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Journal of Cancer 2014, Vol. 5
http://www.jcancer.org
609
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2014; 5(7): 609-624. doi: 10.7150/jca.9002
Research Paper
Pyrroloquinoline Quinone Induces Cancer Cell
Apoptosis via Mitochondrial-Dependent Pathway and
Down-Regulating Cellular Bcl-2 Protein Expression
Zhihui Min1,2,3*, Lingyan Wang1*, Jianjun Jin1,2, Xiangdong Wang1,2,3, Bijun Zhu1, Hao Chen4,, Yunfeng
Cheng1,3,5,6
1. Biomedical Research Center, Zhongshan Hospital, Fudan University, Shanghai 200032, China;
2. Biomedical Research Center, Zhongshan Hospital Qingpu Branch, Shanghai, 201700 China;
3. Shanghai key laboratory of organ transplantation, Shanghai, 200032, China;
4. Department of Cardiothoracic Surgery, Tongji Hospital, Tongji University, Shanghai 200065, China;
5. Department of Hematology, Zhongshan Hospital, Fudan University, Shanghai 200032, China;
6. Department of Hematology, Zhongshan Hospital Qingpu Branch, Shanghai, 201700 China.
* These authors contributed equally to this work.
Corresponding author: Yunfeng Cheng, MD, PhD. Department of Hematology, Biomedical Research Center, Zhongshan Hospital, Fudan
University, 180 Fenglin Rd, Shanghai 200032, China. Email: yfcheng@fudan.edu.cn Hao Chen, MD, PhD. Department of Cardiothoracic
Surgery, Tongji Hospital, Tongji University, 389 Xincun Rd, Shanghai 200065, China. Email: h.chen@fudan.edu.cn Tel: +86-21- 64041990 ext.
2295 Fax: +86-21-64041990 ext. 2295.
© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/
licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
Received: 2014.03.03; Accepted: 2014.06.16; Published: 2014.07.29
Abstract
Pyrroloquinoline quinone (PQQ) has been reported as a promising agent that might contribute to
tumor cell apoptosis and death, yet little is known on its mechanisms. In current study, the effect
of PQQ on cell proliferation and mitochondrial-dependent apoptosis were examined in 3 solid
tumor cell lines (A549, Neuro-2A and HCC-LM3). PQQ treatment at low to medium dosage
exhibited potent anti-tumor activity on A549 and Neuro-2A cells, while had comparably minimal
impact on the viabilities of 2 human normal cell lines (HRPTEpiC and HUVEC). The apoptosis of
the 3 tumor cell lines induced by PQQ were increased in a concentration-dependent manner,
which might be attributed to the accumulation of intracellular reactive oxygen species (ROS),
decline in ATP levels and dissipation of mitochondrial membrane potential (MMP), in conjunction
with down-regulation of Bcl-2 protein expression, up-regulation of activated caspase-3, and dis-
turbed phosphorylated MAPK protein levels. PQQ induced tumor cells apoptosis was significantly
alleviated by pan-caspase inhibitor Z-VAD-FMK. The present work highlights the potential capa-
bility of PQQ as an anti-tumor agent with low toxicity towards normal cells through activating
mitochondrial-dependent apoptosis pathways, and warrants its development for cancer therapy.
Key words: pyrroloquinoline quinone (PQQ); apoptosis; MAPK; mitochondrial membrane poten-
tial; bcl-2.
Introduction
Pyrroloquinoline quinone (PQQ) was primarily
identified in methylotrophic bacteria as a novel bac-
terial enzymatic cofactor [1] and was considered to be
a new B group of vitamins [2]. As an essential nutri-
ent, PQQ is a water-soluble and thermal stable small
molecular organic matter, exists in various plant and
animal cells, at pM to nM levels [3, 4]. Researches of
PQQ have been focused on its activities as an antiox-
Ivyspring
International Publisher
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610
idant and redox modulator[5], cardio- and neu-
ro-protectant [6, 7], and its radioprotective effects on
hemopoietic system [8].
Recently, studies showed that PQQ could induce
apoptosis in human promonocytic leukemia U937 and
lymphoma EL-4 cells, as well as Jurkat cell pro-
grammed death [5]. The underlying mechanism
might be relevant to the increase of intracellular reac-
tive oxygen species (ROS) and depletion of glutathi-
one [9]. Another study in vivo indicated that at nano-
to micro-mole levels of PQQ intake in animal diets
could affect the cell signaling, especially activation of
MAPK-related families and JAK/STAT3 signaling in
the livers of rat [10]. In addition, PI3K/Akt,
ras-related ERK1/2 [7] and phosphorylation of
JNK signaling pathways were proved to be associated
with the neuro-protective effect of PQQ in hippo-
campal neurons [11].
These findings suggested that PQQ not only
regulates redox status of the cells, but also poses im-
pact on the cellular signaling pathways. However, to
date, there is no study that has investigated the effect
of PQQ on directly inducing solid tumor cell apopto-
sis except for the hematological tumors [5, 9]. The
underlying molecular mechanism of PQQ’s anticancer
effect remains to be elucidated. Inasmuch, this work
aimed to determine whether PQQ has apopto-
sis-inducing effect in solid tumor cells, and to explore
the potential mechanisms.
Materials and methods
Chemicals and cell lines
Pyrroloquinoline quinine (PQQ) was obtained
from Changmao Biochemical Engineering Co., LTD
(Changzhou, China). PQQ stock solution (10mM) was
prepared in DMEM medium, stored in -20˚C. Ben-
zyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylke-
tone (Z-VAD-FMK) was acquired from Enzo Life
Sciences, Inc (Lausen, Switzerland). A549 (human
non-small cell lung adenocarcinoma) and Neuro-2A
(mouse neuroblastoma) cell lines were purchased
from the cell bank of Chinese Academy of Sciences
(Shanghai, China). HRPTEpiC (human renal proximal
tubular epithelial cells) was purchased from ScienCell
research laboratories (Carlsbad, California, USA).
HUVEC (human umbilical vein endothelial cells) and
HCC-LM3 (human hepatocellular carcinoma) cell
lines were kindly provided by the Liver Cancer Re-
search Institute of Zhongshan Hospital, Fudan Uni-
versity (Shanghai, China), and maintained on the ba-
sis of ATCC guidelines at our center. All cells were
cultured in Dulbecco’s modified Eagle’s medium
(DMEM/High Glucose, Thermo Scientific HyClone,
Logan, Utah, USA) containing 10% fetal bovine serum
(FBS), 1% (v/v) penicillin-streptomycin (Gibco Invi-
trogen, Grand Island, NY, USA) at 37˚C in a humidi-
fied atmosphere with 5% CO2. Cells were treated for
up to 48h with PQQ at designated concentrations,
another cell culture without PQQ treatment was
served as control.
Cell bio-behaviors assay with a continuous cell
culturing platform (CELL-IQ)
The cell bio-behaviors including total cell num-
ber, cell differentiation and cell movement were
measured by a real-time cell monitoring system,
Cell-IQ cell culturing platform (Chip-Man Technolo-
gies, Tampere, Finland), equipped with a
phase-contrast microscope (Nikon CFI Achromat
phase contrast objective with 10× magnification). The
equipment was controlled by Cell-IQ image software
(Chip-Man Technologies). Analysis was carried out
with a freely distributed Image software (McMaster
Biophotonics Facility, Hamilton, ON, Canada), using
the Manual Tracking plugin created by Fabrice Cor-
deliéres (Institut Curie, Orsay, France). Cell-IQ system
uses machine vision technology for monitoring and
recording time-lapse data, and it can also analyze and
quantify cell functions and morphological parameters
[12]. This system was used to discriminate cell stage
(dividing/stable stage) and calculate cell numbers of
each stage during proliferation. Besides, Cell-IQ was
programmed to quantify the movement of each indi-
vidual cell in the image field. The distance of total cell
movement indicates the high migratory intention of
cancer cells.
In the current study, cells treated with PQQ at
different concentrations were cultured in Cell-IQ sys-
tem with 24-well plates (8 × 103 cells /well) for up to
48h. Images were captured at 5 min intervals for up to
48h. Cell stages, total cell number, cell differentiation
and cell movement were then automatically analyzed.
Cell viability assay
The cell viability of PQQ was evaluated using
Cell Counting Kit-8 (CCK-8) assay (Dojindo Labora-
tories, Kumamoto, Japan), per instruction of the
manufacturer. In brief, cells were seeded in 96-well
plates at 1 × 104 cells/well and allowed an overnight
period for attachment. After treatment for 48h with
PQQ at serial concentrations, CCK-8 solution (10 µl)
was added to each well, followed by 3h of incubation
at 37°C. The absorbance wavelength at 450 nm was
recorded for each well in a FlexStation 3 microplate
reader (Molecular Devices, Sunnyvale, California,
USA), and cell viability was then accessed according
to the manufacturer instruction.
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Detection of apoptosis with Annexin
V-FITC/PI staining
Cell apoptosis was determined by the Annexin
V-fluorescein isothiocyanate (FITC)/Propidium Io-
dide (PI) Apoptosis Detection Kit (EMD Biosciences,
La Jolla, USA). Cells were cultured in 24-well plate at
a density of 5 × 104 cells/ml and treated with PQQ for
24h. Cells were harvested and resuspended in binding
buffer, and then stained with 2.5 µl Annexin V-FITC
and 5 µl propidium iodide (PI), and incubated for 15
min at room temperature in the dark. The stained cells
were detected within 30 minutes with BD FACS Aria
II flow cytometer (BD biosciences, San Jose, Califor-
nia, USA).
Reactive oxygen species (ROS) measurement
The intracellular ROS levels of 3 tumor cells were
measured by reactive oxygen species assay kit (Be-
yotime Institute of Biotechnology, Shanghai, China).
Briefly, cells were seeded in 6-well plates (1 × 105
cells/well) followed by overnight incubation and
treatment with indicated concentration of PQQ for
additional 4h, 12h and 24h. Cells were then tryp-
sinized and washed with PBS buffer before loading
with 5 µM fluorescent probe 2', 7'-dichloro-
dihydrofluorescein diacetate (H2DCFDA) at 37˚C for
30 minutes in dark. The fluorescent intensity was an-
alyzed with flow cytometer (FACS Aria II, BD bio-
sciences).
Cellular ATP release assay
Cells were cultured in the 96-well plates with 100
μl medium per well. After treatment with different
concentrations of PQQ for 4h, 12h, and 24h,
CellTiter-Glo Reagent (Promega, Madison, Wisconsin,
USA) was added to each well for the plate. The rea-
gent and culture medium were mixed for 2 minutes
on an orbital shaker and the optical density (O.D.) was
measured using a microplate reader (Flexstation3,
Molecular Devices). All experiments were done in
triplicate and repeated three times independently.
Detection of active caspase-3
Cells were treated with PQQ for 24h on the in-
dicated concentrations, and collected, followed by
cells were fixating, permeabilizating and intracellular
staining with anti-active Caspase-3 V450 antibody
(BD biosciences), according to the manufacturer’s
instructions. The activity of caspase-3 was presented
as mean fluorescent intensity of cells and performed
by flow cytometer (BD FACS Aria II).
Cell cycle analysis
After PQQ treatment, cells were harvested for
cell cycle phase distributions detected by BD LSR
Fortessa flow cytometry (BD biosciences). Stained
cells were tested on flow cytometer according to the
instructions of Cycle TestTM Plus DNA Reagent Kit
(BD biosciences), and then analyzed using Flowjo
software version 7.6.2. (Tritar Inc., San Carlos, Cali-
fornia, USA).
Measurement of Mitochondrial Membrane
Potential
Mitochondrial membrane potential was assessed
using the fluorescent probe JC-1 (MitoProbeTM, Invi-
trogen, Grand Island, NewYork, USA), which was a
cationic dye that displayed potential-dependent ac-
cumulation and normal formation of red fluorescent
J-aggregates in mitochondria, indicated by a fluores-
cence emission shift from green (~529 nm) to red
(~590 nm). A green fluorescent (JC-1 as a monomer at
low membrane potentials) and a red fluorescent (JC-1
as “J-aggregates” at higher membrane potentials)
were monitored under flow cytometer (BD FACS Aria
II). Samples treated with Carbonyl cyanide
m-chlorophenylhydrazone (CCCP), a mitochondrial
uncoupling agent, were used as positive-control to
perform standard compensation. Consequently, de-
polarization of mitochondrial membrane potential is
indicated by a decrease in the red/green fluorescence
intensity ratio. Cells were suspended in 1ml PBS at
approximately 1 × 106 cells/ml for each sample. For
the positive-control tube, added 1µl of 50mM CCCP
and incubated the cells at 37°C for 5 min. The sample
cells were incubated at 37°C for 20 min with 2 µM
JC-1, then washed once with 2ml PBS and placed in
500 µl PBS until analyzed by flow cytometer.
Measurement of Bcl-2, ERK2, MEK2, pERK1/2
and p38MAPK proteins
To investigate the underlying mechanisms of
apoptosis induced by PQQ, we tested the effect of
PQQ on key signaling pathway proteins in A549 and
neuro-2A cells. These 2 cell lines were treated with
15-
360 μM of PQQ for 24h. The expression levels of
Bcl-2, ERK2, MEK2, pERK1/2 and p38 MAPK were
analyzed by flow cytometer and presented as mean
fluorescent intensity (MFI). In brief, cells were seeded
in 24-well plates and treated with designated concen-
trations of PQQ for 24h. After 24h culture, cells were
fixed and permeabilized (BD CytofixTM Fixation
Buffer Cat. No. 554655, and Phosflow Perm Buffer III,
Cat. No. 558050, BD biosciences), and intracellularly
stained with anti-Human Bcl-2 FITC (BD biosciences),
anti-ERK2 Alexa Fluor 488, anti-MEK2 PE, an-
ti-pERK1/2 pT202/pY204 PerCP-Cy5.5, anti-p38
MAPK pT180/pY182 PE-Cy7 (Phosflow, BD biosci-
ences) according to the manufacturer's instructions.
The expression levels were tested on BD FACS Aria II
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612
flow cytometer and then analyzed using Flowjo soft-
ware version 7.6.2.
Statistical analysis
All analyses were performed with SPSS software
version 13.0 (SPSS Inc., Chicago, USA). Data were
expressed as mean ± SEM. Normality was assessed by
Shapiro-Wilk W test. Student t test and Wilcoxon
rank-sum (Mann-Whitney) test were used for data
fulfilled normal distribution and for those did not,
respectively. When multiple groups were compared,
One Way ANOVA and Kruskal Wallis test were used
for data fulfilled normal distribution and for those did
not, respectively. Two-sided p values less than 0.05
were considered statistically significant.
Results
The proliferation of cancer cell lines
suppressed by PQQ
The cells were seeded in 24-well plates (3 × 104
cells per well) overnight and then treated with PQQ at
different concentrations respectively or without PQQ
treatment (control group). As shown in Figure 1A and
1B, the cell growth curve generated by Cell-IQ moni-
toring system displayed notably inhibitory effects of
PQQ in a dose- and time-dependent manner on
treated A549 and Neuro-2A cells, with IC50 at 24h of
50.16 µM and 56.21 µM, respectively. Compared with
the control group, the proliferation inhibitory effects
of PQQ were significant at 24h and 48h in both cell
lines. The inhibitory effects were significant from 30
µM to 600 µM in A549 cells, and from 30 µM to 360
µM in Neuro-2A cells overtime. Therefore, we chose
the time-point of 24h at low (15-30 µM), medium
(IC50) (60-75 µM) and high (120-300 µM) concentra-
tions for further studies. There were no significant
inhibitory effects observed on HCC-LM3 cells prolif-
eration at 15-150 µM comparing with the control
group, the IC50 at 24h was more than 300 µM. How-
ever, higher dose PQQ treatments (300-1200 µM) in-
hibited the proliferation of HCC-LM3 cells (Figure
1C).
Figure 1. The cells proliferation and viability. PQQ inhibited proliferations of 3 cancer cell lines for up to 48h were measured by Cell-IQ assay. Cell number of each
microscopic field at different time points was imaged and counted. Data obtained from nine different microscopic fields in each group were showed with mean ± SEM and error
bar for A549 cells (panel A), Neuro-2A cells (panel B) and HCC-LM3 cells (panel C). Panel D illustrates the viability of the 3 cancer cells and 2 normal cells (HRPTEpiC, HUVEC)
assessed by CCK-8 assay after PQQ treatment for 48h.
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In order to evaluate the possible cytotoxicity of
PQQ to normal cells, we also examined 2 normal cell
lines (HRPTEpiC and HUVEC). Cell viability was
determined by the CCK-8 assay. Under the same
conditions, these 2 cell lines were exposed to various
concentrations of PQQ (0–300 µM) for 48h. As shown
in Figure 1D, HRPTEpiC cells maintained excellent
viability after incubation with 0–300 µM of PQQ for
48h. HUVEC cells maintained good viability
(100%-80% comparing to the control) until PQQ con-
centration reached 240-300 µM. In contrast, the viabil-
ities of A549 and Neuro-2A cells were remarkably
compromised in a dose-dependent manner, began at
30 µM and became significant from 60µM of PQQ. In
consistent with the Cell-IQ results, the reduced via-
bilities of HCC-LM3 cells were only observed at
higher concentrations (240-300 µM).
Images of each microscopic field were captured
every 20-minute for 48 hours. Cell-IQ system auto-
matically calculated total cell number of each image
by specialized software. As shown in Figure 2, treat-
ment with PQQ at 30-300 µM displayed typical poor
growth and apoptosis morphological changes such as
membrane blebbing, cellular shrinkage and chromatin
condensation in A549 and Neuro-2A cells in a dose-
and time-dependent pattern, whereas the cells of the
control group proliferated rapidly and reached suffi-
cient confluence. Apoptosis changes were also ob-
served in HCC-LM3 cells at higher doses (300-600
µM) of PQQ overtime. These data indicated that A549
and Neuro-2A cells are sensitive to PQQ treatment,
while HCC-LM3 cells response to higher concentra-
tions of PQQ. Therefore, some of the subsequent
studies were selectively focused on A549 and Neu-
ro-2A cells.
Apoptosis of cancer cell lines induced by PQQ
Apoptosis of A549, Neuro-2A and HCC-LM3
cells was measured by flow cytometry analysis after
treatment with PQQ at different concentrations for
24h. Increase in cellular surface staining with Annexin
V-FITC serves as a marker for early apoptosis while
staining with PI indicates loss of cell membrane in-
tegrity. A cell-permeable, irreversible pan-caspase
inhibitor, Z-VAD-FMK, was used to block caspase
activation of apoptotic cells. After 24h treatment with
PQQ, the percentage of apoptosis in A549 cells were
significantly increased when compared to the control
(7.45 ± 0.45% at 30 µM, 12.80 ± 0.38% at 75 µM, 17.88 ±
0.68% at 150 µM, 37.65 ± 0.68% at 300 µM, and 80.87 ±
0.95% at 600 µM, vs 3.12 ± 0.20% of control, p < 0.01
respectively) (Figures 3A and 3C). The percentage of
apoptosis in Neuro-2A cells were also significantly
increased in dose-dependent pattern (11.69 ± 0.47% at
30 µM, 12.62 ± 0.56% at 60 µM, 47.75 ± 1.16% at 120
µM, 65.13 ± 1.05% at 180 µM, 83.15 ± 0.80% at 300 µM,
and 86.92 ± 1.42% at 360 µM, vs 4.15 ± 0.44% of the
control, p < 0.01, respectively) (Figures 3D and 3F). As
shown in Figures 3B and 3C, been pretreated for 3h
with 10µM of Z-VAD-FMK prior to the indicated PQQ
treatment, the percentages of apoptosis of A549 cells
were significantly reduced (2-fold change) when
compared with the corresponding cells that were not
pretreated (3.28 ± 0.52% vs 7.45 ± 0.45% at 30 µM, and
17.38 ± 3.46% vs 37.65 ± 0.68% at 300 µM, p < 0.05,
respectively). Comparing to the corresponding un-
treated cells, the percentages of apoptosis of
Z-VAD-FMK pretreated Neuro-2A cells were simi-
larly decreased (8.33 ± 0.61% vs 11.69 ± 0.47% at 30
µM, 40.79 ± 4.03% vs 83.15 ± 0.80% at 300 µM, p < 0.05,
respectively, Figures 3E and 3F).
After PQQ treatment at different concentrations
for 24h, the percentages of apoptosis HCC-LM3 cells
were gradually increased from 4.92 ± 0.28% (30 µM) to
11.18 ± 0.21% (300 µM) and 21.05 ± 0.92% (1200 µM) in
a dose-dependent manner, compared to the negative
control (1.60 ± 0.35%, p < 0.01, respectively). (Figures
3G and 3I). When compared with the corresponding
untreated cells, the percentages of apoptosis of
Z-VAD-FMK pretreated HCC-LM3 cells were de-
creased (2.71 ± 0.55% vs 4.92 ± 0.28% at 30 µM, and
7.80 ± 1.21% vs 10.81 ± 0.53% at 600 µM, p < 0.05, re-
spectively, Figures 3H and 3I).
Increased reactive oxygen species after PQQ
treatment
The generation of intracellular ROS was quanti-
tatively analyzed by flow cytometer using H2DCFDA
dye after 4h, 12h, and 24h of PQQ treatment. In addi-
tion, separate cell cultures were treated with 500µM of
H2O2 (hydrogen peroxide) 15 minutes prior to the
assessment (positive controls). PQQ treatment re-
sulted in a time- and concentration-dependent ROS
accumulation in A549 and HCC-LM3 cells compared
with the negative controls. Significantly increased
ROS generation were observed at 4h and maintained
at a high level through 24h in most of the concentra-
tion groups (p < 0.01 compared to the corresponding
controls, Figures 4A and 4C). While A549 cells exhib-
ited a rapid and sustained generation of ROS, the ac-
cumulation of ROS in HCC-LM3 cells was a relatively
mild process. In contrast, although the production of
ROS in Neuro-2A cells was also significantly en-
hanced in most of the concentration groups (p < 0.01
compared to the corresponding controls, Figure 4B),
this effect reached peak at 12h.
PQQ treatment reduced cellular ATP release
ATP release levels of all the 3 tumor cell lines
decreased in the presence of PQQ in a dose and time
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614
dependent manner. In particular, a perfect dose de-
pendent trend was presented in A549 cells with PQQ
treatment at 24h (Figure 5A). Compared with the
control, the reduction of ATP was 27% at 75 μM, and
80% at 300 μM (19967.7±2724.2 vs 27463.7±1588.4 at 75
μM, p < 0.05; 17914.7±1982.0 vs 27463.7±1588.4 at 150
μM, p < 0.05; and 5542.4±2407.7 vs 27463.7±1588.4 at
300 μM, p < 0.01). Compared with the control, in
Neuro-2A cells with 24h PQQ treatment, the reduc-
tion of ATP was significant when PQQ dose was 120
μM and above (1359.3±237.3 vs 10559.2±1522.8 at 120
μM, p < 0.01; 836.3±184.7 vs 10559.2±1522.8 at 180 μM,
p < 0.01; 695.8±52.3 vs 10559.2±1522.8 at 300 μM, p <
0.01) (Figure 5B). Again, HCC-LM3 cells responded to
higher concentrations of PQQ at 24h (7490.2±103.5 vs
10585.4±817.2 at 300 μM, p < 0.05; PQQ vs control,
3367.8±587.1 vs 10585.4±817.2 at 600μM, p < 0.01)
(Figure 5C).
Figure 2. Morphological changes induced by PQQ. PQQ inhibited the growth of A549, Neuro-2A and HCC-LM3 cells measured by real time Cell-IQ cell culture platform.
The 3 cell lines were seeded in 24-well plates (8 × 103 cells per well) overnight and then treated with PQQ at different concentrations respectively or without PQQ (control).
Cell proliferation in the given microscopic field was continuously photographed at 20-minute intervals for 48h (magnification 100×). White arrows represent chromatin
condensation and dotted arrows indicate membrane blebbing.
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Figure 3. Apoptosis induced by PQQ. Treatment with PQQ
caused dose-dependent apoptosis in 3 tumor cell lines for 24h. Early
and late apoptosis were detected using Annexin V-FITC/PI-double
staining analyzed by flow cytometry. Involvement of the caspase
apoptosis pathway was confirmed using pan-caspase inhibitor
Z-VAD-FMK pretreatment for 3h prior to the indicated PQQ
treatment. The percentage of cells was described in each quadrant.
The values represent the mean ±SEM of at least 3 independent
experiments. PQQ induced apoptosis in A549 cells in
dose-dependent manner, and Z-VAD-FMK markedly reduced the
apoptosis (panels A, B and C). PQQ induced apoptosis in Neuro-2A
cells in dose-dependent manner, and Z-VAD-FMK noticeably
reduced the apoptosis (panels D, E and F). HCC-LM3 cells mani-
fested the similar trend as well (panels G, H and I). ** p < 0.01 as
compared with control group; *p < 0.05 as compared with PQQ
treatment group.
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Figure 4. Exposure to PQQ triggers
intracellular ROS accumulation. Three
cancer cell lines, A549 cells (panel A), Neu-
ro-2A cells (panel B) and HCC-LM3 cells
(panel C) were exposed to different doses of
PQQ at 4h, 12h and 24h. The generation of
ROS was quantitatively measured by the flow
cytometer using H2DCFDA dye. Separate cell
cultures treated with 500 µM of H2O2 15
minutes prior to the assessment were used as
positive controls. All values are showed as
means ± SEM from three-independent ex-
periments.*p < 0.05 as compared with con-
trol group; **p < 0.01 as compared with
control group.
Figure 5. Reduced cell ATP levels by
PQQ treatment. The intracellular ATP
level in A549 cells (panel A), Neuro-2A cells
(panel B) and HCC-LM3 cells (panel C) in the
presence of different doses of PQQ at 4h,
12h and 24h. Quantitative analysis recorded
the optical density (O.D.) values of cellular
ATP that were reduced when exposed to
PQQ. All values are showed as means ± SEM
from three-independent experiments.
*p < 0.05 as compared with control group;
**p < 0.01 as compared with control group.
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Increased activity of intracellular caspase-3
after PQQ treatment
The expression levels of activated caspase-3 after
24h PQQ treatment increased in dose-dependent
manner in the 3 tumor cell lines (Figure 6). Compared
to the negative control, the fluorescent intensity of
activated caspase-3 in A549 cells showed a trend of
enhancing (15.50 ± 0.89 vs 15.50 ± 0.89 at 15 µM, p >
0.05; 18.43 ± 1.19 vs 15.50 ± 0.89 at 30 µM, p < 0.05;
22.90 ± 1.54 vs 15.50 ± 0.89 at 75 µM, p < 0.01; 42.83 ±
3.15 vs 15.50 ± 0.89 at 300 µM, p < 0.01) (Figures 6A
and 6B). A similar trend was observed in Neuro-2A
cells (Figures 6C and 6D). The fluorescent intensity of
activated caspase-3 cells was significant increased in
all PQQ treatment groups compared to the control
(5.23 ± 0.08 vs 5.02 ± 0.05 at 15 µM, p < 0.05; 8.54 ± 0.59
vs 5.02 ± 0.05 at 60 µM, p < 0.01; 27.47 ± 1.12 vs 5.02 ±
0.05 at 300 µM, p < 0.01). PQQ treatment increased the
expression levels of caspase-3 in HCC-LM3 cells too.
As shown in Figures 6E and 6F, a gradually rise of the
fluorescent intensity of caspase-3 was seen in a
dose-dependent manner (9.80 ± 1.66 vs 8.86 ± 0.07 at
30 µM, p > 0.05; 12.30 ± 0.53 vs 8.86 ± 0.07 at 75 µM, p
< 0.01; 17.30 ± 0.70 vs 8.86 ± 0.07 at 600 µM, p < 0.01).
Effects of PQQ on cell cycle regulation in A549
and Neuro-2A cells
The cell cycle status of the A549 and Neuro-2A
cancer cells were measured after PQQ treatment of
15-360 µM at 24h respectively. Cell cycle distribution
analysis revealed an increasing rate of both tumor
cells in sub-G0/G1 phase, with reduced counts of cells
in S and G2M phases (Figure 7). After PQQ treatment
for 24h, the sub-G0/G1 population significantly in-
creased from 0.47 ± 0.07% (control) to 17.55 ± 1.73%
(150 µM, p < 0.05, Figures 7A and 7B) in A549 cells;
and from 2.89 ± 0.66% (control) to 42.93 ± 0.94% (360
µM, p < 0.05, Figures 7C and 7D) in Neuro-2A cells.
The percentage of A549 cells in G0/G1 phase in-
creased from 47.81 ± 1.23% (control) to 59.64 ± 0.68%
(150 µM, p < 0.05). Correspondingly, the percentage of
cells in S and G2M phase decreased from 36.66 ± 1.13%
(control) to 15.63 ± 1.07% (150 µM, p < 0.05) in A549
cells; and from 14.90 ±0.57% (control) to 7.19 ± 0.34%
(150 µM, p < 0.05) in A549 G2M phase cells. However,
no obvious rise in the percentage of G0/G1, S and
G2M phase was found in Neuro-2A cells.
Figure 6. Activity of caspase-3 in the 3 cancer cell lines treated with PQQ. Effects of PQQ on caspase-3 expression in the 3 cancer cells for 24h. Levels of the activated
caspase-3-V450 were quantitatively measured by flow cytometer and presented as mean fluorescent intensity (MFI). The overlay histograms (panels A, C and E) and histograms
(panels B, D and F) respectively indicated caspase-3 expression at protein level after treatment with various concentrations of PQQ for 24h in the 3 cancer cells. The data are
represented as means ± SEM from three-independent experiments. *p < 0.05 as compared with control group; **p < 0.01 as compared with control group.
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Figure 7. Effects of PQQ on cell cycle regulation in A549 and Neuro-2A cells. Flow cytometry analysis of cell cycle distribution of A549 and Neuro-2A cells. The 2 cell
lines were treated with PQQ at the indicated concentrations for 24h. Effects of PQQ on cell cycle were investigated using PI (Propidium Iodide) staining subjected to flow
cytometry (panels A and C). The peaks from left to right represent sub-G0/G1 phase, G0/G1 phase, S phase and G2M phase respectively. Represent cells in different phases from
three independent experiments (panels B and D).
Decreased mitochondrial membrane potential
after PQQ treatment
A549 and Neuro-2A cancer cells stained with
JC-1 emitted mitochondrial red fluorescence with a
little green fluorescence, indicative of normal polari-
zation state. JC-1 is a cationic dye that manifests mi-
tochondrial polarization by shifting its fluorescence
emission from green (~525 nm) to red (~590 nm).
When cell apoptosis occurs, the mitochondrial mem-
brane potential depolarization is produced. JC-1
probe was released from mitochondria and red fluo-
rescence intensity decreased, while the monomer of
green fluorescence mainly existed in the cytoplasm.
The JC-1 aggregates were dispersed to the monomeric
form (green fluorescence) in both the cancer cells
subjected to PQQ for 24 h in a concentra-
tion-dependent manner. As shown in figures 8A and
8C, red fluorescent percentages of JC-1 of A549 cells
significantly decreased from 79.5% (15 µM PQQ) to
18.7% (300 µM PQQ), compared with the negative
control group (92.7%), p < 0.01 respectively; the ratio
of JC-1 red/green MFI was indicated to a significant
reduction starting from the dose of 15µM PQQ (0.47 ±
0.01), compared to the negative control (0.70 ± 0.03), p
< 0.01. Similarly, in Neuro-2A cells, the percentage of
JC-1 aggregates significantly decreased from 94.2% of
the control group to 91.2% of 15µM PQQ and 10.3% of
360µM PQQ group. The ratios of JC-1 red/green MFI
were decreased with the increasing of PQQ concen-
trations from 0.80 ±0.02 (negative control) to 0.66 ±
0.05 (15 µM, p < 0.05), and 0.11 ± 0.02 (360 µM, p <
0.01) (Figures 8B and 8D).
Decreased expression of Bcl-2 by PQQ
treatment
In order to delineate the possible mechanisms by
which PQQ induced apoptosis we quantitatively
examined the cytoplasmic levels of the Bcl-2 protein
by flow cytometer. As shown in Figure 9, compared to
the controls, the Bcl-2 levels in both tumor cells were
decreased after treatment with different doses of PQQ
for 24h. In A549 cells, except for the 15 μM PQQ group
that the MFI of Bcl-2 protein was higher than that of
the control group (19.23 ± 0.40 vs 17.77 ± 0.72, p < 0.05),
with the PQQ concentrations increased from 30 μM to
300 μM, the mean fluorescent intensity (MFI) of Bcl-2
protein decreased (11.57 ± 0.86 vs 17.77 ± 0.72 at 30
μM, p < 0.01; 10.01 ± 0.45 vs 17.77 ± 0.72 at 300 μM, p <
0.01) (Figures 9A and 9B). Similar pattern was found
in neuro-2A cells, the expression levels of Bcl-2 sig-
nificantly decreased with the increasing of PQQ con-
centrations, beginning at 15 μM (Figures 9C and 9D).
Effects of PQQ on ERK2, MEK2, pERK1/2 and
p38 MAPK pathway
As shown in Figure 10, with the increasing of
PQQ concentrations, increased expression levels of
ERK2 (13.00 ± 1.45 vs 9.16 ± 0.57 at 30 μM, p < 0.01;
35.30 ± 1.86 vs 9.16 ± 0.57 at 300 μM, p < 0.01), MEK2
(36.70 ± 1.08 vs 18.30 ± 0.55 at 15 μM, p < 0.01; 47.00 ±
1.74 vs 18.30 ± 0.55 at 300 μM, p < 0.01), pERK1/2
(28.57 ± 0.87 vs 16.23 ± 0.35 at 15 μM, p < 0.01; 39.11 ±
0.56 vs 16.23 ± 0.35 at 300 μM, p < 0.01) and p38 MAPK
(16.53 ± 1.07 vs 9.60 ± 0.24 at 15 μM, p < 0.01; 23.93 ±
Journal of Cancer 2014, Vol. 5
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619
0.59 vs 9.60 ± 0.24 at 300 μM, p < 0.01) in A549 cells
were detected after 24h PQQ treatment, compared
with the control group. The MFIs of ERK2 and MEK2
in neuro-2A cells were significantly decreased and the
MFI of pERK1/2 were significantly increased, when
compared to the control group.
Figure 8. Decreased mitochondrial membrane potential after PQQ treatment. PQQ decreased mitochondrial membrane potential in A549 and Neuro-2A cells.
Density diagram of flow cytometry analysis showed the distribution of JC-1 aggregates (red) and JC-1 monomer (green) in the mitochondrial membrane with A549 and Neuro-2A
cell lines, respectively (panels A and B). A549 and Neuro-2A cells without PQQ treatment served as controls and CCCP used as positive-control. Both the cell lines were treated
with different concentrations of PQQ (from 15 µM to 360 µM) for 24h and then stained with JC-1 before flow cytometry analysis. Quantitative analysis of the shift of mito-
chondrial red fluorescence to green fluorescence among groups was calculated as red/green fluorescence intensity value (panels C and D). All values are showed as means ± SEM
from three-independent experiments. *p < 0.05 as compared with control group; **p < 0.01 as compared with control group.
Figure 9. Decreased expression of Bcl-2 by PQQ treatment. Effects of PQQ on Bcl-2 expression in A549 and Neuro-2A cells for 24h. Levels of the cytoplasmic Bcl-2
protein were quantitatively measured by flow cytometer and presented as mean fluorescent intensity (MFI). The overlay histograms (panels A and C) respectively indicated Bcl-2
expression at protein level of the 2 cell lines after treatment with various concentrations of PQQ for 24h. Bar diagram (panels B and D) represent MFI values from
three-independent experiments as means ± SEM. *p < 0.05 as compared with control group; **p < 0.01 as compared with control group.
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620
Figure 10. Effects of PQQ on ERK2, MEK2, pERK1/2 and p38 MAPK pathway. Effects of PQQ on biomarkers of apoptotic signal pathway in A549 and Neuro-2A cells
at 24h. Phosphorylated ERK1/2 and p38 MAPK, ERK2 and MEK2 levels were represented as mean fluorescent intensity (MFI) measured by flow cytometer (panels A to D, and
panels I to K). The overlay graphs respectively indicated the levels of proteins related to the signal transduction pathways in the 2 cell lines after treatment with various
concentrations of PQQ for 24h (panels E to H, and panels L to N). The bar diagram represented the corresponding mean fluorescent intensity of protein level. All values are
showed as means ± SEM from three-independent experiments. *p < 0.05 as compared with control group; **p < 0.01 as compared with control group.
Discussion
PQQ, as an antioxidant nutrient, has the effects
of radioprotection and neuroprotection. Previous
study focused on the scavenging of the production of
intracellular ROS [13], which was due to compara-
tively higher reactive electron density and the struc-
ture of indole and pyrrole derivatives. Thus
PQQ exhibits strong antioxidant feature [5]. Recently,
study showed that higher concentrations of PQQ
(50-100 μM) could result in 2-5 fold increase in apop-
tosis-inducing of U937 human promonocytic lym-
phoma cells through the depletion of cellular gluta-
thione and the increasing of intracellular ROS [9].
Other studies also demonstrated that PQQ could reg-
ulate cell function of different cells and induce cell
apoptosis, directly or indirectly [5, 10, 11]. However,
there is no study that has investigated the effect of
PQQ on directly inducing solid tumor cell apoptosis
and the associated mechanisms.
In this study, the apoptosis of three tumor cell
lines (Neuro-2A, A549 and HCC-LM3) induced by
PQQ were measured and the potential mechanisms
were investigated. The result showed that the prolif-
erations of three cell lines were suppressed by PQQ in
a dose- and time-dependent manner. PQQ exhibited
Journal of Cancer 2014, Vol. 5
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621
potent anti-cancer activity while imposed comparably
minimal cytotoxicity to 2 normal cell lines (HRPTEpiC
and HUVEC), a finding concordant with previous
reports that PQQ would stimulate the normal cell
lines (A431 and NIH3T3) proliferation [14, 15]. Typi-
cal apoptotic cellular morphological changes such as
poor growth, membrane blebbing, cellular shrinkage
and chromatin condensation were observed with re-
al-time cell monitoring system. Moreover, flow cy-
tometry and Annexin V-FITC/PI staining showed
that the percentages of apoptosis caused by PQQ in
A549 and Neuro-2A cells were increased in a
dose-dependent manner. PQQ-induced apoptosis of
the 3 tumor cell lines were all alleviated by the treat-
ment of Z-VAD-FMK, a pan-caspase inhibitor (Figure
3), which confirmed the participation of caspase in
PQQ-induced apoptosis. The fact that Z-VAD-FMK
could not completely block PQQ-induced apoptosis,
suggesting other pathways involvement in the
PQQ-induced cell apoptotic process. However, the
caspase-dependent pathway might play a leading
role, considering the significantly 2-fold decrease of
apoptosis in A549 cells after Z-VAD-FMK treatment.
PQQ triggered apoptotic process at lower con-
centrations (30-75 μM), and the apoptotic effects be-
came significant at higher concentrations (120-300
μM). This finding is concordant with a previous study
that PQQ induced programmed Jurkat cell death by
increasing the caspase-3 activity [5]. The proliferation
inhibition and apoptosis-inducing effects of PQQ
were less remarkable in HCC-LM3 cells at lower
concentrations than that in A549 and Neuro-2A cells.
It suggested that different tumor cells have different
sensitivities to PQQ. Nonetheless, the results indi-
cated that the apoptosis of HCC-LM3 cell induced by
PQQ was also probably via the caspase-dependent
pathway. Taken together, the growth inhibitory ef-
fects of PQQ on solid tumor cells of A549, Neuro-2A
and HCC-LM3 were demonstrated to be largely re-
sulted from apoptosis.
The current study measured intracellular ROS
production, ATP release, mitochondrial membrane
potential, cell cycle distribution and related protein
signaling pathways to explore the anti-tumor and
apoptosis-inducing mechanisms of PQQ. ROS plays a
vital role in apoptosis induction under both physio-
logical and pathological conditions. The major source
of intracellular ROS is mitochondria [16]. Numerous
evidences suggest that impaired mitochondria could
stimulate ROS generation, continuous accumulation
of ROS triggers apoptosis via the intrinsic pathway by
opening of mitochondrial permeability transition pore
(PTP) and collapsing of mitochondrial membrane
potential, ultimately caused caspase-3 activation and
cell death [17, 18]. Our data indicated that PQQ
treatment resulted in an enhanced generation of ROS
in time- and dose-dependent manner in all 3 tumor
cells. In Neuro-2A cells the accumulation of ROS
preceded the other 2 cell lines. It might suggest that
Neuro-2A cells are more sensitive to PQQ treatment
on ROS production. Collectively, these data suggested
that the PQQ-induced apoptosis is associated with
ROS generation, which is in agreement with previous
report [9].
Cellular ATP content is associated with the
number of metabolically viable cells. Our data
showed that PQQ treatment decreased the cellular
content of ATP in a time- and dose-dependent manner
in all the 3 tumor cells. Decreased ATP contents of
HCC-LM3 cells were only apparent when exposed to
higher concentrations of PQQ, suggesting that the
liver cancer cells might have a higher tolerance to
PQQ. Our results also indicated that PQQ could dis-
sipate mitochondrial membrane potential of A549 and
Neuro-2A cells in a concentration-dependent manner,
which might be related with decreased cellular ATP
content and enhanced generation of ROS after PQQ
treatment. Previous study [19] reported that in apop-
totic cells, the loss of mitochondrial ATP synthesis
and the increase of inter-membrane creatine phos-
phate concentrations might be consequences of the
loss of mitochondrial membrane potential caused by
the translocation of proapoptotic BH3 proteins to the
mitochondria. The mitochondria play a crucial role in
cell intrinsic apoptosis pathway. The mitochondrial
membrane permeability transition is a critical step in
the induction of intrinsic apoptosis and often repre-
sents the earliest apoptotic signal [20]. During apop-
tosis, mitochondria suffer specific damages that result
in loss of function, and release of cytochrome c that
can potentially halt the electron transfer, leading to
failure in maintaining the mitochondrial membrane
potential and ATP synthesis [21].
The sub-G0/G1 phase, appearing left to the
G0/G1 peak in DNA distribution, represents the cell
population undergoing apoptosis. The current results
showed that with the increasing concentration of
PQQ, significant apoptotic peak appeared in A549
and Neuro-2A cells. The cell cycle data suggested that
the number of A549 and Neuro-2A cells entering the
cell cycle decreased with the increasing of PQQ con-
centration (Figure 7A-7D). This pointed to G0/G1
arrest as one of the mechanisms for PQQ inhibition of
A549 cell growth. Studies reported that Usnic Acid
and Tanshinone treated A549 cells also resulted in
G0/G1 arrest, which possibly could be associated
with decreased expression of cyclin-dependent kinase
(CDK)4, CDK6, and Cyclin D1, and inhibition of Cy-
clin A and Cyclin B [22, 23]. In Neuro-2A cells, no
significant cell cycle arrest was observed. However,
Journal of Cancer 2014, Vol. 5
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622
combined with the results of ROS and ATP experi-
ments, of which the production of ROS reached the
peak at 12h and fall at 24h, and the ATP production
significantly declined from 12h to 24h, implying that
the occurrence of ROS accumulation and ATP de-
clining might happen prior to the cell cycle changes.
This supports a major role of the ROS-mediated mi-
tochondrial pathway in the PQQ-induced Neuro-2A
cell apoptosis, concordant with previous studies [24,
25].
Bcl-2 is an anti-apoptotic protein known as
a mitochondrial membrane protein which can block
programmed cell death and tumorigenesis upon ac-
tivation [26]. The Bcl-2 protein family controls the
integrity of the outer-mitochondrial membrane. Dur-
ing apoptosis, the change of anti- and pro-apoptotic
proteins of Bcl-2 family causes the mitochondrial
membrane potential across the membrane to col-
lapse, leading to the release of cytochrome c into the
cytoplasm, the formation of the apoptosome and an
increase in effector caspase activities, thereby result-
ing in apoptosis [27-29]. Caspase activation is gener-
ally considered a key hallmark of apoptosis in these
pathways [30]. Our results demonstrated that the
expression levels of activated caspase-3 were in-
creased in a dose-dependent manner in 3 cell lines,
which implied the participation of caspase-3 in the
PQQ-induced apoptosis. Given the fact that caspases
play a central role in the apoptotic cascade, these re-
sults strongly supported the hypothesis that PQQ
treatment causes the collapses of the mitochondrial
membrane potential and the reductions of Bcl-2 pro-
tein, and initiates the corresponding elevation of ac-
tivated caspase-3. This finding was backup by recent
studies reporting that grape seed proanthocyanidins
induces apoptosis of A549 by loss of mitochondrial
membrane potential, G1 phase arresting,
down-regulation of the anti-apoptotic proteins Bcl-2
and activation of caspases 9 and 3 [31]. The activation
of A549 cellular apoptosis signal is conducted via
Bcl-2 suppression and p53 and Bax activation [32].
The butein-induced Neuro-2A cells apoptosis is
characterized by increased intracellular reactive oxy-
gen species (ROS) levels and reduced Bcl-2/Bax ratio
[25]. Taken together, we propose that the apopto-
sis-inducing effect of PQQ on A549 and Neuro-2A
cells is partly through the loss of mitochondrial
membrane potential and reduction of Bcl-2 protein.
Aberrant activation of the signaling pathway
was frequently observed in human tumor cells, how-
ever, little is known about the effect of PQQ on tumor
cell signaling pathway. There are 4 mitogen-activated
protein kinase (MAPK) families categorized by se-
quence homology and functions: ERK1/2 (extracel-
lular signal-regulated kinase), p38, JNK (c-Jun
N-terminal kinase), and ERK5 [33]. Activation of
p38MAPK is predominantly implicated in inducing
cancer cell apoptosis and displaying pro-apoptosis by
several chemotherapeutic drugs in experimental set-
tings [34-36]. PQQ seemed to modulate cell apoptosis
and mitochondrial assembly signaling pathways [37,
38]. In the current study, the expressions of
p38MAPK, pERK1/2, ERK2 and MEK2 in A549 cells
were markedly up-regulated by PQQ at 15-300 µM.
The results were also supported by the study report-
ing that activation of JNK and p38MAPK caused
phosphorylation and translocation of Bax into mito-
chondria to cause apoptosis via intrinsic pathway
[39]. ERK is well known as one of the MAPKs and for
its prominent role in controlling proliferation, differ-
entiation and cell survival [40]. The ERK1/2 are acti-
vated by external and internal stimuli in numerous
cell types and played a central role in many signal
transduction pathways, which can directly phos-
phorylate many target proteins including transcrip-
tion factors (c-Jun, c-Myc, P53), leading to the induc-
tion of many cell cycle proteins (p21, Cyclin D1, cdk1)
and then the activation of transcription factor and
apoptotic factors (caspase 9, bad, Bim), and finally
cause cell death [41, 42].
Our data demonstrated that the expression levels
of intracellular ERK2 were notably increased 3-fold
compared to that of the control group in A549 cells,
which would suggest that ERK2, probably activated
by PQQ treatment, induces p53 up-expression and
Bcl-2 suppression [31, 32], Cyclin D1 and CDK4,6 in-
hibition [22], while blocking cell cycle progression,
eventually promotes the caspase cascade reaction,
resulting in cell apoptosis. This inference is in con-
cordance with a previous report [43]. However, using
probes to quantify the levels of these MAPKs in
Neuro-2A cells exhibited that PQQ treatment caused
significant decrease in levels of ERK2 and MEK2, but
increase of pERK1/2 levels, and have no effect on
p38MAPK levels (data not shown), suggesting that
this effect may be cell type specific. Previous study
reported that the anti-epileptic drug valproic acid
increased the degree of ERK1/2 phosphorylation in
Neuro-2A and BT4Cn cells as well [44]. These results
are in agreement with other recent works [45, 46].
These observations permit us to propose that PQQ
concentration greater than 15-30 μM may exert its
apoptosis-inducing effect on A549 and Neuro-2A cells
via the aforementioned mechanisms. However, other
molecular mechanism for the pro-apoptosis effects of
PQQ remains to be determined.
The authors acknowledge that these findings
need to be further investigated in in vivo settings, and
in the current study no positive controls were in-
cluded which might jeopardize the power of the re-
Journal of Cancer 2014, Vol. 5
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623
sults. It might be argued that currently there is no
compound with similar molecular structures in clini-
cal application that could be used as a reference. Thus,
the current study focused primarily on the anti-tumor
effects of PQQ and highlighted the associated poten-
tial mechanisms. In summary, despite the limitations,
this work does for the first time report the effects of
PQQ on solid tumor cells of A549, Neuro-2A and
HCC-LM3 in vitro, that PQQ could outstandingly
suppress the A549 and Neuro-2A cell proliferation
through the mitochondrial-dependent apoptosis
pathway while have comparably minimal toxicity
towards normal cells. The underlying mechanisms
might include, but not limited to, cell cycle arrest at
G0/G1 phase, accumulation of intracellular ROS, de-
cline in ATP levels, disruption of mitochondrial
membrane potential in conjunction with
down-regulation of Bcl-2 protein expression and en-
hancement of the activated caspase-3 expression, and
the enrichment of signal pathway proteins of
p38MAPK, pERK1/2, ERK2 and MEK2 in A549 cells;
reduction of ERK2 and MEK2 in Neuro-2A cells. The
significance of our findings is the possibility of de-
veloping new venues to target cancers. However, as
the anti-tumor activity of PQQ is cell type specific,
why some tumor cells are sensitive to it and others are
not needs to be further investigated. Future studies on
the mechanistics and the potential role of PQQ in
cancer prevention and treatment are warranted.
Acknowledgments
We thank Mr Qunye Tang for providing the ATP
reagent.
This study is supported by National Science
Foundation of China (Grant No. 30972737 and
81170473), Young Scholars of Fudan University
(Grant No. 431), and Pujiang talent research grant
(Grant No. 10PJ1403600) from the Shanghai Commit-
tee of Science and Technology.
Competing Interests
The authors have declared that no competing
interest exists.
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