International Journal of

Molecular Sciences

Article
Ciprofloxacin Enhances TRAIL-Induced Apoptosis
in Lung Cancer Cells by Upregulating the
Expression and Protein Stability of Death
Receptors through CHOP Expression
Eun Jin Lim 1,† , Yu Jeong Yoon 1,† , Jeonghoon Heo 1 , Tae Hwa Lee 2 and Young-Ho Kim 1,3, *
1 Department of Molecular Biology and Immunology, College of Medicine, Kosin University,
Busan 49267, Korea; eunjin1025@hanmail.net (E.J.L.); yuuun33@naver.com (Y.J.Y.);
jeonghoonheo@kosin.ac.kr (J.H.)
2 Department of Obstetrics and Gynecology, College of Medicine, Kosin University, Busan 49267, Korea;
leehula@kosin.ac.kr
3 Institute for Medical Sciences, College of Medicine, Kosin University, Busan 49267, Korea
* Correspondence: kimyh@kosin.ac.kr; Tel.: +82-51-990-6505
† The authors have contributed equally in this work.




Received: 10 September 2018; Accepted: 13 October 2018; Published: 16 October 2018


Abstract: Ciprofloxacin (CIP) is a potent antimicrobial agent with multiple effects on host cells
and tissues. Previous studies have highlighted their proapoptotic effect on human cancer cells.
The current study showed that subtoxic doses of CIP effectively sensitized multiple cancer cells to
tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Although TRAIL
alone mediated the partial proteolytic processing of procaspase-3 in lung cancer cells, co-treatment
with CIP and TRAIL efficiently restored the complete activation of caspases. We found that treatment
of lung cancer with CIP significantly upregulated the expression and protein stability of death
receptor (DR) 5. These effects were mediated through the regulation of transcription factor CCAT
enhancer-binding protein homologous protein (CHOP) since the silencing of these signaling molecules
abrogated the effect of CIP. Taken together, these results indicated that the upregulation of death
receptor expression and protein stability by CIP contributed to the restoration of TRAIL-sensitivity in
lung cancer cells.

Keywords: ciprofloxacin; TRAIL; death receptor; CHOP

1. Introduction
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF
superfamily, is a potent apoptosis-inducing cytokine. TRAIL appears to specifically kill a wide
variety of cancer cells in cultured and xenografted tumors but has little or no effect on normal
cells [1–3]. TRAIL-induced apoptosis is associated with the interaction of TRAIL with two closely
related membrane receptors, TRAIL-R1 and TRAIL-R2. This interaction results in the cooperation with
the adaptor molecule Fas-associated protein with death domain (FADD), leading to the recruitment
and cleavage of the initiator caspase-8 and the consequent activation of an effector caspase, such as
caspase-3 [4,5]. However, many types of cancer develop TRAIL resistance, which is related to the
high expression levels of decoy receptors and antiapoptotic proteins, mutations in TRAIL receptors,
and dysregulation of death-inducing signaling complex (DISC) formation [6–8]. Cancer cells acquire
TRAIL resistance through multiple unknown mechanisms. Therefore, it is important to identify
therapeutic agents capable of sensitizing resistant cancer cells to TRAIL-induced apoptosis.

Int. J. Mol. Sci. 2018, 19, 3187; doi:10.3390/ijms19103187 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 12

Ciprofloxacin (CIP) is a food and drug administration (FDA)-approved fluoroquinolone (FQ)

Int. J. Mol. Sci. 2018, 19, 3187 2 of 12
widely used as a broad spectrum antibiotic. It is effective against various gram-positive and gram-
negative bacteria, specifically by targeting bacterial DNA gyrase and topoisomerase [9,10]. Besides
having antimicrobial
Ciprofloxacin (CIP)activity,
is a foodCIP
andreportedly exerts immunomodulatory
drug administration effects in rodent
(FDA)-approved fluoroquinolone (FQ) models
widely
and human
used [11,12],
as a broad spectrumimproving a wide
antibiotic. spectrum
It is effective of conditions,
against including thrombocytopenia
various gram-positive [13–15],
and gram-negative bacteria,
specifically by targeting
Crohn’s disease [16,17],bacterial DNA gyrase
and rheumatoid and topoisomerase
arthritis [9,10]. Besides
[18,19]. As reported having antimicrobial
by previous studies, FQs,
activity,
alone or in combination with other chemotherapeutic agents, have the ability and
CIP reportedly exerts immunomodulatory effects in rodent models human
to induce [11,12],
apoptosis
improving a wide spectrum of conditions, including thrombocytopenia [13–15], Crohn’s
and cell cycle arrest in various cancer cell lines, rendering them unique among other antibiotic disease [16,17],
and rheumatoid arthritis [18,19]. As reported by previous studies, FQs, alone or in combination with
family members [20–24]. In addition, although CIP was shown to stimulate TRAIL treatment in the
other chemotherapeutic agents, have the ability to induce apoptosis and cell cycle arrest in various
lung cancer
cancer cell rendering
cell lines, line A549,them
the molecular
unique among basis other
by which CIP sensitizes
antibiotic TRAIL-mediated
family members [20–24]. In apoptosis
addition,
was not fully
although investigated
CIP was shown toyet.
stimulate TRAIL treatment in the lung cancer cell line A549, the molecular
basis The current
by which CIPstudy reported
sensitizes for the first time
TRAIL-mediated that CIP
apoptosis sensitized
was not fullycancer cells toyet.
investigated TRAIL-induced
apoptosis and that the expression and stability of TRAIL receptor contributed
The current study reported for the first time that CIP sensitized cancer cells to TRAIL-inducedto this sensitization.
We showed
apoptosis andthat
thatthethe CIP-induced
expression and expression
stability ofand stability
TRAIL of TRAIL
receptor receptor
contributed wassensitization.
to this required to
We showed that the CIP-induced expression and stability of TRAIL receptor was requiredthis
significantly enhance the sensitivity of cancer cells to TRAIL-induced apoptosis. Therefore, to
study showed the importance of death receptors as targets for CIP to enhance
significantly enhance the sensitivity of cancer cells to TRAIL-induced apoptosis. Therefore, this study
the sensitivity of
cancer cells to TRAIL-induced apoptosis.
showed the importance of death receptors as targets for CIP to enhance the sensitivity of cancer cells
to TRAIL-induced apoptosis.
2. Results
2. Results
2.1. Ciprofloxacin Potentiated TRAIL-Induced Apoptosis in Human Lung Cancer Cells
2.1. Ciprofloxacin Potentiated TRAIL-Induced Apoptosis in Human Lung Cancer Cells
To investigate the effect of CIP on TRAIL-induced apoptosis, A549 cells were exposed to 10 to
To investigate
50 ng/mL of TRAILthe effect
with or of CIP onCIP
without TRAIL-induced apoptosis, A549
at various concentrations. cells were
Neither exposed
CIP nor TRAIL to alone
10 to
50 ng/mL of TRAIL with or without CIP at various concentrations. Neither CIP nor
had any effect on cell death, but combined treatment with both CIP and TRAIL markedly enhancedTRAIL alone had
any
cell effect
death onin acell death, but combined
dose-dependent mannertreatment with both
and also induced CIP and TRAIL
morphological markedly
changes in theenhanced cell
cells (Figure
death in a dose-dependent manner and also induced morphological changes
1A,B). Next, we examined whether combined treatment with CIP and TRAIL induces DNA in the cells (Figure 1A,B).
Next, we examined
fragmentation. In thewhether combined
cells that receivedtreatment with CIP
the combined and TRAIL
treatment with induces
CIP andDNA fragmentation.
TRAIL, we detected
In
the typical apoptotic nuclei (Figure 1C) and analyzed the DNA fragmentation (Figure the
the cells that received the combined treatment with CIP and TRAIL, we detected 1D).typical
These
apoptotic nuclei (Figure 1C) and analyzed the DNA fragmentation (Figure 1D).
data indicated that combined treatment with CIP- and TRAIL-induced cell death in human These data indicated
lung
that combined
cancer cells. treatment with CIP- and TRAIL-induced cell death in human lung cancer cells.

Figure 1. Cont.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 12
Int. J. Mol. Sci. 2018, 19, 3187 3 of 12
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 12

Figure 1. CIP enhanced TRAIL-induced A549 cell death. (A) Cells were pretreated with various
concentrations of CIP for 20 h before exposure to TRAIL (10 and 50 ng/mL) for 4 h. Cell viability was
analyzed1. by
Figure 1.
Figure CIP
CIP trypan blue TRAIL-induced
enhanced
enhanced exclusion assay.A549
TRAIL-induced Data cell
A549 represent
cell death.the
death. (A) Cells±
(A)mean
Cells SD pretreated
were
were of 3 samples.
pretreated * pvarious
with
with < 0.05
various
concentrations
compared to the
concentrations of CIP
of CIP for for 20 h before
+ TRAIL-treated exposure
cells. (B)
20 h before exposure to TRAIL
Cells were
to TRAIL (10 and
treated
(10 and 50 ng/mL)
with TRAIL
50 ng/mL) for 4
for 4 h.inCell h. Cell viability
theviability
presencewas or
was analyzed
absence
analyzed of
byCIP by for
trypantrypan
24 blue
blue exclusion
h.exclusion
After assay.
treatment,
assay. DataData
change representthethe
in cell
represent mean ±±
morphology
mean SDSD of3 3samples.
ofwas samples. pp <<light
detected* *by 0.05
0.05
compared toScale
microscopy.
compared
the CIP
to the CIPbar ++ μm. (C) Microscopic
TRAIL-treated
= TRAIL-treated
20 cells. (B) Cells were treated
cells. (B) examination
with TRAIL
was performed
Cells were treated with TRAIL
in the presence by
to detect
in theapoptosis
presence or
or
absence of
nuclear of CIP
stainingfor 24 h.
with After treatment, change in cell morphology was detected by light microscopy.
absence CIP for 24 DAPI.
h. AfterThe images change
treatment, shown in arecell
representatives
morphology was of three
detectedindependent
by light
Scale bar = 20 µm. (C) Microscopic examination was performed to detect apoptosis by nuclear staining
experiments. Scale bar = 10 μm.
microscopy. Scale bar = 20 μm. (C) Microscopic examination was performed to detect apoptosis
(D) Cells were treated with TRAIL for 4 h in the presence or absenceby
with DAPI. The images shown are representatives of three independent experiments. Scale bar = 10 µm.
of CIP forstaining
nuclear 20 h. Forwithanalyzing
DAPI.DNA The fragmentation,
images shown fragmented DNA was separated
are representatives of three byindependent
using 1.5%
(D) Cells were treated with TRAIL for 4 h in the presence or absence of CIP for 20 h. For analyzing
agarose gel. Scale bar = 10 μm. (D) Cells were treated with TRAIL for 4 h in the presence or absence
experiments.
DNA fragmentation, fragmented DNA was separated by using 1.5% agarose gel.
of CIP for 20 h. For analyzing DNA fragmentation, fragmented DNA was separated by using 1.5%
CIP
2.2. CIP Sensitized
Sensitized
agarose TRAIL-Induced Apoptosis through Caspase Pathway
gel. TRAIL-Induced
To evaluate
To evaluate the
the mechanism
mechanism of of CIP
CIP and
and TRAIL-induced
TRAIL-induced apoptosis
apoptosis activation,
activation, poly
poly (ADP-ribose)
(ADP-ribose)
2.2. CIP Sensitized
polymerase (PARP)TRAIL-Induced
cleavage and Apoptosis
caspase through
activity Caspase
were Pathway in the presence of TRAIL, CIP,
determined
polymerase (PARP) cleavage and caspase activity were determined in the presence of TRAIL, CIP,
or both.
or both. Figure
Figure 2A shows
shows that
that in
in the
the presence
presence ofof TRAIL,
TRAIL, PARP
PARP was was cleaved,
cleaved, yielding
yielding a characteristic
characteristic
To evaluate2Athe mechanism of CIP and TRAIL-induced apoptosis activation, polya (ADP-ribose)
85 kDa fragment.
85 kDa fragment. The
The combination
combination treatment
treatment of TRAIL
of TRAIL and CIP also resulted in elevated activation
polymerase (PARP) cleavage and caspase activity were and CIP alsoin
determined resulted in elevated
the presence activation
of TRAIL, CIP,
of
of caspase-8,
caspase-8, caspase-9,
caspase-9, and
and caspase-3.
caspase-3. In
In addition,
addition, we
we showed
showed that
that TRAIL-
TRAIL- and
and CIP-induced
CIP-induced
or both. Figure 2A shows that in the presence of TRAIL, PARP was cleaved, yielding a characteristic
apoptosis was
apoptosis was blocked
blocked by by Benzyl
Benzyl carbonyl-Val-Ala-Asp-fluoromethyl
carbonyl-Val-Ala-Asp-fluoromethyl ketone ketone (z-VAD-fmk) peptide,
peptide,
85 kDa fragment. The combination treatment of TRAIL and CIP also resulted (z-VAD-fmk)
in elevated activation
a general
a general caspase
caspase inhibitor
inhibitor (Figure 2B). We also found that z-VAD-fmk prevented the increase in
of caspase-8, caspase-9, and(Figure 2B). We
caspase-3. also foundwe
In addition, that z-VAD-fmk
showed prevented
that TRAIL- andtheCIP-induced
increase in
apoptotic
apoptotic wasDNA accumulation
DNAblocked
accumulation due
due to treatment with CIP and
to treatment with CIP and TRAIL TRAIL (Figure
(Figure 2C). These results
2C). Thesepeptide,
results
apoptosis by Benzyl carbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk)
provided further
provided further evidence
evidence that
that TRAIL
TRAIL induced
induced the the sensitization
sensitization ofof cancer
cancer cells
cells to
to CIP
CIP through
through aa
a general caspase inhibitor (Figure 2B). We also found that z-VAD-fmk prevented the increase in
caspase-dependent pathway.
caspase-dependent pathway.
apoptotic DNA accumulation due to treatment with CIP and TRAIL (Figure 2C). These results
provided further evidence that TRAIL induced the sensitization of cancer cells to CIP through a
caspase-dependent pathway.

Figure 2. Cont.
Int. J. Mol. Sci. 2018, 19, 3187 4 of 12
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 12

Figure 2.
Figure CIP treatment-induced
2. CIP treatment-induced caspase
caspase activation
activation inin A549 cells. (A)
(A) The
The protein
protein expression
expression of
caspase-3, caspase-8,
caspase-3, caspase-8,caspase-9, caspase-7,
caspase-9, and PARP
caspase-7, and PARPafter treatment with different
after treatment with doses of CIP+TRAIL
different doses of
for 24 h. The total cells were collected and the lysates were subjected to western blotting with
CIP+TRAIL for 24 h. The total cells were collected and the lysates were subjected to western blotting specific
with specific antibodies. Actin was used as a loading control. The proteolytic cleavages in PARP,cas-7,
antibodies. Actin was used as a loading control. The proteolytic cleavages in PARP, cas-3, cas-8, cas-
and
3, cas-9
cas-8, are indicated
cas-7, and cas-9 byarearrows.
indicated(B)byA549 cells(B)
arrows. were
A549 incubated
cells werewith 50 µM with
incubated z-VAD-fmk for 1 h
50 µM z-VAD-
before treatment with CIP + TRAIL. Equal amounts of cell lysates (40 µg) were electrophoresed and
fmk for 1 h before treatment with CIP + TRAIL. Equal amounts of cell lysates (40 µg) were
analyzed for PARP-1 by western blotting. The proteolytic cleavage of PARP is indicated by an arrow.
electrophoresed and analyzed for PARP-1 by western blotting. The proteolytic cleavage of PARP is
(C) For analyzing DNA fragmentation, fragmented DNA was separated by using 1.5% agarose gel.
indicated by an arrow. (C) For analyzing DNA fragmentation, fragmented DNA was separated by
2.3. CIP
usingUpregulated
1.5% agarose Death
gel. Receptors Expression in Various Cancer Cells

We determined whether the modulation of DR4 and/or DR5 protein levels was involved in
2.3. CIP Upregulated Death Receptors Expression in Various Cancer Cells
the sensitizing effect of CIP on TRAIL-induced apoptosis in lung cancer cells. Figure 3 shows that
CIP-regulated, TRAIL-induced
We determined whether the apoptosis
modulation corresponded with upregulation
of DR4 and/or of DR4was
DR5 protein levels andinvolved
DR5. DR4 inand
the
DR5 expression levels in lung cancer cells were increased in a time- and dose-dependent
sensitizing effect of CIP on TRAIL-induced apoptosis in lung cancer cells. Figure 3 shows that CIP- manner
by CIP treatment
regulated, (Figure 3A).
TRAIL-induced Reversecorresponded
apoptosis transcriptionwith
(RT)-PCR analysis of
upregulation showed thatDR5.
DR4 and CIP treatment
DR4 and
slightly increased DR5 mRNA levels in a dose- and time-dependent manner,
DR5 expression levels in lung cancer cells were increased in a time- and dose-dependent mannerbut not those of DR4by
(Figure 3B). We(Figure
CIP treatment also investigated whether
3A). Reverse the CIP-induced
transcription (RT)-PCR upregulation of DR5 that
analysis showed and DR4 is specific
CIP treatment
to A549 increased
slightly cells or also occurs
DR5 mRNA in other
levelslung
in acancer
dose- cell
andtypes (Figure S2). manner,
time-dependent Prostate but
cancer
notcells
those (PC3 and
of DR4
LNCaP), colon cancer cells (HCT116 and HT29), cervical cancer cells (HeLa and
(Figure 3B). We also investigated whether the CIP-induced upregulation of DR5 and DR4 is specificCaski), and breast
cancer
to A549cells
cells(MDA231) wereinexposed
or also occurs other lung to CIP (100
cancer µg/mL)
cell for 24 hS2).
types (Figure andProstate
then examined for (PC3
cancer cells DR5 and
and
DR4 protein expression. CIP induced the expression of DR5 (Figure 3C, middle panel)
LNCaP), colon cancer cells (HCT116 and HT29), cervical cancer cells (HeLa and Caski), and breast in the LNCaP,
HCT116,
cancer HeLa,
cells and Caski
(MDA231) cells.
were No significant
exposed induction
to CIP (100 μg/mL)of DR5
for 24expression
h and thenoccurred in the
examined forPC3,
DR5HT29,
and
and MDA 231 cells. These findings suggested that the CIP-induced upregulation of DR5 and DR4 is
DR4 protein expression. CIP induced the expression of DR5 (Figure 3C, middle panel) in the
not cell type-specific.
LNCaP, HCT116, HeLa, and Caski cells. No significant induction of DR5 expression occurred in the
PC3, HT29, and MDA 231 cells. These findings suggested that the CIP-induced upregulation of DR5
and DR4 is not cell type-specific.
Int. J. Mol. Sci. 2018, 19, 3187 5 of 12
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 12

Figure 3. CIP-induced
Figure 3. CIP-inducedDR5 DR5andandDR4DR4 expression.
expression. (A) (A)A549
A549cells
cells
werewere treated
treated withwith various
various
concentrations of CIP
concentrations of CIP(left) and
(left) andwith
withCIP
CIP100 µg/mL for
100µg/mL forvarious
varioustimetime periods
periods (right).
(right). WholeWhole
cell cell
extracts werewere
extracts analyzed
analyzedforfor
DR4DR4andandDR5
DR5expression
expression by by western
westernblotting.
blotting.(B)(B) CIP-induced
CIP-induced DR4 DR4
and and
DR5 gene expression.
DR5 gene expression.A549A549cells were
cells weretreated
treatedwith
with various concentrations
various concentrations of CIP
of CIP (left)
(left) and and
withwith
CIP CIP
100 µg/mL
100 µg/mL for various timetime
for various periods (right).
periods Total
(right). RNA
Total RNA was
wasextracted
extractedand
andexamined
examined forfor DR4
DR4 and
and DR5
expression by RT-PCR.
DR5 expression Actin was
by RT-PCR. used
Actin as used
was an internal controlcontrol
as an internal to show equalequal
to show RNARNA loading. (C) (C)
loading. Various
Various cancer cell lines were treated with CIP for 24 h and whole cell extracts
cancer cell lines were treated with CIP for 24 h and whole cell extracts were analyzed by western were analyzed by
western blotting. Equal amounts of protein (40 µg) were separated
blotting. Equal amounts of protein (40 µg) were separated by SDS-PAGE and immunoblotted. by SDS-PAGE and
immunoblotted.
2.4. Death Receptor Was Required by CIP to Enhance TRAIL-Induced Apoptosis
2.4. Death Receptor Was Required by CIP to Enhance TRAIL-Induced Apoptosis
To determine the role of DR5 and DR4 in TRAIL-induced apoptosis, we used siRNAs specific to
DR5 and DR4 to downregulate
To determine the role oftheir
DR5 expression. The siRNAs for
and DR4 in TRAIL-induced DR4 andwe
apoptosis, DR5
usedreduced
siRNAsCIP-induced
specific
DR4 to
andDR5DR5andexpression, respectively,
DR4 to downregulate more
their than that
expression. ThebysiRNAs
the control siRNA
for DR4 and (Figure 4A). However,
DR5 reduced CIP-
inducedand
DR4 siRNA DR4DR5
and siRNA
DR5 expression, respectively,
had minimal effects more
on thethan that by the upregulation
CIP-induced control siRNAof (Figure
DR4 and4A). DR5.
Next,However,
by usingDR4 siRNA and DR5
immunoblotting siRNA 4A)
(Figure had and
minimal effectsVonstaining
Annexin the CIP-induced upregulation
assay (Figure 4B), weofexamined
DR4
and DR5.
whether Next, by usingofimmunoblotting
the suppression DR4 and/or (Figure
DR4 by 4A)siRNA
and Annexin V stainingthe
can abrogate assay (Figure 4B),
sensitizing we of
effects
examined whether the suppression of DR4 and/or DR4 by siRNA can abrogate the sensitizing
CIP on cancer cells to TRAIL-induced apoptosis. The results revealed that the effect of CIP on
effects of CIP on cancer cells to TRAIL-induced apoptosis. The results revealed that the effect of CIP
TRAIL-induced apoptosis was effectively abolished in cells transfected with both DR5 and DR4
on TRAIL-induced apoptosis was effectively abolished in cells transfected with both DR5 and DR4
siRNAs, whereas treatment with only DR4 siRNA or only DR5 siRNA had no effect on TRAIL-induced
apoptosis. The silencing of both receptors abolished apoptosis to the same degree as the silencing of
either DR4 or DR5, suggesting that both DR4 and DR5 played major roles in TRAIL-induced apoptosis.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 12

siRNAs, whereas treatment with only DR4 siRNA or only DR5 siRNA had no effect on TRAIL-
induced apoptosis. The silencing of both receptors abolished apoptosis to the same degree as the
silencing of 2018,
Int. J. Mol. Sci. either
19, DR4
3187 or DR5, suggesting that both DR4 and DR5 played major roles in TRAIL-
6 of 12
induced apoptosis.

Figure 4.
4. Effect
Effectofofdeath
deathreceptors
receptors knockdown
knockdown onon CIP-induced
CIP-induced sensitization
sensitization to TRAIL.
to TRAIL. (A) A549
(A) A549 cells
cells
werewere transfected
transfected withwith
DR5DR5 siRNA,
siRNA, DR4DR4 siRNA,
siRNA, andand combinedDR5
combined DR5and
andDR4DR4siRNA.
siRNA. After
After 48 h,h,
the
the cells were pretreated with CIP for 20 h and then treated with TRAIL for 4 h. Whole cell extracts
were analyzed by western blotting using antibodies against PARP, DR4, and DR5. (B) Cell death was
determined by by Annexin
AnnexinV/PI
V/PI staining.
staining.Scale
Scalebar μm.
bar= =2020µm. The barbar
The represents thethe
represents meanmean ± SD.
± SD. * p <* 0.01
p<
indicates a significant difference between the untreated control and CIP + TRAIL-treated
0.01 indicates a significant difference between the untreated control and CIP + TRAIL-treated samples.
samples.
2.5. CHOP Mediated CIP-Induced Death Receptors Upregulation and Sensitization to TRAIL
Because
2.5. CHOP CCAAT/enhancer-binding
Mediated CIP-Induced Death Receptorsprotein homologous
Upregulation and protein (CHOP)
Sensitization is known to be a
to TRAIL
prominent endoplasmic reticulum (ER) stress marker and an important transcription factor of DR5,
Because
we next CCAAT/enhancer-binding
determined whether CIP inducesprotein
CHOP homologous
expression. To protein
further(CHOP)
investigateis known to be a
the underlying
prominent endoplasmic reticulum (ER) stress marker and an important
mechanism of DR5 expression by CIP-induced CHOP upregulation, the protein levels of DR5transcription factor of DR5,
were
we next determined whether CIP induces CHOP expression. To further investigate
also determined using CHOP siRNA. Our results showed that CHOP protein levels were significantly the underlying
mechanism
increased inof DR5 expression
a dose- by CIP-induced
and time-dependent manner CHOP
(Figureupregulation, the protein
5A). In a parallel levels of
experiment, weDR5 were
observed
also
similardetermined
results whenusing
otherCHOP
tumor siRNA.
cells were Our resultstreated
similarly showed
withthat CHOPnotprotein
CIP (data shown).levels were
In order to
significantly increased in a dose- and time-dependent manner (Figure 5A). In a parallel
determine the effect of CHOP on CIP-induced apoptosis, we demonstrated the effects of CIP on CHOP experiment,
we observed similar
siRNA-induced DR5 andresults
DR4 when other To
expression. tumor
clarifycells were similarly
the functional role oftreated
CHOP with CIP (data DR5
in CIP-induced not
shown).
and DR4Inupregulation,
order to determine
CHOP the effect
siRNA of CHOP
was on CIP-induced
also tested. apoptosis, we
DR5 was upregulated by demonstrated the
CIP in A549 cells
effects of CIP on CHOP siRNA-induced DR5 and DR4 expression. To clarify
transfected with scrambled negative control RNA, but transfection with CHOP siRNA significantlythe functional role of
CHOP in CIP-induced DR5 and DR4 upregulation, CHOP siRNA was also
abrogated the upregulation of DR5 (Figure 5B). In addition, treatment with CIP did not affect actin, tested. DR5 was
upregulated
which was usedby CIP
as ainhousekeeper
A549 cells transfected
gene in thesewith scrambled negative
experiments. As expected,control RNA, but transfection
CIP/TRAIL-induced cell
with CHOP siRNA significantly abrogated the upregulation of DR5
death was substantially blocked by siCHOP (Figure 5C). These findings indicated that (Figure 5B).CIP-enhanced
In addition,
treatment with and
DR5 expression CIPTRAIL-induced
did not affectapoptosis
actin, which
throughwas usedactivation.
CHOP as a housekeeper gene in these
experiments. As expected, CIP/TRAIL-induced cell death was substantially blocked by siCHOP
2.6. CIP Sustained
(Figure 5C). TheseDeath Receptor
findings Protein Stability
indicated that CIP-enhanced DR5 expression and TRAIL-induced
apoptosis through CHOP activation.
To characterize the mechanism underlying CIP-induced DR4/DR5 upregulation, we examined the
effect of CIP on DR4/DR5 protein stability in A549 cells. After the treatment with CIP for 12 h, the cells
were treated with cycloheximide (CHX), an inhibitor of de novo protein synthesis in the presence or
absence of CIP. CHX gradually decreased DR4 and DR5 protein expression, but co-treatment with
CHX and CIP sustained DR4 and DR5 protein expression (Figure 6). When DR5 is degraded via the
ubiquitin-proteasome pathways, ubiquitination of DR5 by Cbl E3 ligase is important [25]. We found
that the protein expression of Cbl, one of the E3 ligases that target DR5, was not markedly decreased
in a time-dependent manner in CIP-treated cells (data not shown). These findings suggested that CIP
induced the upregulation of death receptors expression at the post-translational level.
Int. J. Mol. Sci. 2018, 19, 3187 7 of 12
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 12

Figure 5. CIP exposure led to the regulation of CHOP proteins expression. (A) The effects of CIP on
the expression levels of CHOP proteins. A549 cells were treated with various concentrations of CIP.
Equal amounts (40 µg) of cell lysates were separated by SDS-PAGE. The images shown are
representatives of two additional experiments which yielded similar results. (B) A549 cells were
transfected with CHOP siRNA. After 48 h, the cells were pretreated with CIP for 24 h. Whole cell
extracts were analyzed by western blotting using antibodies against CHOP, DR4, and DR5. (C) A549
cells were transfected with CHOP siRNA. After 48 h, cells were treated with CIP + TRAIL for 24 h.
Cell death was determined by Annexin V/PI staining. The bar represents the mean ± SD. * p < 0.01
indicates a significant difference between the untreated group and CIP + TRAIL-treated samples.

2.6. CIP Sustained Death Receptor Protein Stability

To characterize the mechanism underlying CIP-induced DR4/DR5 upregulation, we examined

the effect of CIP on DR4/DR5 protein stability in A549 cells. After the treatment with CIP for 12 h,
CIPexposure
Figure 5. CIP exposureled ledtotothe
theregulation
regulationofofCHOP
CHOPproteins
proteins expression.
expression. (A)(A)
TheThe effects
effects of CIP
of CIP on
the cells
on the
were treatedlevels
expression
with ofcycloheximide
CHOP proteins.
(CHX), an inhibitor of dewith
novovarious
protein synthesis in the
the expression levels of CHOP proteins. A549 A549 cells treated
cells were were treated
with various concentrations
concentrations of CIP.
presence orEqual
absence of CIP. CHXofgradually decreased DR4 and DR5 protein expression, but co-
of CIP.
Equal (40 µg)(40
amountsamounts of µg) cell lysates
cell lysates were were separated
separated by SDS-PAGE.
by SDS-PAGE. The The images
images shown shown
are
treatment with CHX and
are representatives CIP additional
of two sustained experiments
DR4 and DR5 protein
which yieldedexpression
similar (Figure(B)
results. 6). When DR5 is
representatives of two additional experiments which yielded similar results. (B) A549 A549 cells
cells were
degraded
were via the
transfected ubiquitin-proteasome
with CHOP pathways, ubiquitination of DR5 by Cbl E3 ligase
h. is
transfected with CHOP siRNA.siRNA.
After 48After
h, the48cells
h, the
werecells were pretreated
pretreated with CIP for with
24 CIP for 24cell
h. Whole
important
Whole [25]. We found that the protein expression of Cbl, one of the E3 ligases that target
DR5.DR5,
extractscell extracts
were wereby
analyzed analyzed
westernby western
blotting blotting
using using antibodies
antibodies against
against CHOP, CHOP,
DR4, DR4, (C)
and DR5. andA549
was (C)
not markedly decreased inwith
a time-dependent manner in CIP-treated cellsCIP
(data not shown).
cellsA549
werecells were transfected
transfected with CHOP CHOP siRNA.
siRNA. After 48After 48 h,
h, cells cells
were were treated
treated with CIPwith+ TRAIL+ TRAIL
for 24forh.
These24 findings suggested
h. Cell death that CIP
was determined inducedV/PI
by Annexin the staining.
upregulation
The barof death receptors
represents the mean ±expression
SD. * p < 0.01at the
Cell death was determined by Annexin V/PI staining. The bar represents the mean ± SD. * p < 0.01
post-translational level. difference between the untreated group and CIP + TRAIL-treated samples.
indicates a significant
indicates a significant difference between the untreated group and CIP + TRAIL-treated samples.

2.6. CIP Sustained Death Receptor Protein Stability

To characterize the mechanism underlying CIP-induced DR4/DR5 upregulation, we examined

the effect of CIP on DR4/DR5 protein stability in A549 cells. After the treatment with CIP for 12 h,
the cells were treated with cycloheximide (CHX), an inhibitor of de novo protein synthesis in the
presence or absence of CIP. CHX gradually decreased DR4 and DR5 protein expression, but co-
treatment with CHX and CIP sustained DR4 and DR5 protein expression (Figure 6). When DR5 is
degraded via the ubiquitin-proteasome pathways, ubiquitination of DR5 by Cbl E3 ligase is
important [25]. We found that the protein expression of Cbl, one of the E3 ligases that target DR5,
was not markedly decreased in a time-dependent manner in CIP-treated cells (data not shown).
These findings suggested that CIP induced the upregulation of death receptors expression at the
post-translational level.

Figure 6.
Figure 6. Effect
Effectof
ofCIP
CIPon
onDR4
DR4and
andDR5
DR5 protein
protein stability.
stability. (A)(A) A549
A549 cells
cells were
were treated
treated withwith or without
or without CIP
for 24 h in the presence or absence of CHX for the indicated time periods. DR5, DR4, and actin protein
levels were determined by western blotting. Actin was used as a loading control. The band intensity of
DR5 (B) and DR4 (C) proteins was measured using the public domain JAVA image-processing program
Image J. Each value represents the mean ± SD of three independent experiments. * p < 0.05 compared
to that of CHX.

3. Discussion
This study showed for the first time that CIP enhanced TRAIL-mediated apoptosis in cancer
cells. We revealed an effect of CIP on apoptosis signaling in the A549 lung cancer cells. We also
found that CIP and TRAIL, at physiologically relevant doses currently being used for the treatment of

Figure 6. Effect of CIP on DR4 and DR5 protein stability. (A) A549 cells were treated with or without
Int. J. Mol. Sci. 2018, 19, 3187 8 of 12

antibacterial infections in human [26], caused cell cytotoxicity and apoptosis in A549 cells in a dose-
and time-dependent manner.
CIP is an FDA-approved fluoroquinolone widely used as an antimicrobial. CIP has various
anticancer activities, including growth-inhibitory and apoptosis-inducing activities. CIP inhibits cell
growth mainly in colon, leukemia, prostate, and bladder cancers [21,27,28], and induces G2/M arrest
mainly in bladder and prostate cancer cells [26,29]. CIP also induces apoptosis through the activation
of p21 in bladder cells [26]. We showed by flow cytometry that CIP-induced G2/M arrest in lung
cancer cells (data not shown). However, CIP is used as a sensitizer or anticancer drug-enhancer against
chemotherapy-resistant cancer cells.
Although TRAIL is one of the potent cytokines with the potential to kill cancer cells selectively,
its use has limitations because of the resistance that certain cancer types develop [30]. Therefore,
safe agents that can sensitize cancer cells to TRAIL are needed. In this study, we provided evidence
that CIP, an antimicrobial agent, sensitized human lung cancer cells to TRAIL. The overexpression
of DR5 in TRAIL-resistant cancer cells restored TRAIL sensitivity [31]. Our results showed that CIP
caused a significant increase in DR5 protein levels in lung cancer cells. Interestingly, CIP treatment
did not affect the levels of other IAP (XIAP, cIAP1, and cIAP2) (data not shown), emphasizing the
specific effect of CIP on DR5 expression. The increased level of DR5 expression, which led to the
stimulation of the death receptor pathway, appeared to be the cause of the activation of caspase-3,
caspase-8, caspase-9, and PARP (Figure 2). However, the enhancement of TRAIL-induced apoptosis by
CIP-induced DR5 upregulation is associated with the increased activation of the caspase pathway [32].
We found that one of the most important upstream signals required for the upregulation of
death receptors was reactive oxygen species (ROS). We found that CIP did not affect the generation
of ROS in lung cancer cells that ROS scavengers did not downregulate the CIP-induced expression
of DR5, and that ROS scavengers also did not abrogate the potentiation of TRAIL-induced apoptosis
by CIP (data not shown). CHOP is thought to be one of the most important transcription factors
that can directly bind to the DR5 promoter region and dynamically regulate DR5 expression [33].
We investigated whether the transcription factor CHOP is involved in the increase of DR5 expression
by CIP. CIP increased translocation into the nucleus of CHOP. (Figure S1) We further revealed that the
induction of DR5 by CIP is mediated through CHOP upregulation. These findings are supported by
previous studies reporting the CHOP-upregulating and TRAIL effects-enhancing abilities of CIP [34].
Taken together, our study showed that CHOP might play a crucial role in the CIP-induced DR5
upregulation and cancer cell death. Hence, CIP could be an attractive candidate as a therapeutic agent
for cancer chemotherapy. Future clinical studies in relevant animal models, however, are needed to
completely elucidate the potential use of this fascinating molecule in the prevention and treatment
of cancer.

4. Materials and Methods

4.1. Materials
RPMI 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, and all other tissue culture
reagents were obtained from GIBCO/BRL Life Technologies (Grand Island, NY, USA). CIP was
acquired from Aldrich (Milwaukee, WI, USA). Antibodies against PARP-1, Bcl-2, Bcl-xL, Mcl-1,
and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-JNK,
anti-phospho-p38, and anti-phospho-ERK were purchased from Cell Signaling (Beverly, MA,
USA). Benzyl carbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) was purchased from Biomol
(Plymouth Meeting, PA, USA), 2’,7’-dichlorodihydrofluorescein diacetate (H2 DCFDA) was purchased
from Molecular Probes (Eugene, OR, USA). N-acetylcysteine (NAC) and all other chemicals used in
this study were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Int. J. Mol. Sci. 2018, 19, 3187 9 of 12

4.2. Cell Culture and Chemical Treatments

Human lung cancer cell line A549 was obtained from the American Type Culture Collection
(Rockville, MD, USA). The A549 cells were cultured in RPMI 1640 medium supplemented
with heat-inactivated 10% fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin
(100 units/mL) at 37 ◦ C in a humidified incubator with 5% CO2 and 95% air. When the cells were
subconfluent, the medium was replaced with fresh medium, 25–100 ng/mL of CIP was added to the
culture medium, and the cells were incubated for 24 h.

4.3. Cellular Viability Assay

For the morphological evaluation of cell death, approximately 5 × 105 A549 cells were plated into
each well of 60-mm cell culture dishes overnight. For the trypan blue exclusion assay, trypsinized cells
were pelleted and resuspended in 0.2 mL of medium, 0.5 mL of 0.4% trypan blue solution, and 0.3 mL
of phosphate-buffered saline solution (PBS). The samples were mixed thoroughly, incubated at room
temperature for 15 min, and examined under a light microscope. At least 300 cells were counted for
each survival determination.

4.4. Flow Cytometric Assay of DNA Content

After the treatment of cells with the indicated agent, the cells were harvested via trypsinization,
fixed with 70% (v/v) alcohol at 4 ◦ C for 30 min, and washed with PBS. After centrifugation, the cells
were incubated in 0.1 mL of phosphate-citric acid buffer (0.2 M NaHPO4 , 0.1 M citric acid, pH 7.8)
for 30 min at room temperature. Next, the cells were centrifuged and resuspended with 0.5 mL of
50 µg/mL PI diluted in phosphate-citric acid buffer. DNA content was analyzed by using a FACScan
flow cytometer (Beckman Coulter, Inc., Hialeah, FL, USA)

4.5. Analysis of Cytochrome C Release

A total of 2 × 106 cells was harvested, washed once with ice-cold PBS, and gently lysed for 2 min
in 100 µL ice-cold lysis buffer. Lysates were centrifuged at 13,000× g at 4 ◦ C for 20 min to obtain the
supernatants (mitochondria-free cytosolic extracts) and the pellets (mitochondria-containing fractions).
The resulting cytosolic fractions were used for western blot analysis with an anticytochrome c antibody.

4.6. Western Blot Analysis

For western blot analysis, A549 cells were lysed with 1× Laemmli lysis buffer (2.4 M glycerol,
0.14 M Tris, pH 6.8, 0.21 M SDS, 0.3 mM bromophenol blue) and boiled for 10 min. Protein content
was measured with the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). The samples were
diluted with 1× Laemmli lysis buffer containing 1.28 M β-mercaptoethanol and equal amounts of
protein were loaded on 8–12% SDS-polyacrylamide gels. Proteins were separated by SDS-PAGE
and electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose membrane
was blocked with 5% nonfat dry milk in PBS-Tween 20 (0.1%, v/v) for 1 h. The membrane was
incubated with a primary antibody (diluted according to the manufacturer’s instructions) at room
temperature for 1.5 h. Horseradish peroxidase-conjugated antirabbit or antimouse IgG was used as
secondary antibodies. Immunoreactive protein was visualized by the chemiluminescence protocol
(ECL, Amersham, Arlington Heights, IL, USA). To ensure equal protein loading, each nitrocellulose
membrane was stripped and reprobed with an antiactin antibody after the experiment was complete.

4.7. DNA Fragmentation and 4’,6’-Diamidino-2-Phenylindole (DAPI) Staining Assay

To assess DNA fragmentation after CIP with TRAIL for 24 h, approximately 1 × 106 treated A549
cells were lysed for 30 min on ice in buffer containing 10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA,
and 0.5% Triton X-100. Lysates were vortexed and cleared by centrifugation at 13,000× g for 30 min.
Fragmented DNA in the supernatant was extracted with an equal volume of a mixture of neutral
Int. J. Mol. Sci. 2018, 19, 3187 10 of 12

phenol:chloroform:isoamyl alcohol (25:24:1) and electrophoresed on 1.5% agarose gels containing

0.1 g/mL EtBr. The cells were fixed on slide glass through the application of 4% paraformaldehyde for
30 min at room temperature. After washing with PBS, 300 nM DAPI was added to the fixed cells for
10 min. The cells were examined by fluorescence microscopy. Apoptotic cells were identified by the
condensation and fragmentation of their nuclei. All microscopic examination of the DAPI-stained cells
were performed in duplicate.

4.8. Data Analysis

All experiments were repeated three or more times. The data are represented as the
mean ± standard deviation (SD). The difference between two mean values was analyzed using
Student’s t-test and was considered statistically significant when p < 0.05.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/10/

3187/s1.
Author Contributions: Y.-H.K., E.J.L. and Y.J.Y. conceived and designed the experiments; E.J.L. and Y.J.Y.
performed the experiments; E.J.L., Y.J.Y., J.H., T.H.L. and Y.-H.K. analyzed the data; Y.-H.K. contributed
reagents/materials/analysis tools; E.J.L. and Y.-H.K. wrote the paper.
Funding: This research received no external funding.
Acknowledgments: This work was supported by Basic Science Research Program through the NRF funded by
the Ministry of Education, Science and Technology (2015R1D1A1A01058476).
Conflicts of Interest: The authors declare no conflicts of interest.

Abbreviations
TRAIL Tumor necrosis factor (TNF)-related apoptosis-inducing ligand
FADD Fas-associated protein with death domain
DISC Death-inducing signaling complex
FQ Fluoroquinolone
CHOP CCAAT/enhancer-binding protein homologous protein
CHX Cycloheximide
PARP Poly (ADP-ribose) polymerase
PI Propidium iodide
NAC N-acetyl-l-cyteine
ROS Reactive oxygen species

References
1. Sheridan, J.P.; Marsters, S.A.; Pitti, R.M.; Gurney, A.; Skubatch, M.; Baldwin, D.; Ramakrishnan, L.; Gray, C.L.;
Baker, K.; Wood, W.I.; et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors.
Science 1997, 277, 818–821. [CrossRef] [PubMed]
2. Ashkenazi, A.; Pai, R.C.; Fong, S.; Leung, S.; Lawrence, D.A.; Marsters, S.A.; Blackie, C.; Chang, L.;
McMurtrey, A.E.; Hebert, A.; et al. Safety and antitumor activity of recombinant soluble Apo2 ligand.
J. Clin. Investig. 1999, 104, 155–162. [CrossRef] [PubMed]
3. Walczak, H.; Miller, R.E.; Ariail, K.; Gliniak, B.; Griffith, T.S.; Kubin, M.; Chin, W.; Jones, J.; Woodward, A.;
Le, T.; et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat. Med.
1999, 5, 157–163. [CrossRef] [PubMed]
4. Nagata, S. Apoptosis by death factor. Cell 1997, 88, 355–365. [CrossRef]
5. Ashkenazi, A.; Dixit, V.M. Death receptors: Signaling and modulation. Science 1998, 281, 1305–1308.
[CrossRef] [PubMed]
6. Song, J.J.; An, J.Y.; Kwon, Y.T.; Lee, Y.J. Evidence for two modes of development of acquired tumor necrosis
factor-related apoptosis-inducing ligand resistance. Involvement of Bcl-xL. J. Biol. Chem. 2007, 282, 319–328.
[CrossRef] [PubMed]
7. Zhang, L.; Fang, B. Mechanisms of resistance to TRAIL-induced apoptosis in cancer. Cancer Gene Ther. 2005,
12, 228–237. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2018, 19, 3187 11 of 12

8. Maksimovic-Ivanic, D.; Stosic-Grujicic, S.; Nicoletti, F.; Mijatovic, S. Resistance to TRAIL and how to
surmount it. Immunol. Res. 2012, 52, 157–168. [CrossRef] [PubMed]
9. Drlica, K. Mechanism of fluoroquinolone action. Curr. Opin. Microbiol. 1999, 2, 504–508. [CrossRef]
10. Shen, L.L.; Baranowski, J.; Pernet, A.G. Mechanism of inhibition of DNA gyrase by quinolone antibacterials:
Specificity and cooperativity of drug binding to DNA. Biochemistry 1989, 28, 3879–3885. [CrossRef] [PubMed]
11. Dalhoff, A.; Shalit, I. Immunomodulatory effects of quinolones. Lancet Infect. Dis. 2003, 3, 359–371. [CrossRef]
12. Dalhoff, A. Immunomodulatory activities of fluoroquinolones. Infection 2005, 33, 55–70. [CrossRef] [PubMed]
13. Shoenfeld, Y.; Sherer, Y.; Fishman, P. Interleukin-3 and pregnancy loss in antiphospholipid syndrome. Scand. J.
Rheumatol. Suppl. 1998, 107, 19–22. [CrossRef] [PubMed]
14. Savion, S.; Blank, M.; Shepshelovich, J.; Fishman, P.; Shoenfeld, Y.; Toder, V. Ciprofloxacin affects pregnancy
loss in CBA/JxDBA/2J mice possibly via elevation of interleukin-3 and granulocyte macrophage-colony
stimulating factor production. Am. J. Reprod. Immunol. 2000, 44, 293–298. [CrossRef] [PubMed]
15. Blank, M.; George, J.; Fishman, P.; Levy, Y.; Toder, V.; Savion, S.; Barak, V.; Koike, T.; Shoenfeld, Y.
Ciprofloxacin immunomodulation of experimental antiphospholipid syndrome associated with elevation of
interleukin-3 and granulocyte-macrophage colony-stimulating factor expression. Arthritis Rheum. 1998, 41,
224–232. [CrossRef]
16. Stein, R.B.; Hanauer, S.B. Medical therapy for inflammatory bowel disease. Gastroenterol. Clin. N. Am. 1999,
28, 297–321. [CrossRef]
17. Rath, H.C.; Schultz, M.; Freitag, R.; Dieleman, L.A.; Li, F.; Linde, H.J.; Scholmerich, J.; Sartor, R.B. Different
subsets of enteric bacteria induce and perpetuate experimental colitis in rats and mice. Infect. Immun. 2001,
69, 2277–2285. [CrossRef] [PubMed]
18. Lewis, A.J.; Keft, A.F. A review on the strategies for the development and application of new anti-arthritic
agents. Immunopharmacol. Immunotoxicol. 1995, 17, 607–663. [CrossRef] [PubMed]
19. Breban, M.; Fournier, C.; Gougerot-Pocidalo, M.A.; Muffat-Joly, M.; Pocidalo, J.J. Protective effects of
ciprofloxacin against type II collagen induced arthritis in rats. J. Rheumatol. 1992, 19, 216–222. [PubMed]
20. Gurbay, A.; Osman, M.; Favier, A.; Hincal, F. Ciprofloxacin-Induced Cytotoxicity and Apoptosis in HeLa
Cells. Toxicol. Mech. Methods 2005, 15, 339–342. [CrossRef] [PubMed]
21. Herold, C.; Ocker, M.; Ganslmayer, M.; Gerauer, H.; Hahn, E.G.; Schuppan, D. Ciprofloxacin induces
apoptosis and inhibits proliferation of human colorectal carcinoma cells. Br. J. Cancer 2002, 86, 443–448.
[CrossRef] [PubMed]
22. Mondal, E.R.; Das, S.K.; Mukherjee, P. Comparative evaluation of antiproliferative activity and induction of
apoptosis by some fluoroquinolones with a human non-small cell lung cancer cell line in culture. Asian Pac.
J. Cancer Prev. 2004, 5, 196–204. [PubMed]
23. Reuveni, D.; Halperin, D.; Shalit, I.; Priel, E.; Fabian, I. Quinolones as enhancers of camptothecin-induced
cytotoxic and anti-topoisomerase I. effects. Biochem. Pharmacol. 2008, 75, 1272–1281. [CrossRef] [PubMed]
24. Reuveni, D.; Halperin, D.; Fabian, I.; Tsarfaty, G.; Askenasy, N.; Shalit, I. Moxifloxacin increases anti-tumor
and anti-angiogenic activity of irinotecan in human xenograft tumors. Biochem. Pharmacol. 2010, 79,
1100–1107. [CrossRef] [PubMed]
25. Song, J.J.; Szczepanski, M.J.; Kim, S.Y.; Kim, J.H.; An, J.Y.; Kwon, Y.T.; Alcala, M.A., Jr.; Bartlett, D.L.; Lee, Y.J.
c-Cbl-mediated degradation of TRAIL receptors is responsible for the development of the early phase of
TRAIL resistance. Cell Signal. 2010, 22, 553–563. [CrossRef] [PubMed]
26. Aranha, O.; Grignon, R.; Fernandes, N.; McDonnell, T.J.; Wood, D.P.J.; Sarkar, F.H. Suppression of human
prostate cancer cell growth by ciprofloxacin is associated with cell cycle arrest and apoptosis. Int. J. Oncol.
2003, 22, 787–794. [CrossRef] [PubMed]
27. Jun, Y.T.; Kim, H.J.; Song, M.J.; Lim, J.H.; Lee, D.G.; Han, K.J.; Choi, S.M.; Yoo, J.H.; Shin, W.S.;
Choi, J.H. In vitro effects of ciprofloxacin and roxithromycin on apoptosis of jurkat T lymphocytes.
Antimicrob. Agents Chemother. 2003, 47, 1161–1164. [CrossRef] [PubMed]
28. Lawrence, J.W.; Claire, D.C.; Weissig, V.; Rowe, T.C. Delayed cytotoxicity and cleavage of mitochondrial
DNA in ciprofloxacin-treated mammalian cells. Mol. Pharmacol. 1996, 50, 1178–1188. [PubMed]
29. Aranha, O.; Wood, D.P., Jr.; Sarkar, F.H. Ciprofloxacin mediated cell growth inhibition, S/G2-M cell cycle
arrest, and apoptosis in a human transitional cell carcinoma of the bladder cell line. Clin. Cancer Res. 2000, 6,
891–900. [PubMed]
Int. J. Mol. Sci. 2018, 19, 3187 12 of 12

30. Dida, F.; Li, Y.; Iwao, A.; Deguchi, T.; Azuma, E.; Komada, Y. Resistance to TRAIL induced apoptosis caused
by constitutional phosphorylation of Akt and PTEN in acute lymphoblastic leukemia cells. Exp. Hematol.
2008, 36, 1343–1353. [CrossRef] [PubMed]
31. Reuss, D.E.; Mucha, J.; Hagenlocher, C.; Ehemann, V.; Kluwe, L.; Manutner, V.; von Deimling, A. Sensitivity of
malignant peripheral nerve sheath tumor cells to TRAIL is augmented by loss of NF1 through modulation of
MYC/MAD and is potentiated by curcumin through induction of ROS. PLoS ONE 2013, 8, e57152. [CrossRef]
[PubMed]
32. Tian, X.; Ye, J.; Alonso-Basanta, M.; Hahn, S.M.; Koumenis, C.; Dorsey, J.F. Modulation of CCAAT/enhancer
binding protein homologous protein (CHOP) dependent DR5 expression by nelfinavir sensitizes glioblastoma
multiforme cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). J. Biol. Chem. 2011, 286,
29408–29416. [CrossRef] [PubMed]
33. Yamaguchi, H.; Wang, H.G. CHOP is involved in endoplasmic reticulum stress-induced apoptosis by
enhancing DR5 expression in human carcinoma cells. J. Biol. Chem. 2004, 279, 45495–45502. [CrossRef]
[PubMed]
34. Sung, B.; Prasad, S.; Ravindran, J.; Yadav, V.R.; Aggarwal, B.B. Capsazepine, a TRPV1 antagonist, sensitizes
colorectal cancer cells to apoptosis by TRAIL through ROS-JNK-CHOP-mediated upregulation of death
receptors. Free Radic. Biol. Med. 2012, 53, 1977–1987. [CrossRef] [PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).