Novel Triapine Derivative Induces Copper-Dependent Cell Death in Hematopoietic Cancers

Article abstract of DOI:10.1021/acs.jmedchem.8b01996

Triapine, an iron chelator that inhibits ribonucleotide reductase, has been evaluated in clinical trials for cancer treatment. Triapine in combination with other chemotherapeutic agents shows promising efficacy in certain hematologic malignancies; however, it is less effective against many advanced solid tumors, probably due to the unsatisfactory potency and pharmacokinetic properties. In this report, we developed a triapine derivative IC25 (10) with potent antitumor activity. 10 Preferentially inhibited the proliferation of hematopoietic cancers by inducing mitochondria reactive oxygen species production and mitochondrial dysfunction. Unlike triapine, 10 executed cytotoxic action in a copper-dependent manner. 10-Induced up-expression of thioredoxin-interacting protein resulted in decreased thioredoxin activity to permit c-Jun N-terminal kinase and p38 activation and ultimately led to the execution of the cell death program. Remarkedly, 10 showed good bioavailability and inhibited tumor growth in mouse xenograft models. Taken together, our study identifies compound 10 as a copper-dependent antitumor agent, which may be applied to the treatment of hematopoietic cancers.

Full text of DOI:10.1021/acs.jmedchem.8b01996

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Article
A novel triapine derivative induces copper-
dependent cell death in hematopoietic cancers
Ge Chen, Chunyi Niu, Jianhua Yi, Lin Sun, Hengyi Cao, Yanjia Fang,
Taijie Jin, Ying Li, Chunli Lou, Jingwu Kang, Wanguo Wei, and Jidong Zhu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01996 • Publication Date (Web): 05 Mar 2019
Downloaded from http://pubs.acs.org on March 6, 2019
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A novel triapine derivative induces copper-
dependent cell death in hematopoietic
cancers
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Ge Chen,a,b,c,g Chunyi Niu, Jianhua Yi, Lin Sun, Hengyi Cao,
a
a
a
a,g
Yanjia Fang,a
Taijie Jin,a,g Ying Li,a Chunli Lou,
d,e
Jingwu Kang, d Wanguo Wei,b, f Jidong Zhua,*
a
Interdisciplinary Research Center on Biology and Chemistry, Center for
Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 201203, China.
b
School of Life Science and Technology, ShanghaiTech University, Shanghai
2
01210, China.
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c
CAS Center for Excellence in Molecular Cell Science, Shanghai Institutes of
Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031,
China.
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State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for
Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 201203, China.
e
School of Physical Science and Technology, ShanghaiTech University, Shanghai
2
01210, China.
fShanghai Advanced Research institute, Chinese Academy of Sciences, Shanghai,
201210, China
g
University of Chinese Academy of Sciences, Beijing 100049, China.
KEYWORDS: hematopoietic cancers, triapine, copper, mitochondrial ROS, TXNIP.
ABSTRACT
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Triapine, an iron chelator that inhibits ribonucleotide reductase, has been
evaluated in clinical trials for cancer treatment. Triapine in combination with other
chemotherapeutic agents shows promising efficacy in certain hematologic
malignancies; however, it is less effective against many advanced solid tumors,
probably due to the unsatisfactory potency and pharmacokinetic properties. In this
report, we developed a triapine derivative IC25 (10) with potent anti-tumor activity.
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0 preferentially inhibited the proliferation of hematopoietic cancers by inducing
mitochondria ROS production and mitochondrial dysfunction. Unlike triapine, 10
executed cytotoxic action in a copper-dependent manner. 10-induced up-
expression of TXNIP resulted in decreased TRX activity to permit JNK and p38
activation and ultimately led to execution of cell death program. Remarkedly, 10
showed good bioavailability and inhibited tumor growth in mouse xenograft models.
Taken together, our study identifies compound 10 as a copper-dependent anti-
tumor agent, which may be applied to the treatment of hematopoietic cancers.
INTRODUCTION
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Acute leukemia is one of the most prevalent cancer types in both early
childhood and adult population. Depending on the various subtypes of leukemia
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and the underlying genetic mutations, the long-term survival varies considerably.1,
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Over last decades, the treatment of leukemia has progressed slowly and the
mainstay is still chemotherapy. In spite of intensive combination conventional
standard therapies and allogeneic hematopoietic stem cell transplantation,
hematopoietic cancers remain difficult to be treated.3-5 Consequently, there is an
urgent need for the development of more effective therapies for leukemia, with a
view to boosting overall survival and improving treatment outcomes.
3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, triapine) shows
broad-spectrum antitumor activity in vitro and is currently being evaluated in
multiple clinical trials. Early results revealed that administration of triapine alone
could not yield survival benefits for cancer patients,6-9 and this may be attributed
to its weak potency. A combination of triapine with other chemotherapeutic agents,
however, has shown promising disease control in myeloproliferative neoplasms,
cervical and vaginal cancers.10-12 Several hypotheses have been advanced
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concerning the mechanisms involved in the antitumor activity of triapine. Among
them, what is more important is that triapine can promote reactive oxygen species
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ROS) production mediated by forming complex with iron.13-16 ROS, as natural
(
products of cellular metabolism, can damage DNA, proteins and lipids.
Accumulating evidence suggests that regulation of ROS level and causing
oxidative stress are intelligent strategies for cancer therapy.17-18
Although triapine is a well-known chelator of transition metal ions such as iron
(
Fe), copper (Cu) and zinc (Zn), the exact metal ion partners relevant to the
biological activities is yet to be identified.19-20 Cu complexes have long been
predicted as biologically active forms due to its intracellular redox cycling potential.
Copper is a redox-active transition metal which plays important roles as cofactors
in various enzymes that function in antioxidant defense, mitochondrial respiration,
neurotransmitter synthesis and a variety of biochemical processes.21-22 However,
the uncontrolled accumulation of copper can also be harmful to cells by inducing
oxidative stress, which damages cellular components and ultimately leads to cell
death. Thorough systems of copper regulation and trafficking are required to
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satisfy the cellular copper requirement and simultaneously minimize the potential
toxicity.23-25
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Cellular thiols are susceptible to several oxidants, and the thioredoxin (TRX)
has a conserved catalytic site containing cysteine that can be oxidized to cysteine
disulfide reversibly so as to regulate thiol redox balance. TRX is a scavenger of
ROS, protecting cells from oxidation-induced apoptosis.26-28 The reduced form of
TRX directly binds to the N-terminal non-catalytic region of apoptosis signaling
kinase-1 (ASK1) and inhibits its kinase activity. The oxidation of reduced TRX
disjoins ASK1 and activates c-Jun N-terminal kinase (JNK) and p38 MAPK
pathway.29-33 Thioredoxin-interacting protein (TXNIP) is a pro-oxidative stress, pro-
inflammatory and pro-apoptotic protein that binds to TRX and inhibits its thiol-
reducing and oxidant-scavenging activity, thereby triggering cellular oxidative
stress and apoptosis.29, 33 Translocation of TXNIP to mitochondria is the other
well-known event following ROS inducer, and TXNIP binds to TRX2 leading to
mitochondrial dysfunction.34-36
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In this study, we designed and synthesized a series of triapine analogs to
improve its potency. Among them, compound 10 exhibited potent anti-tumor effect
and favorable PK properties. 10-induced cell death was associated with increased
levels of mitochondrial ROS mediated by copper. Moreover, it further induced up-
expression of TXNIP and inhibited TRX activity, along with mitochondrial
dysfunction, and then activated JNK and p38 MAPK pathway. Importantly, 10
exhibited anti-tumor activity in MV4;11 leukemia xenograft models in vivo,
suggesting the potential of 10 for the treatment of hematopoietic cancers.
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RESULTS
Design novel triapine derivatives with potent anti-tumor activity. Triapine has
been proposed as a metal chelator through N*-N*-S* terdentate coordination
system (Figure 1A). Based on the binding mode, we designed three triapine
derivatives, 1, 2 and 3 (Scheme 1), by cyclization of the NH2 group and
aromatization of the thiocarbamide moiety (Figure 1A). We then compared the
antitumor activity of the three compounds to that of triapine in two hematopoietic
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tumor cell lines KG1a and KMS11 and two solid tumor cell lines A549 and Hela.
Compound 1 and 2 inhibited the proliferation of tumor cells more potent than
triapine, while 3 showed much diminished inhibition, suggesting the formation of
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-member ring disrupted the antitumor activity (Figure 1B). Moreover, both 1 and
2 were more effective for hematopoietic tumor cells than solid tumor cells (Figure
B). We further profiled 1 and 2 in additional four leukemia cell lines and revealed
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that the antiproliferative activity of 1 was more potent than that of 2, suggesting
that benzothiazole is more favorable than benzimidazole (Figure 1C).
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Figure 1. The antiproliferative activity of triapine derivatives 1, 2 and 3. (A)
Chemical structures of compound 1, 2 and 3. (B) The antiproliferative activities
of 1, 2, 3 and triapine were evaluated in two hematopoietic tumor cell lines
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(
KG1a and KMS11) and two solid tumor cell lines (A549 and Hela) with a
CellTiter-GloTM Luminescent cell viability assay 48 h post-treatment, and the cell
viability was measured compared to that of vehicle-treated cells. (C) SEM, HL60,
KOPN8 and MV4;11 cells were treated with compound 1 and 2 for 48 h, cell
viability was analyzed.
We next investigated the structure-activity relationship (SAR) of 1 (Table 1).
Compound 4, which lacks the N*-N*-S* terdentate coordination system, totally
abolished the cytotoxicity, indicating that chelation with metal is indispensable for
the antitumor activity of triapine analogs. Replacement of fused cyclohexane with
tetrahydropyran resulted in 5, which is much less potent and less selective for
MV4;11 cells over normal human umbilical vein endothelial cells (HUVEC). A
methyl substitution on hydrazine (6) led to complete loss of antitumor activity.
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Table 1. Antiproliferative activities of triapine derivatives
Y
R1
N
N
N
X
S
IC50 (nM,
MV4;11)
IC50 (nM,
HUVEC)
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SFa
3.6
Compou
nd
X
Y
R1
Triapine
1
967.6 ± 108.4 3,448 ± 538
N
C
N
N
CH2
CH2
O
H
H
8.9 ± 0.3
908.1 ± 29.4 102.0
1
5,445 ±
04
4
5
6
> 50,000
< 0.31
3.5
6
H
197.1 ± 33.7
> 50,000
698 ± 28
22,272 ±
CH2
CH3
< 0.45
3
41
a: SF (selectivity factor): IC50 values in HUVEC/ IC50 values in MV4;11.
Given these results, we mainly focused on the optimization of benzothiazole in
the right part (Table 2, Scheme 1). Installation of methoxyl (7) or fluoride (13) to
6
-position (R ) of benzothiazole dramatically compromised the cytotoxic activity,
2
suggesting that 6-position substitution was not tolerated. Fluoro or methoxyl
substitution on other positions of benzothiazole slightly decreased the potency (8,
9
, 14-16). Interestingly, dimethoxyl substitution 10 was more potent (IC50 = 3.6
nM) and the selectivity over HUVEC was much better than 1. 1,3-dioxolane
analog 11 exhibited similar selectivity as 1, however, the potency was slightly
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decreased. EWG group trifluoromethoxyl (12) significantly compromised the
potency to 179.5 nM. Alkyl groups such as methyl (17), dimethyl (18) and indane
analog (19) did not benefit the potency either.
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Scheme 1. Synthesis of triapine derivatives
R4
R4
R4
a
S
N
S
N
b
NH2
NHNH2
R3
NH2
R3
R3
e
R2
R2
R2
H
N
N
R2
R4
N
N
S
R3
R5
R5
e
R5
R5
d
S
N
F
c
S
N
Cl
NHNH2
NH2
3
7
Reagents and conditions: (a) KSCN, Br , AcOH, 0 ℃-rt, 3 h. (b) 80% Hydrazine
2
hydrate, conc. HCl, Ethylene glycol, 150 ℃, 5 h.38 (c) NaCS OEt, DMF, 5 ℃, 4
2
3
9
h; SOCl , CH Cl , 0 ℃-rt, 2 h. (d) 80% Hydrazine hydrate, EtOH, 70 ℃, 5 h.
2
2
2
(e) 6,7-dihydroquinolin-8(5H)-one, MeOH, reflux, 5 h.
Table 2. SRA study of triapine derivatives
H
N
N
R2
R4
N
IC50 (nM,
MV4;11)
IC50 (nM,
HUVEC)
N
S
R3
SFa
R5
cmpd
R2
R3
R4
R5
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H
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1,436 ± 37
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OCH
427.4 ±
H
H
H
11.5
31.1
256.8
1
21.5
3
OCH
H
1,040 ± 92
3
OCH OCH
924.6 ±
80.6
1
0
3
3
1
1
1
1
2
3
H
H
F
H
H
H
-OCH O-
H
H
H
H
H
F
22.3 ± 3
179.5 ± 49.4
1,988 ± 43
33.5 ± 8.2
23.0 ± 2.2
60.9 ± 7.16
2,171 ± 117
609 ± 21
4,977 ± 121
2,649 ± 418
767.2 ± 16
1,335 ± 211
587.9 ±
97.4
3.4
2.5
79.1
33.4
21.9
2
H
H
F
H
H
OCF3
H
H
F
14
1
1
5
6
H
17
H
H
H
H
CH3
CH3
H
H
H
45.3 ± 0.9
57.1 ± 11.6
156.8 ± 4.3
13.0
18.9
8.3
1
8.9
1
8
CH3
1,076 ± 41
-
19
1,300 ± 84
CH CH CH -
2
2
2
a: SF (selectivity factor): IC50 values in HUVEC/ IC50 values in MV4;11.
Considering the bioactivity of the compounds and the diversity of substitution
groups, we selected the compounds 1, 9, 10, 15, and 17 to test their microsome
stability (Table 3). Unfortunately, un- or mono-substituted benzothiazolyl
compounds showed poor microsome stability. To our delight, 10 demonstrated
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good stability in mouse liver microsome (Clint,mouse, 69.6 mL/min/Kg) with longer
T1/2 (78.4 min) compared to triapine. Moreover, mouse pharmacokinetic data
indicated that this compound had satisfactory ADME properties (AUC: 734
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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2
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5
5
5
5
5
6
0
1
2
3
4
5
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7
8
9
0
1
2
3
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8
9
0
1
2
3
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8
9
0
1
2
3
4
5
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7
8
9
0
hr·ng/ml, T : 1.4 hrs and bioavailability: 25%) (Table 4).
1/2
Table 3. Microsome stability of triapine derivatives
compound
T1/2 (minute)
Clint (mL/min/Kg)
Triapine
10.5
3.9
5.6
78.4
3.2
2.2
518.1
1396.1
972.4
69.6
1706.9
2518.8
1
9
10
1
1
5
7
Table 4. Mouse pharmacokinetic parameters of 10
IV (1
PO (10
T
(hr)
0.59
3.39
——
1.40
——
673
734
1
/2
Cl (l/hr/kg)
Cmax (μg/L)
AUClast
294
F (%)
25.0%
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Journal of Medicinal Chemistry
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1
0 inhibits the growth of hematopoietic cancer cells in vitro and in vivo. Given
the potent activity and its favourable pharmacokinetic properties, we further
evaluated the anticancer spectrum of 10. We examined its activity in 25 cancer
cell lines and compared it with that of triapine. Generally, 10 was 10 to 100-fold
more potent than triapine (Figure 2A, Supplementary Table 1). Moreover, the
selectivity of 10 against MV4;11 cells over HUVEC is more than 250 folds,
superior to that of triapine (3.6 folds, Table 1 and 2). 10 was more effective at
inhibiting the viability of hematopoietic tumor cells than that of solid tumor cells
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
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9
0
1
2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Figure 2B). Among these cell lines, MV4;11 was the most sensitive cell to the
cytotoxic effect of 10. We therefore evaluated 10 in MV4;11 tumor xenograft
mouse model. Treatment with 10 orally at a dose of 30 mg/kg significantly
inhibited tumor growth (Figure 2C), with no obvious impact on mouse host body
weight (Figure 2D). Thus, we have identified a novel triapine derivative 10 that
could effectively inhibit the growth of tumor cells, especially leukemia cells.
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0
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0
1
2
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8
9
0
1
2
3
4
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7
8
9
0
Figure 2. The antitumor activity of 10 in vitro and in vivo. (A) All cell lines
were exposed to increasing concentrations of triapine or 10 and the cell viability
was tested using a CellTiter-GloTM Luminescent cell viability assay 48 h post-
treatment. The IC50 value was calculated by non-linear regression analysis and a
standard sigmoidal dose-response model in the GraphPad Prism program, and
the logarithm of mean IC50 values is presented as a histogram. (B) Summary of
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logarithm of mean IC50 of 10 against solid cancer cells (n=11) compared with
hematopoietic cancer cells (n=14). (C) In vivo efficacy study of 10. The tumor
volume was calculated based on the width and length of tumors. The mice
transplanted with MV4;11 leukemia cells were treated with 30 mg/kg 10
administered orally twice daily, while the control mice were treated with vehicle.
0
1
2
3
4
5
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7
8
9
0
1
2
3
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0
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9
0
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8
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0
1
2
3
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5
6
7
8
9
0
*
P<0.05, ** P<0.01. (D) Body weight of mice during 14 days of 10 treatments.
0 induces mitochondrial ROS-dependent cell death. We next analyzed the
1
molecular mechanism underlying the antitumor activity of 10. Treatment of 10 led
to profound G1 arrest in MV4;11 cells after 24 h and cell death after 48 h (Figure
S1A). The cell death was not rescued by Z-VAD or/and Nec-1, suggesting that
the cells did not undergo apoptosis or necroptosis (Figure S1B). We then asked
whether 10 triggers ferroptotic cell death in MV4;11 cells. Ferrostatin-1, which
has been reported to block ferroptosis,41 failed to inhibit 10-induced cell death
either (Figure S1C). Because ROS plays an important role in a variety of cell
deaths induced by iron chelators,16, 26 we next tested whether ROS contributes
to the cytotoxic activity of 10. MV4;11 cells were pretreated with various ROS
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scavengers prior to the addition of 10. Only N-acetyl-L-cysteine (NAC), but not
butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or α-tocopherol
1
1
1
1
1
1
1
1
1
1
2
2
2
2
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2
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2
2
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0
1
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8
9
0
1
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0
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0
1
2
3
4
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8
9
0
(
α-TOC), significantly attenuated 10-induced cell death (Figure 3A). Among the
ROS scavengers, only NAC structurally contains a sulfhydryl group, making us
question whether reduced glutathione (GSH) could block the cell death induced
by 10. Indeed, treatment with GSH was sufficient to restore the cell viability after
10 addition (Figure 3B). We also found that the ratio of GSH/GSSG, an essential
indicator of cellular oxidative stress,42 was decreased in 10-treated MV4;11 cells
(
Figure 3C), suggesting that redox homeostasis was disturbed by the treatment
of 10. We found that 10 treatment elevated the levels of mitochondrial ROS, but
not intracellular ROS in MV4;11 cells (Figure 3D). Pretreatment with 5 mM NAC
1 h before 10 addition led to almost complete inhibition of mitochondrial ROS
generation (Figure 3E). Interestingly, triapine treatment did not increase the levels
of mitochondrial ROS (Figure 3F), suggesting that 10 and triapine induce cell
death through different mechanism.
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Figure 3. 10 selectively induces mitochondrial oxidative stress in MV4;11 cells.
(A) MV4;11 cells were pretreated with 100 μM BHA, 100 μM BHT, 10 μM α-
TOC, 0.5 mM NAC or 5 mM NAC and incubated with 10, and the cell viability
was measured with the CellTiter-GloTM Luminescent cell viability assay. (B)
MV4;11 cells were pretreated with 5 mM GSH and incubated with 10. The cell
viability was measured. (C) Measurement of the ratio of reduced glutathione (GSH)
to oxidized glutathione (GSSG) in MV4;11 cells treated with 10. Cells were treated
with the GSH/GSSG-GloTM assay kit, and the luminescence was measured with
the SpectraMax i3 microplate reader. Data are representative of two independent
experiments. *** P< 0.001. (D) MV4;11 cells in 6-well plates were treated with
1
1
1
1
1
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1
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2
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1
2
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4
5
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8
9
0
5
0 nM 10 for 4 or 8 h then stained with CellROX or MitoSOX. Intracellular ROS
levels or mitochondrial ROS levels were determined with flow cytometry. (E)
MV4;11 cells in 6-well plates were treated with 50 nM 10 for 4 h in the presence
or absence of 5 mM NAC. Mitochondrial ROS levels were determined with flow
cytometry. (F) MV4;11 cells in 6-well plates were treated with 50 nM 10 or 500
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nM triapine for 4h then stained with MitoSOX. Mitochondrial ROS levels were
determined with flow cytometry.
0
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0
Cu2+ cooperates with 10 to inhibit cancer cell survival. It has been shown that
the N*-N*-S* terdentate coordination system in triapine allows chelation with
transition metal ions.19, 43 In order to identify the exact chelation partner of 10,
we treated MV4;11 cells with 10 in the presence of various metal ions including
2
+
2+
3+
2+
2+
Cu , Ca , Fe , Fe , and Mn . We found that only incubation with copper
could potentiate 10-induced death (Figure 4A). Incubation with 10 in the presence
2
+
of Cu caused an increasing proportion of dead cells (Figure 4B) and decreasing
ratio of GSH/GSSG (Figure 4C) compared to the treatment of 10 alone. We next
examined the potential synergy of 10 with Cu2+ on inhibiting MV4;11 cell survival.
The combination index (CI) values showed the strong synergistic effect of Cu2+
and 10 (Figure 4D). Interestingly, copper exhibited antagonistic effect on triapine’s
activity (Figure 4D), suggesting that although 10 and triapine are structurally
related, they utilize different metal ions to execute anti-tumor activity. Then we
evaluated the chelation ability of 10 and triapine with Cu or Fe by UV-vis
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spectroscopic titration. We found that 10 is equipotent to triapine to chelate with
Fe2+ or Fe , however, it is much more potent to chelate with Cu than triapine
3+
2+
1
1
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9
0
(
Figure S2).
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Figure 4. Cu2+ cooperates with 10 to inhibit cancer cell survival. (A) MV4;11
cells were pretreated with 50 μM Cu , Ca , Fe , Fe or Mn2+ and incubated
with indicated concentrations of 10 starting from 100 nM with 3-fold dilution series.
The cell viability was measured with the CellTiter-GloTM Luminescent cell viability
assay. (B) Cells were grown in 12-well plates and treated with 10 for 24 h in
2
+
2+
3+
2+
0
1
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1
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7
8
9
0
2
+
the presence or absence of Cu . Apoptosis was detected by flow cytometry
using Annexin V-FITC and propidium iodide (PI) double staining. (C) Measurement
of the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in
2
+
MV4;11 cells treated with 10 in the absence or presence of Cu . Cells were
treated with the GSH/GSSG-GloTM assay kit, and the luminescence was measured
with the SpectraMax i3 microplate reader. Data are representative of two
independent experiments. * P< 0.05, *** P< 0.001. (D) Analysis of combination
effects between 10 or triapine with Cu2+ for MV4;11 cells. Combination index
analysis was carried out using the Calcusyn software. The logarithm of CI values
is shown. Green indicates synergism while red indicates antagonism.
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0 triggers copper-dependent cell stress and death. We next asked if 10-
induced cell death is dependent on intracellular copper. We found that cell death
induced by 10 treatment was suppressed by co-treatment with copper chelator
bathocuproine disulfonate (BCS) or ammonium tetrathiomolybdate (TM),24,44 but
not iron chelator deferoxamine (DFO) (Figures 5A). Consistently, 10-induced
mitochondrial ROS production was attenuated by BCS treatment, indicating that
intracellular copper is indispensable for the cytotoxic activity of 10 (Figures 5B).
Then we investigated whether copper homeostasis is disturbed by 10. We
employed inductively coupled plasma mass spectrometry (ICP-MS) to measure
the total intracellular copper content. Addition of 10 modestly increased
intracellular copper content, suggesting that the copper homeostasis in the cells
were altered by 10 (Figures 5C). Cotreatment of 10 with Cu2+ significantly
increased intracellular copper content, whereas triapine had minimal effects on
intracellular copper abundance (Figure 5C). Copper chelator BCS completely
1
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0
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+
prevented the increase in copper content after exposure to 10 and Cu (Figures
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C). These data suggested that 10 promoted cell death by inducing intracellular
copper accumulation and copper-dependent ROS production.
0
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Figure 5. 10 triggers copper-dependent cell death. (A) MV4;11 cells were
pretreated with 10 μM TM, 20 μM DFO or 200 μM BCS and incubated with
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indicated concentrations of 10 starting from 5 μM with 3-fold dilution series, and
the cell viability was measured. (B) MV4;11 cells in 6-well plates were treated
with 50 nM compound 10 for 4 h in the presence or absence of 200 μM BCS.
Mitochondrial ROS levels were determined with flow cytometry. (C) The addition
of 10 and Cu2+ lead to an increasing in total cellular copper for 12 h, but BCS
treatment can restore the total copper content. Data are representative of two
independent experiments. * P< 0.05, ** P<0.01, *** P< 0.001.
1
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MAPK. GSH/GSSH ratio was reduced in 10-treated cells, suggesting that the
thiol redox was disrupted by 10 treatment (Figure 3C). Cellular thiols are
susceptible to several oxidants, and the thioredoxin (TRX) is critical for maintaining
intracellular thiol redox balance.29, 32 Indeed, treatment with 10 decreased TRX
activity (Figure 6A). Previous studies have suggested that TXNIP can bind and
inhibit TRX, thus activating JNK and p38 MAPK pathway. Consistently, we
observed an obvious increase in both mRNA and protein levels of TXNIP induced
by 10 treatment in MV4;11 cells (Figures 6B, 6C). Consequently, JNK and p38
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were phosphorylated following 10 treatment (Figure 6C), which may potentiate
the cell oxidative stress effect of 10. However, triapine had no effects on TXNIP
level and the activation of JNK and p38 (Figure S3A).
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Figure 6. 10 increases TXNIP expression and induces the activation of JNK
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experiments. ** P< 0.01. (B) MV4;11 cells were treated with 10 for 24 h, and
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then qPCR assay using two pairs of TXNIP primers was performed. * P< 0.05,
*** P< 0.001. (C) MV4;11 cells were treated with 50 nM 10 w/o NAC for 24 h.
Whole-cell lysates were prepared, and the expression of TXNIP and the
phosphorylated form of JNK and p38 were determined by western blotting.
GAPDH served as a loading control.
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mitochondrial shutting of TXNIP, under oxidative stress, affected mitochondrial
dysfunction and oxidation of mitochondrial DNA (mtDNA).45 As the loss of
mitochondrial membrane potential (MMP, ΔΨm) is associated with the production
of mitochondrial ROS, we examined the effect of 10 treatment on MMP. The
treatment of 10 resulted in loss of MMP detected by flow cytometry using cationic
dye JC-1 (Figure 7A), whereas triapine treatment had no impact (Figure S3B).
mtDNA is physically associated with the inner mitochondrial membrane, and
elevated ROS levels could lead to mtDNA damage.46 In MV4;11 cells, we
detected a significant decrease in mtDNA copy number following treatment with
1
0 (Figure 7B), but with no decrease of mitochondrial mass (Figure 7C).
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Furthermore, 10 markedly reduced oxygen consumption and impaired respiration
on mitochondrial function (Figures 7D). The morphologies of mitochondria in
MV4;11 cells were examined by transmission electron microscope (TEM). 24
hours after the addition of 10, mitochondria with a clear swelling and less number
of cristae or deformed cristae were observed (Figure 7E). These results show
that 10 treatment can reduce mitochondrial oxidative phosphorylation (OXPHOS)
and affect mitochondrial morphology and function.
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Figure 7. 10 triggers the mitochondrial dysfunction pathway. (A) 10 induced the
collapse of ΔΨm. The cells stained with JC-1, followed by FACS analysis. Data
are representative of two independent experiments. ** P< 0.01. (B) Mitochondrial
DNA copy number was assessed as the relative amplification of MT-ND2 (mtDNA
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