Dual Inhibition of Pyruvate Dehydrogenase Complex and Respiratory Chain Complex Induces Apoptosis by a Mitochondria-Targeted Fluorescent Organic Arsenical in vitro and in vivo

Article abstract of DOI:10.1002/cmdc.201900686

Based on the potential therapeutic value in targeting mitochondria and the fluorophore tracing ability, a fluorescent mitochondria-targeted organic arsenical PDT-PAO-F16 was fabricated, which not only visualized the cellular distribution, but also exerted anti-cancer activity in vitro and in vivo via targeting pyruvate dehydrogenase complex (PDHC) and respiratory chain complexes in mitochondria. In details, PDT-PAO-F16 mainly accumulated into mitochondria within hours and suppressed the activity of PDHC resulting in the inhibition of ATP synthesis and thermogenesis disorder. Moreover, the suppression of respiratory chain complex I and IV accelerated the mitochondrial dysfunction leading to caspase family-dependent apoptosis. In vivo, the acute promyelocytic leukemia was greatly alleviated in the PDT-PAO-F16 treated group in APL mice model. Our results demonstrated the organic arsenical precursor with fluorescence imaging and target-anticancer efficacy is a promising anticancer drug.

Full text of DOI:10.1002/cmdc.201900686

Full Papers
DOI: 10.1002/cmdc.201900686
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Dual Inhibition of Pyruvate Dehydrogenase Complex and
Respiratory Chain Complex Induces Apoptosis by a
Mitochondria-Targeted Fluorescent Organic Arsenical
in vitro and in vivo
Yu-Jiao Liu+,[a] Xiao-Yang Fan+,[a] Dong-Dong Zhang,[b] Yin-Zheng Xia,[a] Yan-Jun Hu,[c]
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Feng-Lei Jiang,[a] Fu-Ling Zhou,*[b] and Yi Liu*[a, c, d]
Based on the potential therapeutic value in targeting mitochon-
dria and the fluorophore tracing ability, a fluorescent mitochon-
dria-targeted organic arsenical PDT-PAO-F16 was fabricated,
which not only visualized the cellular distribution, but also
exerted anti-cancer activity in vitro and in vivo via targeting
pyruvate dehydrogenase complex (PDHC) and respiratory chain
complexes in mitochondria. In details, PDT-PAO-F16 mainly
accumulated into mitochondria within hours and suppressed
the activity of PDHC resulting in the inhibition of ATP synthesis
and thermogenesis disorder. Moreover, the suppression of
respiratory chain complex I and IV accelerated the mitochon-
drial dysfunction leading to caspase family-dependent apopto-
sis. In vivo, the acute promyelocytic leukemia was greatly
alleviated in the PDT-PAO-F16 treated group in APL mice
model. Our results demonstrated the organic arsenical precur-
sor with fluorescence imaging and target-anticancer efficacy is
a promising anticancer drug.
Introduction
carry out the pre-clinical trials for the treatment of cancer, such as
leukemia, lymphoma and solid tumor.[6]
Arsenical had been used to treat with psoriasis, syphilis or
rheumatism in China and Greece since two thousand years ago.[1]
To date the most dramatic finding in the history of arsenical drugs
is that arsenic trioxide (ATO) was successfully used to treat acute
promyelocytic leukemia (APL)[2] in 1970 s.[3] Nowadays, ATO and
all-trans retinoic acid (ATRA) have been the first-line treatment
regimens to trigger the cell redifferentiation or apoptosis-inducing
therapy for APL.[1c,4] Besides that ATO was approved to treat APL
by the Food and Drug Administration (FDA) in 2000,[5] organic
arsenicals darinaparsin (DAR) and 4-(N-(S-glutathionylacetyl)amino)
phenylarsonous acid (GSAO) also have been approved by FDA to
Although many researches focused on the anti-tumor
mechanism of ATO in the past several decades,[7] there are still
some problems to be solved. It is widely accepted that ATO can
enhance the SUMOylation and ubiquitination leading to the
degradation of fusion protein PML-RARα (expressed by fusion
gene of promyelocytic leukemia gene and retinoic acid receptor
alpha) and APL cell re-differentiation via the directly binding
with the cysteine residues in RBCC (RING-B-box-coiled-coil)
domain located in zinc finger motif in PML-RARα.[8] However,
the research in vitro has shown that methylation metabolite
MMAIII (mono-methyl-arsonic acid) of ATO has the higher
affinity toward PML-RARα fusion protein than that of ATO or
other methylation metabolites.[9] What’s more, literature showed
that the occurrence of cell re-differentiation may be not
dependent on methylated arsenicals.[9c] These findings indicate
the degradation of PML-RARα fusion protein induced by ATO is
a very complicated process. Besides ATO, As4O6 and As4S4 also
demonstrated the better inhibitory ability to many cancer cell
lines by various signal pathways.[10]
Comparing with the inorganic arsenicals, organic arsenicals
have some unique advantages including the designable structure,
the better biological efficacy and the higher affinity toward the
cysteine residues.[11] Furthermore, organic arsenicals are generally
thought to induce the apoptosis rather than cell re-
differentiation,[12] which means organic arsenicals may carry out
the anti-cancer efficacy toward leukemia and lymphoma, as well
as solid tumors. It has been reported that organic arsenical DAR
can induce the cell cycle arrest, mitochondrial dysfunction, MAPK-
mediated SHP1-dependent cell death or ROS-mediated
apoptosis.[12b,13] Although the signal pathways activated by DAR
are diverse for the different cell lines, the influence on mitochon-
[a] Y.-J. Liu,+ X.-Y. Fan,+ Y.-Z. Xia, F.-L. Jiang, Prof. Y. Liu
Department of Chemistry
Wuhan University
Wuhan 430072 (China)
E-mail: yiliuchm@whu.edu.cn
[b] Dr. D.-D. Zhang, Prof. F.-L. Zhou
Department of Haematology
Zhongnan Hospital
Wuhan University
Wuhan 430071 (China)
E-mail: zhoufuling@whu.edu.cn
[c] Dr. Y.-J. Hu, Prof. Y. Liu
College of Chemistry and Materials Science
Nanning Normal University
Nanning 530001 (China)
[d] Prof. Y. Liu
School of Chemistry and Chemical Engineering
Wuhan University of Science and Technology
Wuhan 430081 (China)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.201900686
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dria is not neglected in all functional mechanisms. Additionally,
organic arsenical GSAO inhibiting the neovascularization and cell
proliferation is closely related to the reaction between the
metabolite CAO of GSAO and adenine nucleotide translocase
(ANT) in mitochondria.[14] Moreover, our former researches also
support the viewpoint that mitochondria play the vital role in the
cytotoxicity of organic arsenicals and many other drugs.[15]
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Based on these reports, we got a mitochondria-targeted
fluorescent organic arsenical to improve the anti-cancer activity
and visualize the distribution in the cells at the same time.
Supported by the literature that F16, a mitochondria-targeted
fluorescent compound, impaired the mitochondrial function to
initiate cellular apoptosis or necrosis,[16] we found that PDT-
PAO-F16 mainly accumulated in mitochondria and displayed
the better inhibitory efficacy. Surprisingly, PDT-PAO-F16 showed
the dual inhibition toward pyruvate dehydrogenase complex
(PDHC) and respiratory chain complexes. After the systematic
mechanism exploration, the observations revealed that PDT-
PAO-F16 triggered the respiration dysfunction, thermogenesis
disorder, membrane swelling and enhanced permeability,
resulting in the release of cytochrome c and the activation of
caspase-dependent ROS-mediated apoptosis. As well, it exhib-
ited good inhibitory ability toward APL in mice model.
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Results and Discussion
Figure 1. A) and B) Spectra characterization of 10 μM PDT-PAO-F16 in PBS. C)
Influence of MK571 on NB4 cellular uptake and cell viability. NB4 cells were
incubated with 0.6 μM PDT-PAO-F16 for 0.5 h, 1 h, 5 h, 12 h and 24 h, followed
by the flow cytometer assessment and the cell viability assessment. D) Confocal
images of PDT-PAO-F16 characterizing its uptake rate. The pictures were
snapped 8, 16, 24, 32, 40, 48, 56, 64 min later after 4.8 μM PDT-PAO-F16 was
added to the cultural medium of NB4 cells. E) NB4 cells were co-incubated with
4.8 μM PDT-PAO-F16 and 1 μM mito-tracker red for 1 hour and PDT-PAO-F16
and mito-tracker red accumulated at the same position which demonstrated
the mito-targeting ability of PDT-PAO-F16. F) NB4 cells were incubated with
1 μM cccp in advance for 10 min, followed by the addition of 4 μM PDT-PAO-
F16 for 3 h and the mitochondrial tracker (MitoTracker® Red CM-H2XRos)
staining. G) NB4 cells were incubated with 4.8 μM PDT-PAO-F16 for 3 h,
followed by the mitochondrial tracker staining. Upper: 1 μM MitoTracker® Red
CMÀ H2XRos; lower: 1 μM MitoTracker® Deep Red FM.
Synthesis and characterization of PDT-PAO-F16
Similar with ATO associated with mitochondrial dysfunction, more
and more reports have shown that organic arsenicals can induce
the mitochondria-mediated or ROS-mediated cell death.[11d,15a] F16
as the cationic form of PVI could accumulate into mitochondria
and cause mitochondria dysfunction. Linking the targeting
compound F16 with the compound PAO-PDT with highly
anticancer efficiency (Scheme 1), we got the targeting compound
PDT-PAO-F16. The UV-vis spectrum ranging from 400 nm to
500 nm characterized the PVI moiety and the absorbance near
270 nm belonged to the SÀ AsÀ S bond (Figure 1A).[17] In addition,
PDT-PAO-F16 showed the maximum excitation at 451 nm and the
maximum emission at 555 nm (Figure 1B).
Cellular uptake and mitochondrial accumulation
In order to assess the biological activity, the half-inhibitory
concentrations (IC50) of PDT-PAO-F16 for five cancer cell lines
(NB4, HL-60, HeLa, MCF-7, SGC7901) were determined by MTT
assay. Illustrated in Table S1, PDT-PAO-F16 showed the best
efficacy toward leukemia cells among the five cell lines, namely
NB4 cells (IC50/24h =0.66�0.14 μM) and HL-60 cells (IC50/24h
=
0.86�0.24 μM). Furthermore, it displayed the toxicity for
normal cells (HEK 293 and GES-1) less than that for leukemia
cells, which indicated that the introduction of PVI increased the
inhibitory capacity, as well as the cellular selectivity. Further-
more, NB4 cells were chose for follow up research.
According to the fluorescent intensity in cells demonstrating
the intracellular uptake of PDT-PAO-F16, PDT-PAO-F16 could enter
into the NB4 cells and even accumulate in mitochondria within an
hour in a time-dependent manner (Figure 1D and 1E) Additionally,
pre-treatment of un-coupler CCCP, resulting in the collapse of
Scheme 1. Synthesis of PDT-PAO-F16 ((E)-1-(3-((4-(1,3,2-dithiarsinan-2-yl)
phenyl)amino)-3-oxopropyl)-4-(2-(1H-indol-3-yl)vinyl)pyridin-1-ium).
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mitochondrial membrane potential, induced the random distribu-
tion of PDT-PAO-F16, as well as the mitochondrial tracker
(Figure 1F). These results demonstrated that PDT-PAO-F16 relied
on the mitochondrial membrane potential as a kind of delocaliza-
tion lipophilic cation (DLC) to mainly target the mitochondria
(Figure 1G). Besides, the compound stopped accumulation after
the long-time incubation (at least 12 h) (Figure 1C). After the
possibility of cell death was ruled out, the most likely reason was
that organic arsenical PDT-PAO-F16 was removed from the NB4
cells. The pre-incubation of multi-drug resistance protein 1 (MRP1)
inhibitor MK571 could maintain the fluorescence intensity of PDT-
PAO-F16 in the NB4 cells even cell death, which indicated the
elimination of PDT-PAO-F16 was mediated by the MRP proteins.
These results were in accordance with the former researches.[18] Of
course, there may be other possibilities for the compound to be
eliminated, such as internal metabolism.
ing the high affinity between arsenic and sulfydryl in LA,[17,21] it
was speculated that PDT-PAO-F16 may affect the E2 activity via
perturbing with the sulfydryl in LA to prevent the acryl group
converted to CoA. The observation that the activity of PDHC E2
was suppressed in the presence of PDT-PAO-F16 and recovered
to some extent by the co-incubation of dimercapto compounds
(DTT (dithiothreitol), DMSA ((r*,s*)-2,3-dimercaptobutanedioic
acid) and DMP (2,3-dimercapto-1-propanol)) indeed supported
the speculation (Figure 2C). Another piece of evidence was
obtained that some dimercapto compounds can also protect
NB4 cells from death to some extent in spite of the existence of
high dosage of PDT-PAO-F16 (Figure S1).
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Effect on mitochondrial respiratory chain complexes
The respiratory chain enzyme complexes are responsible for the
oxygen consumption and ATP synthesis locating at mitochon-
dria. The addition of PDT-PAO-F16 of low dosage mainly had a
remarkable impact on complex I and IV of isolated mitochon-
dria while had negligible impact on complex II and III (Fig-
ure 3A). Complex IV is the terminal enzyme complex in electron
transport chain, where over 90% of oxygen is consumed, thus it
largely controls the mitochondrial respiration.[22] The mitochon-
drial respiration of isolated mitochondria and cells was moni-
tored by Clark Oxygen Electrode. Relatively low respiratory rate
Inhibition of PDHC activity in NB4 cells
The cell viability could be recovered by the incubation together
of lipoic acid (LA) (Figure 2A). LA is not only a powerful
antioxidant but also a necessary disulfhydryl coenzyme for
pyruvate dehydrogenase complex (PDHC). PDHC is a kind of
multi-enzyme complex converting pyruvate into acetyl-CoA
including pyruvate dehydrogenase (E1), dihydrolipoyltransace-
tylase (E2) and dihydrolipoamide dehydrogenase (E3) linking
the glycolysis metabolic pathway to the citric acid cycle.[19,20]
After cells were incubated together with PDT-PAO-F16, there
was a significant decrease in the activity of PDHC in NB4 cells
via a dose- and time-dependent manner (Figure 2B). Consider-
Figure 2. A) The effect of LA on NB4 cell viability with PDT-PAO-F16
incubated for 24 h monitored by trypan blue staining. B) The PDHC activity
of NB4 cells. C) The PDHC E2 activity of NB4 cells. NB4 cells with different
concentrations of PDT-PAO-F16 were incubated for 3 h or 1.2 μM PDT-PAO-
F16 and 500 μM DTT, 300 μM DMP or 100 μM DMSA, then the cells were
sacrificed for the PDHC E2 activity assessment by the kit.
Figure 3. A) The effect on activities of respiratory chain complex I, II, III and
IV. B) The effect of mitochondrial respiration in the presence of PDT-PAO-
F16. Different concentrations of PDT-PAO-F16 were added into isolated
mitochondria. Then the oxygen consumption rate (OCR) of each state was
monitored. C) The OCR of NB4 cells under various conditions.
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for state 4 characterizes integrate mitochondrial inner mem-
brane and the high respiratory rate for state 3 (about 3-fold
higher than state 4) characterizes that respiratory chain
functions well. The isolated mitochondria without PDT-PAO-F16
showed low respiratory rate of state 4 and high of state 3
(Figure 3B). After mitochondrial exposure with different concen-
trations of PDT-PAO-F16, state 4 changed merely until the
concentration of PDT-PAO-F16 reached 8 μM while respiratory
rate of state 3 decreased a lot until this concentration. They
both increased gradually then compared with the 8 μM PDT-
PAO-F16 condition. However, both the changes of state 3 and
state 4 induced a dramatic decline of respiratory control ratio
(RCR=state 3/state 4). The effect on respiratory rate demon-
strated the inhibition of mitochondrial respiration and the
damage of mitochondrial function. As a result, the cellular
oxygen consumption rate was decreased after the 24 h
incubation of PDT-PAO-F16 (Figure 3C). With the permeabiliza-
tion of the plasma membrane achieved with 0.1 mg/mL
digitonin to dissipate the respiratory substrates, all the cells
came to almost respiratory arrest. And then respiration was
initiated with 5 mM NADH substrate to complex I followed by
being provided 5 mM succinate substrate to complex II. Even
with adequate substrate to complex I, cells showed less oxygen
consumption in a dosage-dependent manner under PDT-PAO-
F16 exposure circumstances. While after the addition of
succinate, the oxygen consumption rate decreased in a much
slighter tendency, indicating that PDT-PAO-F16 had much more
inhibitory impact on complex I than complex II and also
impaired other complexes function.
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Figure 4. A) and B) The change of intracellular ROS level after exposure for
24 h (A) or with 1.2 μM for different time. C) and D) The collapse of
mitochondrial membrane potential of NB4 cells with PDT-PAO-F16. E) The
decrease of intracellular ATP level after exposure for 24 h. F) The
thermogenic curves of isolated mitochondria taking pyruvate as substrate.
After incubated with various concentrations of PDT-PAO-F16, the heat
output curves were obtained and analyzed for acquisition of thermogenic
parameters.
Production of ROS in NB4 cells
It has been pointed out in literature that complex I and III are
associated with the generation of ROS.[23] The disturbance of
complex I by PDT-PAO-F16 may lead to the leakage of electrons
so as to excessive production of ROS. The influences were
dependent on the dose of PDT-PAO-F16 and incubation time
(Figure 4A and 4B). The ROS level increased over 2 times even
under 0.6 μM PDT-PAO-F16 exposure for 24 h. And under
1.2 μM PDT-PAO-F16 exposure, the accumulated PDT-PAO-F16
within 1 h could cause ROS burst right away and continued to
24 h. During the initial incubation time (3 h to 6 h), there was a
temporary drop in intracellular ROS level which may be a result
of endogenous antioxidants’ elimination, such as glutathione
system and thioredoxin system. There were researches pointed
that the burst of intracellular ROS can be stimulated by
arsenicals, which further makes the oxidative damage to lipids,
proteins and DNA in cells.[24] Besides, as the scavenge reagent
for intracellular GSH, the pre-incubation of L-buthionine-(S,R)-
sulfoximine (BSO) with NB4 cells decreased the cellular
antioxidant capacity, which further resulted the change that cell
viability dropped down remarkably even exposed with the
nontoxic concentration of PDT-PAO-F16 (Figure S2). This finding
also supports the point that the cancer cells with inherently
lower levels of GSH are more sensitive to the toxic effects of
arsenic.[25] However, the decline of cell viability can’t be
prevented from PDT-PAO-F16 exposure by some antioxidants,
like NAC (N-acetyl-L-cysteine), VC (vitamin C), VE (vitamin E) and
so on, which revealed that the burst of ROS was not the initial
trigger point by PDT-PAO-F16, but the key step in killing NB4
cells mechanism (data not shown).
Damage on mitochondrial membrane
The accumulation of high level ROS can oxidize DNA, lipids and
proteins, especially proteins with some reactive cysteine
residues located in vital complexes essential for mitochondrial
function.[26] This indicated that the burst of ROS relied on the
disturbance of respiratory chain complexes, as well as con-
nected with the other mitochondrial complexes dysfunction, for
instance, the mitochondrial membrane permeability transition
pore complex. Under normal circumstances, mitochondrial
inner membrane has a low permeability to charged species
intrinsically and mitochondrial membrane potential exists.[27]
The isolated mitochondrial membrane permeability to H+ and
K+ enhanced on PDT-PAO-F16 exposure, evidenced by the
decline of absorbance at 540 nm (Figure S3). Thus it caused the
collapse of mitochondrial membrane potential (Figure 4C, 4D
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and Figure S4). As well as the permeabilization of membrane,
swelling as a result of water influx occurred and the absorbance
decreased (Figure S5). Pre-treatment of some specific protective
reagents, such as CsA (cyclosporine A), RR (Ruthenium red),
EGTA (ethyenebis(oxyethylenenitrilo)tetraacetic acid), EDTA
((ethylenediamine)tetraacetic acid), and DTT, could deter the
swelling to some extent, which indicated the swelling induced
by PDT-PAO-F16 may be the result attributed to concomitance
of multiple intricate mechanisms including mitochondrial
membrane permeability transition pore opening and the
oxidation of active sites in vital proteins.[28]
Mitochondria play the vital role on supplying metabolic energy
to the cell in the form of ATP via direct participating in a number
of metabolic reactions, as well as regulating the signal trans-
mission during the apoptosis of cancer cells.[29] After the
mitochondrial damages occurred, the cellular ATP synthesis was
interrupted (Figure 4E) and the isolated mitochondrial thermo-
genesis was interfered monitored (Figure 4F) by microcalorimeter
which indicated the impairment of intracellular “energy factory”.
The relative parameters are concluded in Table S2 and Figure S6.
can activate the downstream signals like caspase family leading
to cell apoptosis. The content of caspase 9 and caspase 3
decreased with the increase of active cleaved-caspase 9 and
cleaved caspase 3 (Figure 5B). And then the cells suffered from
DNA damage indicated by the more and more conversion of
Parp to cleaved-Parp. At last, it was found that PDT-PAO-F16
induced cellular apoptosis monitored by the Annexin V-APC
and 7-AAD double staining via a dose- and time-dependent
manner (Figure 5C), which was consistent with the reports that
the bulk of arsenicals can active the apoptotic signaling
pathway in many kinds of cell lines.[12,15a] Besides, the addition
of pan-caspase inhibitor Z-VAD-fmk in advance protected the
majority of cells from apoptosis, which further confirmed the
caspase family-dependent cell apoptosis induced by PDT-PAO-
F16 (Figure 5D and Figure S7). Moreover, cellular morphology
changed including nuclear shrinkage, chromatin condensation,
and apoptotic body formation in the occurrence of apoptosis
manifested by Hoechst 33342 and PI staining (Figure S8).
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Reducing leukemic burden and inducing long-term survival
in an APL mouse model
Induction of caspase family-dependent intrinsic apoptosis
We further evaluated the PDT-PAO-F16’s efficacy in vivo using a
transplantable APL mouse model (Figure 6A). C1498 cells,
lymphoblast from a female C57BL/6 mouse with acute myeloid
When the outer membrane was ruptured and inner membrane
is damaged, some pro-apoptosis proteins could be released
into cytoplasm, such as cytochrome c, pro-caspases and AIF.
Indeed, the content of released cytochrome c increased by a
dose-dependent way (Figure 5A). The release of cytochrome c
Figure 6. PDT-PAO-F16 reduces leukemia burden in an APL mouse model. A)
Schematic representation of transplantable APL mouse model and treatment
procedure. B) The arsenic content of mice femur. C) White blood cells
counting of the mouse blood before being sacrificed showed reduced
leukemic cells in the blood of APL mice. D) The body weight curve of the
mice recorded every three days. E) The survival curve shows that PDT-PAO-
F16 significantly prolonged the survival compared with the saline group. F)
After the mice being sacrificed, the organs were collected and the organ
coefficients were calculated according to the organ weight and the related
body weight.
Figure 5. The apoptosis caused by PDT-PAO-F16. A) The increase of intra-
cellular Cyt-c level of NB4 cells after incubation for 24 h. B) The expression of
some apoptotic signal pathway related proteins. C) Annexin V-APC/7-AAD
staining for NB4 cells apoptosis induced by PDT-PAO-F16. D) The effect of Z-
VAD-fmk or Nec-1 on apoptosis.
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leukemia, were injected intravenously via the tail vein into
C57BL/6 mice. After the WBC blast, 200 μL PBS with 2.2 mg/kg
PDT-PAO-F16 or not were given intraperitoneally once a week
and continued for 5 weeks. Mouse body weight was measured
every 3 days. As shown in Figure 6C, PDT-PAO-F16 could
alleviate the mice leukemic phenotypes by eliminating abnor-
mal APL cells remarkably compared with the saline group even
after the first treatment. Notably, PDT-PAO-F16 could always
maintain the body weight increase while the saline group
suffered from abnormal body weight loss (Figure 6D) and as a
result, our compound prolonged the overall survival of APL
mice (Figure 6E). After the mice were sacrificed, the relative
organ coefficients demonstrated the transplanted APL cells
caused great damage to the mice organ but our compound
greatly alleviated or even eliminated the damage (Figure 6F).
Besides, the arsenical could accumulate a little in mice femur to
withstand the myeloid invasion of leukemic cells without
cytotoxicity as the arsenic concentration in femur increased
slightly (Figure 6B). Of note: we found the treated group mice
were in good health condition during the 5-week treatment
period and no signs of toxicity. All these results indicated that
PDT-PAO-F16 was a potential candidate for APL.
Keywords: Arsenic · leukemia · mitochondria · PDHC ·
apoptosis
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[1] a) S. Waxman, K. C. Anderson, Oncologist 2001, 6, 3–10; b) S. Gibaud, G.
Jaouen, in Medicinal organometallic chemistry, Springer, 2010, 1–20; c) J.
Zhu, Z. Chen, V. Lallemand-Breitenbach, Nat. Rev. Cancer 2002, 2, 705–
714.
[2] C. Huynh, D. Roth, D. M. Ward, J. Kaplan, N. W. Andrews, Proc. Natl.
Acad. Sci. USA 2004, 101.
[3] M. A. Sanz, P. Fenaux, M. S. Tallman, E. H. Estey, B. Löwenberg, T. Naoe,
E. Lengfelder, H. Döhner, A. K. Burnett, S.-J. Chen, V. Mathews, H. Iland,
E. Rego, H. Kantarjian, L. Adès, G. Avvisati, P. Montesinos, U. Platzbecker,
F. Ravandi, N. H. Russell, F. Lo-Coco, Blood 2019, 133, 1630.
[4] F. Massaro, M. Molica, M. Breccia, Int J Hematol Oncol 2016, 5, 105–118.
[5] B. R. Smith, C. M. Eastman, J. T. Njardarson, J. Med. Chem. 2014, 57,
9764–9773.
[6] a) N. C. Lloyd, H. W. Morgan, B. K. Nicholson, R. S. Ronimus, Angew.
Chem. Int. Ed. 2005, 44, 941–944; Angew. Chem. 2005, 117, 963–966;
b) B. K. Mandal, Y. Ogra, K. T. Suzuki, Chem. Res. Toxicol. 2001, 14, 371–
378.
[7] a) P. J. Dilda, P. J. Hogg, Cancer Treat. Rev. 2007, 33, 542–564; b) V.
Lallemand-Breitenbach, J. Zhu, Z. Chen, Trends Mol. Med. 2012, 18, 36–
42.
[8] X.-W. Zhang, X.-J. Yan, Z.-R. Zhou, F.-F. Yang, Z.-Y. Wu, H.-B. Sun, W.-X.
Liang, A.-X. Song, V. Lallemand-Breitenbach, M. Jeanne, Science 2010,
328, 240–243.
[9] a) H. Naranmandura, N. Suzuki, K. T. Suzuki, Chem. Res. Toxicol. 2006, 19,
1010–1018; b) K. T. Suzuki, A. Katagiri, Y. Sakuma, Y. Ogra, M. Ohmichi,
Toxicol. Appl. Pharmacol. 2004, 198, 336–344; c) Q. Q. Wang, X. Y. Zhou,
Y. F. Zhang, N. Bu, J. Zhou, F. L. Cao, H. Naranmandura, Oncotarget
2015, 6, 25646.
[10] a) M. J. Kim, J. H. Jung, W. S. Lee, J. W. Yun, J. N. Lu, S. M. Yi, H. J. Kim, S.-
H. Chang, G.-S. Kim, S. C. Hong, Oncol. Rep. 2014, 31, 2305–2311; b) L.
Zhang, Y. Tong, X. Zhang, M. Pan, S. Chen, Drug Des. Dev. Ther. 2015, 9,
5851.
[11] a) S. Shen, X.-F. Li, W. R. Cullen, M. Weinfeld, X. C. Le, Chem. Rev. 2013,
113, 7769–7792; b) M. J. Mass, A. Tennant, B. C. Roop, W. R. Cullen, M.
Styblo, D. J. Thomas, A. D. Kligerman, Chem. Res. Toxicol. 2001, 14, 355–
361; c) E. Dopp, U. Von Recklinghausen, R. Diaz-Bone, A. Hirner, A.
Rettenmeier, Environ. Res. 2010, 110, 435–442; d) Y. Liu, D. Duan, J. Yao,
B. Zhang, S. Peng, H. Ma, Y. Song, J. Fang, J. Med. Chem. 2014, 57, 5203–
5211.
[12] a) G.-Q. Chen, L. Zhou, M. Styblo, F. Walton, Y. Jing, R. Weinberg, Z.
Chen, S. Waxman, Cancer Res. 2003, 63, 1853–1859; b) I. Khairul, Q. Q.
Wang, Y. H. Jiang, C. Wang, H. Naranmandura, Oncotarget 2017, 8,
23905.
[13] a) D. Ravi, S. Bhalla, R. B. Gartenhaus, J. Crombie, J. Sharma, I. K. Kandela,
A. P. Mazar, A. M. Evens, Clin. Cancer Res. 2014, 1532; b) A. S. Don, O.
Kisker, P. Dilda, N. Donoghue, X. Zhao, S. Decollogne, B. Creighton, E.
Flynn, J. Folkman, P. J. Hogg, Cancer Cell 2003, 3, 497–509.
[14] a) D. Park, J. Chiu, G. G. Perrone, P. J. Dilda, P. J. Hogg, Cancer Cell Int.
2012, 12, 11; b) M. Elliott, S. Ford, E. Prasad, L. Dick, H. Farmer, P. Hogg,
G. Halbert, Int. J. Pharm. 2012, 426, 67–75.
[15] a) X.-Y. Fan, X.-Y. Chen, Y.-J. Liu, H.-M. Zhong, F.-L. Jiang, Y. Liu, Sci. Rep.
2016, 6; b) M. Picard, D. C. Wallace, Y. Burelle, Mitochondrion 2016, 30,
105–116; c) M. Vuda, A. Kamath, Mitochondrion 2016, 31, 63–74.
[16] V. R. Fantin, P. Leder, Cancer Res. 2004, 64, 329–336.
[17] A. M. Spuches, H. G. Kruszyna, A. M. Rich, D. E. Wilcox, Inorg. Chem.
2005, 44, 2964–2972.
[18] a) L. D. Garbinski, B. P. Rosen, J. Chen, Environ. Int. 2019, 126, 585–597;
b) S. Xu, Y. F. Zhang, M. W. Carew, W. H. Hao, J. F. C. Loo, H.
Naranmandura, X. Chris Le, Arch. Toxicol. 2013, 87, 1013–1023.
[19] E. L. Guevara, N. S. Nemeria, L. Yang, F. Jordan, FASEB J. 2016, 30, 851–
856.
[20] E. Saunier, C. Benelli, S. Bortoli, Int. J. Cancer 2016, 138, 809–817.
[21] a) X. Yan, J. Li, Q. Liu, H. Peng, A. Popowich, Z. Wang, X. F. Li, X. C. Le,
Angew. Chem. Int. Ed. 2016, 55, 14051–14056; Angew. Chem. 2016, 128,
14257–14262; b) Z. Diaz, K. K. Mann, S. Marcoux, M. Kourelis, M.
Colombo, P. B. Komarnitsky, W. H. Miller, Jr., Leukemia 2008, 22, 1853–
1863.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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Conclusion
In conclusion, the newly synthesized organic arsenical PDT-PAO-
F16 visualized the cellular distribution, and exerted anti-cancer
activity via targeting PDHC and respiratory chain complexes in
mitochondria. The introduction of PVI moiety to PDT-PAO-F16
improves anti-cancer activity and selectivity attributed to accumu-
lating in mitochondria in cancer cells as a kind of delocalization
lipophilic cation. It mainly inhibited the activity of PDHC and
respiratory chain complex I and IV leading to the mitochondrial
dysfunction, causing mitochondrial depolarization, ROS genera-
tion, suppression of ATP synthesis and thermogenesis disorder.
Thus it led to the release of cytochrome c and caspase family-
dependent cell apoptosis. Importantly, it could inhibit leukemic
cell blast in APL mice and showed almost no side effect. To sum
up, this study provides insights into the design and the anti-cancer
mechanism of organic arsenical and also provides a promising
viable therapy for APL.
Experimental Section
Experimental details and supporting results are available in the
Supporting Information.
Acknowledgements
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (21673166, 21873075), and
the Fundamental Research Funds for the Central Universities
(2015203020212). We thank Fang Zhou for providing confocal
services (Analytical & Testing Center, IHB, CAS).
[22] A. Birk, W. Chao, C. Bracken, J. Warren, H. Szeto, Br. J. Pharmacol. 2014,
171, 2017–2028.
[23] M. P. Murphy, Biochem. J. 2009, 417, 1–13.
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[24] B. Halliwell, Trends Pharmacol. Sci. 2011, 32, 125–130.
Cell Biol. L. 2017, 1862, 39–48; b) S. Desagher, J.-C. Martinou, Trends Cell
Biol. 2000, 10, 369–377.
1
2
3
4
5
[25] C.-H. Yang, M. Kuo, J. Chen, Y. Chen, Brit. J. cancer 1999, 81, 796.
[26] P. R. Angelova, A. Y. Abramov, Free Radical Biol. Med. 2016, 100, 81–85.
[27] G. Kroemer, L. Galluzzi, C. Brenner, Physiol. Rev. 2007, 87, 99–163.
[28] a) T.-I. Peng, M.-J. Jou, Ann. N. Y. Acad. Sci. 2004, 1011, 112–122; b) M.
González-Durruthy, J. M. Monserrat, L. C. Alberici, Z. Naal, C. Curti, H.
González-Díaz, RSC Adv. 2015, 5, 103229–103245.
Manuscript received: December 7, 2019
Revised manuscript received: February 10, 2020
Version of record online: ■■■, ■■■■
[29] a) A. J. Kastaniotis, K. J. Autio, J. M. Kerätär, G. Monteuuis, A. M. Mäkelä,
R. R. Nair, L. P. Pietikäinen, A. Shvetsova, Z. Chen, J. K. Hiltunen, BBA-Mol.
6
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Y.-J. Liu, X.-Y. Fan, Dr. D.-D. Zhang,
Y.-Z. Xia, Dr. Y.-J. Hu, F.-L. Jiang,
Prof. F.-L. Zhou*, Prof. Y. Liu*
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Dual Inhibition of Pyruvate Dehy-
drogenase Complex and Respira-
tory Chain Complex Induces
Apoptosis by a Mitochondria-
Targeted Fluorescent Organic
Arsenical in vitro and in vivo
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A fluorescent mitochondria-targeted
organic arsenical PDT-PAO-F16 was
fabricated, and was found to accumu-
late in mitochondria within hours and
to inhibit the activity of PDHC and res-
piratory chain complex I and IV,
This further caused caspase-family-
dependent cell apoptosis in vitro and
in vivo. Owing to this apoptotic-
inducing ability, PDT-PAO-F16 allevi-
ated and even diminished acute pro-
myelocytic leukemia cells in mice.
leading to mitochondrial dysfunction.