bgq278 368..380Guggulsterone (GS) inhibits smokeless tobacco and
nicotine-induced NF-kB and STAT3 pathways in head and neck cancer
cells
Muzafar A.Macha, Ajay Matta1, S.S.Chauhan, K.W.Michael Siu1 and
Ranju Ralhan1,2,3,4,
Department of Biochemistry, All India Institute of Medical
Sciences, New Delhi-110029, India, 1Department of Chemistry and
Center for Research in Mass Spectrometry, York University, 4700
Keele Street, Toronto, Ontario, Canada M3J 1P3, 2Alex and Simona
Shnaider Laboratory of Molecular Oncology and Department of
Pathology and Laboratory Medicine, Mount Sinai Hospital, Joseph and
Wolf Lebovic Health Complex, 600 University Avenue, Room 6-500
Toronto, ON, Canada M5G 1X5, 3Joseph and Mildred Sonshine Family
Center for Head and Neck Diseases and Department of
Otolaryngology—Head and Neck Surgery, Mount Sinai Hospital,
Toronto, ON, Canada M5G 1X5 and 4Department of Otolaryngology—Head
and Neck Surgery, University of Toronto, ON, Canada M5G 2N2
To whom correspondence should be addressed. Alex and Simona
Shnaider Laboratory of Molecular Oncology, Department of Pathology
and Laboratory Medicine, Mount Sinai Hospital, Joseph and Wolf
Lebovic Health Complex. 600 University Avenue, Room 6-500, Toronto,
ON, Canada M5G 1X5. Tel: þ1 416 586 4800 6426. Fax: þ1 416 586
8628; Email:
[email protected]
Understanding the molecular pathways perturbed in smokeless
tobacco- (ST) associated head and neck squamous cell carcinoma
(HNSCC) is critical for identifying novel complementary agents for
effective disease management. Activation of nuclear factor- kappaB
(NF-kB) and cyclooxygenase-2 (COX-2) was reported in ST-associated
HNSCC by us [Sawhney,M. et al. (2007) Expres- sion of NF-kappaB
parallels COX-2 expression in oral precancer and cancer:
association with smokeless tobacco. Int. J. Cancer, 120,
2545–2556]. In search of novel agents for treatment of HNSCC, we
investigated the potential of guggulsterone (GS),
(4,17(20)-pregnadiene-3,16-dione), a biosafe nutraceutical, in in-
hibiting ST- and nicotine-induced activation of NF-kB and signal
transducer and activator of transcription (STAT) 3 pathways in
HNSCC cells. GS inhibited the activation of NF-kB and STAT3
proteins in head and neck cancer cells. This inhibition of NF-kB by
GS resulted from decreased phosphorylation and degradation of
nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor, alpha the inhibitory subunit of NF-kB. Impor- tantly,
treatment of HNSCC cells with GS abrogated both ST- and
nicotine-induced nuclear activation of NF-kB and pSTAT3 pro- teins
and their downstream targets COX-2 and vascular endothe- lial
growth factor. Furthermore, GS treatment decreased the levels of
ST- and nicotine-induced secreted interleukin-6 in cul- ture media
of HNSCC cells. In conclusion, our findings demon- strated that GS
treatment abrogates the effects of ST and nicotine on activation of
NF-kB and STAT3 pathways in HNSCC cells that contribute to
inflammatory and angiogenic responses as well as its progression
and metastasis. These findings provide a biologic rationale for
further clinical investigation of GS as an effective complementary
agent for inhibiting ST-induced head and neck cancer.
Introduction
Tobacco is used by .1.3 billion people worldwide (1); it kills .5
million people annually and the World Health Organization estimates
this number to increase to 8 million by 2030 (2). Tobacco use is
the single largest cause of cancer globally (3,4). The number of
adult smokers has declined in the USA (3,4), but the use of
smokeless tobacco (ST) products (e.g. chewing tobacco, khaini and
snuff) is increasing worldwide, and these products can be a gateway
for life- long addiction (1,5). ST consumption is emerging as a
major risk factor for head and neck squamous cell carcinoma (HNSCC)
(6,7), the most common cancer in men in many Asian countries and
the sixth most common cancer in USA (8). The causal association
between smoking and HNSCC has been unequivocally established
(5,9–12). The evidence that ST causes oral cancer was confirmed by
the International Agency for Research on Cancer and ST was
classified as a human carcinogen, with nitrosoamines being one of
the major carcinogenic constituents (13). In a recent study, 23
polycyclic aromatic hydrocarbons have been identified in ST (14).
Nicotine, a major component of ST, besides causing addiction has
been shown to regulate cell proliferation, angiogenesis and inhibit
apoptosis in- duced by anticancer drugs (15). An indepth
understanding of the molecular mechanism(s) underlying ST-related
head and neck carci- nogenesis will lead to the development of
effective strategies to pre- vent and treat ST-related HNSCC.
Studies carried out in our laboratory and others have shown
aberrant expression of genes involved in ST-associated HNSCC
(16–20). ST has been shown to increase oxidative stress (21) plays
a major role in acti- vation of nuclear factor-kappaB (NF-jB) and
pSTAT3 pathways, involved in inflammation, survival and
proliferation of cancer cells (21–24). Our laboratory also reported
exposure of oral premalignant cultures and cancer cells (AMOS III)
to ST resulted in increased cell proliferation and activation of
NF-jB; association of ST consumption with overexpression of NF-jB
and cyclooxygenase-2 (COX-2) was observed in clinical oral squamous
cell carcinoma (SCC) tissue samples also (23–25). The persistent
activation of NF-jB can lead to elevated expression and secretion
of interleukin (IL)-6. Interestingly, blocking NF-jB diminished the
accumulation of active signal transducer and activator of
transcription (STAT) 3 in HNSCC cells, suggesting existence of
cross talks between NF-jB and STAT3 pathways (25). Recently our
group observed nuclear pSTAT3 accumulation in clinical oral
squamous cell carcinoma tissue samples was significantly associated
with ST con- sumption habits (26), indicating a plausible link
between STAT3 and ST. Therefore, it is important to identify agents
that can abrogate these effects of ST in head and neck cancer cells
for developing therapies to prevent and treat ST-related head and
neck cancer.
Guggulsterone (GS), (4,17(20)-pregnadiene- 3,16-dione), derived
from the plant Commiphora mukul, is widely used to treat obesity,
diabetes, hyperlipidemia, atherosclerosis and osteoarthritis (27).
GS suppresses inflammation by inhibiting inducible nitric oxide
synthe- tase (28) and NF-jB induced by various carcinogens and
tumor pro- moters (29). Our group and several other reports have
shown that treatment with GS induces apoptosis and suppress
proliferation of wide variety of human tumor cell types (28–33). GS
inhibits invasion, angiogenesis and metastasis of tumor cells and
reverses chemoresist- ance (31–33). GS has also been shown to
inhibit both constitutive and inducible STAT3 pathways in head and
neck cancer cell lines (34,35). Recently, Leeman-Neill et al. (35)
showed antiproliferative effects of GS are partially dependent on
STAT3 inactivation. They showed knocking down expression of STAT3
using small interfering RNA in head and neck cancer cells reduced
GS-induced cell death in com- parison with the no transfection
controls. In addition, our group in collaboration with Dr Bharat
Aggarwal’s laboratory showed that GS inhibits inducible and
constitutive STAT3 activation through the
Abbreviations: CLSM, confocal laser scan microscopy; COX-2,
cyclooxyg- enase-2; EDTA, ethylenediaminetetraacetic acid; EGTA,
ethyleneglycol-bis (aminoethylether)-tetraacetic acid; HEPES,
N-2-hydroxyethylpiperazine-N#-2- ethanesulfonic acid; HNSCC, head
and neck squamous cell carcinoma; GS, guggulsterone; IL,
interleukin; IkBa, nuclear factor of kappa light polypeptide gene
enhancer in B-cells inhibitor, alpha; NF-jB, nuclear factor-kappaB;
PBS, phosphate-buffered saline; SCC, squamous cell carcinoma; SDS,
sodium dodecyl sulfate; STAT, signal transducer and activator of
transcription; ST, smokeless tobacco; VEGF, vascular endothelial
growth factor.
The Author 2010. Published by Oxford University Press. All rights
reserved. For Permissions, please email:
[email protected] 368
D ow
ecem ber 2021
induction of tyrosine phosphatase, which makes it a potentially
effec- tive suppressor of tumor cell survival and proliferation
(34). We further showed that activation of STAT3 by IL-6 is
completely abrogated by pretreatment with GS in melphalan sensitive
MM.1S cells. Similar results of GS-induced inhibition of STAT3
activation have also been reported in colon cancer cells (36).
Together, these reports clearly suggest inhibition of STAT3
activation is an important mechanism of GS-induced cell death.
Hence, it will be interesting to determine the effect of GS on ST-
and nicotine-induced head and neck carcinogenesis.
In the current study, we determined the effect of ST and/or
nicotine treatment on NF-jB and STAT3 signaling in head and neck
cancer cells. Furthermore, we challenged ST- and/or
nicotine-induced activa- tion of NF-jB and STAT3 signaling by
pretreatment of head and neck cancer cells (SCC4 and HSC2) with GS,
to evaluate its therapeutic potential. This may help us in
designing novel therapies based on such biosafe nutraceutical
agents that target ST-induced inflammation, sur- vival and
proliferation in head and neck cancer cells.
Materials and methods
Antibodies and reagents
Z-GS, nicotine,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and
propidium iodide were purchased from Sigma–Aldrich (St Louis, MO).
Z-GS was dissolved in dimethyl sulfoxide as a 100 mM stock solution
and stored at 20C; Penicillin, streptomycin, Dulbecco’s modified
eagle medium/ F12 medium, fetal bovine serum, L-glutamine, sodium
pyruvate, vitamins and minimum essential medium were obtained from
Invitrogen (Grand Island, NY). Rabbit polyclonal antibodies against
NF-jB (p65 subunit) (sc-109), NF-jB (pp65 subunit) (sc-101749,
Ser-276), nuclear factor of kappa light polypeptide gene enhancer
in B-cells inhibitor, alpha (IkBa) (sc-371), pIkBa (sc-101713, Ser-
32), Jak1 (sc-295), pJak1 (sc-16773, Tyr 1022) and vascular
endothelial growth factor (VEGF) (sc-152); goat polyclonal
antibodies against COX-2 (sc-1746) and pSTAT3 (sc-7993, Tyr 705)
and mouse monoclonal antibodies against STAT3 (sc-8019) and
a-tubulin (sc-5386) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Goat anti-rabbit, anti-goat, anti
mouse-horseradish peroxidase conjugates were purchased from DAKO
Cy- tomations (Glostrup, Denmark), and goat anti-rabbit Alexa 594
was pur- chased from Molecular Probes (Eugene, OR).
Bacteria-derived recombinant human IL-6 was obtained from Biosource
International (Cama- rillo, CA). ST extract was prepared as
described before by us (37).
Cell culture
Head and neck cancer cells (SCC4 and HSC2) were grown in monolayer
cultures in Dulbecco’s modified eagle medium (Sigma, St Louis, MO)
supple- mented with 10% fetal bovine serum (Sigma), 1 mM
L-glutamine, 1 mM minimum essential medium, 100 lg/ml streptomycin
and 100 U/ml penicillin in a humidified incubator (5% carbon
dioxide, 95% air) at 37C as described earlier (38).
Preparation of nuclear extracts
Nuclear extracts were prepared from control, untreated and treated
head and neck cancer cells (SCC4 and HSC2) as described earlier
(39). Briefly head and neck cancer cells (SCC4 and HSC2) (2 107)
cells were treated, harvested, washed with cold phosphate-buffered
saline (PBS, pH 5 7.2) and incubated on ice for 15 min in hypotonic
buffer A [10 mM N-2-hydroxyethylpiperazine- N#-2-ethanesulfonic
acid (HEPES) (pH 5 7.9), 10 mM KCl, 0.1 mM
ethylenediaminetetraacetic acid (EDTA), 0.1 mM
ethyleneglycol-bis(ami- noethylether)-tetraacetic acid (EGTA), 1 mM
dithiothreitol, 0.5 mM phenyl- methylsulfonyl fluoride and 0.6%
NP-40]. Cells were vortexed gently for lysis, and nuclei were
separated from the cytosol by centrifugation at 12 000g for 1 min.
Nuclei were resuspended in buffer C [20 mM HEPES (pH 5 7.9), 25%
glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol and
0.5 mM phenylmethylsulfonyl fluoride] and shaken for 30 min at 4C.
Nuclear extracts were obtained by centrifugation at 12 000g for 10
min and stored at 80C till use.
Western blotting
Western blotting was carried out as described previously by us
(38). Whole-cell lysates were prepared from treated and untreated
control cells SCC4 and HSC2 cells and protein concentration was
determined using the Bradford reagent (Sigma–Aldrich) and equal
amounts of proteins (80 lg/lane) were resolved on 10% sodium
dodecyl sulfate (SDS)–polyacrylamide gel. The proteins were then
electrotransferred onto polyvinylidenedifluoride membrane. After
block- ing with 5% non-fat milk in Tris-buffered saline (0.1 M, pH
5 7.4), blots were
incubated with specific antibodies as per manufacturer’s
recommended pro- tocol at 4) served as a control for protein
loading in each lane. Membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies, (DAKO Cytomation),
diluted at an appropriate dilution in 1% bovine serum albumin, for
2 h at room temperature. After each step, blots were washed three
times with Tween (0.1%)-Tris-buffer saline. Protein bands were
detected by the enhanced chemiluminescence method (Santa Cruz
Biotechnology) on XO- MAT film. The band intensities were measured
as integrated density values using AlphaEase FC Software (version
3.1) with ChemiImager IS-4400 (Alpha Innotech Corporation, San
Leandro, CA).
Confocal laser scan microscopy
For confocal laser scan microscopy (CLSM), 5 103 SCC4 cells were
plated on coverslips and grown for 24 h. Expression and
localization of p65 protein was observed in treated and untreated,
control SCC4 cells using CLSM as described by us (38). Briefly,
treated and control SCC4 cells were, fixed in methanol for 20 min
at 20C, rinsed with PBS (pH 5 7.2) and incubated with p65 antibody
overnight at 4C. After rinsing in PBS, the coverslips were
incubated with biotinylated secondary antibody (LSAB plus Kit; DAKO
Cytomations) for 45 min at 37C followed by incubation with
streptavidin- conjugated fluorochrome, fluorescein isothiocyanate
(DAKO Cytomations). Thereafter, the coverslips were counterstained
with propidium iodide (10 mg/ml; Sigma) for 30 s. Coverslips were
rinsed and mounted in mounting medium. Slides were examined using a
CLSM-LSM510 scanning module (Carl Zeiss Microscopy; Jena GmbH,
Germany).
IL-6 assay
Cell-free supernatants were collected from GS (50 lM), ST extract
(20 lg/ml), nicotine (10 lM) and IL-6-treated SCC4 cells and
filtered through a 0.22 lm low protein-binding polyethylsulfonate
membrane filter. Aliquots of 100 ll were removed, and IL-6 contents
were determined by enzyme-linked immu- nosorbent assay kit
(Biosource International, Camarillo, CA).
Chromatin immunoprecipitation assay
For chromatin immunoprecipitation assay, treated and untreated
control SCC4 cells were cross-linked with 1% formaldehyde at 37C
for 10 min. The cross- linking reaction was quenched by washing the
cells several times with cold PBS, buffer I (0.25% Triton X 100, 10
mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 5 6.5) and buffer II (200 mM
NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 5 6.5). Cells were
centrifuged, resuspended in cold immunoprecipitation buffer (1%
SDS, 10 mM EDTA, 50 mM Tris, pH 5 8.1 and protease inhibitors) and
sonicated to an average fragment size of 300–600 bp. Solubilized
chromatin was clarified by centrifugation at 21 000g, and the
supernatant was preincubated for 2 h with protein-A/G aga- rose
beads. Precleared chromatin was incubated overnight at 4C with 2 lg
anti-STAT3 antibody. Preimmune rabbit serum was used as a negative
control. Immune complexes were bound to protein-A agarose beads at
4C for an additional 2–3 h. Beads were washed sequentially for 10
min each in buffer I (20 mM Tris, pH 8.1, containing 0.1% SDS, 1%
Triton X 100, 2 mM EDTA and 150 mM NaCl), buffer II (20 mM Tris, pH
5 8.1, containing 500 mM NaCl, 0.1% SDS, 1% Triton X 100 and 2 mM
EDTA), buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate 1 mM
EDTA, 10 mM Tris, pH 5 8.1) and 10 mM Tris, pH 8.1 and 2 mM EDTA
(TE buffer). Protein–DNA complexes were eluted from the protein-A
agarose beads with 1% SDS and 0.1M NaHCO3 at room temperature for
10 min with gentle agitation. The supernatant was trans- ferred to
a fresh tube, and cross-links were reversed overnight at 65C.
Samples were treated with Proteinase K (Sigma Chemical Co., St
Louis, MA) at 37C for 2 h and were extracted once with phenol, and
the DNA was precipitated with 2.5 vol ethanol plus 20 lg glycogen
as carrier. Precipitated DNA was pelleted, washed once with 70%
ethanol, dried and resuspended in water. The purified DNA was used
as a template for polymerase chain reaction amplifica- tion using
primers (forward, 5#-TTGGTGCCAAATTCTTCTCC-3#; reverse, 5#-
CACACGTCCTCACTCTC GAA-3#) flanking the putative STAT-binding sites
located at 848 and 630 in human VEGF promoter region. The
polymerase chain reaction products were resolved on 1.2% agarose
and stained with ethi- dium bromide, and the band intensities were
quantified using Chemimager 4400 (Alpha Innotech
Corporation).
Results
Effect of GS on ST- and nicotine-induced phosphorylation and
localization of p65 subunit of NF-jB Phosphorylation of p65 subunit
of NF-jB complex is required for its nuclear translocation and
transcriptional activation. Therefore, we in- vestigated the effect
of GS on ST- and nicotine-induced phosphoryla- tion of p65 in head
and neck cancer cells. SCC4 and HSC2 cells were
GS inhibits NF-jB and STAT3 activation
369
ecem ber 2021
treated with GS (50 lM)/ST (20 lg/ml)/nicotine (10 lM) for
different time intervals, and protein extracts obtained from
cytosolic fraction were checked for phosphorylated p65 protein. As
shown in Figure 1a, GS completely inhibited the phosphorylation of
p65, whereas either ST- or nicotine-induced the p65 phosphorylation
in a time-dependent manner with significant increase of
phosphorylated p65 (pp65) ob- served after 2 h of treatment in SCC4
cells (Figure 1b and c). Interest-
ingly, we observed that treatment with GS (50 lM) for 4 h
completely abrogated the ST- or nicotine-induced phosphorylation of
p65, respec- tively, in both the cell lines (Figure 1d and e).
Thus, GS inhibited ST- or nicotine-induced NF-jB activation by
inhibiting p65 phosphorylation.
GS treatment decreased nuclear accumulation of p65 as compared with
the untreated control (Figure 2a), whereas ST or nicotine in-
creased nuclear accumulation of p65 as shown by western
blotting
Fig. 1. Effect of GS on ST- and nicotine-induced phosphorylation
p65. SCC4 cells were treated with (a) 50 lM GS for 0 and 30 min and
1, 2, 4 and 6 h; (b) ST (20 lg/ml) for 0,1, 2, 4, 6 and 9 h; or (c)
10 lM nicotine for 0, 30 min, 1, 2, 3 and 4 h. SCC4/HSC2 cells were
pretreated with 50 lM GS for 0, 1, 2, 4, 6 and 9 h, followed by ST
for 4 h (d) or with nicotine for 1h (e). Cytoplasmic extracts were
prepared, separated on 10% SDS–PAGE. Proteins were then
electrotransferred on polyvinylidenedifluoride membrane followed by
blocking with 5% non-fat milk overnight. Blots were incubated with
specific antibody against pp65. Protein expression was determined
using enhanced chemiluminescence method. The same blots were
stripped and reprobed with a-tubulin antibody to show equal protein
loading.
M.A.Macha et al.
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Fig. 2. (a–e) Effect of GS on ST- and nicotine-induced nuclear
translocation of p65. SCC4 cells were treated with (a) 50 lM GS for
0, 30 min, 1, 2, 4 and 6 h; (b) ST (20 lg/ml) for 0, 1, 2, 4, 6 and
9 h; or (c) 10 lM nicotine for 0 and 30 min and 1, 2, 3 and 4 h.
SCC4/HSC2 cells were pretreated with 50 lM GS for 0, 30 min, 1, 2,
4, and 6 h, followed by ST for 4 h (d) or with nicotine for 2 h
(e). Cytoplasmic and nuclear extracts were prepared according to
materials and methods and separated on 10% SDS–PAGE. Proteins were
then electrotransferred on polyvinylidenedifluoride membrane
followed by blocking with 5% non-fat milk overnight. Blots were
incubated with specific antibody against p65. Protein expression
was determined using enhanced chemiluminescence method. The same
blots were stripped and reprobed with a-tubulin or laminin B
antibody to show equal protein loading in cytoplasmic and nuclear
fraction respectively. (f) Effect of GS on ST- and nicotine-induced
nuclear translocation of p65 using immunofluorescence. SCC4 cells
grown on coverslips, were kept untreated (i–iii) or treated with GS
(50 lM) for 4 h (iv–vi); ST (20 lg/ml) for 6 h (vii–ix); with
nicotine (10 lM) for 2 h (x–xii); pretreated with 50 lM GS for 4 h
and stimulated with ST (20 lg/ml) for 6 h (xiii–xv) or with
nicotine (10 lM) for 2 h (xvi–xviii).Then immunolabelled with
anti-p65 antibody followed by streptavidin-conjugated
fluorescein
GS inhibits NF-jB and STAT3 activation
371
ecem ber 2021
using cytoplasmic and nuclear fractions obtained from treated and
control SCC4 cells (Figure 2b and c). However, treatment with GS
(50 lM) for 4 h completed abrogated the ST- or nicotine-induced
nuclear translocation, with concomitant increase in cytoplasmic ac-
cumulation of p65 in both SCC4 and HSC2 cells (Figure 2d and e).
These findings were further confirmed by CLSM of p65 subunit using
control and GS-treated SCC4 cells (Figure 2f).
Effect of GS on ST- and nicotine-induced IjBa phosphorylation
The degradation of IjBa and subsequent release of NF-jB (p65 and
p50) requires prior phosphorylation at Ser-32 and Ser-36 residues.
Therefore, to investigate whether the inhibitory effect of GS is
medi- ated through the alteration of IkBa phosphorylation, SCC4
cells were treated with GS (50 lM)/ST (20 lg/ml)/nicotine (10 lM)
for different time intervals and western blotting was performed for
phospho-IjBa
Fig. 2. Continued.
isothiocyanate (FITC)-labeled tertiary antibody (green
fluorescence) and nuclei were counterstained with propidium iodide
(PI) (red fluorescence). Panels (i), (iv), (vii), (x), (xiii) and
(xvi) represents nuclei showing red fluorescence of PI; panels
(ii), (v), (viii), (xi), (xiv) and (xvii) shows Green fluorescence
for p65 protein in cytoplasm and nuclei; panels (iii), (vi), (ix),
(xii), (xv) and (xviii) shows increased Green fluorescence for p65
protein in cytoplasm of SCC4 cells. (original magnification 100 for
all photomicrographs).
M.A.Macha et al.
ecem ber 2021
Fig. 3. Effect of GS on ST- and nicotine-induced IjBa
phosphorylation and IjBa degradation. SCC4/HSC2 cells were treated
with GS (50 lM) for 0, 30 min, 1, 2, 4 and 6 h; ST (20 lg/ml) for
0, 1, 2, 4, 6 and 9 h; or 10 lM nicotine for 0, 30 min, 1, 2, 3 and
4 h. SCC4 cells were pretreated with 50 lM GS for 0, 1, 2, 4 and 6
h, followed by ST for 4 h or with nicotine for 2 h. Whole-cell
extracts were prepared, separated on 10% SDS–PAGE. Proteins were
then electrotransferred on polyvinylidenedifluoride membrane
followed by blocking with 5% non-fat milk overnight. Blots were
incubated with specific antibody against pIjBa (a–e) and IjBa
(f–j). Protein expression was determined using enhanced
chemiluminescence method. The same blots were stripped and reprobed
with a-tubulin antibody to show equal protein loading.
GS inhibits NF-jB and STAT3 activation
373
ecem ber 2021
(Ser32/36). As shown in Figure 3a, GS treatment decreased consti-
tutively expressed phosphorylated IjBa (Ser-32/36) as revealed by
western blotting. However, either ST or nicotine increased the
expres- sion of pIjBa with a significant increase in expression
observed after 4 and 1 h on ST and nicotine treatment, respectively
(Figure 3b and c). Interestingly, treatment with GS (50 lM)
completely abrogated ST/ nicotine-induced phosphorylation of IjBa
in SCC4 and HSC2 cells (Figure 3d and e).
We determined whether the GS inhibition by either ST- or nicotine-
induced NF-jB activation was due to inhibition of IjBa degradation.
GS
induced the expression of IjBa in a time-dependent manner (Figure
3f), with maximum expression observed at 4 h; however, ST- or
nicotine- induced IjBa degradation in a time-dependent manner
(Figure 3g and h). ST induced the degradation of IjBa was observed
within 4 h and complete loss of IjBa expression was found within 6
h of ST treatment. Upon nicotine treatment, degradation started as
early as in 2 h with no detectable expression observed within 4 h.
Notably, pretreatment with GS abrogated the ST-induced IjBa
degradation in both cell lines (Figure 3i). Similarly we observed
nicotine-induced IjBa degradation was abrogated in GS pretreated
SCC4 and HSC2 cells
Fig. 3. Continued.
M.A.Macha et al.
ecem ber 2021
(Figure 3j). Thus, GS pretreatment abolished ST- and
nicotine-induced NF-jB activation by inhibiting the phosphorylation
of IjBa.
GS inhibits upregulation of COX-2 in response to ST and nicotine
treatment
NF-jB activation results in nuclear translocation p65-subunit fol-
lowed by increased expression of its target genes, such as COX-2.
Therefore, we investigated the effect of GS on COX-2
expression.
A time-dependent decrease in COX-2 expression was observed, with
complete absence of COX-2 protein at 6 and 12 h of GS treatment
(Figure 4a). Treatment with STand nicotine increased the
cytoplasmic expression of COX-2 (Figure 4b). Treatment with tumor
necrosis factor-a [TNF-a (0.1 nM, 1 h)] was used as a positive
control for inducing NF-jB-mediated COX-2 upregulation. Notably, GS
treat- ment inhibited the ST- and nicotine-induced expression of
COX-2 in a time-dependent manner (Figure 4b).
Fig. 4. GS inhibits upregulation of COX-2 in response to ST and
nicotine treatment. SCC4 cells (2–3 106 cells) were treated with
(a) GS (50 lM) for 0, 1, 2, 3, 6 and 12 h (b) SCC4 cells (2–3 106
cells) were pretreated with GS (50 lM) for 6 h followed by
treatment with either ST (20 lg/ml) or with nicotine(10 lM) for 4 h
and whole-cell extracts were prepared. Tumor necrosis factor-a
treated cells were used as a positive control for COX-2
overexpression. Sixty micrograms of whole-cell extracts were
resolved on 10% SDS–PAGE, electrotransferred to a
polyvinylidenedifluoride membrane followed by blocking with 5%
non-fat milk overnight. Protein expression was determined using
enhanced chemiluminescence method and probed for the COX-2. Western
blot for a-tubulin was done to show equal loading of protein. (c)
Effect of GS, ST or nicotine on the expression of secretory IL-6.
SCC4 cells were treated with 50 lM GS, 20 lg/ml of ST or 10 lM
nicotine for different time points, and cell-free supernatants were
collected and filtered through a 0.22 mm low protein-binding
polyethylsulfonate membrane filter. Aliquots of 100 ll were
removed, and IL-6 contents were determined by enzyme-linked
immunosorbent assay.
GS inhibits NF-jB and STAT3 activation
375
ecem ber 2021
Effect of GS, ST and nicotine on the expression of secretory
IL-6
The persistent activation of NF-jB can lead to elevated expression
and secretion of IL-6 in HNSCC and may contribute to inflammatory
and angiogenic responses as well as its progression and metastasis.
We determined secreted IL-6 levels in GS, ST- or
nicotine-treated
SCC4 cells using enzyme-linked immunosorbent assay. SCC4 cells were
treated with GS (50 lM), ST (20 lg/ml) and nicotine (10 lM) for
different time intervals, and cell-free supernatants were collected
and filtered through a 0.22 l low protein-binding
polyethylsulfonate membrane filter. Aliquots of 100 ll were used
for IL-6 assay using an
Fig. 5. GS inhibits IL-6 inducible STAT3 phosphorylation in SCC4
cells. (a) SCC4 cells (2–3 106 cells) were treated with IL-6 (10
ng/ml) for the indicated times, and whole-cell extracts were
prepared. (b) SCC4 cells (2–3 106 cells) were pretreated with 50 lM
GS for the indicated durations and stimulated with IL-6 (10 ng/ml)
for 2 h and whole-cell extracts were prepared. (c) GS-induced
inhibition of STAT3 phosphorylation is reversible. SCC4 (2–3 106
cells) were treated with 50 lM GS treated for 4 h and washed with
PBS two times to remove GS before resuspension in fresh medium.
Cells were removed at indicated times and lysed to prepare the
whole-cell extract. (d–h) GS inhibits constitutive and ST or
nicotine induces STAT3 phosphorylation in SCC4 cells. SCC4 cells
(–3 106 cells) were treated with (d) GS (50 lM); ST (20 lg/ml); or
(e) with nicotine (10 lM) (f) for the indicated times, and
whole-cell extracts were prepared. SCC4 cells (–3 106 cells) were
pretreated with 50 lM GS for the indicated durations and stimulated
with (g) ST (20 lg/ml) for 4 h or (h) with nicotine (10 lM) for 2 h
and whole-cell extracts were prepared. Sixty micrograms of
whole-cell extracts were resolved on 7.5% SDS–PAGE,
electrotransferred to a polyvinylidenedifluoride membrane followed
by blocking with 5% non-fat milk overnight. Protein expression was
determined using enhanced chemiluminescence method and probed for
the phosphorylated STAT3 (upper panel) and stripped and reprobed
for STAT3 (lower panel). Western blot for GAPDH was done to show
equal loading of protein.
M.A.Macha et al.
ecem ber 2021
enzyme-linked immunosorbent assay kit. We observed that GS
decreased IL-6 levels in a time-dependent manner with complete loss
of IL-6 within 6 h of GS treatment (Figure 4c). However, both STand
nicotine increased IL-6 secretion in a time-dependent manner
(Figure 4c). These results were in concordance with the expression
levels of NF-jB in SCC4 cells.
GS inhibits IL-6 inducible STAT3 phosphorylation in SCC4
cells
IL-6-induced signals are mediated through STAT3 phosphorylation;
therefore, we determined the effect of GS on IL-6-induced STAT3
phosphorylation. SCC4 cells were treated with IL-6 (10 ng/ml) for
different time intervals and whole-cell lysates were prepared.
Western blot analysis showed that IL-6 induced phosphorylation of
STAT3 in a time-dependent manner with phosphorylation induction as
early as in 10 min and maximum phosphorylation at 2 h (Figure 5a).
SCC4 cells were then preincubated with GS for different time
intervals, followed by IL-6 treatment for 2 h and effect on STAT3
phosphorylation was examined. Figure 5b shows that IL-6-induced
STAT3 phosphorylation was blocked by GS in a time-dependent manner.
Exposure of cells to GS for 4 h was sufficient to completely
suppress IL-6-induced STAT3 phosphorylation. However, GS treatment
alone at these time points had no effect on STAT3 levels in these
cells (Figure 5a).
GS induced inhibition of STAT3 phosphorylation is reversible in
SCC4 cells
We next determined whether GS-induced inhibition of STAT3 phos-
phorylation was reversible. SCC4 cells were first treated with GS
for 4 h, washed twice with PBS and the cells were then cultured in
fresh medium without GS for different time intervals, and the
levels of phos- phorylated STAT3 were measured. GS treatment
induced the suppres- sion of STAT3 phosphorylation (Figure 5c), but
once GS was removed, the levels of phosphorylated STAT3 increased
in a time-bound manner (Figure 5c). The reversal of phosphorylation
was partial at 18 h, with complete reversal at 36 h, without any
changes in the STAT3 levels.
GS inhibited ST/nicotine-induced STAT3 phosphorylation in SCC4
cells
The aberrant function of transcription factor NF-jB can lead to
stim- ulation of STAT3 by an autocrine/paracrine mechanism
involving the release of IL-6, prompting us to determine the effect
of GS on the molecular cross talk between these two pathways. We
investigated whether GS inhibits the constitutive STAT3
phosphorylation in SCC4 cells in accordance with its effect on
NF-jB expression. SCC4 cells were incubated either with GS (50 lM)
for indicated time intervals and compared with the untreated
control cells. Western blot analysis
showed that GS inhibited the constitutively active STAT3 in time-
dependent manner (Figure 5d). GS-induced inhibition was observed as
early as 1 h with complete inhibition of STAT3 phosphorylation
observed at 4 h; however, GS treatment did not alter the overall
STAT3 protein expression (Figure 5d). We also observed similar ef-
fect of GS treatment on the expression of activated upstream STAT3
kinase, pJAK1 (Figure 5d). However, no effect was observed on the
total JAK1 expression at these time points of GS treatment. These
results suggested that GS-induced inhibition of STAT3 phosphoryla-
tion was mediated by inhibiting the upstream kinases.
Furthermore, we investigated the effect of GS on ST- and nicotine-
induced STAT3 activation in vitro in SCC4 cells by western
blotting. Both ST- and nicotine- induced STAT3 phosphorylation
(Figure 5e and f). Increased STAT3 phosphorylation was observed in
a time-dependent manner with maximum phosphorylation at 6 h by ST
and within 4 h by nicotine, respectively. SCC4 cells were then
incubated with GS for different time intervals and examined for ST
and nicotine inducible STAT3 phosphorylation. Both ST- and
nicotine-induced STAT3 phos- phorylation was blocked by GS in a
time-dependent manner (Figure 5g and h). Exposure of cells to GS
for 4 h completely suppressed ST- and nicotine-induced STAT3
phosphorylation. GS, ST or nicotine, alone or in combination, had
no effect on total STAT3 expression levels in these cells. These
results clearly demonstrate that GS abrogated ST- and
nicotine-mediated activation of pSTAT3 in SCC4 cells.
GS inhibited ST- and nicotine-induced expression of VEGF and
recruitment of STAT3 to the VEGF promoter
STAT3 is a transcriptional activator of VEGF and STAT3 inhibition
effectively blocks VEGF expression. STAT3 is being activated by
both ST and nicotine, therefore, we demonstrated the effect of
these compounds on VEGF expression and effect of GS pretreatment on
ST- or nicotine-induced VEGF expression using western blotting.
SCC4 cells were treated with either GS (50 lM) for different time
intervals, or with ST (20 lg/ml)/nicotine (10 lM) for 6 and 12 h.
As shown in Figure 6a, GS downregulated VEGF expression in a
time-dependent manner, whereas both ST- and nicotine-induced VEGF
expression (Figure 6b). Notably, pretreatment of cells with GS for
6 h followed by either ST or nicotine for 12 h, inhibited both ST-
and nicotine- induced VEGF expression (Figure 6b).
To demonstrate the effect of GS on ST- or nicotine-induced inter-
action between STAT3 protein and VEGF promoter in vivo, we carried
out chromatin immunoprecipitation assays using an anti-STAT3 anti-
body. The occupancy of the promoter was analyzed using specific
pairs of primers spanning the STAT3 binding motif of the VEGF
promoter. SCC4 cells were treated with either GS (50 lM) for 6 h;
ST (20 lg/ml)/nicotine (10 lM) 12 h or pretreated with GS for 6
h
Fig. 5. Continued.
377
ecem ber 2021
followed by ST and nicotine for 12 h. Significantly greater binding
of STAT3 to the VEGF promoter was observed in ST- or
nicotine-treated cells (Figure 6c). GS treatment significantly
inhibited both ST- and nicotine-induced recruitment of STAT3 to the
VEGF promoter (Figure 6c).
Discussion
Knowledge of molecular pathways implicated in ST-induced head and
neck cancers is critical for development of novel agents that are
better targeted to ST-induced molecular changes and to ST-related
HNSCC for effective disease management. Herein, we demonstrated
that ST or nicotine activates NF-jB in HNSCC cells (SCC4 and HSC2)
and this activation was mediated through phosphorylation and
degradation of IjBa. ST as well as nicotine-induced IL-6 secretion,
which in turn activated the STAT3 pathway. Therefore, agents that
can suppress NF-jB and STAT3 activation might have potential to
suppress ST/
nicotine-induced head and neck cancer. In this context, we investi-
gated the potential of GS to block ST/nicotine-induced-NF-jB and/or
IL-6/STAT3 activation in HNSCC cells (SCC4/HSC2) and deter- mined
its effect on the abrogation of expression of genes implicated in
carcinogenesis. Our results clearly showed that GS treatment ab-
rogated both ST- and nicotine-induced NF-jB activation by suppress-
ing IjBa phosphorylation and degradation, thereby inhibiting the
phosphorylation and nuclear translocation of the p65 subunit and
sub- sequent inhibition of COX-2 expression. Proteosomal
degradation of IjBa protein is mediated by inhibitor kappa kinase B
dependent phosphorylation. Our results clearly demonstrated
decreased phos- phorylation of IjBa in head and neck cancer cells
on treatment with GS in time-dependent manner, thus reduced
degradation. In support of our study, Shishodia et al. (40) also
showed inhibition of tumor ne- crosis factor-a dependent activation
of inhibitor kappa kinase B in presence of GS. This reduced
phosphorylation of IjBa and inhibited nuclear translocation of p65
subunit of NF-jB. This aberrant
Fig. 6. GS inhibits ST- and nicotine-induced expression of VEGF and
recruitment of STAT3 to the VEGF promoter. SCC4 cells (2–3 106
cells) were treated with (a) with either GS (50 lM) for different
time points (b) ST (20 lg/ml)/nicotine (10 lM) for 6 and 12 h, or
pretreatment of GS with for 6 h followed by either ST or nicotine
for 12 h. Whole-cell extracts were prepared and 60 lg of extracts
were resolved on 10% SDS–PAGE, electrotransferred to a
polyvinylidenedifluoride membrane followed by blocking with 5%
non-fat milk overnight. Protein expression was determined using
enhanced chemiluminescence method and probed for the VEGF and
stripped and reprobed for a-tubulin to show equal loading of
protein. (c) Cross-linked, sheared chromatin was prepared from
treated SCC4 and subjected to immunoprecipitation with the STAT3
antibody. The precipitates were subjected to polymerase chain
reaction analysis using primer pairs spanning the human VEGF
promoter. The control was the polymerase chain reaction product of
chromatin obtained before immunoprecipitation.
M.A.Macha et al.
ecem ber 2021
activation of NF-jB can also stimulate STAT3 by an autocrine/para-
crine mechanism involving the release of IL-6, which confers both
proliferative and survival potential in HNSCC (25,41), prompting us
to determine the effect of GS on molecular cross talk between these
two pathways. We observed GS decreased IL-6 secretion in SCC4
cells, whereas both ST and nicotine increased the secretory IL-6
lev- els, and these changes in IL-6 levels were paralleled by
changes in p65 subunit of NF-jB. We also observed that GS abrogated
both consti- tutive and ST/nicotine-induced STAT3 phosphorylation
in cancer cells, which may be one of the mechanisms for reducing
the prolif- erative potential of HNSCC cells.
Interestingly, Arredondo et al. (42) showed multifold increase in
STAT-3 in oral keratinocytes stimulated with aged and diluted side-
stream cigarette smoke or nicotine, both at the messenger RNA and
protein levels. In addition, the downregulation of NF-jB, const-
itutive and inducible STAT3 by GS in other HNSCC cell lines and
xenografts have been reported (28,31,34,35,43,44). Importantly,
these observations are in accord with clinical studies from our
group and others, showing significant association of pSTAT3
activation with ST consumption habits (26,45). Previously, we have
also shown that GS downregulates the expression of cyclin D1, Bcl2
and Bcl-xL in SCC4 cells (46). These genes are known to be
regulated by both STAT3 and NF-jB (47,48). Notably, we found that
suppression of STAT3 phos- phorylation by GS was reversible,
returning to nearly control values within 36 h of GS
withdrawal.
Activation of NF-jB and COX-2 by ST was previously reported by our
group and others using different HNSCC cell lines, namely AMOS III
and SCC38 (22,24,48). Herein, we showed that ST- and
nicotine-induced NF-jB activation was mediated by the phosphory-
lation and degradation of IjBa. Notably, other forms of ST, such as
snuff extracts, have been shown to stimulate nuclear localization
of p50 and p65 subunits of NF-jB in RAW264.7 cells. Normal human
oral keratinocytes exposed to environmental tobacco smoke or nico-
tine also showed a multifold increase in NF-jB at the messenger RNA
and protein levels. Importantly, GS inhibited constitutive COX-2
ex- pression and treatment with GS abrogated the expression of both
ST- and nicotine-induced COX-2 expression. COX-2 has been
implicated in carcinogenic processes and its overexpression in
malignant cells has been shown to enhance cellular invasion, induce
angiogenesis, regulate anti-apoptotic cellular defenses and augment
immunologic resistance through production of prostaglandin E2
(22,48–50). Our results suggested that nicotine-induced NF-jB
activation resulting in increased expression of COX-2 protein head
and neck cancer cells (SCC4), which can be inhibited by treatment
with GS.
Our study also showed GS-inhibited ST/nicotine-induced STAT3
activation is required for VEGF receptor signaling and neovascula-
ture in human oral cancer (36). In accordance with the STAT3 acti-
vation, we observed an increase in VEGF expression in SCC4 cells
upon treatment with ST or nicotine and GS downregulated the levels
of VEGF protein. The similar time kinetics of STAT3 and VEGF
expression prompted us to determine if STAT3 regulated VEGF ex-
pression by activation of the VEGF promoter. Using co-immunopre-
cipitation and chromatin immunoprecipitation assays, we identified
an increased interaction of STAT3 on the VEGF promoter upon ST and
nicotine treatment, whereas GS treatment inhibited this interac-
tion, suggesting that STAT3 might serve as a transcription
regulator of VEGF promoter. These observations are consistent with
an earlier report demonstrating GS-inhibited STAT3 binding on VEGF
pro- moter (36). Thus, ST-induced STAT3 inhibition by GS and the
sub- sequent reduction in VEGF expression could represent a
potential clinical therapeutic approach for patients with
ST-associated HNSCC.
In conclusion, our studies demonstrated that ST and nicotine acti-
vate both NF-jB and STAT3 pathways in HNSCC cells and their
downstream targets—COX-2 and VEGF. Importantly, GS not only
inhibits the constitutively active NF-jB and STAT3 pathways but
also abrogates ST- and nicotine-induced activation of these
pathways underscoring its putative chemopreventive potential.
Hence, our find- ings provide a biologic rationale for
investigating the chemopreven-
tive and complementary therapeutic potential of GS in ST-associated
head and neck cancers.
Acknowledgements
M.A.M is recipient of Senior Research Fellowship of Council of
Scientific and Industrial Research, New Delhi, India. A.M. is the
recipient of a Mathematics of Information Technology and Complex
Systems Accelerate Fellowship, Ontario, Canada. RR gratefully
acknowledges support from the Joseph and Mildred Sonshine Center
for Head and Neck Diseases, Alex and Simona Shnaider Laboratory of
Molecular Oncology, Temmy Latner/Dynacare and the Department of
Otolaryngology-Head and Neck Surgery, Mount Sinai Hos- pital,
University of Toronto. K.W.M.S. acknowledges funding from the
Ontario Institute for Cancer Research.
Conflicts of Interest: None declared.
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Received May 19, 2010; revised December 2, 2010; accepted December
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