Irreversible EGFR Inhibitor EKB-569 Targets Low-LET γ-Radiation-Triggered Rel Orchestration and Potentiates Cell Death in Squamous Cell Carcinoma

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DOI: 10.1371/journal.pone.0029705 · Source: PubMed
Abstract
EKB-569 (Pelitinib), an irreversible EGFR tyrosine kinase inhibitor has shown potential therapeutic efficiency in solid tumors. However, cell-killing potential in combination with radiotherapy and its underlying molecular orchestration remain to be explored. The objective of this study was to determine the effect of EKB-569 on ionizing radiation (IR)-associated NFκB-dependent cell death. SCC-4 and SCC-9 cells exposed to IR (2Gy) with and without EKB-569 treatment were analyzed for transactivation of 88 NFκB pathway molecules, NFκB DNA-binding activity, translation of the NFκB downstream mediators, Birc1, 2 and 5, cell viability, metabolic activity and apoptosis. Selective targeting of IR-induced NFκB by EKB-569 and its influence on cell-fate were assessed by overexpressing (p50/p65) and silencing (ΔIκBα) NFκB. QPCR profiling after IR exposure revealed a significant induction of 74 NFκB signal transduction molecules. Of those, 72 were suppressed with EKB-569. EMSA revealed a dose dependent inhibition of NFκB by EKB-569. More importantly, EKB-569 inhibited IR-induced NFκB in a dose-dependent manner, and this inhibition was sustained up to at least 72 h. Immunoblotting revealed a significant suppression of IR-induced Birc1, 2 and 5 by EKB-569. We observed a dose-dependent inhibition of cell viability, metabolic activity and apoptosis with EKB-569. EKB-569 significantly enhanced IR-induced cell death and apoptosis. Blocking NFκB improved IR-induced cell death. Conversely, NFκB overexpression negates EKB-569 -induced cell-killing. Together, these pre-clinical data suggest that EKB-569 is a radiosensitizer of squamous cell carcinoma and may mechanistically involve selective targeting of IR-induced NFκB-dependent survival signaling. Further pre-clinical in-vivo studies are warranted.
Irreversible EGFR Inhibitor EKB-569 Targets Low-LET c-
Radiation-Triggered Rel Orchestration and Potentiates
Cell Death in Squamous Cell Carcino ma
Natarajan Aravindan
2,4,5
, Charles R. Thomas J
3
, Sheeja Aravindan
4
, Aswathi S. Mohan
1
, Jamunarani
Veeraraghavan
2,5
, Mohan Natarajan
1
*
1 Department of Otolaryngology, Head and Neck Surgery, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America,
2 Department of Radiation Oncology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America, 3 Dep artment of Radiation
Medicine, Oregon Health and Science University Knight Cancer Institute, Portland, Oregon, United States of America, 4 Department of Pathology, The University of
Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America, 5 Department of Pediatrics, The University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma, United States of America
Abstract
EKB-569 (Pelitinib), an irreversible EGFR tyrosine kinase inhibitor has shown potential therapeutic efficiency in soli d
tumors. However, cell-k illing pot ential in combination with radiotherapy and its underlying molecular orchestration
remain to be ex plored. The objective of this study was to determine the effect of EKB-569 on ionizing radiation (IR)-
associated NFkB-dependent cell death. SCC-4 and SCC-9 cells exposed to IR (2Gy) with and without EKB-569 treatment
were analyzed for transactivation of 88 NFkB pathw ay molecules, NFkB DNA-binding activity, translation of the NFkB
downstream mediators, Birc1, 2 and 5, cell viability, metabolic activity and apo ptosis. Selective targeting of IR-induced
NFkB by EKB-569 and its influence on cell-fate were assessed by ov erexpressing (p50/p65) and silencing (DIkBa)NFkB.
QPCR profiling after IR exposure revealed a significant induction of 74 NFkB signal transduction molecules. Of tho se, 72
were suppressed with EKB-569. EMSA revealed a dose dependent inhibi tion of NFkB by EKB- 569. More importantly, EKB-
569 inhibited IR-induced NFkB in a dose-dependent manner, and this inhibition was sustained up to at least 72 h.
Immunoblotting revealed a significant suppression of IR-in duced Birc1, 2 and 5 by EKB-569. We observed a dose-
dependent inhibition of cell viability, metabolic activity and apoptosis with EKB-569. EKB-569 significantly enhanced IR-
induced cell death and apoptosis. Blocking NFkB improved IR-induced cell death. Conversely, NFkB overexpression
negates EKB-569 -induced cell-killing . Together, these pre-clinical data suggest that EKB-569 is a radiosensitizer of
squamous cell carcinoma and may mechanistically involve selective targeting of IR-induced NFkB-dependent survival
signaling. Further pre-clinical in-vivo studies are warranted.
Citation: Aravindan N, Thomas CR Jr, Aravindan S, Mohan AS, Veeraraghavan J, et al. (2011) Irreversible EGFR Inhibitor EKB-569 Targets Low-LET c-Radiation-
Triggered Rel Orchestration and Potentiates Cell Death in Squamous Cell Carcinoma. PLoS ONE 6(12): e29705. doi:10.1371/journal.pone.0029705
Editor: Christina Lynn Addison, Ottawa Hospital Research Institute, Canada
Received August 8, 2011; Accepted December 1, 2011; Published December 29, 2011
Copyright: ß 2011 Aravindan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported, in whole or in part, by National Institutes of Health Grant R01 CA112175 (to M.N.) and funds from the Office of Science
(Biological and Environmental Research), United States Department of Energy Grant No. DE-FG03-02ER63449 (to M.N.). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: natarajan@uthscsa.edu
Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth
most common cancer in the world and accounts for 90% of
malignant neoplasias of the upper respiratory system [1]. Despite
recent advances in the management of locally advanced HNSCC,
the overall survival of patients has improved only marginally over
the past three decades [2] mainly due to development of therapy-
induced chemo and radioresistance. To that note, in recent years
there has been substantial interest in developing novel therapeutic
agents that specifically target growth factor pathways that, are
dysregulated in tumor cells. Such targeted ‘‘biological’’ agents
might offer alternative treatment options for patients refractive to
chemoradiotherapy. Also, with unique mechanisms of action and
toxic profiles that generally do not overlap, targeted agents and
standard therapies can be used in combinations to enhance overall
treatment efficacies and prevent dose reduction.
Because many solid tumors, including HNSCC have hyper
activated epidermal growth factor receptor (EGFR) [3,4], there
has been great interest in the use of EGFR inhibitors to control
cancer growth. EGFR is a 170 kDa glycoprotein containing an
extracellular ligand binding domain, and an intracellular tyrosine
kinase (TK) domain [5]. Upon binding to ligands such as EGF or
TGFa, EGFR dimerizes with itself (homodimers) or other
members of the family such as c-ErbB-2 (heterodimers). Upon
dimerization, TK activation increases and receptor gets autopho-
sphorylated at tyrosine residues. Phosphorylated EGFR (p-EGFR),
like other activated receptor TKs, involved in phosphorylation and
activation of several signal transduction pathways including
phosphoinositide 3-kinase-AKT, extra cellular signal-regulated
kinase 1and 2 (ERK1/2), and the signal transducer and activator
of transcription 3 (STAT3). Activation of these signal transduction
pathways subsequently activate key transcriptional machineries
such as NFkB that promote tumor growth and progression by
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r.
inducing inhibition of apoptosis, proliferation, maturation,clonal
expansion, invasion, and metastasis.
NFkB is a member of the c-rel proto-oncogene family found
within the promoter and enhancer region of a wide variety of
genes involved in proliferation, cell cycle control [6,7], oncogenic
activation [8], cell growth, differentiation and metastasis [9,10].
NFkB is retained in the cytoplasm by association with the
inhibitory protein IkB. On phosphorylation, IkB is ubiquitinated
and subsequently degraded by the 26S proteasome, resulting in
the liberation of NFkB. NFkB can then enter into the nucleus to
regulate the expression of downstream genes. Elevated NFkB
activity has been linked with tumor resistance to chemotherapy
and IR [11] in a number of cancer types, including head and neck
cancer [12]. Conversely, inhibition of NFkB favors pro-apoptotic
processes, decreases growth and clonogenic survival [13–15] and
enhances chemo/radiosensitivity [16–20]. In addition to this
persistant activation of growth-promoting signaling pathways,
development of HNSCC also involves the accumulation of genetic
and epigenetic alterations in tumor-suppressor proteins.. The
activation of EGFR is a frequent event in HNSCC, and has
provided the molecular basis for current efforts aimed at
evaluating the clinical activity of EGFR inhibitors in HNSCC
[21,22]. However, to date, the role of EGFR-dependent NFkBin
the functional orchestration of HNSCC progression and metastasis
is poorly realized [22,23]. Since NFkB is able to regulate more
than 150 genes, and is able to functionally orchestrate many steps
in carcinogenesis, tumor progression and metastasis, it is important
to delineate the efficacy of potential EGFR-TK inhibitors that
target the NFkB-dependent HNSCC cell survival advantage.
The two most commonly employed strategies in drug
development are introducing covalent (irreversible) binding of
the drug target and and broadening the affected receptor tyrosine
kinase targets of the drug within the cell. Currently, the second
generation of EGFR TKI compounds is emerging from the drug
developmental pipeline and being introduced into clinical trials.
Many of these second-generation compounds form tighter
covalent bonds with their target, which should theoretically
increase their effectiveness by prolonging the inhibition of EGFR
signaling to the entire lifespan of the drug-bound receptor
molecule. In cell culture systems, such irreversibly binding TKIs
can effectively kill cells that have acquired resistance to first-
generation TKIs [24]. As per the other common theme of drug
development, second-generation EGFR TKI have been developed
that, in addition to blocking EGFR signaling, target multiple
kinases in the ErbB family. The signaling network that emerges
from the ErbB family of transmembrane TK receptors (of which
EGFR is a member) is large, interconnected, and redundant, with
many possible routes between the ligand at the cell surface and the
message destination within the nucleus [25]. It is this diversity in
possible signal transduction routes that allows a cell to have
flexibility and, in the case of cancer cells treated with anticancer
agents, facilitates resistant cell clones that bypass the inhibited
receptor [26]. Blocking multiple signaling pathways with either a
combination of agents or a single but multi-targeted agent has
been synergistic in its effects in preclinical models [27]. Second-
generation EGFR TKIs have been developed that target
additional members of the ErbB family or other downstream
or parallel pathways such as the NFkB pathway’. EKB-569
(Pelitinib; WAY-172569), a 4-Dimethylamino-but-2-enoic acid
[4-(3-chloro-4-flurophenylamino)-3-cyano-7ethoxy-quinolin-6-yl]-
amide is one such second generation irreversibly-binding inhibitor
of EGFR TK activity [28]. In this study, we examined the efficacy
of EKB-569 in inhibiting ionizing radiation (IR)-induced NFkB
activity, in modulating the transcription of 88 NFkB-dependent
signal transduction molecules, in activating translation of NFkB-
mediated downstream Birc1, 2 and 5 protein, in reducing cell
viability, and metabolic activity and apoptosis. Further, we
delineated the selective targeting of IR-induced NFkB through
EKB-569 and its direct influence in HNSCC cell-fate.
Materials and Methods
Cell Culture
Human tongue squamous cell carcinoma SCC-4 and SCC-9
cells were obtained from ATCC (Manassas, VA) and maintained
as monolayer cultures in DMEM/F-12 50/50 (Mediatech Inc.,
Herndon, VA) growth medium supplemented with 1.5 g/L
sodium bicarbonate, 2 mM L-glutamine, 15 mM HEPES, 1%
NEAA, 1% MEM vitamins, 5000 I.U/ml penicillin/5000
mg/ml
streptomycin, 1% sodium pyruvate, and 10% FBS (Invitrogen,
Carlsbad, CA). For passage and for all experiments, the cells were
detached using trypsin (0.25%)/EDTA (1%), resuspended in
complete medium, counted (Countess, Invitrogen) and incubated
in a 95% air/5% CO2 humidified incubator.
Irradiation experiments
SCC-4 and SCC-9 cells were exposed to 2Gy using Gamma
Cell 40 Exactor (Nordion International Inc, Ontario, Canada) at a
dose rate of 0.81Gy/min. Irradiated cells were examined for IR-
induced alterations in NFkB signal transduction, selective yet,
sustained NFkB activity, NFkB’s role in survival advantage and to
identify the efficacy of EKB-569 on IR-induced NFkB dependent
HNSCC progression. Mock irradiated cells were treated identical
except that the cells were not subjected to IR. Irradiated cells were
incubated at 37uC for additional 1, 3, 6, 24, 48 and 72 h. All
experiments were repeated at least three times in each group.
Plasmid preparation and DNA Transfection
Transient transfection of NFkB p65 and p50 subunits was
carried out by the lipofection method using Effectene
TM
reagent
(Qiagen, Inc., Valencia, CA) as described in our earlier studies
[29]. NFkB inhibition was achieved using transient transfection of
S32A/S36A double mutant IkBa (DIkBa, Upstate biotechnology,
Lake Placid, NY) as reported in our earlier studies [29] . The
mutated form of IkBa with a serine-to-alanine mutation at
residues 32 and 36 does not undergo signal-induced phosphory-
lation and thus remains bound to NFkB subsequently preventing
nuclear translocation and DNA binding. After 18 h, transfection
medium was replaced with growth medium before IR.
Electrophoretic Mobility Shift Assay (EMSA)
Nuclear protein extraction and electrophoretic mobility shift
assay for NFkB, AP-1 and SP-1 were performed as described in
our earlier studies [29]. Autoradiograms were overexposed in
order to reveal the low inhibitory effects that were below the
constitutive level. Densitometry analysis was performed using a
BioRad Multi-Analyst software package with an integrated density
program. Group-wise comparisons were made using ANOVA
with Tukey’s post-hoc correction. A P value of ,0.05 is considered
statistically significant. For the competition assay, the nuclear
extract was pre-incubated with unlabeled homologous NFkB
oligonucleotide followed by addition of [c-
32
P]-ATP labeled NFkB
probe. Supershift analysis was performed as described earlier [29].
Immunoblotting
Total protein extraction and immunoblotting were performed
as described in our earlier studies [29]. Rabbit polyclonal anti-
IkBa, Birc1, 2, 5 or Bax antibody (Santa Cruz) were used to detect
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the respective protein expression levels between the EKB treated,
IR exposed and control groups. Blots were stripped and reprobed
with mouse monoclonal anti-a-tubulin antibody (Santa Cruz) to
determine equal loading of the samples. One diamentional gel
analysis was performed using a BioRad Multi-Analyst software
package with an integrated density program. Group-wise com-
parisons were made using ANOVA with Tukey’s post-hoc
correction. A P value of ,0.05 is considered as statistically
significant.
Real-Time QPCR profiling of NFkB signaling pathway
molecules
Total RNA extraction and real-time QPCR profiling were
performed as described in our earlier studies [29] . We used human
NFkB signaling pathway profiler (Realtimeprimers.com, Elkins
Park, PA) containing 88 genes representing 8 functional groups
including (i) Rel/NFkB/IkB family, (ii) NFkB responsive genes, (iii)
Ligands & Transmembrane receptors, (iv) Adaptor proteins, (v)
Signal transduction kinases, (vi) Transcription factors, (vii) Cell
death/survival molecules, and (viii) Other factors. We started with
this highly selected QPCR profiler instead of an all-encompassing
gene array because the selected genes entail a well-characterized
profile governing NFkB signal transduction and transcriptional
targets, hence facilitating interpretation of data, simplifying data
acquisition and analysis, and avoiding genes not functionally
characterized. Furthermore, QPCR profiling allows detection and
quantification of gene expression in real-time. Each profiling plate
was also equipped with reverse transcription control, positive PCR
control, genomic DNA control and five housekeeping genes b-
Actin, GAPDH, Rpl13a, HPRT1 and b2M. The DD
ct
values were
calculated by normalizing the gene expression levels to the
expression of the housekeeping genes. The normalized data were
then compared between groups, and the relative expression level of
each gene was expressed as fold change. When comparing each
gene’s signal intensity between groups, we used a twofold or more
($2 fold) increase or decrease to represent ‘‘stringent’’ criteria for
upregulation or downregulation and an increase/decrease of ,2
fold to represent ‘‘less stringent’’ criteria. Classifying gene regulation
criteria in this manner can provide an index of reliability of the gene
expression data [29].
Cell Viability
Trypan blue dye exclusion assay was used to identify IR
modulated cell viability in HNSCC cells and further, to determine
the efficacy of EKB-569 in this setting. Cells exposed to IR alone
and cells pre-treated with EKB-569 followed by exposure to IR,
were sequentially analyzed with the Countess automated cell
counter (Carlsbad, CA). Furthermore, to determine the efficiency
of EKB-569 in targeting IR-induced NFkB dependent cell
viability, trypan blue exclusion assay was performed in NFkB
over-expressed HNSCC cells exposed to EKB-569. Group-wise
comparisons were made using ANOVA with Tukey’s post-hoc
correction. A P value of ,0.05 is considered statistically
significant.
Cell survival by MTT assay
Cell survival was analyzed using MTT assay as described in our
previous studies [29]. HNSCC cells at a density of 1000 cells/
300
ml in a 24-well plate were either (i) mock-irradiated, (ii)
exposed to IR alone, (iii) treated with EKB-569 (0.5, 1.0, 2.0 and
5.0
mg) alone, (iv) pretreated with EKB-569 (5.0 mg) followed by
exposure to IR, (v) prior transfection with DIkBa followed by
exposure to IR, or (vi) prior transfection with p50/p65 treated
with or without EKB-569. The treated and/or exposed cells were
added with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoli-
um bromide (30
mL/well from 5 mg/mL stock) for 4 h after 24,
48 and 72 h of post-IR. Solubilization of converted purple
formazan dye was accomplished by acid-isopropanol with
continuous shaking at 37uC. The reaction product was quantified
by measuring the absorbance at 570 nm using Synergy II micro
plate reader (Biotek). Cell survival response was compared using
ANOVA with Tukey’s post-hoc correction.
Nuclear morphology by dual staining
SCC-4 cells (5610
5
cells in 500 ml of complete growth medium)
grown in 4-well plate (Nunc) were either: 1) sham treated, 2)
treated with EKB-569 (0.5–5.0
mg), 3) exposed to IR with or
without prior EKB-569/DIkBa transfection, or 4) transfected with
p50/p65 subunit with or without prior EKB-569 treatment. The
cells were analyzed for nuclear morphology as described earlier
[30]. In brief, the medium was replaced with a fresh medium
containing reduced serum (2%) without any added growth factors
and incubated further for 16 h at 37uC in air/CO2 incubator.
The cells were then stained with acridine orange (1
mg/ml) and
ethidium bromide (1
mg/ml) and immediately examined for the
morphological characteristics of apoptosis at 2006 magnification
using an Olympus VANOX fluorescent microscope. Four
morphological states were examined: (1) viable cells with normal
nuclei (bright green chromatin with organized structure); (2) viable
cells with apoptotic nuclei (green chromatin which are highly
condensed and/or fragmented); (3) non-viable cells with normal
nuclei (bright orange chromatin with organized structure); and (4)
non-viable cells with apoptotic nuclei (bright orange chromatin
which is highly condensed or fragmented).
Results
EKB-569 selectively inhibits IR-induced persistent
activation of NFkB
The effect of EKB-569 in selectively inhibiting IR-induced
NFkB-DNA binding activity was elucidated using four different
approaches. First, we investigated whether EKB-569 as a stand-
alone compound, could modulate NFkB activity in both SCC-4
and SCC-9 HNSCC cells. Compared to untreated cells, EKB-569
treatment dose-dependently inhibited NFkB DNA binding activity
with a substantial inhibition at 5.0
mg (Fig. 1A & B). Next, to
unveil the radiosensitizing efficacy of EKB-569, HNSCC cells
mock-irradiated, exposed to IR or treated with EKB-569 (0.5, 1.0,
2.0 or 5.0
mg) and then exposed to IR were analyzed for
alterations in NFkB activity. Unlike the mock-IR controls, IR at
2 Gy significantly (P,0.001) induced NFkB-DNA binding activity
in both SCC-4 and SCC-9 cells (Figure 1 A & B, bottom panel).
This IR-induced NFkB activity was drastically (P,0.001) inhibited
with EKB-569 treatment in a dose dependent manner (Fig. 1D) in
both cell types. It is interesting to note that at 5.0
mg
concentration, EKB-569 completely suppressed IR-induced NFkB
activity even below the constitutive (mock-IR) levels in this setting.
Further, to delineate whether EKB-569 persistently inhibits IR-
induced NFkB or there is recovery of IR-induced NF-kB activity
over time, SCC-4 cells pretreated with EKB-569 and exposed to
IR were examined for 3 days post-radiation exposure. EKB-569-
induced inhibition of IR-induced NF kB DNA-binding activity
remained at the same decreased level at all time points investigated
(Figure 1E). Densitometric analysis revealed a significant
(P,0.001) inhibition of IR-induced NFkB DNA-binding activity
up to at least 3 days post-radiation exposure. (Fig. 1F). To confirm
the specificity of the EMSA band seen in Figure 1 A and B, a
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competition binding assay was performed. The NFkB DNA-
binding activity was competitively reduced to 47% and 36.4% by
the addition of 0.02 and 0.2 pmol of homologous unlabeled NFkB
specific-double stranded oligonucleotide, respectively. Supershift
analysis with p50 and p65 antibodies further confirmed that the
gel shifted bands are indeed NFkB (data not shown).
Figure 1. Effect of EKB-569 on radiation modulated NFkB, AP1 and SP1 DNA binding activity. A representative autoradiograms showing
the NFkB-DNA binding activity in the nuclear extracts of human SCC-4 cells (A) or SCC-9 cells (B) that are either treated with EKB-569 alone (upper
panel) or in combination with IR (Lower panel). NF-kB-specific bands are indicated by an arrow head. Autoradiogram was slightly overexposed to
reveal EKB-inhibited NF-kB-specific bands. Densitometric analysis of three independent experiments showing dose-dependent inhibition of NFkB-
DNA binding activity in SCC-4 cells (C) and SCC-9 cells (D). (E) Time-dependent inhibition of NFkB-DNA binding activity in human SCC-4 cells by EKB-
569 (5.0
mg) in the presence or absence of IR exposure. EMSA was carried out in the nuclear extract at 1, 3, 6, 24, 48 and 72 h post-exposure. (F)
Representative autoradiogram from three independent experiments showing AP-1 DNA binding activity in SCC-4 cells treated with EKB-569 (1.0, 2.0
and 5.0
mg) or exposed to IR in the presence or absence of EKB-569. (G) Representative autoradiogram from three independent experiments showing
SP-1 DNA binding activity in SCC-4 cells treated with EKB-569 (0.5, 1.0, 2.0 and 5.0
mg) or exposed to IR in the presence or absence of EKB-569.
doi:10.1371/journal.pone.0029705.g001
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Next, to demonstrate that the inhibition of NFkB signaling
pathway is not a EKB-569 compound-specific effect and that the
proposed combination (IR and EGFR inhibition) can be carried
out on to the clinic with any other EGFR compound, we
incubated the SCC-4 cells with other commonly used irreversible
EGFR blockers, afatinib and neratinib (HKI-272). Afatinib and
neratinib dose-dependently inhibit NF-kB DNA-binding activity
(Figure 2 A, B, C & D). The inhibition of NFkB was found to be
persistent up to at least 72 h (Figure 2H). To further validate
whether EKB-569 directly inhibits NFkB activity, we examined
the inhibitory effect on the activity of unpstream kinases. Data
presented in Figure 2G shows EKB-569 and other related EGFR
inhibitors, afatinib (300 nM) and neratinib (200 nM) significantly
block the expression of IR-induced upstream IkB kinase beta
(IKK-b). Additionally, we confirmed that EKB-569-mediated
inhibition of NF-kB is EGFR-dependent. EGFR-knockdown
experiments with a widely used specific EGFR inhibitor,
PD153035 were performed to confirm the EGFR-mediated NFkB
Figure 2. Effect of EGFR inhibitors on NFkB DNA binding activity, EGFR mRNA and, EGFR and IKKb protein levels. (A) Representative
autoradiogram showing the NFkB-DNA binding activity in the nuclear extracts of human SCC-4 cells exposed to IR (2Gy) or treated with 50, 100 or
200 nM HKI-272 (neratinib) prior to IR exposure. Neratinib treatment significantly inhibited IR-induced NFkB DNA binding activity (Left panel).
Representative autoradiogram showing the NFkB-DNA binding activity in human SCC-4 cells exposed to 50, 100 or 200 nM neratinib (Right panel).
Compared to the mock-IR cells, neratinib induced a dose-dependent suppression of NFkB activity in these cells. (B) Representative autoradiogram
showing the NFkB-DNA binding activity in human SCC-4 cells exposed to IR with or without Neratinib (200 nM) and harvested after 1, 3, 6, 24, 48 and
72 h. Neratinib persistently inhibited IR-induced NFkB-DNA binding activity at all time points investigated. (C) Representative autoradiogram
showing the NFkB-DNA binding activity in human SCC-4 cells exposed to 100, 200 or 300 nM afatinib. Compared to the mock-IR cells, afatinib
induced a dose-dependent suppression of NFkB activity (Left panel). Representative autoradiogram showing the NFkB-DNA binding activity in SCC-4
cells exposed to IR or treated with 100, 200 or 300 nM afatinib and exposed to IR. Afatinib treatment significantly inhibited IR-induced NFkB DNA
binding activity (Right panel). (D) Representative autoradiogram showing the NFkB-DNA binding activity in human SCC-4 cells exposed to IR with or
without afatinib (300 nM) and harvested after 1, 3, 6, 24, 48 and 72 h. Afatinib treatment persistently inhibited IR-induced NFkB-DNA binding activity
at all time points investigated. (E) Representative autoradiogram showing the NFkB-DNA binding activity in human SCC-4 cells exposed to IR or
treated with 50, 75 or 100 nM PD 153035 hydrochloride (a potent EGFR-TK inhibitor) and exposed to IR. PD153035 treatment induced a significant
dose-dependent inhibition of IR-induced NFkB DNA binding activity. (F) Real-time QPCR analysis showing EGFR mRNA levels in SCC-4 cells mock-
irradiated, exposed to 2Gy and in cells treated either with EKB-569 (5.0
mg) or PD153035 (50 nM) and exposed to IR. (G) Immunoblot showing
complete suppression of radiation induced EGFR and IKKb levels in SCC-4 cells pretreated with EKB-569 (5.0
mg), afatinib (300 nM), neratinib (200 nM)
or PD153035 (75 nM). (H) QPCR analysis showing complete and sustained (up to 72 h) suppression of radiation induced EGFR transcriptional levels in
SCC-4 cells treated with either afatinib (300 nM) or neratinib (200 nM).
doi:10.1371/journal.pone.0029705.g002
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inhibition. Cells incubated with PD153035 at concentrations 50,
75 and 100 nM clearly showed a significant decrease in radiation-
induced NFkB DNA binding activity and mRNA expression
similar to the cells incubated with EKB-569 (Figure 2 E&F).
In order to determine whether EKB-569 selectively targets
NFkB or the global transcription machinery in general, we
analyzed the effect of EKB-569 on IR-modulated AP-1 and SP-1
transcription factors. SCC-4 cells mock-irradiated, treated with
EKB-569 (0.5–5.0
mg), exposed to IR or, treated with EKB-569
(0.5–5.0
mg) and then exposed to IR were examined for AP-1 and
SP-1 DNA binding activity (Figure 1 F&G). In contrast to the NF-
kB pathway response, EKB-569 by itself, without radiation
exposure, fails to inhibit the constitutive levels of AP-1 DNA-
binding activity. On the other hand, with regard to SP-1, EKB-
569 inhibits its activity at the lower concentrations of 1 and 2 ug,
but not at the higher (5 ug) concentration. More interestingly, with
the addition of EKB-569 further increased the activation of AP-1
and the SP-1 induced by IR exposure. These results confirmed
that the mechanism of EKB-569-mediated radiosensitization is
acting specifically through NF-kB pathway.
EKB-569 inhibits IR-induced transcriptional modulation of
NFkB signal transduction and pathway molecules in
HNSCC cells
To further to substantiate our findings of IR-induced NFkB
activation and EKB-569 associated selective targeting, SCC-4 cells
mock-irradiated, exposed to IR or pretreated with EKB-569
(5.0
mg) and then exposed to IR were examined for transcriptional
changes in 88 NFkB signal transduction and downstream target
genes (Figure S1). Compared to mock-IR controls, IR exposure
upregulated 74 genes, down regulated two genes, while having no
effect on the expression of 12 genes. Though, originally we
intended to classify the gene expression implying less stringent
(overall) and stringent ($2 fold) criteria, there is only one gene,
Myd88 showed less than 2 fold (1.4) while remaining 73 genes
showed significant ($2 fold) upregulation compared to untreated
control. Conversely, EKB-569 pre-treatment profoundly inhibited
72 of 74 IR-induced genes in this setting (Figure 3). Interestingly,
expression of two genes, TLR4 and Ppm1A were significantly
increased with EKB-569. A plethora of scientific literature
demonstrates the functional significance of these NFkB-dependent
signaling and target molecules in tumor cell radioresistance
suggesting that inhibitory approaches of these molecules may
benefit radiosensitization.
EKB-569 regulates NFkB dependent downstream Birc 1, 2
and 5 and upregulates pro-apoptotic Bax in HNSCC cells
QPCR profiling demonstrated a significant inhibition of IR-
induced NFkB-dependent downstream pro-survival protein, Birc 2
and 5 upon EKB-569 treatment (Figure 3). In order to confirm the
IR-induced modulations and to validate the functional significance
of EKB-569-mediated regulation, we investigated whether the
transcriptional machinery modulation is in fact translated to the
protein level. First, immunoblotting analysis confirmed the
involvement of post-translational modification of IkB in IR-
induced NFkB. Further, we observed a significant setback of IR-
inhibited IkBa levels upon EKB-569 treatment. This correlated
well with induced NFkB activity data (Figure 1 A–D). Compared
to mock-IR controls, we observed a significant induction of BIRC
2 and 5 levels (Figure 4 A&B) reflecting and correlating well with
their mRNA expression levels. More importantly, treatment with
EKB-569 completely (P,0.001) inhibited IR-induced BIRC2 and
5 in SCC-4 cells. Though IR did not show induced expression of
BIRC 1 in this setting, we observed a conferring inhibition of this
protein with EKB-569. Conversely, we observed a significant
induction of pro-apoptotic Bax in cells pre-treated with EKB-569.
EKB-569 confers radiosensitization in HNSCC cells
To identify the efficacy of EKB-569 at the cellular or tissue level
of HNSCC radiosensitization, we examined their potential in
conferring functional endpoints like cell viability, survival and
apoptotic death. First, trypan blue exclusion assay demonstrated
that EKB-569 as a stand-alone compound induced dose-
dependent inhibition of SCC-4 cell viability with a maximum
(P,0.001) inhibition at 5.0
mg concentration (Figure 4C). Simi-
larly, unlike the mock-irradiated control, cells exposed IR
significantly (P,0.001) inhibited HNSCC cell viability
(Figure 4D). More importantly, compared to IR exposed cells,
EKB-569 (5.0
mg) treatment significantly (P,0.001) conferred IR-
inhibited cell viability. Substantiating our cell viability data, MTT
analysis revealed a dose dependent inhibition of metabolic activity
with EKB-569 treatment (Figure 4E). To that end, at low
concentration (0.5
mg) we did not see any significant inhibition
of cell survival. However, with increase in EKB-569 concentration
we observed a significant (1.0
mg, P,0.05; 2.0 mg, P,0.01 and
5.0
mg, P ,0.001) inhibition of cell survival in this setting. On the
other hand, compared to mock-irradsiated, cell exposed to IR
showed significant (P,0.01) suppression of cell survival (Figure 4E).
Addition of EKB-569 significantly conferred IR-inhibited cell
survival in a dose dependent fashion. Even concentrations as low
as 0.5
mg significantly conferred IR-induced cell death and we
observed a complete inhibition of cell survival in IR-exposed cells
with 5.0
mg demonstrating the radiosensitizing potential of EKB-
569 in HNSCC cells. Further, nuclear morphology with dual
staining showed bright green chromatin with organized structures
in untreated control cells indicating viable cells with normal nuclei
(Figure 4F). Where as, cells treated with EKB-569 showed typical
apoptotic features of bright orange chromatin with blebbing,
nuclear condensation, and fragmentation. We observed a dose
dependent increase in apoptosis after 0.5, 1.0, 2.0 and 5.0
mgof
EKB-569. Consistent with our cell viability and survival data, we
observed an induced cell death in cells exposed to IR with bright
orange chromatin with blebbing, nuclear condensation, and
fragmentation. More importantly, compared to IR alone, cells
pre-treated with EKB-569 (5.0
mg) and exposed to IR showed
extensive apoptotic characteristics and demonstrated a radiosen-
sitizing potential in HNSCC cells (Figure 4F).
EKB-569 targets IR-induced NFkB- regulated
radiosensitization
To further identify whether targeting IR-induced NFkB
orchestrates EKB-569-induced radiosensitization in HNSCC cells,
we adopted two approaches. First, we determined whether IR-
induced NFkB regulates induced radioprotection in SCC-4 cells.
To achieve this we investigated the alterations in cell viability,
survival and death after muting IR-induced NFkB. Ecotopic
expression of IR-induced NFkB was inhibited by transient
transfection of DIkBa. Knocking-out IR-induced NF kB was
confirmed with EMSA (Figure 5A&B). Compared to vector
controls, knocking out IR-induced NFkB with DIkBa significantly
(P,0.001) conferred IR-inhibited cell survival (Figure 5C), cell
viability (Figure 5D) and enhanced IR-induced cell death (evident
with bright orange chromatin with blebbing, nuclear condensa-
tion, and fragmentation) dictating the role of IR-induced NFkBin
radioresistance. Next, to identify that EKB-569 induced radiosen-
sitization occurs at least in part by targeting IR-induced NFkB,
p50/p65 over-expressed SCC-4 cells were treated with EKB-569
EKB Radiosensitizes Squamous Cell Carcinoma
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EKB Radiosensitizes Squamous Cell Carcinoma
PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29705
and analyzed for cell viability, survival and death. EMSA analysis
(Figure 5A) confirmed the robust NFkB DNA-binding activity in
p50/p65 transfected SCC-4 cells (Figure 5B). Further, over
expression of NFkB in these cells significantly (P ,0.001) induced
cell survival (Figure 5C) and showed bright green chromatin with
organized nuclear morphology (Figure 5E) and, served as positive
controls for our study. Consequently, treatment with EKB-569
significantly (P,0.001) inhibited cell viability, survival and showed
bright orange chromatin with blebbing, nuclear condensation, and
fragmentation in these NFkB over-expressed cells (Figure 5C–E)
Figure 3. Real time QPCR profiling: Histograms showing IR-induced NFkB-dependent downstream signal transduction molecules
and the effect of EKB-569 (5.0
mg) on these IR-modulated genes in human SCC-4 cells.
doi:10.1371/journal.pone.0029705.g003
Figure 4. Effect of EKB-569 on radiation modulated prosurvival signaling molecules, cell viability, survival and/or death. (A)
Representative immunoblot showing expression levels of IkBa, pro-apoptotic Bax and anti-apoptotic Birc1, 2 and 5 in human SCC-4 cells exposed to
IR or treated with EKB-569 (5.0
mg) prior to IR exposure. a-tubulin was used to show equal loading of protein samples. (B) Semi-quantitative 1D gel
analysis showing increased IkBa and Bax levels in EKB-569 treated cells. EKB-569 treatment significantly suppressed Birc1, 2 and 5 in these IR-exposed
SCC-4 cells. (C) Histograms showing the percent cell viability in cells treated with EKB-569 (0.5, 1.0, 2.0 and 5.0
mg). EKB-569 inflicted a dose
dependent inhibition of cell viability in this setting. (D) Histograms showing the percent cell viability in cells either mock-irradiated, exposed to
IRexposure or treated with EKB-569 (5.0
mg) and exposed to IR. Compared to the mock-IR cells, IR resulted in reduced cell viability. Relatively, EKB-569
treatment significantly conferred IR-inhibited cell viability. Cell viability was measured using Trypan-blue dye exclusion assay and counted in
automated countess cell counter. (E) Cell survival in mock-IR, EKB-569 (0.5, 1.0, 2.0 and 5.0
mg) treated and in irradiated cells with or without EKB-569
treatment. MTT assay was used to analyze the induced cytotoxicity and the reaction product was quantified by measuring the absorbance at 570 nm.
Percent cell survival was calculated as (mean of test wells/mean of control wells) 6100 and compared using ANOVA. EKB-569 induced a dose
dependent inhibition of cell survival. Like-wise IR suppressed cell survival and this IR-inhibited cell survival was further inhibited with EKB-569ina
dose dependent fashion. (F) Nuclear morphology with dual staining showing apoptotic characteristics in cells either mock-IR, treated with EKB-569
(0.5, 1.0, 2.0, 5.0
mg), exposed to IR, or treated with EKB-569 and exposed to IR.
Insert:
High magnification photomicrographs showing chromatin with
organized structures indicating viable cells with normal nuclei in untreated control cells and, chromatin with blebbing, nuclear condensation, and
fragmentation indicating typical apoptotic characteristics in cells treated with 5.0
mg of EKB-569 and exposed to IR.
doi:10.1371/journal.pone.0029705.g004
EKB Radiosensitizes Squamous Cell Carcinoma
PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e29705
delineating that EKB-569 target NFkB and potentiate cell death in
this setting.
Discussion
Primary and acquired resistance to conventional chemotherapy
and radiotherapy represent the central therapeutic challenge in
oncology today. Resistance may develop through varied mecha-
nisms, including increased expression of cellular drug efflux
pumps; mutation of the therapeutic target; increased activity of
DNA repair mechanisms and altered expression of genes involved
in apoptotic pathways. To overcome these resistance mechanisms,
conventional cancer treatments are increasingly combined with
molecularly targeted therapies. Because cytotoxic and targeted
therapies have distinct biologic effects and toxicity profiles, such
combinations are both rational and well tolerated. To date, the
molecular pathway most frequently targeted in combination with
conventional chemotherapy or radiotherapy is that of the EGFR.
After activation by binding of the EGF and other natural ligands,
EGFR activates prosurvival, pro-angiogenic, and anti-apoptotic
pathways that may confer resistance to cytotoxic therapies.
Interestingly, all these aforementioned functional pathways are
known to be controlled by transcriptional master switch regulator,
NFkB that also happens to be a downstream target for EGFR. In
Figure 5. IR-induced NFkB regulates radioresistance in HNSCC cells. (A) Representative autoradiogram of EMSA analysis showing complete
muting of NFkB DNA binding activity in IR-induced or NFkB overexpressed cells with DIkBa.(B) Densitometric analysis of NFkB-DNA binding activity
showing significant NFkB silencing with DIkBa and significant activation with p50/p65 transfection with NFkB over expression vectors, p50 and p65.
(C) Histograms showing the results of MTT analysis in p50/p65 over-expressed cells treated with EKB-569 (5.0
mg). NFkB over-expression robustly
induced SCC-4 cell survival. Conversely, treating NFkB over-expressed cells with EKB-569 completely (P,0.001) inhibited NFkB-induced SCC-4 cell
survival. Like-wise, muting NFkB (with DIkBa) completely inhibited IR-induced cell survival. (D) Histograms showing cell viability in NFkB muted cells
exposed to IR or NFkB overexpressed cells treated with EKB-569. Silencing NFkB significantly inhibited IR-induced cell viability. Like-wise, treating
NFkB overexpressed cells with EKB-569 (5.0
mg) completely inhibited NFkB-induced cell viability. (E) Nuclear morphology with dual staining showing
typical yet increased apoptotic characteristics in NF kB muted cells exposed to IR. NFkB overexpressed cells displayed chromatin with organized
structures indicating good viability with normal nuclei. However, treatment with EKB-569 (5.0
mg) significantly inflicted chromatin with blebbing,
nuclear condensation, and fragmentation in these NFkB overexpressed cells.
doi:10.1371/journal.pone.0029705.g005
EKB Radiosensitizes Squamous Cell Carcinoma
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this study, we investigated the specific inhibitory effect of EGFR
TK inhibitor EKB-569 on the regulation of NFkB-dependent
survival advantage and elucidated its influence in potentiating
radiotherapy for head and neck cancers. To our knowledge, for
the first time, we have demonstrated the specific inhibition of IR-
induced NFkB with irreversible EGFR TK inhibitor, EKB-569
and dissected out the functional downstream signaling that
orchestrate in promoting radiosensitization at least in head neck
cancer.
Our results indicate that radiation at clinically relevant doses
activated NFkB pathway in SCC-4 cells through the mechanism
that interacted with EGFR. To that note, activation of EGFR
intrinsic receptor protein TK and tyrosine autophosphorylation
results in the activation of a number of key signaling pathways
[31]. One major downstream signaling route is via Ras-Raf-
MAPK pathway [32] where activation of Ras initiates a multistep
phosphorylation cascade that leads to the activation of ERK1 and
2 [33] that regulate transcription of molecules that are linked to
cell proliferation, survival, and transformation [33]. Another
important target in EGFR signaling is PI3K and the downstream
protein-serine/threonine kinase Akt [34,35] which transduces
signals that trigger a cascade of responses from cell growth and
proliferation to survival and motility [35]. One more route is via
the stress-activated protein kinase pathway, involving protein
kinase C and Jak/Stat. Interestingly, the activation of these
pathways converges into distinct transcriptional program involving
NFkB that mediate cellular responses, including cell division,
survival (or death), motility, invasion, adhesion, and cellular repair
[25]. QPCR profiling revealed a significant increase in these
EGFR dependent NFkB activating molecules viz. Akt1, Jun,
Map3K1, Raf1 after IR and, EKB-569 treatment resulted in
complete suppression of these molecules and serve as the positive
controls for the study.
Trans formed cells have been shown to possess deregulated
apoptotic machinery [36]. Transcriptional regulator s that regu-
late pro-apoptotic and/or activate anti-apoptotic pr oteins play a
key role in switching the therapy associa ted balance of apoptotic
cell death. In this regard, EGFR blockers appear to inhibit tumor
cell death via multiple mechanis ms. EGFR-mediated signaling
via the Ras-Raf-MAPK, PI3-K/Akt or PKC-Jak/STAT path-
ways leads to the activation of NFkB which in turn imbalance the
pro/anti-apoptotic protein ex pression. As is evident from our
data, IR-induced NF kBandNFkB-dependent met abolic activity,
cell viability and cell death indicate NFkB’s direct role in induced
radio resistance. Consist ently , in multiple tumor cells, we and
others have extensively documented that RT induces NFkB
activity and delineated its direct role in induced radioresistance
[29,37–43]. Conversely, muting NFkB function has been shown
to restore apoptosis [44] and confer apoptotic effect in chemo
and/or ra diores istan t tumor cells [45]. Consistently, we observed
a complete inhibition of IR-induced NFkB activity with EKB-569
desig nating that this compound may rectify IR-induced aberrant
apoptotic machinery. These results though confirmed that the
mecha nism of EKB-569-mediated radios ensitization of squamous
cell carcinoma is acting specifically through NF-kB pathway, it is
interesting to note an induction in the activity of other
transcription factors, AP-1 and SP-1. This differential mechanism
in the activation of NFkB versus AP-1 and SP-1 may be
speculated partly as cell type- and/or stimuli-specific. However,
addressing the complete mechanism involved in the induction of
IR-induced AP-1 and SP-1 with EKB-569 treatment and its
impact on radiosensitization compa red to other EGFR-T K
inhibitors may help in ascertain the complexity in the
combination treatments.
It is also interesting to note form this study that the inhibition of
NFkB signaling pathway is not a EKB-569 compound-specific
effect. Other commonly used irreversible EGFR blockers, afatinib
and neratinib (HKI-272) dose-dependently inhibit NFkB DNA-
binding activity. The inhibition of NFkB by these two related
compounds was found to be persistent up to at least 72 h as seen
with EKB-569 treatment. Similarly, all three EGFR inhibitors,
EKB-569, afatinib and neratinib directly inhibit NFkB activity by
blocking the activity of IR-induced upstream IkB kinase beta
(IKK-
b). This direct action of inhibition of NF-kB is EGFR-
dependent. EGFR-knockdown experiments with a widely used
specific EGFR inhibitor, PD153035 confirmed the EGFR-
mediated inhibition of NFkB DNA-binding activity and mRNA
expression in the irradiated cells. Therefore the proposed
combination of IR and EGFR/NFkB inhibition can be carried
out on to the clinic with any EGFR inhibitor compounds other
than EKB-569.
To further substantiate our findings, we analyzed the efficacy of
EKB-569 in IR-modulated NFkB signaling pathway transcrip-
tional response. Interestingly, EKB-569 robustly modulates the
transcriptional response of NFkB signal transduction and
downstream mediators of this pathway in SCC-4 cells. To that
note, EKB-569 inhibited IR-induced transcription of pro-survival
molecules in this setting. Disruption of aberrantly regulated
survival signaling mediated by NFkB has recently become an
important task in the therapy of several chemoresistant and
radioresistant cancers [46]. Anti-apoptotic molecules are expressed
at high levels in many tumors and have been reported to
contribute to the resistance of cancers to RT [47]. Because
activation of caspases plays a central role in the apoptotic
machinery [47], therapeutic modulation of molecules such as
IAPs could target the core control point that overturn the cell fate
and determine sensitivity to RT [48–51]. A recent body of
evidence has emphasized a central role for NFkB in the control of
cell proliferation and survival. NF kB enhances cell survival by
switching on the activation of pro-survival molecules that dampen
pro-apoptotic signals and attenuate apoptotic response to
anticancer drugs and IR [52,53]. In this perspective, we recently
demonstrated that muting IR-induced NFkB regulates NFkB
dependent pro-survival molecules and potentiate radiosensitization
at least in breast cancer and neuroblastoma models. To our
knowledge, the present study for the first time throws light on the
efficacy of EKB-569 in regulating IR altered NFkB signal
transduction and downstream effector molecules in HNSCC cells.
This insight into the comprehensive regulation of IR-induced
survival transcription recognizes EKB-569 as ‘‘potential radiosen-
sitizer’’ and further allows us to identify the role of EGFR
dependent NFkB mediated orchestration of radioresistance at least
in HNSCC.
Though a plethora of studies dissected out the EGFR
downstream signaling (some of them discussed above) and
suggested that these signaling converge at transcriptional machin-
ery, there remained a paucity of information on the role of specific
transcriptional switch in orchestrating EGFR dependent tumor
progression. Not only, this study throws light on the molecular
blue print that underlies after clinical doses of IR in HNSCC, this
study also identifies the potential of the EGFR TK, EKB-569 in
selectively targeting IR-induced NFkB and subsequent tumor
progression. In this regard, p65 subunit of NFkB is constitutively
activated in 70% of HNSCC and IR-induced NFkB plays an
important role in HNSCC resistance to RT. Though constitutive
and RT-induced NFkB has been causally linked to induced-
radioresistance, its precise participation in RT-induced cell death
orchestration is poorly understood. In this regard, results of the
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PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e29705
present study exhibit that ecotopically muting IR-induced NFkB
with DIkBa robustly induced cell death in HNSCC cells
demonstrating that IR-induced NFkB regulates cell death at least
in this setting. Furthermore, to causally delineate that EKB-569
dependent silencing of NFkB mediates the induced radiosensiti-
zation, we analyzed their effect on NFkB overexpressed cells. For
the first time, the results of the present study imply that EKB-569
inhibits HNSCC cell survival and viability by selectively targeting
NFkB.
In summary, these results demonstrate that EKB-569 signifi-
cantly inhibits IR-induced NFkB activity in human HNSCC cells.
Furthermore, this study identifies the EKB-569-associated inhibi-
tion of NFkB pathway survival signaling blue print, more precisely
to the regimen of the treatment modality, in this case IR.
Evidently, treatment with EKB-569 profoundly conferred IR-
inhibited HNSCC cell survival and viability. Consistently, this
EGFR TK significantly enhanced IR-induced HNSCC apoptosis.
More importantly, NFkB over expression and knockout studies
demonstrated that EKB-569-associated targeting of IR-induced
NFkB mediates cell death in HNSCC cells. Taken together, these
data strongly suggest that EKB-569 may exert radiosensitization at
least in part by selectively targeting IR-induced NFkB dependent
survival signaling, that potentiate radiotherapy in effective
HNSCC cell killing. Further in-depth in vivo studies are warranted
to verify this suggestion and are presently under investigation in
our laboratory.
Supporting Information
Figure S1 QPCR profiling amplification charts and heat
map showing transcriptional changes in 88 NFkB-
dependent downstream target genes in SCC-4 cells. Cells
were either mock-irradiated, exposed to IR or pretreated with
EKB-569 (5 ug) and then exposed to IR. Real-time QPCR
profiling was performed using human NFkB signaling pathway
profiler (Realtimeprimers.com, Elkins Park, PA).
(TIF)
Author Contributions
Conceived and designed the experiments: MN CRT NA. Performed the
experiments: MN JV SA ASM. Analyzed the data: MM JV ASM.
Contributed reagents/materials/analysis tools: MN ASM. Wrote the
paper: MN ASM JV.
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EKB Radiosensitizes Squamous Cell Carcinoma
PLoS ONE | www.plosone.org 12 December 2011 | Volume 6 | Issue 12 | e29705
    • "Recently, a Phase I trial was conducted to determine the dosing for acceptable toxicity levels and induced therapy responses of EKB-559 [129]. Pre-clinical studies, using squamous cell carcinoma suggest that EKB-569 increases sensitivity to radiotherapy [130]. At present, no clinical trial has been conducted in HNSCC patients. "
    [Show abstract] [Hide abstract] ABSTRACT: Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that activates downstream signaling pathways, including the Ras-MEK-Erk and PI3K-AKT pathways, leading to cell proliferation, resistance to apoptosis, angiogenesis and the ability to metastasize. EGFR overexpression is a significant finding in cancer, particularly in head and neck cancer, where it is also associated with a poor prognosis. In recent years, several molecules have been designed to inhibit EGFR activation. Among the many available anti-EGFR drugs, only cetuximab was approved for the treatment of head and neck cancers. However, no predictive biomarkers of cetuximab response are currently known. In the present review, we provide an updated assessment of EGFR biology and its clinical impact in head and neck cancers. A special emphasis is placed on novel patents of EGFR-inhibitors that are anticipated to diversify the anti-EGFR therapies available to treat head and neck cancers. In particular, we outline a new class of irreversible multi-target inhibitors (e.g. afatinib, icotinib, CUDC-101), which may significantly contribute to new head and neck cancer therapies.
    Full-text · Article · Mar 2016
    • "Total RNA extraction and real-time quantitative PCR (QPCR) profiling were performed as described earlier [38, 39]. We used custom-made transcriptome profilers [40] pertaining to stemness and epithelial-to-mesenchymal transition (EMT) signaling. "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction: Therapy-associated onset of stemness-maintenance in surviving tumor-cells dictates tumor relapse/recurrence. Recently, we recognized the anti-pancreatic cancer (PC) potential of seaweed polyphenol manifolds and narrowed down three superior drug-deliverables that could serve as adjuvants and benefit PC cure. Utilizing the PC- cancer stem cells (PC-CSCs) grown ex vivo and mouse model of residual-PC, we investigated the benefits of seaweed polyphenols in regulating stemness-maintenance. Methods: ALDH(+)CD44(+)CD24(+) PC-CSCs from Panc-1, Panc-3.27, MiaPaCa-2, or BxPC-3 cells-derived xenografts grown ex vivo were either mock-irradiated, exposed to fractionated irradiation (FIR, 2Gy/D for 5 days), treated with polyphenols (100 μg/ml) of Hormophysa triquerta (HT-EA), Spatoglossum asperum (SA-EA) or Padina tetrastromatica (PT-EA) with/without FIR were examined for cell viability, transcription of 93 stem-cell-related molecules (QPCR profiling). Polyphenol-dependent regulation of FIR-transactivated Oct4, Zic3, EIF4C, Nanog, and LIF (QPCR) and functional translation of Nanog, SOX2, and OCT3/4 (immunoblotting) were examined in Panc-1/Panc-3.27/MiaPaCa-2/BxPC-3-xenografts derived PC-CSCs. Effect of seaweed-polyphenols in the regulation of EMT (N-Cadherin), pluripotency- (SOX2, OCT3/4, Nanog) and stemness-maintenance (PI3KR1, LIF, CD44) in therapy (FIR, 2Gy/D for 5D/wk for 3-weeks) resistant residual tumors were examined by tissue microarray construction and automated immunohistochemistry. Results: Ex vivo exposure of PC-CSCs to SA-EA, PT-EA and HT-EA exhibit dose-dependent inhibition of cell viability. FIR amplified the transcription of 69, 80, 74 and 77 stem-cell related genes in MiaPaCa-2-, Panc-1-, Panc-3.27- and BXPC3-established xenograft-derived ALDH(+)CD44(+)CD24(+)PC-CSCs. Treatment with SA-EA, PT-EA, or HT-EA completely suppressed FIR-activated stem-cell transcriptional machinery in ALDH(+)CD44(+)CD24(+)PC-CSCs established from MiaPaCa-2, Panc-1, Panc-3.27 and BXPC3 xenografts. QPCR validated EIF4C, OCT3/4, Nanog, LIF, and ZIC3 transcriptional profile outcomes. Nanog, Sox2, and OCT3/4 immunoblotting affirmed the PC-CSC radiosensitizing benefit of seaweed polyphenols. Residual-PC tissues microarrayed and immunostained after in vivo treatments recognized complete regulation of FIR-induced SOX2, OCT3/4, Nanog, LIF, CD44, PIK3R1, N-Cadherin, and E-Cadherin with SA-EA, PT-EA, and HT-EA. Conclusions: These data, for the first time, documented the EMT/stemness-maintenance in therapy-resistant PC-CSCs. Further, the data suggest that seaweed polyphenols may inhibit PC relapse/recurrence by targeting therapy-orchestrated stem-cell signaling in residual cells.
    Full-text · Article · Sep 2015
    • "The expression of CCL2 and CCL7 mRNA are found to be elevated in irradiated mice compared with age-matched controls 26 weeks after irradiation. In some other studies, it was found that CCL2 mRNA was up-regulated in tumor cell lines (SCC-4, SCF-7, IOMM Lee, SF-3061, or A549) receiving X-ray or γ-ray irradiation [50,51,70,71]. All these previous results indicate that the expression level of CCL2 significantly increases regardless of in vitro or in vivo exposure. "
    [Show abstract] [Hide abstract] ABSTRACT: Using quantitative real-time polymerase chain reaction (PCR) array, we explored and compared the expression changes of inflammation-related genes in human peripheral blood irradiated with 0.5, 3, and 10 Gy doses of X-rays 24 h after exposure. Results indicated that the expression of 62 out of 84 genes was significantly altered after X-ray radiation. Among these 62 genes, 35 (such as TNFSF4) are known to be associated with radiation response, but others are novel. At a low radiation dose (0.5 Gy), 9 genes were up-regulated and 19 were down-regulated. With further increased dose to 3 Gy, 8 unique genes were up-regulated and 19 genes were down-regulated. We also identified 48 different genes that were differentially expressed significantly after 10 Gy of irradiation, and among these transcripts, up-regulated genes accounted for only one-third (16 genes) of the total. Of the 62 genes, 31 were significantly altered only at a specific dose, and a total of 10 genes were significantly expressed at all 3 doses. The dose- and time-dependent expression of CCL2 was confirmed by quantitative real-time reverse-transcription PCR. A number of candidate genes reported herein may be useful molecular biomarkers of radiation exposure in human peripheral blood.
    Full-text · Article · Nov 2014
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