Potential involvement of the 18 kDa translocator protein and reactive oxygen species in apoptosis of THP-1 macrophages induced by sonodynamic therapy
Potential involvement of the 18 kDa translocator protein and reactive oxygen species in apoptosis of THP-1 macrophages induced by sonodynamic therapy
Xin Sun 0 1 2
Shuyuan Guo 0 2
Wei Wang 0 2
Zhengyu Cao 0 2
Juhua Dan 0 2
Jiali Cheng 0 2
Wei Cao 0 2
Fang Tian 0 2
Wenwu Cao 0 1 2
Ye Tian 0 2
0 Funding: This study was supported by the National Natural Science Foundation of China (NSFC) (81400339 and 81701848), the State Key Program of NSFC (81530052), Natural Science Foundation of Heilongjiang Province (QC2016121), and Medical Scientific Research Foundation of Harbin Medical University (2016LCZX57). The funders had
1 Laboratory of Photoand Sono-theranostic Technologies, Harbin Institute of Technology , Harbin, Heilongjiang , China , 2 Department of Cardiology, the First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University , Harbin, Heilongjiang , China , 3 Department of Pathophysiology, the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education , Harbin, Heilongjiang , China , 4 Materials Research Institute, The Pennsylvania State University , University Park, Pennsylvania , United States of America
2 Editor: Yi-Hsien Hsieh, Institute of Biochemistry and Biotechnology , TAIWAN
Sonodynamic therapy (SDT) with exogenous protoporphyrin IX (PpIX) or endogenous PpIX derived from 5-aminolevulinic acid (ALA) has been carried out to produce apoptotic effects on macrophages, indicating a potential treatment methodology for atherosclerosis. Our previous studies have found that mitochondria damage by reactive oxygen species (ROS) plays a major role in the SDT-induced apoptosis. This study aimed at investigating the potential involvement of the mitochondrial 18 kDa translocator protein (TSPO) and ROS in the pro-apoptotic effects of SDT on THP-1 macrophages. THP-1 macrophages were divided into control and SDT groups, and went through pretreatment of the specific TSPO ligand PK11195 and ROS scavengers N-Acetyl Cysteine (NAC), then compared with groups without pretreatment. Application of PK11195 reduced intracellular accumulation of endogenous PpIX. PK11195 and NAC reduced the generation of intracellular ROS and oxidation of cardiolipin induced by SDT, respectively. PK11195 and NAC also reduced SDT-induced mitochondrial membrane potential (ΔΨm) loss, the translocation of cytochrome c and cell apoptosis. PpIX accumulation, ROS generation and cell apoptosis were also attenuated by siTSPO. Our findings indicate the pivotal role of TSPO and ROS in SDT-induced cardiolipin oxidation, ΔΨm collapse, cytochrome c translocation and apoptosis in THP-1 macrophages.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
5-Aminolevulinic acid (ALA) is a natural precursor of protoporphyrin IX (PpIX) in the heme
biosynthesis pathway. Over several intermediate enzyme steps outside the mitochondria, a
ring system with four pyrrol-rings is synthesized from eight ALA molecules, yielding PpIX
no role in study design, data collection and
analysis, decision to publish, or preparation of the
inside the mitochondria and finally heme via the addition of a ferrous ion to the center of
the pyrrol-ring structure [
]. By adding ALA, the naturally occurring porphyrins PpIX may
selectively accumulate in the mitochondria of cancer cells and inflammatory cells due to the
limited capacity of porphobilinogen deaminase and ferrochelatase [
]. Such selectivity has
been exploited in sonodynamic therapy (SDT) of tumor, a modality that involves the systemic
administration of a tumor-localizing sensitizer and its subsequent activation by ultrasound
through many mechanisms, such as sonoluminescence and sonochemistry, resulting primarily
in reactive oxygen species (ROS)-induced apoptotic cell death [
Macrophages participate in the lipid metabolism and inflammatory processes, and play a
pivotal role in the progression and destabilization of atherosclerotic plaque [
]. In a
ballooninjured rabbit carotid artery model, we characterized the selective accumulation of ALA-PpIX
in the atherosclerotic plaque. Moreover, ALA-PpIX in plaques was positively correlated with
macrophage content, which provides a macrophage-selective treatment agent . We also
found that SDT with exogenous PpIX or endogenous ALA-PpIX induced apoptosis of
macrophages in vitro [
] and in vivo [
], indicating a promising therapy for the treatment of
atherosclerosis. However, the mechanism remains unclear.
The 18 kDa mitochondrial translocator protein (TSPO), formerly known as
peripheraltype benzodiazepine receptor (PBR), can be found in glial cells of the brain and in cells of the
peripheral tissues, including macrophages [
]. It is localized to outer mitochondrial
membrane and physically associated with voltage dependent anion channel (VDAC) and adenine
nucleotide translocator (ANT) that are the core components of the mitochondrial permeability
transition pore (mPTP). It has been confirmed that TSPO has a high-affinity recognition site
for porphyrins, particularly PpIX [
]. ROS, the main SDT-induced cytotoxic agent, can
diffuse only approximately 0.01±0.02 μm in its lifetime [
]. The mPTP is thus expected to be one
of the primary targets of ALA-mediated SDT. The damaged mPTP may trigger apoptotic
processes by disruption of the mitochondrial transmembrane potential (ΔCm) and release of
mitochondrial pro-apoptotic factors [
In this study, we used specific TSPO ligand PK11195 and siTSPO, as well as ROS scavengers
N-Acetyl Cysteine (NAC) to investigate the role of TSPO and ROS in SDT treatment of
Materials and methods
Cell culture and experimental conditions
A human leukemic cell line, THP-1 cell (American Type Culture Collection, ATCC, Manassas,
VA, USA), was cultured in RPMI 1640 medium containing 10% fetal bovine serum, 20 μg/ml
penicillin and 20 μg/ml streptomycin at 37ÊC in a humidified atmosphere with 5% CO2. The
cells were differentiated into macrophages by adding 100 ng/ml PMA for 72 hours. THP-1
macrophages were seeded in 96-well plates and incubated with ALA-PpIX, with or without
PK11195 pretreatment. It has been confirmed that neither ALA administration nor ultrasound
exposure alone shows significant effects on THP-1 macrophage apoptosis [
]. In this case, to
characterize the roles of TSPO and ROS in the SDT-induced apoptotic process, THP-1
macrophages were cultured in 35-mm Petri dishes and received no treatment or SDT treatment, and
with or without PK11195 and NAC.
ALA (1 mM) containing RPMI 1640 medium was added to the cultured THP-1 macrophages
and incubated in the dark for 3 hours. The medium was then replaced by RPMI 1640 without
ALA. For PK11195 and NAC pretreatment, the cells were seeded into the 35-mm Petri dishes.
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In the preliminary studies, the concentration of PK11195 (25 μM) was determined by
examining the cytotoxicity of PK11195 (0±200 μM) on THP-1 macrophages and the effects of
PK11195 (25 and 50 μM) on SDT-induced cell apoptosis (S1 Fig). The concentration of NAC
(20 mM) has been determined in our previous study [
Ultrasonic exposure system
After ALA incubation, the cells were exposed to the ultrasound, as previously described [
Briefly, the ultrasonic transducer, pulse generator and power amplifier used in this study were
designed and assembled by the Harbin Institute of Technology (Harbin, China). The
homemade ultrasonic transducer (diameter: 35 mm; resonance frequency: 1.0 MHz; duty factor:
10%; repetition frequency: 100 Hz) was placed in a water bath and 30 cm under the cells. The
ultrasonic intensity used was 0.5 W/cm2, as measured by a hydrophone (Onda Corp.,
Sunnyvale, CA, USA).
Fluorescence detection of endogenous ALA-PpIX
Intracellular PpIX was identified by a fluorescence microscope (Olympus, Tokyo, Japan).
Fluorescence intensity of PpIX was measured by a fluorescence microplate reader (Titertek
Fluoroscan II, Flow Laboratories, McLean, VA, USA) at 405 nm excitation and 635 nm
Reactive oxygen species detection
DCFH-DA was added to the medium of cells at a final concentration of 20 μM and incubated
at 37ÊC for 30 minutes. The cells were observed using fluorescence microscopy before and at
different time (0, 1, 2 and 3 hours) after SDT treatment. Then the cells were washed carefully
with phosphate buffered solution (PBS). A total of 1×106 cells were collected, resuspended in
serum-free medium and measured using a fluorospectrophotometer (USB2000, Ocean Optics
Inc., USA) at 488 nm excitation and 525 nm emission wavelengths.
Cardiolipin oxidation analysis
Cardiolipin oxidation in mitochondria was determined with 10-N-Nonyl-Acridine Orange
(NAO). Before and at different time (0, 1, 2 and 3 hours) after SDT treatment, macrophages
were incubated with 10 μg/ml NAO for 15 minutes at 37ÊC in the dark and monitored by the
fluorescence microscope. Then the cells were washed carefully with PBS twice. A total of 1×106
cells were collected and measured by the fluorospectrophotometer at 480 nm excitation and
525 nm emission wavelengths.
The ΔCm was assessed using fluorescent probe jc-1. The jc-1 is a dual emission
potential-sensitive probe. Red fluorescence (Ex/Em = 425/590 nm) attributes to a potential-dependent
aggregation in the mitochondria, and green fluorescence (Ex/Em = 490/530 nm), reflecting
the monomeric form of jc-1, which appears in the mitochondria after ΔCm loss. The emission
spectra of jc-1 shift from green to red with increasing concentration (i.e. aggregation) in the
mitochondria, thus, allows for a dual-color assessment of ΔCm. Before and at different time
(0, 1, 2 and 3 hours) after SDT treatment, macrophages were incubated with 10 mg/ml jc-1 for
20 minutes at 37ÊC in the dark. Cells from each sample were then analyzed by a FacsCalibur
flow cytometer (Becton-Dickinson, USA).
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Cytosolic and mitochondrial cytochrome c measurement
Western blot analysis was performed to measure cytosolic and mitochondrial cytochrome c.
Three hours after the SDT treatment, cells were collected. The mitochondrial and cytosolic
fractions were obtained for Western blot analysis. Primary antibody was goat polyclonal
anticytochrome c antibody (1:600). Secondary antibody was AP-IgG (1:500). The protein bands
were quantified by a Bio-Rad ChemiDocTM EQ densitometer and Bio-Rad Quantity One
software (Hercules, CA, USA). Actin was used as a loading control for the cytosolic fraction. HSP
60 was used as a loading control for the mitochondrial fraction.
Cell apoptosis assay
Cell apoptosis was assessed by the Annexin V-FITC apoptosis kit according to the
manufacturer's instructions. Three hours after the treatments, the cells were incubated with 5 μl Annexin
V and 5 μl PI for 10 minutes at room temperature in the dark. Cells from each sample were
then analyzed by the flow cytometer. The data were analyzed using the CELLQuest software
(Becton Dickinson, Franklin Lakes, NJ, USA). Cells in the lower-right quadrant
(AnnexinV+/PI-) represent early apoptotic cells.
RNA interference of TSPO
The small interference RNA of TSPO (siTSPO) was based on previously published paper:
siTSPO 5’-CACUCAACUACUGCGUAUG-3’ [
]. SiTSPO and the scramble siRNA were
synthesized by GenePharma (Shanghai, China) and were transfected into THP-1 derived
macrophages with X-tremeGENE siRNA transfection reagent according to the routine process.
Assessment of silencing efficiency was performed by western blot with the protein collected 48
hours after the transfection.
Cell viability assay
During the experiment, the cells were seeded into the 35 mm Petri dishes and incubated with
different concentrations of PK11195 (0±200 μM) for 24 hours. The survival rate of the cells
was measured by MTT assay. Experiments were repeated three times independently.
Isolation of murine peritoneal macrophages
Peritoneal macrophages were isolated from C57BL/6 mice (6±8 weeks old) 3 days after the
intraperitoneal injection of 2 ml of 3% thioglycollate (Sigma-Aldrich). Five million peritoneal
cells were plated in Petri dishes with RPMI 1640 medium containing 10% FBS and allowed to
adhere for 4 hours. The purity of macrophages was identified by immunofluorescence staining
Mitochondrial membrane potential was assessed using fluorescent probe jc-1. Macrophages
were incubated with 10 mg/mL jc-1 for 20 minutes at 37ÊC in the dark. The fluorescence
intensity was measured using a fluorospectrophotometer (Varian Australia Pty Ltd,
Melbourne, Victoria, Australia) at 488 nm excitation and 530 nm (green) and 590 nm (red)
emission wavelengths. Experiments were repeated three times independently.
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All data were reported as mean value ± standard deviation. A Shapiro-Wilk test was first used
to test the normality of the data. One-way analysis of variance followed by
Student-NewmanKeuls testing was used to determine the difference among the groups. Statistical evaluation
was performed using Statistical Analysis System software (version 9.2, SAS institute, Cary,
NC). Differences with P < 0.05 were considered statistically significant.
PK11195 attenuated endogenous ALA-PpIX accumulation
The fluorescence microscope detection showed that PpIX red fluorescence was observed in
macrophages after incubation with ALA for 3 hours, which was decreased in cells pretreated
with PK11195 (Fig 1A). The fluorescence intensity of PpIX was increased by 3.8 folds in cells
incubation with ALA (P < 0.001), as compared with control. Pretreatment with PK11195
decreased endogenous ALA-PpIX fluorescence intensities by 24% (P < 0.01) (Fig 1B).
Effects of PK 11195 and NAC on SDT-induced ROS generation
Intracellular ROS generation was assessed by measuring the conversion of non-fluorescent
DCFH-DA to fluorescent DCF. The green fluorescence of DCF was significantly increased at
Fig 1. Effects of PK 11195 on endogenous ALA-PpIX accumulation. THP-1 macrophages were incubated for 3 hours
with 5-aminolevulinic acid (ALA) with or without PK 11195. (A) Red fluorescence of intracellular PpIX was identified
by fluorescence microscope. Scar bar: 0.1 mm. (B) Fluorescence intensity of PpIX was measured by fluorescence
microplate reader. P < 0.001 compared to no treatment group, ##P < 0.01 compared to ALA treated group.
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0, 1, 2 and 3 hours after SDT (S2 Fig). The green fluorescence of DCF was present in few
control cells and cells co-treated with SDT and NAC, but in a small portion of cells co-treated with
SDT and PK11195, and most of the SDT-treated cells (Fig 2A). In accordance with this, the
fluorescence intensity of DCF was increased (P < 0.001) in the SDT-treated macrophages, in
comparison to the untreated controls. The generation of ROS by SDT was prevented by
cotreatment with NAC and decreased by co-treatment with PK11195 (Fig 2B).
Effects of PK 11195 and NAC on SDT-induced cardiolipin oxidation
Cardiolipin oxidation in mitochondria was assessed by using NAO. The fluorescence
microscope detection showed that NAO green fluorescence was observed within macrophages
surrounding the nucleus. NAO green fluorescence was decreased at 0 hour after SDT, and
further decreased at 1 hour (S2 Fig). In the control group, most cells showed a high level of
NAO labeling. However, in the SDT group, cells showed a decreased level of NAO labeling
as an indication of increased cardiolipin oxidation, which was prevented by co-treatment
with PK11195 or NAC (Fig 3A). In accordance with this, the fluorescence intensity of NAO
was reduced by 45% (P < 0.001) in the SDT-treated macrophages, in comparison to the
untreated controls. Pretreatment with PK11195 and NAC, cells showed respectively 43% and
75% increase in the fluorescence intensity of NAO in comparison to SDT treatment alone
Effects of PK 11195 and NAC on SDT-induced ΔCm loss
A ΔCm-sensitive dye, JC-1, was used to examine whether loss of ΔCm is associated with
SDTinduced apoptosis. Red fluorescence of JC-1 was decreased at 0 hour after SDT, and further
decreased at 1 hour (S2 Fig). As shown in Fig 4A, cells with ΔCm loss were present in the
lower right quadrant. Three hours after the treatment, ΔCm loss was seen in 20.91 ± 3.16% of
the control cells, which was increased to 72.31 ± 3.52% in SDT-treated cells, and 40.87 ± 3.46%
and 21.55 ± 2.72% in SDT-treated cells that co-treated with PK11195 and NAC respectively
Effects of PK 11195 and NAC on SDT-induced cytochrome c translocation
The level of cytochrome c in the cytosol and mitochondria were determined by Western
blotting. As shown in Fig 5A, the cytosolic cytochrome c level was low in the control cells. Low
level of cytosolic cytochrome c was also present in SDT-treated cells pretreated with PK11195
and NAC, whereas high level of cytochrome c could be seen in the cytosol of SDT-treated cells.
The mitochondrial cytochrome c level was high in the control cells. High level of
mitochondrial cytochrome c was also present in SDT-treated cells pretreated with PK11195 and NAC,
whereas low level of cytochrome c could be seen in the mitochondria of SDT-treated cells.
Quantitative analysis revealed that the SDT-treated cells showed a 106% (P < 0.001) increase
in cytosolic cytochrome c and a 50% (P < 0.01) decrease in mitochondrial cytochrome c as
compared with the control cells, which was prevented by co-treatment with PK11195 and
NAC (Fig 5B).
Effects of PK 11195 and NAC a on SDT-induced macrophage apoptosis
Cell apoptosis was measured using flow cytometry with double staining of Annexin V and PI.
As shown in Fig 6A, early apoptosis was seen in 13% of the control cells. It was increased to
39% in the SDT-treated cells at 3 hours after the treatment. This increase was prevented in cells
pretreated with PK11195 and NAC. Quantitative analysis showed that early apoptosis rate in
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Fig 2. Effects of PK 11195 and NAC on SDT-induced ROS generation using the dye DCFH-DA. THP-1
macrophages were treated with 5-aminolevulinic acid mediated sonodynamic therapy (ALA-SDT), with or without
pretreatment of PK 11195 or NAC. (A) Immediately after treatment, ROS production in THP-1 macrophages was
observed under fluorescence microscope. The ALA-SDT treated cells showed increased green fluorescence level of
ROS as compared with the untreated controls, and this effect was inhibited by adding PK 11195 or NAC before
sonication. Scar bar: 0.1 mm. (B) Fluorescence intensity of ROS was measured using fluorospectrophotometer.
P < 0.001 compared to no treatment group, ###P < 0.001 compared to ALA-SDT treated group.
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Fig 3. Effects of PK 11195 and NAC on SDT-induced cardiolipin oxidation. THP-1 macrophages were treated for 1
hours with 5-aminolevulinic acid mediated sonodynamic therapy (ALA-SDT), with or without pretreatment of PK
11195 or NAC, and cardiolipin oxidation was determined with 10-N-Nonyl-Acridine Orange (NAO). (A) Green NAO
fluorescence was monitored by fluorescence microscope. Cell nuclei were stained with Hoechst. Scar bar: 0.1 mm. (B)
Fluorescence intensity of NAO was measured using fluorospectrophotometer. P < 0.001 compared to no treatment
group, ##P < 0.01 compared to ALA-SDT treated group, ###P < 0.001 compared to ALA-SDT treated group.
the control group was 16.34 ± 1.88%. The SDT-treated cells displayed a 109% (34.21 ± 6.44%,
P < 0.001) increase in early apoptosis rate in comparison to the untreated controls, whereas
only 38% increase was observed in the cells pretreated with PK11195 (22.58 ± 4.50%) and 5%
increase with NAC (17.25 ± 4.68%) (Fig 6B).
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Fig 4. Effects of PK 11195 and NAC on SDT-induced ΔCm loss, as determined with JC-1 by flow cytometer.
THP1 macrophages were treated for 1 hours with 5-aminolevulinic acid mediated sonodynamic therapy (ALA-SDT), with
or without pretreatment of PK 11195 or NAC. (A) Cells with polarized mitochondria are found in the upper right
quadrant, corresponding to high emission of fluorescence at both 590 nm (FL2-H, orange-red) and 527 nm (FL1-H,
green), whereas cells with depolarized mitochondria are present in the lower right quadrant. (B) Quantifications of
percentage of cells with low mitochondrial membrane potential (ΔCm) in the indicated groups. P < 0.001
compared to no treatment group, ###P < 0.001 compared to ALA-SDT treated group.
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Fig 5. Effects of PK 11195 and NAC on SDT-induced cytochrome c translocation. THP-1 macrophages were
treated for 3 hours with 5-aminolevulinic acid mediated sonodynamic therapy (ALA-SDT), with or without
pretreatment of PK 11195 and NAC. (A) Cytosolic translocation of mitochondrial cytochrome c was examined by
western blot of the cytosol and mitochondria. Actin and HSP60 were used as loading controls. (B) Quantitative
representation of the Western blot. P < 0.01, P < 0.001 compared to no treatment group, #P < 0.05 compared to
ALA-SDT treated group, ##P < 0.01 compared to ALA-SDT treated group.
Knockdown of TSPO attenuated PpIX accumulation, ROS generation and
cell apoptosis by SDT
The expression level of TSPO was significantly decreased in cells treated by siTSPO (Fig 7A).
Treatment of siTSPO decreased intracellular ALA-PpIX fluorescence intensity by 40%
(P < 0.001) (Fig 7B). The fluorescence intensity of DCF was decreased by 41% (P < 0.001) in
the SDT-treated siTSPO macrophages, compared with non-siTSPO macrophages (Fig 7C). In
addition, early apoptosis rate was decreased by 36% (P < 0.001) in the SDT-treated siTSPO
macrophages, compared with non-siTSPO macrophages (Fig 7D).
This study demonstrates a TSPO-related pathway for macrophage apoptosis triggered by SDT.
Previously, several other mechanisms have been reported. For example, SDT triggers DNA
fragmentation and apoptosis in a syngeneic colon cancer model [
]. SDT damages
mitochondria, activates pro-apoptotic factors Bax and cytochrome c in a human tongue squamous
carcinoma SAS cell line [
]. Excessive intracellular ROS production followed by lipid peroxidation
increase due to SDT has also been described in the SAS cells [
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Fig 6. Effects of PK 11195 and NAC on SDT-induced macrophage apoptosis. THP-1 macrophages were treated for 3 hours with
5-aminolevulinic acid mediated sonodynamic therapy (ALA-SDT), with or without pretreatment of PK 11195 and NAC. Cell apoptosis and
necrosis were assessed by flow cytometry with double staining of Annexin V and propidium iodide (PI). (A) Cells in the lower-right quadrant
(Annexin-V+/PI-) represent early apoptotic cells. (B) Quantifications of early apoptosis rate in the indicated groups. P < 0.001 compared to
no treatment group, ##P < 0.01 compared to ALA-SDT treated group, ###P < 0.001 compared to ALA-SDT treated group.
In this study, by applying TSPO ligand PK11195 and TSPO siRNA, we demonstrated that
TSPO was involved in the process of SDT-induced macrophage apoptosis, including PpIX
accumulation, ROS generation, cardiolipin oxidation, ΔCm disruption and cytochrome c
translocation. The similar effects were observed in the isolated of murine peritoneal
macrophages (S3 Fig).
It has been reported that TSPO is involved in transport of porphyrins, like
coproporphyrinogen III and PpIX across the mitochondrial membrane [
]. Several studies have also been
published showing interactions of PpIX and TSPO in various cell models [
interaction was suggested to mediate the action of porphyrin based photosensitization in
photodynamic therapy (PDT) of tumors . Furthermore, it was demonstrated that selectively
increasing TSPO expression in tumor cells by low-level light treatment facilitated
ALA-PDTinduced tumor cell death [
]. In the present study, pretreatment with PK11195 or siRNA, the
accumulation of PpIX was partially attenuated in macrophages (Figs 1B and 7B), which was in
accordance with the previous studies in various tumor cells [
The decrease of ROS generation in the SDT-treated cells in the presence of PK11195 was
expected from the reduction of intracellular ALA-PpIX accumulation. Intracellular ROS
generation was also decreased in the presence of NAC, indicating that SDT produced ROS in
macrophages (Fig 2). In addition, generation of ROS by SDT within mitochondria was confirmed
by assessing cardiolipin oxidation (Fig 3). Likewise, siTSPO decreased ROS generation by SDT
Because the targets of SDT are the sites where the ROS is produced, the mPTP associated
with TSPO is among the targets for SDT. Previous studies have shown that opening of mPTP
by ROS is involved in TSPO activation-induced apoptosis [
]. When the mPTP opens, the
ΔCm collapses as a consequence of the dissipation of the proton gradient generated in the
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Fig 7. Effect of knockdown of TSPO on PpIX accumulation, SDT-induced ROS generation and cell apoptosis. (A)
Knockdown of TSPO in THP-1 macrophages was examined by western blot. (B) Fluorescence intensity of PpIX
was measured by fluorescence microplate reader. (C) Fluorescence intensity of ROS was measured using
fluorospectrophotometer. (D) Quantifications of early apoptosis rate in the indicated groups. P < 0.001 compared
to no treatment group, ##P < 0.01 compared to ALA-SDT treated group, ###P < 0.001 compared to ALA-SDT treated
mitochondrial intermembrane space, which is an early event of the apoptotic cascade [
agreement with these data, we showed that the ΔCm was disrupted as early as 1 hour after
SDT. PK11195 and NAC attenuated this effect (Fig 4), suggesting the involvement of TSPO
and ROS in the SDT-induced ΔCm disruption.
Disruption of ΔCm has been reported to result in swelling of the mitochondrial matrix,
mechanical rupture of the outer membrane, and release of inter-membrane proteins, such as
cytochrome c and apoptosis-inducing factor [
]. Once released into the cytosol, these
mitochondrial proteins mediate either a caspase-dependent apoptotic pathway or translocate
further into the nucleus to induce a caspase-independent apoptotic pathway [
]. In the present
study, we found that cytochrome c in the cytosol of SDT-treated macrophages was
significantly increased (Fig 5), suggesting the translocation of cytochrome c from the mitochondria.
This is in accordance with the fact that ROS generated by SDT induced dissociation of
cytochrome c from cardiolipins after oxidized. Other studies have also found that ROS-induced
VDAC alterations induce mitochondrial membrane permeability selective for cytochrome c
]. Increase of VDAC pore size via phosphorylation by protein kinase A, can be a
mechanism of allowing cytochrome c release [
]. Moreover, assemblage of VDAC molecules
into groups of up to 20 or even larger aggregates, including hexagonal packing, may play a role
in cytochrome c release [
After cytosolic translocation, cytochrome c works with Apaf-1 and procaspase-9 in the
presence of dATP or ATP to initiate apoptotic process by activating the downstream effector
]. This is in accordance with our finding that early apoptotic cells were increased
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Fig 8. Schematic summary illustrating cytochrome c (Cytc) release due to reactive oxygen species (ROS)
generation following activation of 5-aminolevulinic acid derived protoporphyrin IX (ALA-PpIX) binding to the
18 kDa mitochondrial translocator protein (TSPO) and subsequent opening of mitochondrial permeability
transition pore (mPTP).
after the SDT treatment (Fig 6). Furthermore, SDT-induced cytochrome c translocation and
cell apoptosis was almost abolished by PK11195 and siTSPO, as well as NAC, indicating the
pivotal role of TSPO and ROS.
In conclusion, this study provides evidence that TSPO and ROS are involved in THP-1
macrophage apoptosis by SDT treatment. As shown in Fig 8, activation of ALA-PpIX binding
to TSPO by ultrasound leads to ROS generation, resulting in the release of cytochrome c from
oxidated cardiolipins at the inner mitochondrial membrane and the increase of permeability
of the outer mitochondrial membrane, allowing cytochrome c translocation, which in turn
induces macrophages apoptosis.
S1 Fig. Effects of PK11195 with different concentrations on cell viability and SDT-induced
apoptosis in THP-1 macrophages. (A) Cytotoxicity of PK11195 on THP-1 macrophages with
different concentrations was analyzed by MTT assay. (B) SDT-induced apoptosis was assessed
by flow cytometry with double staining of Annexin V and propidium iodide (PI). P < 0.001
compared to no treatment group, ###P < 0.001 compared to SDT treated group.
S2 Fig. Effects of SDT on ROS generation, cardiolipin oxidation and mitochondrial
membrane potential loss in THP-1 macrophages. (A) Fluorescence intensity of ROS was
measured by using fluorospectrophotometer with the staining of fluorescent probe DCFH-DA.
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(B) Cardiolipin oxidation was determined by using fluorospectrophotometer with the staining
of NAO. (C) mitochondrial membrane potential was assessed by using
fluorospectrophotometer with the staining of jc-1. P < 0.01 compared to baseline,
P < 0.001 compared to baseline. (TIF)
S3 Fig. Effects of PK11195 on ALA-PpIX accumulation, and ROS generation and cell
apoptosis by SDT in isolated murine peritoneal macrophages. (A) Peritoneal macrophages
were isolated from C57BL/6 mice and confirmed by immunofluorescent staining with CD68
antibodies. Scale bar represents 0.1 mm. (B) Fluorescence intensity of PpIX in the indicated
groups detected by a fluorescence microplate reader. (C) Intracellular ROS generation in the
indicated groups detected by fluorospectrophotometer with the staining of fluorescent probe
DCFH-DA. (D) Quantifications of early apoptosis rate in the indicated groups measured by
flow cytometry with double staining of Annexin V and PI.
group. ##P < 0.01, ###P < 0.001 compared to SDT group.
P < 0.001 compared to control
The authors gratefully thank Jianting Yao and Weiwei Gao for excellent technical assistance,
as well as Dr. Jing Shen critical reading of the manuscript.
Conceptualization: Xin Sun.
Data curation: Xin Sun.
Formal analysis: Xin Sun.
Cao, Fang Tian.
Investigation: Xin Sun, Shuyuan Guo, Wei Wang, Zhengyu Cao, Juhua Dan, Jiali Cheng, Wei
Methodology: Xin Sun, Shuyuan Guo, Wei Wang, Juhua Dan, Jiali Cheng, Wei Cao, Fang
Resources: Wenwu Cao, Ye Tian.
Supervision: Wenwu Cao, Ye Tian.
Writing ± original draft: Xin Sun, Zhengyu Cao.
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