Antiproliferative effects of chalcones on T cell acute lymphoblastic leukemia-derived cells: Role of PKCβ

Article abstract of DOI:10.1002/ardp.202000062

In this study, a series of 20 chalcone derivatives was synthesized, and their antiproliferative activity was tested against the human T cell acute lymphoblastic leukemia-derived cell line, CCRF-CEM. On the basis of the structural features of the most active compounds, a new library of chalcone derivatives, according to the structure–activity relationship design, was synthesized, and their antiproliferative activity was tested against the same cancer cell line. Furthermore, four of these derivatives (compounds 3, 4, 8, 28), based on lower IC₅₀ values (between 6.1 and 8.9 μM), were selected for further investigation regarding the modulation of the protein expression of RACK1 (receptor for activated C kinase), protein kinase C (PKC)α and PKCβ, and their action on the cell cycle level. The cell cycle analysis indicated a block in the G0/G1 phase for all four compounds, with a statistically significant decrease in the percentage of cells in the S phase, with no indication of apoptosis (sub-G0/G1 phase). Compounds 4 and 8 showed a statistically significant reduction in the expression of PKCα and an increase in PKCβ, which together with the demonstration of an antiproliferative role of PKCβ, as assessed by treating cells with a selective PKCβ activator, indicated that the observed antiproliferative effect is likely to be mediated through PKCβ induction.

Full text of DOI:10.1002/ardp.202000062

Received: 2 March 2020
Revised: 18 April 2020
Accepted: 22 April 2020
|
|
DOI: 10.1002/ardp.202000062
F U L L P A P E R
Antiproliferative effects of chalcones on T cell acute
lymphoblastic leukemia‐derived cells: Role of PKCβ
1
2
1
3
Emanuela Corsini
| Giorgio Facchetti
| Sara Esposito | Ambra Maddalon
|
2
2
Isabella Rimoldi
| Michael S. Christodoulou
1
Laboratory of Toxicology, Dipartimento di
Scienze Politiche ed Ambientali, Università
degli Studi di Milano, Milano, Italy
Abstract
In this study, a series of 20 chalcone derivatives was synthesized, and their
antiproliferative activity was tested against the human T cell acute lymphoblastic
leukemia‐derived cell line, CCRF‐CEM. On the basis of the structural features of the
most active compounds, a new library of chalcone derivatives, according to
the structure–activity relationship design, was synthesized, and their anti-
proliferative activity was tested against the same cancer cell line. Furthermore, four
of these derivatives (compounds 3, 4, 8, 28), based on lower IC50 values (between
2
DISFARM, Sezione di Chimica Generale e
Organica “A. Marchesini”, Università degli
Studi di Milano, Milano, Italy
3
Laboratory of Toxicology, Dipartimento di
Scienze Farmacologiche e Biomolecolari,
Università degli Studi di Milano, Milano, Italy
Correspondence
Isabella Rimoldi and Michael S. Christodoulou,
DISFARM, Sezione di Chimica Generale e
Organica “A. Marchesini,” Università degli
Studi di Milano, Via Venezian, 21, 20133
Milano, Italy.
6
.1 and 8.9 μM), were selected for further investigation regarding the modulation of
the protein expression of RACK1 (receptor for activated C kinase), protein kinase
C (PKC)α and PKCβ, and their action on the cell cycle level. The cell cycle analysis
indicated a block in the G0/G1 phase for all four compounds, with a statistically
significant decrease in the percentage of cells in the S phase, with no indication of
apoptosis (sub‐G0/G1 phase). Compounds 4 and 8 showed a statistically significant
reduction in the expression of PKCα and an increase in PKCβ, which together with
the demonstration of an antiproliferative role of PKCβ, as assessed by treating cells
with a selective PKCβ activator, indicated that the observed antiproliferative effect
is likely to be mediated through PKCβ induction.
Email: isabella.rimoldi@unimi.it (I. R.) and
michail.christodoulou@unimi.it (M. S. C.)
K E Y W O R D S
anticancer activity, antiproliferative agents, cancer treatment, inhibitors, substituent effect
1
| INTRODUCTION
worldwide are projected to reach over 13 million in 2030 (World
Health Organization, https://www.who.int/cancer/resources/keyfacts/
Chalcones are naturally occurring compounds produced by plants,
precursors of flavonoids; their structure includes two aromatic rings
joined by an α,β‐unsaturated carbonyl system (Figure 1). They have
been reported to have a wide application in the pharmacological
area, due to their diverse biological activities, such as antimicrobial,
antibacterial, antifungal, anti‐inflammatory, antinociceptive proper-
ties, as well as antitumor activity that has been observed in different
types of cancer, including leukemia, non‐small‐cell lung cancer, colon
en/, visited Jan 8, 2020). The new generations of anticancer drugs are
designed to target signals that promote or regulate the cell cycle,
growth factors or their receptors, and pathways affecting DNA repair
[2]
and cell death, rather than directly targeting DNA synthesis. Many
different cellular targets of chalcones were highlighted in cancer cells,
[3]
as repressing Aurora Kinase A gene in breast cancer or revealing
their potential reversal activity against P‐gp‐mediated multidrug re-
[4,5]
sistence, involved in resistance mechanism,
as inducing poly(ADP‐
[
1,2]
cancer, prostate cancer, and breast cancer.
ribose) polymerase cleavage and stabilizing p53 in a dose‐dependent
[6]
Cancer is a matter of concern in medicinal chemistry and an in-
creasing burden to the population, where the deaths from cancer
manner in colorectal carcinoma. Recently, chalcone derivatives have
[7,8]
been shown to inhibit Notch signaling in CCRF‐CEM.
Arch Pharm. 2020;e2000062.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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antiproliferative effects and possible modulation of PKC and RACK1
expression in the human T‐ALL‐derived cell line, CCRF‐CEM.
2
| RESULTS AND DISCUSSION
FIGURE 1 The general structure of chalcone and synthesized
2.1 | Chemistry
derivatives
Chalcone derivatives were synthesized using the synthetic metho-
[
30,31]
The T cell acute lymphoblastic leukemia (T‐ALL) is one of the
most aggressive blood cancers, which account for approximately 15%
of pediatric and 25% of adult acute lymphoblastic leukemias, which is
dology described previously.
Briefly, an aqueous solution of
sodium hydroxide was added slowly to a methanol solution with an
appropriate amount of acetophenone. After the solution was cooled
to room temperature, an appropriate amount of appropriate ben-
zaldehyde was added. The mixture was stirred at room temperature
overnight to provide the corresponding chalcones in good yields
(Tables 1 and 2). The synthesis of products was easy to achieve, with
a good grade of purity. On the contrary, in this study, as well as in
[
9]
curable in most cases. However, chemotherapy resistance was
observed in patients with T‐ALL, leading to treatment failure and
early relapse, and the causes of this resistance are not completely
[
10]
elucidated.
In a recent study, it has been shown that the over-
expression of the receptor of activated C kinase 1 (RACK1) was
responsible for chemotherapy resistance of T‐ALL, increasing the
activity of isoform α of protein kinase C (PKCα), which led to a re-
2
previous studies, the chalcone derivative with a p‐NO group on ring
A could not be synthesized. All the products were characterized by
the nuclear magnetic resonance (NMR) analysis, in which the pre-
sence of the olefinic double‐bond protons with a coupling constant J
of 15.6 Hz confirmed the trans stereochemistry.
[11]
duction in the level of Apaf‐1, caspase 3, and FEM1b.
RACK1 was first identified in the early 1990s, and it has been
[
12]
named so due to its interaction with protein kinase C (PKC).
RACK1 is now recognized as a multiple‐target‐scaffolding protein
involved in several key biological events, including development,
[
13,14]
immune response, neuronal activity, and cancer.
Due to its
2.2 | Biological activity
plethora of interaction proteins, RACK1 controls essential cellular
processes, such as transcription and translation, cell proliferation
The antiproliferative effects of the first library of chalcone deriva-
tives 1–20 were evaluated on CCRF‐CEM cells, that is, T lympho-
blasts (T‐ALL) from a 4‐year‐old girl isolated in 1964. For this blood
tumor cell line, a duplication time of approximately 24 hr was re-
[
13]
and growth, as well as cell spreading and cell–cell interactions.
Regarding PKC, RACK1 could serve as a receptor for activated
PKCβ and other PKC isoforms, including PKCδ and PKCμ. The
binding of RACK1 to PKC leads to an increase in kinase activity
and the shuttling of activated PKC to its correct cellular location.
It has been early recognized that PKC activity and/or modulation
of its isoenzyme expression have been implicated in the regulation,
both increase and decrease, of malignant cell proliferation, apop-
[
32]
ported.
Cells were treated for 72 hr, allowing three cell cycles,
with increasing noncytotoxic concentrations of the synthesized
chalcones (0.16, 0.8, 4, 20, and 100 μM) or dimethyl sulfoxide
(DMSO) as vehicle control. The absence of cytotoxicity was assessed
by lactate dehydrogenase leakage in a preliminary experiment,
testing compounds at a 100‐μM concentration (data are not shown).
After 72 hr, cell numbers were counted at the Coulter Counter, and
the IC50 values were calculated by the linear regression analysis of
data (IC50 is the concentration resulting in 50% inhibition of cell
proliferation); the results are presented in Table 1.
[
15‐18]
tosis, tumor invasiveness, and resistance phenotype.
It is
currently recognized that PKC isoforms have a complex, not uni-
vocal, role in cancer, meaning that they can have both detrimental
or protective roles, depending on the type of tumor considered.
Therefore, PKC modulators must be used with caution, taking into
account the specific role of the different isoforms in the different
tumors. Many PKC inhibitors have indeed entered clinical trials,
but they have yielded very limited success so far. Also, as more
recent studies have demonstrated that PKC can also function as
tumor suppressors, future clinical efforts should focus on restor-
Cells were treated for 72 hr with increasing noncytotoxic con-
centrations of the synthetic chalcones. Cell proliferation was as-
sessed by cell counting at the Coulter Counter. The IC50 (μM) values
were calculated by the linear regression analysis of data in three
independent experiments. IC50 is reported as mean ± standard
deviation (SD).
[
19]
ing, rather than inhibiting, PKC activity.
Similar to PKC, RACK1
aberrant expression, prooncogenic or antioncogenic effects, and
contribution to the various stages of cell migration and invasion
On the basis of the results of the aforementioned screening and
considering the antiproliferative activity of compounds 3, 4, 5, 8, and
9, which showed the lowest IC50 values (ranging from 6.1 to 8.9 μM),
the second library of chalcone derivatives was created, compounds
21–32, based on the presence of the same electron‐withdrawing or
electron‐donating substituents of compounds 3, 4, 5, 8, and 9 on the
aromatic rings. This second library will elucidate the importance of
[
14,20,21]
have been described in various cancers.
Continuing our research in the synthesis of anticancer com-
[22‐29]
pounds,
the aim of this study was to synthesize chalcone deri-
vatives containing electron‐withdrawing or electron‐donating
substituents on both the aromatic rings and evaluate their

CORSINI ET AL.
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TABLE 1 The effect of chalcone derivatives 1–20 on the proliferation of CCRF‐CEM cells
1
2
1
2
Compound
R
R
IC50 (μM)
>30
Compound
R
R
IC50 (μM)
>30
1
2
3
4
5
6
7
8
9
o‐F
H
11
12
13
14
15
16
17
18
19
20
p‐Me
p‐Me
p‐Me
p‐Me
p‐Me
p‐OMe
p‐OBn
p‐Cl
o,p‐Cl
2
H
>30
p‐Ph
>30
m‐NO
o‐CF
2
H
8.9 ± 1.2
7.5 ± 1.4
7.9 ± 1.2
>30
p‐Me
p‐OMe
>30
3
H
>30
p‐OMe
H
p‐NO
p‐Me
p‐Me
p‐Me
2
>30
2‐naphthyl
H
>30
H
p‐Me
m‐NO
>30
>30
H
2
6.1 ± 1.1
6.8 ± 1.1
>30
m‐NO
2
>30
H
o‐OMe
p‐Br
p‐OMe
p‐Cl
m‐NO
2
>30
1
0
p‐Me
p‐OMe
>30
the single substituent in the antiproliferative activity, based on
structure–activity relationships (SARs). The antiproliferative activity
of the new library against CCRF‐CEM cells is presented in Table 2.
The partition coefficient, calculated for all the new compounds and
for 3, 4, 5, 8, and 9, showed good values for almost all of them.
Cells were treated for 72 hr with increasing noncytotoxic con-
centrations of the synthetic chalcones. Cell proliferation was as-
sessed by cell counting at the Coulter Counter. The IC50 (μM) values
were calculated by linear regression analysis of data in three in-
dependent experiments. IC50 is reported as mean ± SD.
position, and the OMe group at the para position (compounds 3, 4,
1
and 5, respectively), as R substituents, was observed, whereas the
2 3
presence of the NO at the meta position, the CF at the ortho po-
sition, and the OMe group at the ortho position (compounds 8, 28,
2
and 9, respectively), as R substituents, was observed, which is
mandatory for the highest inhibition.
From the new library, we considered chalcones 3, 4, 8, and 28,
which contained the same substituents with resonance or inductive
electron‐withdrawing effect at the same positions but on the oppo-
site aromatic rings. These compounds were chosen for further ana-
lyses with the aim to investigate the mechanism underlying their
antiproliferative effects.
The new SAR experiments showed that for the lower IC50 values,
2 3
the presence of the NO at the meta position, the CF at the ortho
TABLE 2 The effect of chalcone derivatives 21–32 on the proliferation of CCRF‐CEM cells
1
2
1
2
Compound
R
R
IC50 (μM)
44.7 ± 22.9
8.9 ± 1.2
LogP
4.33
4.57
3.82
6.29
6.48
5.69
4.48
5.03
4.51
Compound
R
R
IC50 (μM)
14.7 ± 0.5
6.1 ± 1.1
LogP
3.41
5.19
4.87
5.9
2
3
2
4
2
2
2
2
5
1
o‐NO
m‐NO
p‐NO
2
H
H
H
H
H
H
H
H
H
27
8
H
H
H
H
H
H
H
H
o‐NO
m‐NO
o‐CF
m‐CF
p‐CF
2
2
2
2
2
26.2 ± 20.1
7.5 ± 1.4
28
29
30
9
3
11.4 ± 2.2
14.8 ± 3.2
49.5 ± 21.0
6.8 ± 1.1
o‐CF
3
3
3
4
5
6
m‐CF
3
14.0 ± 2.2
15.3 ± 1.2
13.7 ± 1.7
27.1 ± 1.8
7.9 ± 1.2
3
5.7
p‐CF
3
o‐OMe
m‐OMe
p‐OMe
4.58
5.26
3.99
o‐OMe
m‐OMe
p‐OMe
31
32
14.0 ± 3.7
20.4 ± 9.4

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Next, we investigated the effect of the selected compounds on
proliferation was evaluated at 72 hr. Results are shown in Figure 3.
A statistically significant decrease in PKCα expression was observed
for compounds 4 and 8 (Figure 3a), an increase in PKCβ expression
was observed for compounds 3, 4, and 8, and no changes in RACK1
were observed for all four compounds, indicating a selective action
rather than an unspecific effect on protein synthesis. To understand
the possible implication of the increased expression of PKCβ, Pseudo
RACK1, a peptide that has been demonstrated both in vitro and in
the cell cycle. The cell cycle was evaluated by flow cytometry, as-
sessing cellular DNA content after cell staining with propidium iodide
and deconvolution of the cellular DNA content frequency histo-
[
33]
grams,
which allow to investigate the distribution of cells in three
major phases of the cycle (G0/G1 vs. S vs. G2/M) and make it possible
to detect apoptotic cells with fractional DNA content (pre‐G0). The
results are presented in Figure 2. Consistent with the lack of cyto-
toxicity, there was no increase in % of pre‐G0 cells (Figure 2a),
whereas cell cycle arrest in G0/G1 was observed for all selected
compounds, characterized by a statistically significant increase in the
[34]
vivo to bind and activate PKC in the absence of PKC activators,
was used. Cells were treated for 72 hr with an increasing con-
centration of Pseudo RACK1, and cells were counted at the end of
the experiment (Figure 3d). Dose‐dependent inhibition of cell pro-
liferation was observed, suggesting that the increase in PKCβ in-
duced by compounds 3, 4, and 8 may probably explain the observed
decrease in cell proliferation. The significance of the reduction of
%
of cells in the G0/G1 phase (Figure 2b) and a concomitant re-
duction of the cells in the S phase (Figure 2c). Results of the cell cycle
analysis are consistent with the cytostatic effect, rather than the
induction of apoptosis.
In CCRF‐CEM the involvement of RACK1/PKC in chemotherapy
PKCα expression requires further studies, but consistent with the
[11]
[
11,17]
resistance has been described.
This, together with the role of
data published,
a possible hypothesis could be a loss of che-
[
14,19]
RACK1/PKC in cancer,
has led us to investigate the effect of
motherapy resistance, which could be of extreme importance in the
compounds 3, 4, 8, and 28 on the protein expression of PKCα, PKCβ,
and RACK1, and the role of PKCβ in cell proliferation by using the
selective activator Pseudo RACK1. For protein expression, com-
pounds were tested with the IC50 values for 48 hr. This early time
point was chosen to observe if changes in the expression levels
occurred before the time point of 72 hr, which was used to assess the
antiproliferative effect. The effect of Pseudo RACK1 on cell
treatment of T‐ALL.
3 | CONCLUSION
The preparation of two different libraries of chalcone derivatives
(the first for an initial screening and the second for a SAR
FIGURE 2 The cell cycle analysis after 72 hr of treatment with the selected compounds in CCRF‐CEM. Cells were treated for 72 hr with the
selected compounds tested with the IC50 values or with dimethyl sulfoxide as a vehicle control. Cells were fixed and processed according to the
propidium iodide (PI) staining protocol listed in Section 4. In total, 30,000 cells were analyzed using the flow cytometer. The cell cycle analysis
calculated pre‐G0, G0/G1, S, and G2 phases from PI histograms. Each value represents mean ± standard deviation of three independent
experiments. The statistical analysis was performed by analysis of variance, followed by Dunnett's multiple comparison test with *p < 0.05 and
**p < 0.01 versus control cells

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FIGURE 3 The effect of the selected chalcone derivatives on the α and β isoforms of protein kinase C (PKC), RACK1 expression, and
antiproliferative effect of PKCβ activation on CCRF‐CEM. (a–c) Effects on PKCα (a), PKCβ (b), and RACK1 (c) protein expression. Cells were
treated for 48 hr with compounds 28, 3, 4, and 8 with the IC50 values or with dimethyl sulfoxide as vehicle control. β‐Tubulin expression was
used to normalize expression. The image is a representative Western blot analysis. Each value represents the mean ± standard deviation (SD),
n = 3 independent experiments. The statistical analysis was performed with Dunnett's multiple comparison test with *p < 0.05 and **p < 0.01
versus control (Cont). (d) The effect of PKCβ activation on CCRF‐CEM proliferation. Cells were treated for 72 hr with increasing concentrations
of Pseudo RACK1 (0.5–1.5 μM), a selective peptide activator of PKCβ. Cell proliferation was assessed by cell counting at the Coulter Counter.
Each value represents the mean ± SD, n = 3 independent experiments. The statistical analysis was performed with Dunnett's multiple
comparison test with *p < 0.05 and **p < 0.01 versus control
identification), containing electron‐withdrawing or electron‐
donating substituents, was described in this study, and the
compounds were tested for their antiproliferative activity against
CCRF‐CEM cells. From the first screening, compounds 3, 4, 5, 8,
and 9, which showed the highest antiproliferative activity,
were selected as lead compounds for the synthesis of the second
library. After the second screening, chalcones 3, 4, 8, and 28,
bearing electron‐withdrawing substituents at the same positions
but on the opposite aromatic rings, were chosen for further
investigation. The cell cycle analysis of these compounds
expression was detected for compounds 3, 4, and 8. However,
no changes were observed in RACK1 for all four compounds,
suggesting a selective action rather than an effect on protein
synthesis. Dose‐dependent inhibition of the cell proliferation
with the peptide Pseudo RACK1 suggested that the increase in
PKCβ induced by compounds 3, 4, and 8 could explain the
decrease in cell proliferation. Even if further experiments
are needed to better characterize the effect on PKC isoforms,
the mechanisms underlying the observed modulatory effects,
that is, pretranscriptional or posttranscriptional effects, and
their implication in terms of multidrug resistance, compounds
4 and 8 have been identified as candidates for further in-
vestigation and development as an additional therapeutic option
in T‐ALL.
demonstrated
a cytostatic effect, rather than induction of
apoptosis. Moreover, regarding the protein expression of PKCα,
PKCβ, and RACK1, a significant decrease in PKCα expression was
observed for compounds 4 and 8, whereas an increase in PKCβ

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4
4
4
| EXPERIMENTAL
3
127.86, 123.88, and 122.12 ppm. MS (ESI) for C15H11NO : m/z
+
2
54.48 [M+H] .
.1 | Chemistry
(
1
E)‐1‐Phenyl‐3‐[2‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one (4)
.1.1 | General procedures
3
H NMR (300 MHz, CDCl ) δ = 8.14 (dd, J = 2.1 Hz, 15.6 Hz 1H),
8.03–7.95 (m, 2H), 7.82 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H),
All reactions were monitored by thin‐layer chromatography on silica
7.63–7.58 (m, 2H), 7.54–7.48 (m, 3H), and 7.43 (d, J = 15.6 Hz, 1H)
13
1
13
gel, with detection by ultraviolet light (254 nm). H NMR and
C
3
ppm. C NMR (75 MHz, CDCl ) δ = 190.28, 140.18, 140.16, 137.66,
NMR spectra were recorded with a Varian Oxford 300‐MHz spec-
trometer at 300 and 75 MHz, respectively. Mass spectrometry (MS)
analyses were performed by using a Thermo Finnigan (MA) LCQ
Advantage system MS spectrometer with an electrospray ionization
source and an “Ion Trap” mass analyzer. The MS spectra were
obtained by the direct infusion of a sample solution in MeOH under
ionization (ESI positive).
134.03, 132.99, 132.06, 129.63, 129.42, 129.01, 128.64, 127.93,
126.62, 126.35, 126.28, 126.20, 126.13, 125.75, and 122.12 ppm. MS
+
(ESI) for C16
11
H F
3
O: m/z 277.08 [M+H] .
(E)‐1‐Phenyl‐3‐[3‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one (23)
1
3
H NMR (300 MHz, CDCl ) δ = 8.04 (d, J = 7.2 Hz, 2H), 7.89–7.79
1
3
3
(m, 3H), and 7.68–7.50 (m, 6H) ppm. C NMR (75 MHz, CDCl )
The InChI keys of the investigated compounds, together with
some activity data, are provided as Supporting Information.
δ = 189.96, 142.74, 137.82, 135.72, 133.03, 131.74, 131.54, 131.31,
129.48, 128.69, 128.52, 126.83, 126.78, 126.73, 126.69, 125.59,
124.73, 124.68, 124.63, 124.58, 123.73, and 121.98 ppm. MS (ESI)
+
for C16H
11
F
3
O: m/z 277.44 [M+H] .
4
.1.2 | General procedure for the synthesis of
α,β‐unsaturated ketones
(E)‐1‐Phenyl‐3‐[3‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one (24)
1
3
H NMR (300 MHz, CDCl ) δ = 8.28 (d, J = 9.0 Hz, 2H), 8.05–8.02
To a methanol solution (30 ml) of acetophenone (5.0 mmol), an aqu-
eous solution of potassium hydroxide (5%, 25 ml) was slowly added at
(m, 2H), 7.80–7.78 (m, 2H), 7.67 (s, 1H), 7.63–7.60 (m, 1H), and
1
3
7.56–7.51 (m, 3H) ppm.
3
C NMR (75 MHz, CDCl ) δ = 188.21,
0
°C. An appropriate amount of benzaldehyde (6.0 mmol) was added
149.98, 139.94, 136.62, 133.54, 131.89, 131.06, 128.14, 128.02,
127.92, 127.64, 126.89, 126.55, 126.13, 126.09, 125.99, 125.93,
and the new solution was stirred overnight at room temperature. The
resulting solution was evaporated in vacuum, followed by the addi-
tion of EtOAc. Then the organic phase was washed with water and
11 3
125.01, and 121.87 ppm. MS (ESI) for C16H F O: m/z 277.36
+
[M+H] .
2 4
brine, dried with Na SO , filtered, and evaporated. The desired
product was obtained after purification by flash column
(E)‐3‐(2‐Methoxyphenyl)‐1‐phenylprop‐2‐en‐1‐one (25)
[
30]
1
chromatography.
3
H NMR (300 MHz, CDCl ) δ = 8.10 (d, J = 15.9 Hz, 1H), 7.90
(d, J = 7.3 Hz, 2H), 7.83 (d, J = 7.6 Hz, 1H), 7.75–7.68 (m, 1H),
(
1
E)‐3‐(2‐Nitrophenyl)‐1‐phenylprop‐2‐en‐1‐one (21)
7.55–7.48 (m, 2H), 7.41–7.31 (m, 3H), 7.23 (d, J = 15.7 Hz, 1H), and
13
3
H NMR (300 MHz, CDCl ) δ = 8.24–8.15 (m, 4H), 7.88 (d, J = 6.0 Hz,
3
3.86 (s, 3H) ppm. C NMR (75 MHz, CDCl ) δ = 188.55, 157.12,
1H), 7.84–7.82 (m, 1H), 7.76–7.71 (m, 1H), and 7.56–7.47 (m, 4H)
143.56, 137.19, 137.16, 135.66, 131.86, 131.13, 129.56, 128.18,
1
3
ppm. C NMR (75 MHz, CDCl
3
) δ = 188.13, 157.34, 148.30, 139.69,
121.99, 121.00, 115.91, 112.87, and 54.93 ppm. MS (ESI) for
+
1
1
36.71, 129.75, 129.61, 129.28, 128.80, 127.55, 127.42, 127.17,
C H
16 14
O
2
: m/z 239.41 [M+H] .
3
26.24, and 118.96 ppm. MS (ESI) for C15H11NO : m/z 254.37
+
[
M+H] .
(E)‐3‐(3‐Methoxyphenyl)‐1‐phenylprop‐2‐en‐1‐one (26)
1
H
3
NMR (300 MHz, CDCl ) δ = 8.03 (d, J = 6.9 Hz, 2H), 7.78
(
1
E)‐3‐(3‐Nitrophenyl)‐1‐phenylprop‐2‐en‐1‐one (3)
(d, J = 15.9 Hz, 1H), 7.54–7.46 (m, 4H), 7.35–7.30 (m, 1H), 7.25–7.23
H NMR (300 MHz, CDCl
3
) δ = 8.49 (s, 1H), 8.23 (d, J = 9.0 Hz, 1H),
(m, 1H), 7.16 (t, J = 1.5 Hz, 1H), 6.98–6.94 (m, 1H), and 3.84 (s, 3H)
1
3
8
7
1
1
2
.03 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 9.0 Hz, 1H), 7.84–7.79 (m, 1H), and
3
ppm. C NMR (75 MHz, CDCl ) δ = 190.47, 159.96, 144.70, 138.19,
1
3
.67–7.50 (m, 5H) ppm.
C NMR (75 MHz, CDCl
3
) δ = 189.58,
136.27, 132.76, 129.93, 128.60, 128.49, 122.41, 121.07, 116.29,
+
41.56, 137.56, 136.65, 134.24, 133.26, 130.70, 130.01, 128.77,
113.47, and 55.32 ppm. MS (ESI) for C16
H
14
O
2
: m/z 239.19 [M+H] .
3
28.56, 124.61, and 122.33 ppm. MS (ESI) for C15H11NO : m/z
+
54.61 [M+H] .
(E)‐3‐(4‐Methoxyphenyl)‐1‐phenylprop‐2‐en‐1‐one (5)
1
H
3
NMR (300 MHz, CDCl ) δ = 8.01 (d, J = 8.1 Hz, 2H), 7.79
(
1
E)‐3‐(4‐Nitrophenyl)‐1‐phenylprop‐2‐en‐1‐one (22)
(d, J = 15.9 Hz, 1H), 7.62–7.47 (m, 5H), 7.41 (d, J = 15.6 Hz, 1H), 6.93
13
H
NMR (300 MHz, CDCl
3
)
δ = 8.28 (d, J = 9.0 Hz, 2H), 8.03
3
(d, J = 8.7 Hz, 2H), and 3.85 (s, 3H) ppm. C NMR (75 MHz, CDCl )
(d, J = 9.0 Hz, 2H), 7.80 (d, J = 2.4 Hz, 2H), 7.78 (s, 1H), 7.67 (s, 1H),
δ = 190.55, 161.68, 144.65, 138.53, 132.49, 130.18, 129.04, 128.52,
1
3
and 7.63–7.51 (m, 3H) ppm. C NMR (75 MHz, CDCl
3
) δ = 188.85,
128.38, 128.00, 127.64, 119.85, 114.42, 113.61, and 55.62 ppm. MS
+
142.16, 137.33, 136.47, 133.98, 132.85, 130.96, 129.08, 128.52,
(ESI) for C16
H O
14 2
: m/z 239.33 [M+H] .

CORSINI ET AL.
7 of 9
|
(
1
E)‐1‐(2‐Nitrophenyl)‐3‐phenylprop‐2‐en‐1‐one (27)
(d, J = 15.6 Hz, 1H), 7.42–7.38 (m, 4H), 7.14 (dd, J = 3.0 Hz, 8.4 Hz),
13
H
NMR (300 MHz, CDCl
3
)
δ = 8.18 (d, J = 9.3 Hz, 1H), 7.76
3
and 3.85 (s, 3H) ppm. C NMR (75 MHz, CDCl ) δ = 189.72, 159.88,
(t, J = 8.7 Hz, 1H), 7.65 (t, J = 8.1 Hz, 1H), 7.52–7.48 (m, 3 H), 7.41–7.37
143.87, 138.02, 136.83, 133.07, 129.98, 128.73, 128.05, 123.41,
13
(m, 3H), 7.25 (d, J = 16.2 Hz, 1H), and 7.00 (d, J = 16.2 Hz, 1H) ppm.
C
14 2
122.14, 116.55, 114.71, and 54.97 ppm. MS (ESI) for C16H O : m/z
+
3
NMR (75 MHz, CDCl ) δ = 192.83, 146.25, 136.31, 133.98, 133.95,
239.45 [M+H] .
131.02, 130.56, 128.98, 128.79, 128.67, 128.52, 126.25, and
+
1
24.51 ppm. MS (ESI) for C15
H
11NO
3
: m/z 254.56 [M+H] .
(E)‐1‐(4‐Methoxyphenyl)‐3‐phenylprop‐2‐en‐1‐one (32)
1
3
H NMR (300 MHz, CDCl ) δ = 8.05–8.02 (m, 2H), 7.78 (d, J = 15.6 Hz,
(
1
E)‐1‐(3‐Nitrophenyl)‐3‐phenylprop‐2‐en‐1‐one (8)
1H), 7.65–7.61 (m, 2H), 7.53 (d, J = 15.6 Hz, 1H), 7.44–7.39 (m, 3H),
13
H NMR (300 MHz, CDCl
3
) δ = 8.83 (s, 1H), 8.38 (dd, J = 8.1 Hz,
3
6.98–6.96 (m, 2H), and 3.83 (s, 3H) ppm. C NMR (75 MHz, CDCl )
27.6 Hz, 2H), 7.89 (d, J = 15.6 Hz, 1H), 7.74, 7.66–7.56 (m, 3H), 7.51
δ = 189.12, 162.73, 145.87, 139.77, 133.12, 131.85, 129.65, 129.01,
1
3
(
d, J = 15.6 Hz, 1H), and 7.46–7.44 (m, 3H). C NMR (75 MHz,
CDCl ) δ = 187.97, 148.43, 146.73, 139.51, 134.31, 134.04, 131.17,
29.88, 129.08, 128.71, 127.01, 123.23, and 120.68 ppm. MS (ESI)
128.77, 128.45, 127.93, 118.85, 114.32, 113.87, and 54.69 ppm. MS
+
3
(ESI) for C16
H O
14 2
: m/z 239.17 [M+H] .
1
+
3
for C15H11NO : m/z 254.43 [M+H] .
4
.1.3 | Log Pow determination
(
E)‐3‐Phenyl‐1‐[2‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one (28)
1
H NMR (300 MHz, CDCl
H), 7.55–7.53 (m, 1H), 7.50 (dd, J = 2.0 Hz, 7.5 Hz, 2H), 7.43
d, J = 7.6 Hz, 1H), 7.40–7.35 (m, 3H), 7.31 (d, J = 15.6 Hz, 1H), 7.02
3
) δ = 7.97 (d, J = 8.9 Hz, 1H), 7.58 (t, J = 7.4 Hz,
A reversed‐phase high‐performance liquid chromatography (HPLC)
analysis was performed to correlate the hydrophobicity of the
compounds with their retention times. The chromatograms were
registered using a Partisil C18‐ODS reversed‐phase HPLC column at
25°C and water/acetonitrile (50:50) as mobile phase with KI as an
internal standard (flow rate: 1 ml/min; λ = 254 nm). The calibration
curve was realized in comparison with reference compounds chosen
1
(
1
3
(
d, J = 15.4 Hz, 1H) ppm. C NMR (75 MHz, DMSO) δ = 187.89, 146.12,
1
1
1
38.93, 138.51, 137.66, 135.05, 133.12, 131.67, 130.85, 130.03,
29.85, 129.66, 129.43, 129.12, 129.03, 128.88, 127.56, 125.13,
+
25.00, and 122.02 ppm. MS (ESI) for C16
H F
11 3
O: m/z 277.17 [M+H] .
[
35‐37]
in OECD guideline TG 107 (OECD TG 107).
(
1
E)‐3‐Phenyl‐1‐[3‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one (29)
H NMR (300 MHz, CDCl
3
) δ = 8.26 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H),
7
7
.89–7.84 (m, 2H), 7.69–7.64 (m, 2H), 7.51 (d, J = 15.6 Hz, 1H),
4.2 | Biological assays
4.2.1 | Cells
1
3
.45–7.44 (m, 2H), and 7.23–7.14 (m, 2H) ppm. C NMR (75 MHz,
DMSO) δ = 188.89, 145.56, 139.33, 138.73, 137.36, 134.95, 132.93,
1
1
31.33, 130.55, 129.93, 129.90, 129.85, 129.58, 129.34, 128.70,
28.20, 127.06, 125.35, 125.30, and 122.07 ppm. MS (ESI) for
For all experiments, the CCRF‐CEM cell line was used (CEM/C1;
ATCC CRL‐2265™, gift from Luca Mazzarella, European
+
C
16
11
H F
3
O: m/z 277.31 [M+H] .
a
Institute of Oncology, Department of Experimental Oncology,
5
(
E)‐3‐Phenyl‐1‐[4‐(trifluoromethyl)phenyl]prop‐2‐en‐1‐one (30)
Milan, Italy). Cells were seeded at a density of 6 × 10 cells/ml in
1
H NMR (300 MHz, CDCl
H), 7.54–7.40 (m, 3H), and 7.25 (d, J = 7.8 Hz, 2H) ppm. C NMR
75 MHz, CDCl ) δ = 187.51, 143.82, 139.92, 137.72, 134.15, 131.89,
3
) δ = 8.78–8.73 (m, 2H), 8.13 (d, J = 8.1 Hz,
Rosewell Park Memorial Inistitute 1640 medium containing 2 mM
L‐glutamine, 0.1 mg/ml streptomycin, 100 IU/ml penicillin, and
0.1% gentamycin, supplemented with 10% heat‐inactivated fetal
1
3
2
(
3
1
1
31.56, 127.94, 127.02, 127.00, 126.84, 125.94, 125.65, 125.82,
2
calf serum (media), and cultured at 37°C in 5% CO for 48 hr for
25.01, 124.99, 124.83, 124.06, and 122.12 ppm. MS (ESI) for
Western blot analysis (3 ml per sample) and for 72 hr for cell
proliferation or cell cycle analysis (1 ml per sample). Cell culture
media and all supplements were obtained from Sigma‐Aldrich Co.
(St. Louis, MO). Also, 50‐mM stock solutions were prepared for
each compound in DMSO. DMSO was used as vehicle control in
all experiments (0.1% final concentration in a culture medium).
Pseudo RACK1 activator of protein kinase Cβ (Pseudo RACK) was
obtained from Tocris Bioscience (Bristol, UK). The pseudosub-
strate consists of a peptide derived from the C2 domain of PKCβ
linked by a disulfide bridge to the Antennapedia domain vector
peptide, which ensures a rapid and effective uptake of the acti-
vator peptide. Once inside the cell, the disulfide bonds were
subjected to a reduction in the cytoplasm, leading to the release
of the activator peptide.
+
C
16
11
H F
3
O: m/z 277.54 [M+H] .
(
1
E)‐1‐(2‐Methoxyphenyl)‐3‐phenylprop‐2‐en‐1‐one (9)
H NMR (300 MHz, CDCl
3
) δ = 7.82 (d, J = 15.5 Hz, 1H), 7.65 (dd,
J = 2.0 Hz, 7.5 Hz, 1H), 7.58–7.55 (m, 2H), 7.48–7.44 (m, 1H), 7.38–7.33
13
(m, 4H), 7.04–7.01 (m, 1H), 6.98–6.93 (m, 1H), and 3.86 (s, 3H) ppm.
C
NMR (75 MHz, CDCl
3
) δ = 189.43, 156.97, 143.58, 138.18, 137.65,
136.44, 131.99, 131.76, 129.88, 128.10, 122.14, 121.14, 116.27,
+
14 2
112.76, and 55.32 ppm. MS (ESI) for C16H O : m/z 239.34 [M+H] .
(
1
E)‐1‐(3‐Methoxyphenyl)‐3‐phenylprop‐2‐en‐1‐one (31)
H NMR (300 MHz, CDCl
3
) δ = 8.03 (d, J = 15.6 Hz, 1H), 7.65–7.62
(m, 2H), 7.59 (dd, J = 1.8 Hz, 7.5 Hz, 1H), 7.56–7.54 (m, 1H), 7.52

8
of 9
CORSINI ET AL.
|
4
.2.2 | Cell proliferation assay
(Amersham, Little Chalfont, UK). Proteins were visualized using some
primary antibodies against PKCα, PKCβII, and RACK1, and developed
using enhanced chemiluminescence (Bio‐Rad). The images of the
blots were acquired with the Molecular Imager Gel Doc XR (Bio‐Rad).
The optical density of bands was calculated and analyzed by means of
the Image Lab version 4.0.1 (Bio‐Rad), normalized by β‐tubulin, and
results are expressed as a percentage of control.
Preliminary experiments were conducted to assess cell viability;
5
6
7
× 10 cells/ml were treated with 100 μM of each compound for
2 hr. Cell viability was evaluated by assessing lactate dehy-
drogenase leakage using a commercially available kit (Takara Bio Inc.,
Japan), and no cytotoxicity was observed (data are not shown). Cells
5
were seeded at a density of 6 × 10 cells/ml and incubated for 72 hr
with five concentrations of the compounds (maximum concentration
tested: 100 μM and four 1:5 dilutions) or DMSO as vehicle control.
After incubation, cells were counted at the Coulter Counter (Coulter
Electronics, Ltd., Luton, UK). From the dose–response curve and by
4.2.5 | Statistical analysis
All experiments were repeated at least three times. Data are ex-
pressed as mean ± SD. The statistical analysis was performed using
InStat software version 7.0 (GraphPad Software, La Jolla, CA). The
statistical significance was determined by analysis of variance, fol-
lowed by a multiple comparison test as indicated in the legends. The
effects were designated significant at p < 0.05.
the linear regression analysis of data, the IC50 values (IC50
=
effective chemical concentration required for inhibition reduced cell
proliferation by 50%, compared with vehicle‐exposed cultures) were
calculated for each compound.
5
To assess the role of PKCβ on cell proliferation, 6 × 10 cells/ml
were treated with increasing concentrations of Pseudo RACK for
72 hr. After incubation, cells were counted at the Coulter Counter,
CONFLICT OF INTERESTS
6
and results are expressed as a number of cells × 10 /ml.
The authors declare that there are no conflict of interests.
ORCID
4
.2.3 | Cell cycle analysis by DNA content (PI)
Emanuela Corsini
Giorgio Facchetti
Isabella Rimoldi
http://orcid.org/0000-0002-6927-5956
http://orcid.org/0000-0002-1260-1335
http://orcid.org/0000-0002-6210-0264
After 72‐hr incubation, cells were centrifuged at 1,200 rpm for 5 min
at 5°C, culture media were discarded, cells were washed with
phosphate‐buffered saline (PBS), and they were fixed overnight at
Michael S. Christodoulou
http://orcid.org/0000-0002-5098-3143
−
20°C in 70% ethanol in PBS. Cells were then centrifuged at
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How to cite this article: Corsini E, Facchetti G, Esposito S,
Maddalon A, Rimoldi I, Christodoulou MS. Antiproliferative
effects of chalcones on T cell acute lymphoblastic leukemia‐
derived cells: Role of PKCβ. Arch Pharm. 2020;e2000062.
https://doi.org/10.1002/ardp.202000062
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