American Journal of Molecular Biology, 2013, 3, 248-254 AJMB Published Online October 2013 (
TLR5 involvement in attenuated IL-8 production in
nuclear decorin silenced oral mucosal dysplastic
keratinocytes and squamous cell carcinoma*
Nyla Dil1,2#, Abhijit G. Banerjee1,3
1Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Canada
2Department of Immunology, and Department of Medical Microbiology and Infectious Diseases, Faculty of Medicine, University of
Manitoba, Winnipeg, Canada
3Center for Genomic Bio-Medicine and Research Institute, Durg, Chhattisgarh, India
Received 7 August 2013; revised 3 September 2013; accepted 27 September 2013
Copyright © 2013 Nyla Dil, Abhijit G. Banerjee. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Head and neck cancer is one of the most prevalent
cancers in the world. Roughly half of these malignan-
cies originate from oral mucosa and constitute oral
squamous cell carcinomas. Despite many advances in
diagnostic and therapeutic regimens, five-year sur-
vival rate remains at roughly 50%, indicating the
need for in depth understanding of the oral squamous
cell carcinoma immunobiology. We have previously
shown that in human dysplastic oral keratinocytes
(DOK) and malignant squamous cell carcinoma
(SCC-25), multifunctional proteoglycan decorin is
aberrantly expressed and localized in the nucleus
bound to nuclear EGFR. In vitro nuclear decorin
knockdown significantly reduced IL-8 and IL8-de-
pendent migration, invasion and angiogenesis in these
cells. Since toll-like receptor (TLR) signalling leads to
IL-8 production, we examined here if these receptors
played a role in decorin silencing mediated reduction
in IL-8 levels in oral mucosal d ysplast ic keratino cytes
and squamous carcinoma cells. Decorin silenced DOK
and SCC-25 cells showed a marked diminution of
TLR5 mRNA and protein expression compared with
respective controls that translated into the loss of func-
tion in response to appropriate TLR ligand. In these
mucosal oral epithelia , decorin stable kno ckdown sig-
nificantly down-regulated IL-8 production following
activation with TLR5 ligand flagellin. These data sug-
gest that decorin silencing interferes with IL-8 pro-
duction, in part, by altering TLR5 expression and
signaling in dysplastic and malignant oral epithelia.
This study highlights the significance of TLR5 ex-
pression and signaling and its plausible interactions
with proteoglycans in mucosal cancers.
Keywords: Oral Cancer; Mucosal Cancer; Nuclear
Decorin; TLR5; IL-8; Flagellin; PAMP; DAMP; SLRP;
Head and neck squamous cell carcinoma (HNSCC) is a
devastating disease that accounts for roughly 1.6 million
newly diagnosed cases and 330,000 deaths each year
worldwide [1,2]; half of these cancers are localized in the
oral cavity and are termed oral squamous cell carcinoma.
A multifactorial mucosal cancer, oral squamous cell car-
cinoma, is primarily associated with chronic tobacco,
alcohol use and betel chewing; however, chronic in-
flammation, viral infections (human papillomavirus),
genetic predisposition and poor oral hygiene have also
been strongly implicated in oral tumorigenesis [3-5].
The majority of inflammatory conditions are triggered
by Toll-like receptors (TLRs) which are a large family of
at least 11 evolutionarily conserved proteins character-
ized by ligand specific activation and complex down-
stream signaling [6-8]. TLRs are predominantly ex-
pressed on various immune and structural cells where
they serve as innate cell surface pathogen sensors and
promote local inflammation by triggering the induction
of various proinflammatory mediators, including IL-8.
Recently, TLR expression or up-regulation has been de-
tected in various tumour types, especially in epithelium
derived cancers [9-11]. Expression of TLRs varies in
different cancerous cell types; however, evidence indi-
*Competing Interests: No financial or non-financial competing interests
#Corresponding author.
N. Dil, A. G. Banerjee / American Journal of Molecular Biology 3 (2013) 248-254 249
cates that TLR expression is functionally associated with
tumorigenesis. It has been suggested that TLR expression
might promote malignant transformation of epithelial
cells [11,12]. Engagement of TLRs promotes tumor de-
velopment and metastasis, protects the cancerous cells
from immune attack and induces resistance to apoptosis
and chemo-resistance in some malignancies [10,12-14].
In addition to its putative proinflammatory functions,
IL-8 is a potent angiogenic factor and plays an important
role in cancer progression and metastasis [15-18]. IL-8 is
responsible for most of the angiogenic activity induced
by human oral mucosal dysplastic and malignant epithe-
lial cells [19,20]. Previously, we have shown that in hu-
man dysplastic oral keratinocytes (DOK) and malignant
squamous carcinoma cells (SCC-25), multifunctional
proteoglycan decorin is atypically expressed and local-
ized in the nucleus bound to nuclear epidermal growth
factor receptor (EGFR) [18,21]. Post-transcriptional nu-
clear decorin knock-down resulted in significantly re-
duced IL-8 and IL8-dependent migration, invasion and
angiogenic potential in these dysplastic and malignant
oral mucosal epithelia [18,20]. Since TLR signaling is an
upstream event in the majority of cytokines/chemokine
expression, in this current study, we examined if TLR
pathway is implicated in the aberrant nuclear decorin
silencing mediated attenuated IL-8 production.
2.1. Cell Lines and Culture Conditions
Oral mucosa origin, premalignant-Dysplastic Oral Kera-
tinocytes (DOK) and malignant-Squamous Carcinoma
(SCC-25) cell lines were routinely maintained in
DMEM/F12 (Hyclone, Logan, Utah) supplemented with
10% Foetal Calf Serum for use as in vitro model in our
studies, as described previously [20,22,23]. Silencing of
decorin gene expression was achieved using short hairpin
RNA (shRNA) technology as described earlier [18].
Briefly, oligonucleotides targeting decorin transcript va-
riants A1 and A2 and scrambled sequence control were
custom synthesized, annealed, and cloned into shRNA
expression vector pGeneClip PuroTM (Promega) by Super
Array Bioscience Corporation (Frederick, MD). BLAST
queries were performed to ensure that the sequences have
no significant homology with any other human genes.
Transformation grade shRNAi plasmids were transfected
into DOK and SCC-25 cells using Effectene™ transfec-
tion reagent following manufacturer’s protocol (Qiagen,
Valencia, CA). The stable transfectants were selected for
puromycin (Calbiochem, San Diego, CA) resistance at
2.5 µg/ml optimal concentration. Pools of stable trans-
fectants (maintained at 1 µg/ml of puromycin) were used
in all experiments to avoid clone-specific differences.
Decorin knock down was confirmed at transcript and
protein level by quantitative real-time reverse transcrip-
tion-PCR and Western blotting, respectively. Pooled
decorin-shRNA transfected DOK or SCC-25 clones
showed more than 80% reduction in decorin mRNA ex-
pression and almost complete abrogation of decorin pro-
tein expression in nuclear lysates and/or in whole cell
lysates when compared to control-shRNA transfected
clones or no transfection wild type DOK [18]. Herein,
untransfected DOK and SCC-25 cells will be referred to
as wild type (WT), scrambled shRNA stable transfectants
as control (or Ctrl-shRNA in figures), and decorin
shRNA stable transfectants as decorin silenced (or DCN-
shRNA in figures).
2.2. Multiplex PCR
The transcript expression levels of innate immune TLRs
and co-regulatory molecules were quantified in decorin
silenced, control, and WT DOK and SCC25 cells using
multiplex PCR (MPCR) kit for human signaling receptor
set-2 (TLR1, TLR2, TLR3, TLR5, TLR6, TLR9 and
CD14) from Maxim Biotech, San Francisco, CA. This
set also included housekeeping gene-GAPDH, as internal
cDNA loading control in each reaction. MPCR was car-
ried out according to the manufacturer’s instructions.
Briefly, 1X MPCR buffer, 2.5 units of Taq DNA poly-
merase, and cDNA template from DOK and SCC25 cells
were mixed in a 25 µl reaction and subjected to 35 cycles
of PCR, with denaturing, annealing, and extension tem-
peratures at 96˚C, 67˚C, and 70˚C, respectively. Follow-
ing MPCR, the DNA amplicons were fractionated elec-
trophoretically on 2% agarose gel containing 0.5 µg/ml
ethidium bromide.
2.3. Real-Time PCR
RNA was extracted from DOK and SCC-25 cells using
RNeasy Plus mini kit (Qiagen, Valencia, CA) and 2.5 g
of total RNA was used to synthesize cDNA, using Su-
prescript III Reverse Transcriptase (Invitrogen, San
Diego, CA). Quantitative RT-PCR was performed using
QuantiTect SYBR Green PCR kit (Qiagen, Valencia,
CA) on the Mini Opticon Real-Time PCR system
(BioRad, Hercules, CA) as per manufacturer’s protocol.
Quantitative PCR primer pairs were designed for SYBR
Green chemistry based detection of amplicons for TLR5
used as relative house-keeping gene expression control to
normalize for sample variations.
2.4. Western Blot Analysis
Cells were rinsed with ice-cold PBS and were lysed in a
Copyright © 2013 SciRes. OPEN ACCESS
N. Dil, A. G. Banerjee / American Journal of Molecular Biology 3 (2013) 248-254
buffer containing 20 mM Tris, pH 7.6, 0.1% SDS, 1%
Triton-X, 1% deoxycholate, 100 µg/ml PMSF, and pro-
tease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO).
Lysates were centrifuged at 20,000 x g for 20 min at 4˚C.
Protein concentration was determined by Bis-Cinchonic
Acid (BCA) protein assay (Pierce, Rockford, IL) and
subjected to 10% SDS-PAGE analysis, followed by
transfer to polyvinylidene difluoride membrane (Bio-Rad,
Hercules, CA). The membranes were immunoprobed
with 1:500 dilution of monoclonal antibody to human
TLR5 (Alexis Biochemicals, San Diego, CA) or 1:1000
dilution of anti-human beta-tubulin polyclonal antibody.
Western blots were developed with appropriate horserad-
ish peroxidase conjugated secondary antibodies (Bio-Rad)
and ECL Plus chemiluminescence system (Amersham,
Arlington Heights, IL) and exposed to auto radiographic
films. Radiographs were scanned and densitometry
analysis was done using AlphaEase FC software (Alpha
Innotech Corporation, San Leandro, CA).
2.5. ELISA for IL-8 Quantification
Decorin silenced, control and WT DOK and SCC-25
cells (5 × 105 cells/well) were cultured in complete me-
dium in 24-well flat-bottom plates at a final volume of a
500 µl. Cells were stimulated with varying concentra-
tions of flagellin (Alexis Biochemicals, San Diego, CA);
100 ng/ml concentration was found to be optimal. Cul-
ture supernatants were collected after 24 h, 48 h or 72 h
of incubation and IL-8 was assayed with 100 μl of cell
free culture supernatant using DuoSet IL-8 ELISA kit (R
& D Systems, Minneapolis, MN) according to manufac-
turer’s instructions. Absorbance was read at 450 nm with
the SPECTRAMax 190 microplate spectrophotometer
and results were analyzed by SOFTMax Pro software
(Molecular Devices, Sunnyvale, CA). Sample concentra-
tions were determined by interpolation from the standard
curve. IL-8 lower detection limit was found to be 5.6
pg/ml. Samples were read in triplicate.
2.6. Statistical Analysis
Student’s paired t test was used to determine the statisti-
cal significance of the data. Statistical analysis was per-
formed on Graph Pad Prism Software. Significance was
evaluated at p values:
*p < 0.05, **p < 0.01, ***p < 0.001.
3.1. TLR5 Expression down Regulation in
Decorin Silenced DOK and SCC25 Cells
Toll-like receptor expression has been described in many
cancers especially epithelial derived tumours and has
been linked to tumour progression [24]. Many of the
TLRs are known to trigger IL-8 expression. We sought to
determine whether nuclear decorin silencing has an ef-
fect on any or all of the TLRs expression in dysplastic
and malignant oral epithelial cells. Multiplex PCR analy-
sis showed that out of an array of TLRs, TLR5 expres-
sion was significantly reduced in decorin silenced DOK
and SCC-25 cells compared to respective WT and con-
trol cells (Figure 1). Interestingly, no difference was
observed in the expression of TLR1, TLR2, TLR3, TLR6,
TLR9 and CD14 between decorin silenced and WT/con-
trol DOK or SCC-25 cells (Figure 1). We confirmed this
observation by Real time PCR analysis using specific
primers; no difference was observed in the expression of
TLR1, TLR2, TLR3, TLR4, TLR6, TLR8 and TLR9
between decorin silenced and WT or controls dysplastic
or malignant epithelial cells (data not shown). However,
Real time PCR analysis using TLR5 specific primers
showed more than 75% reduction in TLR5 expression in
decorin silenced DOK and SCC-25 cells (Figure 2(a)).
To investigate if this reduction in TLR5 expression
translates into lower TLR5 receptor levels, we performed
Western blot analysis and observed similar TLR5 protein
reduction in decorin silenced DOK and SCC-25 cells in
comparison to TLR5 protein expression in respective WT
and/or control cells (F igures 2(b) and (c)).
3.2. Attenuated Flagellin-Induced IL-8
Production in Decorin Silenced DOK
and SCC25 Cells
We have shown previously that nuclear decorin silencing
results in attenuated constitutive IL-8 production in oral
mucosal DOK and SCC-25 cells. Here, we sought to
determine if nuclear decorin silencing-mediated TLR5
down regulation has an effect on IL-8 induction in these
dysplastic and malignant oral epithelial cells. Flagellin is
a known ligand for TLR5 and flagellin stimulation of
Figure 1. Toll-like receptors and CD14 expression in decorin
silenced and WT dysplastic and malignant oral epithelial cells.
DOK and SCC-25 cells were stably transfected with decorin-
shRNA (DCN-shRNA), or scrambled sequence-shRNA (Ctrl-
shRNA) or no transfection control (WT). RNA was extracted
and cDNA was subjected to multiplex RT-PCR to detect mul-
tiple pattern recognition receptor transcripts. MPCR products
were fractionated electrophoretically on a 2% agarose gel con-
taining 0.5mg/ml ethidium bromide, visualized under UV
light and photographed. The results shown are representative
of three independent experiments.
Copyright © 2013 SciRes. OPEN ACCESS
N. Dil, A. G. Banerjee / American Journal of Molecular Biology 3 (2013) 248-254 251
Figure 2. TLR5 down regulation in decorin silenced DOK
and SCC25 cells. RNA was extracted from WT, control and
decorin silenced DOK and SCC-25 cells and (a) cDNA was
subjected to quantitative RT-PCR, normalized TLR5 expres-
sion from one representative experiment of three. (b) Cell
lysates were collected as described in materials and methods
and subjected to SDS-PAGE followed by immunoblotting
using anti-TLR5 and antitubulin antibodies. (c) Densitomet-
ric analysis is presented as a histogram of TLR5 relative
band density from 3 experiments. ***p < 0.001 compared to
respective controls.
epithelial cells results in increased IL-8 production. To
investigate the TLR5 link in IL-8 mitigation in decorin
silenced dysplastic and malignant epithelia, we deter-
mined and compared the levels of IL-8 production upon
flagellin stimulation in these cells. Briefly, cells were
stimulated with flagellin for 24, 48 and 72 h; 24 h time
point was considered optimal for IL-8 production. Con-
sistent with down regulation of TLR5 expression levels,
we found a significant reduction in flagellin stimulated
IL-8 production in decorin silenced cells compared to
WT or ctrl-shRNA treated DOK or SCC-25 cells (Figure
3). It is interesting to note that SCC-25 cells produce
much higher levels of flagellin stimulated IL-8 compared
to dysplastic oral keratinocytes.
A number of functions have been assigned to small leu-
cine-rich proteoglycan (SLRPs) decorin. The most in-
triguing function is the ability to inhibit growth and me-
tastasis of a range of tumors. Accordingly, decorin is
being studied extensively as a naturally occurring poten-
tial oncosuppressive agent [25]. Decorin is normally pre-
sent in the extracellular stromal compartment and analy-
ses of various tumors indicate that it is rarely expressed
by cancer cells. However, studies by us and others indi-
cate that there are exceptions to this prevailing decorin
expression and decorin mediated cancer growth and sup-
pression model [21,26,27]. Ectopic decorin expression
has been linked to increased vascular endothelial growth
factor expression and increased angiogenesis [28]. In our
previous studies of oral precancerous and cancerous le-
sions and cellular models of oral cancer progression, we
demonstrated that decorin is aberrantly expressed and
localized in the nucleus bound to nuclear epidermal
growth factor receptor (EGFR) in the dysplastic and ma-
Figure 3. Reduced Flagellin-induced IL-8 production in
decorin Silenced DOK and SCC25. Cells were cultured with
100 ng/ml flagellin and IL-8 was measured in 24 h culture
supernatants using ELISA. Data are presented as mean ± SD
of three replicates of one representative experiment of four.
***p < 0.001 compared to respective controls.
Copyright © 2013 SciRes. OPEN ACCESS
N. Dil, A. G. Banerjee / American Journal of Molecular Biology 3 (2013) 248-254
lignant oral epithelial cells [18,21]. Stable knock down of
nuclear decorin resulted in significant down regulation of
IL-8 and IL-8 dependent migration, invasion and angio-
genic potential in this oral cancer cell line model. Here,
we show that reduced IL-8 in nuclear decorin silenced
DOK and SCC-25 cells is mediated, in part, by reduced
TLR5 expression and signaling.
Toll-like receptors are a major component of an evolu-
tionarily conserved, classic pattern recognition system,
which underpins pathogen recognition, mediates inflam-
matory responses and bridges innate and adaptive immu-
nity. Historically, TLR agonists have been used as adju-
vant in anti cancer immunotherapy. However, a growing
body of clinical and experimental studies suggest that the
neoplastic process may actually subvert TLR signalling
pathway to enhance cancer progression. Hence, the TLR
system appears to play the role of a double-edged sword
in the neoplastic development.
Increased expression of various TLRs has been found
to be associated with a variety of cancers of gastrointes-
tinal, oral and genital mucosa [11,29]. Of all the TLRs,
TLR5 is the most prominent TLR that appears to have a
role in mucosal neoplastic processes. Enhanced TLR5
expression has been reported in gastric and colorectal
malignancies, as well as in precancerous lesions of uter-
ine cervix [30,31]. Stimulation with TLR5-ligand, flagel-
lin, increased proliferation and induced TLR5 mediated
IL-8 production in various gastric cancer cell lines [32].
A role for TLR5 has also been described in mucosal head
and neck cancers. In biopsy sample of 119 oral tongue
squamous cell carcinoma patients, TLR5 expression was
found to be more abundant and widespread in cancer
cells than in the adjacent healthy lingual epithelial tissue
[33]. In this oral cancer cohort, TLR5 expression levels
predicted prognosis in terms of recurrence and patient
survival. In salivary gland adenocarcinoma, flagellin ac-
tivation of TLR5 promoted migration and invasion of
cancer cells [12]. Hence, increased TLR5 expression is
correlated with mucosal cancer progression and, there-
fore, has been proposed as a biomarker for gastric and
cervical dysplasia as well as oral cancer and might help
identify dysplastic lesions in mucosal epithelium [30,31,
The canonical function of proteoglycans is the main-
tenance and regulation of the architecture of various ex-
tracellular matrices (ECM). Decorin is an important
structural component of the ECM and a fundamental
regulator of collagen fibrillogenesis that got its eponym
based on its ability to decorate collagen fibrils. However,
a growing body of recent evidence indicates that secreted
proteoglycans in particular those from the small leucine-
rich family (including decorin), hyaluronan-binding gene
family as well as the glycosaminoglycan hyaluronan act
as endogenous ligand for TLR2, TLR4 and TLR6 in ad-
dition to their above mentioned widely recognized struc-
tural function [34,35]. These proteoglycans and other
endogenous molecules act as “danger signals” and can
activate pattern recognition receptors and thus are re-
ferred to as damage-associated molecular patterns
(DAMPs) [36]. Any direct or indirect interaction be-
tween decorin and TLR5 has hitherto not been described.
We show here for the first time that under-expressing
aberrant nuclear localized decorin results in down regu-
lation of TLR5 and TLR5 mediated IL-8 production in
dysplastic and malignant oral epithelial cells. This might
be due to nuclear decorin interactions with its binding
partner nuclear EGFR (which acts as a transcription fac-
tor) or additional interactions with other nuclear and/or
cytosolic factors. Further studies are warranted to deci-
pher these interactions and to explore how exactly the
gene expression of TLR5 is regulated by nuclear local-
ization of decorin in these cells.
Using shRNA expression plasmid mediated in vitro sta-
ble nuclear decorin silencing, we present here a study of
the involvement of TLRs in attenuated IL-8 production
upon nuclear decorin knockdown in dysplastic and ma-
lignant oral mucosal epithelial cells. We demonstrate that
nuclear decorin knockdown significantly inhibits TLR5
(a putative mucosal cancer biomarker) and TLR5 medi-
ated IL-8 production in these cells. These findings and
our previous work on nuclear decorin suggest that nu-
clear decorin silencing suppresses major tumor devel-
opment and progression processes such as IL-8 depend-
ent migration, invasion, and angiogenesis in part by miti-
gating the expression of TLR5. This study also highlights
the significance of TLR5 expression and signaling in de-
velopment and progression of mucosal cancers. It might
be potentially useful in developing a therapeutic regimen
for patients presenting with premalignant or malignant
oral mucosal lesion accompanied with poor oral health
associated mucosal inflammation.
ND contributed to study concept, performed, and ana-
lyzed the experiments, interpreted data and wrote the
manuscript. AGB conceptualized nuclear decorin study
and designed and BLAST decorin targeting oligonucleo-
tides, analyzed results and interpreted data, and approved
the final manuscript and directed the research program.
All authors reviewed and approved the final manuscript.
The authors gratefully acknowledge Dr. J.K. Vishwanatha, University
of North Texas, Fort Worth for providing the oral cancer progression
model cell lines and Linda Delmage, Charlton Cooper, Susan Pylypas,
Copyright © 2013 SciRes. OPEN ACCESS
N. Dil, A. G. Banerjee / American Journal of Molecular Biology 3 (2013) 248-254 253
University of Manitoba for technical assistance. This project was sup-
ported by research grants form Manitoba Health Research Council and
Manitoba Medical Service Foundation and by funds from Department
of Oral Biology and Faculty of Dentistry, University of Manitoba.
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