Anti-leukemic properties of aplysinopsin derivative ee-84 alone and combined to bh3 mimetic a-1210477

Article abstract of DOI:10.3390/md19060285

Aplysinopsins are a class of marine indole alkaloids that exhibit a wide range of biological activities. Although both the indole and N-benzyl moieties of aplysinopsins are known to possess antiproliferative activity against cancer cells, their mechanism of action remains unclear. Through in vitro and in vivo proliferation and viability screening of newly synthesized aplysinopsin analogs on myelogenous leukemia cell lines and zebrafish toxicity tests, as well as analysis of differential toxicity in noncancerous RPMI 1788 cells and PBMCs, we identified EE-84 as a promising novel drug candidate against chronic myeloid leukemia. This indole derivative demonstrated drug-likeness in agreement with Lipinski’s rule of five. Furthermore, EE-84 induced a senescent-like phenotype in K562 cells in line with its cytostatic effect. EE-84-treated K562 cells underwent morphological changes in line with mitochondrial dysfunction concomitant with autophagy and ER stress induction. Finally, we demonstrated the synergistic cytotoxic effect of EE-84 with a BH3 mimetic, the Mcl-1 inhibitor A-1210477, against imatinib-sensitive and resistant K562 cells, highlighting the inhibition of antiapoptotic Bcl-2 proteins as a promising novel senolytic approach against chronic myeloid leukemia.

Full text of DOI:10.3390/md19060285

marine drugs
Article
Anti-Leukemic Properties of Aplysinopsin Derivative EE-84
Alone and Combined to BH3 Mimetic A-1210477
Sungmi Song 1,†, Sua Kim 1,†, Eslam R. El-Sawy
2,3
, Claudia Cerella 1,4 , Barbora Orlikova-Boyer 1,4
,
Gilbert Kirsch 3 , Christo Christov , Mario Dicato and Marc Diederich *
5
4
1,
1
Department of Pharmacy, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu,
Seoul 08626, Korea; sson35@snu.ac.kr (S.S.); suakim@snu.ac.kr (S.K.); claudia.cerella@lbmcc.lu (C.C.);
barbora.orlikova@lbmcc.lu (B.O.-B.)
Chemistry Department of Natural Compounds, National Research Centre, Dokki, 12622 Giza, Egypt;
eslamelsawy@gmail.com
2
3
4
UMR CNRS 7565 SRSMC, Université du Lorraine, 57070 Metz, France; gilbert.kirsch@univ-lorraine.fr
Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, 9, Rue Edward Steichen,
2
540 Luxembourg, Luxembourg; dicato.mario@chl.lu
Service d’Histologie, Faculté de Médicine, Université de Lorraine, INSERM U1256 NGERE,
4000 Nancy, France; christo.christov@univ-lorraine.fr
5
5
*
†
Correspondence: marcdiederich@snu.ac.kr; Tel.: +82-2-880-8919
These authors equally contributed to this work.
Abstract: Aplysinopsins are a class of marine indole alkaloids that exhibit a wide range of biological
activities. Although both the indole and N-benzyl moieties of aplysinopsins are known to possess
antiproliferative activity against cancer cells, their mechanism of action remains unclear. Through
in vitro and in vivo proliferation and viability screening of newly synthesized aplysinopsin analogs
on myelogenous leukemia cell lines and zebrafish toxicity tests, as well as analysis of differential
toxicity in noncancerous RPMI 1788 cells and PBMCs, we identified EE-84 as a promising novel drug
candidate against chronic myeloid leukemia. This indole derivative demonstrated drug-likeness in
agreement with Lipinski’s rule of five. Furthermore, EE-84 induced a senescent-like phenotype in
K562 cells in line with its cytostatic effect. EE-84-treated K562 cells underwent morphological changes
in line with mitochondrial dysfunction concomitant with autophagy and ER stress induction. Finally,
we demonstrated the synergistic cytotoxic effect of EE-84 with a BH3 mimetic, the Mcl-1 inhibitor A-
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Song, S.; Kim, S.; El-Sawy,
E.R.; Cerella, C.; Orlikova-Boyer, B.;
Kirsch, G.; Christov, C.; Dicato, M.;
Diederich, M. Anti-Leukemic
Properties of Aplysinopsin Derivative
EE-84 Alone and Combined to BH3
Mimetic A-1210477. Mar. Drugs 2021,
1
9, 285. https://doi.org/10.3390/
md19060285
1
210477, against imatinib-sensitive and resistant K562 cells, highlighting the inhibition of antiapoptotic
Academic Editor:
Bcl-2 proteins as a promising novel senolytic approach against chronic myeloid leukemia.
Orazio Taglialatela-Scafati
Keywords: aplysinopsin analogs; indole alkaloids; marine source; chronic myeloid leukemia;
BH3 mimetics
Received: 1 March 2021
Accepted: 13 May 2021
Published: 21 May 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1
. Introduction
Chronic myeloid leukemia (CML) is characterized by the oncogenic BCR-ABL1 fusion
gene expression, which codes for a leukemogenic tyrosine kinase [ ]. The first-line therapy
1
for CML is imatinib, a tyrosine kinase inhibitor (TKI) that selectively inhibits the activity
of the BCR-ABL fusion protein. Since the discovery of imatinib, the overall survival rate
of patients with CML has drastically increased, with patients showing durable responses
after imatinib treatment [2]. Leukemia cells, however, develop resistance mechanisms
to escape chemotherapy; therefore, despite the high remission rate, a significant number
of patients develop resistance or become intolerant to imatinib treatment. Furthermore,
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
3
3% of patients who receive imatinib treatment do not achieve a complete cytogenetic
response (CCyR) [ ]. Thus, alternative strategies for the management of CML are needed
3
to combat chemoresistance, for which compounds of natural origin have shown promise
as potential therapeutic agents by their capacity to induce cellular stress mechanisms
sensitizing leukemia cells against cytotoxic treatments.
4
.0/).
Mar. Drugs 2021, 19, 285. https://doi.org/10.3390/md19060285
https://www.mdpi.com/journal/marinedrugs

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The endoplasmic reticulum (ER) is responsible for protein translocation, proper fold-
ing, and protein post-translational modifications [
mation may disrupt this balance and result in ER stress that can trigger the unfolded
protein response (UPR) [ ]. The UPR represents a series of adaptive cellular mechanisms
designed to restore protein homeostasis [ ], and ER stress activates apoptotic cell death
under severe or chronic stress conditions [ ]. Autophagy is also a stress-induced cell
survival program that involves a catabolic process to degrade large protein aggregates and
damaged organelles in autophagosomes [ ]. Although autophagy and ER stress function
4]. Altered cell metabolism and inflam-
5
2
6
7
independently, increasing evidence supports that these processes can be coactivated [8].
Aplysinopsin and its derivatives possess rich structural diversity and have been
reported to exhibit a wide range of medicinal and biological activities. For example,
they act as neuromodulators [9] and possess antineoplastic [9], antiplasmodial [10], and
antimicrobial activities [11]. Interestingly, aplysinopsins display cytotoxicity against a
range of cancer cell lines [12]. However, their anticancer potential in leukemic cell lines
and the corresponding molecular mechanisms remain to be further investigated.
Here we evaluated the anti-leukemic activity of aplysinopsin (EE-115) and analogs
EE-31, EE-80, EE-84, and EE-92 (Scheme 1) against chronic myeloid leukemia cell lines. EE-
8
4 exhibited drug-like properties in line with Lipinski’s rule of five and showed a stronger
cytostatic and cytotoxic effect on leukemia cells than healthy cell models. Furthermore, its
safety profile was validated in vivo by using developing zebrafish larvae. Mechanistically,
EE-84 induced a senescent-like phenotype in line with its cytostatic activity, triggered
autophagy, ER stress, metabolic alterations, and mitochondrial dysfunction. In addition,
EE-84 sensitized imatinib-sensitive and -resistant K562 cells against the Mcl-1 inhibitor
A-12101477 to induce caspase-dependent apoptosis. Altogether, this study warrants further
investigation of the aplysinopsin analog EE-84 as a preclinical drug candidate against
chronic myeloid leukemia.
Scheme 1. Synthetic pathway for the preparation of aplysinopsin EE-115 and its analogs EE-31, EE-80, EE-84, and EE-92.
Reagents and conditions: ( ) benzyl chlorides, NaH, DMF; ( ) oxalyl chloride, dry ethyl ether, heating; ( ) the reactant
a
b
c
amines 2-cyanoacetohydrazide and 1-(2-amino-5-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)ethan-1-one, dry THF,
◦
TEA, stirring, 3 h; (d) POCl DMF, 0 C, NaOH; (e) piperidine, reflux, 4 h.
3
2. Results
2.1. Aplysinopsin Analogs Display Cytostatic Activities in Myeloid Leukemia Cells
Aplysinopsin (EE-115) and its analogs EE-31, EE-80, EE-84, and EE-92 (Figure 1) were
tested for their anti-leukemic effects on the myeloid leukemia cell line K562, using the
1
trypan blue exclusion test (Tables 1 and 2 and Figure S1). NMR spectrum data of H of the
compounds EE-31, EE-80, EE-84, EE-92, and EE-115 (Figure S2) and 1 C NMR spectrum
3
data of the compounds EE-31, EE-80, EE-84, and EE-92 (Figure S3) were provided.

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Figure 1. Chemical structures. (A) Aplysinopsin (EE-115) and analogs (EE-31, EE-80, EE-84, EE-92). (B) A-1210477.
Table 1. GI50 of EE-84 in CML cell lines. GI50
(µM) values were calculated after indicated times
of treatment.
GI50 (µM)
Compound
Cell Line
24 h
48 h
72 h
EE-84
KBM5
MEG01
>50
>50
33.95 ± 1.89
28.14 ± 1.68
48.40 ± 5.02
28.89 ± 1.52
K562IR
KBM5IR
>100
>100
95.63 ± 16.56
32.77 ± 2.25
55.09 ± 3.28
29.15 ± 2.05
GI50 values were calculated after trypan blue staining and represent the mean
experiments. GI50 indicates the concentration required to cause 50% overall growth inhibition.
±
S.D. of three independent
Compounds EE-31, EE-80, EE-92, and EE-115 only weakly affected the overall growth
(
Figure S1A) and viability (Figure S1B) of K562 cells at concentrations up to 50
Interestingly, EE-84 inhibited the growth of K562 with a GI50 of 32.22 3.91 M and
9.07 ± 0.80 µM after 48 and 72 h, respectively (Figure S1A).
Based on the GI50 results in the K562 cell line, we selected EE-84 for further investi-
µM.
±
µ
1
gations compared to EE-115, the parental compound. To generalize the antiproliferative
effect of EE-84 in CML, GI50 values of EE-84 were calculated on imatinib-sensitive (KBM5,
MEG01) and -resistant (IR) (K562IR and KBM5IR) CML cells (Table 1). We next validated
the cytostatic profiles of EE-84 by Methocult colony formation assays (CFA) to assess the
compounds’ effects on the clonogenic potential in a 3D culture environment (Figure 2A–E).
EE-84 reduced the total surface area and average size of K562 colonies after a 48-h pretreat-
ment (Figure 2A). Similar results were observed for other CML cell lines (Figure 2A–E).

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Figure 2. Cont.

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Figure 2. Differential anti-leukemic effects observed by CFA after aplysinopsin treatments (
from three independent experiments of clonogenic assays after 48 h pretreatment of EE-84 (
) KBM5IR, ( ) K562IR cells are shown on the left. Corresponding quantifications (number of colonies, the total surface
A–
E). Representative pictures
A
) K562, ( ) KBM5, ( ) MEG01,
B
C
(
D
E
area of colonies, and average size of colonies) are indicated on the right. Statistical analysis was performed by one-way
ANOVA, followed by Sidak’s multiple comparisons test. Differences were considered significant when * p < 0.05, ** p < 0.01,
*
** p < 0.001 compared to control. n/s: not significant.
Considering the moderate cytotoxic effect of 50
cells (Figure S1), we quantified cell death induction using Hoechst/PI staining by fluores-
cent microscopy (Figure 3A). EE-84 reduced K562 cell viability leading to 11.0 4.23% of
µM EE-84 and EE-115 at 72 h in K562
±
propidium iodide (PI)-positive cells without nuclear fragmentation. A similar percentage
of cell death was shown after quantification of Annexin V APC/PI by FACS (Figure 3B).
EE-84 induced approximately 16.5% cell death (Table 2) at the highest concentration of
5
0
µ
M. Imatinib (1
µ
M) was used as a control. Cell cycle analysis showed an accumulation
1.51% after 48 h and 13.03 1.19% after 72 h with EE-84
M), concomitant with a reduction of the G1 population from 58.40 2.81% at 48 h to
2.03 ± 2.99% at 72 h (Figure 3C and Table 3). Celecoxib (40 µM) was used as a control.
of cells in the sub-G1 phase of 8.34
±
±
(50
µ
±
5
Table 2. Percentage of cell death after treatment of K562 cells with increasing concentrations of EE-84. (Data from Figure 3B).
EE-84 (µM)
Ima (µM)
1
0
10
20
30
40
50
AnnV+/PI− (%)
AnnV+/PI+ (%)
5.12 ± 1.19
2.68 ± 0.77
7.05 ± 1.46
2.50 ± 0.62
5.60 ± 1.98
4.40 ± 0.94
6.65 ± 2.29
7.51 ± 2.70
7.71 ± 2.10
8.97 ± 1.03
8.22 ± 3.12
8.25 ± 1.95
51.77 ± 2.29
31.27 ± 5.24
Combined cell
death (%)
7
.80 ± 1.73
9.55 ± 3.22
10.00 ± 0.85
14.16 ± 0.61
16.68 ± 0.89
16.46 ± 0.02
83.03 ± 14.50
Results are the mean ± S.D. of three independent experiments.

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Figure 3. Percentage of cell death after increasing concentrations of EE-84 treatment in K562 cells. (A) Representative mi-
croscopy images of Hoechst 33342/PI double-stained nuclei of K562 cells exposed to different concentrations of aplysinopsin
derivatives at indicated times are shown. The percentage of apoptotic cells was calculated from three independent experi-
ments. (
cell cycle analysis, K562 cells were treated with 10, 30, and 50
determined by FACS. All data were expressed as mean SD of three independent experiments. Statistical analysis was
B) Results obtained after Annexin V APC/PI staining and quantification by FACS. Ima: imatinib (1
µM). (C) For
µM EE-84 for 48 and 72 h. Cell cycle phase distribution was
±
performed by two-way ANOVA, followed by Dunnett’s multiple comparisons test (Microscopy, Cell cycle); by Sidak’s
multiple comparisons test (Annexin V/PI); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls.

Mar. Drugs 2021, 19, 285
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Table 3. Cell cycle phase distribution after treatment of K562 cells with increasing concentrations of EE-84. (Data from
Figure 3C).
4
7
8 h Treatment
EE-84 (µM)
Celecoxib (µM)
%
0
10
30
50
40
Sub-G1
G1
1.78 ± 0.91
59.81 ± 2.29
18.02 ± 2.49
20.39 ± 2.12
1.48 ± 0.81
4.65 ± 2.42
61.11 ± 1.58
20.99 ± 0.83
13.25 ± 0.32
8.34 ± 1.51
58.40 ± 2.81
20.41 ± 1.84
12.86 ± 1.46
2.79 ±1.90
64.77 ± 3.09
15.00 ± 0.93
17.44 ± 2.13
59.59 ± 1.12
20.30 ± 1.68
18.64 ± 0.75
S
G2/M
2 h Treatment
EE-84 (µM)
Celecoxib (µM)
%
0
10
30
50
40
Sub-G1
G1
6.07 ± 2.88
62.33 ± 2.13
15.50 ± 2.14
16.10 ± 2.50
4.08 ± 2.80
60.83 ± 2.33
17.52 ± 4.24
17.57 ± 0.90
6.33 ± 0.74
55.09 ± 2.26
21.55 ± 1.64
17.03 ± 2.69
13.03 ± 1.19
52.03 ± 2.99
19.85 ± 1.33
15.08 ± 2.89
3.41 ± 2.04
68.43 ± 4.67
11.20 ± 2.37
16.96 ± 3.04
S
G2/M
Results are the mean ± S.D. of three independent experiments.
2.2. Drug-Likeness of Compounds EE-84 and EE-115
Next, we examined the drug-likeness of the two compounds according to Lipinski’s
rule of five (Table 4) [13]. A molecule is orally active when there are no more than 5 H
bond donors and no more than 10 H bond acceptors, when the molecular mass is less
than 500 Daltons, and when the octanol–water partition coefficient LogP is not greater
than 5. EE-115 did not violate any of the conditions mentioned above. For EE-84, The
LogP corresponds to 5.91; however, this aplysinopsin derivative can still be considered a
drug-like candidate since one exception is acceptable.
Table 4. In silico prediction for the drug-likeness of EE-84 and EE-115. Drug-likeness of the
aplysinopsin derivatives were calculated and interpreted based on Lipinski’s rule of five.
EE-84
EE-115
Mass
500.00
1
254.00
2
Hydrogen bond donor
Hydrogen bond acceptor
LogP
5
4
5.91
142.51
1.85
74.54
Molar reactivity
2.3. Effect of EE-84 and EE-115 on Healthy Cells and Zebrafish Embryos
We then assessed the effect of EE-84 and EE-115 on healthy models. Even though
EE-84 affected the proliferation of RPMI 1788 cells dose-dependently (Figure S4A), this
modulation was evident at later times (as early as 72 h vs. 48 h in cancer cells), with a higher
GI50 (40.10
Altogether, EE-84 generated significant differential toxicity of RPMI-1788/K562 cells at
0 and 50 M (Figure 4A). On the other hand, EE-115 inhibited the growth of normal
±
3.79 in RPMI 1788 vs. 19.07
±
0.80 µM after 72 h in K562 cells; Tables 1 and 5).
4
µ
RPMI 1788 cells (Table 5) more than K562 cells (Figure S4A). Moreover, EE-84 did not
significantly affect the viability of RPMI 1788 cells, whereas EE-115 reduced the viability of
RPMI 1788 cells at the highest concentrations (Table S1 and Figure S4B). Altogether EE-84
appeared to have a more advantageous safety profile.

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Figure 4. Effect of aplysinopsin derivatives on healthy cell models. (
was determined by the trypan blue exclusion test. ( ) Toxicity test of EE-84 in nonproliferating and proliferating PBMCs.
After 24, 48, and 72 h of EE-84 treatment, viability was determined by the trypan blue exclusion test. Zebrafish toxicity
assays were performed by treatment with aplysinopsins at indicated concentrations. ( ) Representative pictures of zebrafish
are shown. Zebrafish viability ( ), relative body length ( ), and heartbeats/min ( ) were measured. Data represents a
total of 15 fish per group. Trypan blue exclusion test data represents the mean SD of three independent experiments.
Statistical analysis: two-way ANOVA with Dunnett’s multiple comparison test (trypan blue exclusion test); * p < 0.05,
* p < 0.01 *** p < 0.001 compared to controls. Ordinary one-way ANOVA followed by Dunnett’s post hoc test (Zebrafish
assay) revealed significant differences indicated by *** p < 0.001 compared to control. n/s: not significant.
A) Differential toxicity (RPMI 788/K562) of EE-84
B
C
D
E
F
±
*
,

Mar. Drugs 2021, 19, 285
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Table 5. GI50 of aplysinopsins on RPMI 1788. GI50
(
µM) values were evaluated after indicated times
of compound treatment.
GI50 (µM)
Compound
24 h
48 h
72 h
EE-84
EE-115
>50
>50
>50
30.36 ± 1.95
40.10 ± 3.79
27.72 ± 2.30
GI50 values were calculated after trypan blue staining and represent the mean
experiments. GI50 indicates the concentration required to cause 50% overall growth inhibition.
±
S.D. of three independent
We generalized our findings and validated the absence of cytotoxic/cytostatic effects
of EE-84 on nonproliferating and proliferating peripheral blood mononuclear cells (PBMCs)
from healthy donors. In addition, EE-84 did not induce toxic effects in any of the two cell
models (Figure 4B).
We then treated zebrafish embryos 24 h post-fertilization (hpf) to validate the in vivo
safety profiles of the aplysinopsin compounds EE-84 and EE-115. The zebrafish were
observed after 24 h of exposure to different concentrations of aplysinopsins. No significant
morphological changes were notable at the different concentrations of the compounds used
(Figure 4C). Concentrations up to 100 µM of EE-84 and EE-115 did not affect the survival
rate (Figure 4D) or growth (Figure 4E) of the treated zebrafish. A moderate decrease in the
heart rate (Figure 4F) was noted for EE-84.
Altogether, based on these results, we further investigated the molecular and cellular
effects of EE-84, considering the observed differential toxicity.
2
.4. EE-84 Induced Morphological Changes and Cell Stress Responses in CML Cells
EE-84 (50 M) increased the cell size (Figure 5A) and the number of intracellular
vacuoles up to 96 h. These morphological changes were quantified by flow cytometry
Figure 5B). Compared to DMSO-treated controls, EE-84-treated K562 cells showed a
µ
(
progressive increase in forward scatter values (FSC-H; a.u.) and side scatter (SSC-H; a.u.)
as measurements of cell size and granularity, respectively. The increase in cell size and
granularity and the cytostatic effects were considered part of a cellular stress response
driving cells into senescence. We then examined the senescent-like phenotype using stress-
associated (SA)-
positive cells after treatment with EE-84 at 50
induce senescence, was used as a bona fide control (Figure 5C).
β
-galactosidase staining. We measured increased cell size and SA-
β-gal
µM for 72 h. Doxorubicin (80 nM), known to
In addition, morphological changes were examined by transmission electron mi-
croscopy (TEM). K562 cells treated with EE-84 underwent mitochondrial damage after 24,
4
8, and 72 h of compound exposure as well as mitophagy (Figure 5D). In addition, we
assessed the mitochondrial metabolism in K562 after treatment with EE-84 at 30 M for
2 h by using the Agilent Seahorse XFp Cell Mito Stress test. The oxygen consumption rate
OCR) significantly decreased after treatment with EE-84 compared to control (Figure 5E).
µ
7
(
Thus, our results confirmed that EE-84 induced cell stress and reduced mitochondrial activ-
ity in agreement with morphological alterations of the mitochondria observed by TEM.

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Figure 5. Cont.

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Figure 5. Morphological changes of K562 cells treated with EE-84. ( M EE-84 for 24,
A) K562 cells were treated with 50
µ
4
8, 72, and 96 h. Light microscopy at magnification 200 was used for morphological observation. (B) K562 cells were
×
treated with indicated concentrations of EE-84 for up to 96 h. At the indicated time points, cells were harvested and
analyzed by flow cytometry using forward (relative cell sizes; FSC-H) and side scatter (granularity; SSC-H) measurements.
For morphological analysis of EE-84, exposed K562 cell debris were excluded. (
treated with 50 M of EE-84 for 72 h. The quantitative analysis of the incidence of the cells with positive
Doxo: doxorubicin (80 nM). ( ) Transmission electron microscopy at magnifications of 8000 or 10,000 (whole cells) and
0,000 or 25,000 (cell details). Cells were treated with 30 M EE-84 and were exposed for up to 72 h. Phagophores,
autophagosomes, injured mitochondria, and incidence of mitophagy were highlighted using red, black, blue, and yellow
arrows, respectively. DMSO-treated K562 cells at 24 h were used as control. ( ) Measurement of mitochondrial function in
K562 cells. The experiments were conducted using the Seahorse XFp Cell Mito Stress test, and the OCR measurement is
visualized. Results are the mean SD of three independent experiments. Statistical analysis was performed by two-way
ANOVA, followed by Dunnett’s multiple comparisons test (Cell size by FACS); by Tukey’s multiple comparisons test
Senescence); by Sidak’s multiple comparisons test (Seahorse XFp Cell Mito Stress test); * p < 0.05, ** p < 0.01, *** p < 0.001
compared to controls.
C
) SA-
β-gal staining of the K562 cells
µ
β-gal staining.
D
×
×
2
×
×
µ
E
±
(
2
.5. Autophagy and Endoplasmic Reticulum (ER) Stress Were Triggered by EE-84 as Cellular
Stress Responses in K562 Cells
Autophagy is a catabolic pathway activated in response to different cellular stres-
sors, such as damaged organelles, accumulation of misfolded or unfolded proteins, ER
stress, and DNA damage [14]. Based on the TEM results, we then investigated the ex-
pression levels of the microtubule-associated protein-1 light chain 3 (LC3I) conversion to
the phosphatidylethanolamine-conjugated form of LC3I (LC3II) by Western blot. Results
showed that levels of LC3 II gradually increased over time after treatment with EE-84
at 30
µM in K562 at 24, 48, and 72 h, respectively (Figure 6A). In addition, we observed
the formation of cytoplasmic vacuoles in EE-84 treated K562 cells after 48 h at 30
µM by
Diff-Quik staining, suggestive of autophagy induction. To validate the involvement of
EE-84 in autophagy induction, we post-treated bafilomycin A1 (Baf-A1), an inhibitor of the
+
vacuolar-type H -ATPase which blocks the late phase of autophagy by preventing lysosomal
acidification, at 40 nM for 8 h (cells were treated or not with EE-84 for 40 h before addition of
Baf-A1 for 8 h), resulting in significant inhibition of vacuole formation after 48 h at 30 µM
EE-84 (Figure 6B). To see the effect of autophagy inhibition on cell viability, we conducted
a trypan blue assay test of EE-84 treated K562 cells with or without Baf-A1 post-treatment.
89.0% of cell viability was observed in EE-84 treated K562 cells with Baf-A1 post-treatment
compared to 98.7% of cell viability in EE-84 treated K562 cells alone (Figure 6B).
In addition, studies have shown that autophagy and endoplasmic reticulum (ER) stress
are closely related [8]. ER stress is considered a protective stress response in eukaryotic
cells [15]. We then assessed whether EE-84 induces ER stress and subsequently regulates
autophagy. We measured sensor proteins such as PERK, ATF6, and GRP78 that play a
role in activating the unfolded protein response (UPR) in response to ER stress. PERK and
phosphorylated eIF2
α were significantly increased after treatment with EE-84 at 30 and
5
0
µM in K562 cells after 72 h. The abundance of ATF4, an effector of PERK and eIF2α,

Mar. Drugs 2021, 19, 285
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also increased, which indicated that the PERK-eIF2
α
-ATF4-CHOP pathway was activated
under EE-84-induced ER stress (Figure 6C). Since GRP78 is involved in glycolysis [16],
we then assessed the glycolytic flux levels in K562 after treatment with EE-84 at 50
µM
for 72 h by using the Agilent seahorse XFp Glycolysis Stress test. The glycolysis and
glycolytic capacity measured by the extracellular acidification rate (ECAR) significantly
decreased after treatment with EE-84 compared to control (Figure 6D). We also confirmed
the glycolytic flux levels in K562IR cells after treatment with EE-84 at 100 µM for 72 h.
The results showed that the glycolytic capacity of ECAR also significantly decreased after
treatment with EE-84 (Figure S5). Altogether, these data demonstrated that the prolonged
treatment with EE-84 induced the PERK-eIF2
EE-84-induced autophagy.
α-ATF4-CHOP UPR pathway involved in
Figure 6. Cont.

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Figure 6. EE-84 induced autophagy and ER stress. (
Western blot analysis of the LC3B protein. B-actin was used as a loading control. Quantification of LC3B protein bands
through normalization by -actin protein bands. ( ) Diff-Quik staining of K562 cells after 48 h of EE-84 treatment (30 M)
with or without Baf-A1 post-treatment. Baf-A1: bafilomycin A1 (40 nM). ( ) ER stress-related proteins were detected by
Western blot analysis. B-actin was used as a loading control. After quantifying the bands, p-eiF2 levels were normalized to
total eIF2 , and all other proteins were normalized to -actin. TSG: thapsigargin (300 nM). ( ) Measurement of glycolytic
capacity in K562 cells. 2-DG: 2-deoxy-D-glucose. The experiments were conducted using the Seahorse XFp Glycolysis Stress
test, and the flow chart and bar graph show the measurement of ECAR. Results are the mean SD of three independent
experiments. Statistical analysis was performed by two-way ANOVA, followed by Tukey’s multiple comparisons test
autophagy Western blot, Diff-Quik staining); by Dunnett’s multiple comparisons test (ER stress Western blot); by Sidak’s
multiple comparisons test (Seahorse XFp Glycolysis Stress test); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls.
A) K562 cells were treated with 30 µM EE-84 for 24, 48, and 72 h.
β
B
µ
C
α
α
β
D
±
(
2
.6. EE-84 Sensitized K562 Cells against Mcl-1 Inhibitor A-1210477 and Showed Synergistic
Cytotoxicity in K562 and K562IR Cells
Conditions of ER stress may promote Mcl-1 protein stabilization via mechanisms
involving UPR and ATF4 upregulation [17,18]. Considering the cytostatic potential but
limited cytotoxicity of EE-84 along with the induction of ER stress, we then investigated
the expression levels of the antiapoptotic protein, Mcl-1, which may be responsible for
the apoptotic blockage. Based on the increase of antiapoptotic Mcl-1 expression in EE-
8
4-treated K562 cells after 24 h compared to control (Figure 7A), we speculated that the
combined treatment of the specific Mcl-1 inhibitor A-1210477 with EE-84 might sensitize
K562 cells to apoptotic cell death. Using subtoxic concentrations of EE-84 (20 and 30 M)
and A-1210477 (10 M), we first assessed the combinatory effects of these compounds using
Hoechst/PI staining after 24 h (Figure 7B). We observed 46.89 21.84% and 56.00 25.46%
µ
µ
±
±
induction of apoptotic cell death in combinatory treatments. The combination index (CI) of
each compound-pair was calculated, and the combinatory treatment showed a synergistic
effect (Figure 7C and Table 6). We also compared viability between EE-84 alone and in
combination with A-1210477 after 24 h (Table 7). Our results showed that the Mcl-1-specific
inhibitor A-1210477 sensitized EE-84-treated K562 and K562IR cells. Next, we confirmed
the apoptotic cell death mechanism triggered by the combination treatment. As shown in
Figure 7D, about 14.5% of apoptotic cell death was induced by A-1210477 alone, whereas
over 40% of apoptosis was observed by a combination of EE-84 and A-1210477 after 24 h.
z-VAD pretreatment completely prevented apoptosis induction in K562 cells in single and
combined treatments (Figure 7D). The caspase-dependent nature of cell death modality was
also confirmed by Annexin V APC/PI staining after 24 h in K562 cells (Figure 7E). Results
showed that EE-84 (30
early apoptosis (AnnV+/PI
after 24 h of treatment (Table 8). Again, zVAD completely protected against cell death in
all instances. Imatinib (1 M) was used as a control. In addition, the cytotoxic effect of
subtoxic concentrations of EE-84 (30 and 50 M) in combination with A-1210477 (20 M)
µ
M) in combination with A-1210477 (10
µ
M) induced 23.03
±
4.08%
−
) and 23.07 5.32% late apoptosis (AnnV+/PI+) in K562 cells
±
µ
µ
µ
was also tested on K562IR cells after 24 h of treatment, using the trypan blue exclusion
test (Figure 7F). Results showed that viability was reduced by 25.4% when cells were

Mar. Drugs 2021, 19, 285
14 of 29
cotreated with 20
and 20
effects of these compounds using Annexin V APC/PI staining after 24 h in K562IR cells
Figure 7G). Altogether, our results indicated that the specific Mcl-1 inhibitor A-1210477
µM A-1210477 and 30
µM of EE-84. A co-treatment with 50
µM EE-84
µM A-1210477 induced 40% of cell death. We further confirmed the combinatory
(
sensitized K562 and K562IR cells to EE-84, significantly increasing cell death induction.
Since mitochondrial dysfunction can trigger apoptosis, we measured the percentage of
mitochondrial membrane potential (MMP) loss due to the combined effects of EE-84 with
A-1210477 in K562 after 24 h. Results showed that the percentage of MMP loss significantly
increased in K562 after cotreatment with EE-84 and A-1210477 (Figure 7H). In addition, we
confirmed the combination effects of EE-84 with A-1210477 in K562 by colony formation
assays (CFA); EE-84 combined with A-1210477 reduced the number of colonies, the total
surface area, and the average size of colonies in K562 (Figure 7I). The obtained values for
the two compound combinations denoted synergism, and 30
A-1201477 were selected for further investigations.
µM of EE-84 with 10 µM of
Table 6. Combination indexes obtained by Compusyn software after treatment of K562 with EE-84
and A-1201477.
EE-84 (µM)
A-1210477 (µM)
Fa
CI
2
3
0
0
10
10
0.483
0.563
0.66
0.57
Figure 7. Cont.

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Figure 7. Cont.

Mar. Drugs 2021, 19, 285
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Figure 7. EE-84 sensitized CML cells to BH3 mimetics. (
compared to DMSO-treated control. K562 cells were treated with varying concentrations of EE-84 for 24 h. The effects
on Mcl-1 and -actin were determined by Western blot analysis. Western blot data interpreted in terms of fold changes
in protein expression compared to control at the corresponding time point. The values represented the average of three
independent experiments. ( ) Cotreatment analysis of 20 and 30 M EE-84 with 10 M A-1210477 using Hoechst 33258/PI
staining showed an increase in apoptotic cell death compared to single treatments of compounds after 24 h in K562 cells.
) The plot representing the fraction affected-combination index (Fa-CI) of the treatment of K562 cells with EE-84 and
A-1210477 after 24 h was obtained with Compusyn software. ( ) The optimal compound combination of 30 M EE-84 and
M A-1210477 was analyzed by Hoechst/PI staining. z-VAD-fmk was used as a pan-caspase inhibitor. ( ) The compound
combination of 30 M EE-84 and 10 M A-1210477 treated K562 cells after 24 h. After 24 h of treatment, the type of cell death
A) EE-84 increased expression levels of antiapoptotic protein Mcl-1
β
B
µ
µ
(
C
D
µ
10
µ
E
µ
µ
triggered by EE-84 with or without A-1210477 was characterized by FACS after Annexin V APC/propidium iodide (PI)
staining. Pictures are representative of three independent experiments (left panel), and the corresponding quantification
(
right panel) is shown. Ima: imatinib (1
staining of K562IR analyzed by FACS. Results are the mean
membrane potential (MMP) of the combination of 30 M EE-84 and 10
percentage of cells exhibiting MMP loss was calculated (right panel). z-VAD-fmk was used as a pan-caspase inhibitor. Eto:
etoposide (100 M). ( ) Differential anti-leukemic effects observed by CFA after EE-84 treatments with A-1210477 on K562.
Corresponding quantifications (the number of colonies, the total surface area of colonies, and the average size of colonies)
are indicated on the right. Results are the mean SD of three independent experiments. Statistical analysis was performed
µ
M). (
F
) K562IR cell viability by trypan blue exclusion assay. (
SD of three independent experiments. (
M A-1210477 was analyzed by FACS, and the
G
) Annexin V APC/PI
±
H
) Mitochondrial
µ
µ
µ
I
±
by one-way or two-way ANOVA, followed by Dunnett’s multiple comparisons test (Microscopy), by Tukey’s multiple
comparisons test (colony formation assay, MMP, Annexin V/PI); * p < 0.05, ** p < 0.01, *** p < 0.001 compared to controls,
&
&&
&&&
p < 0.05,
p < 0.01,
p < 0.001 between indicated conditions.

Mar. Drugs 2021, 19, 285
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Table 7. Viability of K562 and K562IR cells (%) after treatment with EE-84 alone or in combination
with A-1210477. Viability (%) was calculated after indicated times of compound treatment.
Viability (24 h)
Treatment
Cell Line
%
K562
98.0 ± 1.0
EE-84 (30 µM)
K562IR
95.3 ± 0.6
K562
K562IR
96.0 ± 0.7
97.0 ± 2.7
EE-84 (50 µM)
A-1210477 (10 µM)
A-1210477 (20 µM)
K562
79.3 ± 1.5
73.0 ± 3.6
K562IR
EE-84 (30 µM) + A-1210477 (10 µM)
EE-84 (30 µM) + A-1210477 (20 µM)
K562
K562IR
69.0 ± 3.0
74.7 ± 7.1
EE-84 (50 µM) + A-1210477 (20 µM)
Results are the mean ± S.D. of three independent experiments.
K562IR
60.0 ± 7.2
Table 8. Cell death induction (%) after treatment with EE-84 alone or in combination with A-1210477. (Data represent
Figure 7E).
0
EE-84 30 µM
6.00 ± 1.43
2.98 ± 0.23
8.98 ± 2.14
A-121047710 µM
9.31 ± 1.78
EE-84 30 µM +A-121047710 µM
23.03 ± 4.08
Ima 1 µM
37.77 ± 4.07
6.97 ± 0.88
44.74 ± 21.78
AnnV+/PI− (%)
AnnV+/PI+ (%)
5.36 ± 1.14
2.53 ± 0.28
7.90 ± 2.00
4.73 ± 0.17
23.07 ± 5.32
Combined cell death (%)
14.04 ± 3.24
z-VAD-fmk (50 µM)
46.10 ± 0.02
0
EE-84 30 µM
A-121047710 µM
6.04 ± 0.99
EE-84 30 µM +A-121047710 µM
5.24 ± 1.15
Ima 1 µM
10.86 ± 3.57
2.93 ± 0.15
13.79 ± 5.60
AnnV+/PI− (%)
AnnV+/PI+ (%)
4.46 ± 0.68
3.61 ± 0.13
8.07 ± 0.60
5.33 ± 1.35
3.13 ± 0.21
8.46 ± 1.55
2.81 ± 0.21
3.84 ± 0.28
Combined cell death (%)
8.85 ± 2.28
9.08 ± 0.99
Results are the mean ± S.D. of three independent experiments.
2
.7. Synergistic Induction of Caspase-Dependent Apoptosis by Combination Treatment of EE-84
and A-1210477 in K562 Cells
The cytotoxic effect and caspase-dependent apoptotic activity of the combination
treatment were also validated by quantification of intracellular ATP levels. ATP levels
decreased significantly after combination treatments compared to the compounds alone
(
Figure 8A). The 1 h pretreatment of z-VAD-fmk showed an increase in cell viability with
the single treatment of 10 M A-1210477 and the combined treatment. The addition of the
caspase inhibitor showed no significant difference in the ATP levels for the single treatment
of 30 M EE-84. To further validate our data, the activity of caspase-3/7 was measured.
µ
µ
There was a significant increase in the caspase-3/7 activity by 5.52-fold after the combined
treatment of EE-84 and the Mcl-1 inhibitor compared to the untreated control (Figure 8B).
To examine the detailed mechanism by which the combined treatment acts, we studied the
intrinsic and extrinsic apoptotic pathways in K562 cells by Western blot (Figure 8C–E). First,
the expression level of caspase-8, a cysteine protease that initiates apoptotic signaling via
the extrinsic pathway, was detected. Cleavage of pro-caspase-8 in the combined treatment
was observed (Figure 8C). Our results showed a reduction of pro-caspase-9, the initiator
caspase critical for the intrinsic pathway, concomitant with the appearance of a cleaved
fragment at 18 kDa after the combined treatment. In line with the previous observation
of increased caspase-3/7 activity for the combination treatment, we observed a strong
cleavage of pro-caspase-3 and cleaved fragments of caspase-7 for the combined treatment
(
Figure 8D). Our evaluation of antiapoptotic Mcl-1 and Bcl-xL expression levels showed an
increase of both in EE-84 (30 M) and A-1210477 (10 M) single treatments in K562 cells as
well as in the combined treatment (Figure 8E).
µ
µ

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Figure 8. Caspase-dependent induction of apoptosis in K562 cells by EE-84 in synergism with the Mcl-1 inhibitor A-1210477.
) K562 cell viability was determined by the measurement of cellular ATP content using the CellTiter-Glo assay. K562 cells
were pretreated at the concentration of 50 M z-VAD-fmk for 1 h before exposure to the compounds. ( ) Effects of single or
(A
µ
B
combination treatments of EE-84 and A-1210477 with or without z-VAD-fmk on caspase-3/7 activity in K562 cells. Three
separate experiments were performed for both CellTiter-Glo and caspase-3/7 assays, and each condition was measured in
triplicates. Values are the mean
from the control. Statistical analysis was performed by one-way or two-way ANOVA, followed by Dunnett’s multiple
±
SD of three independent experiments. The asterisk indicates a value significantly different
&
&&
comparisons test (ATP assay, caspase-3/7 assay); *** p < 0.001 compared to controls;
p < 0.001 between indicated
) Western blots showing expression
levels of apoptosis-related proteins after single or combination treatments of EE-84 and A-1210477 in K562 cells. ( ) Results
of expression levels of initiator caspases, caspase-8 and -9; ( ) expression levels of effector caspases, caspase-3 and -7,
and PARP-1; ( ) expression levels of antiapoptotic proteins Mcl-1 and Bcl-xL are displayed. Protein expression levels of
#
##
conditions;
p < 0.001 compared to corresponding treatment without z-VAD-fmk. (C–E
C
D
E
procaspase-8, -9, -3, and -7 are quantified and expressed in fold changes relative to control. Protein expression levels of
cleaved PARP-1, Mcl-1, and Bcl-xL are quantified and expressed in fold changes relative to control. The values represent the
average of three independent experiments.

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3. Discussion
Many products of marine organisms have been identified as modulators of cell death,
exerting cytotoxic effects on cancer cells as activators of apoptosis, autophagy, or onco-
sis [19]. Currently, seven marine-based drugs have been approved, 23 compounds are
undergoing phase I–III clinical trials, and thousands of compounds have been isolated
from marine life and are undergoing preclinical studies [20]. Among these, four are used
®
for cancer treatment: cytarabine (Cytosar-U , Vitaris, Canonsburg, PA, USA), trabectedin
®
®
(
Yondelis , PharmaMar, Colmenar Viejo, Madrid, Spain), eribulin mesylate (Halaven ,
®
Eisai Inc., Tokyo, Japan) and the conjugated antibody brentuximab vedotin (Acentris ,
Takeda Oncology, Cambridge, MA, USA) [21]. Cytosine arabinoside (cytarabine), originally
isolated from the sponge Cryptothethya crypa, and now produced synthetically, is one of
the most effective drugs to treat acute myeloid leukemia [21–23]. In addition, trabectedin,
a marine metabolite of Ecteinascidia turbinata, is used for the treatment of soft tissue sar-
coma [24]. However, despite the broad array of marine compounds clinically available or
investigated, there is still an enormous library of natural products that remains untapped.
Aplysinopsins, a class of marine indole alkaloids, comprise two main distinct moieties—
an indole and an imidazolidinone ring—and are isolated from a variety of marine organ-
isms, including sponges [25], corals [26], anemone [27], and mollusks [28,29]. Aplysinopsins
were first isolated by Kazlauskas et al., and initially identified as the major metabo-
lite of eight Indo-Pacific sponge species of the genera Thorecta [12]. Since then, more
aplysinopsin derivatives have been discovered and extracted from Verongia spengeli, Derci-
tus sp., Smenospongia aurea, Verongular rigida, Dictyoceratida sp., Aplysinopsis reticulata,
Aplysina sp., Hyrtios erecta, and Thorectandra [28], among others. In line with the effort
to identify new aplysinopsins with therapeutic potential, we report a set of synthetic
aplysinopsin derivatives that possess anti-leukemic effects. In addition, aplysinopsin
derivatives displayed a range of cytostatic effects, some inducing antiproliferating effects
on myeloid leukemia cells more than others. We identified EE-84 as having the most potent
antiproliferative effect in several chronic myeloid leukemia cell lines (K562, KBM5, MEG01,
K562IR, and KBM5IR) as shown by trypan blue exclusion tests, CFAs, Hoechst/PI, and
Annexin V/PI staining assays.
Identifying lead candidates from a library of compounds and characterizing their
safety profiles by testing their toxicity in healthy models is essential in the drug discovery
process. In addition, it helps predict clinical adverse effects in the future [28]. EE-84 showed
differential toxicity in the noncancerous cell model, RPMI 1788, compared to K562, and
was nontoxic to PBMCs. In the zebrafish model. EE-84 was also well-tolerated. The safe
profile and drug-likeness of EE-84, supported by Lipinski’s rule of five, warranted further
investigation of this compound.
EE-84 induced morphological changes in K562 cells as shown by diff-Quik staining
and TEM imaging. In parallel with the cytostatic effect, EE-84 triggered a significant
increase in the cell size together with the appearance of mitochondrial damage after 48 h
of treatment. Furthermore, evaluation of mitochondrial dysfunction by Agilent Seahorse
XFp Cell Mito Stress test showed significant inhibition of mitochondrial function after
7
2 h of 30 µM treatment. Interestingly, EE-84-treated K562 cells displayed a senescent-
like phenotype, suggesting an interplay between mitochondrial dysfunction and cellular
senescence in the EE-84-induced antiproliferative effect.
Cellular senescence is one of the many defense mechanisms that cells undergo to
combat extrinsic and/or intrinsic stresses by halting cell cycle progression. Drug-mediated
cellular stress leading to senescence is often accompanied by senescence-associated se-
cretory phenotype (SASP). Many studies revealed that in senescent cells, mitochondrial
function is significantly affected [30]. For example, Galanos et al. showed perturbations
in the mitochondrial morphology in p21-inducible precancerous and cancerous cellular
models (Li-Fraumeni and Saos-2 cell lines), characterized by the enlargement of the mito-
chondria and the damaged morphology of cristae [31
,32]. Mitochondria were elongated or
branched, and the cristae were abnormally distributed or lost, suggesting a disturbance

Mar. Drugs 2021, 19, 285
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of mitochondrial dynamics in the senescent cell. Wiley et al. also reported a distinct type
of senescence associated with mitochondrial damage called “Mitochondrial Dysfunction
Associated-Senescence” (MiDAS). They observed that mitochondrial dysfunction induces
senescence and differs from the senescence caused by genotoxic or oncogenic stress by
analyzing the secretome [33].
Senolytic compounds specifically induce apoptosis in senescent cells [34]. Dasatinib
has been approved to treat CML as one of the second-generation tyrosine kinase inhibitors
used in imatinib resistance and/or intolerance [35]. The senolytic drug combination
of dasatinib and quercetin decreased senescent cells in human adipose tissue [36]. In
addition, BH3 mimetic ABT-263 (Navitoclax) also induced apoptosis in mouse senescent
bone marrow hematopoietic stem cells (HSCs) as a potent senolytic drug [37]. Hence,
these studies support the idea that EE-84 triggered a senescent-like phenotype in response
to mitochondrial dysfunction, autophagy, and prolonged ER stress as cellular metabolic
alteration. In the present study, we showed that EE-84 induces a senescent-like phenotype.
Interestingly, the combination of BH3 mimetic A-1210477 with EE-84 further triggers
canonical apoptotic cell death in K562 and K562IR. We speculate that under these conditions,
Mcl-1 inhibitor A-1210477 may act as a senolytic compound able to eradicate imatinib-
sensitive and resistant CML cells. The effect of EE-84 as a single agent points at the ability
of this compound to act as a general stress inducer. New effective combinatorial anticancer
treatments also include stress inducers forcing cancer cells to rely more on a prosurvival
factor. Targeting this specific protein promotes the eradication of cancer cells. Interestingly,
some of the cases reported do involve prosurvival Bcl-2 family proteins. Dexamethasone,
for example, is leading to Bcl-2 dependence and sensitization to venetoclax in multiple
myeloma by altering the balance between pro- and antiapoptotic Bcl-2 protein members
(increased Bim and decreased Mcl-1 vs. Bcl-2 upregulation), making Bcl-2 expression
essential for survival [38]. Kapoor and colleagues report that CLL chronically resistant to
ibrutinib (a Bruton tyrosine kinase (BTK) inhibitor) are sensitized to venetoclax partly also
by a STAT3-mediated Bcl-2 upregulation in resistant CLL, implying a shift in the type of
Bcl-2 family protein dependence [39]. At this level of investigation, we might speculate
that EE-84 contributes to a similar scenario. Mcl-1 protein upregulation (or stabilization?)
might signalize an increased reliance of CML cells on Mcl-1 as part of a stress response
induced by EE-84. Further investigations will be required to elucidate any modulatory
effects of EE-84 on the expression level of proapoptotic Bcl-2 proteins. In this view, EE-84
might be a compound to consider in combinatorial regimens.
Next, we investigated whether autophagy and ER stress occur concurrently with
inhibition of proliferation by EE-84 in K562 cells. Accumulation of LC3-II, a standard
marker for autophagosomes, was detected increasingly over time after 24, 48, and 72 h of
EE-84 treatment, as seen in our Western blotting results. There was a significant increase
in the formation of cytoplasmic vacuoles after 48 h EE-84 treatment at 30 µM, and this
phenomenon was abrogated by a post-treatment with Baf-A1. Furthermore, 98.7% of K562
cells were viable after 48 h EE-84 treatment alone; however, the viability of K562 cells
significantly decreased with Baf-A1 post-treatment, implying that EE-84 drives the K562
cells to undergo autophagy as a cell survival mechanism. ER stress was also activated
by EE-84, given that sensor proteins of UPR, such as PERK and ATF6, and downstream
effectors such as CHOP and ATF4 were upregulated to varying degrees. GRP78 has
been initially characterized as a glucose-regulated protein [16]. Our Western blot results
showed decreased levels of GRP78. These results are in line with data obtained by using
the Seahorse glycolysis stress test. We observed reduced glycolysis levels, supporting
the hypothesis of an impairment of the glycolytic function by EE-84 treatment in K562
and K562IR cells. Altogether, EE-84 induced autophagy, ER stress, and mitochondrial
alterations as cell stress reactions, further leading to apoptosis. Overall, EE-84 plays a
potential role in inhibiting CML cell activities via induction of cellular stress modalities.
The evasion of apoptotic cell death by cancer cells can impair responses to anticancer
therapy. Prosurvival B-cell lymphoma 2 (BCL-2) proteins play a role of perpetrators in

Mar. Drugs 2021, 19, 285
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this scenario because they prevent apoptosis by keeping the cell death effectors like BAX
and BAK under control [40]. BH3 mimetics offer a solution to this as they are designed to
inhibit antiapoptotic BCL2 family proteins, leading to BAX and BAK activation, and thus
promoting apoptosis [41]. Mcl-1 became a popular therapeutic target because it is one of
the most frequently amplified genes across all human cancers. Moreover, an increase in
Mcl-1 expression is commonly associated with chemotherapy resistance [42]. This study
evaluated the synergistic effect of EE-84, a cytostatic marine compound, with the Mcl-1
inhibitor A-1210477 against CML K562 and K562 imatinib-resistant cells. We showed that
the cotreatment of the marine compound and the Mcl-1 inhibitor induced apoptotic cell
death along with the activation of caspase activity. These results are in line with other
studies in which BH3 mimetics like ABT199 showed synergism with cell stress inducers
like cardiac glycosides [43,44] and coumarin derivatives [44].
In the present study, we investigated the preclinical use of aplysinopsins as anti-
leukemic agents. Our results identify EE-84 as a potential drug candidate for CML as
it possesses drug-like properties and is well-tolerated in healthy models in vitro and
in vivo. Mechanistically, EE-84 induces antiproliferative effects associated with the complex
interplay between mitochondrial dysfunction, a senescent-like phenotype, autophagy, and
ER stress, potentially inducing a condition favorable for senolysis by synergizing with
senolytic compounds. To potentiate the anti-leukemic effects of EE-84, we suggest the
cotreatment of this aplysinopsin derivative with the BH3 mimetic A-1210477 in K562
and K562IR, as it significantly increases cell death of malignant cells. In conclusion,
the combination of EE-84 with BH3 mimetics is efficient and highly synergistic. Future
investigations will determine whether this combinatory approach can become a therapeutic
opportunity against resistant forms of CML.
4
4
4
. Materials and Methods
.1. Chemistry
.1.1. General Information
All reagents and solvents were of commercial grade. Melting points were determined
on the digital melting point apparatus (Electro thermal 9100, Electro thermal Engineering
Ltd., serial No. 8694, Rochford, UK) and are uncorrected. Elemental analyses were
performed on a FlashSmart
™ Elemental Analyzer (Thermo Scientific, Courtaboeuf, France)
1
13
and were found within 0.4% of the theoretical values. H and C NMR spectra were
±
measured with a Bruker Avance spectrometer (Bruker, Germany) at 400 and 101 MHz,
respectively, using TMS as the internal standard. Hydrogen coupling patterns are described
as (s) singlet, (d) doublet, (t) triplet, (q) quartet, and (m) multiplet. The chemical shifts were
defined as parts per million (ppm) relative to the solvent peak. The reaction progress was
checked by pre-coated TLC Silica gel 0.2 nm F254 nm [Fluka], visualized under UV lamp
2
5
54 and 365 nm. 2-Cyanoacetohydrazide [45]; N-benzyl indoles [46] methyl creatinine [47];
-methoxy indole-3-aldehyde [48] were prepared as reported. 1-(2-Amino-5-methyl-4,5,6,7-
tetrahydrobenzo[b]thiophen-3-yl)ethan-1-one [49] was provided by Ahmed B. Abdelwahab,
Ph.D., UMR CNRS 7565 SRSMC, Université de Lorraine, 57070 Metz, France.
4
.1.2. General Procedure for the Preparation of EE-31, EE-80, EE-84, EE-92
◦
To a solution at 0 C of oxalyl chloride (0.44 mL, 5.1 mmol) in dry ethyl ether (25 mL)
was added dropwise a solution of indole (4.14 mmol) in dry ethyl ether (5 mL). The
resulting solution was refluxed for 2 h. After removing the solvent under vacuo, the
◦
residue was dissolved in dry tetrahydrofuran (20 mL) and cooled to 0 C. To the THF
solution was added slowly the amine (9.73 mmol) in dry tetrahydrofuran (20 mL). After
complete addition, 1 mL of triethylamine was added, and the reaction was left to stir
overnight. The precipitate obtained was filtered off, washed several times with water,
dried, and recrystallized from acetone.
0
2
-(1-Benzyl-1H-Indol-3-yl)-N -(2-Cyanoacetyl)-2-Oxoacetohydrazide EE-31

Mar. Drugs 2021, 19, 285
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◦
1
Yield 0.45g, 86%; mp 264–6 C; H NMR (400 MHz, DMSO)
δ: 10.63 (s, 1H), 8.90 (s,
1
(
1
2
4
H), 8.26 (s, 1H), 7.72–7.50 (m, 5H), 7.41–7.21 (m, 4H), 5.46 (s, 2H), 3.83 (s, 2H); 13C NMR
101 MHz, DMSO) : 162.16, 141.47, 136.79, 136.70, 129.14, 128.97, 128.56, 128.26, 128.01,
27.73, 127.65, 127.29, 126.98, 124.17, 123.56, 122.53, 121.92, 116.04, 112.14, 111.95, 50.28,
4.10; Anal. Calcd for C H N O (360.37): C, 66.66; H, 4.48; N, 15.55; found: C, 66.87; H,
δ
20
16
4
3
.50; N, 15.42.
0
2
-(1-Benzyl-5-Methoxy-1H-Indol-3-yl)-N -(2-Cyanoacetyl)-2-Oxoacetohydrazide EE-80
◦
1
Yield (0.31g, 63%); mp 199–201 C; H NMR (400 MHz, DMSO)
s, 1H), 7.76 (s, 1H), 7.57–7.43 (d, H), 7.41–7.20 (m, 5H), 7.06–6.77 (m, 2H), 5.44 (s, 2H), 3.87
m, 3H), 3.33 (s, 2H); 13C NMR (101 MHz, DMSO)
161.34, 162.32, 156.47, 136.51, 131.22,
28.74, 127.83, 127.60, 127.33, 115.52, 113.07, 112.52, 103.72, 55.37, 50.06, 23.80; Anal. Calcd
for C H N O (390.40): C, 64.61; H, 4.65; N, 14.35; found: C, 64.55; H, 4.70; N, 14.32.
δ: 10.62 (s, 1H), 8.78
(
(
1
δ
21
18
4
4
(N-(3-Acetyl-4,5,6,7-Tetrahydro-5-Methylbenzo[b]Thiophen-2-yl)-2-(1-Benzyl-5-Methoxy-1H-
Indol-3-yl)-2-Oxoacetamide EE-84
◦
1
Yield (0.38g, 61%); mp 165–7 C; H NMR (400 MHz, CDCl )
δ
: 13.58 (s, 1H), 9.07
s, 1H), 8.08 (d, J = 2.5 Hz, 1H), 7.35–6.90 (m, 8H), 5.38 (s, 2H), 3.92 (d, J = 6.4 Hz, 3H),
.82–2.80 (dd, J = 16.1, 5.0 Hz, 1H), 2.58–2.55 (m, 2H), 2.41 (s, 3H), 1.91–1.87 (m, 2H), 1.15
d, J = 6.4 Hz, 2H), 1.10 (d, J = 6.6 Hz,3H); 13C NMR (101 MHz, CDCl3)
: 196.38, 194.06,
3
(
2
(
δ
1
64.12, 157.30, 140.94, 135.40, 131.26, 130.59, 130.42, 129.06, 128.98, 128.29, 128.24, 126.91,
1
23.08, 117.26, 115.78, 114.52, 111.48, 104.33, 55.81, 51.54, 36.56, 35.96, 31.01, 30.65, 29.25,
2
4.52, 21.76; Anal. Calcd for C H N O S (500.61): C, 69.58; H, 5.64; N, 5.60; S, 6.40; found:
29 28 2 4
C, 69.60; H, 5.56; N, 5.60; S, 6.48.
0
N -(2-Cyanoacetyl)-2-(1-(2,4-Dichlorobenzyl)-5-Methoxy-1H-Indol-3-yl)-2-Oxoacetohydrazide
EE-92
◦
Yield (0.25g, 56%); mp 242–4 C; 1H NMR (400 MHz, DMSO)
δ: 10.74 (s, 1H), 10.39
(
5
1
5
s, 1H), 8.70 (s, 1H), 7.75 (dd, J = 17.6, 2.3 Hz, 2H), 7.58–7.26 (m, 2H), 7.09–6.83 (m, 2H),
.65 (s, 2H), 3.82 (s, 3H), 3.34 (s, 2H); 13C NMR (101 MHz, DMSO) : 180.68, 161.35, 156.58,
40.97, 133.49, 133.29, 132.90, 131.21, 130.50, 129.22, 127.88, 113.29, 112.27, 111.62, 103.79,
5.39, 47.52, 23.80; Anal. Calcd for C21H16Cl2N4O4 (459.28): C, 54.92; H, 3.51; Cl, 15.44; N,
2.20; found: C, 54.93; H, 3.46; Cl, 15.54; N, 12.33.
δ
1
4.1.3. General Procedure for the Preparation of EE-115
1,3-Dimethyl creatinine (2.8 g, 22.7 mmol) and indole-3-aldehydes (22.7 mmol) were
heated under reflux in piperidine (30 mL) for 4 h. After cooling, the reaction mixture
was poured into water (200 mL) and then stirred for 30 min. The precipitate was filtered,
washed several times with water, air dried and crystallized from methanol.
(
1
Z)-5-((1H-Indol-3-yl)Methylene)-1,3-Dimethylimidazolidine-2,4-Dione (Aplysinopsin) EE-
15 [50]
◦
◦
1
Yield 80%, mp 236–8 C (reported mp 236 C); H NMR (400 MHz, CDCl )
δ: 8.87 (d,
3
J = 2.7 Hz, 1H), 8.54 (s, 1H), 7.85–7.66 (m, 1H), 7.45 (dd, J = 6.7, 1.4 Hz, 1H), 7.33–7.18 (m,
3H), 6.42 (s, 1H), 3.34 (s, 3H), 3.23 (s, 3H).
4.2. Cell Lines and Cell Cultures
The human chronic myeloid leukemia K562 (ATCC, CCL-243, Manassas, VA, USA),
MEG01 (ATCC, CRL-2021, Manassas, VA, USA), and the normal B lymphocyte RPMI 1788
cell lines (KCLB, 10156, Seoul, Korea) were cultured in Roswell Park Memorial Institute
(
RPMI) 1640 medium (Lonza, Basel, Switzerland), supplemented with 10% heat-inactivated
fetal bovine serum (FBS) (Biowest, Riverside, CA, USA) and 1% penicillin-streptomycin
solution (100 ) (GenDEPOT, Katy, TX, USA). KBM-5 cells were kindly donated by Dr.
Bharat B. Aggarwal. Imatinib-resistant KBM5 cells (KBM5R) cells were obtained by sequen-
tially increasing the concentration of imatinib from 0.25 to 1 M imatinib in IMDM media
×
µ

Mar. Drugs 2021, 19, 285
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supplemented with 10% (v/v) fetal calf serum and 1% (v/v) antibiotic–antimycotics [51].
Imatinib-resistant K562 (K562IR) cells were a gift of the Catholic University of Seoul and
cultured in RPMI 1640 medium with 25 mM HEPES (Lonza) supplemented with 10% (v/v)
FCS and 1% (v/v) antibiotic–antimycotics. Both resistant cell types were cultured with 1 µM
◦
of imatinib and washed three times before each experiment. Cells were maintained at 37 C
TM
and 5% of CO in a humified atmosphere. Mycoplasma detection by Mycoalert (Lonza)
2
was performed every 30 days, and cells were used within three months after thawing.
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient cen-
trifugation using Ficoll-Hypaque (GE Healthcare, Roosendaal, The Netherlands) from
freshly collected buffy coats as previously described [51], obtained from healthy adult
human volunteers (Red Cross, Luxembourg City, Luxembourg) after ethical approval as
well as written informed consent from each volunteer. After isolation, cells were incubated
6
overnight at 2
×
10 cells/mL in RPMI 1640 (supplemented with 1% antibiotic–antimycotic
◦
and 10% FCS (BioWhittaker, Verviers, Belgium) at 37 C and 5% CO in a humidified
atmosphere. The day after, cell concentration was adjusted at 1
same fresh complete medium and then treated as indicated.
2
6
×
10 cells/mL using the
4
.3. Compounds
Mcl-1 inhibitor, A-1210477 (S7790, Selleckchem, Seoul, Korea) was used in single
and combination treatments. Etoposide (E1383, Sigma-Aldrich, Seoul, Korea), Imatinib
SML 1027, Sigma-Aldrich, St. Louis, MO, USA), and Celecoxib (PZ0008, Sigma-Aldrich,
(
Seoul, Korea) were used as positive controls. Caspase inhibitor 1 (z-VAD-fmk, 187389-52-2,
Calbiochem, Seoul, Korea) served to inhibit caspase-dependent apoptosis.
4.4. Cell Proliferation and Viability
Cell proliferation and viability were assessed by the trypan blue exclusion method
(Lonza), and viable cells were counted using a hematocytometer (Marienfeld, Lauda-
Königshofen, Germany). Differential toxicity was calculated by comparing the viability
of RPMI 1788 cells to the viability of cancer cells (normal/cancer cells). The difference in
viability was expressed in terms of fold change.
4
.5. Colony Formation Assay
5
For 48 h pretreatment of EE-84 colony formation assays, approximately 3 × 10 cells
were seeded in each well of a 24-well plate, treated with EE-84 at indicated concentra-
tions, and incubated at 37 C and 5% of CO in a humidified atmosphere for 48 h. Af-
◦
2
ter 48 h, 1000 cells were counted and grown in a semisolid methylcellulose medium
(Methocult H4230, StemCell Technologies Inc., Vancouver, BC, Canada) supplemented
with 10% FBS. Colonies were detected after 10 days of culture by adding 1 mg/mL of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Sigma-Aldrich)
and were analyzed by Image J 1.8.0 software (U.S. National Institute of Health, Bethesda,
MD, USA).
4.6. Quantification of Apoptosis and Necrosis
The percentage of apoptotic cells was quantified as the fraction of cells showing
fragmented nuclei, as assessed by fluorescence microscopy (Nikon, Tokyo, Japan) after
staining with Hoechst 33342 (Sigma-Aldrich) and propidium iodide (Sigma-Aldrich). In
addition, apoptosis was confirmed by Annexin V APC (Biolegend, 640919)/propidium
iodide (BD Biosciences, 556547) staining and fluorescence-activated cell sorter (FACS)
analysis according to the manufacturer protocol (BD Biosciences, 556547).
4.7. In Silico Drug Likeliness Properties
Lipinski’s ‘rule of five’ for drug likeliness properties was evaluated using the SCFBio
website (http://www.scfbio-iitd.res.in/).

Mar. Drugs 2021, 19, 285
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4.8. Zebrafish Toxicity
For toxicity assays, embryos were treated with 0.003% phenylthiourea (PTU) 14 h
before the assay to remove pigmentation. Then, 2 h before the assay, the embryo’s shell
was eliminated and then treated for up to 24 h with aplysinopsin compounds at indicated
concentrations in 24-well plates. Viability and abnormal development were assessed
after 24 h of treatment under light microscopy (Carl Zeiss Stereomicroscope DV4, Seoul,
Korea). Pictures were taken by fixing embryos onto a glass slide with 3% methylcellulose
(
Sigma-Aldrich).
4.9. Cell Cycle Analysis
Cells were collected and fixed in 70% ethanol. DNA was stained with propidium
iodide (PI) solution (1
µ
g/mL, Sigma-Aldrich, St. Louis, MO, USA) in 1XPBS (Biosesang,
◦
Seongnam, Korea) for 30 min at 37 C, supplemented with RNase A (100
µg/mL, Roche,
Basel, Switzerland). Samples were analyzed by flow cytometry using the FACSCaliburTM
system, Becton Dickinson (BD) Biosciences (San Jose, CA, USA). Data were recorded
statistically (10,000 events/sample) using the Cell Quest software (BD Biosciences) and
analyzed using Flow-Jo 8.8.5 software (Tree Star, Inc., Ashland, OR, USA).
4.10. Cell Morphology/Wright-Giemsa Staining
Diff Quik staining was used to analyze the morphological features of compound-
5
treated cells. Approximately 3
×
10 cells were seeded in each well of a 24-well plate
and treated with EE-84 at the indicated concentrations for the indicated time. Cells were
then spun onto a microscope glass slide for 5 min at 500 g using a cytopad with caps
Elitech Group Inc., Puteaux, France). Cells were fixed, air-dried, and then stained with the
×
(
Diff-Quik staining kit (Sysmex, Kobe, Japan). The stained cells were examined, and images
were captured with an inverted microscope (Nikon Eclipse Ti2).
4.11. SA-β-Gal Assay
The senescence-associated (SA)-β-Gal activity was measured as previously reported [52].
K562 cells treated with 80 nM doxorubicin 72 h were used as a positive control for senes-
cence induction.
4.12. Analysis of Cell Size and Complexity (Granularity)
Flow cytometry acquisitions of FSC-H (forward) vs. SSC-H (size) were performed
as a method to monitor the cell size and granularity (cell complexity) in untreated vs.
TM
EE-84-treated K562 (FACSCalibur ; BD Biosciences). Data (10,000 events) were recorded
using the Cellquest Pro software (BD Biosciences) and further analyzed using FlowJo
software (Treestar, Ashland, OR, USA).
4
.13. Analysis of Mitochondrial Membrane Potential (MMP) Levels
◦
To monitor mitochondrial membrane potential (MMP), cells were incubated at 37 C
for 30 min with 50 nM MitoTracker Red CMXRos (all from Molecular Probes, Invitrogen,
Grand Island, NY, USA) and then analyzed by flow cytometry. Data were recorded
statistically (10,000 events/sample) using the CellQuest Pro software. Data were analyzed
using the Flow-Jo 8.8.7 software, and results were expressed as cells with MMP loss (%).
4.14. Transmission Electron Microscopy (TEM)
Cells were pelleted and fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences,
Hatfield, PA, USA) diluted in 0.1 M sodium cacodylate buffer, pH 7.2 (Electron Microscopy
Sciences) overnight. Cells were then rinsed twice with sodium cacodylate buffer, postfixed
for 2 h in 2% osmium tetroxide at room temperature, washed with distilled water, and
◦
stained with 0.5% uranyl acetate at 4 C overnight. Samples were then dehydrated in
successive ethanol washes, followed by infiltration of 1 (100% ethanol):1 (Spurr’s resin).
Samples were kept overnight embedded in 100% Spurr’s resin, mounted in molds, and

Mar. Drugs 2021, 19, 285
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◦
left to polymerize in an oven at 56 C for 48 h. Ultrathin sections (70–90 nm) were cut
with an ultramicrotome, EM UC7 (Leica Microsystems Ltd., Seoul, Korea). Sections were
stained with uranyl acetate and lead citrate and subsequently viewed using a JEM1010
transmission electron microscope (JEOL Korea Ltd., Seoul, Korea).
4.15. Determination of the Oxygen Consumption Rate and Glycolysis Stress
The oxygen consumption rate (OCR) was measured using a Seahorse XFp Cell Mito
Stress Assay (#103010-100, Agilent Technologies, Seoul, Korea) ran on a Seahorse XFp
analyzer (Agilent Technologies, Seoul, Korea) according to the manufacturer’s instructions.
Briefly, cells were seeded at 30,000 cells per well and treated with EE-84 for 48 h in 175
µ
◦
L
medium. Before measurements, plates were equilibrated in a CO -free incubator at 37 C
2
for 1 h. The analysis was performed using 1.5
trifluoromethoxy)phenylhydrazone (FCCP), and 1
Data were analyzed using the Seahorse XF Cell Mito Stress Test report generator software
Agilent). The extracellular acidification rate (ECAR) was measured in response to the
sequential injection of 10 mM glucose, 2 M oligomycin (H+-ATP-synthase inhibitor), and
0 mM 2-deoxy-d-glucose (2DG) (hexokinase inhibitor) to detect non-glycolytic acidifica-
µM oligomycin, 0.5
µM carbonyl cyanide-4-
(
µM rotenone/antimycin A as indicated.
(
µ
5
tion, glycolysis, maximal glycolytic capacity, and glycolytic reserve using a Seahorse XFp
analyzer with a Glycolysis Stress Test kit (Agilent, Santa Clara, CA, USA).
4.16. Measurement of Intracellular ATP Content
To quantify metabolically active cells, intracellular ATP levels were measured by
CellTiter-Glo Luminescent Cell Viability Assay (Promega, Cosmogenetech, Seoul, Korea)
following the manufacturer’s protocol.
4.17. Measurement of Caspase-3/7 Activity
According to the manufacturer’s instructions, the activation of caspase-3/7 was mea-
sured using the Caspase-Glo 3/7 Assay (Promega, Madison, WI, USA). The caspase-3/7
reagent was added to the sample volume at a 1:1 ratio, and the cells were incubated for
1
h at room temperature. The luminescence of triplicate samples was measured using a
microplate reader.
4.18. Whole-Cell Extracts and Western Blotting
For the preparation of whole-cell extracts, cells were harvested, washed in cold 1xPBS,
TM
and lysed in Mammalian Protein Extraction Reagent (M-PER , Thermo Fisher, Waltham,
MA, USA) supplemented with a 1
Roche, Basel, Switzerland) according to the manufacturer’s instructions. Protein concentra-
tion was measured using the Bradford assay. Proteins were aliquoted and stored at
Afterward, proteins were subjected to sodium dodecyl sulfate (SDS)–polyacrylamide gel
electrophoresis (PAGE) and transferred to PVDF membranes (GE Healthcare, Little Chal-
font, UK). Membranes were incubated with selected primary antibodies: anti-caspase-7
×
protease inhibitor cocktail (Complete, EDTA-free,
◦
−
80 C.
(
9494S), anti-caspase-9 (9502S), anti-caspase-8 (9746), anti-Mcl-1 (4572S), anti-LC3B (2775),
ATF4 (11815), ATF6 (65880), Bip/GRP78 (C50B12), CHOP (L63F7), eIF2 (9722) and PERK
3192) from Cell Signaling (Danvers, MA, USA), anti-caspase-3 (sc-56053) and anti-PARP-1
sc-53643) from Santa Cruz Biotechnology (Dallas, TX, USA), anti-Bcl-xL (610212) from BD
α
(
(
Pharmingen (San Jose, CA, USA), eIF2a (Phospho-Ser51) (11279) from Signalway Antibody
Co. (College Park, MD, USA) and anti- -actin (5441) from Sigma Aldrich (St. Louis, MO,
USA). Blots were probed in PBS-T containing the appropriate blocking agent (5% milk or
% BSA) for 1 h. Membranes were prehybridized overnight with the indicated primary
β
5
antibodies. After washing, blots were incubated with species-appropriate HRP-conjugated
secondary antibody (Santa Cruz) in PBS-T containing 5% milk. Proteins of interest were
detected with ECL Plus Western blotting Detection System reagent (GE Healthcare) using
ImageQuant LAS 4000 mini system (GE Healthcare).

Mar. Drugs 2021, 19, 285
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4.19. Statistical Analysis
Data are expressed as the mean
way or two-way ANOVA (analysis of variance) followed by either Dunnett’s multiple
comparison test or Sidak’s multiple comparison test, unless otherwise stated, using Prism
±
SD and significance was estimated by using one-
8
software, GraphPad Software (La Jolla, CA, USA). p-values were considered statistically
significant when p < 0.05. Legends are represented as follows: * p < 0.05, ** p < 0.01,
** p < 0.001.
*
5. Conclusions
Through the screening of several aplysinopsin analogs, we selected EE-84 as an
interesting anti-leukemic agent. EE-84 exhibited a safety profile as it had minimal impact
on healthy models in vitro and in vivo. We also found that the treatment of K562 with EE-
8
4 induced an antiproliferative effect concomitant with autophagy and ER stress induction
as well as senescence. In addition, mitochondrial dysfunction was observed in line with
altered K562 cell morphology after EE-84 treatment. Treatment of EE-84 combined with the
BH3 mimetic A-1210477 (specific for Mcl-1) potentialized apoptotic cell death. We suggest
this cotreatment as a promising preclinical approach to therapeutic failure, specifically in
resistant CML.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.
3390/md19060285/s1, Table S1: IC50 of aplysinopsin analogs in the noncancerous cell line RPMI
1
788. Figure S1: Effect on proliferation and viability of aplysinopsin-treated on K562 cells. Figure S2:
1
H NMR spectral data of (A) EE-31, (B) EE-80, (C) EE-84, (D) EE-92 and (E) EE-115. Figure S3:
1
3
C NMR spectral data of (A) EE-31, (B) EE-80, (C) EE-84, and (D) EE-92. Figure S4: Effect on the
proliferation and viability of EE-84 and EE-115 on noncancerous cell line RPMI1 788. Figure S5:
Measurement of glycolytic capacity in K562IR cells. Figure S6: EE-84 sensitizes K562IR cells to BH3
mimetics after 24 h.
Author Contributions: S.S. and S.K.: equally performed the experiments; S.S., S.K., B.O.-B., C.C.
(
Claudia Cerella), C.C. (Christo Christov), G.K., M.D. (Mario Dicato) and M.D. (Marc Diederich): ana-
lyzed/discussed the data; E.R.E.-S.: designed and prepared aplysinopsin and its analogs; G.K.: pro-
vided the compounds; S.K. and M.D. (Marc Diederich) conceived the experiments and wrote/edited
the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: S.K., S.S. and M.D. (Marc Diederich) were supported by National Research Foundation
(
NRF) [Grant Number 019R1A2C1009231] and by a grant from the MEST of Korea for Tumor
Microenvironment Global Core Research Center (GCRC) [Grant Number 2011-0030001]. Support
from Brain Korea (BK21) FOUR program and Creative-Pioneering Researchers Program at Seoul
National University [Funding number: 370C-20160062] are acknowledged. C.C. (Claudia Cerella)
and B.O.-B. were supported by Télévie Luxembourg. LBMCC is supported by “Recherche Cancer et
Sang” Foundation, “Recherches Scientifiques Luxembourg”, “Een Häerz fir kriibskrank Kanner”,
Action Lions “Vaincre le Cancer”, and Télévie Luxembourg. E.R.E.-S. was supported by French and
Egyptian governments through a cofinanced fellowship granted by the French Embassy in Egypt
(Institut Français d’Egypte) and the Science and Technology Development Fund (STDF).
Institutional Review Board Statement: The study was conducted in agreement with the guidelines
of the Institutional Animal Care and Use Committee (IACUC) of Seoul National University.
Informed Consent Statement: PBMCs were obtained from Red Cross Luxembourg with informed
consent obtained from all subjects (donators of PBMC blood products) involved in the study. Specif-
ically, agreement LBMCC-2019-0001: Assessment of differential toxicity of new drugs or drug
combinations in preclinical development in ex-vivo proliferating peripheral blood mononuclear cells
vs. proliferating cancer cells, agreement LBMCC-2019-0002: Assessment of toxicity of new drugs or
drug combinations in preclinical development in non-proliferating peripheral blood mononuclear
cells (systemic acute toxicity).
Data Availability Statement: Data supporting reported results can be found here: https://data.
mendeley.com/datasets/z2rkrgwbm9/2; https://data.mendeley.com/datasets/bs3r484nfn/1; https:
/
/data.mendeley.com/datasets/ym9mcsm2fg/1, accessed on 19 May 2021.

Mar. Drugs 2021, 19, 285
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Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
AML
Bcr-Abl
BH
acute myeloid leukemia
breakpoint cluster region-Abelson
Bcl-2 homology domain
BSA
bovine serum albumin
CCyR
CFA
CML
DMSO
FBS
complete cytogenetic response
colony formation assay
chronic myeloid leukemia
dimethyl sulfoxide
fetal bovine serum
FDA
FLT3
GI
food and drug administration
FMS-like tyrosine kinase 3
growth inhibition
HPF
IC
hours post fertilization
inhibitory concentration
ITD
internal tandem duplication
octanol–water partition coefficient
mammalian protein extraction reagent
myeloid cell leukemia 1
mitochondrial membrane potential
3-(4,5-dimethylthiazol-2-1)-2,5,-diphenyltetrazolium bromide
poly(ADP-ribose)polymerase
peripheral blood mononuclear cell
phosphate buffered saline
propidium iodide
LogP
MPER
Mcl-1
MMP
MTT
PARP-1
PBMC
PBS
PI
PMSF
PTU
RPMI
SA
phenylmethylsulphonyl fluoride
phenylthiourea
Roswell Park Memorial Institute
surface area
SD
standard deviation
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
TEM
TKD
UPR
transmission electron microscopy
tyrosine kinase domain
unfolded protein response
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