Access to this full-text is provided by Taylor & Francis.
Content available from OncoTargets and Therapy
This content is subject to copyright. Terms and conditions apply.
© 2018 Lu et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php
and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you
hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission
for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
OncoTargets and Therapy 2018:11 7213–7227
OncoTargets and erapy Dovepress
submit your manuscript | www.dovepress.com
Dovepress 7213
ORIGINAL RESEARCH
open access to scientific and medical research
Open Access Full Text Article
http://dx.doi.org/10.2147/OTT.S184337
Changyu Lu
Xiaolei Chen
Qun Wang
Xinghua Xu
Bainan Xu
Department of Neurosurgery,
Chinese PLA General Hospital,
Beijing 100853, China
Background and objective: The present study was designed to explore the roles of
mitochondrial fission and MAPK–ERK–YAP signaling pathways and to determine their
mutual relationship in TNFα-mediated glioblastoma mitochondrial apoptosis.
Materials and methods: Cellular viability was measured via TUNEL staining, MTT assays,
and Western blot. Immunofluorescence was performed to observe mitochondrial fission. YAP
overexpression assays were conducted to observe the regulatory mechanisms of MAPK–ERK–
YAP signaling pathways in mitochondrial fission and glioblastoma mitochondrial apoptosis.
Results: The results in our present study indicated that TNFα treatment dose dependently
increased the apoptotic rate of glioblastoma cells. Functional studies confirmed that TNFα-
induced glioblastoma apoptosis was attributable to increased mitochondrial fission. Excessive
mitochondrial fission promoted mitochondrial dysfunction, as evidenced by decreased mito-
chondrial potential, repressed ATP metabolism, elevated ROS synthesis, and downregulated
antioxidant factors. In addition, the fragmented mitochondria liberated cyt-c into the cytoplasm/
nucleus where it activated a caspase-9-involved mitochondrial apoptosis pathway. Furthermore,
our data identified MAPK–ERK–YAP signaling pathways as the primary molecular mechanisms
by which TNFα modulated mitochondrial fission and glioblastoma apoptosis. Reactivation of
MAPK–ERK–YAP signaling pathways via overexpression of YAP neutralized the cytotoxicity
of TNFα, attenuated mitochondrial fission, and favored glioblastoma cell survival.
Conclusion: Overall, our data highlight that TNFα-mediated glioblastoma apoptosis stems
from increased mitochondrial fission and inactive MAPK–ERK–YAP signaling pathways, which
provide potential targets for new therapies against glioblastoma.
Keywords: glioblastoma, apoptosis, mitochondrion, TNFα, mitochondrial fission, MAPK-
ERK-YAP signaling pathways
Introduction
Although glioblastoma multiforme (GBM) is a rare tumor whose incidence is less than
3.19/100,000 in the population globally, its poor prognosis with a median survival
of 15 months and inevitable recurrence after a median survival time of 32–36 weeks
make it a heavy burden on the health care system. Unfortunately, little is known about
the etiology of GBM, although several risk factors have been proposed, such as age,
exposure to radiation, and family history. Notably, excessive hyperplasia of glial cells
is the primary pathogenesis of GBM.1 Accordingly, several approaches have been
attempted to induce the death of glial cells, especially TNFα-based therapy.
Correspondence: Bainan Xu
Department of Neurosurgery,
Chinese PLA General Hospital,
28 Fuxing Road, Beijing 100853, China
Tel +86 138 0127 1711
Email dr_lcy@126.com
Journal name: OncoTargets and Therapy
Article Designation: Original Research
Year: 2018
Volume: 11
Running head verso: Lu et al
Running head recto: TNFα promotes glioblastoma A172 cell
DOI: 184337
RETRACTED ARTICLE: TNFα promotes
glioblastoma A172 cell mitochondrial apoptosis via
augmenting mitochondrial fission and repression of
MAPK–ERK–YAP signaling pathways
RETRACTED
This article was published in the following Dove Press journal:
OncoTargets and Therapy
OncoTargets and Therapy 2018:11
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7214
Lu et al
A gene delivery strategy to induce TNFα overexpres-
sion has been attempted to increase the apoptotic index of
glioblastoma cells.2 The effectiveness of the TNFα-based
therapy is later validated by several clinical studies.3 Ample
in vivo and in vitro evidence potentially implies that TNFα
considerably augments the apoptosis of glioblastoma cells.4
This information indicates that TNFα-based therapy is a
promising tool for the treatment of glioblastoma. However,
the molecular mechanisms of TNFα involved in glioblastoma
cell death have not been fully described.
Mitochondria control an array of subcellular functions,
such as energy metabolism, ROS production, cell prolif-
eration, calcium balance, and cell death.5,6 Previous studies
have provided molecular insight into the mitochondrial
etiology in GBM and have identified mitochondria as a
potentially therapeutic target to modulate the growth of
gliomas.7 In addition, TNFα-based therapy has been linked
to mitochondrial dysfunction in GBM. For example, TNFα
promotes mitochondrial oxidative stress via the JNK–
NF–κB pathways.8 Some researchers have demonstrated
that TNFα induces mitochondrial apoptosis via increas-
ing tBid stability.9 In addition, other studies suggest that
Bnip3-related mitochondrial necrotic death is activated by
TNFα.10 This information indicates that TNFα potentially
targets mitochondria in glioblastoma cells. Recently, mito-
chondrial fission has been thought to be the early feature
of mitochondrial abnormalities and to promote the death
of several kinds of tumors, such as breast cancer,11 ovarian
cancer,12 pancreatic cancer,13 and bladder cancer.14 TNFα has
been found to be associated with Drp1 activation during the
inflammation-mediated cardiomyocyte injury.15 However, no
studies have investigated the role of mitochondrial fission in
TNFα-treated glioblastoma cells. In the present study, we ask
whether mitochondrial fission is required for TNFα-mediated
mitochondrial apoptosis in glioblastoma cells.
The MAPK–ERK signaling pathway has been found to
be the upstream inhibitor of mitochondrial fission. In liver
cancer, defective ERK signaling upregulates FAK expression
and the latter promotes mitochondrial fission.16 Moreover,
in neuroblastoma N2a cells, increased ERK signaling inhib-
its mitochondrial fission and sustains cellular viability.17
Furthermore, in-depth studies have indicated that ERK
modulates mitochondrial fission via YAP. Increased YAP
suppresses mitochondrial fission in human rectal cancer,18
cerebral ischemia-reperfusion injury,19 and dendritic cells.20
These findings uncover the critical role played by ERK–YAP
signaling in inhibiting mitochondrial fission. Considering
that ERK is also the classical antiapoptotic signal for cancer,21
we ask whether TNFα handles mitochondrial fission via
repressing the MAPK–ERK–YAP signaling pathways. Alto-
gether, the aim of our study was to investigate the therapeutic
effects of TNFα on glioblastoma cells and determine its
influence on mitochondrial fission and the MAPK–ERK–
YAP signaling pathways.
Materials and methods
Cell culture and treatment
Human glioblastoma cell line A172 (ATCC® CRL 1620™)
was purchased from American Type Culture Collection.
These cells were cultured with l-DMEM supplemented
with 10% FBS (Biowest, Mexico City, Mexico, USA) and
1% penicillin/streptomycin in a humidified atmosphere with
5% CO2 at 37°C. Different doses of TNFα were added to the
medium of A172 cells for 12 hours to induce cell damage
(0–20 ng/mL). This concentration of TNFα was chosen
according to a previous study.22 Cells were exposed to
10 mM mitochondrial division inhibitor-1 (Mdivi-1; Sigma-
Aldrich Co., St Louis, MO, USA; EMD Millipore, Billerica,
MA, USA) to inhibit the activity of mitochondrial fission.
In contrast, to activate mitochondrial fission, 5 µm FCCP
(Selleck Chemicals, Houston, TX, USA) was pretreated for
40 minutes at 37°C in a 5% CO2 atmosphere.23
MTT assay, TUNEL staining, and LDH
release assay
The cell viability was determined by MTT assays (Sigma-
Aldrich Co.). Briefly, cells were seeded onto 96-well plates,
and then 20 µL of MTT at a concentration of 5 mg/mL was
added to the medium. The plates were placed for 4 hours in
the dark at 37°C and 5% CO2. After that, the medium was
removed and 100 µL of dimethyl sulfoxide (DMSO) was
added into the medium for 15 minutes in the dark at 37°C and
5% CO2. Then, the samples were observed at a wavelength of
570 nm. The relative cell viability was recorded as a ratio to
that of the control group. Apoptotic cells were quantified using
a one-step TUNEL kit (Beyotime Institute of Biotechnology,
Haimen, China) according to the manufacturer’s instructions.24
Cells were seeded onto the 12-well plates and incubated with
fluorescein–dUTP (Beyotime Institute of Biotechnology) for
30 minutes at 37°C in a 5% CO2 atmosphere. After being
labeled with DAPI, the cells were observed using a laser confo-
cal microscope (TcS SP5; Leica Microsystems, Inc., Buffalo
Grove, IL, USA). LDH was released into the medium when
cellular membranes ruptured. To evaluate the levels of LDH in
the medium, an LDH Release Detection kit (Beyotime Institute
of Biotechnology) was used according to manufacturer’s proto-
col. Cells treated with PBS were used as the control group for
MTT assay, LDH release assay, and TUNEL staining.
RETRACTED
OncoTargets and Therapy 2018:11 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7215
TNFα promotes glioblastoma A172 cell
Measurement of mitochondrial
membrane potential and mitochondrial
permeability transition pore (mPTP)
opening rate
Mitochondrial potential was evaluated using 5,5′,6,6′-
tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine
chloride (JC-1) staining. Cells were seeded onto 12-well
plates. After washing with PBS three times, the cells were
treated with the JC-1 probe for 30 minutes in the dark at
37°C and 5% CO2. Then, cells were washed with PBS three
times to remove the free JC-1. After replaced with fresh
DMEM, the cells were observed using a laser confocal
microscope (TcS SP5). At least 30 cells were randomly
chosen.25 To measure the mPTP opening, cells were loaded
with PBS containing 25 nM tetramethylrhodamine, methyl
ester (TMRM, T668; Thermo Fisher Scientific, Waltham,
MA, USA). After 30 minutes, cells were washed with
PBS again to remove the free TMRM. Then, samples were
observed at a wavelength of 480 nm using a microplate
reader (Epoch 2; BioTek Instruments, Inc., Tokyo, Japan).
Cells treated with PBS were used as the control group for
MTT assay and TUNEL staining.
Western blots
Total proteins were extracted using RIPA Lysis Buffer
(Cat. No: P0013E; Beyotime, Beijing, China). After that,
proteins were rapidly centrifuged (20,000 rpm) for 10 min
at 4°C to pellet cell debris. Supernatant was collected and
quantified using an Enhanced BCA Protein Assay Kit
(Beyotime, Cat. No: P0009). Then, proteins (45–60 µg)
were loaded in a 10%–15% SDS-PAGE gel and transferred
to PVDF membranes (Bio-Rad, Hercules, CA, USA). Sub-
sequently, membranes were blocked with 5% skim milk
for 45 minutes at room temperature. After washing with
tris buffered saline with tween 20 (TBST) three times at
room temperature, the membranes were incubated with
the primary antibodies at 4°C overnight.26 After washing,
horseradish peroxidase-conjugated secondary antibodies
were incubated with membranes for 50 minutes at room
temperature. Then, the bands were observed using an ECL
Prime Western Blotting Detection Reagent (GE Healthcare,
Buckinghamshire, UK). The primary antibodies used in the
present study are described as follows: p-ERK (1:1,000,
#ab176660; Abcam, Cambridge, MA, USA), t-ERK
(1:1,000, #ab54230; Abcam), Yap (1:1,000; #14074; Cell
Signaling Technology, Danvers, MA, USA), complex III
subunit core (CIII-core2, 1:1,000, #459220; Invitrogen,
Merck KGaA, Darmstadt, Germany), complex II (CII-30,
1:1,000, #ab110410; Abcam), complex IV subunit II
(CIV-II, 1:1,000, #ab110268; Abcam), Drp1 (1:1,000,
#ab56788; Abcam), Fis1 (1:1,000, #ab71498; Abcam),
Opa1 (1:1,000, #ab42364; Abcam), Mfn2 (1:1,000,
#ab57602; Abcam), Mff (1:1,000, #86668; Cell Signaling
Technology), Tom20 (1:1,000, #ab186735; Abcam), Bcl-2
(1:1,000, #3498; Cell Signaling Technology), Bax (1:1,000,
#2772; Cell Signaling Technology), Bcl-2 (1:1,000, #3498;
Cell Signaling Technology), Bad (1:1,000, #ab90435;
Abcam), and x-IAP (1:1,000, #ab28151; Abcam).
Immunouorescence
Cells were seeded onto poly-d-lysine-coated coverslips.
Then, methanol-free 4% paraformaldehyde was used
to fix cells for 15 minutes at room temperature. Subse-
quently, samples were blocked with 5% goat serum at
room temperature for 45 minutes. After washing with
TBST, samples were incubated with primary antibody at
4°C overnight. The primary antibodies used in the present
study were as follows: p-ERK (1:1,000, #ab176660), Yap
(1:1,000, #14074), Tom20 (1:1,000, #ab186735), and cyt-c
(1:1,000, #ab90529; Abcam). Subsequently, samples were
rinsed three times with TBST for 15 minutes and followed
by incubation with secondary antibody for 45 minutes at
room temperature; after rinsing three times for 5 minutes
using TBST, the samples were labeled with DAPI to tag
the nuclei. Cells were observed using a laser confocal
microscope (TcS SP5).27
Transfection
The pDC315–YAP vector was designed and purchased
from Vigene Biosciences, Inc. (Rockville, MD, USA).
Then, the plasmid was transfected in 293 T cells using
Lipofectamine 2000®. After 48 hours, the supernatant was
collected and amplified to obtain adenovirus-YAP (Ad-YAP).
Subsequently, A172 cells were infected with Ad-YAP using
Lipofectamine 2000® for 6 hours at 37°C and 5% CO2.
Western blot was performed to observe the overexpression
efficiency.28
ROS and antioxidant factors
quantication
ROS generation was quantified using flow cytometry. Cells
were seeded onto the 12-well plates. After washing with
PBS, dihydroethidium (DHE) staining was added into the
medium and the cells were incubated with the DHE probe
for 30 minutes in the dark at 37°C and 5% CO2. Then, PBS
was used to wash cells to remove the free DHE probe. Sub-
sequently, 0.25% trypsin was applied to collect the cell. Flow
RETRACTED
OncoTargets and Therapy 2018:11
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7216
Lu et al
cytometry analysis was performed using the BD FACSCanto
II cytometer (BD, San Diego, CA, USA). Analysis of the data
was performed using FACSDiva software (BD). Besides, the
ROS production was also observed using a laser confocal
microscope (TcS SP5). The concentration of cellular anti-
oxidant factors such as GSH (Glutathione Reductase Assay
Kit, Cat. No: S0055; Beyotime), SOD (Total Superoxide
Dismutase Assay Kit, Cat. No: S0101; Beyotime), and GPX
(Cellular Glutathione Peroxidase Assay Kit, Cat. No: S0056;
Beyotime) was measured via ELISA according to the manu-
facture’s guidelines.29 Cells treated with PBS were used as
the control group for MTT assay and TUNEL staining.
Caspase-3/9 activities and Trypan Blue
staining
Caspase-3 and caspase-9 activities were measured using the
Caspase-3 Activity Assay Kit (Cat. No: C1115; Beyotime)
and Caspase-9 Activity Assay Kit (Cat. No: C1158; Beyo-
time) following the manufacturer’s instructions.30 Briefly,
cells were seeded onto 96-well plates. Then, 100 µL of
caspase-3 and caspase-9 reagents were added to each sample.
After incubation for 30 minutes in the dark at 37°C and 5%
CO2, the samples were measured at a wavelength of 570 nm
using the microplate reader (Epoch 2). The relative caspase
activity was recorded as the ratio to that of the control group.
Trypan Blue staining was conducted using 0.4% Trypan Blue
probe, which was treated with cells for 2 minutes. Then,
the number of Trypan Blue-positive cells was calculated by
counting at least three random separate fields.
Cellular ATP level detection
Cellular ATP levels were measured using the Enhanced
ATP Assay Kit (Cat. No: S0027; Beyotime) following the
supplier’s specifications.31 Briefly, cells were seeded onto
96-well plates at a density of 1×104/well. Subsequently,
100 µL of staining solution (Enhanced ATP Assay Kit, Cat.
No: S0027) was added to each well and incubated with the
cells for 4 hours in the dark at 37°C and 5% CO2. The rela-
tive ATP production was recorded using a microplate reader
(Epoch 2) at a wavelength of 570 nm.
Statistical data analyses
The results are presented as the mean±standard error (SE)
from at least three independent experiments using SPSS
16.0 software (SPSS Inc., Chicago, IL, USA). One-way
ANOVAs were carried out for comparisons between control
and treated groups. Pairwise comparisons were made by post
hoc Tukey’s test. Differences were considered as significant
at P,0.05.
Results
TNFα dose-dependently promotes
glioblastoma cell apoptosis in vitro
In the present study, glioblastoma cells were incubated
with different doses of TNFα for 12 hours. Then, the cel-
lular viability was measured via MTT assay. As shown in
Figure 1A, with increasing concentrations of TNFα, the
viability of glioblastoma cells decreased progressively.
Reduction in cellular viability may result from cell death.
To analyze the cellular death rate, the LDH release assay
was performed. Compared to the control group, TNFα dose
dependently elevated the content of LDH in the medium
(Figure 1B), indicating that TNFα promoted glioblastoma
cell death. This finding was further supported via Trypan Blue
and TUNEL staining, which exhibited an increased number
of Trypan-positive (Figure 1C and D) and TUNEL-positive
cells (Figure 1E and F) in the presence of TNFα stress.
At the molecular levels, cell death is primarily executed via
caspase-3 activation, which cleaves DNA into fragments.
Accordingly, caspase-3 activity was measured, and the
results shown in Figure 1G illustrate that caspase-3 activity
was drastically increased with the rise in TNFα. Altogether,
our data indicate that TNFα treatment dose dependently pro-
motes glioblastoma cell apoptosis. Notably, no significant
difference was observed between the control group and the
1 ng/mL TNFα group. The minimal proapoptotic dose of
TNFα is 5 ng/mL; accordingly, 5 ng/mL TNFα was used in
the following studies.
Mitochondrial ssion is activated by
TNFα treatment
Several thorough studies from many laboratories have
reported that mitochondrial fission is an early event leading
to cell death.13 In the present study, we explored the functional
role of TNFα in mitochondrial fission. The immunofluores-
cence assay in Figure 2A demonstrated that mitochondria
are highly connected networks. However, after TNFα
treatment, mitochondria become small, roundish fragments
that are characteristic of mitochondrial fission. To quantify
mitochondrial fission, we measured the average length of
mitochondria with or without TNFα treatment. In the control
group, the length of mitochondria was ~8.9 µm. Interest-
ingly, TNFα treatment (5 ng/mL) reduced the mitochondrial
length to ~2.3 µm (Figure 2B). This information indicated
RETRACTED
OncoTargets and Therapy 2018:11 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7217
TNFα promotes glioblastoma A172 cell
that mitochondrial fission was activated by TNFα treatment
in glioblastoma cells. To provide additional evidence for the
role of TNFα in triggering mitochondrial division, Mdivi-1,
an antagonist of mitochondrial division was used. Mean-
while, a mitochondrial fission agonist was administered to the
normal glioblastoma cells to activate mitochondrial fission,
which was used as the positive control group. Then, Western
blot was performed to analyze alterations in protein levels
related to mitochondrial fission.32,33 When compared to the
control group, TNFα treatment increased the levels of Drp1,
Mff, and Fis1, the key elements in executing mitochondrial
fission (Figure 2C–H). In contrast, inhibitors of mitochondrial
fission such as Mfn2 and Opa1 were significantly downregu-
lated in response to TNFα treatment (Figure 2C–H). This
effect of TNFα was similar to the action of FCCP, which
caused an imbalance between mitochondrial fission factors
(Figure 2C–H). Interestingly, Mdivi-1 could abrogate the
promotive effects of TNFα on mitochondrial fission-related
proteins (Figure 2C–H). Altogether, our data confirm that
TNFα promotes mitochondrial fission activation in glio-
blastoma cells.
TNFα-mediated mitochondrial ssion
promotes mitochondrial dysfunction
Abnormal mitochondrial fission plays a decisive role in medi-
ating mitochondrial dysfunction. To verify whether TNFα
induces mitochondrial damage in glioblastoma cells via
mitochondrial fission, mitochondrial function was measured.
First, cell ROS production was determined via flow cytom-
etry. When compared to the control group, TNFα treatment
significantly increased ROS production in glioblastoma cells
(Figure 3A and B), and this effect was similar to the results
obtained via administering FCCP (Figure 3A and B). Interest-
ingly, TNFα-mediated ROS production was mostly negated
by Mdivi-1 (Figure 3A and B). Because of the cellular ROS
outburst, the concentration of cellular antioxidants such as
Figure 1 TNFα promotes glioblastoma cell apoptosis in a dose-dependent manner.
Notes: (A) Different doses of TNFα were added into the media of glioblastoma cells, and then, the cellular viability was measured via MTT assay. (B) An LDH release assay
was performed to detect cell death. (C and D) Trypan Blue staining for cell death. The number of Trypan Blue-positive cell was recorded. (E and F) A TUNEL assay was
used to determine the rate of apoptosis. The number of TUNEL-positive cells was measured. (G) Caspase-3 activity was measured to determine the activation level of the
caspase-3 protein. *P,0.05 vs the control group. The 0 ng/mL TNFα was used as the control group.
077DVVD\
71)αQJP/
$
/'+UHOHDVH
IROGV
71)αQJP/
%
7U\SDQ%OXH
SRVLWLYHFHOOV
71)αQJP/
&
781(/
SRVLWLYHFHOOV
71)αQJP/
)
&DVSDVHDFWLYLW\
IROGV
71)αQJP/
*
&RQWURO
71)αQJP/
'
7U\SDQ%OXH
P
71)αQJP/
(
781(/
0HUJHG
P
&RQWURO
RETRACTED
OncoTargets and Therapy 2018:11
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7218
Lu et al
Figure 2 TNFα activates mitochondrial ssion in glioblastoma cells.
Notes: (A) An immunouorescence assay for mitochondria using mitochondrial specic antibody Tom20. (B) The average length of mitochondria was measured, which was
used to analyze the extent of mitochondrial ssion. (C–H) Western blot was performed to analyze protein expression of mitochondrial ssion-related factors. To perform
the loss- and gain-of-function assays for mitochondrial ssion, Mdivi-1, a pharmacological antagonist was used in TNFα-treated cells to inhibit the activation of mitochondrial
ssion. FCCP, an agonist for mitochondrial ssion, was administered to the control group, which was used as the positive control group. Drp1, Fis1, and Mff are mitochondrial
ssion activators whose levels were upregulated in response to TNFα treatment and downregulated by Mdivi-1. By contrast, Mfn2 and Opa1 are the mitochondrial ssion
inhibitors whose expression levels were repressed by TNFα stress and were increased by Mdivi-1. *P,0.05.
Abbreviation: Mdivi-1, mitochondrial division inhibitor-1.
10
*
8
6
4
2
Mitochondrial
length (µm)
0
Control
TNFα (1 ng/mL)
TNFα (5 ng/mL)
AB
C
2.50
**
2.00
1.50
1.00
Drp1 expression
0.50
0.00
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Mdivi-1
Control FCCP
1.25
**
1.00
0.75
0.50
Mfn2 expression
0.25
0.00
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Mdivi-1
Control FCCP
1.25
**
1.00
0.75
0.50
Opa1 expression
0.25
0.00
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Mdivi-1
Control FCCP
3.00
**
2.00
1.00
Mff expression
0.00
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Mdivi-1
Control FCCP
D
GH
4.00
**
3.00
2.00
1.00
Fis1 expression
0.00
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Mdivi-1
Control FCCP
EF
β-actin
Drp1
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Mdivi-1
Control+FCCP
Fis1
Mff
Mfn2
Opa1
Control
MitochondriaMagnification
10 µm
TNFα (1 ng/mL) TNFα (5 ng/mL)
10 µm
RETRACTED
OncoTargets and Therapy 2018:11 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7219
TNFα promotes glioblastoma A172 cell
GSH, SOD, and GPX was obviously reduced in response to
TNFα treatment (Figure 3C–E). However, Mdivi-1 could
reverse the levels of GSH, SOD, and GPX (Figure 3C–E).
The abovementioned data suggested that TNFα-mediated
mitochondrial oxidative stress via mitochondrial fission.
The core function of mitochondria is to produce ATP,
which is required for cellular metabolism. Interestingly,
the content of ATP was significantly reduced in the pres-
ence of TNFα treatment (Figure 3F), similar to the results
obtained after administering FCCP. However, Mdivi-1
supplementation abrogated the inhibitory effects of TNFα
on ATP production (Figure 3F). At the molecular level,
mitochondria produce ATP via the mitochondrial respiratory
complex. Notably, the protein expression of mitochondrial
respiratory complex was significantly repressed by TNFα
(Figure 3G–J), and this effect was negated by Mdivi-1. This
information indicated that TNFα-mediated mitochondrial
fission reduced the levels of the mitochondrial respiratory
complex. Altogether, our data confirm that TNFα treatment
causes an obvious mitochondrial malfunction that occurs, at
least in part, through mitochondrial fission.
TNFα-mediated mitochondrial
ssion activates a caspase-9-related
mitochondrial apoptotic pathway
Damaged mitochondria initiate cellular apoptosis programs.34
Based on this, we explored whether TNFα-mediated mito-
chondrial fission accounted for glioblastoma cell apoptosis. An
early molecular feature of mitochondrial apoptosis is a drop
in the mitochondrial potential. As shown in Figure 4A and B,
Figure 3 TNFα-initiated mitochondrial ssion contributes to glioblastoma mitochondrial injury.
Notes: (A and B) ROS levels were measured using DHE probe, and ow cytometry was performed to quantify the content of cell ROS. To perform the loss- and gain-of-
function assays for mitochondrial ssion, Mdivi-1, a pharmacological antagonist was used in TNFα-treated cells to inhibit mitochondrial ssion activation. FCCP, an agonist
for mitochondrial ssion was administered to the control group, which was set as the positive control group. (C–E) ELISAs for cellular antioxidants such as GSH, SOD,
and GPX. (F) Cellular ATP production was measured using a commercial kit. (G–J) Western blot was conducted to analyze the protein expression of the mitochondrial
respiratory complex. *P,0.05.
Abbreviations: DHE, dihydroethidium; Mdivi-1, mitochondrial division inhibitor-1; CIII-core2, complex III subunit core; CII-30, complex II; CIV-II, complex IV subunit II.
*6+OHYHOV
QPROPJSURWHLQ
&RQWURO
&RQWURO)&&3
71)α
71)α0GLYL
&
62'DFWLYLW\
8PJSURWHLQ
&RQWURO
&RQWURO)&&3
71)α
71)α0GLYL
'
*3;DFWLYLW\
8PJSURWHLQ
&RQWURO
&RQWURO)&&3
71)α
71)α0GLYL
(
$73SURGXFWLRQ
RIFRQWURO
&RQWURO
&RQWURO)&&3
71)α
71)α0GLYL
)
&RQWURO
&RQWURO)&&3
71)α
71)α0GLYL
&,9,,
H[SUHVVLRQ
&RQWURO
&RQWURO)&&3
71)α
71)α0GLYL
&,,
H[SUHVVLRQ
&,,,FRUH
H[SUHVVLRQ
&RQWURO
&RQWURO)&&3
71)α
71)α0GLYL
7RP
&,9,,
&,,
&,,,FRUH
&RQWURO
&RQWURO)&&3
71)α
71)α0GLYL
*+,-
526
&RQWURO)&&3
&RQWURO
526
71)α0GLYL
&RQWURO
526
71)α
&RQWURO
$
526IOXRUHVFHQFH
IROGV
&RQWURO
&RQWURO)&&3
71)α
71)α0GLYL
%
RETRACTED
OncoTargets and Therapy 2018:11
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7220
Lu et al
compared to the control group, TNFα markedly reduced the
mitochondrial potential as evidenced by decreased red fluo-
rescence and increased green fluorescence. Interestingly, this
alteration could be abrogated by Mdivi-1 (Figure 4A and B),
suggesting that inhibition of mitochondrial fission protected
the mitochondrial potential in the presence of TNFα treat-
ment. The collapse of the mitochondrial potential indicates
hyperpermeability of the mitochondrial outer membrane.35
Accordingly, we evaluated the opening rate of the mPTP.
Compared to the control group, TNFα treatment increased
the opening rate of mPTP (Figure 4C), similar to the results
obtained via administration of FCCP. However, Mdivi-1
supplementation significantly blocked the mPTP opening
(Figure 4C). Excessive opening of mPTP could facilitate
mitochondrial proapoptotic cyt-c translocation into the cyto-
plasm where cyt-c interacts with and activates caspase-9.36
The immunofluorescence assay for cyt-c indicated that TNFα
treatment promoted cyt-c migration to the nucleus (Figure 4D
and E), and this effect was negated by Mdivi-1. In response to
the cyc-c liberation, the activity of caspase-9 was increased
in TNFα-treated cells, whereas Mdivi-1 treatment prevented
caspase-9 activation (Figure 4F).
In addition, we also found that the expression levels
of mitochondrial proapoptotic proteins such as Bax and
Bad were significantly upregulated in TNFα-treated cells
(Figure 4G–K), similar to the results obtained via adding
FCCP. By comparison, the levels of antiapoptotic proteins
such as Bcl-2 and x-IAP were downregulated in response to
TNFα stress (Figure 4G–K). Interestingly, Mdivi-1 treatment
reversed the levels of antiapoptotic factors. These results
indicated that mitochondrial apoptosis was activated by
TNFα via mitochondrial fission.
TNFα modulates mitochondrial ssion
via MAPK–ERK–YAP signaling pathways
Subsequently, we explored the molecular mechanism by
which TNFα controlled mitochondrial fission in glioblastoma
cells. Previous studies have suggested that mitochondrial
fission is negatively regulated by the MAPK–ERK–YAP
signaling pathways.37,38 In the present study, we noted abun-
dant p-ERK expression in the control group via Western blot
(Figure 5A–C). However, TNFα treatment significantly sup-
pressed p-ERK expression (Figure 5A–C), indicative of ERK
inactivation in response to TNFα stimulus. Moreover, the
TNFα-mediated decrease in p-ERK expression was closely
associated with a drop in YAP expression (Figure 5A–C),
suggesting that TNFα inactivated MAPK–ERK–YAP path-
ways in glioblastoma cells. PD98059 was used to inhibit ERK
activity, which was used to mimic the inhibitory effects of
TNFα on ERK pathways. This finding was further supported
Figure 4 (Continued)
&RQWURO)&&371)α0GLYL71)α&RQWURO
5HG
IOXRUHVFHQFH
*UHHQ
IOXRUHVFHQFH0HUJHG
P
&RQWURO
71)α
71)α0GLYL
&RQWURO)&&3
P373RSHQLQJ
UDWHIROGV
&RQWURO
71)α
71)α0GLYL
&RQWURO)&&3
5DWLRRIUHGWR
JUHHQIOXRUHVFHQFH
LQWHQVLW\
$%
&
RETRACTED
OncoTargets and Therapy 2018:11 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7221
TNFα promotes glioblastoma A172 cell
Figure 4 TNFα-activated caspase-9 apoptosis is regulated by mitochondrial ssion.
Notes: (A and B) Mitochondrial potential was observed via JC-1 staining. Red uorescence of the JC-1 probe indicates the normal mitochondrial potential, whereas green
uorescence of the JC-1 probe means a defective mitochondrial potential. The red-to-green uorescence intensity was recorded to quantify the mitochondrial potential.
To perform the loss- and gain-of-function assays for mitochondrial ssion, Mdivi-1, a pharmacological antagonist was used in TNFα-treated cells to inhibit mitochondrial
ssion activation. FCCP, an agonist for mitochondrial ssion, was administered to the control group, which was set as the positive control group. (C) mPTP opening was
measured in response to TNFα stress and/or mitochondrial ssion inhibition. (D and E) Immunouorescence assay for mitochondrial cyt-c translocation into nucleus. Nuclei
were labeled by DAPI, and the colocalization of cyt-c and DAPI indicates the migration of mitochondrial cyt-c into nucleus. The relative expression of nuclear cyt-c was
monitored. (F) Caspase-9 activity was determined via ELISA. TNFα-mediated caspase-9 activation could be abrogated by Mdivi-1. (G–K) Western blot was performed to
analyze the alterations in mitochondrial apoptotic proteins. Bax and Bad are proapoptotic proteins, whereas Bcl-2 and x-IAP are antiapoptotic proteins. TNFα regulated the
balance of proapoptotic and antiapoptotic proteins via mitochondrial ssion. *P,0.05.
Abbreviation: Mdivi-1, mitochondrial division inhibitor-1.
&RQWURO)&&371)α0GLYL71)α&RQWURO
&\WF'$3,0HUJHG
P
&RQWURO
71)α
71)α0GLYL
&RQWURO)&&3
&DVSDVH
DFWLYLW\IROGV
&RQWURO
71)α
71)α0GLYL
&RQWURO)&&3
1XFOHDU
H[SUHVVLRQRIF\WF
&RQWURO
71)α
71)α0GLYL
&RQWURO)&&3
%DG
%D[
%FO
[,$3
*$3'+
&RQWURO
71)α
71)α0GLYL
&RQWURO)&&3
%FOH[SUHVVLRQ
&RQWURO
71)α
71)α0GLYL
&RQWURO)&&3
[,$3H[SUHVVLRQ
&RQWURO
71)α
71)α0GLYL
&RQWURO)&&3
%DGH[SUHVVLRQ
&RQWURO
71)α
71)α0GLYL
&RQWURO)&&3
%D[H[SUHVVLRQ
'(
)
+,
.
-
*
RETRACTED
OncoTargets and Therapy 2018:11
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7222
Lu et al
Figure 5 TNFα handles mitochondrial ssion via MAPK–ERK–YAP pathways.
Notes: (A–C) The expression values of ERK and YAP were determined via Western blot. Phosphorylated ERK and YAP expressions were both downregulated by TNFα.
Subsequently, Ad-YAP was transfected into cells to overexpress YAP in TNFα-treated cells. PD98059 was used to inhibit ERK activity, which was used to mimic the inhibitory
effects of TNFα on ERK pathways. (D and E) Immunouorescence of p-ERK and YAP in cells treated with TNFα or transfected with Ad-YAP or Ad-ctrl. (F and G). The
overexpression efciency of Ad-YAP infection. Western blot was performed to analyze the protein expression of YAP in cells treated with Ad-YAP or Ad-ctrl. (H and I)
Mitochondrial ssion was determined via immunouorescence using mitochondrial-specic Tom20 antibody. The average length of mitochondria was evaluated to quantify
mitochondrial ssion. *P,0.05.
Abbreviation: Ad, adenovirus.
$
&RQWURO
&RQWURO3'
71)αQJP/
71)αQJP/
$GFRQWURO
71)αQJP/
$G<$3
S(5.
W(5.
<$3
βDFWLQ
&RQWURO
&RQWURO3'
71)αQJP/
71)αQJP/
$GFRQWURO
71)αQJP/
$G<$3
S(5.
H[SUHVVLRQ
%
&RQWURO
&RQWURO3'
71)αQJP/
71)αQJP/
$GFRQWURO
71)αQJP/
$G<$3
<$3
H[SUHVVLRQ
&
&RQWURO
&RQWURO
3'
71)α
QJP/
71)αQJP/
$GFRQWURO
71)αQJP/
$G<$3
P
+
0LWRFKRQGULD0DJQLILFDWLRQ
&RQWURO
&RQWURO3'
71)αQJP/
71)αQJP/
$GFRQWURO
71)αQJP/
$G<$3
0LWRFKRQGULDO
OHQJWKP
,
'
*
)
&RQWURO
&RQWURO3'
71)αQJP/
71)αQJP/
$GFRQWURO
71)αQJP/
Y$G<$3
<$3
IOXRUHVFHQFH
LQWHQVLW\
(
&RQWURO
$GFRQWURO
$G<$3
<$3
H[SUHVVLRQ
&RQWURO
$G<$3
$GFRQWURO
<$3
βDFWLQ
&RQWURO
&RQWURO
3'
71)α
QJP/
71)αQJP/
$GFRQWURO
71)αQJP/
$G<$3
S(5.0HUJHG <$3
P
RETRACTED
OncoTargets and Therapy 2018:11 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7223
TNFα promotes glioblastoma A172 cell
via immunofluorescence (Figure 5D and E). The fluorescence
intensities of p-ERK and YAP in the TNFα-treated cells
decreased by ~65% and ~50% of the control levels, respec-
tively. To demonstrate whether MAPK–ERK–YAP signaling
pathways were required for TNFα-mediated mitochondrial
fission, we overexpressed YAP in TNFα-treated cells. The
transfection efficiency was verified via immunofluorescence
assay (Figure 5D and E) and Western blot (Figure 5F and G).
Then, mitochondrial fission was evaluated again. As shown
in Figure 5H and I, TNFα treatment promoted the forma-
tion of fragmented mitochondria whose length was shorter
when compared to that of the control group. Interestingly,
TNFα-mediated mitochondrial division could be inhibited
by YAP overexpression (Figure 5H and I). Altogether, our
results confirm that MAPK–ERK–YAP signaling pathways
are required for TNFα-controlled mitochondrial fission.
MAPK–ERK–YAP signaling pathways
are also involved in mitochondrial
malfunction and glioblastoma cell death
We explored whether MAPK–ERK–YAP signaling path-
ways are involved in TNFα-mediated mitochondrial injury
and cell death. First, ROS production was measured via
immunofluorescence assay. Compared to the control group,
TNFα treatment elevated the levels of cell ROS (Figure 6A
and B), and this effect was reversed by YAP overexpression.
In addition, cyt-c translocation from the mitochondria into
the cytoplasm/nucleus was exacerbated by TNFα stress
and was repressed by YAP overexpression (Figure 6C
and D). In response to cyt-c leakage, caspase-9 activity was
augmented in TNFα-treated cells and was reduced to
near-normal levels with YAP overexpression (Figure 6E).
Altogether, this information indicated that TNFα-mediated
mitochondrial injury could be interrupted via activation of
the MAPK–ERK–YAP axes.
With respect to cell apoptosis, TUNEL assays were con-
ducted to observe the apoptotic cells. Compared to the control
group, TNFα treatment elevated the number of TUNEL-
positive cells (Figure 6F and G), and this effect was abro-
gated by YAP overexpression. Similarly, the LDH cytotoxic
test also indicated that TNFα-mediated LDH release could
be suppressed by YAP overexpression (Figure 6H). Col-
lectively, the above data demonstrate that TNFα-mediated
mitochondrial damage and cell death are mainly regulated
by the MAPK–ERK–YAP axes.
Discussion
The treatment of glioblastomas currently remains difficult
due to inevitable recurrence and rapid progression.39 Current
treatment options include radiation therapy in addition to
surgery or surgery combined with chemotherapy.40 In the
present study, we found that TNFα treatment significantly
reduced the viability of glioblastoma cells in a dose-dependent
manner. Functional investigations revealed that TNFα
supplementation activated mitochondrial fission and that
mitochondrial fission subsequently mediated mitochondrial
injury and initiated caspase-9-involved mitochondrial apop-
tosis. Inhibition of mitochondrial fission could abrogate the
proapoptotic effects of TNFα on glioblastoma cells. Further-
more, we showed that TNFα-induced mitochondrial fission
was modified by the MAPK–ERK–YAP signaling pathways.
TNFα treatment repressed the activity of MAPK–ERK–YAP
signaling pathways, leading to an increase in the content of
mitochondrial fission factors such as Drp1. Reactivation of
MAPK–ERK–YAP signaling pathways could inhibit TNFα-
mediated mitochondrial fission and provide a prosurvival
advantage for glioblastomas cells. Collectively, this is the
first study to demonstrate that TNFα regulates glioblastoma
cell viability and mitochondrial homeostasis by modulating
mitochondrial fission through MAPK–ERK–YAP-dependent
signaling pathways (Figure 7). Our results lay the foundation
to help us understand the molecular mechanisms of TNFα-
mediated cancer cytotoxicity.
TNFα, an inflammatory cytokine, is of significant
importance in regulating cancer progression in many types
of malignant tumors.41,42 This fact led to several animal
experiments and clinical studies to explore the detailed role
of TNFα in retarding the progression of glioblastomas.
Early studies have demonstrated that gene transduction of
a human TNFα-vector substantially increased the apoptotic
index and reduced the growth rate in human glioblastoma
cells.2 Subsequent studies determined that TNFα supplemen-
tation enhanced the susceptibility of human glioblastoma
cells to natural killer cells.43 In addition, TNFα treatment
also reduced the adhesion capacity, evoked cellular oxidant
stress,44,45 and suppressed tumor angiogenesis46 in primary
or recurrent glioblastomas. In the current study, our results
demonstrated that TNFα stress was closely associated with
mitochondrial damage in glioblastoma cells. In response
to TNFα stimulus, mitochondrial ROS production was
increased, which was accompanied by a drop in the levels
of antioxidant factors. In addition, mitochondrial ATP pro-
duction was also impaired, which may result from TNFα-
mediated downregulation of the mitochondrial respiratory
complex. More importantly, decreased mitochondrial poten-
tial, extended mPTP opening time, and more cyt-c liberation
into the nucleus were noted in TNFα-treated cells. These
alterations worked together to initiate caspase-9-related mito-
RETRACTED
OncoTargets and Therapy 2018:11
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7224
Lu et al
AB
D
E
H
2.50
**
2.00
1.50
1.00
ROS
production (fold)
0.50
0.00
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Ad-control
TNFα (5 ng/mL) +
Ad-YAP
4.00
**
3.00
2.00
Nuclear cyt-c
expression (folds)
1.00
0.00
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Ad-control
TNFα (5 ng/mL) +
Ad-YAP
4.00
**
3.00
2.00
Caspase-9 activity
(folds)
1.00
0.00
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Ad-control
TNFα (5 ng/mL) +
Ad-YAP
4.00
**
3.00
2.00
LDH release
(folds)
1.00
0.00
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Ad-control
TNFα (5 ng/mL) +
Ad-YAP
60
* *
50
40
30
Number of
TUNEL + cell (%)
20
10
0
Control
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Ad-control
TNFα (5 ng/mL) +
Ad-YAP
C
FG
Control
ROS
45 µm
TNFα (5 ng/mL)
TNFα (5 ng/mL) +
Ad-control
TNFα (5 ng/mL) +
Ad-YAP
DAPICyt-cMerged
10 µm
ControlTNFα (5 ng/mL)
TNFα (5 ng/mL) +
Ad-control
TNFα (5 ng/mL) +
Ad-YAP
TUNELDAPIMerged
60 µm
ControlTNFα (5 ng/mL)
TNFα (5 ng/mL) +
Ad-control
TNFα (5 ng/mL) +
Ad-YAP
Figure 6 MAPK–ERK–YAP pathways also participate in the regulation of mitochondrial homeostasis and cell death.
Notes: (A and B) ROS production was measured using immunouorescence. Ad-YAP was transfected into cells to reactivate MAPK–ERK–YAP pathways. (C and D)
Immunouorescence assays for cyt-c. The cellular location of cyt-c was determined, and DAPI was used to label the nucleus. (E) Caspase-9 activity was examined to
determine the role of MAPK–ERK–YAP pathways in caspase-9-mediated mitochondrial apoptosis. (F and G) TUNEL staining for apoptotic cells. The ratio of TUNEL-positive
cells was recorded. (H) An LDH release assay was used to analyze cell death. The ratio of relative LDH release was recorded compared to the control group. *P,0.05.
Abbreviation: Ad, adenovirus.
RETRACTED
OncoTargets and Therapy 2018:11 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7225
TNFα promotes glioblastoma A172 cell
chondrial apoptotic pathways, accounting for glioblastoma
cell death. Our findings are similar to previous studies that
indicated that TNFα treatment promoted mitochondrial dys-
function in glioblastoma cells.47 This information identifies
mitochondria as a primary target for TNFα-based therapy.
Based on this, the discovery of other drugs principally act-
ing on mitochondria may provide more clinical benefits for
patients with glioblastoma.
The novel finding in our study is that we show that
TNFα induces mitochondrial damage via mitochondrial
fission. Notably, mitochondrial fission has been suggested
as a chief cause of cell death by inducing mitochondrial
damage in several diseases. In cardiac ischemia-reperfusion
injuries, aberrant mitochondrial fission exacerbates cardio-
myocyte death via promoting mPTP opening and cardio-
lipin oxidation.5,6,48 Moreover, uncontrolled mitochondrial
fission also participates in fatty liver disease by disrupting
hepatocyte mitochondrial metabolism.49 In addition, in pan-
creatic cancer,13 breast cancer,11 ovarian cancer,12 and liver
cancer,50 mitochondrial fission exerts negative effects on
mitochondrial homeostasis and has been proposed to be a
primary apoptotic trigger. In the present study, we show for
the first time that mitochondrial fission induces mitochon-
drial damage, which precedes cell apoptosis in a caspase-9-
dependent manner. This is the first study to define the role
of mitochondrial fission in glioblastoma. Considering the
detrimental effects of mitochondrial fission on cell viability,
approaches to activate mitochondrial fission are of utmost
importance when designing antitumor therapies. Notably,
several studies have also found that TNFα treatment also
activated mitochondrial fusion in human kidney-2 cells51 and
cardiomyocytes.52 These results establish the various effects
of TNFα on mitochondrial fission and mitochondrial fusion.
This seems to be dependent on cell types. However, more
researches are required to validate our concept.
At the molecular level, we found that TNFα activated
mitochondrial fission via repression of MAPK–ERK–YAP
signaling pathways. First, more robust data concerning
the inhibitory effects of MAPK–ERK pathways on mito-
chondrial fission have been provided by several in vitro
and in vivo studies.53 More importantly, the MAPK–ERK
pathway, as the classical antiapoptotic pathway, has been
demonstrated to send beneficial signals to cells under
various states of stress.54 As a new downstream effector of
MAPK–ERK pathways, YAP was originally identified as
a proto-oncogene.55 Higher YAP expression is closely cor-
related with cancer progression and tumor metastasis.18 In
addition, increased YAP effectively controls mitochondrial
fission and sustains mitochondrial integrity,18 favoring cell
metabolism and growth. Based on this finding, several
researchers propose that MAPK–ERK–YAP pathways
are the upstream inhibitors of mitochondrial fission. This
conclusion is supported by our results. We found that reac-
tivation of MAPK–ERK–YAP pathways repressed mito-
chondrial fission and abrogated TNFα-mediated cell death.
Accordingly, our results combined with previous findings
highlight the molecular mechanisms by which TNFα regu-
lates mitochondrial fission. At the molecular levels, several
researchers have investigated the mechanism by which YAP
modulated mitochondrial fission. Increased YAP reduces
the transcription and expression of Mff and Drp1, strongly
attenuating mitochondrial fission. Moreover, YAP has an
ability to modify the phosphorylation of Drp1. In addition,
YAP overexpression also reverses mitochondrial fusion via
upregulating the expression of mitochondrial fusion factors
such as OPA1 and Mfn2. These results explain the inhibitory
effect of YAP on mitochondrial fission.
The clinical implication that can be drawn from our
study is multifold. Our data provide a piece of evidence for
the role of mitochondrial fission in glioblastoma viability.
This information indicates that mitochondrial fission would
be considered as a potential target to prevent glioblastoma
progression via promoting mitochondrial fission-mediated
cell apoptosis. On the other hand, our findings identify
MAPK–ERK–YAP pathways as novel regulators for han-
dling mitochondrial function and glioblastoma viability. This
may highlight a new entry point for treating glioblastoma by
targeting the MAPK–ERK–YAP signaling axes.
Figure 7 TNFα treatment elevates the apoptotic rate of glioblastoma in vitro by
initiating fatal mitochondrial ssion and interrupting MAPK–ERK–YAP signaling
pathways.
71)α
0$3.±(5.LQDFWLYDWLRQ
<$3GRZQUHJXODWLRQ
0LWRFKRQGULDOILVVLRQ
2[LGDWLYHLQMXU\
&DVSDVHGHSHQGHQW
FHOODSRSWRVLV
0LWRFKRQGULDO
G\VIXQFWLRQ
&\WFOLEHUDWLRQ
RETRACTED
OncoTargets and Therapy 2018:11
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
7226
Lu et al
Limitation
The primary limitation of our study is that only one cell line
was used in the present study to explore the roles of TNFα
and mitochondrial fission in cell viability. Animal studies
and clinical researches are required to further verify our
findings.56
Conclusion
Altogether, our results show that TNFα treatment elevates
the apoptotic rate of glioblastoma in vitro by initiating fatal
mitochondrial fission and interrupting MAPK–ERK–YAP
signaling pathways. These findings define mitochondrial
fission as a novel tumor suppressor that acts by inducing
mitochondrial damage, with potential implications for new
approaches to glioblastoma treatment.
Availability of data and materials
The datasets used and/or analyzed during the current study
are available from the corresponding author on reasonable
request.
Acknowledgment
No funding was received.
Author contributions
CL, XC, and QW made substantial contributions to the
concept and design of the present study, QW, XX, BX,
and CL contributed to the performance of experiments,
data analysis and interpretation, and manuscript writing.
All authors contributed to data analysis, drafting and
revising the article, gave final approval of the version to
be published, and agree to be accountable for all aspects
of the work.
Disclosure
The authors report no conflicts of interest in this work.
References
1. Groneberg DA, Addicks AM, Bendels MH, Quarcoo D, Jaque J,
Brüggmann D. Glioblastoma research: US and international network-
ing achievements. Oncotarget. 2017;8(70):115730–115735.
2. Walther W, Stein U, Pfeil D. Gene transfer of human TNF alpha into
glioblastoma cells permits modulation of mdr1 expression and potentia-
tion of chemosensitivity. Int J Cancer. 1995;61(6):832–839.
3. Zhang W, Sun Y, Liu L, Li Z. Prognostic Significance of TNFR-Associated
Factor 1 and 2 (TRAF1 and TRAF2) in Glioblastoma. Med Sci Monit.
2017;23:4506–4512.
4. Ge H, Mu L, Jin L, et al. Tumor associated CD70 expression is involved
in promoting tumor migration and macrophage infiltration in GBM. Int J
Cancer. 2017;141(7):1434–1444.
5. Zhou H, Shi C, Hu S, Zhu H, Ren J, Chen Y. BI1 is associated with micro-
vascular protection in cardiac ischemia reperfusion injury via repress-
ing Syk-Nox2-Drp1-mitochondrial fission pathways. Angiogenesis.
2018;21(3):599–615.
6. Zhou H, Wang J, Zhu P, et al. NR4A1 aggravates the cardiac micro-
vascular ischemia reperfusion injury through suppressing FUNDC1-
mediated mitophagy and promoting Mff-required mitochondrial fission
by CK2α. Basic Res Cardiol. 2018;113(4):23.
7. Cho SY, Kim SH, Yi MH, et al. Expression of PGC1α in glioblastoma
multiforme patients. Oncol Lett. 2017;13(6):4055–4076.
8. Gupta P, Dixit D, Sen E. Oncrasin targets the JNK-NF-κB axis to sen-
sitize glioma cells to TNFα-induced apoptosis. Carcinogenesis. 2013;
34(2):388–396.
9. Unterkircher T, Cristofanon S, Vellanki SH, et al. Bortezomib primes
glioblastoma, including glioblastoma stem cells, for TRAIL by increas-
ing tBid stability and mitochondrial apoptosis. Clin Cancer Res. 2011;
17(12):4019–4030.
10. Kim JY, Kim YJ, Lee S, Park JH. BNip3 is a mediator of TNF-induced
necrotic cell death. Apoptosis. 2011;16(2):114–126.
11. Lucantoni F, Düssmann H, Llorente-Folch I, Prehn JHM. BCL2 and
BCL(X)L selective inhibitors decrease mitochondrial ATP produc-
tion in breast cancer cells and are synthetically lethal when combined
with 2-deoxy-D-glucose. Oncotarget. 2018;9(40):26046–26063.
12. Noack S, Raab M, Matthess Y, et al. Synthetic lethality in CCNE1-
amplified high grade serous ovarian cancer through combined inhibition
of Polo-like kinase 1 and microtubule dynamics. Oncotarget. 2018;
9(40):25842–25859.
13. Pan L, Zhou L, Yin W, Bai J, Liu R. miR-125a induces apoptosis,
metabolism disorder and migration impairment in pancreatic cancer
cells by targeting Mfn2-related mitochondrial fission. Int J Oncol. 2018;
53(1):124–136.
14. Huang L, Luan T, Chen Y, et al. LASS2 regulates invasion and chemore-
sistance via ERK/Drp1 modulated mitochondrial dynamics in bladder
cancer cells. J Cancer. 2018;9(6):1017–1024.
15. Shen YL, Shi YZ, Chen GG, et al. TNF-α induces Drp1-mediated
mitochondrial fragmentation during inflammatory cardiomyocyte
injury. Int J Mol Med. 2018;41(4):2317–2327.
16. Sun X, Cao H, Zhan L, et al. Mitochondrial fission promotes cell migra-
tion by Ca2+/CaMKII/ERK/FAK pathway in hepatocellular carcinoma.
Liver Int. 2018;38(7):1263–1272.
17. Qiao P, Zhao F, Liu M, Gao D, Zhang H, Yan Y. Hydrogen sulfide inhib-
its mitochondrial fission in neuroblastoma N2a cells through the Drp1/
ERK1/2 signaling pathway. Mol Med Rep. 2017;16(1):971–977.
18. Li H, He F, Zhao X, et al. YAP Inhibits the Apoptosis and Migration of
Human Rectal Cancer Cells via Suppression of JNK-Drp1-Mitochondrial
Fission-HtrA2/Omi Pathways. Cell Physiol Biochem. 2017;44(5):
2073–2089.
19. Geng C, Wei J, Wu C. Yap-Hippo pathway regulates cerebral hypoxia-
reoxygenation injury in neuroblastoma N2a cells via inhibiting
ROCK1/F-actin/mitochondrial fission pathways. Acta Neurol Belg.
Epub 2018 May 23.
20. Du X, Wen J, Wang Y, et al. Hippo/Mst signalling couples metabolic
state and immune function of CD8α+ dendritic cells. Nature. 2018;
558(7708):141–145.
21. Zhang S, Shi R, Chen S, Wei X, Zhou Q, Wang Y. All-trans retinoic
acid inhibits the proliferation of SGC7901 cells by regulating caveolin-1
localization via the ERK/MAPK signaling pathway. Oncol Lett.
2018;15(2):1523–1528.
22. Yan H, Xiao F, Zou J, et al. NR4A1-induced increase in the sensitivity
of a human gastric cancer line to TNFα-mediated apoptosis is associated
with the inhibition of JNK/Parkin-dependent mitophagy. Int J Oncol.
2018;52(2):367–378.
23. Ackermann M, Kim YO, Wagner WL, et al. Effects of nintedanib on
the microvascular architecture in a lung fibrosis model. Angiogenesis.
2017;20(3):359–372.
24. du GQ, Shao ZB, Wu J, et al. Targeted myocardial delivery of GDF11 gene
rejuvenates the aged mouse heart and enhances myocardial regeneration
after ischemia-reperfusion injury. Basic Res Cardiol. 2017;112(1):7.
25. Schock SN, Chandra NV, Sun Y, et al. Induction of necroptotic cell
death by viral activation of the RIG-I or STING pathway. Cell Death
Differ. 2017;24(4):615–625.
RETRACTED
OncoTargets and erapy
Publish your work in this journal
Submit your manuscript here: http://www.dovepress.com/oncotargets-and-therapy-journal
OncoTargets and Therapy is an international, peer-reviewed, open
access journal focusing on the pathological basis of all cancers, potential
targets for therapy and treatment protocols employed to improve the
management of cancer patients. The journal also focuses on the impact
of management programs and new therapeutic agents and protocols on
patient perspectives such as quality of life, adherence and satisfaction.
The manuscript management system is completely online and includes
a very quick and fair peer-review system, which is all easy to use. Visit
http://www.dovepress.com/testimonials.php to read real quotes from
published authors.
OncoTargets and Therapy 2018:11 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
Dovepress
7227
TNFα promotes glioblastoma A172 cell
26. Li J, Chen L, Xiong Y, et al. Knockdown of PD-L1 in Human Gastric
Cancer Cells Inhibits Tumor Progression and Improves the Cytotoxic
Sensitivity to CIK Therapy. Cell Physiol Biochem. 2017;41(3):907–920.
27. Zhang Y, Zhou H, Wu W, et al. Liraglutide protects cardiac microvas-
cular endothelial cells against hypoxia/reoxygenation injury through
the suppression of the SR-Ca(2+)-XO-ROS axis via activation of the
GLP-1R/PI3K/Akt/survivin pathways. Free Radic Biol Med. 2016;95:
278–292.
28. Yu S, Wang X, Geng P, et al. Melatonin regulates PARP1 to control
the senescence-associated secretory phenotype (SASP) in human fetal
lung fibroblast cells. J Pineal Res. 2017;63(1):e12405.
29. Hernansanz-Agustín P, Ramos E, Navarro E, et al. Mitochondrial
complex I deactivation is related to superoxide production in acute
hypoxia. Redox Biol. 2017;12:1040–1051.
30. Zhu H, Jin Q, Li Y, et al. Melatonin protected cardiac microvascular
endothelial cells against oxidative stress injury via suppression of
IP3R-[Ca2+]c/VDAC-[Ca2+]m axis by activation of MAPK/ERK signal-
ing pathway. Cell Stress Chaperones. 2018;23(1):101–113.
31. Randriamboavonjy V, Kyselova A, Elgheznawy A, Zukunft S, Wittig I,
Fleming I. Calpain 1 cleaves and inactivates prostacyclin synthase
in mesenteric arteries from diabetic mice. Basic Res Cardiol. 2017;
112(1):10.
32. Zhou H, Hu S, Jin Q, et al. Mff-Dependent Mitochondrial Fission
Contributes to the Pathogenesis of Cardiac Microvasculature Ischemia/
Reperfusion Injury via Induction of mROS-Mediated Cardiolipin
Oxidation and HK2/VDAC1 Disassociation-Involved mPTP Opening.
J Am Heart Assoc. 2017;6(3):e005328.
33. Jin Q, Li R, Hu N, et al. DUSP1 alleviates cardiac ischemia/reperfu-
sion injury by suppressing the Mff-required mitochondrial fission and
Bnip3-related mitophagy via the JNK pathways. Redox Biol. 2018;14:
576–587.
34. Fuhrmann DC, Brüne B. Mitochondrial composition and function under
the control of hypoxia. Redox Biol. 2017;12:208–215.
35. Li R, Xin T, Li D, Wang C, Zhu H, Zhou H. Therapeutic effect of
Sirtuin 3 on ameliorating nonalcoholic fatty liver disease: The role of
the ERK-CREB pathway and Bnip3-mediated mitophagy. Redox Biol.
2018;18:229–243.
36. Zhou H, Yue Y, Wang J, Ma Q, Chen Y. Melatonin therapy for diabetic
cardiomyopathy: A mechanism involving Syk-mitochondrial complex
I-SERCA pathway. Cell Signal. 2018;47:88–100.
37. Zhou H, Li D, Zhu P, et al. Inhibitory effect of melatonin on necroptosis
via repressing the Ripk3-PGAM5-CypD-mPTP pathway attenuates
cardiac microvascular ischemia-reperfusion injury. J Pineal Res. 2018:
e12503.
38. Zhou H, Wang J, Zhu P, Hu S, Ren J. Ripk3 regulates cardiac microvas-
cular reperfusion injury: The role of IP3R-dependent calcium overload,
XO-mediated oxidative stress and F-action/filopodia-based cellular
migration. Cell Signal. 2018;45:12–22.
39. Tamimi AF, Juweid M. Epidemiology and outcome of glioblastoma.
In: De Vleeschouwer S, editor. Glioblastoma. Brisbane, AU: Codon
Publications; 2017.
40. Mann J, Ramakrishna R, Magge R, Wernicke AG. Advances in Radio-
therapy for Glioblastoma. Front Neurol. 2017;8:748.
41. Perez JG, Tran NL, Rosenblum MG, et al. The TWEAK receptor Fn14
is a potential cell surface portal for targeted delivery of glioblastoma
therapeutics. Oncogene. 2016;35(17):2145–2155.
42. Xu Q, Choksi S, Liu Z. Switching from TNF-induced inflammation to
death signaling. Mol Cell Oncol. 2018;5(1):e1392402.
43. Kondo S, Yin D, Takeuchi J, et al. Tumour necrosis factor-alpha induces
an increase in susceptibility of human glioblastoma U87-MG cells to
natural killer cell-mediated lysis. Br J Cancer. 1994;69(4):627–632.
44. Sawada M, Kiyono T, Nakashima S, et al. Molecular mechanisms
of TNF-alpha-induced ceramide formation in human glioma cells:
P53-mediated oxidant stress-dependent and -independent pathways.
Cell Death Differ. 2004;11(9):997–1008.
45. Zhou H, Wang S, Hu S, Chen Y, Ren J. ER-Mitochondria Microdomains
in Cardiac Ischemia-Reperfusion Injury: A Fresh Perspective. Front
Physiol. 2018;9:755.
46. Jazayeri S, Feli A, Bitaraf MA, Solaymani Dodaran M, Alikhani M,
Hosseinzadeh-Attar MJ. Effects of Copper Reduction on Angiogenesis-
Related Factors in Recurrent Glioblastoma Cases. Asian Pac J Cancer
Prev. 2016;17(10):4609–4614.
47. Zhang C, Jiang H, Wang P, Liu H, Sun X. Transcription factor NF-kappa
B represses ANT1 transcription and leads to mitochondrial dysfunc-
tions. Sci Rep. 2017;7:44708.
48. Zhou H, Zhu P, Wang J, Zhu H, Ren J, Chen Y. Pathogenesis of cardiac
ischemia reperfusion injury is associated with CK2α-disturbed mito-
chondrial homeostasis via suppression of FUNDC1-related mitophagy.
Cell Death Differ. 2018;25(6):1080–1093.
49. Zhou H, du W, Li Y, et al. Effects of melatonin on fatty liver disease:
The role of NR4A1/DNA-PKcs/p53 pathway, mitochondrial fission,
and mitophagy. J Pineal Res. 2018;64(1):e12450.
50. Tak H, Kang H, Ji E, Hong Y, Kim W, Lee EK. Potential use of TIA-1,
MFF, microRNA-200a-3p, and microRNA-27 as a novel marker for
hepatocellular carcinoma. Biochem Biophys Res Commun. 2018;497(4):
1117–1122.
51. Liu N, Jiang Z, Liu Y, et al. Human trypsin inhibitor reduces the apop-
tosis of lipopolysaccharide-induced human kidney-2 cells by promot-
ing mitochondrial fusion. Mol Med Rep. 2017;16(3):2899–2906.
52. Nan J, Hu H, Sun Y, et al. TNFR2 Stimulation Promotes Mitochondrial
Fusion via Stat3- and NF-kB-Dependent Activation of OPA1 Expres-
sion. Circ Res. 2017;121(4):392–410.
53. Tomer D, Chippalkatti R, Mitra K, Rikhy R. ERK regulates mitochon-
drial membrane potential in fission deficient Drosophila follicle cells
during differentiation. Dev Biol. 2018;434(1):48–62.
54. Kochetkova EY, Blinova GI, Bystrova OA, Martynova MG, Pospelov VA,
Pospelova TV. Targeted elimination of senescent Ras-transformed
cells by suppression of MEK/ERK pathway. Aging. 2017;9(11):
2352–2375.
55. Yan H, Qiu C, Sun W, et al. Yap regulates gastric cancer survival
and migration via SIRT1/Mfn2/mitophagy. Oncol Rep. 2018;39(4):
1671–1681.
56. Kusaczuk M, Krętowski R, Stypułkowska A, Cechowska-Pasko M.
Molecular and cellular effects of a novel hydroxamate-based HDAC
inhibitor – belinostat – in glioblastoma cell lines: a preliminary report.
Invest New Drugs. 2016;34(5):552–564.
RETRACTED
Available via license: CC BY-NC
Content may be subject to copyright.
