A mitochondria-targeted organic arsenical accelerates mitochondrial metabolic disorder and function injury

Article abstract of DOI:10.1016/j.bmc.2019.01.008

Considering the vital role of mitochondria in the anti-cancer mechanism of organic arsenical, the mitochondria-targeted precursor PDT-PAO-TPP was designed and synthesized. PDT-PAO-TPP, as a delocalization lipophilic cation (DLCs) which mainly accumulated

Full text of DOI:10.1016/j.bmc.2019.01.008

Accepted Manuscript
A mitochondria-targeted organic arsenical accelerates mitochondrial metabolic
disorder and function injury
Xiao-Yang Fan, Yu-Jiao Liu, Yu-Meng Cai, An-Dong Wang, Yin-Zheng Xia,
Yan-Jun Hu, Feng-Lei Jiang, Yi Liu
PII:
S0968-0896(18)31994-1
https://doi.org/10.1016/j.bmc.2019.01.008
BMC 14700
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
23 November 2018
31 December 2018
14 January 2019
Please cite this article as: Fan, X-Y., Liu, Y-J., Cai, Y-M., Wang, A-D., Xia, Y-Z., Hu, Y-J., Jiang, F-L., Liu, Y.,
A mitochondria-targeted organic arsenical accelerates mitochondrial metabolic disorder and function injury,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.008
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A mitochondria-targeted organic arsenical accelerates
mitochondrial metabolic disorder and function injury
a#
a#
a
a
a
Xiao-Yang Fan , Yu-Jiao Liu , Yu-Meng Cai , An-Dong Wang , Yin-Zheng Xia ,
b
a
a,b,c,*
Yan-Jun Hu , Feng-Lei Jiang , Yi Liu
a
State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for
Biology and Medicine (MOE), College of Chemistry and Molecular Sciences,
Wuhan University, Wuhan 430072, P. R. China
b
College of Chemistry and Chemical Engineering, Hubei Normal University,
Huangshi 435002, P. R. China
c
Key Laboratory of Coal Conversion and New Carbon Materials of Hubei Province,
College of Chemistry and Chemical Engineering, Wuhan University of Science and
Technology, Wuhan 430081, P. R. China
#
These authors contributed equally to this work
*
Corresponding author: Prof. Dr. Yi Liu, Department of Chemistry, College of
Chemistry and Molecular Sciences, Wuhan University,
Wuhan 430072, P. R. China.
Fax: +86 27 68754067 Tel: +86 27 68753465
E-mail: yiliuchem@whu.edu.cn

Abstract
Considering the vital role of mitochondria in the anti-cancer mechanism of organic
arsenical, the mitochondria-targeted precursor PDT-PAO-TPP was designed and
synthesized. PDT-PAO-TPP, as a delocalization lipophilic cation (DLCs) which
mainly accumulated in mitochondria, contributed to improve anti-cancer efficacy and
selectivity towards NB4 cells. In detail, PDT-PAO-TPP inhibited the activity of
PDHC resulting in the suppression of ATP synthesis and thermogenesis disorder.
Additionally, the inhibition of respiratory chain complex I and IV by short-time
incubation of PDT-PAO-TPP also accelerated the respiration dysfunction and
continuous generation of ROS. These results led to the release of cytochrome c and
activation of caspase family-dependent apoptosis. Different from the mechanism of
PDT-PAO in HL-60 cells, it mainly induced the mitochondrial metabolic disturbance
resulting in the intrinsic apoptosis via inhibiting the activity of PDHC in NB4 cells,
which also implied that the efficacy exertion of organic arsenical was a complex
process involved in many aspects of cellular function. This study systematically
clarifies the anti-cancer mechanism of mitochondria-targeted organic arsenical
PDT-PAO-TPP and confirms the new target PDHC of organic arsenicals, which
further supports the organic arsenical as a promising anticancer drug.
Keywords: mitochondria; organic arsenical; PDH; TPP; apoptosis

Introduction
Arsenic trioxide (ATO) has been approved by the U.S. FDA for the successful
1
-4
clinical treatment of acute promyelocytic leukemia . Evidence has shown that for
the high affinity between arsenic atom and sulfhydryl group, arsenicals have various
targets among intracellular proteins and endogenous or exogenous antioxidants, such
as thioredoxin, thioredoxinreductase, DNA repair enzymes, hemoglobin, peroxide
reductase, glutathione reductase, pyruvate dehydrogenase complex, peroxiredoxin and
2
-5
so on . For their better biological compatibility and outstanding designability,
6
arsenicals had aroused much interest and shed light on the antitumor mechanism
7
.
ATO can give rise to the oligomerization, sumoylation and degradation of
8
,9
PML-RARα fusion protein . While, organic arsenicals are generally thought to
induce the apoptosis rather than cell re-differentiation, which indicates that organic
arsenicals may hold the anti-cancer efficacy towards leukemia and lymphoma, as well
1
0-12
as solid tumors
. Similarly, organic arsenicals can consume the endogenous
antioxidants, disturb the intracellular redox balance, impair the mitochondrial function,
6
,7,13-15
as well as react with sulfhydryl group in vital active sites
. Mitochondrial
damage mediated apoptosis induced by organic arsenicals is a common pathway.
Mitochondria as the indispensable organelles can provide the energy in the form of
ATP through the process of oxidative phosphorylation, and a mass of metabolic
1
6-18
intermediates for the biosynthesis like lipid metabolism
. Besides, they also
19
participate in regulating multiple signal pathways including cell apoptosis .
Compared to the normal mitochondria, the mitochondria in cancer cells have the
higher content of ROS, higher level of membrane potential and down-regulated
expression of apoptosis-related proteins supporting the rapid cell proliferation and
2
0-22
escape from death
These differences gradually become the potential targets of
2
2,23
mitochondria-targeted anti-tumor drugs
. A report has shown that dichloroacetic
acid can inhibit the activity of pyruvate dehydrogenase kinase (PDK) resulting in the
+
disturbance of glycolysis, the burst of ROS and activation of K channel to promote
24,25
apoptosis and to inhibit cancer growth
. Besides, there are some drugs, such as

ABT-737, ABT-199 and ATO, which can activate the apoptosis signaling pathways
through affecting the activities of related enzymes and regulating the level of
2
6,27
intracellular ROS
. In addition, delocalization lipophilic cation (DLCs) can target
cancer cells mitochondria selectively for its higher membrane potential, which puts
2
8-31
forward the feasible ideas about the design of mitochondria-targeted drugs
.
We designed a mitochondria-targeted organic arsenical based on the former finding
that mitochondria play the vital role in the anti-cancer mechanism of organic arsenical
PDT-PAO, PIM-PAO-PDT and PAM-PAO-PDT. As a type of mitochondria-targeted
3
2
group , TPP was introduced to bind with organic arsenical PDT-PAO to yield
mitochondria-targeted precursor PDT-PAO-TPP. In the cytotoxicity screening, organic
arsenical PDT-PAO-TPP showed the better efficacy and selectivity towards NB4 cells.
After the systematical study, it was confirmed that majority of PDT-PAO-TPP
accumulated in mitochondria, which immediately induced the mitochondrial
depolarization. What’s more, PDT-PAO-TPP showed the dual inhibitions towards
pyruvate dehydrogenase complex (PDHC) and respiratory chain complexes, resulting
in the release of cytochrome c and the activation of caspase-dependent ROS-mediated
apoptosis.
Results and Discussion
Synthesis and cytotoxicity
In order to improve the mitochondria-targeted property, TPP was introduced to the
organic arsenical PDT-PAO. As shown in Scheme 1, linking the TPP with PDT-PAO
via amide bond yielded final product PDT-PAO-TPP. TPP has been reported to enter
3
3
into the mitochondria selectively with negligible cellular toxicity . The design of
PDT-PAO-TPP combined the structural simplicity and better anti-cancer bioactivity.
Seen from Table 1, several kinds of cancer cell lines (NB4, HL-60, HeLa, MCF-7,
SGC7901) were chosen to assess the killing ability of organic arsenicals by the MTT
assay. Comparing with PDT-PAO, compound PDT-PAO-TPP retained the better
inhibitory activity towards all of cancer cell lines with half-inhibitory concentrations

(
IC50) at below 10 µM. In details, PDT-PAO-TPP displayed the best efficacy towards
NB4 cells with IC50 value of 0.65 ± 0.05 µM (24 h) and 0.51 ± 0.01 µM (48 h). Thus
the newly synthesized compound PDT-PAO-TPP had the better inhibitory capacity.
Scheme 1.The synthetic routes of PDT-PAO and PDT-PAO-TPP.
a
Table 1. Inhibition effects of PDT-PAO and PDT-PAO-TPP on different cell lines.
PDT-PAO
PDT-PAO-TPP
Cell lines
2
4h
48h
24h
48h
NB4
HL-60
1.25±0.02
1.12±0.02
2.51±0.97
6.44±0.31
9.00±2.17
6.27±0.73
0.82±0.15
0.65±0.05
3.58±0.01
9.05±2.46
4.63±0.48
4.44±1.46
4.63±0.48
0.51±0.01
1.01±0.18
1.02±0.24
2.19±0.13
3.36±0.38
2.72±0.26
3.23±0.32
3.91±1.05
0.99±0.09
1.46±0.16
2.08±0.35
HeLa
SGC7901
MCF-7
HEK293
a
The data (IC50) are expressedas the mean ± SD of three independent experiments and the unit
-
1
is µmol·L .

Assessment of mitochondria-targeted property
TPP moiety as the mitochondria-targeted group in PDT-PAO-TPP made us to
speculate that it can accumulate mainly in the mitochondria. The obvious collapse of
mitochondrial membrane potential followed by slight decrease of cell viability was
induced by PDT-PAO-TPP after the short-time incubation, whereas there was only
slight variation in the intracellular ROS level under the same conditions (Figure 1).
However, the addition of PDT-PAO made the negligible difference on membrane
potential, minor alterations on ROS content in NB4 cells, as well as slight decline on
cell viability. The observations indirectly indicated that compound PDT-PAO-TPP
mainly targeted mitochondria to exert its bioactivity via immediately triggering the
collapse of the membrane potential.

Figure 1. Effects on NB4 cells after short-time incubation. (A) The variation of mitochondrial
membrane potential. NB4 cells were incubated with 1.2 µM PDT-PAO or 0.6 µM PDT-PAO-TPP,
followed by JC-1 staining. The change of intracellular ROS level by PDT-PAO-TPP (B) or
PDT-PAO (C) was shown. NB4 cells were incubated with 1.2 µM PDT-PAO or 0.6 µM
PDT-PAO-TPP for 1 h, 3 h and 6 h, followed by DCFH-DA staining. The decrease of cell viability
(MTT assay) by PDT-PAO-TPP (D) or PDT-PAO (E) was shown.
Inhibition of pyruvate dehydrogenase complex (PDHC) activity
Pyruvate dehydrogenase complex (PDHC) as a kind of multi-enzyme complex
including pyruvate dehydrogenase (E1), dihydrolipoyltransacetylase (E2) and

dihydrolipoamide dehydrogenase (E3), links the glycolysis metabolic pathway to the
3
4,35
citric acid cycle and fatty acid synthesis
. Treatment of PDT-PAO-TPP or
PDT-PAO can suppress the PDHC activity by a concentration and time dependent
3
7
manner (Figure 2A, 2B and Figure S1). Together incubation with LA can protect
NB4 cells from death (Figure 2C and Figure S2), which means that PDT-PAO-TPP
may disturb the dihydrolipoyltransacetylase (E2) in PDHC via interacting with the
sulfydryl in DHLA to prevent the acryl group converted to CoA. The observation
(Figure 2D) that the activity of PDHC E2 was inhibited by the treatment of
PDT-PAO-TPP and recovered by the dimercapto compounds incubation in advance
implied PDHC-targeted of PDT-PAO-TPP. And these dimercapto compounds (DTT,
DMSA and DMP) recovered the cell viability to some extent despite of the treatment
of PDT-PAO-TPP or PDT-PAO (Figure S3).
Figure 2. The inhibition of PDHC and E2 activity. The decrease of PDHC activity by a
concentration- (A) and time- (B) dependent way. NB4 cells with different concentrations of
PDT-PAO-TPP were incubated for 0.5 h, 2.5 h or 5 h, then the cells were sacrificed to assess the
PDHC activity. (C) The effect of LA on cell viability (trypan blue staining). (D) The suppression
of PDHC E2 activity. NB4 cells with different concentrations of PDT-PAO-TPP and some other

regents (75 µM LA, 500 µM DTT, 300 µM DMP and 150 µM DMSA) were incubated for 3 h for
the PDHC E2 activity assessment.
Influence on mitochondrial energy metabolism and thermogenesis
36
Mitochondria is called the intracellular “energy factory” . As revealed in Figure
3
A and 3B, intracellular ATP level declined after the treatment of PDT-PAO-TPP or
PDT-PAO, which accorded with the decrease of membrane potential (Figure 3C, 3D
and 3E). To avoid interfering from other organelles, isolated mitochondrial
thermogenesis was determined with the help of microcalorimeter (Figure 3F and 3G).
There are four phases in the thermogenic curve including lag phase, activity recovery
phase, stationary increase phase and decline phase. It is the lag phase to adapt to the
physiological environment in the initial 20 h. Then mitochondria undergo a short
activity recovery phase taking about 10 h and the following duration time of
stationary phase lasts approximately 15 h. Finally, because the oxygen and nutrition
are exhausted, there is a quick decline phase. Partial metabolic parameters were
3
7
obtained based on the thermokinetic equation below :
ln P = ln P + k·t
where P and P
t
0
(1)
t
0
are the heat output power at specific time and initial time,
respectively. k represents the rate constant of log phase including activity recovery
phase (k ), stationary increase phase (k ) and decline phase (k ). Other parameters
were seen from the heat output curve. All the parameters were shown in Table 2 and it
was suggested that PDT-PAO-TPP or PDT-PAO can slow down the rate constant (k
1
2
3
2
3
8
and k
3
) and reduce the maximum power resulting in the suppression of the heat
output.

Figure 3. The influence on mitochondrial metabolism. The decrease of intracellular ATP level
after 24 h incubation((A) and (B)) (C) (D) The collapse of mitochondrial membrane potential by
JC-1 staining. Green: percentage of cells with decreased membrane potential and Red for the
normal. (E) Confocal images about mitochondrial membrane potential by JC-1 staining. The

positive sample was treated by 1 µM cccp for 10 min in advance. (F) and (G) The thermogenic
curves of isolated mitochondria taking pyruvate as substrate incubated with various concentrations
of PDT-PAO-TPP (F) or PDT-PAO (G) for a long time.
Table 2. Influences of PDT-PAO and PDT-PAO-TPP on mitochondrial metabolism
a
parameters.
PDT-PAO
PDT-PAO-TPP
c (µM)
0
50
100
3.08
0.99
0.66
0.99
200
4.29
0.99
0.62
0.99
0
25
50
100
3.88
0.99
0.41
0.99
-9.13
0.99
-
3
-1
k
k
k
1
2
3
(10 min )
7.33
0.99
1.18
0.99
4.07
0.99
0.19
0.98
5.15
0.99
1.00
0.99
3.82
0.99
0.72
0.99
4.82
0.99
0.46
0.98
-9.35
0.99
2
R
1
-3
-1
(10 min )
2
R
2
-3
-1
(10 min ) -24.13 -21.47 -16.17 -16.52 -20.47 -14.05
2
R
3
0.99
141.64
49.89
10.32
0.99
88.2
0.98
96.9
0.94
94.7
0.99
0.99
Pm (W)
(h)
Q (J)
153.92 152.01 106.55 112.34
t
m
65.18
11.62
73.17
12.23
86.43
11.44
45.07
10.36
45.41
10.82
47.57
10.24
51.77
11.23
a
k
1
: rate constant in activity recovery phase; k
2
: rate constant in stationary increase phase; k
3
:
rate constant in decline phase; R: correlation coefficient; P
maximum power output time; Q: total heat output.
m
: maximum power output; t
m
:
Destruction on mitochondrial respiration
Making use of Clark Oxygen Electrode, the mitochondrial respiration process was
monitored. It has been known that the ideal isolated mitochondria have the relatively
low respiratory rate for state 4 characterizing mitochondrial inner membrane integrity
and the high respiratory rate for state 3 (about 3-fold than state 4) meaning the normal
function of respiratory chain. The exposure of PDT-PAO-TPP brought a decrease in
state 3 and an increase in state 4 collectively resulting in a significant decline in
respiratory control ratio (RCR = state 3/ state 4)(Figure 4A). However, the short-time

incubation of PDT-PAO made the negligible effect on mitochondrial respiration
Figure S4). Activities of complex I and IV in the mitochondrial respiratory chain had
(
an obvious inhibition (Figure 4B) and only high dose of PDT-PAO-TPP suppressed
the activity of complex III, whereas complex II still maintained the initial catalytic
activity.
Figure 4. The impairment on mitochondrial respiration. (A) The effect on isolated mitochondrial
respiration in the presence of PDT-PAO-TPP. (B) The effect on activities of respiratory chain
complex I, II, III and IV by organic arsenical PDT-PAO-TPP. The specific inhibitors added for
four complexes are 5 µM rotenone (CI), 10 mM sodium malonate (CII), 4 µM antimycin (CIII),
3
and 3 mM NaN (CIV), respectively. After incubated with compound PDT-PAO-TPP for 3-5
minutes, the absorbance at specific wavelength was monitored for at least 3 minutes.
Assessment of intracellular ROS and thiols level
It has been pointed out that complex I and III are associated with the generation of
3
9
ROS . As shown in Figure 5A and Figure S5A, the accumulation of ROS was
apparent after the long-time incubation with PDT-PAO-TPP or PDT-PAO.
Intracellular redox balance is regulated by the variation between free sulfydryl and
disulfides in endogenic antioxidants. The generation of excessive ROS and
synchronously moderate decrease of total thiols (Figure 5B and Figure S5B)
suggested that the redox homeostasis was off and the oxidation status environment in
NB4 cells was built. In addition, as the scavenging reagent for intracellular GSH,
BSO pre-added can decrease the cellular antioxidant capacity. The cell viability
remarkably dropped down even exposed with the nontoxic concentration of

PDT-PAO-TPP or PDT-PAO (Figure 5C and Figure S5C), which confirmed the
evidence that reducing inherent GSH level in cancer cells will contribute to enhance
the sensitivity for the arsenical drugs.
Figure 5. Assessment of intracellular redox state. (A) The variation of intracellular ROS level in
the presence of PDT-PAO-TPP by DCFH-DA or DHE staining. (B) The decrease of intracellular
total thiols level. (C) The cell viability in the presence of 50 µM BSO or without BSO (MTT
assay).
Impairment on mitochondrial membrane and induction of apoptosis
Besides, it is necessary to maintain the mitochondrial membrane integrity for
normal function exertion. When exposed with PDT-PAO-TPP, mitochondrial swelling
was induced manifesting as the decreased absorbance at 540 nm due to the enhanced
permeability to water (Figure 6A), which can be partially deterred by the
pre-incubation of CsA, RR and EGTA (Figure S6). Not only that but the membrane
+
+
permeability to H and K was also enhanced by PDT-PAO-TPP, evidenced by the
decline of absorbance at 540 nm (Figure S7). Comparatively, the influences on
mitochondrial swelling and permeability by PDT-PAO were slight (Figure S8).
Furthermore, the TEM pictures intuitively revealed the declined electron density in
mitochondria, the irregular shape and even ruptured membrane after incubated with
PDT-PAO-TPP (Figure S9). The defective mitochondrial membrane will prompt some
pro-apoptosis proteins to release into cytoplasm, such as cytochrome c, pro-caspases
4
0,41
and AIF . Actually, the release of cytochrome c indeed had a remarkable increase
via a dose-dependent way (Figure 6B and Figure S10A). Commonly, once
cytochrome c located in the mitochondria leaks out, the downstream signals like
4
2
caspase family can be active resulting in cell apoptosis . Just like shown in Figure 6C

and Figure S10B, apoptotic cells proportion (FITC+/PI- and FITC+/PI+) ascended
significantly after the addition of PDT-PAO-TPP in a dose- and time-dependent
manner, whereas the induction of apoptosis were blocked when the cells pre-cultured
4
3
with Z-VAD-fmk, a pan-caspase inhibitor (Figure 6D and Figure S10C). Moreover,
when the cells underwent apoptosis, the hallmarks including smaller cell size,
condensed nuclei, and DNA fragmentation were displayed in Figure S11 evidenced
by the blue light point in NB4 cells. Additionally, the death cells were shown as the
red light stained by PI.
Figure 6. The induction of apoptosis through the mitochondrial pathway. (A) Isolated
2
+
mitochondrial swelling in the presence of PDT-PAO-TPP in the presence of 4 μM Ca . (B) The
increase of intracellular Cyt-c level for 24 h. (C) Apoptosis induced by PDT-PAO-TPP for 12 h
and 24h. (D) The protective effect of 20 µM Z-VAD-fmk or 490 nM Nec-1 on apoptosis.
The newly synthesized organic arsenical PDT-PAO-TPP showed the better
inhibitory efficacy to NB4 cells than PDT-PAO, which was attributed to the
mitochondria-targeted effect of TPP moity. In the initial 3 h, the majority of
PDT-PAO-TPP entered into mitochondria in NB4 cells and immediately affected the
membrane potential, which accorded with our assumption about design of
PDT-PAO-TPP. It has been reported that PDT-PAO can induce the oxidative
6
stress-mediated apoptosis via targeting thioredoxinreductase in HL-60 cells .

Nevertheless, both of PDT-PAO-TPP and PDT-PAO made no apparent differences on
intracellular ROS level in the initial 6 h co-culture. Even if the cells were treated with
NAC, VE, VC or GSH in advance, the addition of PDT-PAO-TPP or PDT-PAO still
induced the decline of cell viability, which indicated the different anti-cancer
mechanism.
LA is not only a powerful antioxidant but also a necessary disulfhydryl coenzyme
for dihydrolipoyltransacetylase (E2) in pyruvate dehydrogenase complex (PDHC). As
expected, whether PDHC or E2 in NB4 cells showed a significant decrease on its
catalytic activity after exposure with PDT-PAO-TPP, which supported the result that
PDT-PAO-TPP may interact with sulfydryl in DHLA located in the
dihydrolipoyltransacetylase (E2) in PDHC to prevent the acryl group converted to
CoA in the mitochondria. Moreover, when the As-sulfydryl bond was reduced by the
exogenous dimercapto compounds (DTT, DMP and DMSA) to free sulfydryl, the
activity of E2 and cell viability of NB4 cells was recovered subsequently. It has been
known that tumor cells consume glucose at a dramatically high rate compared to
normal cells, which means that they have the priority for aerobic glycolysis for
4
4-46
proliferation
. Although it seems that tumor cells have the less dependency on
citric acid cycle to produce ATP, citric acid cycle still plays the key role in cellular
4
6
proliferation acting as a hub for biosynthesis . The suppression of PDHC activity
subsequently inhibited citric acid cycle to provide continuous intermediates, like
NADH and FADH, which are associated with the mitochondrial respiratory chain and
36,47
oxidative phosphorylation process
. Both PDT-PAO-TPP and PDT-PAO can
induce the mitochondrial metabolic disturbance after the long-time exposure via
inhibiting the activity of PDHC. Getting rid of other organelles, mitochondria are
extracted for further exploration. Based on the assessment of oxygen consumption in
isolated mitochondria, the restrained respiration process by PDT-PAO-TPP was
revealed clearly. Furthermore, the terminal enzyme complex IV in the respiratory
chain passes the electron from cytochrome oxidase to oxygen yielding water, thus it
4
8
controls the mitochondrial respiration . There is no doubt that the inhibition for
complexes by PDT-PAO-TPP was linked tightly to the respiration dysfunction, as

4
9,50-51
well as the less ATP production through the proton leak
. The proton leak means
the escape of electrons from the electron transport chain to randomly hit the nearby
molecules and ultimately produce free radicals leading to inevitable ROS. The
inhibition of complex I and III by PDT-PAO-TPP induced the leak of electrons and
led to the generation of ROS with a higher rate in the mitochondria. Short-time
incubation with PDT-PAO-TPP not PDT-PAO can trigger the mitochondrial
respiratory depression. Moreover, in the later period of co-incubation with
PDT-PAO-TPP or PDT-PAO, the accumulation of ROS and depletion of intracellular
thiols can intensify electron escaping, the leakage of protons gradient and
disappearance of membrane potential. Besides, enhanced generation of ROS may also
attack some reactive cysteine residues located in vital proteins or some important
4
8
reactions necessary for cellular normal function exertion to trigger a vicious cycle .
Mitochondrial membrane permeabilization is thought to be the decisive event
5
0
delimiting the frontier between cell survival and cell death . Some hallmarks in
mitochondrial membrane to indicate cell death appeared after treated by
PDT-PAO-TPP. For one thing, it can give rise to mitochondrial swelling as a
consequence of increase of IM permeability, which further demonstrated MPTP
2
+
opening via a Ca -dependent way confirmed by the almost complete protection from
5
2
swelling by CsA, RR and EGTA . Intuitively, this swelling brought about the
distension and the disorganization of the cristae as well as the reduction of the
electron density of the matrix. For another, the presence of PDT-PAO-TPP enhanced
+
+
the IM permeability to H and K , as well as the interruption of ATP synthesis.
Although the effects on mitochondrial membrane by PDT-PAO were relatively slight,
it also triggered the mitochondrial dysfunction after the long-time incubation.
Additionally, the released cytochrome c from mitochondria can promote the
formation of “apoptosome” to active the caspase-9, which in turn catalyzes the
5
3
proteolytic activation of the effector caspases like caspase-3 . Based on these
observations, both of PDT-PAO-TPP and PDT-PAO induced the mitochondrial
dysfunction and imbalance of redox state leading to the caspase family-dependent
intrinsic apoptosis via the inhibition towards PDHC, which is different from the

mechanism of PDT-PAO in HL-60 cells due to the different cell properties. The major
anti-tumor mechanism difference between PDT-PAO-TPP and PDT-PAO was that
organic arsenical PDT-PAO-TPP can work faster with higher efficacy via impairing
the mitochondria in the cancer cells as a mitochondria-targeted compound.
Conclusion
In conclusion, a mitochondria-targeted organic arsenical PDT-PAO-TPP was
synthesized by the introduction of TPP to PDT-PAO. Compared to PDT-PAO,
PDT-PAO-TPP showed the better efficacy towards NB4 cells. The accumulation of
PDT-PAO-TPP in mitochondria improved the effective concentration of organic
arsenical to exert the influence on mitochondria faster. The dual inhibition of PDHC
and respiratory chain complexes accelerated the thermogenesis disorder and
respiratory dysfunction, as well as the oxidative stress injury. The further damage on
the mitochondrial membrane integrity stimulated the release of cytochrome c, which
activated the caspase family-dependent mitochondrial apoptosis pathway. These
findings provide deep insights into the design and the anti-tumor mechanism of
organic arsenical, which indicates the complexity and diversity in the research about
organic arsenical as a promising anti-cancer drug.
Experimental
All the details were placed in Supporting Information.
Acknowledgment
We gratefully acknowledge financial support from the National Natural Science
Foundation of China (21673166).
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