Identification of H2S/NO-donating artemisinin derivatives as potential antileukemic agents

Article abstract of DOI:10.1039/c9ra08239e

Three H₂S/NO-donating artemisinin derivatives were designed and synthesized. Their antiproliferative activities were evaluated against human acute myeloid leukemia (AML) cell lines of K562 and K562/ADR and human normal liver cells of LO2. Biological evaluation indicated that NO-donating compound 10c exhibited the most potent cytotoxicity against leukemia cells, similar to the bioactivity of clinical drug of homoharringtonine, but showed less toxicity than homoharringtonine against LO2 cells. Further mechanism studies revealed that 10c could enhance the levels of intracellular NO and ROS, induce apoptosis and S phase cell cycle arrest, and disturb the mitochondrial membrane potential in K562 and K562/ADR cells. Western blot results demonstrated that 10c noticeably promoted autophagy by up-regulating the levels of Beclin1 and L3-II expression, inhibited the AKT signaling, and stimulated the AMPK and JNK signaling in both leukemia cell lines. Overall, 10c exhibited the potential to be a promising candidate for the therapy of AML.

Full text of DOI:10.1039/c9ra08239e

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Identification of H S/NO-donating artemisinin
2
derivatives as potential antileukemic agents†
Cite this: RSC Adv., 2020, 10, 501
a
b
cd
Runmei Tian, Yan Chen,*b
b
Xuemei Chen, Pei Huang,
Jing Wang,
cd
cd
and Zhigui Ma*a
Yongzheng Chen,
Lei Zhang
*
2
Three H S/NO-donating artemisinin derivatives were designed and synthesized. Their antiproliferative
activities were evaluated against human acute myeloid leukemia (AML) cell lines of K562 and K562/ADR
and human normal liver cells of LO2. Biological evaluation indicated that NO-donating compound 10c
exhibited the most potent cytotoxicity against leukemia cells, similar to the bioactivity of clinical drug of
homoharringtonine, but showed less toxicity than homoharringtonine against LO2 cells. Further
mechanism studies revealed that 10c could enhance the levels of intracellular NO and ROS, induce
apoptosis and S phase cell cycle arrest, and disturb the mitochondrial membrane potential in K562 and
K562/ADR cells. Western blot results demonstrated that 10c noticeably promoted autophagy by up-
regulating the levels of Beclin1 and L3-II expression, inhibited the AKT signaling, and stimulated the
AMPK and JNK signaling in both leukemia cell lines. Overall, 10c exhibited the potential to be
a promising candidate for the therapy of AML.
Received 10th October 2019
Accepted 16th December 2019
DOI: 10.1039/c9ra08239e
rsc.li/rsc-advances
Artemisinin (1, Fig. 1), a naturally occurring sesquiterpene
lactone isolated mainly from Artemisia annua L., had been used
Introduction
Leukemia, a cancer of the blood or bone marrow, is the ninth as a traditional Chinese medicine for treating malaria hundreds
1
4
most common cancer in the United States. Leukemia can be years ago. Nowadays, numerous derivatives of artemisinin have
clinically classied into four major kinds: acute lymphoblastic been synthesized, and several artemisinin derivatives with less
leukemia (ALL), chronic lymphocytic leukemia (CLL), acute toxicity, improved solubility and enhanced antimalarial activity,
myeloid leukemia (AML), and chronic myeloid leukemia (CML). such as dihydroartemisinin (2, Fig. 1) and artesunate (3, Fig. 1),
AML is the most common type of acute leukemia in adults. have been used as rst-line antimalarial drugs widely. Besides,
There are several possible treatments for AML, such as alloge- artemisinin and its derivatives have been shown to exhibit
5
neic bone marrow transplantation, which involves high-dose signicantly antineoplastic activities in vitro and in vivo.
chemotherapy and radiation, as well as a high risk of death. Furthermore, they showed antiproliferative activities towards
Chemotherapy is another countermeasure for newly diagnosed various human cancer cells, such as U937, NB-4, PC-3, MDA-
6
patients with AML, such as cytotoxic drugs (adriamycin and MB-231 and MCF-7. Particularly, dihydroartemisinin and
homoharringtonine). However, the resistance to chemotherapy artesunate were promising candidates for leukemia treatment
2
drugs has developed in a signicant portion of AML patients.
due to its potential anticancer activity in leukemic cell lines and
7,8
Therefore, it is necessary to develop a novel strategy or drug for murine models. Recently, our group also reported that arte-
3
treatment of AML.
sunate and its derivative displayed anticancer activity against
9,10
leukemia cells in vitro and in vivo. In addition, some studies
have shown that artemisinin and its derivatives could be used as
sensitizers to improve the cytotoxicity of other chemothera-
11,12
a
peutic drugs.
However, up to now, there were no artemisinin
Department of Pediatric Hematology, West China Second University Hospital,
Sichuan University, Chengdu 610041, PR China. E-mail: zhiguima@scu.edu.cn
b
Department of Pediatrics, Affiliated Hospital of Zunyi Medical University, Zunyi
5
63003, PR China. E-mail: cyz600@163.com
c
Key Laboratory of Biocatalysis & Chiral Drug Synthesis of Guizhou Province, School of
Pharmacy, Zunyi Medical University, Zunyi 563003, PR China. E-mail: lzhang@zmu.
edu.cn
d
Key Laboratory of Basic Pharmacology of Ministry of Education, Joint International
Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical
University, Zunyi 563003, PR China
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c9ra08239e
1
Fig. 1 The structures of artemisinin and its derivatives.
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28
derivatives or artemisinin derivatives in combination chemo- an IC50 value of 48 nM in HT-29 cells. In 2017, Gu et al.
therapy had entered into clinical trials for the treatment of synthesized a series of NO-releasing bifendate derivatives, and
leukemia.
further biological evaluation showed that the most potent
Hydrogen sulde (H S), nitric oxide (NO) and carbon compound signicantly inhibited the proliferation of resistant
2
monoxide (CO), are increasingly recognized as important K562/ADR cells in vitro and in vivo by releasing high levels of
29
endogenous mediators of diverse physiological processes, such NO.
13
as vasodilation and neuromodulation.
H
2
S was reported to
2
Given that both H S/NO and artemisinin derivatives possess
regulate protein kinases such as p38 mitogen-activated protein anticancer activity against various cancer cell lines, we
14
kinase and trigger cell apoptosis at concentration of 20 mM.
2
hypothesized that the H S/NO-donating artemisinin derivatives
There are several kinds of donor molecules which can release could exert potent cytotoxicity. In this study, to nd valid
H S under physiological conditions, such as inorganic salts candidates for the treatment of AML, we designed and synthe-
2
(
NaHS and Na S). The drawback in using salts as H S donors is sized three H S/NO-donating artemisinin derivatives by
2
2
2
the uncontrolled release prole. Therefore, many small organic coupling artesunate with H
compounds have been designed and synthetized for releasing NO donor (ISMN) using the molecular hybridization strategy
S via endogenous enzymatic and/or non-enzymatic pathways, (Fig. 3). We further evaluated these H S/NO-donating artemisi-
2
S donors (ADT-OH and TBZ) and
H
2
2
including 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT- nin derivatives for their antileukemic activities against AML cell
OH, 4, Fig. 2) and 4-hydroxy benzothiazamide (TBZ, 5, lines, K562 and K562/ADR.
1
5
Fig. 2). Some reports had pointed out that H S donors also
2
16
displayed antitumor effects.
Meanwhile, a number of
synthetic H S releasing molecules have been shown to exhibit
anticancer activities in vitro and in vivo. Moreover, NO is
2
Results and discussion
17
The synthesis of hybrid molecules 10a–10c is outlined in
Scheme 1. Firstly, intermediate 3 was prepared according to the
method described previously via sodium borohydride (NaBH )
4
catalyzed reduction reaction of artemisinin (1) and 4-dimethy-
laminopyridine (DMAP) catalyzed esterication reaction. Then,
the target molecules were obtained in good yields by reacting 3
a renowned gasotransmitter with a variety of physiological
18
roles. Previous studies have reported that high levels of NO
could inhibit proliferation and survival of tumor cells, and
sensitize tumors to chemotherapy in vitro and in vivo, by inhi-
bition of key transcription factors, DNA repair enzymes and
30
19,20
drug efflux pumps.
Like H S donors, NO donors also have
2
2
and different H S donors (4 and 5) or NO donor (7) via EDCI and
gained great attention for decades owing to the high reactivity
and inconvenient handling, which contain organic nitrates,
DMAP catalyzed esterication reaction. The desired hybrid
compounds were puried using column chromatography on
21
diazeniumdiolates, S-nitrosothiols and furoxans. For example,
nitroglycerin (6), isosorbide 5-mononitrate (ISMN, 7) and iso-
sorbide dinitrate (ISDN, 8) (Fig. 2) are organic nitrates, well-
known vasodilators used in the therapy and prevention of
1
13
silica gel and further characterized via H NMR, C NMR and
high-resolution mass spectra (HRMS).
The antiproliferative activities of target hybrids (10a–10c)
were performed against AML cell lines of K562 and K562/ADR
and human normal hepatic LO2 cells by the CCK-8 assay in
vitro initially, compared with lead compounds artemisinin (1),
22
angina pectoris by releasing NO, as well as exhibit antitumor
23,24
and enhance chemosensitivity.
Chen et al. demonstrated
that the bioconversion of nitroglycerin to release NO occurred
through mitochondrial enzyme aldehyde dehydrogenase 2
dihydroartemisinin (2), artesunate (3), H
and 5), NO donor (7) and positive controls, homoharringtonine
HHT) and adriamycin (ADR). The IC50 values of test
compounds were summarized in Table 1. All three H S/NO-
2
S releasing moieties (4
25
(
ALDH2). Moreover, JS-K (9, Fig. 2), an NO-donating dia-
(
zeniumdiolate, released high levels of NO when suitably acti-
vated by glutathione or GST, and exhibited anticancer activity
2
26,27
donating artemisinin derivatives were notably effective in
inhibiting the growth of two leukemic cell lines, which dis-
played more potent antiproliferative activities than three lead
against various human cancer cell lines.
Particularly, Kodela
and co-workers reported that NOSH-aspirin bearing both NO
and H S-releasing moieties suppressed eleven different human
2
cancer cell lines, and NOSH-1 was the most potent hybrid, with
Fig. 2 The structures of H
2
S/NO-donating agents.
2
Fig. 3 Design of H S/NO-donating artemisinin derivatives.
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K562/ADR cells. In K562 cell line, 10c showed similar inhibitory
activity (IC50 ¼ 0.316 ꢀ 0.021 mM) to clinical drug HHT (IC
¼
50
0
.367 ꢀ 0.011 mM) and exhibited superior cytotoxic activity to
positive control ADR (IC ¼ 0.672 ꢀ 0.056 mM). Meanwhile, the
5
0
inhibitory activity of hybrid 10c (IC50 ¼ 6.634 ꢀ 0.795 mM)
against resistant K562/ADR cells was slightly stronger than that
of ADR (IC50 ¼ 9.161 ꢀ 0.658 mM), but slightly weaker than that
of HHT (IC50 ¼ 3.256 ꢀ 0.082 mM). Furthermore, in LO2 cells,
NO releasing artemisinin derivative 10c also showed lower
cytotoxicity (IC50 ¼ 3.077 ꢀ 0.281 mM) than two clinical drugs
HHR (IC50 ¼ 0.915 ꢀ 0.149 mM) and ADR (IC ¼ 2.736 ꢀ 0.123
50
mM), suggesting that target compound 10c may be a promising
candidate for further investigation.
Compound 10c was composed of artesunate moiety (3) and
ISMN moiety (7) (Scheme 1), and the IC50 values (shown in Table
1
) of 10c for K562 (IC50 ¼ 0.316 ꢀ 0.021 mM) and K562/ADR cells
(
IC50 ¼ 6.634 ꢀ 0.795 mM) were signicantly less than that of
individual moieties, 3 (IC ¼ 10.17 ꢀ 0.135 and 36.433 ꢀ 2.902
5
0
mM) and 7 (IC50 > 200 and 200 mM), respectively. Furthermore,
in order to investigate the antiproliferative activity of the
combination of 3 and 7, the growth inhibition of compounds 3,
7, and 3 + 7 against K562 and K562/ADR cells were determined
by the CCK-8 assay. As seen in Fig. 4, it was notable that
compounds 3 and 7 at 0.3 and 6 mM displayed less potent
activity in both K562 and K562/ADR cell lines, respectively,
Scheme
2
1 Synthesis of H S/NO-donating artemisinin derivatives.
ꢁ
Reagents and conditions: (a) NaBH
4
, MeOH, 0–5 C; (b) succinic however, conjugate 10c exhibited 2.98-fold and 2.67-fold
anhydride, DMAP, DCM, rt; (c) EDCI, DMAP, DMF, rt.
increase in its antiproliferative activity on K562 and K562/ADR
cells comparable to the combination of 3 and 7, respectively.
These results suggested that the antitumor activity of hybrid 10c
compounds, artemisinin, dihydroartemisinin and artesunate.
Nevertheless, all H S/NO donors showed weak inhibitory
2
activity with IC50 values more than 200 mM. Interestingly, hybrid
10c incorporated with ISMN showed stronger activity than 10a
and 10b with H S releasing moieties against both K562 and
2
Table
2
1 Antiproliferative activity of H S/NO-donating artemisinin
derivatives in vitro
a
IC50 (mM)
Compd
K562
K562/ADR
LO2
1
2
3
4
5
7
11.085 ꢀ 0.832
8.666 ꢀ 0.277
10.17 ꢀ 0.135
>200
>200
>200
>200
83.341 ꢀ 1.988
19.893 ꢀ 1.66
19.864 ꢀ 0.331
>200
>200
>200
28.068 ꢀ 1.095
36.433 ꢀ 2.902
>200
>200
>200
1
1
1
0a
0b
0c
2.981 ꢀ 0.391
1.031 ꢀ 0.116
0.316 ꢀ 0.021
0.367 ꢀ 0.011
0.672 ꢀ 0.056
16.632 ꢀ 2.058
8.479 ꢀ 1.081
6.634 ꢀ 0.795
3.256 ꢀ 0.082
9.161 ꢀ 0.658
9.862 ꢀ 0.479
2.244 ꢀ 0.359
3.077 ꢀ 0.281
0.915 ꢀ 0.149
2.736 ꢀ 0.123
HHT
ADR
Fig. 4 Compounds 3, 7, 10c and 3 + 7 inhibited the proliferation of
K562 and K562/ADR cells. K562 cells were incubated with vehicle, 3
(0.3 mM), 7 (0.3 mM), 10c (0.3 mM) and 3 + 7 (0.3 mM + 0.3 mM) for 72 h,
and K562/ADR cells were incubated with vehicle, 3 (6 mM), 7 (6 mM),
a
The cells were treated with the indicated concentrations of each test
compound for 72 h by the CCK-8 assay. The IC50 value (drug
concentration inhibiting 50% of the cell proliferation) for each
compound was calculated and expressed as mean IC50 (mM) ꢀ SD
from three independent experiments.
10c (6 mM) and 3 + 7 (6 mM + 6 mM) for 72 h, respectively. The cell
proliferation was measured by CCK-8. Data were expressed as the
means ꢀ SD from three independent experiments. **P < 0.01 vs. the
###
control group;
P < 0.001 vs. the 10c group.
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may be attributed to the synergic effects of artesunate and NO
donor moiety in cancer cells.
Reactive oxygen species (ROS) are cellular metabolites and
play vital roles in cellular homeostasis. In addition, more
It was reported that high levels of NO could result in nitro- evidences suggested that the excessive generation of ROS
31
sative stress (NS) and induce tumor cell apoptosis. In order to contributed to apoptosis and autophagy in various tumor
33
verify the effect of NO on the anticancer activity of 10c, the cells. Extensive studies have showed that artemisinin and its
intracellular NO levels were rst determined using DAF-FM DA derivatives induced apoptosis by ROS generation mediated by
5
as a cell permeable uorescent probe. K562 and K562/ADR cells endoperoxide bridges. Therefore, in this study, the generation
were incubated with vehicle, 10c (1 mM, 10 mM and 100 mM), 3 of intracellular ROS in K562 and K562/ADR cells was investi-
(100 mM) and 7 (100 mM) for 3 h, respectively. And we measured gated by uorescence microscopy and ow cytometer aer
NO generation by using uorescence microscopy (Fig. 5A and C) DCFH-DA staining, respectively. Cells were incubated with
and microplate reader (Fig. 5B and D), respectively. The results vehicle, 10c (1 mM, 5 mM and 10 mM) and 3 (10 mM) for 24 h,
showed that hybrid 10c promoted the generation of NO in both respectively. As shown in Fig. 6A and D, 10c-treated groups gave
K562 and K562/ADR cells in a dose-dependent manner rise to increase in green uorescence intensity in a dose-
compared with the control group. Notably, the parents 3 and 7 dependent manner, suggesting that 10c heightened the levels
also increased the intracellular NO levels, however, the effects of of intracellular ROS. Simultaneously, as the increased concen-
3
and 7 were signicantly weaker than that of 10c in both K562 trations of 10c, the ROS production and percentage of ROS were
and K562/ADR cells, respectively, suggesting that the levels of gradually increased in K562 and K562/ADR cells (Fig. 6B, C, E
intracellular NO induced by different compounds might be and F). Moreover, incubation with 10c caused higher levels of
positively correlated with their antiproliferative activities. ROS production in sensitive K562 cells compared with that in
Furthermore, it was observed that treatment with test resistant K562/ADR cells. Nevertheless, NO and ROS were re-
34,35
compounds caused lower levels of NO production in resistant ported to react to form peroxynitrite (ONOO–).
So, the
K562/ADR cells, which might be associated with their anti- correlation between NO and ROS induced by 10c needs our
cancer activities in sensitive and resistant cancer cell lines. further studies.
Interesting, parent 3 without NO donor also enhanced the
Cell cycle progression is vital to the cell processes, such as
intracellular NO levels, which may be related to previous study cell division and replication. Cell cycle arrest could inhibit
that the production of NO could be induced by inducible nitric cancer cell proliferation. Accumulating reports had reported
32
oxide synthase (iNOS).
that artemisinin derivatives inhibited cancer cells via cell cycle
arrest. To examine the antineoplastic mechanism of 10c on
36
Fig. 5 The generation of intracellular NO in K562 and K562/ADR cells. Cells were incubated with vehicle, 10c (1 mM, 10 mM and 100 mM), 3 (100
mM) and 7 (100 mM) for 3 h, respectively. (A and C) Intracellular NO was measured using DAF-FM DA staining by fluorescence microscopy. (B and
D) NO production was assessed by microplate reader. The data were expressed as the mean ꢀ SD from three independent experiments. **P <
0
.01 vs. the control group.
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Fig. 6 The generation of intracellular ROS in K562 and K562/ADR cells. Cells were incubated with vehicle, 10c (1 mM, 5 mM and 10 mM) and 3 (10
mM) for 24 h, respectively. (A and D) Intracellular ROS was measured using DCFH-DA staining by fluorescence microscopy. (B, C, E and F) ROS
production and percentage of ROS were measured by flow cytometer, respectively. The data were expressed as the mean ꢀ SD from three
independent experiments. **P < 0.01 vs. the control group.
37
cell cycle, cell cycle distribution was measured using ow apoptosis. To uncover the involvement of mitochondria
cytometry aer staining with PI. K562 and K562/ADR cells were pathway in 10c induced apoptosis, the changes of mitochon-
incubated with vehicle, 10c (1 mM, 5 mM and 10 mM) and drial membrane potentials were assessed in this paper. K562
3
(10 mM) for 24 h, respectively. As shown in Fig. 7, treatment of and K562/ADR cells were incubated with vehicle, 10c (1 mM, 5
K562 and K562/ADR cells with 10c resulted in accumulation at mM and 10 mM) and 3 (10 mM) for 24 h, respectively, and further
the S phase a concentration-dependent manner, respectively, stained with uorochrome JC-1 dye followed by ow cytometry
compared with the control group. Meanwhile, the levels of the analysis. Fig. 9 showed that 10c obviously disturbed the mito-
accumulation of K562 cells in the S phase aer treatment with chondrial membrane and decreased MMP in a dose-dependent
10c were slightly higher than the levels of the accumulation of manner in K562 and K562/ADR cells, which indicated that 10c
K562/ADR cells. could induce cell apoptosis through mitochondria pathway. At
Previous studies have shown that artemisinins and its the same time, mitochondrial membrane potential in K562 cells
derivatives can induce cancer cell apoptosis by modulating the was more sensitive to 10c than that in resistant K562/ADR cells.
expression of apoptosis-related regulators and signal events.
In an effort to explore the molecular mechanisms underlying
Accordingly, to further conrm the effect of 10c on induction of the 10c-induced cell cycle arrest and apoptosis, we determined
cancer cell apoptosis, K562 and K562/ADR cells were incubated the regulatory effects of 10c on the cycle- and apoptosis-related
with vehicle, 10c (1 mM, 5 mM and 10 mM) and 3 (10 mM) for 24 h, proteins in K562 and K562/ADR cells. Cells were incubated with
respectively, and the percentages of apoptotic cells were vehicle, 10c (1 mM, 5 mM and 10 mM) and 3 (10 mM) for 24 h,
measured by ow cytometry aer staining with Annexin V-FITC respectively, and the relative levels of CDK1, CDK2, cleaved
and 7-AAD. As shown in Fig. 8, treatment with 10c induced caspase-3 and cleaved PARP expression were measured by
apoptosis in both K562 and K562/ADR cells in a dose-dependent western blot. Aer 24 h treatment with 10c, suppression of
manner, and 10c at 10 mM showed signicantly stronger effect CDK1 and CDK2 proteins expression, and up-regulation of
than that of 3 in both K562 and K562/ADR cell lines.
cleaved caspase-3 and cleaved PARP proteins expression were
It was reported that the loss of mitochondrial membrane observed in a concentration-dependent manner compared with
potential (MMP) was a signicant marker of mitochondrial the control group (Fig. 10), and the effects of 10c were signi-
dysfunction and played a key role in the process of cell cantly stronger in K562 cells than those in K562/ADR cells.
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Fig. 7 Cell cycle arrest effects of compound 10c. K562 and K562/ADR cells were incubated with vehicle, 10c (1 mM, 5 mM and 10 mM) and 3 (10
mM) for 24 h, respectively, and cell cycle distribution was measured using flow cytometry after staining with PI. The data were expressed as the
mean ꢀ SD from three independent experiments. (A and C) Flow cytometry analysis. (B and D) Quantitative analysis. *P < 0.05, **P < 0.01 vs. the
control group.
Fig. 8 Compound 10c induced leukemia cells apoptosis. K562 and K562/ADR cells were incubated with vehicle, 10c (1 mM, 5 mM and 10 mM) and
3
(10 mM) for 24 h, respectively, and the percentages of apoptotic cells were measured by flow cytometry after staining with Annexin V-FITC and
-AAD. The data were expressed as the mean ꢀ SD from three independent experiments. (A and C) Flow cytometry analysis. (B and D)
Quantitative analysis. **P < 0.01 vs. the control group.
7
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Fig. 9 Effects of 10c on the mitochondrial membrane potential. K562 and K562/ADR cells were incubated with vehicle, 10c (1 mM, 5 mM and 10
mM) and 3 (10 mM) for 24 h, respectively, and stained with JC-1 dye followed by flow cytometry analysis. The data were expressed as the mean ꢀ
SD from three independent experiments. (A and C) Flow cytometry analysis. (B and D) Quantitative analysis. **P < 0.01 vs. the control group.
Besides apoptosis, autophagy is another programmed cell mediated by activation of mTOR, which is a negative regulatory
38
44
death, which plays a dual role in the cancer therapy. Auto- axis of autophagy. To further explore the molecular mecha-
phagy could induce cell death or protect cells depending upon nisms underlying the antiproliferative activity of 10c, we
39
the cell type and context. Therefore, to assess whether auto- investigated the regulatory effects of treatment with 10c on the
phagy contributes to 10c-mediated inhibition of leukemia cell AMPK, JNK and AKT signaling in K562 and K562/ADR cells.
proliferation, we investigated the effects of 10c on levels of the Cells were incubated with vehicle, 10c (1 mM, 5 mM and 10 mM)
autophagy-related proteins in K562 and K562/ADR cells. Cells and 3 (10 mM) for 24 h, respectively, and the relative levels of
were incubated with vehicle, 10c (1 mM, 5 mM and 10 mM) and 3 AMPK, JNK and AKT phosphorylation were determined by
(10 mM) for 24 h, respectively, and the relative levels of Beclin1 western blot using b-actin as internal standard. As shown in
and LC3-II expression were determined by western blot. Aer Fig. 12, incubation with 10c signicantly inhibited the AKT
treatment with 10c, the Beclin1 and cleavage of LC3 (LC3-II) phosphorylation and stimulated the AMPK and JNK phos-
levels were increased in a dose-dependent manner in K562 phorylation in a dose-dependent manner in both cancer cell
and K562/ADR cells (Fig. 11). We found that the level of auto- lines. Similarly, treatment with 3 also clearly reduced the levels
phagy in resistant K562/ADR cells was lower than that in of AKT phosphorylation and up-regulated the levels of AMPK
sensitive K562 cells, and the 10c-induction of autophagy were and JNK phosphorylation, while the regulatory effects of parent
weaker in K562/ADR cells than those in K562 cells.
3 (10 mM) in both cell lines were noticeably less than that of 10c
The crosstalk between apoptosis and autophagy is quite (10 mM).
40
complex and remains controversial. Apoptosis and autophagy
may be activated by the common upstream signals, giving rise
to the activation of the combination of autophagy and Experimental
41
apoptosis, such as AMPK, JNK and AKT signaling. Autophagy
and apoptosis can be promoted by AMP activated protein kinase
General
Commercially available reagents and solvents were analytical
grade and used without further purication. H NMR and
(
AMPK), which is a key energy sensor and regulates cellular
1
13
42
C
metabolism to maintain energy homeostasis, and c-Jun N-
terminal kinase (JNK), which is a stress-activated protein
kinase (SAPK) of the mitogen activated protein kinase (MAPK)
NMR spectra were recorded with an Agilent 400 NMR spec-
trometer, using TMS as an internal standard. High resolution
mass spectrometry (HRMS) were obtained on an Agilent 6520
Accurate-Mass Q-TOF. Column chromatography was carried out
on silica gel (200–300 mesh).
43
family. Meanwhile, AKT is a serine/threonine protein kinase
showing antiapoptotic activity, as well as inhibiting autophagy
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Fig. 10 Effects of 10c on the cycle- and apoptosis-related proteins. K562 and K562/ADR cells were incubated with vehicle, 10c (1 mM, 5 mM and
0 mM) and 3 (10 mM) for 24 h, respectively, and the relative levels of CDK1, CDK2, cleaved caspase-3 and cleaved PARP expression were
1
determined by western blot using b-actin as internal standard. The data were selected images and expressed as the mean ꢀ SD from three
independent experiments. (A and F) western blot analysis, (B–E, G–J) quantitative analysis. *P < 0.05, **P < 0.01 vs. the control group.
1
General procedure for the preparation of hybrid compounds
0a–10c
Hybrid compound 10b: yellow solid, yield 78%; H NMR
1
(400 MHz, CDCl
3
) d 7.90 (d, J ¼ 8.8 Hz, 2H), 7.75 (s, 1H), 7.38 (s,
1
1
2
1
0
H), 7.15 (d, J ¼ 8.8 Hz, 2H), 5.82 (d, J ¼ 10.0 Hz, 1H), 5.46 (s,
Intermediate 3 (0.26 mmol) was dissolved in dry N,N-dime-
thylformamide (3 mL), then 1-(3-(dimethylamino)propyl)-3-
ethylcarbodiimide hydrochloride (EDCI) (2.0 eq.), 4-dimethyla-
minopyridine (DMAP) (2.4 eq.), and appropriate donor (0.9 eq.
for 4, 0.95 eq. for 5 and 7) were added. The reaction mixture was
stirred at room temperature for 3–5 h. The reaction solution was
quenched by adding water (20 mL), and the crude was ltered
and dried in vacuum oven overnight. The crude residue was
puried by silica gel ash column chromatography
H), 2.95–2.81 (m, 4H), 2.60–2.54 (m, 1H), 2.42–2.34 (m, 1H),
.06–2.02 (m, 1H), 1.90–1.87 (m, 1H), 1.80–1.70 (m, 3H), 1.65–
.60 (m, 1H), 1.54–1.46 (m, 1H), 1.42 (s, 3H), 1.39–1.26 (m, 3H),
1
3
.97 (d, J ¼ 5.6 Hz, 3H), 0.86 (d, J ¼ 7.2 Hz, 3H); C NMR (100
MHz, CDCl ) d201.37, 170.96, 170.43, 153.36, 136.68, 128.48,
3
1
3
(
21.52, 104.53, 92.44, 91.51, 80.12, 51.48, 45.14, 37.23, 36.15,
4.00, 31.76, 29.09, 25.90, 24.53, 21.95, 20.18, 12.05; HRMS-ESI
+
m/z): calcd for C26
H
34NO
8
S [M + H] 520.2000, found
5
20.2001.
(dichloromethane/methanol 100 : 1 for 10a, AcOEt/PE 1 : 4 for
1
Hybrid compound 10c: white solid, yield 73%; H NMR (400
1
0b and 1 : 8 for 10c) to afford the pure compound.
Hybrid compound 10a: red solid, yield 63%; H NMR (400
1
MHz, CDCl
3
) d 5.80 (d, J ¼ 9.6 Hz, 1H), 5.44 (s, 1H), 5.37–5.34
(
m, 1H), 5.24 (d, J ¼ 2.8 Hz, 1H), 5.00 (t, J ¼ 5.2 Hz, 1H), 4.51 (d, J
MHz, CDCl ) d 7.69 (d, J ¼ 8.4 Hz, 2H), 7.40 (s, 1H), 7.28 (d, J ¼
3
¼
5.2 Hz, 1H), 4.06–3.97 (m, 3H), 3.93–3.89 (m, 1H), 2.73 (t, J ¼
8
4
1
3
.4 Hz, 2H), 5.84 (d, J ¼ 10 Hz, 1H), 5.46 (s, 1H), 2.99–2.84 (m,
H), 2.61–2.56 (m, 1H), 2.43–2.35 (m, 1H), 2.06–2.02 (m, 1H),
.93–1.88 (m, 1H), 1.80–1.64 (m, 4H), 1.43 (s, 3H), 1.40–1.30 (m,
H), 1.25 (s, 1H), 0.98 (d, J ¼ 6.0 Hz, 3H), 0.87 (d, J ¼ 6.8 Hz, 3H);
6
1
1
.4 Hz, 2H), 2.68–2.60 (m, 2H), 2.57–2.52 (m, 1H), 2.41–2.33 (m,
H), 2.05–2.01 (m, 1H), 1.92–1.87 (m, 1H), 1.80–1.60 (m, 4H),
.53–1.45 (m, 1H), 1.43 (m, 3H), 1.39–1.24 (m, 3H), 0.97 (d, J ¼
1
3
1
3
6.0 Hz, 3H), 0.86 (d, J ¼ 7.2 Hz, 3H); C NMR (100 MHz, CDCl
3
)
3
C NMR (100 MHz, CDCl ) d 215.45, 171.77, 170.81, 170.26,
d 171.08, 104.45, 92.27, 91.48, 86.43, 81.46, 81.30, 80.10, 77.60,
1
8
2
53.49, 135.98, 129.19, 128.21, 122.96, 104.50, 92.42, 91.51,
0.09, 51.48, 45.15, 37.25, 36.15, 34.01, 31.77, 29.67, 29.14,
7
2
3.32, 69.22, 51.49, 45.17, 37.24, 36.16, 34.02, 31.79, 29.12,
8.88, 25.89, 24.53, 21.94, 20.19, 11.99; HRMS-ESI (m/z): calcd
9.12, 25.93, 24.54, 21.96, 20.19, 12.07; HRMS-ESI (m/z): calcd
+
+
for C25H
39
N
2
O
13 [M + NH
4
] 575.2447, found 575.2443.
for C28
H
33
O
8
S
3
[M + H] 593.1332, found 593.1335.
508 | RSC Adv., 2020, 10, 501–511
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Fig. 11 Effects of 10c on the autophagy-related proteins. K562 and
K562/ADR cells were incubated with vehicle, 10c (1 mM, 5 mM and 10
mM) and 3 (10 mM) for 24 h, respectively, and the relative levels of
Beclin1 and LC3 expression were determined by western blot using b-
actin as internal standard. The data were selected images and Fig. 12 Effects of 10c on the AMPK, JNK and AKT signaling. K562 and
expressed as the mean ꢀ SD from three independent experiments. (A K562/ADR cells were incubated with vehicle, 10c (1 mM, 5 mM and 10
and D) western blot analysis, (B, C, E and F) quantitative analysis. *P < mM) and 3 (10 mM) for 24 h, respectively, and the relative levels of AMPK,
0
.05, **P < 0.01 vs. the control group.
JNK and AKT phosphorylation were determined by western blot using
b-actin as internal standard. The data were selected images and
expressed as the mean ꢀ SD from three independent experiments. (A
and E) western blot analysis, (B–D and F–H) quantitative analysis. *P <
Pharmacology
0
.05, **P < 0.01 vs. the control group.
Cytotoxicity assay. The cytotoxicity of target compounds were
tested against human acute myeloid leukemia K562 and K562/
ꢁ
ADR cells and human normal hepatic LO2 cells in vitro by test compounds 37 C for 24 h. Subsequently, cells were
CCK-8 assay, respectively. The K562, K562/ADR and LO2 cell collected and washed with PBS, and then resuspended in
lines used in present study were purchased from KeyGen incubation buffer containing uorescein probe DCFH-DA
ꢂ1
ꢁ
Biotech (Nanjing, China). Cells (2500–3000 cells per well) were (10 mmol L ) at 37 C for 20 min. The cells were washed with
ꢁ
cultured in 96-well plates and incubated at 37 C for 24 h, and PBS and intracellular NO was analyzed by uorescence
treated in triplicate with different concentrations of individual microscopy and ow cytometer, respectively. Each experiment
compounds for 72 h, and 0.1% DMSO for control. Then, CCK-8 was done three times.
(
3
10 mL) was added to each well and plates were incubated at
7 C for 2 h. The absorbance at 450 nm was measured using 6-well plates and incubated overnight. Then cells were treated
Cell cycle analysis. K562 and K562/ADR cells were planted on
ꢁ
a microplate reader. The IC50 value of each compound was with 0.1% DMSO or different concentrations of test compounds
ꢁ
calculated by GraphPad Prism version 6.01.
37 C for 24 h. Cells were collected, washed twice with PBS, xed
ꢁ
Measurement of intracellular NO. K562 and K562/ADR cells with 70% ethanol at 4 C for 2 h. Subsequently, cells were
were incubated with 0.1% DMSO or different concentrations of incubated with RNase A (100 mL) at 37 C for 30 min, and then
test compounds at 37 C for 3 h. Then cells were harvested and stained with propidium iodide (400 mL) at 4 C in the dark for
washed with PBS and then resuspended in incubation buffer 30 min. The cell cycles were detected by ow cytometry. Each
containing uorescein probe DAF-FM DA (50 mmol L ) at 37 C experiment was done three times.
ꢁ
ꢁ
ꢁ
ꢂ1
ꢁ
for 30 min. The cells were washed with PBS and intracellular NO
Apoptosis analysis. K562 and K562/ADR cells were planted
was analyzed by uorescence microscopy and microplate on 6-well plates and incubated overnight. Next, cells were
reader, respectively. Each experiment was done three times.
Measurement of intracellular ROS. K562 and K562/ADR cells compounds at 37 C for 24 h. Cells were collected, washed twice
were incubated with 0.1% DMSO or different concentrations of with PBS and resuspended in binding buffer (500 mL).
treated with 0.1% DMSO or different concentrations of test
ꢁ
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Subsequently, cells were stained with Annexin V-FITC (5 mL) and Department of Science and Technology of Zunyi City and Affil-
-AAD (5 mL) at room temperature in the dark for 15 min. Cell iated Hospital of Zunyi Medical University ([2018]56 and [2018]
7
apoptosis was analyzed by ow cytometer. Each experiment was 62).
done three times.
Mitochondrial membrane potential (MMP) analysis. K562
and K562/ADR cells were planted on 6-well plates and incubated
Notes and references
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ꢁ
concentrations of test compounds at 37 C for 24 h. Cells were
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There are no conicts to declare.
2
Acknowledgements
This work was nancially supported by the National Natural
Science Foundation of China (81860622); Joint Fund of the
510 | RSC Adv., 2020, 10, 501–511
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