result of chemical synthesis [6,7]. The most commonly used chemical
synthesis method is the Claisen–Schmidt condensation of the corresponding aromatic aldehyde and
0
2
-hydroxyacetophenones in alkaline or acid catalysis [8–10].
The great interest in chalcones is influenced by the ease of synthesis of these compounds and
the extensive biological activity characteristics they exhibit: in particular antiviral, anthelmintic,
antibacterial, antiprotozoal, insecticidal, antiulcer, cytotoxic, and anticancer [11 13]. Due to the
–
confirmed therapeutic effect of chalcones on human cancer, we decided to check their activity against
canine cancer cells.
Cancer in dogs is a major challenge in modern veterinary medicine and research into the search
for new cancer therapies is highly needed [14]. On the other hand, of the various animal species that
develop cancer (e.g., cats, horses, rabbits, ferrets), dogs turned out to be the most appropriate model
for comparative research due to their body size, life expectancy, cancer incidence and sharing of living
conditions with humans. Moreover, dogs together with mice and rats, are popular laboratory animals
necessary for different types of toxicological studies [15].
In conclusion, the presented study examined the antitumor activity of new chalcone analogues
with the methoxy group on selected canine lymphoma and leukemia cell lines. After the hydroxyl group,
the methoxy group is the second most commonly identified group in natural flavonoid compounds,
and because of this, we decided to test a series of chalcones containing a different amount and places
of methoxy group substitution. The cytotoxic, antiproliferative and proapoptotic effects of newly
synthesized compounds were analyzed. The study showed that the obtained analogues could have
anticancer potential in the treatment of canine lymphoma or leukemia.
2
. Results
2
.1. Chalcone Analogues with the Methoxy Group Have Selective Antiproliferative Activity on Canine Cancer
Cell Lines
First, an assay was performed to determine whether the tested chalcones have antiproliferative
activity against normal cells, and if the position of the substituents in the structure has an influence
on the potency of such effects. The study showed that normal cells, represented by the NIH/3T3 and
J774E.1 cell line, were less sensitive to the antiproliferative action of the tested chalcones than cancer
cells. The IC50 value (concentration that inhibits the proliferation of 50% of cells) was determined only
for two compounds in case of the NIH/3T3 cell line and four compounds in case of the J77.4.E cell
line. These four compounds (1, 2, 6 and 7) proved to be the most active also in research with the use of
cancer cell lines. The curves showing the inhibition of normal cell growth by individual compounds
are shown in Figure 1A,B.
While the tested compounds did not show a significant inhibitory effect on the growth of normal
cells, their potency in relation to cancer cell lines was visibly different. All the tested compounds had a
particularly strong effect on the CLBL-1 cell line, representing the most common type of lymphoma
in dogs—diffuse large B-cell lymphoma (DLBCL). Other B lymphocytes, from chronic lymphocytic
leukemia (CLB70) were also more sensitive than the others. Natural killer (NK) cells obtained from the
rare and refractory form of canine lymphoma were found to be the most resistant. Similarly, but slightly
more strongly, the tested compounds acted on the GL-1 cell line, representing canine acute leukemia.

Molecules 2020, 25, 4362
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Detailed results in the form of a table containing IC50 values for all tested compounds on all
tested cell lines are presented in Table 1. Growth inhibition curves of canine cancer cell lines are shown
in Figure 1C–F. % start a new page
Figure 1. Concentration-dependent curves presenting the effects of the tested chalcones on viability of
normal (A,B) and canine cancer cell lines (C–F) after 48 h of incubation with different concentrations
of the individual chalcones (1.5, 3.125, 6.25, 12.5, 25 and 50
independent experiments.
µM). The values are means from three

Molecules 2020, 25, 4362
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Table 1. IC50 values (µM concentration of the tested compounds that inhibits the proliferation of 50% of cells) for all tested compounds and all cell lines used in the
study, obtained by the MTT test after 48 h treatment. The results are presented as mean
each. Statistical differences were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. Values without common letters (a, b, c, d) in the
superscript differ statistically (p < 0.05). The table shows differences in the strength of action of each compound on selected cell lines. N.A.—not achieved.
±
standard deviation (SD) of three separate experiments, with three wells
Compound/Cell Line
GL-1
30.33 ± 5.51
33.89 ± 7.00
N.A.
CLBL-1
CNK-89
CLB-70
24.09 ± 6.23
30.66 ± 9.39
43.81 ± 0.66
N.A.
3T3
N.A.
N.A.
N.A.
N.A.
J774
37.83 ± 3.40 a
0
0
0
00
ac
ac
11.88 ± 1.58 b
37.30 ± 6.57 a
c
c
2
2
2
-hydroxy-2 -methoxychalcone (1)
00
b
42.61 ± 3.60 a
ac
-hydroxy-3 -methoxychalcone (2)
13.34 ± 3.04
36.50 ± 3.87
00
a
b
-hydroxy-4 -methoxychalcone (3)
18.78 ± 2.36
N.A.
N.A.
0
00 00 00
2
-hydroxy-3 ,4 ,5 -trimethoxychalcone (4)
N.A.
15.62 ± 1.52
N.A.
N.A.
0
0
0
00 00 00
2
-hydroxy-4 ,6 ,3 ,4 ,5 -pentamethoxychalcone (5)
N.A.
33.57 ± 9.96
N.A.
N.A.
N.A.
N.A.
0
00 00
-hydroxy-2 ,5 -dimethoxychalcone (6)
27.52 ± 4.59 a
31.18 ± 6.32
40.58 ± 3.92
9.76 ± 0.99 b
40.83 ± 3.00 c
38.63 ± 8.05
48.51 ± 1.52
24.47 ± 5.26 a
40.40 ± 3.02 c
29.82 ± 10.04 a
2
0
0
0
ac
b
ad
c
46.11 ± 4.66 d
ac
2
-hydroxy-4 ,6 -dimethoxychalcone (7)
9.18 ± 0.75
26.78 ± 9.03
32.17 ± 8.69
0
a
8.04 ± 1.08 b
c
38.94 ± 6.02 a
2
-hydroxychalcone (8)
N.A.
N.A.

Molecules 2020, 25, 4362
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2
.2. Chalcone Analogues with the Methoxy Group, at Least Partially, Kill Cancer Cells through Apoptosis
The substances with the strongest activity in the MTT test were selected for further research on the
nature of cell death induced by the studied compounds. To determine the type of cell death induced by
the tested chalcones, in the first place, the assay for detection of early signs of apoptosis, which is the
externalization of phosphatidylserine, was performed. The study used the two cell lines which were
the most sensitive to the tested compounds: CLBL-1 and CLB70 and the compounds with the strongest
0
00 00
0 0 0
6), 2 -hydroxy-4 ,6 -dimethoxychalcone
action: 2 -hydroxy-2 ,5 -dimethoxychalcone (compound
compound ) and initial compound (compound ). It was shown that cell death takes place by
apoptosis, as confirmed by the statistical analysis of the results. After 48 h of incubation with the
tested chalcone analogues at relatively low concentrations of 5 and 10 M (concentrations harmless to
(
7
8
µ
normal cells), both the CLBL-1 and CLB70 cells were found to have a positive reaction with Annexin V,
which is evidence of phosphatidylserine externalization and thus the ongoing process of apoptosis.
As a result of the actions of the tested compounds, in addition to the population of apoptotic cells, the
presence of cells that have already lost the integrity of the cell membrane was found (such a population
of cells can also be referred as necrotic). The presence of cells staining positively with propidium
iodide, a compound permanently staining the DNA of dead cells, can be explained by the relatively
long incubation time used in the study (some of the cells that died by apoptosis had already lost their
integrity too). Another explanation assumes that apoptosis is only one of the processes that cells
undergo under the influence of the tested chalcones. Detailed results and representative dot plots are
shown in Figure 2.
Figure 2. Percentage of annexin V positive (apoptotic) cells after 48 h of incubation with the culture
medium alone (0) or 5 and 10 µM of the tested chalcones. On the right: representative dot-plots of
annexin V/PI staining. The values are means of four independent experiments. * Considered significant
in comparison to the control (p < 0.05).
To better explain the type of cell death that cancer cells undergo after treatment with the tested
compounds, specific assays were carried out to determine the activity of caspases—the most important
enzymes involved in the process of apoptosis. By flow cytometry, it was found that in CLBL-1
and CLB70 cells, after treatment with the tested chalcones, activation of the effector caspase 3 and
initiator caspase 8 occur. The observed activation of caspase 3, the most important caspase in the
caspase-dependent pathway of apoptosis was not massive and covered only a small percent of cells
(
about 15%). This result confirms the induction of apoptosis in the cells, but at the same time, indicates
that apoptosis is not the only type of cell death of cells treated with the tested compounds. This
result is consistent with the result of the Annexin V staining, in which similar percentage of necrotic
0
00 00
cells was observed. 2 -Hydroxy-2 ,5 -dimethoxychalcone (6) and initial compound activated caspase

Molecules 2020, 25, 4362
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0
0
0
3
the most strongly, whereas 2 -hydroxy-4 ,6 -dimethoxychalcone (
7) had a slightly weaker effect.
More pronounced activation of caspase 3 occurred in the CLBL-1 cell line. Similar results were obtained
for caspase 8, which was more strongly activated in the CLBL-1 cells, showing a statistically significantly
higher level after treatment with all three compounds. Detailed results along with representative
histograms are presented in Figure 3 (caspase 3 activation) and Figure 4 (caspase 8 activation).
Figure 3. Percentage of cells with active caspase 3/7 after 48 h of incubation with the culture medium
alone (0) or 5 and 10
µM of the tested chalcones. On the right: representative histograms for caspase
3
/7 activation. The values are means of four independent experiments. * considered significant in
comparison to the control (p < 0.05).
Figure 4. Percentage of cells with active caspase 8 after 48 h of incubation with the culture medium
alone (0) or 5 and 10
µM of the tested chalcones. On the right: representative histograms for caspase
8
activation. The values are means of four independent experiments. * considered significant in
comparison to the control (p < 0.05).
2
.3. Tested Chalcone Analogues Trigger DNA Damage in Canine Lymphoma/Leukemia Cell Lines
Because the degree of apoptosis induction detected in Annexin V binding assay was lower than
expected and did not correlate perfectly with the results of cytotoxicity tests, we decided to check
the level of DNA damage as additional marker of potentially lethal events in the cells (leading to
cell death and the initiation of the apoptosis process). The study showed that the incubation of

Molecules 2020, 25, 4362
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0
00 00
CLB70, CNK89 and even the resistant GL-1 cell lines with 2 -hydroxy-2 ,5 -dimethoxychalcone
0 0 0 0
(6), 2 -hydroxy-4 ,6 -dimethoxychalcone (7) and initial 2 -hydroxychalcone (8) provoked the DNA
damaged manifested by increased histone H2AX phosphorylation. Detailed results are presented in
Figure 5.
Figure 5. Western blot analysis for phosphorylated H2AX of CNK89, CLB70 and GL-1 cell lines after
4
8 h of incubation with different concentrations (5 and 10 µM) of compound 6–8 (A–C).
3
. Discussion
The aim of the presented study was to obtain chalcone analogues with better anticancer properties
compared to the parent compound. Because a significant problem in the use of various flavonoids
is their poor availability, it was decided to focus on improving properties such as the ability to enter
the cell. Therefore, the modification that was made was to create analogues containing a methoxy
group instead of a hydroxyl group. Such a change can have a dual effect. Often, the conversion of a
hydroxyl to a methoxy group results in a decrease in the biological activity of the flavonoid compound
in vitro [16,17]. On the other hand, it was proven that methoxylated flavonoids have the ability to
easily penetrate the cells [18]. For example, it was demonstrated that after transferring lung cell lines
to the medium containing dimethoxyflavones, within five minutes, the cells accumulated 30–50 times
more test compounds than were present in the surrounding buffer [19]. The intracellular transport of
5
,7-dimethoxyflavone was approximately 10-fold higher than that of chrysin (5,7-dihydroxyflavone).
Moreover, chrysin was rapidly metabolized by the human liver (S9 fraction), with no parent compound
remaining after a 20-min incubation. In contrast, 5,7-dimethoxyflavone was metabolically stable
over the whole 60-min time-course studied [20,21]. Better absorption and distribution, as well as the

Molecules 2020, 25, 4362
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prolonged metabolism of methoxylated flavonoids, makes them even more promising compounds.
Furthermore, their oral bioavailability exceeds the bioavailability of hydroxylated flavonoids, which
makes them easy to administer [22]. Based on the knowledge of this phenomenon, we decided to
create derivatives with a methoxy group and see how such modification affects the antitumor activity
in vitro.
In our research, we first decided to check whether the obtained analogues show selective
antiproliferative activity against cancer cells. As shown in Figure 2, it turned out that in fact,
canine leukemia/lymphoma cells are more sensitive to the action of the obtained chalcones than
the normal cell lines used in the study. The potency of the antiproliferative activity of the
tested compounds was higher in relation to the cancer cell lines, but the same compounds that
most strongly inhibited the proliferation of cancer cells were also the most toxic to normal cells.
0
00
0
00
These compounds were: 2 -hydroxy-2 -methoxychalcone (
2
1
), 2 -hydroxy-3 -methoxychalcone (
2
),
0
00 00
0
0
0
-hydroxy-2 ,5 -dimethoxychalcone (
6) and 2 -hydroxy-4 ,6 -dimethoxychalcone (
7), the most active
of which were the last two. Analyzing the differences in the chemical structure of individual compounds,
at this stage of research, it can be concluded that the presence of two methoxy groups, regardless of their
position, is responsible for the strength of the cytotoxic activity on normal cell lines. Regarding cancer
cell lines, the differences in the potency of all four compounds mentioned above are too small to find
conclusions. It seems, however, that the strongest antitumor activity is shown by compounds containing
one methoxy group, but only in the position 2 or 3 of the B ring. Derivatives containing a methoxy group
in 4 position or containing 3 or more such groups – are characterized by weaker antiproliferative activity.
Looking at the table with IC50 values, one more interesting fact can be seen—the lowest IC50 value
0
was observed in relation to the CLBL-1 cell line, for the parent compound—2 -hydroxychalcone (
8).
This effect was observed only for the CLBL-1 cell line and this value does not differ statistically from
0
00 00
0 0 0
the values for 2 -hydroxy-2 ,5 -dimethoxychalcone (6) and 2 -hydroxy-4 ,6 -dimethoxychalcone (7).
The explanation for this observation may be the fact that the CLBL-1 cell line was characterized by
very strong sensitivity to all the tested compounds, therefore it is impossible in this case to capture
the influence of small differences in the structure of the compounds on their activity against this
cell line. However, the observed high sensitivity of the CLBL-1 cell line to the flavonoids may be a
valuable therapeutic indication. This cell line was obtained from a dog with the most common type of
lymphoma, and if it reflects the sensitivity of the cells of this type of disease then it may be possible to
consider more thorough studies on the potential use of this group of compounds in therapy in dogs.
The proapoptotic effect of flavonoids or specifically chalcones is still a frequently discussed subject
of scientific research. Numerous scientific reports describe the different strengths and mechanisms
of proapoptotic action of compounds from this group [23–26]. In our research, we focused not
on studying the mechanism of the induced apoptosis, but on comparing the strength with which
various methoxy derivatives of chalcones induce suicidal cell death. Comparing proapoptotic
0
00 00
0
0
0
activity 2 -hydroxy-2 ,5 -dimethoxychalcone (
6
), 2 -hydroxy-4 ,6 -dimethoxychalcone (
7) and initial
0
2
5
2
-hydroxychalcone (8) it was noted that the presence of methoxy groups in positions 2 and
in the B ring, affects the potency of the proapoptotic action of the obtained derivatives.
-Hydroxy-2 ,5 -dimethoxychalcone (6) showed a clearly stronger proapoptotic activity than the
0
00 00
parent compound both in the annexin V and in caspase 3/7 and 8 activity tests. In the case of the
CLBL-1 cell line, this difference was less visible, and in relation to caspase 8 the parent compound
0
0
more strongly caused its cleavage. The presence of two methoxy groups, but at positions 4 and 6
compound ) also increased the proapoptotic activity compared to the parent compound, as clearly
(
7
seen in studies using the CLB70 cell line. However, this difference could not be confirmed by test
using the CLBL-1 cell line. Another important observation was that the potency of the cytotoxic
activity of the obtained derivatives with the methoxy group observed in the MTT test significantly
exceeded that observed in the apoptosis tests. Because the MTT assay assesses the antiproliferative
activity of the tested compounds, it can be concluded that the obtained chalcone derivatives strongly
inhibited the proliferation of canine lymphoma/leukemia cells and the induction of apoptosis itself was

Molecules 2020, 25, 4362
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slightly weaker. Such action of various chalcones has already been described. In addition to apoptosis,
the compounds belonging to the chalcones have also been found to cause cell cycle arrest, which
may be both the cause and the effect of the proapoptotic action of this group of compounds [27
Because the mechanism of antitumor action of chalcones, apart from inhibiting cell proliferation and
induction of apoptosis, also includes regulation of cell survival, invasiveness or angiogenesis [31 32],
–30].
,
we decided to additionally check how the introduction of the methoxy group impact DNA damage.
One of the mechanisms identified for this action is related to the chalcones’ ability to selectively target
of the ubiquitin-proteasome system (UPS) [31
of an -unsaturated carbonyl group is the key molecular determinant conferring UPS- and DUBs
deubiquitinating enzymes)-inhibitory activity of different chalcones [33]. Because DUBs are crucial in
,32]. Published evidence suggests that the presence
α,β
(
regulating a variety of cellular pathways, including cell growth and proliferation, apoptosis, protein
quality control, DNA repair and transcription [34], the ability to inhibit them is an important feature of a
potential anti-cancer compound. Inhibiting DUBs activity impairs 20S proteasome proteolytic activities
and the cellular deubiquitinating enzymes, leading to increased accumulation of ubiquitinated proteins
and consequently causes endoplasmic reticulum stress and cell death [35,36]. The effect may be also
DNA damage. In the present study it has been clearly demonstrated that both the parent compound
and the two strongest derivatives cause DNA damage observed as an increase in histone H2AX
phosphorylation. Despite the use in this assay of cell lines (CNK89 and GL-1) slightly less sensitive
to the action of chalcones it cannot be unequivocally stated that the obtained derivatives acted more
strongly than the parent compound. Such observations can be explained by the lack of influence of
methoxy groups on the formation of DNA damage (the presence of an α,β-unsaturated carbonyl group
is rather responsible for such action) and the fact that the observed DNA damage was to some extent
an effect of the ongoing apoptosis. However, the strength and extent of the DNA damage found, even
in the cancer cell lines more resistant to chalcones, indicates that at least in part, the DNA damage was
the cause of the death of the canine lymphoma/leukemia cells.
4
. Materials and Methods
4
.1. Compounds
The chalcone analogues used in the study are numbered from 1 to 8. All were obtained
via a Claisen–Schmidt condensation of an appropriate 2-hydroxyacetophenone and a substituted
benzaldehyde dissolved in methanol in an alkaline environment at high temperature according to the
procedure described previously [37–39]. The structures of the tested chalcones are shown in Figure 6.
1
O
O
O
6'
2"
5"
1'
1
3
1"
3"
H
NaOH, MeOH 5'
reflux przez 2h
2
+
2'
4'
6"
4"
OH
OH
3'
8
O
O
O
O
O
O
O
O
OH
OH
OH
O
OH
1
2
3
4
O
O
O
O
O
O
O
O
O
O
O
OH
OH
O
OH
5
O
6
7
Figure 6. General scheme for the synthesis of chalcones and structures of obtained methoxy derivatives.
The numbering of carbon atoms has been placed on compound 8.

Molecules 2020, 25, 4362
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The resulting compounds were characterized by the following NMR spectral data (Supplementary
Materials Figures S1–S29):
0
00
1
2
1
-Hydroxy-2 -methoxychalcone (
1
). H NMR (600 MHz) (CDCl )
δ
(ppm): 3.94 (s 3H, -OCH ), 6.94 (td,
3
3
0
00
00
H, J = 7.8, 0.6 Hz, H-5 ), 6.96 (d, 1H, J = 8.8 Hz, H-3 ), 7.01 (t, 1H, J = 7.5 Hz, H-5 ), 7.04 (dd, 1H,
J = 8.3, 0.6 Hz, H-3 ), 7.41 (ddd, 1H, J = 8.6, 7.9, 1.5 Hz, H-4 ), 7.49 (t, 1H, J =8.8, 7.8, 1.4 Hz, H-4 ); 7.65
0
00
0
0
0
0
(
1
1
dd, 1H, J = 7.6, 1.3 Hz, H-6 ), 7.78 (d, 1H, J = 15.6 Hz, H-2), 7.93 (dd, 1H, J = 8.0, 1.3 Hz, H-6 ), 8.23 (d,
1
3
00
H, J = 15.6 Hz, H-3); 12.95 (s, 1H, -OH). C NMR (151 MHz, CDCl ) δ = 55.73 (-OCH ), 111.45 (C-3 ),
3 3
0
0
0
00
00
0
18.67 (C-3 ), 118.88 (C-5 ), 120.33 (C-1 ), 120.94 (C-2), 120.94 (C-5 ), 123.76 (C-1 ), 129.77 (C-6 ), 129.84
C-6 ), 132.34 (C-4 ), 136.28 (C-4 ), 141.27 (C-3), 159.17 (C-2 ), 163.71 (C-2 ), 194.44 (C-1).
00 00 0 00 0
(
0
00
1
2
(
-Hydroxy-3 -methoxychalcone (
2
). H NMR (600 MHz) (CDCl )
δ
(ppm): 3.87 (s, 3H, -OCH ), 6.95
3
3
0
00
ddd, 1H, J = 8.1, 7.1, 1.1 Hz, H-5 ), 6.99 (dd, 1H, J = 8.2, 2.4 Hz, H-4 ), 7.04 (dd, 1H, J = 8.4, 0.9 Hz,
H-3 ), 7.17 (dd, 1H, J = 2.2, 1.1 Hz, H-2 ), 7.27 (d, 1H, J = 7.7 Hz, H-6 ), 7.36 (t, 1H, J = 7.9 Hz, H-5 ),
0
00
00
00
0
7
.51 (ddd, 1H, J = 8.5, 7.2, 1.3 Hz, 1H, H-4 ), 7.64 (d, 1H, J = 15.5 Hz, H-2), 7.89 (d, 1H, J = 15.5 Hz, H-3),
0
13
7.92 (dd, 1H, J = 8.1, 1.4 Hz, H-6 ), 12.80 (s, 1H, -OH). C NMR (151 MHz, CDCl )
δ
= 55.55 (-OCH ),
3
3
00
00
0
0
0
00
1
(
13.87 (C-2 ), 116.76 (C-4 ), 118.80 (C-3 ), 119.01 (C-5 ), 120.16 (C-1 ), 120.58 (C-2), 121.43 (C-6 ), 129.81
C-6 ), 130.19 (C-5 ), 136.12 (C-1 ), 136.58 (C-4 ) 145.53 (C-3), 160.14 (C-3 ), 163.75 (C-2 ), 193.86 (C-1).
0
00
00
0
00
0
0
00
1
2
(
-Hydroxy-4 -methoxychalcone (
3
). H NMR (600 MHz) (CDCl )
δ
(ppm): 3.87 (s, 3H, -OCH ), 6.94
3
00
3
0
00
0
ddd, 1H, J = 8.1, 7.1, 1.0 Hz, H-5 ), 6.94–6.97 (m, 2H, H-3 , H-5 ), 7.02 (dd, 1H, J = 8.3, 0.8 Hz, H-3 ),
0
00
00
7
.49 (ddd, 1H, J = 8.5, 7.1, 1.5 Hz, H-4 ), 7.54 (d, 1H, J = 15.4 Hz, H-2), 7.63 (m, 2H, H-2 , H-6 ), 7.90
0
13
(
d, 1H, J = 15.0 Hz, H-3), 7.93 (dd, 1H, J = 8.0, 1.3 Hz, H-6 ), 12.95 (s, 1H, -OH). C NMR (151 MHz,
00 00 0 0 0
δ = 55.59 (-OCH ), 114.66 (C-3 ,-5 ), 117.74 (C-2), 118.72 (C-3 ), 118.89 (C-5 ), 120.26 (C-1 ),
3
CDCl3)
00 0 00 00 0 00 0
27.49 (C-1 ), 129.67 (C-6 ), 130.69 (C-2 , C-6 ), 136.28 (C-4 ), 145.50 (C-3), 162.17 (C-4 ), 163.69 (C-2 ),
1
1
93.82 (C-1).
0
00 00 00
1
2
-Hydroxy-3 ,4 ,5 -trimethoxychalcone (
4
). H NMR (600 MHz) (CDCl )
δ
(ppm): 3.91 (s, 3H,
3
00
00
00
00
00
C-4 -OCH ), 3.94 (s, 6H, C-3 -OCH and C-5 -OCH ), 6.89 (s, 2H, H-2 and H-6 ), 6.96 (ddd, 1H,
J = 8.0, 7.2, 1.0 Hz, H-5 ), 7.04 (dd, 1H, J = 8.4, 1.0 Hz, H-3 ), 7.51 (ddd, 1H, J = 8.3, 7.1, 1.4 Hz, H-4 ),
3
3
3
0
0
0
0
7
.54 (d, 1H, J = 15.4 Hz, H-2), 7.85 (d, 1H, J = 15.5 Hz, H-3), 7.93 (dd, 1H, J = 8.1, 1.5 Hz, H-6 ), 12.84 (s,
H, -OH). 1 C NMR (151 MHz, CDCl )
3
δ
= 56.42 (C-2 -OCH and C-6 -OCH ), 61.19 (C-4 -OCH ),
00
00
0
00
1
1
1
3
3
3
3
00
00
00
0
0
0
06.08 (C-2 , C-6 ), 118.81 (C-3 ), 118.95 (C-5 ), 119.41 (C-2), 120.16 (C-1 ), 129.74 (C-6 ), 130.20 (C-1 ),
36.52 (C-4 ), 140.97 (C-4 ), 145.79 (C-3), 153.68 (C-3 , C-5 ), 163.73 (C-2 ), 193.67 (C-1).
0
00
00
00
0
0
0
0
00 00 00
1
2
-Hydroxy-4 ,6 ,3 ,4 ,5 -pentamethoxychalcone (
5
). H NMR (600 MHz) (CDCl )
δ
(ppm): 3.84 (s, 3H,
3
0
00
0
00
00
C-4 -OCH ), 3.90 (s, 3H, C-4 -OCH ), 3.91 (s, 3H, C-6 -OCH ), 3.91 (s, 6H, C-2 -OCH and C-6 -OCH ),
3
3
3
3
3
0
0
00
00
5.96 (d, 1H, J = 2.3 Hz, H-5 ), 6.11 (d, 1H, J = 2.3 Hz, H-3 ), 6.84 (s, 2H, H-2 , H-6 ), 7.70 (d, 1H,
13
J = 15.5 Hz, H-3), 7.80 (d, 1H, J = 15.5 Hz, H-2), 14.31 (s, 1H, -OH). C NMR (151 MHz, CDCl )
C-4 -OCH ), 55.94 (C-6 -OCH ), 56.27 (C-2 -OCH and C-6 -OCH ), 61.15 (C-4 -OCH ), 91.46 (C-5 ),
δ
= 55.75
3
0
0
00
00
00
0
(
3
3
00
3
3
3
00
0
00
0
00
93.97 (C-3 ), 105.69 (C-2 , C-6 ), 106.45 (C-1 ), 127.06 (C-2), 131.28 (C-1 ), 140.21 (C-4 ), 142.54 (C-3),
00
00
0
0
0
1
53.54 (C-3 , C-5 ), 162.53 (C-6 ), 166.34 (C-4 ), 169.56 (C-2 ), 192.49 (C-1).
0
00 00
1
00
2
-Hydroxy-2 ,5 -dimethoxychalcone (
6
). H NMR (600 MHz) (CDCl )
δ
(ppm): 3.83 (s, 3H, C-5 -OCH ),
3
3
0
0
00
0
3
.90 (s, 3H, C-2 -OCH ), 6.90 (d, 1H, J = 9.0 Hz, H-3 ), 6.94 (ddd, 1H, J = 9.0, 7.2, 0.9 Hz, H-5 ), 6.97 (dd,
3
0
0
0
00
1
1
1
(
1
H, J = 9.0, 3.1 Hz, H-4 ), 7.02 (dd, 1H, J = 8.4, 1.0 Hz, H-3 ), 7.17 (d, 1H, J = 3.1 Hz, H-6 ), 7.49 (ddd,
0
0
H, J = 8.3, 7.1, 1.5 Hz, H-4 ), 7.75 (d, 1H, J = 15.6 Hz, H-2), 7.92 (dd, 1H, J = 8.1, 1.6 Hz, H-6 ), 8.19 (d,
13
00
H, J = 15.6 Hz, H-3), 12.92 (s, 1H, -OH). C NMR (151 MHz, CDCl ) δ = 56.02 (C-5 -OCH ), 56.27
3
3
00 00 00 00 0 0 0
C-2 -OCH ), 112.63 (C-3 ), 114.32 (C-6 ), 117.80 (C-4 ), 118.70 (C-3 ), 118.91 (C-5 ), 120.31 (C-1 ),
3
0
0
0
0
00
21.17 (C-2), 124.31 (C-1 ), 129.85 (C-6 ), 136.34 (C-4 ), 141.05 (C-3), 153.65 (C-5 ), 153.73 (C-2), 163.71
C-2 ), 194.35 (C-1).
0
(
0
0
0
1
0
2
-Hydroxy-4 ,6 -dimethoxychalcone (
7
). H NMR (600 MHz) (CDCl )
δ
(ppm): 3.83 (s, 3H, C-4 -OCH ),
3
3
0
0
0
3.92 (s, 3H, C-6 -OCH ), 5.97 (d, 1H, J = 2.4 Hz, H-5 ), 6.11 (d, 1H, J = 2.4 Hz, H-3 ), 7.36–7.43 (m, 3H,
3

Molecules 2020, 25, 4362
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0
0
00
00
00
00
H-3 , H-4 , H-5 ), 7.58–7.62 (m, 2H, H-2 , H-6 ), 7.79 (d, 1H, J = 15.6 Hz, H-3), 7.91 (d, 1H, J = 15.6 Hz,
13
00 0
δ = 55.72 (C-4 -OCH ), 55.99 (C-6 -OCH ), 91.41
3 3
H-2), 14.29 (s, 1H, -OH). C NMR (151 MHz, CDCl )
3
0 0 0 00 00 00 00
C-5 ), 93.93 (C-3 ), 106.47 (C-1 ), 127.66 (C-2), 128.48 (C-2 and C-6 ), 129.00 (C-3 and C-5 ), 130.18
(
(
0
0
00
0
0
0
C-4 ), 135.70 (C-1 ), 142.45 (C-3), 162.64 (C-6 ), 166.37 (C-4 ), 168.53 (C-2 ), 192.77 (C-1).
0
1
2
-Hydroxychalcone (
8
). H NMR (600 MHz) (CDCl )
δ
(ppm): 3.94 (s 3H, -OCH ), 6.94 (td, 1H, J = 7.8,
3
3
0
00
00
0.6 Hz, H-5 ), 6.96 (d, 1H, J = 8.8 Hz, H-3 ), 7.01 (t, 1H, J = 7.5 Hz, H-5 ), 7.04 (dd, 1H, J = 8.3, 0.6 Hz,
0
00
0
H-3 ), 7.41 (ddd, 1H, J = 8.6, 7.9, 1.5 Hz, H-4 ), 7.49 (t, 1H, J =8.8, 7.8, 1.4 Hz, H-4 ); 7.65 (dd, 1H, J = 7.6,
00
0
1
.3 Hz, H-6 ), 7.78 (d, 1H, J = 15.6 Hz, H-2), 7.93 (dd, 1H, J = 8.0, 1.3 Hz, H-6 ), 8.23 (d, 1H, J = 15.6 Hz,
13
00 0
H-3); 12.95 (s, 1H, -OH). C NMR (151 MHz, CDCl ) δ = 55.73 (-OCH ), 111.45 (C-3 ), 118.67 (C-3 ),
3
3
0 0 00 00 0 00
18.88 (C-5 ), 120.33 (C-1 ), 120.94 (C-2), 120.94 (C-5 ), 123.76 (C-1 ), 129.77 (C-6 ), 129.84 (C-6 ), 132.34
1
(
00 0 00 0
C-4 ), 136.28 (C-4 ), 141.27 (C-3), 159.17 (C-2 ), 163.71 (C-2 ), 194.44 (C-1).
4
.2. Cell Lines and Cell Culture
The study involved the following normal, non-cancerous NIH/3T3 (fibroblasts) and J774E.1
(macrophages) murine cell lines and a panel of canine cancer cell lines: CLBL-1 (B-cell lymphoma),
GL-1 (B/T-cell leukemia), CLB70 (B-cell chronic lymphocytic leukemia) and CNK89 (NK-cell lymphoma).
The NIH/3T3 and J774E.1 cell lines were bought from the American Type Culture Collection (ATCC,
Rockville, MD, USA). CLBL-1 was obtained from Barbara C. Ruetgen from the Institute of Immunology,
Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria [40], GL-1 was
obtained from Yasuhito Fujino and Hajime Tsujimoto from the University of Tokyo, Department of
Veterinary Internal Medicine [41,42] while CLB70 [43] and CNK89 (Grudzie n´ et al., in press) were
established in our laboratory.
The cell lines were maintained in RPMI 1640 (J774E.1, NIH/3T3, CLBL-1 and GL-1) (Institute of
Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland) or Advanced
RPMI (Gibco, Grand Island, NY, USA) (CLB70 and CNK89) culture medium supplemented with 2 mM
L-glutamine (Sigma Aldrich, Steinheim, Germany), 100 U/mL penicillin, 100
Sigma Aldrich, Steinheim, Germany), and 10–20% heat-inactivated fetal bovine serum—FBS (Gibco,
Grand Island, NY, USA).
µg/mL streptomycin
(
4
.3. Cell Proliferation Assay
The cell proliferation was determined using the MTT test (Sigma Aldrich, Steinheim, Germany).
4
5
In brief, 1
×
10 (NIH/3T3 and J77.4.E) or 1
×
10 (canine cancer cell lines) cells per well were seeded
in a 96-well-plate (Thermo Fisher Scientific, Denmark), and the tested chalcones were added in the
increasing concentrations (1.5, 3.125, 6.25, 12.5, 25 and 50 M). After incubation for 48 h, 20 L of MTT
µ
µ
solution (5 mg/mL) were added to each well. After the contents dissolved, the optical density of wells
was measured with a microplate reader (Spark, Tecan) at a reference wavelength of 570 nm. The values
were means from three independent experiments (three wells each).
4
.4. Western Blotting
6
2
For Western blot analysis the cells were seeded in a total of 5
×
10 cells per 25 cm cell culture
flasks and two selected concentration of the compounds were added. After 48 h incubation the cells
were harvested, rinsed with cold PBS, suspended in a lysis buffer (50 mM Tris–HCl pH 7.5, 100 mM
NaCl, 1% NP-40 and protease inhibitors set) and incubated for 20 min on ice. The suspensions were
centrifuged at 10,000 rpm at 4
added to clear supernatants, and the samples were boiled at 95
on 10–15% gel. For the tests, the resolved proteins were transferred to a PVDF membrane (Millipore,
Billerica, MA, USA), using Semidry Transfer Cell (Bio-Rad, Hercules, CA, USA). After the transfer, the
◦
C for 12 min. Then, a sodium dodecyl sulfate (SDS) sample buffer was
◦
C for 5 min and subjected to SDS-PAGE
membrane was blocked overnight with 1% casein in TBS at 4
antibody (dilution 1:2000) (Santa Cruz Biotechnology, USA) at room temperature for 1 h, followed
◦
C, and then incubated with primary

Molecules 2020, 25, 4362
12 of 15
by secondary horseradish peroxidase-labelled antibody (Dako, Denmark). The bound antibodies
were visualized using ChemiDoc Touch Instruments (BioRad, Hercules, CA, USA). The anti- H2A.X
ab26350) antibody was from Abcam (Cambridge, UK) while the anti- actin (C-4) antibody was from
Santa Cruz Biotechnology (Santa Cruz, CA, USA).
γ
(
β
4
.5. Apoptosis Assays by Flow Cytometry
After seeding at the same density as for cytotoxicity tests in 96-well plates (TPP, Trasadingen,
Switzerland) the cells were incubated for 48 h with two selected concentrations of the tested chalcones
5 and 10 µM). For the phosphatidylserine externalization and membrane integrity test, cells were
collected, suspended in a binding buffer and stained with Annexin V-FITC and PI (final PI concentration
g/mL). To evaluate caspases 3/7 and 8 the cells were harvested, washed twice with PBS and
(
1
µ
stained according to the manufacturer’s instructions. Briefly, for active caspase 3/7 detection
CellEventCaspase-3/7 Green Detection Reagent was added to the samples. For the detection of
active caspase 8, FITC-IETD-fmk was added to the samples and the cells were incubated for 0.5 h.
Then, the cells were washed twice and re-suspended in wash buffer. Flow cytometric analysis was
immediately performed using a flow cytometer (FACS Calibur; Becton Dickinson, Biosciences, San Jose,
CA, USA). CellQuest 3.lf. Software (Becton Dickinson, San Jose, CA, USA) was used for data analysis.
4
.6. Statistical Analysis
All data are shown as means with standard deviations (SD). Statistical differences were analyzed
using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical analysis was
performed with STATISTICA version 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA).
The results were considered significant at p < 0.05.
5
. Conclusions
Modification of the chemical structure of 2 -hydroxychalcone leads to the formation of a derivatives
0
with various antitumor activity in vitro. Such modification consists of creation the analogues containing
a methoxy group instead of a hydroxyl group caused both a reduction and an increase in the antitumor
strength of the action depending on the number of groups added and their positions. However, the
basic mechanism of action did not change, and all the derivatives obtained exerted antiproliferative
and proapoptotic activity and, have the capacity to induce DNA damage, but to varying degrees.
The cytotoxic effect of the parent compound and derived derivatives was stronger in relation to cancer
cell lines than to normal ones. Further research is needed into the possibility of using chalcones as an
adjuvant treatment of canine lymphoma or leukemia.
1
0
00
Supplementary Materials: The following are available online. Figure S1: H NMR spectral of 2 -hydroxy-2 -
1
0 00
methoxychalcone (1) (CDCl3, 600 MHz), Figure S2: Part of the H NMR spectral 2 -hydroxy-2 -methoxychalcone
) (CDCl3, 600 MHz), Figure S3: C NMR spectral of 2 -hydroxy-2 -methoxychalcone (
1) (CDCl3, 151 MHz), Figure S5: HSQC spectral
of 2 -hydroxy-2 -methoxychalcone (1) (CDCl3, 151 MHz), Figure S6: H NMR spectral of 2 -hydroxy-3 -
methoxychalcone ( ) (CDCl3, 600 MHz), Figure S7: Part of the H NMR spectral 2 -hydroxy-3 -methoxychalcone
1
3
0
00
(
1
1
) (CDCl3, 151 MHz),
0
00
Figure S4: COSY spectral of 2 -hydroxy-2 -methoxychalcone (
0
00
1
0 00
1
0
00
2
1
3
0
00
(
2
) (CDCl3, 600 MHz), Figure S8:
C NMR spectral of 2 -hydroxy-3 -methoxychalcone (
2
) (CDCl3,
) (CDCl3, 151 MHz), Figure S10:
) (CDCl3, 600 MHz), Figure S11: Part of the
0
00
1
51 MHz), Figure S9: HSQC spectral of 2 -hydroxy-3 -methoxychalcone (
2
1
1
0
00
H NMR spectral of 2 -hydroxy-4 -methoxychalcone (
3
0
00
13
H NMR spectral 2 -hydroxy-4 -methoxychalcone (
3
) (CDCl3, 600 MHz), Figure S12:
C NMR spectral
0
00
0 00
3) (CDCl3, 151 MHz), Figure S13: COSY spectral of 2 -hydroxy-4 -
of 2 -hydroxy-4 -methoxychalcone (
0 00
) (CDCl3, 151 MHz), Figure S14: HSQC spectral of 2 -hydroxy-4 -methoxychalcone
methoxychalcone (
(
(
3
1
0 00 00 00
H NMR spectral of 2 -hydroxy-3 ,4 ,5 -trimethoxychalcone (4)
3
) (CDCl3, 151 MHz), Figure S15:
1
0
00 00 00
0 00 00 00
C NMR spectral of 2 -hydroxy-3 ,4 ,5 -trimethoxychalcone (4)
CDCl3, 600 MHz), Figure S16: Part of the H NMR spectral 2 -hydroxy-3 ,4 ,5 -trimethoxychalcone
13
(
4
) (CDCl3, 600 MHz), Figure S17:
0
00 00 00
(
CDCl3, 151 MHz), Figure S18: COSY spectral of 2 -hydroxy-3 ,4 ,5 -trimethoxychalcone (
4
) (CDCl3,
) (CDCl3, 151 MHz),
5) (CDCl3, 600 MHz),
0 00 00 00
51 MHz), Figure S19: HSQC spectral of 2 -hydroxy-3 ,4 ,5 -trimethoxychalcone (4
1
1
0
0
0
00 00 00
Figure S20: H NMR spectral of 2 -hydroxy-4 ,6 ,3 ,4 ,5 -pentamethoxychalcone (

Molecules 2020, 25, 4362
13 of 15
1
0
0
0
00 00 00
Figure S21: Part of the H NMR spectral 2 -hydroxy-4 ,6 ,3 ,4 ,5 -pentamethoxychalcone (
5
5
) (CDCl3,
) (CDCl3,
13
0
0
0
00 00 00
6
1
1
00 MHz), Figure S22:
C NMR spectral of 2 -hydroxy-4 ,6 ,3 ,4 ,5 -pentamethoxychalcone (
0
0
0
00 00 00
51 MHz), Figure S23: HSQC spectral of 2 -hydroxy-4 ,6 ,3 ,4 ,5 -pentamethoxychalcone (
5) (CDCl3,
0
0
0
00 00 00
51 MHz), Figure S24 HMBC spectral of 2 -hydroxy-4 ,6 ,3 ,4 ,5 -pentamethoxychalcone (
5
) (CDCl3, 151 MHz),
) (CDCl3, 600 MHz), Figure S26:
) (CDCl3, 600 MHz), Figure S27:
) (CDCl3, 151 MHz), Figure S28: COSY
) (CDCl3, 151 MHz), Figure S29: HSQC spectral of
-hydroxy-2 ,5 -dimethoxychalcone (6) (CDCl3, 151 MHz).
1
0
00 00
00 00
Figure S25: H NMR spectral of 2 -hydroxy-2 ,5 -dimethoxychalcone (
6
1
0
Part of the H NMR spectral 2 -hydroxy-2 ,5 -dimethoxychalcone (
6
1
3
0
00 00
C NMR spectral of 2 -hydroxy-2 ,5 -dimethoxychalcone (
6
0
00 00
spectral of 2 -hydroxy-2 ,5 -dimethoxychalcone (
6
0
00 00
2
Author Contributions: Conceptualization, methodology software validation, formal analysis, data curation, A.P.;
investigation, A.P., M.H., B.H.S. and M.Ł.; resources, M.Ł. and E.K.; writing-original draft preparation, A.P. and
E.K.; writing—review and editing, T.J.; visualization, M.H. and B.H.S.; supervision, B.O.-M. and T.J.; project
administration, A.P.; funding acquisition, A.P. All authors have read and agreed to the published version of
the manuscript.
Funding: This publication was financed under the Leading Research Groups support project from the subsidy
increased for the period 2020–2025 in the amount of 2% of the subsidy referred to in Art. 387 (3) of the Law of
2
0 July 2018 on Higher Education and Science, obtained in 2019.
Acknowledgments: We would like to thank B.C. Ruetgen (Institute of Immunology, Department of Pathobiology,
University of Veterinary Medicine Vienna) for providing CLBL-1 cell line and Y. Fujino and H. Tsujimoto (University
of Tokyo, Department of Veterinary Internal Medicine) for providing GL-1 cell line.
Conflicts of Interest: The authors disclose nonfinancial or personal relationships with other people or organizations
that could influence (bias) their work.
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