Mitoapocynin, a mitochondria targeted derivative of apocynin induces mitochondrial ROS generation and apoptosis in multiple cell types including cardiac myoblasts: a potential constraint to its therapeutic use

Article abstract of DOI:10.1007/s11010-020-04039-4

Mitoapocynin is a triphenylphosphonium conjugated derivative of apocynin that specifically locates to the mitochondria. It has been developed as a mitochondrially targeted therapeutic antioxidant. We attempted to attenuate the mitochondrial ROS induced in

Full text of DOI:10.1007/s11010-020-04039-4

Molecular and Cellular Biochemistry (2021) 476:2047–2059
https://doi.org/10.1007/s11010-020-04039-4
Mitoapocynin, a mitochondria targeted derivative of apocynin induces
mitochondrial ROS generation and apoptosis in multiple cell types
including cardiac myoblasts: a potential constraint to its therapeutic
use
Amena Mahmood1,3 · Padmini Bisoyi1 · Rajkumar Banerjee2 · Md Yousuf2,4,5 · Shyamal K. Goswami1
Received: 17 September 2020 / Accepted: 22 December 2020 / Published online: 30 January 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Mitoapocynin is a triphenylphosphonium conjugated derivative of apocynin that specifcally locates to the mitochondria. It
has been developed as a mitochondrially targeted therapeutic antioxidant. We attempted to attenuate the mitochondrial ROS
induced in H9c2 cardiac myoblast cells treated with norepinephrine. Mitoapocynin was a poor quencher of total ROS as
detected by the fuoroprobe DCFH-DA. Using mitochondrial superoxide specifc probe MitoSoxRed, we found that 5–10 µM
mitoapocynin itself induces superoxide over and above that is generated by the norepinephrine treatment. A supposedly con-
trol molecule to mitoapocynin, the synthetic compound PhC11TPP, having the triphenylphosphonium group and a benzene
moiety with C11 aliphatic chain spacer was also found to be a robust inducer of mitochondrial ROS. Subsequent assays with
several cell lines viz., NIH3T3, HEK293, Neuro2A, MCF-7 and H9c2, showed that prolonged exposure to mitoapocynin
induces cell death by apoptosis that can be partially prevented by the general antioxidant N-acetyl cysteine. Analyses of
mitochondrial electron transport complexes by Blue Native Polyacrylamide gel electrophoresis showed that both mitoapoc-
ynin and PhC11TPP disrupt the mitochondrial Complex I and V, and in addition, PhC11TPP also damages the Complex IV.
Our data thus highlights the limitations of the therapeutic use of mitoapocynin as an antioxidant.
Keywords Oxidative stress · Mitochondria · Mitoapocynin · NOX
Abbreviations
SIRT1
Sirtuin 1
ROS
Reactive oxygen species
DCFH-DA Dichloro-dihydro-fuoresceindiacetate
RET
Reverse electron transport
NAC
Nox
YFP
PI
N-acetyl cysteine
NADPH oxidase
Yellow fuorescent protein
Propidium Iodide
Amena Mahmood and Padmini Bisoyi have contributed equally to
this work.
Nrf2
The nuclear factor erythroid 2-related factor
2
* Md Yousuf
TPP
Triphenylphosphonium
usuf.chem@gmail.com
* Shyamal K. Goswami
skgoswami@mail.jnu.ac.in
Introduction
1
School of Life Sciences, Jawaharlal Nehru University,
New Delhi 110067, India
Mitochondria metabolize carbon sources generating NADH,
and then transfer its electrons to molecular oxygen, pro-
ducing ATP. During this process, it ensure that there is no
unwarranted release of electrons or other secondary free
radicals that are toxic to the surrounding biomolecules.
However, certain electron transporting complexes selectively
release electrons at a lower intensity that interact with the
2
Applied Biology Division, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India
3
DS-Kothari Fellow, UGC, New Delhi, India
4
DST, SERB-National Postdoctoral Fellow, New Delhi, India
5
Present Address: Department of Chemistry, Ramnagar
College, Purba Medinipur, Shikarbar, West Bengal 721453,
India
Vol.:(0123456789)
1 3

2048
Molecular and Cellular Biochemistry (2021) 476:2047–2059
free oxygen, producing superoxide. Superoxide dismutase
almost instantaneously converts it to hydrogen peroxide.
These reactive oxygen species (ROS) are the mediators
of the retrograde and anterograde signalling under physi-
ological conditions [1]. Until date, eleven enzymatic and
non-enzymatic sources of ROS have been identifed in the
mitochondria. These are located in the Complex I, II and
III of the electron transport chain [2]. Under physiological
conditions, superoxide and hydrogen peroxide generated
from these sites play a role in cell growth, diferentiation,
and metabolic regulations [3]. Certain pathological condi-
tions induce metabolic reprogramming and overproduction
of ROS from these sites [4]. Such dysregulated generation of
ROS plays a major role in the diseases like cancer, neurode-
generation, diabetes, atherosclerosis, ischemia–reperfusion
Injury and other cardiovascular disorders [5–9]. Mitochon-
dria also produce ROS by another mechanism called reverse
electron transport (RET) that occurs when electrons from
reduced ubiquinone are transferred to NAD+ through the
complex I, and reducing it to NADH. RET is involved in
physiological processes like the activation of macrophages
against bacterial infection, re-organization of the electron
transport chain, oxygen sensing etc. [10]. Dysfunctional
RET has also been associated with several diseases [11].
Due to the critical role of ROS in health and diseases, the
generation and attenuation of mitochondrial ROS remains
under tight control. There are several antioxidant enzymes
viz., catalase, superoxide dismutase, thioredoxin peroxi-
dase and redox proteins like thioredoxin and glutaredoxin
ensuring the homeostatic control of the mitochondrial ROS
generation [2].
their therapeutic uses are questionable bioavailability,
unknown metabolic intermediates, poorly defned target(s)
of action and toxicity, if any [16]. Certain non-polyphenolic
dietary constituents from fruits and vegetables also boost
the cellular antioxidant status by activating various signal-
ling kinases, epigenetic regulators, and microRNAs, which
converge through the antioxidant transcription factor Nrf2
[17]. Paradoxically, depending upon their doses and the tis-
sue contexts; several of these dietary phytochemicals exhibit
both anti-oxidant and pro-oxidant activities. Therefore, upon
indiscriminate consumption, these phytochemicals might
scavenge the ROS under certain conditions while facilitat-
ing its generation under certain other, severely limiting their
therapeutic potential [18].
Due to the immense importance of mitochondrial ROS in
the pathobiology of numerous diseases, vis-à-vis the benef-
cial efects of many phytochemicals; eforts have also been
made to deliver of some of those molecules directly to the
mitochondria [19]. A common approach to such delivery is
to covalently tag the lipophilic cations like triphenylphos-
phonium [20].
Apocynin is a plant derived nontoxic antioxidant that
inhibits nicotinamide adenine dinucleotide phosphate oxi-
dase (NADPH oxidase or Nox), an enzyme that produces
superoxide from oxygen. Human and mice have multiple
isoforms of Nox(s) that are widely expressed in various tis-
sues. ROS generated from these Nox(s) regulate the down-
stream redox signalling associated with neuronal dysfunc-
tion, infammation and other pathological conditions [21].
Apocynin has been extensively studied for its pharmaco-
logical properties [22]. In certain cell types, apocynin is
enzymatically converted into its dimer Diapocynin, which
is more efective than apocynin on the Nox complex. It also
inhibits the expression of the mRNA for gp91 (phox) subu-
nit of Nox and suppresses the production of TNF-alpha and
IL-10 [23]. When injected intraperitoneally in rats, apocynin
is converted to its glycosyl derivative in plasma, liver and
brain [24]. Taken together, despite the consensus that apo-
cynin and its derivatives exert their antioxidant efects by
the inhibition of Nox, complete mechanism of its action is
poorly understood as yet.
Since excessive generation of ROS in the mitochondria
has been associated with many diseases, their diminution
by therapeutic antioxidants have also been explored. Die-
tary constituents such as vitamin E & C, carotenoids, and
polyphenols have long been shown to boost the antioxidant
defence [12]. However, these polyphenols from various plant
sources are mostly pleiotropic in nature and often exert their
benefcial efects through multiple mechanisms. As an exam-
ple, dietary polyphenols resveratrol, quercetin, catechins
etc., exert anti-infammatory efects via the modulation of
signalling pathways and activating SIRT1, a histone deacety-
lase [13]. Green tea polyphenol epigallocatechin 3-gallate
is a widely studied antioxidant that prevents infammation,
weight loss, several heart and brain diseases. It interacts
with proteins and phospholipids in the plasma membrane
and regulates signalling pathways, mitochondrial function,
DNA methylation etc. [14]. Interestingly, in presence of Fe3+
it produces hydrogen peroxide, thus acting as a pro-oxidant
as well [15]. Therefore, despite the evidences that therapeu-
tic antioxidants are efective for the management of certain
degenerative diseases; their utility, especially against com-
plex diseases remains limited. Other issues that also restrict
Due to the efcacy of apocynin against various neuro-
degenerative and infammatory disorders, eforts have also
been made for directly delivering it into the mitochondria for
quenching the ROS. In several experimental models, mitoap-
ocynin, a triphenylphosphonium derivative of apocynin that
easily permeate into the mitochondria, improves the neu-
ronal functions impaired due to oxidative stress [25–28].
We have earlier demonstrated that when H9c2 cardiac
myoblasts are treated with norepinephrine (NE), it induces
ROS from Nox2 [29]. We further observed that the ROS gen-
erated by NE is compartmentalized; while H2O2 is generated
in the cytosol, superoxide is generated in the mitochondria
1 3

Molecular and Cellular Biochemistry (2021) 476:2047–2059
2049
[29]. To assess the role of these two ROS in the downstream
signalling, we wanted to selectively quench the mitochon-
drial superoxide load by mitoapocynin. To that purpose, we
synthesized mitoapocynin-C11 (apocynin conjugated to a
mitochondria-targeting triphenylphosphonium cation moi-
ety [TPP+]) via an alkyl chain consisting of eleven carbon
atoms (Fig. 1). The presence of a highly lipophilic and delo-
calized cationic moiety in mitoapocynin-C11makes it more
cell-permeable and selectively target mitochondria [25].
Specifcally, its sequestration into mitochondria is facilitated
by TPP+conjugation via long carbon–carbon side chains.
We unexpectedly observed that rather than reducing the ROS
load, it triggers substantial ROS generation in the mitochon-
dria, followed by apoptosis. Our study thus highlights the
serious limitations of the therapeutic use of these mitochon-
dria targeted antioxidant.
the synthesis of mitoapocynin were purchased from Sigma-
Aldrich, USA; Avra chemicals, India and Spectrochem,
India.
General analytical procedures
Silica gel (100–200 mesh) was used for column chroma-
tography. Thin-layer chromatography was performed on
Merck-precoated silica gel 60-F254 plates. Chemicals and
solvents obtained from commercial sources were purifed
using standard methods. The 1H and 13C NMR spectra were
recorded on a Bruker-Avance 300 MHz/400 MHz/500 MHz
(see the Supporting Information). Chemical shifts (δ) are
reported in parts per million, using TMS (δ=0) as an inter-
nal standard in CDCl3. Data for1H NMR is reported as fol-
lows: chemical shift (δ in ppm), multiplicity (s-singlet, br.
s-broad singlet, d-doublet, t-triplet, q-quadruplet, m-multi-
plet), coupling constant (Hz), integration. ESI mass spectra
were recorded on a Finnigan LCQ Advantage max spectrom-
eter. All products reported showed 1H NMR and 13C NMR
spectra in agreement with the assigned structures. All tested
compounds yielded data consistent with a purity of at least
95% compared with the theoretical values.
Materials and methods
Cell culture and other reagents
H9c2 cardiac myoblasts were procured from Sigma-Aldrich,
USA (originally from ECACC, UK). NIH3T3 (murine fbro-
blast), HEK293 (human embryonic kidney), Neuro 2A (Neu-
roblastoma) and MCF-7 (human breast cancer) cells were
procured from the national cell repository (NCCS), Pune,
India (originally from the ATCC, USA).
Synthesis of (11‑Hydroxyundecyl)
triphenylphosphonium bromide (2)
A solution of 11-bromoundecane-1-ol (1.0 g, 3.98 mmol) in
dry acetonitrile (200 mL) was treated with triphenylphos-
phine (1.05 g, 4.01 mmol) in round bottom fask. The solu-
tion was refuxed with stirring for 48 h. The solvent was
removed in a vacuum, and the crude product was subjected
to fash column chromatography on silica gel (mesh size
All reagents used in this study were purchased from
Sigma Aldrich, USA unless mentioned otherwise. Foe-
tal bovine serum was obtained from Gibco (USA origin),
Hydrogen peroxide sensitive YFP plasmid pHyPer-Cyto
was from Evrogen (Moscow, Russia), and MitoSoxRed dye
was obtained from Invitrogen, USA. Chemicals used for
Fig. 1 Synthetic Route to Mitoapocynin and PhC11TPP -Control
molecule. In mitoapocynin-C11, apocynin is conjugated to mitochon-
dria-targeting triphenylphosphonium cationic moiety [TPP+]) via
eleven carbon aliphatic chain. PhC11TPP, the control molecule is a
synthetic compound having a benzene ring conjugated to the triphe-
nylphosphonium cationic moiety via the same eleven carbon aliphatic
chain
1 3

2050
Molecular and Cellular Biochemistry (2021) 476:2047–2059
100–200) with methanol-DCM (5–7%) to yield the com-
pound 2 as a colourless oil (1.43 g, 70%).
on silica gel (mesh size 100–200) with, MeOH-DCM
(2–5%) to yield desired compound 6 as a colourless gummy
oil (364 mg, 72%).
1H NMR (300 MHz, MeOD) δ (ppm) 8.14 – 7.66 (m,
15H), 3.60 – 3.43 (m, 4H), 1.83 – 1.45 (m, 6H), 1.30 (s,
12H) 0.13C NMR (75 MHz, MeOD) δ (ppm) 136.34,
136.31, 134.99, 134.85, 131.72, 131.56, 120.65, 119.51,
114.21, 63.06, 55.07, 50.01, 49.72, 49.44, 49.15, 48.87,
48.59, 48.30, 33.74, 31.75, 31.54, 30.74, 30.62, 30.56,
30.43, 30.00, 27.01, 23.68, 23.63, 23.17, 22.50.
1H NMR (400 MHz, CDCl3) δ (ppm) 8.03 (d, J=6.8 Hz,
2H), 7.83 – 7.74 (m, 8H), 7.71 (d, J=6.9 Hz, 7H), 7.55 (t,
J = 7.4 Hz, 1H), 7.43 (t, J = 7.6 Hz, 2H), 4.33 – 4.26 (m,
2H), 3.61 (s, 2H), 1.82 – 1.69 (m, 2H), 1.59 (s, 4H), 1.40
(s, 2H), 1.23 (d, J=15.6 Hz, 10H). 13C NMR (126 MHz,
CDCl3) δ (ppm) 166.71, 135.07, 133.58, 133.50, 132.83,
130.57, 130.48, 129.82, 129.50, 128.33, 118.69, 118.01,
77.39, 77.13, 76.88, 65.11, 30.51, 30.38, 29.69, 29.39,
29.19, 29.10, 28.69, 25.99, 22.64, 22.42, 22.02. ESI-MS:
m/z [M]+ calculated for C36H42O2P+: 537.2917, found:
537.35.
Synthesis of mitoapocynin (5)
To a stirred solution of vanillic acid (3) (250 mg, 1.48 mmol)
in dry dimethylformamide (DMF) (20 mL) was treated with
a solution of N, N-dicyclohexylcarbodiimide (DCC; 307 mg,
1.48 mmol) in dry DMF (5 mL) in an argon atmosphere.
The mixture was cooled to 0 °C, and a solution of com-
pound 2 (840 mg, 1.63 mmol) in DMF (5 mL) was added
slowly. To the resulting mixture, catalytic amounts of N,
N-dimethylpyridin-4-amine (DMAP) was added. The reac-
tion was stirred for overnight at room temperature, and any
resulting precipitate was removed by fltering. The solvent
was then removed, producing a brown residue that was sub-
jected to fash column chromatography on silica gel (mesh
size 100–200) with, MeOH-DCM (5–10%) to yield desired
compound 5 as a light-yellow gummy oil (511 mg, 52%).
1H NMR (400 MHz, CDCl3) δ (ppm) 7.81 – 7.65
(m, 15H), 7.56 (d, J = 8.3 Hz, 1H), 7.51 (s, 1H), 7.02 (d,
J=8.3 Hz, 1H), 4.26 (t, J=6.4 Hz, 2H), 3.90 (s, 3H), 3.46
(d, J = 2.2 Hz, 2H), 2.04 (s, 1H), 1.78 – 1.67 (m, 2H),
1.62 – 1.45 (m, 4H), 1.45 – 1.35 (m, 2H), 1.33 – 1.26 (m,
2H), 1.28 – 1.14 (m, 8H). 13C NMR (126 MHz, CDCl3)
δ (ppm) 166.67, 150.91, 146.79, 135.12, 133.51, 133.43,
130.59, 130.49, 128.85, 123.97, 121.96, 118.63, 117.94,
114.73, 112.22, 77.35, 77.10, 76.85, 71.81, 64.91, 56.11,
30.52, 30.39, 29.70, 29.26, 29.14, 29.01, 28.55, 26.07,
22.63, 22.31, 21.91, 19.18. ESI-MS: m/z [M] +calculated
for C37H44O4P+: 583.29, found: 583.55.
Cell culture
H9c2, MCF-7, NIH3T3, Neuro 2A and HEK 293 cells
were cultured as monolayer in Dulbecco’s modifed Eagle’s
medium (DMEM; 500 mg/l glucose, 2 mmol/l glutamine)
supplemented with 10% foetal bovine serum (FBS), 90 units/
ml Penicillin, 90 µg/ml Streptomycin and 5 µg/ml ampho-
tericin B at 37 °C in a humidifed incubator with 5% CO2.
Cell viability assay
Cell viability were assayed by the MTT [3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent [30].
Cells were seeded in a 96 well plate at a density of 3×103
cells/well and kept overnight, followed by a 12–14 h of
serum starvation. The cells were then treated with various
concentrations of mitoapocynin or PhC11TPP and incubated
for 12 h at 37 °C. In case of checking cell viability in pres-
ence of N-Acetyl Cysteine (NAC), selected groups were pre-
treated with 500 µM or 1 mM of NAC for 1hour, followed
by exposure with mitoapocynin or PhC11TPP at 37 °C for
4 h. MTT reagent (20 µL) at concentration of 5 µg/ml was
then added to each well and the plates were incubated for 3 h
at 37 °C followed by solubilisation of the purple formazan
crystals in DMSO. The absorbance was measured at 570 nm
in a microplate reader (Thermo Scientifc Varioskan Flash).
Synthesis of PhC11TPP‑control (6)
To a stirred solution of benzoic acid (4) (100 mg,
0.818 mmol) in dry dimethylformamide (DMF) (10 mL)
was treated with a solution of N, N-dicyclohexylcarbodiim-
ide (DCC; 169 mg, 0.818 mmol) in dry DMF (2.5 mL) in
an argon atmosphere. The mixture was cooled to 0 °C, and
a solution of compound 2 (462 mg, 0.900 mmol) in DMF
(5 mL) was added slowly. To the resulting mixture, catalytic
amounts of N, N-dimethylpyridin-4-amine (DMAP) was
added. The reaction was stirred for overnight at room tem-
perature, and any resulting precipitate was removed by flter-
ing. The solvent was then removed, producing a colourless
residue that was subjected to fash column chromatography
Measuring apoptosis
DNA content was measured by Propidium Iodide (PI,
Sigma-Aldrich P4170) staining. Briefy, cells were seeded
in 35 mm culture dishes. Post a 12–14 h of serum starvation,
the cells were treated with 5–50 µM M-Apo/PhC11TPP for
8 h. Cells were then harvested and suspended in 1 mL ice
cold 1X PBS. After a centrifugation at 1500 rpm for 5 min,
PBS was removed and cells were further suspended in 75%
ethanol and stored at 4 °C for one overnight. The ethanol
was removed post centrifugation at 3000 rpm for 5 min,
1 3

Molecular and Cellular Biochemistry (2021) 476:2047–2059
2051
followed by 3 washes with 1X PBS. Cells were incubated in
dark for 30 min with 500 µL ice-cold FACS bufer (5 mg/
mL PI, 10 µg/mL RNase, 0.1% Triton X 100). Cells were
analysed by fow cytometer using FACS Aria, Fusion (BD
Biosciences, MountainView, CA, USA) at 535 nm excita-
tion maxima.
transfection reagent. Twenty-four hours after transfection,
cells were kept in serum free media for 12–14 h. Cells were
then labelled with MitoSoxRed (2.5 µM) and mitoapocynin
or PhC11TPP for 15 min at 37 °C followed by washing
thrice with 1X PBS to remove excess MitoSoxRed. Cells
were incubated for fve more minutes with mitoapocynin or
PhC11TPP and live cell images were captured in a chamber
with 5% CO2 at 37 °C in an A1R HD confocal microscope
(Nikon) with excitation maxima of 510 nm and 488 nm laser
line. The fuorescence intensity was quantifed by NIS-ele-
ment AR-ver 4.000 software.
Monitoring of ROS generation
H9c2 cardiac myoblasts were seeded in 35 mm cell culture
dishes at a density of 3×105 cells per dish. After a 12–14 h
of serum starvation, the cells were labelled with 2′-7′-dichlo-
rodihydrofuorescein diacetate (DCFH-DA, 10 µM) along
with apocynin or mitoapocynin. After 15 min, the unbound
DCFH-DA was washed of with 1X PBS, followed by addi-
tion of apocynin or mitoapocynin, together with the adr-
energic agonist norepinephrine (2.5 µM) for 10 min. The
live cells were then visualized and captured at an excitation
maxima of 488 nm under an A1R HD confocal microscope
(Nikon). The mean forescence intensity was measured by
NIS-Element software (Nikon).
Estimation of mitochondrial ROS
MCF7 cells were seeded in 96 well black wall plates at a
density of 3×103 cells per well. Cells were grown overnight
and then kept in serum free medium for 12–14 h followed
by treatment with mitoapocynin or PhC11TPP at desired
doses for various durations at 37 °C. Prior to estimation,
cells were treated with MitoSoxRed for 15 min and then
washed thrice with 1X PBS and suspended in phenol free
media. Absorbance was measured using microplate reader
(Thermo Scientifc Varioskan Flash) at excitation maxima
of 510 nm.
Monitoring superoxide generation
in the mitochondria
Cells were seeded at a density of 3×105 in 35 mm glass bot-
tom dishes. After an overnight growth, the cells were serum
starved for 12–14 h, and then labelled for ffteen minutes
with MitoSoxRed (2.5 µM) along with apocynin/mitoapoc-
ynin/PhC11TPP/vehicle. After 15 min, the unbound Mito-
SoxRed was washed of thrice with 1X PBS [31]. Cells were
further incubated for an additional 10 min with apocynin/
mitoapocynin/PhC11TPP with or without the adrener-
gic agonist norepinephrine (2.5 µM) for 10 min. Live cell
images were captured in a live cell chamber with 5% CO2
at 37 °C with excitation maxima of 510 nm laser line in an
A1R HD confocal microscope (Nikon). For the time lapse
imaging of superoxide generation, cells were treated with
mitoapocynin or PhC11TPP along with MitoSoxRed for
15 min at 37 °C [31]. The excess dye was then removed by
washing thrice with 1X PBS. Cells were then replenished
with mitoapocynin or PhC11TPP in phenol free media in 5%
CO2 at 37 °C. Imaging was performed at various time points
i.e., 0 min, 5 min, 10 min, 30 min, 60 min and 120 min in an
A1R HD confocal microscope (Nikon).
Analysis of mitochondrial respiratory chain complexes
by blue native PAGE
The mitochondrial electron transport complexes were ana-
lysed by the Blue Native PAGE as described by Konovalova
S [32]. Briefy, MCF cells were treated with mitoapocynin or
PhC11TPP for 2 and 4 h, cells were harvested and permeabi-
lized by digitonin (1.65 mM). The mitochondrial complexes
were released from mitochondrial membrane with the help
of 10% lauryl maltoside. The complexes (10 µg of mitochon-
drial preparation) were then separated on the basis of their
mass using a blue native gradient gel and transferred onto
a PVDF membrane. To visualize the complexes, the PVDF
membranes were incubated with a cocktail of antibodies
corresponding to the selected subunits of the fve electron
transport complexes.
Statistical analysis
Measurement of H2O2 generated in the cytosol
Each experiment was performed in triplicate and the results
are expressed as mean SEM. The experimental groups
were compared using one-way ANOVA followed by Tuk-
ey’s multiple comparison tests using the statistics module
of graph pad prism version 7. A value of P<0.05 was con-
sidered signifcant.
Generation of cytosolic H2O2 was monitored by the recom-
binant fuorescent proteins HyPer-Cyto, specifcally sensi-
tive to H2O2. MCF7 cells grown in 35 mm glass bottom
culture dishes to a density of 3×105 cells were transfected
with 2 µg pHyPer-Cyto plasmid using Lipofectamine 3000
1 3

2052
Molecular and Cellular Biochemistry (2021) 476:2047–2059
molecules were characterized by 1H and 13C NMR spec-
troscopy and ESI-MS mass spectrometry.
Results
Synthesis of mitoapocynin
Mitoapocynin induces superoxide generation
in the mitochondria
The published method for the synthesis of mitoapocynin
(5) involved a large number of steps [25]. We found it dif-
fcult and not adaptable to scale-up. We thus optimized and
adopted a simple two-step protocol that is economic and
gives better yield. The details are given in the Methods and
the outline is given in Fig. 1. Briefy, (11-Hydroxyunde-
cyl) triphenylphosphonium bromide (2) were synthesized
by mixing triphenylphosphine and 11-bromo-undecane-
1-ol (1) in acetonitrile solvent under refuxing condition.
Finally, this salt was used with vanillic acid under stand-
ard ester coupling condition to generate target compound
mitoapocynin (5) with moderate to good yield. We also
synthesized a control molecule, PhC11TPP (6) where a
phenyl ring was inserted instead of vanillin ring. This
TPP product was synthesized to compare its bio-activity
directly with mitoapocynin. The mitoapocynin and control
In H9c2 cardiac myoblasts, activation of the adrenergic
receptors by NE co-stimulates Nox2 that generates ROS
in the cytosol and the mitochondria [29, 33]. Consider-
ing the importance of mitochondrial ROS in cardiovascu-
lar diseases, we wanted to selectively quench it and study
the downstream efects [34]. Cells were treated with NE
(2.5 μM) along with mitoapocynin (5 and 10 µM) to selec-
tively quench the mitochondrial ROS [22, 25, 35]. Apocynin
(5 and 10 µM) was also included as a general antioxidant and
an inhibitor of Nox2 for a comparison. Generation of ROS
was monitored by the probe DCFH-DA under a fuorescent
microscope. As shown in Fig. 2a, apocynin itself slightly
increased the ROS levels by 1.5–2.0-fold above baseline but
moderately quenched the ROS generated by NE. We con-
sistently observed that the lower dose of apocynin (5 μM)
-
+
-
+
NE (2.5µM)
M-APO (µM)
-
+
5
-
+
-
+
a
5
5
10
10
APO (µM)
5
10
10
10
5
10
5
10
5
****
****
****
***
***
+
**
***
**
-
0
0
0
NE
M-APO (µM)
-
5
+
5
-
-
+
5
+
NE
-
NE
+
10 10
5
10 10
APO (µM)
NE (2.5µM)
-
+
-
+
b
-
+
-
+
-
5
5
*
10
10 M-APO (µM)
APO (µM)
5
5
10
10
5.0
2.5
0.0
5.0
2.5
0.0
5.0
2.5
0.0
***
***
**
*
*
*
*
NE
NE
-
5
+
5
-
+
-
+
-
+
-
NE
M-A PO (µM)
5
5
10 10
10 10
APO (µM)
Fig. 2 Mitoapocynin induces superoxide in the mitochondria in
H9c2 cells: a H9c2 cells were labelled with redox sensitive fuoro-
probe DCFH-DA (10 µM) along with apocynin (APO) or mitoapo-
cynin (M-APO) at for 15 min, washed and then treated with the
adrenergic agonist NE (2.5 µM) for 10 min. ROS generation were
monitored by imaging the live cells under a fuorescence micro-
scope at excitation maxima of 488 nm. b Superoxide generation in
the mitochondria were monitored by the mitochondria specifc fuo-
roprobe MitoSoxRed (2.5 µM) for 15 min together with APO or
M-APO for 15 min. After washing of the forescent probe, cells were
treated with NE (2.5 µM) together with APO or M-APO for 10 min.
Images of live cells were captured with the excitation maxima of
510 nm laser line at 60X magnifcation with A1R confocal micro-
scope (Nikon). Relative forescence intensity (fold change) expressed
as the Mean SEM of three independent experiments performed in
duplicate. ***P≤0.001 versus control, **P≤0.01 versus control,
*P≤0.05 versus control, RFI Relative forescence intensity (fold
change)
1 3

Molecular and Cellular Biochemistry (2021) 476:2047–2059
2053
was slightly more efective than the higher dose i.e., 10 μM
in quenching ROS (~50% and ~35% respectively). Notice-
ably, mitoapocynin itself substantially increased the ROS
generation with ~ 6 and 4.5-fold increase above the back-
ground, and the lower dose (5 μM) was a stronger inducer.
When added together with NE, it was moderately efective
in quenching the ROS as~30–35% reduction was seen for
the two concentrations used. The fuoroprobe DCFH-DA
detects superoxide, hydrogen peroxide and a several other
secondary ROS in the cytosol and other cellular compart-
ments [36]. We thus thought that mitoapocynin might be
quenching only a subset of ROS and thus showing lesser
efcacy with this probe [25, 26]. We thus further examined
the ability of mitoapocynin in selectively quenching the
mitochondrial superoxide. Cell treated with NE and apo-
cynin or mitoapocynin were probed with MitoSoxRed, a
mitochondria specifc probe for superoxide [31]. As shown
in Fig. 2b, apocynin itself slightly increased (~ 1.5-fold)
the superoxide level at 10 µM dose and quenched the ROS
induced by NE by~20–30%. Also, in conformity with the
data shown in Fig. 2a, the lower dose was slightly more
efective quencher than the higher dose. On the contrary,
both doses of mitoapocynin alone induced superoxide gen-
eration by~2.5 and 3.5-fold respectively. Quite unexpect-
edly, when mitoapocynin was added together with NE, the
ROS level was even higher than that seen with NE alone.
Particularly, 10 µM mitoapocynin together with NE gener-
ated twice the amount of superoxide generated by NE alone.
This suggest that mitoapocynin and NE generated superox-
ide in an additive manner. This result was surprising and
we further probed whether the generation of superoxide by
mitoapocynin in the mitochondria is an intrinsic function of
the whole molecule, or it is due to the triphenylphosphonium
moiety that is attached to apocynin for targeting it to the
mitochondria. As shown in Fig. 3a, superoxide generation
by both mitoapocynin and the triphenylphosphonium moiety
PhC11TPP was quite robust, but mitoapocynin was slightly
stronger than PhC11TPP. Although MitoSoxRed is a specifc
probe for mitochondrial superoxide, we further confrmed
it by treating cells with the general antioxidant N-acetyl
cysteine (NAC) and the SOD mimetic MnTMPyP. As shown
in Fig. 3b, while 10 µM NAC quenched the ROS genera-
tion by mitoapocynin up to ~ 75%, MnTMPyP completely
quenched it even at 25 µM dose. Such result is expected
because, while NAC indirectly acts as an antioxidant by
increasing the level of glutathione, MnTMPyP mimics SOD
that directly destroys the superoxide pool [37, 38].
treatment, cells start rounding up and detaching from
the dish, typical signs of apoptosis. We therefore tested
whether mitoapocynin induces cell death due to oxida-
tive stress. Number of commonly used cell lines of mul-
tiple origins viz., MCF-7 (Human breast cancer), NIH3T3
(mouse embryonic fbroblast), HEK293 (Human embryonic
kidney), N2A (Mouse neural crest), and H9c2 (Rat heart)
were treated with several concentrations of mitoapocynin
till 12 h. Parallel treatments were also done with equiva-
lent doses of PhC11TPP, the triphenylphosphonium tag for
mitoapocynin. As shown in Fig. 4a, at the lower doses (5
and 10 µM), both mitoapocynin and PhC11TPP killed the
cells in a progressive manner with increasing time. Also, in
MCF-7 and NIH3T3 cells, mitoapocynin was more toxic as
compared to PhC11TPP. With the higher doses i.e., 20 and
50 µM, the death rate was faster in MCF-7, NIH3T3 and
HEK293 cells as substantial cell death were seen within four
hours. At 20 µM dose, while mitoapocynin was more toxic
for NIH3T3 and H9c2 cells, PhC11TPP was more noxious
for the HEK293 cells. Unlike for other cells, in Neuro2A
and H9c2 cells, the death rate was progressive, irrespective
of the concentration of mitoapocynin or PhC11TPP. Notice-
ably, among all these cell lines tested, the Neuro2A cells
were least sensitive to both mitoapocynin and PhC11TPP as
more than 60% of the cells remained viable with the highest
dose (50 µM) even after 12 h. To confrm that the cell death
were due to apoptosis, we treated cells with mitoapocynin/
PhC11TPP, stained with propidium iodide and measured the
DNA content in sub-G1 fraction by fow cytometry (Fig. 4b).
We also examined whether the generation of superoxide by
mitoapocynin is a transient burst as it is seen in cells treated
with growth factors, cytokines and hormones [39, 40]. As
shown in Fig. 4c, when MCF-7 cells were treated with
10 µM mitoapocynin or PhC11TPP, superoxide generation
continued till 2 h, the last time point we had tested. Also,
to ensure that the cell death is due to oxidative stress, we
treated MCF-7 cells with 0.5–1 mM NAC followed by treat-
ment with mitoapocynin or PhC11TPP (20 µM). As shown
in Fig. 4d, NAC partially protected the cells from apoptosis
induced by either mitoapocynin or PhC11TPP.
In mitoapocynin treated cells H2O2 does
not accumulate in the cytosol
Superoxide generated by the mitochondrial electron
transport chain, is immediately converted into hydrogen
peroxide by the mitochondrial superoxide dismutases.
The hydrogen peroxide thus generated often difuse to
the other cellular compartment, especially the cytosol, to
elicit physiological responses [41]. We thus examined if
mitoapocynin causes any increase in cytosolic hydrogen
peroxide. MCF-7 cells were transfected with the plasmid
Mitoapocynin induces apoptosis in multiple cell
lines
While experimenting on the generation of superoxide
by mitoapocynin, we noticed that after several hours of
1 3

2054
Molecular and Cellular Biochemistry (2021) 476:2047–2059
5 µM
APO
10 µM
-
5 µM
5 µM
10 µM
10 µM
-
-
a
Phc11TPP
M-APO
4.0
4.0
3.0
2.0
1.0
0.0
4.0
3.0
2.0
1.0
0.0
***
**
3.0
2.0
1.0
0.0
**
**
5
-
5
10 APO (µM)
-
10
M-APO (µM)
-
5
10 Phc11TPP (µM)
b
4.0
3.0
2.0
1.0
0.0
** **
M-APO (10 µM)
NAC
-
-
-
+
-
+
+
+
5 µM
2.5 µM 5 µM
10 µM
-
-
5
-
-
2.5
5
10 NAC (µM)
10 10 10 10 M-APO (µM)
4.0
3.0
2.0
1.0
0.0
M-APO (10 µM)
MnTMPyP (µM )
-
-
-
50
+
-
+
25
+
50
+
100
**
-
5
-
-
2.5
5
10
MntmPyp (µM)
M-APO (µM)
-
10 10
10 10
Fig. 3 The triphenylphosphonium tag used for delivering apoc-
ynin to the mitochondria induces robust ROS generation. a H9c2
cells were labelled with MitoSoxRed together with APO/M-APO/
PhC11TPP for 15 min. Excess MitoSoxRed was washed of and
cells were re-incubated with APO/M-APO/Phc11TPP for 10 min.
Live cells images were captured in a chamber with 5% CO2 at 37 °C
using A1R HD confocal microscope (Nikon) with excitation maxima
of 510 nm at 60X. b H9c2 cells were treated with the antioxidant
N-acetyl cysteine (NAC) or the SOD mimetic MnTMPyP along with
MitoSoxRed for 20 min. Post wash, cells were again incubated with
M-APO (10 µM) for 15 min and images were captured with excita-
tion maxima of 510 nm laser line by A1R HD confocal microscope
(Nikon). Relative forescence intensity (fold change) was expressed
as the Mean SEM of three independent experiments performed in
duplicate. ****P≤0.0001 versus control; ***P≤0.001 versus con-
trol, **P≤0.01 versus control, *P≤0.05 versus control. RFI Relative
forescence intensity (fold change)
Hyper-Cyto that encodes a cytosolic hydrogen peroxide
sensing fuorescent protein. Cells were then treated with
mitoapocynin and MitoSoxRed. As shown in Fig. 5, there
were no appreciable increase in fuorescence in the cyto-
solic Hyper-Cyto in mitoapocynin or PhC11TPP treated
cells. This suggest that the superoxide that is generated by
mitoapocynin largely remain in the mitochondria and does
not increase the level of cytosolic H2O2.
Mitoapocynin causes disassembly
of the mitochondrial electron transport complexes
In a study using C elegans, it has been demonstrated that
mitochondrial SOD-2 & 3 remains associated with the
electron transport complexes, protecting those from oxida-
tive damage. In worms defcient in sod-2, supercomplexes
are damaged due to oxidative stress [42]. Considering that
mitoapocynin generates ROS followed by cell death, we
1 3

Molecular and Cellular Biochemistry (2021) 476:2047–2059
2055
looked into the possible damage to the mitochondrial elec-
tron transport complexes. MCF-7 cells were treated with
mitoapocynin or PhC11TPP for 2 and 4 h, mitochondrial
fractions were isolated and resolved on a Blue Native gra-
dient PAGE. Immunoblotting was done with the antibody
cocktail recognizing electron transport complexes. As shown
in Fig. 6, Complex I and V are sharply decreased in mitoapo-
cynin treated cells and the decrease was sharp for the Com-
plex I at 4 h. In PhC11TPP-treated cells, there were also a
decrease in Complex I and V, but unlike in mitoapocynin
treated cells, even after 4 h, some of the Complex V was still
remaining. Also, in PhC11TPP treated cells, Complex IV
was not visible at 4 h. These data suggest that upon treatment
with mitoapocynin and PhC11TPP, mitochondrial electron
transport complexes, particularly Complex I and V are dam-
aged and this is likely to be the cause of cell death induced
by these two reagents.
multiple ROS, some of which have not been characterized
as yet and a few of those might not be quenched by apocynin
[33]. Thirdly, although numerous studies have shown that
apocynin is both an antioxidant and an inhibitor of Nox2;
its ability to inhibit Nox in non-leukocytes have been con-
tested [47]. It has been shown that apocynin even gener-
ates ROS in mouse embryonic stem cells [48]. Therefore,
depending upon the cell type, it might have multiple mode of
actions [49]. Rather, what was perplexing is, mitoapocynin
not only failed to neutralize the mitochondrial superoxide
generated by NE, it increased the ROS level on its own in an
additive manner. This was contrary to the prevailing under-
standing of the therapeutic value of delivering apocynin to
the mitochondria. Like DCFH-DA, the accuracy of Mito-
SoxRed in measuring superoxide in the mitochondria has
also been debated. These probes are prone to generation of
secondary free radicals and might have an additional oxida-
tion product [50]. Nevertheless, despite such limitations, if
there is any in our assays, the generation of mitochondrial
superoxide by mitoapocynin, either alone or in presence of
NE was convincing as it is substantial and dose dependent.
Further perplexing was the observation that slightly lower
but still substantially high level of ROS was also generated
by PhC11TPP, the triphenylphosphonium cationic moiety
used for targeting apocynin to the mitochondria. Anticipat-
ing that these unexpected results could be due to certain spe-
cifc characteristics of the H9c2 cells, we extended our study
to several other cell lines. Five other cell lines of diverse
tissue origins also showed ROS generation followed by cell
death with both mitoapocynin and PhC11TPP. Since tools
and techniques for measuring ROS generation is tissues are
not available, we could not extend our study in vivo. Nev-
ertheless, considering least commonality between the cell
lines we have tested, we believe that the in vivo efects of
mitoapocynin would not be very diferent from the ex vivo
results. To be especially noted that we also observed sub-
tle diferences in the kinetics and the extent of toxicity of
PhC11TPP and mitoapocynin between themselves for a
given cell line; as well as between the cell lines. The killing
efects of mitoapocynin or PhC11TPP was rather less on
Neuro2a cells as compared to the other cell lines tested. It
is thus likely that in the previous studies on the protecting
efects of mitoapocynin neurodegeneration in experimental
animals, the non-specifc cytotoxicity of mitoapocynin might
have gone unnoticed [26–28]. Although these results suggest
that the primary source of ROS generation is PhC11TPP, it
needs to be answered why apocynin could not mitigate it?
Available literature suggests that upon oxidation by the ROS,
apocynin is converted into its di- and trimers [51]. So, it is
possible that, due to its covalent linking to the triphenylphos-
phonium group, its di- or trimerization is prevented. To be
noted that in mouse embryonic stem cells, apocynin induces
ROS rather than quenching it [49]. So, it is also possible that
Discussion
Mitochondria is second only to the nucleus for its structural
and functional complexity. Besides carbon metabolism and
ATP generation, it also play key roles in calcium homeo-
stasis, redox signalling, apoptosis etc. It is thus a common
target for the antioxidants; diagnostic probes; drugs for can-
cer, cardiovascular, and neurodegenerative diseases [43].
The lipophilic triphenylphosphonium cation has widely
been used for delivering anticancer compounds, antioxi-
dants, micoRNAs, and photosensitizers into the mitochon-
dria [44, 45]. Delivery of several natural antioxidants viz.,
co-enzyme Q [Mitoquinone], vitamin E [Mitochromanol],
SOD mimetic [MitoTEMPOL] and apocynin [mitoapocynin]
have shown promising results [44]. The objective of our
study was to attenuate the mitochondrial ROS generated in
H9c2 cells upon treatment with NE and to follow its physi-
ological consequences. Since our earlier study have clearly
demonstrated that apocynin can mitigate the hypertrophic
efects of NE on rat heart, we wanted to have a mechanistic
insight by selectively targeting it to the mitochondria, the
primary source of ROS in NE treated cells [35]. Although
mitoapocynin have been used for mitigating neuronal dys-
functions [25–28], its efcacy on the cardiac muscle cells
have not been examined. However, alleviation of the adverse
efects of the β-adrenergic agonist isoprenaline by deliver-
ing Puerarin, an isofavone; to the mitochondria has been
reported [46]. Our observation that apocynin was moder-
ately efective in quenching the DCFH-DA sensitive ROS
was not surprising as there are certain inherent limitations
in the assay. Firstly, the chemistry of oxidation of DCFH-
DA is quite complex and under certain condition this probe
itself could be a source of secondary ROS generation [36].
Secondly, upon treatment with NE, H9c2 cells generate
1 3

2056
Molecular and Cellular Biochemistry (2021) 476:2047–2059
a
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
NIH/3T3
MCF7
HEK293
100
80
*
**
**
**
**
**
**
**
**
*
**
**
**
**
**
**
**
**
**
**
60
40
20
0
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
4hrs
8hrs
12hrs
4hrs
8hrs
12hrs
4hrs
8hrs
12hrs
H9C2
120
100
80
60
40
20
0
120
N2a
100
80
60
40
20
0
Cont
5 µM
10 µM
20 µM
50 µM
**
**
**
**
**
Phc11TPP
M-APO
**
**
**
**
**
**
12hrs
12hrs
b
MCF7
3T3
100
80
60
40
20
0
***
***
100
80
60
40
20
0
***
***
***
***
***
***
***
***
***
***
***
***
***
*
Phc11TPP
5 min
M-APO
c
0 min
10 min
30 min
60 min
120 min
4.0
3.0
2.0
1.0
0.0
**
**
**
M-APO
(10 µM)
**
**
**
**
**
Phc11TPP
(10 µM)
d
120
100
80
60
40
20
0
**
M-APO Phc11TPP
**
**
**
***
***
***
-
-
-
-
0.5
-
-
1
-
-
0.5
1
-
-
0.5
-
1
NAC (mM)
20 20 20
-
M-APO (µM)
-
-
-
20 20 20
Phc11TPP (µM)
1 3

Molecular and Cellular Biochemistry (2021) 476:2047–2059
2057
◂Fig. 4 Mitoapocynin and PhC11TPP induces cell death. a MCF7,
NIH3T3, H9c2, Neuro2A, and HEK 293 cells were incubated with
M-APO or PhC11TPP (5–50 µM) for diferent durations till 12 h
in a tissue culture incubator. Upon completion of incubation for the
desired durations, cell viability was measured by the MTT reagent as
described in the Methods. The purple formazan crystals were solu-
bilized in DMSO and the absorbance was measured at 570 nm in a
microplate reader (Thermo Scientifc Varioskan Flash). b Cells were
incubated with M-APO or PhC11TPP (5–50 µM) for 8 h, followed by
trypsinisation and centrifugation to make single cell suspensions. The
cells were then stained with Propidium iodide and analysed at 535 nm
Ex max. The graph represents Sub G1 population as apoptotic cells.
Cells were captured by BD FACS Aria Fusion (the raw data is given
in supplementary Fig S1). c MCF7 cells were labelled with Mito-
SoxRed along with 10 µM of M-APO/PhC11TPP for 15 min. The
unbound dye was then washed of and cells were re-treated with
M-APO/PhC11TPP. At diferent time points as marked, live cell
images were captured by A1R HD confocal microscope (Nikon) with
excitation maxima of 510 nm laser line. Relative forescence intensity
(fold change), was expressed as the Mean SEM of three independ-
ent experiments performed in duplicate (d). MCF7 cells were seeded
in a 96 well plate and a subset were pre-treated with NAC (0.5 and
1 mM) of for 1 hr followed by a treatment with M-APO/PhC11TPP
(20 µM) for 4 h. Cell viability was assayed by the MTT reagent. Data
expressed as the Mean SEM of three independent experiments per-
formed in duplicate. ***P≤0.001 versus control; **P≤0.01 versus
control, *P≤0.05 versus control. RFI Relative forescence intensity
(fold change)
in some cell lines, while PhC11TPP acts as a strong inducer
of ROS, apocynin group attached to it adds to the ROS gen-
eration instead of quenching. This possibility is in tune with
our consistent observation that the lower dose of apocynin
(5 µM) is a better quencher of ROS than the higher dose
i.e., 10 µM. Taken together, these results do not necessarily
contradict what is established about apocynin, i.e., it is an
antioxidant both in vivo, and ex vivo. It rather suggest that a
cautionary approach is required while assessing its benefcial
efects of mitoapocynin in quenching mitochondrial ROS in
various tissues. Since metabolic conversion of apocynin is
necessary for its antioxidant function in vivo, any structural
constrains imposed upon it while conjugating it to a vector
molecule ought to be carefully assessed.
Although the details analyses of the structure–function
relationship triphenylposphonium conjugated apocynin
is beyond the scope of this study, to reiterate the novelty
of our observations, we have done another independent
assay that shows both PhC11TPP and mitoapocynin dam-
ages the electron transport complexes in the mitochondria.
This observation is highly novel and future study would be
required to test whether the ROS generation by mitoapoc-
ynin and PhC11TPP cause damage to the electron transport
complexes or it is due to the damage of the electron transport
Phc11TPP
M-APO
3.0
20 µM
20 µM
10 µM
10 µM
Control
**
2.5
**
2.0
HyPer-Cyto
*
1.5
1.0
0.5
0.0
MitoSox Red
+
10µM 20µM 10µM 20µM
M-APO
HyPer-Cyto
Phc11TPP
MitoSox Red
Fig. 5 Mitoapocynin or Phc11TPP does not induce cytosolic hydro-
gen peroxide MCF 7 cells were transiently transfected with the plas-
mid pHyPer-Cyto that expresses hydrogen peroxide sensitive fuores-
cence protein in the cytosol. Cells were labelled with MitoSoxRed
(2.5 µM) together with M-APO/PhC11TPP for 15 min. Unbound dye
was then washed of, followed by incubation with M-APO/PhC11TPP
for 10 more minutes. Live cell images were captured at 60X with
an Ex max at 488 nm and 510 nm for Hyper-Cyto and MitoSoxRed
respectively. Quantifcation of the fuorescence intensity normal-
ized to control was performed (right panel). Data expressed as the
Mean SEM of three independent experiments performed in dupli-
cate. **P≤0.01 versus control, *P≤0.05 versus control. RFI Rela-
tive forescence intensity (fold change)
1 3

2058
Molecular and Cellular Biochemistry (2021) 476:2047–2059
References
M-APO Phc11TPP
2h
1. Brand MD (2016) Mitochondrial generation of superoxide and
hydrogen peroxide as the source of mitochondrial redox signaling.
Free Radic Biol Med 100:14–31
4h
-
2h 4h
2. Mailloux RJ (2018) Mitochondrial antioxidants and the mainte-
nance of cellular hydrogen peroxide levels. Oxid Med Cell Longev
2018:7857251. https://doi.org/10.1155/2018/7857251
3. Quiros PM, Mottis A, Auwerx J (2016) Mitonuclear communica-
tion in homeostasis and stress. Nat Rev Mol Cell Biol 17:213–226
4. Sena LA, Chandel NS (2012) Physiological roles of mitochondrial
reactive oxygen species. Mol Cell 48:158–167
C I
C V
C III
5. Chen Y-R, Zweier JL (2014) Cardiac mitochondria and reactive
oxygen species generation. Circ Res 114:524–537
6. Badrinath N, Yoo SY (2018) Mitochondria in cancer: in the
aspects of tumorigenesis and targeted therapy. Carcinogenesis
39:1419–1430
C IV
7. Franco-Iborra S, Vila M, Perier C (2018) Mitochondrial quality
control in neurodegenerative diseases: focus on Parkinson’s dis-
ease and Huntington’s disease. Front Neurosci 12:342
8. Lefranc C, Friederich-Persson M, Palacios-Ramirez R, Cat AND
(2018) Mitochondrial oxidative stress in obesity: role of the min-
eralocorticoid receptor. J Endocrinol 238:R143–R159
9. Kiyuna LA, e Albuquerque RP, Chen C-H, et al (2018) Targeting
mitochondrial dysfunction and oxidative stress in heart failure:
challenges and opportunities. Free Radic Biol Med 129:155–168
10. Onukwufor JO, Berry BJ, Wojtovich AP (2019) Physiologic impli-
cations of reactive oxygen species production by mitochondrial
complex I reverse electron transport. Antioxidants 8:285
11. Scialò F, Fernández-Ayala DJ, Sanz A (2017) Role of mitochon-
drial reverse electron transport in ROS signaling: potential roles
in health and disease. Front Physiol 8:428
Fig. 6 Mitoapocynin or PhC11TPP damages mitochondrial elec-
tron transport complexes MCF-7 cells were treated with M-APO or
PhC11TPP (20 µM) for 2–4 h and the mitochondrial fractions were
isolated. Eighty micrograms of mitochondrial preparations were
resolved on a gradient Blue Native Polyacrylamide Gel followed by
immunoblotting using an antibody cocktail of antibodies for the mito-
chondrial electron transport complexes (NDUFB8 subunit of Com-
plex I, the 30 kDa subunit of Complex II, the Core protein two of
Complex IV, and the α subunit of Complex V)
12. Wojcik M, Burzynska-Pedziwiatr I, Wozniak L (2010) A review of
natural and synthetic antioxidants important for health and longev-
ity. Curr Med Chem 17:3262–3288
complexes by these compounds, ROS is generated as a by-
product. We thus would like to conclude our study with a
cautionary note on the limitations of using triphenylphos-
phonium group for delivering therapeutics to the mitochon-
dria and other organelles.
13. Chung S, Yao H, Caito S et al (2010) Regulation of SIRT1 in
cellular functions: role of polyphenols. Arch Biochem Biophys
501:79–90
14. Kim H-S, Quon MJ, Kim J (2014) New insights into the mecha-
nisms of polyphenols beyond antioxidant properties; lessons from
the green tea polyphenol, epigallocatechin 3-gallate. Redox Biol
2:187–195
Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s11010-020-04039-4.
15. Elbling L, Weiss R-M, Teufelhofer O et al (2005) Green tea extract
and (–)-epigallocatechin-3-gallate, the major tea catechin, exert
oxidant but lack antioxidant activities. FASEB J 19:807–809
16. Barrajón-Catalán E, Herranz-López M, Joven J et al (2014)
Molecular promiscuity of plant polyphenols in the management
of age-related diseases: far beyond their antioxidant properties.
Adv Exp Med Biol 824:141–159
Acknowledgements Authors thankfully acknowledge funding support
from the Science & Engineering Research Board (SERB), Govern-
ment of India, under number EMR/2016/001832 to SKG. AM is a
recipient of DS-Kothari Fellowship from the University Grants Com-
mission, India. PB is a recipient of Senior Research Fellowship from
the Council of Scientifc and Industrial Research, India. MY acknowl-
edges postdoctoral fellowship support from SERB-DST (DST File
No: PDF/2017/000439), New Delhi, India. Sumona Ghosh, Technical
Assistant performed some of the experiments shown in Figs. 3 and
4. We thank Director, CSIR-IICT (Ms. No. IICT/Pubs./2020/196) for
providing necessary facilities to synthesize mitoapocynin.
17. Lee JH, Khor TO, Shu L et al (2013) Dietary phytochemicals
and cancer prevention: Nrf2 signaling, epigenetics, and cell
death mechanisms in blocking cancer initiation and progres-
sion. Pharmacol Ther 137:153–171
18. Fernando W, Rupasinghe HV, Hoskin DW (2019) Dietary
phytochemicals with anti-oxidant and pro-oxidant activities: a
double-edged sword in relation to adjuvant chemotherapy and
radiotherapy? Cancer Lett 452:168–177
Compliance with ethical standards
19. Oyewole AO, Birch-Machin MA (2015) Mitochondria-targeted
antioxidants. FASEB J 29:4766–4771
Conflict of interest The authors declare that they have no confict of
interest.
20. Smith RA, Hartley RC, Cocheme HM, Murphy MP (2012) Mito-
chondrial pharmacology. Trends Pharmacol Sci 33:341–352
21. Altenhöfer S, Radermacher KA, Kleikers PW et al (2015)
Evolution of NADPH oxidase inhibitors: selectivity and
mechanisms for target engagement. Antioxid Redox Signal
23:406–427
1 3

Molecular and Cellular Biochemistry (2021) 476:2047–2059
2059
H2S and sulfane sulfur production. Cell Chem Biol 25:447-459.
e4. https://doi.org/10.1016/j.chembiol.2018.01.011
22. Simonyi A, Serfozo P, Lehmidi TM et al (2012) The neuropro-
tective efects of apocynin. Front Biosci Elite Ed 4:2183–2193
23. Kanegae MP, Condino-Neto A, Pedroza LA et al (2010) Diapoc-
ynin versus apocynin as pretranscriptional inhibitors of NADPH
oxidase and cytokine production by peripheral blood mononu-
clear cells. Biochem Biophys Res Commun 393:551–554
24. Wang Q, Smith RE, Luchtefeld R et al (2008) Bioavailability
of apocynin through its conversion to glycoconjugate but not to
diapocynin. Phytomedicine 15:496–503
38. Xu J, Wu J, Zong C et al (2013) Manganese porphyrin-dsDNA
complex: a mimicking enzyme for highly efcient bioanalysis.
Anal Chem 85:3374–3379. https://doi.org/10.1021/ac4000688
39. Spencer NY, Engelhardt JF (2014) The basic biology of redox-
osomes in cytokine-mediated signal transduction and implications
for disease-specifc therapies. Biochemistry 53:1551–1564. https
://doi.org/10.1021/bi401719r
40. Nian M, Paul L, Khaper N, Liu P (2004) Infammatory cytokines
and postmyocardial infarction remodeling. Circ Res 94:1543–
1553. https://doi.org/10.1161/01.RES.0000130526.20854.fa
41. Neal A, Rountree A, Kernan K et al (2016) Real-time imaging of
intracellular hydrogen peroxide in pancreatic islets. Biochem J
473:4443–4456. https://doi.org/10.1042/BCJ20160481
42. Suthammarak W, Somerlot BH, Opheim E et al (2013) Novel
interactions between mitochondrial superoxide dismutases and
the electron transport chain. Aging Cell 12:1132–1140. https://
doi.org/10.1111/acel.12144
25. Dranka BP, Giford A, McAllister D et al (2014) A novel mito-
chondrially-targeted apocynin derivative prevents hyposmia
and loss of motor function in the leucine-rich repeat kinase 2
(LRRK2R1441G) transgenic mouse model of Parkinson’s dis-
ease. Neurosci Lett 583:159–164
26. Langley M, Ghosh A, Charli A et al (2017) Mito-apocynin pre-
vents mitochondrial dysfunction, microglial activation, oxida-
tive damage, and progressive neurodegeneration in mitopark
transgenic mice. Antioxid Redox Signal 27:1048–1066
27. Brenza TM, Ghaisas S, Ramirez JEV et al (2017) Neuronal pro-
tection against oxidative insult by polyanhydride nanoparticle-
based mitochondria-targeted antioxidant therapy. Nanomedicine
Nanotechnol Biol Med 13:809–820
43. Battogtokh G, Choi YS, Kang DS et al (2018) Mitochondria-tar-
geting drug conjugates for cytotoxic, anti-oxidizing and sensing
purposes: current strategies and future perspectives. Acta Pharm
Sin B 8:862–880
28. Ghosh A, Langley MR, Harischandra DS et al (2016) Mitoapo-
cynin treatment protects against neuroinfammation and dopa-
minergic neurodegeneration in a preclinical animal model of
Parkinson’s disease. J Neuroimmune Pharmacol 11:259–278
29. Saleem N, Goswami SK (2017) Activation of adrenergic receptor
in H9c2 cardiac myoblasts co-stimulates Nox2 and the derived
ROS mediate the downstream responses. Mol Cell Biochem
436:167–178
44. Wang JY, Li JQ, Xiao YM et al (2020) Triphenylphosphonium
(TPP)-based antioxidants: a new perspective on antioxidant
design. ChemMedChem 15(5):4040–4410
45. Kafkova A, Trnka J (2020) Mitochondria-targeted compounds in
the treatment of cancer. Neoplasma 67:450–460
46. Li W, Wu J, Xiang D et al (2019) Micelles loaded with puerarin
and modifed with triphenylphosphonium cation possess mito-
chondrial targeting and demonstrate enhanced protective efect
against isoprenaline-induced H9c2 cells apoptosis. Int J Nano-
medicine 14:8345–8360
30. Giordano G, Hong S, Faustman EM, Costa LG (2011) Measure-
ments of cell death in neuronal and glial cells. In vitro neurotoxi-
cology. Humana Press, Totowa NJ, pp 171–178
47. Heumüller S, Wind S, Barbosa-Sicard E et al (2008) Apocynin is
not an inhibitor of vascular NADPH oxidases but an antioxidant.
Hypertension 51:211–217
31. Wojtala A, Bonora M, Malinska D et al (2014) Methods to moni-
tor ROS production by fuorescence microscopy and fuorometry.
Methods Enzymol 542:243–262. https://doi.org/10.1016/B978-0-
12-416618-9.00013-3
48. Kučera J, Binó L, Štefková K et al (2015) Apocynin and diphe-
nyleneiodonium induce oxidative stress and modulate PI3K/Akt
and MAPK/Erk activity in mouse embryonic stem cells. Oxid Med
Cell Longev 2016:7409196. https://doi.org/10.1155/2016/74091
96
32. Konovalova S (2019) Analysis of mitochondrial respiratory chain
complexes in cultured human cells using blue native polyacryla-
mide gel electrophoresis and immunoblotting. JoVE J Vis Exp
144:e59269. https://doi.org/10.3791/59269
49. Graton ME, Potje SR, Troiano JA et al (2019) Apocynin alters
redox signaling in conductance and resistance vessels of spontane-
ously hypertensive rats. Free Radic Biol Med 134:53–63
50. Xiao Y, Meierhofer D (2019) Are hydroethidine-based probes reli-
able for reactive oxygen species detection. Antioxid Redox Signal
31:359–367
33. Thakur A, Alam MJ, Ajayakumar M et al (2015) Norepinephrine-
induced apoptotic and hypertrophic responses in H9c2 cardiac
myoblasts are characterized by diferent repertoire of reactive
oxygen species generation. Redox Biol 5:243–252
34. Jindal E, Goswami SK (2011) In cardiac myoblasts, cellular redox
regulates FosB and Fra-1 through multiple cis-regulatory mod-
ules. Free Radic Biol Med 51:1512–1521
51. Ximenes VF, Kanegae MP, Rissato SR, Galhiane MS (2007) The
oxidation of apocynin catalyzed by myeloperoxidase: proposal for
NADPH oxidase inhibition. Arch Biochem Biophys 457:134–141
35. Saleem N, Prasad A, Goswami SK (2018) Apocynin prevents iso-
proterenol-induced cardiac hypertrophy in rat. Mol Cell Biochem
445:79–88
36. Kalyanaraman B, Darley-Usmar V, Davies KJ et al (2012) Meas-
uring reactive oxygen and nitrogen species with fluorescent
probes: challenges and limitations. Free Radic Biol Med 52:1–6
37. Ezeriņa D, Takano Y, Hanaoka K et al (2018) N-Acetyl cysteine
functions as a fast-acting antioxidant by triggering intracellular
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
1 3