Ogni antibiotico è efficace in relazione a un determinato gruppo di microrganismi
comprare ampicillina online in italiain caso di infezioni oculari vengono scelte gocce ed unguenti.
Mcr-13-0239 1279.129
Signal Transduction
Minocycline Targets the NF-kB Nexus through Suppressionof TGF-b1-TAK1-IkB Signaling in Ovarian Cancer
Parvin Ataie-Kachoie1, Samina Badar1,2, David L. Morris1,2, and Mohammad H. Pourgholami2
Substantial evidence supports the critical role of NF-kB in ovarian cancer. Minocycline, a tetracycline, has
been shown to exhibit beneficial effects in this malignancy through regulation of a cohort of genes thatoverlap significantly with the NF-kB transcriptome. Here, it was examined whether or not the molecularmechanism could be attributed to modulation of NF-kB signaling using a combination of in vitro and in vivomodels. Minocycline suppressed constitutive NF-kB activation in OVCAR-3 and SKOV-3 ovarian carci-noma cells and was correlated with attenuation of IkBa kinase (IKK) activation, IkBa phosphorylation anddegradation, and p65 phosphorylation and nuclear translocation. The inhibition of IKK was found to beassociated with suppression of TGF-b-activated-kinase-1 (TAK1) activation and its dissociation from TAK1-binding-protein-1 (TAB1), an indispensable functional mediator between TGF-b and TAK1. Further studiesdemonstrated that minocycline downregulated TGF-b1 expression. Enforced TGF-b1 expression inducedNF-kB activity, and minocycline rescued this effect. Consistent with this finding, TGF-b1 knockdownsuppressed NF-kB activation and abrogated the inhibitory effect of minocycline on this transcription factor.
These results suggest that the minocycline-induced suppression of NF-kB activity is mediated, in part,through inhibition of TGF-b1. Furthermore, the influence of minocycline on NF-kB pathway activation wasexamined in female nude mice harboring intraperitoneal OVCAR-3 tumors. Both acute and chronicadministration of minocycline led to suppression of p65 phosphorylation and nuclear translocationaccompanied by downregulation of NF-kB activity and endogenous protein levels of its target gene products.
These data reveal the therapeutic potential of minocycline as an agent targeting the pro-oncogenic TGF-b–NF-kB axis in ovarian cancer.
Implications: This preclinical study lends support to the notion that ovarian cancer management would benefitfrom administration of minocycline. Mol Cancer Res; 11(10); 1279–91. 2013 AACR.
molecules such as ICAM-1, VEGF, matrix metalloprotei-
Uncontrolled activation of NF-kB signaling has been
nases (MMP), and COX-2 (reviewed in ref. 5). On the basis
implicated in propagation of a large number of malignancies
of the detrimental consequences of NF-kB activity in
including ovarian cancer (1, 2). Intrinsically or constitutively
ovarian cancer, it is imperative to assume that the blockade
active NF-kB has been defined critical in the promotion of
of this transcription factor is a potential therapeutic target for
cell proliferation, angiogenesis, metastasis (3), and develop-
this malignancy.
ment of drug resistance (4) in ovarian cancer. It is well
established that NF-kB exerts these effects through regula-
a safe antibiotic from the second-generation tetracycline
tion of numerous gene products including cell-cycle regu-
family. There is compelling preclinical evidence that min-
latory genes (e.g., cyclin D1 and c-Myc), antiapoptotic genes
ocycline along with a number of other tetracyclines have
(e.g., survivin, Bcl-2, and Bcl-x
potent antitumorigenic and antimetastatic properties in a
L), and genes encoding
inflammatory cytokines such as interleukin (IL)-6, adhesion
variety of tumors including leukemia (6), melanoma (7),renal, prostate (8), and breast cancer (9). Along this line, wehave recently reported the preliminary results showing that
Authors' Affiliations: 1Department of Surgery and 2Cancer Research
minocycline inhibits growth of human ovarian cancer xeno-
Laboratories, St George Hospital, University of New South Wales, Sydney,
grafts (10, 11) and also suppresses ovarian cancer-induced
malignant ascites formation (10). We have also established
Corresponding Author: David L. Morris, Professor and Head of Depart-
that minocycline treatment of preclinical models of ovarian
ment of Surgery, Level 3 Pitney Building, St. George Hospital, Gray St.,Kogarah, Sydney, NSW 2217, Australia. Phone: 612-91132590; Fax: 612-
cancer modulates specific transcriptional and signal trans-
91133997; E-mail:
[email protected]
duction cascades that lead to cell-cycle arrest, apoptosis,
and downregulation of angiogenesis and metastasis (10–12).
2013 American Association for Cancer Research.
The exact mechanism by which minocycline mediates these
Ataie-Kachoie et al.
anticancer effects remains to be elucidated. However, our
D1, c-Myc, MMP-9 and ICAM-1 (Santa Cruz Biotechnol-
very recent investigations have proved its ability to inhibit
ogy), and mouse monoclonal anti-b-actin (R&D Systems).
the proinflammatory cytokine IL-6 (12), VEGF (10), and
Secondary antibodies were goat anti-rabbit or anti-mouse
MMP-2 and 9 (12), which are consistently involved in
immunoglobulin G (IgG) conjugated with horseradish per-
oxidase (HRP; Santa Cruz Biotechnology) and goat anti-
Because we have observed that minocycline influences the
rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen).
expression of IL-6, VEGF, and MMPs, which are regulatedby NF-kB and also suppresses tumorigenic cellular processes
which are, at least in part, NF-kB mediated, we postulated
The human ovarian cancer cell lines, OVCAR-3 and
that minocycline exerts its antitumor effects through mod-
SKOV-3, were obtained from American Type Culture
ulation of the NF-kB pathway. NF-kB is present in the
Collection. These cell lines were authenticated by DNA
cytosol as an inactive form bound to the inhibitory protein
short tandem repeat profiling, and experiments were carried
IkBa. When stimulated by appropriate extracellular signals,
out within 6 months of resuscitation. Cells were maintained
IkBa is phosphorylated by inhibitor of IkB kinase (IKK),
in RPMI-1640 medium with 2 mmol/L L-glutamine, 2 g/L
which results in proteasome-mediated degradation of IkBa.
sodium bicarbonate, 4.5 g/L glucose, 10 mmol/L HEPES, 1
Then the active complex of NF-kB gets librated and trans-
mmol/L sodium pyruvate (Invitrogen) supplemented with
locates to nucleus to mediate transcription of its target genes
10% heat-inactivated FBS and penicillin–streptomycin (50
(13). IKK complex is activated either through autopho-
U/mL) at 37C in a humidified atmosphere containing 5%
sphorylation or via phosphorylation by a series of mitogen-
CO2 as suggested by the manufacturer. Cell lines were
activated kinase kinase kinases (MAP3K) including TGF-
routinely assessed by cell morphology and their average
b–activated kinase-1 (TAK1). TAK1 has emerged as the
doubling time. At 60% to 80% confluency, cells were treated
most central IKK kinase (14), which gets activated in
with the indicated concentrations of minocycline (ranging
conjunction with its activator proteins, TAK1-binding pro-
from 0 to 100 mmol/L) dissolved in 5% FBS media. Treat-
tein (TAB)-1, TAB2, and TAB3 in response to divergent
ments were carried out for 24 hours.
stimuli such as lipopolysaccharides, IL-1b, TNF-a, andTGF-b (15).
The purpose of this study was to determine the influ-
Transfection efficiencies were monitored by transfection
ence of minocycline on NF-kB signal transduction path-
with a plasmid-encoding GFP (pEGFP; Clontech). For
way in ovarian cancer. Toward this goal, we used ovarian
TGF-b1–forced expression, the plasmid pCMV6 contain-
carcinoma cell lines, OVCAR-3 and SKOV-3, both har-
ing the full-length cDNA of human TGF-b1 (pCMV6-
boring constitutively activated NF-kB (16, 17). It was
TGF-b1), and the control vector (pCMV6-Neo) both
observed that minocycline decreased NF-kB activation in
designed by Origene were used. For silencing TGF-b1, a
these cells as assessed by luciferase reporter and DNA-
pool of 3 target-specific lentiviral plasmids each encoding
binding assays. To characterize the molecular mechanism
19 to 25 nt (plus hairpin) short hairpin RNA (shRNA)
behind this inhibitory effect, we used immunocytochem-
designed to knock down TGF-b1 gene expression (TGF-b1
ical and Western blot analysis together with stable knock-
shRNA plasmid) and a control nonsilencing shRNA plas-
down and overexpression strategies to show that minocy-
mid-A (Santa Cruz Biotechnology) were used. For luciferase
cline exerts this effect through suppression of TGF-b1
reporter studies to measure NF-kB activity, a NF-kB lucif-
which results in blockade of TAB1/TAK1/IKK/NF-kB
erase reporter plasmid (pNF-kB-Luc), which contains 6
p65 axis. To further confirm our in vitro observations,
copies of the NF-kB consensus binding sequence, was used
next we conducted in vivo-based investigations revealing
for the first time the inhibition of NF-kB activation alongwith the downregulation of NF-kB–regulated gene pro-
Stable transfection
ducts by minocycline in an experimental model of ovarian
OVCAR-3 and SKOV-3 cells were transfected with
cancer in mice. The evidence presented here clearly shows
pCMV6-TGF-b1, pCMV6-Neo, TGF-b1 shRNA, scram-
the huge potential of minocycline to dismantle the NF-kB
bled shRNA, or pEGFP by electroporation using Nucleo-
oncogenic pathway in ovarian cancer.
fector I Device (Lonza). In brief, cells were washed with PBSand detached with trypsin. A total of 1 106 cells were
Materials and Methods
suspended in 100 mL of Nucleofector Solution Mix. After
Chemicals and antibodies
addition of plasmid (2 mg), cells were transferred into the
Unless otherwise stated, all drugs and chemicals used in
electroporation cuvette and then pulsed (pulse program,
this study were obtained from Sigma-Aldrich (Australian
EN-138 for OVCAR-3 and EH-100 for SKOV-3 cells;
subsidiary). The following primary antibodies were used
solution, SF). Electroporated cells were plated, grown for
throughout this study: rabbit polyclonal antibodies specific
24 hours, and then cultured for 12 to 14 days in the presence
for NF-kB p65, phospho-NF-kB p65 (Ser536), IkBa, phos-
of 500 mg/mL genticine (for TGF-b1 plasmid) or 10 mg/mL
pho-IkBa (Ser32/36), IKKb, phospho-IKKa/b (Ser176/180),
puromycine (for TGF-b1 shRNA plasmid). Single-cell
TAK1, phospho-TAK1 (Thr187), TAB1 (Cell Signaling
clones were picked using sterile cotton buds after low-density
Technology), Histone H1, TGF-b1, Bcl-2, Bcl-xL, cycline
seeding in Petri dishes and were expanded to establish
Mol Cancer Res; 11(10) October 2013
Molecular Cancer Research
Regulation of NF-kB Pathway By Minocycline
independent cell lines. Established single-cell clones were
Preparation of cytosolic and nuclear extracts
screened for functionality by Western blot analysis using
At the end of each minocycline treatment, cells or crushed
tumors were washed twice with ice-cold PBS and harvestedin buffer A (10 mmol/L HEPES, pH 7.9; 1 mmol/L EDTA,
Luciferase reporter assay
1 mmol/L EGTA, 10 mmol/L KCl, 1 mmol/L DL-Dithio-
The effect of minocycline on NF-kB–dependent reporter
threitol (DTT), 0.5 mmol/L phenylmethylsulfonylfluoride;
gene transcription was analyzed by luciferase reporter assay.
PMSF) containing 120 mmol/L NaCl and protease inhibitor
Briefly, cells were plated at a subconfluent density in 6-well
cocktail (Sigma). Then they were lysed at 4C with 0.2 mL
tissue culture plates and grown for 24 hours. Cells were then
buffer A supplemented with 0.5% Nonidet P-40 and under
transiently cotransfected with pEGFP (Clontech) and pNF-
continuous shaking. After centrifugation, the supernatant
kB–Luc plasmid using GeneJuice transfection reagent
80C (cytosolic extract) and the pellets
(Novagen) according to the manufacturer's protocol. Trans-
were resuspended in ice-cold nuclear extraction buffer
fection efficiency was determined by fluorescence micros-
(20 mmol/L HEPES, pH 7.9, 0.4 mol/L NaCl, 1 mmol/
copy. Twenty-four hours posttransfection, cells were treated
L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L
with different concentration of minocycline (0–100 mmol/
PMSF, 2.0 mg/mL leupeptin, and 2.0 mg/mL aprotinin)
L) or pyrrolidine dithiocarbamate (PDTC; 50 mmol/L) and
and incubated at 4C for 15 minutes with occasional mixing.
incubated for another 24 hours. Cell lysates were collected in
Nuclear protein extracts were obtained by centrifugation at
30 mL of the Promega passive cell lysis buffer, and luciferase
13000 g for 15 minutes, and the supernatant was stored at
activity was assayed using a Luciferase assay system (Pro-
80C. Protein content was assayed with the Bio-Rad
mega) and a TD-20/20 luminometer (Turner Biosystems).
protein reagent. All cell fractionation steps were carried out
To avoid generating artifacts, which might occur when using
normalization to another reporter construct (e.g., Renillaluciferase; ref. 18), we have only used data from experiments
Immunoprecipitation and Western blotting
in which transfection efficiencies were comparable and also
For Western blot analysis, 50 mg of nuclear, cytosolic, or
relative light units were normalized for protein concentra-
cellular lysate (collected as previously described; ref. 10) were
tion of each lysate as determined using the Bio-Rad Protein
subjected to SDS-PAGE. Following electrotransfer, poly-
Assay. The relative amount of luciferase activity in the
vinylidene difluoride membrane (Millipore Corporation)
untreated cells was designated 1.
was probed with specific antibodies. Immune complexeswere detected using HRP conjugated with either anti-mouse
NF-kB DNA-binding assay
or anti-rabbit followed by chemiluminescence detection
DNA-binding activity of NF-kB was quantified using
(Perkin Elmer Cetus). To show equal protein loading, blots
the TransAM NF-kB p65 Transcription Factor Assay Kit
were stripped and reprobed with specific antibodies recog-
(Active Motif), according to the manufacturer's protocol.
nizing b-actin.
Briefly, nuclear extracts were added to each well of a 96-
For immunoprecipitations, cells lysates were precleaned
well plate coated with NF-kB consensus oligonucleotides.
with rabbit IgG with protein G-agarose. Then they were
NF-kB p65 proteins bound to the target sequence were
transferred to fresh tube and reacted with normal rabbit IgG
detected with primary antibodies specific to p65 and an
or anti-TAK1 antibody before undergoing precipitation
HRP-conjugated secondary antibody. Optical density was
with protein G-agarose. The immunoprecipitates were col-
measured at 450 nm as a relative measure of protein-
lected and washed 3 times with the lysis buffer and subjected
bound NF-kB. Specificity was confirmed by using wild-
to Western blotting.
type and mutant NF-kB oligonucleotide competitors.
Results are expressed as fold change in optical density
Establishment of intraperitoneal xenografts and
values of each sample comparing with its corresponding
assessment of minocycline molecular activity
Female nude athymic Balb C nu/nu mice (6 weeks old)
were purchased from Biological Resources (Faculty of Med-
icine, University of New South Wales, Sydney, NSW,
Immunofluorescent staining was conducted as previously
Australia). The mice were housed and maintained in laminar
described (10). Immunostaining of p-p65 and TGF-b1 was
flow cabinets under specific pathogen-free conditions in
carried out in OVCAR-3 and SKOV-3 cells seeded on
facilities approved by the University of New South Wales
sterilized cover slips and treated with minocycline (100
Animal Care and Ethics Committee (ACEC). All procedures
mmol/L) or PDTC (50 mmol/L) for 24 hours. Nuclear
carried out on mice were in strict accordance with the
translocation of p65 was examined in cells collected from
protocol approved by ACEC (approval number: 9/23B),
the peritoneal cavity of ovarian cancer-bearing mice sub-
and all efforts were made to minimize suffering. Intraper-
jected to acute or chronic minocycline treatment. Immu-
itoneal tumors were established according to the method
nostained cells were analyzed for protein expression or
previously described (19). Briefly, 10 106 log-phase
localization using Olympus IX71 laser scanning microscopy
growing OVCAR-3 cells suspended in 0.5 mL PBS were
with 60 oil immersion lens. Images were acquired and
injected intraperitoneally to each mouse. For acute treat-
processed with FV1200 software.
ment, on day 28 after cell inoculation, mice were randomly
Mol Cancer Res; 11(10) October 2013
Ataie-Kachoie et al.
assigned to one of the treatment or control groups (8 mice
per group). Minocycline was dissolved in sterile normal
Minocycline suppresses constitutive NF-kB activation in
saline (0.6 mg/mL). Mice were injected intraperitoneally
ovarian cancer cells in a concentration-dependent
with a single dose of minocycline (30 mg/kg). Control group
received sterile normal saline instead. Chronic treatment is
A wide variety of tumor cell types are known to harbor
described in details in our previous report (10). In brief, 10
constitutively active form of NF-kB, which often conveys
days after cell inoculation, mice were randomly divided into
poor clinical outcome (20). Ovarian cancer cell lines
3 groups (12 per group) and were treated for 28 days with 0.3
OVCAR-3 and SKOV-3 are known to express constitu-
mg/mL or 0.6 mg/mL minocycline containing drinking
tively active NF-kB (16, 17). Whether minocycline affects
waters. Control group received drug-free sterile drinking
NF-kB activity in these cells was examined by means of
water. At the end of both the treatment periods, peritoneal
luciferase reporter assay. It was found that minocycline
cavity was washed according to the procedure explained
suppressed NF-kB activation in these cells in a concen-
earlier (10). Then animals were euthanized using Lethabarb
tration-dependent manner (Fig. 1A). The highest sup-
R (100 mg/kg) intraperitoneal (i.p.) injection (VIRBAC)
pression occurred at a concentration of 100 mmol/L
and tumors were immediately dissected and preserved at
minocycline with percentage reduction relative to vehi-
80C for later analysis. Cells harvested from the peritoneal
cle-treated cells: 65 4% for OVCAR-3 and 55 4.5%
cavity and were analyzed by confocal imaging for nuclear
for SKOV-3 (P < 0.001). PDTC (a standard pharmaco-
translocation of NF-kB p65. NF-kB activity was evaluated
logic inhibitor of p65) used as experimental control,
in the nuclear fraction of tumor cell lysates. Whole-cell and
decreased the NF-kB transcriptional activity in both
fractionated tumor lysates were used to determine NF-kB
ovarian cancer cell lines, indicating that the assay was
p65 phosphorylation and nuclear translocation. The expres-
sion of NF-kB–responsive gene products was examined in
Moreover, consistent with decreased transcriptional activ-
tumor whole-cell lysates.
ity of NF-kB, the NF-kB factor p65 DNA-binding activitywas suppressed in a concentration-dependent fashion in the
Statistical analysis
minocycline-treated cells, as measured by using an ELISA-
All statistical analyses were conducted using GraphPad
based kit. The maximum inhibition was observed at a
Prism Software version 6.0. Data are presented as mean
concentration of 100 mmol/L minocycline with percentage
SD. The student t test was used to compare 2 independent
inhibition relative to vehicle-treated cells: 75 7.5% for
group means. One-way ANOVA was used to determine the
OVCAR-3 (P < 0.01) and 54 6% for SKOV-3 (P < 0.05).
statistical differences between more than 2 groups; a signif-
PDTC as experimental control decreased p65 DNA-binding
icant interaction was interpreted by a subsequent simple
activity (Fig. 1B).
effects analysis with Bonferroni correction. Statistical signif-
These results indicate that minocycline is effective in
icance was established at the P < 0.05 level.
suppressing constitutively active NF-kB.
Figure 1. Minocycline suppressesconstitutive NF-kB activation in aconcentration-dependent manner.
A, NF-kB–mediated luciferaseactivity in OVCAR-3 and SKOV-3cells transfected with a NF-kB–responsive luciferase reporterplasmid and subjected to indicatedtreatments. Results are normalizedrelative to protein concentrationand expressed as fold changecomparing with vehicle-treatedcells. B, DNA-binding activity ofNF-kB p65 in the nuclear extractsof OVCAR-3 and SKOV-3 cellsafter indicated treatments asmeasured by an ELISA-basedTransAM kit. The results arepresented as fold of the vehicle-treated control. Columns, means of3 independent experiments done intriplicate; bars, SD. , P < 0.05;
, P < 0.01; and , P < 0.001versus vehicle-treated cells.
Mol Cancer Res; 11(10) October 2013
Molecular Cancer Research
Regulation of NF-kB Pathway By Minocycline
Figure 2. Minocycline inhibitsNF-kB p65 phosphorylation andnuclear translocation. A, confocalimaging analysis of NF-kB p65(green) in OVCAR-3 and SKOV-3cells under control conditions andexposed to specified treatments.
Cells were also stained withpropidium iodide (PI; red). Imageswere obtained at 60magnification. The scale barsrepresent 50 mm. B, cellular levelsof total and phosphorylated p65 inOVCAR-3 and SKOV-3 cellstreated with increasingconcentrations of minocycline asexamined by Western blotting.
C, Western blot analysis of thesubcellular localization of p65 inOVCAR-3 and SKOV-3 cellsexposed to varying concentrationsof minocycline. Densitometricanalysis of immunoblotting datawas carried out using the QuantityOne image program (Bio-Rad). Thevalues are normalized to b-actin orHistone H1 as loading controls andare expressed as the percentage ofvehicle-treated group. Numbersopposite column indicators,concentration of minocycline(mmol/L); Columns, means of 3independent experiments; bars,SD. , P < 0.01 and , P < 0.001versus vehicle-treated cells.
Minocycline abrogates NF-kB p65 phosphorylation and
Results obtained from Western blot analysis also show
nuclear translocation
significant inhibitory effect of minocycline at 50 and 100
NF-kB transcriptional activity correlates with Ser536
mmol/L concentrations on constitutive Ser536 phosphoryla-
phosphorylation of the NF-kB subunit, p65, and its sub-
tion of p65 in OVCAR-3 and SKOV-3 cells (P < 0.01). p65
sequent nuclear translocation (21). We therefore sought to
expression in the whole-cell extract remained unchanged
investigate the effect of minocycline on these processes.
after minocycline treatment (Fig. 2B). However, as shown
Immunofluorescent staining revealed that minocycline
in Fig. 2C, treatment with minocycline decreased nuclear
(100 mmol/L) suppressed the expression of phosphorylated
translocation of p65. Retention of p65 in the cytosol of
p65 (Ser536) in the nucleus of both OVCAR-3 and SKOV-3
minocycline-treated cells was significant even at 10 mmol/L
cell lines (Fig. 2A). PDTC as experimental control also
minocycline treatment (P < 0.001 for OVCAR-3 and P <
blocked p-p65 (Ser536) expression.
0.01 for SKOV-3).
Mol Cancer Res; 11(10) October 2013
Ataie-Kachoie et al.
These results support the conclusion that minocycline
phosphorylation of TAK1 at Thr-187 is inhibited by min-
inhibits phosphorylation and nuclear translocation of p65.
ocycline in a concentration-dependent manner in both cellslines.
Minocycline inhibits IkBa degradation and
When activated, TAK1 mediates phosphorylation of IKK
in association with its activator proteins TAB1 and TAB2 or
Translocation of NF-kB to the nucleus succeeds the
the structurally related TAB3 (15). Among these activator
proteolytic degradation of the NF-kB–associated inhibi-
proteins, TAB1 has been recognized to be sufficient for the
tory protein; IkBa (22). Therefore, we next examined
association with and the induction of the catalytic activity of
whether minocycline-induced NF-kB inhibition is asso-
TAK1 (25) and also prevention of its dephosphorylation and
ciated with inhibition of IkBa degradation. Figure 3A
inactivation (26). Therefore, we next investigated whether
shows that under conditions used in this study, minocy-
minocycline affects the interaction of TAK1 with its acti-
cline attenuates the degradation of IkBa in OVCAR-3
vator protein TAB1. For this, we carried out immunopre-
and SKOV-3 cells. These results indicate that minocycline
cipitation with the anti-TAB1 antibody. Western blot anal-
mediates its effect through the suppression of IkBa
ysis of immunoprecipitants revealed that treatment with
degradation, which in turn leads to the suppression of
minocycline decreased the association of TAK1 with TAB1
the activation of NF-kB.
in a concentration-dependent manner consistent with
Given that IkBa phosphorylation is necessary for IkBa
decreased phosphorylation of TAK1. Given that total TAK1
degradation (22), we investigated the effect of minocycline
levels remained equivalent, a reduction of TAB1-bound
on IkBa phosphoryation by using the proteasome inhibitor
TAK1 indicates that more TAK1 has become unbound and
N–cbz–Leu–Leu–leucinal (MG-132), which prevents IkBa
is dissociated from TAB1 (Fig. 3E).
degradation (23). As shown in Fig. 3B, minocycline sup-
Collectively, these results suggest that minocycline abro-
pressed IkBa phosphorylation at serine 32/36 in the pres-
gates TAK1 activation and its interaction with the activator
ence of the proteasome inhibitor. The percentage inhibition
protein TAB1.
of IkBa phosphorylation observed with 100 mmol/L min-ocycline was determined at 80 4.5% for OVCAR-3 and
Minocycline downregulates TGF-b1 expression
75 5% for SKOV-3 cells as compared with corresponding
Because of the observed inhibition of TAB1/TAK1/IKK
controls (P < 0.01). These results suggest that minocycline
axis by minocycline and because TAB1 has been shown to
negatively modulates IkBa phosphorylation leading to
have specificity for TGF-b–induced NF-kB signaling (27,
inhibition of its degradation.
28), we next investigated whether minocycline-inducedinhibition of TAK1 activity is mediated through down-
Minocycline blocks IkBa kinase activation
regulation of TGF-b. Immunocytochemical staining (Fig.
Because minocycline inhibits the phosphorylation of
4A) and Western blot analysis (Fig. 4B) revealed that
IkBa, we next tested its effect on IKK a/b complex
minocycline markedly reduced the endogenous expression
activation, which is required for phosphorylation of IkBa.
of TGF-b1 (Fig. 4A).
Activation of the IKK complex is dependent on the phos-phorylation of 2 serine residues in at least one of its subunits
Suppression of NF-kB by minocycline is mediated
(Ser176/180 of IKKa or Ser177/181 of IKKb subunit;
through inhibition of TGF-b1
ref. 13). Therefore, we evaluated the activation status of
The downregulation of TGF-b1 expression by minocy-
the IKKa/b complex by Western blot analysis with a
cline coupled with inhibition of NF-kB pathway was a good
phosphospecific antibody that detects the endogenous levels
indication that the effect of minocycline on NF-kB pathway
of IKKa and IKKb only when phosphorylated at Ser176 and
might be mediated through modulation of TGF-b1. To
Ser180 or Ser177 and Ser181, respectively. As shown in Fig.
investigate this hypothesis, we stably transfected OVCAR-3
3C, minocycline treatment resulted in a concentration-
and SKOV-3 cells with either a TGF-b1–expressing
dependent inhibition of IKKa/b complex phosphorylatin
(pCMV-TGF-b1) or a control plasmid (pCMV-Neo). As
at these serine residues, whereas the total levels of IKKa and
expected, TGF-b1 protein levels were increased in TGF-b1–
IKKb did not change.
dominant cells compared with control nontransfected orpCMV-Neo–transfected cells (Fig. 4C). Consistent with
Minocycline suppresses TGF-b–activated kinase 1
this, NF-kB activity was increased by 4.5-fold in TGF-
activation and its association with TAB1
b1-OVCAR-3 cells (P < 0.001) and 3.5-fold in TGF-b1-
Because TAK1 has been known as the major regulator of
SKOV-3 cells (P < 0.01) comparing with empty vector–
IKK phosphorylation and activation (14), we sought to
transfected cell lines. Furthermore, minocycline could sig-
examine whether minocycline inhibition of IKK activation
nificantly suppress NF-kB activity in TGF-b1–dominant
is rendered through modulation of TAK1 activation. Phos-
OVCAR-3 (P < 0.001) and SKOV-3 cells (P < 0.01; Fig.
phorylation of Thr-187 is strongly linked with TAK1 kinase
4D). These results suggest that ectopic TGF-b1 induces NF-
activity (24). We thus examined the activation status of
kB activity and minocycline inhibits this activation. There-
TAK1 by Western blot analysis using a phosphospecific
fore, it is likely that the effect of minocycline on NF-kB
antibody that detects endogenous levels of TAK1 only when
pathway is at least in part mediated through inhibition of
phosphorylated at threonine 187. As shown in Fig. 3D, the
Mol Cancer Res; 11(10) October 2013
Molecular Cancer Research
Regulation of NF-kB Pathway By Minocycline
Figure 3. Minocycline abrogatesTAK1/TAB1/IKK/IkBa signaling. A,effects of minocycline ondegradation of IkBa. Cytoplasmicextracts of OVCAR-3 and SKOV-3cells treated with minocycline(0–100 mmol/L) were subjected toWestern blot analysis using IkBaantibody. B, effects of minocyclineon phosphorylation of IkBa.
Cytoplasmic extracts of cellstreated with indicatedconcentrations of minocycline inthe presence or absence of MG-132 (10 mmol/L) were subjectedto Western blot analysis usingIkBa and p-IkBa antibodies.
C and D, minocycline inhibits thephosphorylation of IKK and TAK1.
Whole-cell extracts after treatmentwith minocycline (0–100 mmol/L)were analyzed by Westernblotting using the indicatedantibodies. b-actin, loadingcontrol. Densitometric values ofthe bands obtained by using theQuantity One image program(Bio-Rad) were corrected andexpressed relative to that of controlcells which was set as 100.
Columns, means of 3 independentexperiments; bars, SD. , P < 0.05;
, P < 0.01; and , P < 0.001versus vehicle-treated cells. E,whole-cell extracts after indicatedtreatments with minocycline wereisolated and immunoprecipitatedwith normal rabbit IgG or anti-TAB1antibody. Coimmunoprecipitationswere analyzed using anti-TAK1antibody. The experiment wasrepeated 3 times with the sameresults.
Mol Cancer Res; 11(10) October 2013
Ataie-Kachoie et al.
Figure 4. Influence of minocycline on TGF-b1 expression and TGF-b1–induced NF-kB activation in ovarian cancer cells. A, confocal immunocytochemistryof TGF-b1 (green) in OVCAR-3 and SKOV-3 cells treated with minocycline in comparison with control cells. The nuclei are counterstained with propidiumiodide (PI; red). Images were obtained at 60 magnification. The scale bars represent 100 mm. B, the expression levels of TGF-b1 in cells treated withminocycline (0–100 mmol/L) as estimated by Western blot analysis. The densitometric values quantified using the Quantity One image program (Bio-Rad) werecorrected on the basis of b-actin and were expressed as the percentage of the values corresponding to control cells. Columns, means of 3 independentexperiments; bars, SD. , P < 0.01 versus control cells. C, TGF-b1 expression in cells either untransfected or stably transfected with pCMV6-TGF-b/pCMV6-Neo as assessed by Western blotting. b-actin, loading control. D, NF-kB–dependent luciferase activity of TGF-b1–dominant cells treated with vehicle/minocycline (100 mmol/L) expressed as fold change comparing pCMV6-Neo–transfected cells. Results were standardized relative to protein concentration.
Columns, means of 3 independent experiments; bars, SD. , P < 0.01 and , P < 0.001 versus pCMV6-Neo–transfected cells. E, TGF-b1 expressionin cells either untransfected or stably transfected with Scrambled shRNA/TGF-b1-shRNA as assessed by Western blotting. b-actin, loading control. F, NF-kBactivity of Scrambled shRNA/TGF-b1-shRNA–transfected cells treated with vehicle/minocycline (100 mmol/L) assessed by luciferase reporter assay. Resultswere standardized relative to protein concentration and expressed as fold change comparing with vehicle-treated cells in each group. Columns, means of3 independent experiments; bars, SD. , P < 0.01 and , P < 0.001 versus vehicle-treated cells in each group.
Mol Cancer Res; 11(10) October 2013
Molecular Cancer Research
Regulation of NF-kB Pathway By Minocycline
Figure 5. Minocycline suppressesNF-kB activation in female BALB/cathymic nude mice bearingintraperitoneal OVCAR-3 tumors.
DNA-binding activity of NF-kB p65in the nuclear extracts of tumorcells excised from mice subjectedto either A, a single dose (30 mg/kg,i.p. for 24 hours) or B, chronic (0, 0.3or 0.6 mg/mL in drinking water for 4weeks) minocycline treatment asmeasured by an ELISA-basedTransAM kit. The results arepresented as fold of the controlgroup. Bars, SD. , P < 0.05 and
, P < 0.01 versus controlgroup. C and D, representativeconfocal images showing nucleartranslocation of p65 (green) in cellscollected from the peritoneal cavityof mice subjected to a single doseor chronic minocycline treatment.
The nuclei are counterstained withpropidium iodide (PI; red).
Images were obtained at 60magnification. The scale barsrepresent 50 mm. E and F, whole-cell or fractionated protein lysatesof tumor tissues excised fromcontrol mice or mice undergoneacute or chronic minocyclinetreatment were subjected toWestern blotting for the expressionof p-p65 and p65. Thedensitometric values quantifiedusing the Quantity One imageprogram (Bio-Rad) were correctedon the basis of b-actin orHistone H1 and were expressedas the percentage of the valuescorresponding to control group.
Bars, SD. , P < 0.05; , P < 0.01;and , P < 0.001 versus control.
To reinforce this notion, next we knocked down TGF-b1
treatment on NF-kB activity in an experimental model
expression by shRNA (Fig. 4E). Although minocycline
of ovarian cancer in mice. As shown in Fig. 5A, a single
treatment significantly inhibited the activity of NF-kB in
dose of minocycline suppressed the NF-kB activity in
OVCAR-3 (P < 0.001) and SKOV-3 cells (P < 0.01) stably
tumors excised from mice by 65 6% (P < 0.05).
transfected with the scrambled shRNA, ablation of TGF-b1
Analyzing the samples from our previous animal experi-
completely abrogated this inhibitory effect of minocycline
ment where minocycline was administered in drinking
on NF-kB activity (Fig. 4F). These findings suggest that
water for a period of 4 weeks (10), we also observed a
TGF-b1 is a key player in the influence of minocycline on
significant dose-dependent decline in NF-kB activity in
the transcription factor NF-kB.
tumors collected from these chronically treated mice.
Compared with control group, NF-kB activity was
Minocycline inhibits NF-kB and NF-kB–regulated gene
reduced by 42 7% (P < 0.05) in the 0.3 mg/mL
products in ovarian tumor-bearing athymic nude mice
minocycline-treated mice. Obviously, the effect was more
To assess whether the inhibitory effect of minocycline
pronounced in the group receiving the larger minocycline
on NF-kB activity in ovarian cancer cells could occur
dose where NF-kB activity decreased by 63 3% (P <
in vivo, we next evaluated the effect of minocycline
0.01; Fig. 5B). In addition, confocal microscopy showed
Mol Cancer Res; 11(10) October 2013
Ataie-Kachoie et al.
Figure 6. Minocyclinedownregulated the expression ofNF-kB–dependent gene productsin ovarian cancer-bearing mice.
Protein lysates of tumor tissuesexcised from mice subjected to (A)single dose (30 mg/kg, i.p., 24hours) or (B) chronic (0, 0.3 or 0.6mg/mL in drinking water for 4weeks) minocycline treatmentwere analyzed by Western blottingfor the expression of Bcl-2, Bcl-xL,cyclin D1, C-Myc, MMP-9, andICAM-1 proteins. Densitometricvalues obtained by using theQuantity One image program(Bio-Rad) were normalized tob-actin and their relativedifferences with the correspondingcontrol values (arbitrarily set at 100)are shown. , P < 0.05; , P < 0.01;and , P < 0.001 versuscontrol.
cytoplasmic retention of p65 in tumor cells harvested from
peritoneal cavity of mice subjected to both single-dose
Although numerous studies have indicated that minocy-
(Fig. 5C) and chronic (Fig. 5D) minocycline treatment. In
cline exhibits antitumor activities, its precise mechanism of
line with this, phosphorylation and nuclear translocation
action is not well defined yet. Because NF-kB pathway has
of p65 were completely blocked in tumors excised from
been linked with cancer and due to the inhibitory effect of
single-dose minocycline-treated animals (P < 0.001; Fig.
minocycline on NF-kB–regulated gene products, it is pos-
5E). These effects were also observed in a dose-dependent
sible that modulation of NF-kB pathway is one of the major
fashion in mice subjected to chronic treatment where 0.6
mechanisms of action of minocycline. The major aim of the
mg/mL minocycline could completely abrogate p65 phos-
current study was to determine the effects of minocycline on
phorylation and nuclear translocation (P < 0.001; Fig.
NF-kB pathway in ovarian cancer. In vitro, using ovarian
cancer cells, we found that minocycline inhibited constitu-
To further confirm the inhibitory effect of minocycline on
tive activation of NF-kB which was confirmed by the
NF-kB transcriptional activity in vivo, we next examined the
observed inhibition of NF-kB p65 phosphorylation, nuclear
effect of minocycline on the expression of NF-kB–regulated
translocation, DNA binding, and transcriptional activity in
gene products including Bcl-2 and Bcl-xL, which have been
minocycline-treated cells. This was followed by acute (24
linked to tumor survival; cyclin D1 and c-Myc, which are
hours) treatment of ovarian tumor-bearing mice with min-
involved in tumor cell proliferation; and MMP-9 and
ocycline. Analysis of ascites cells and tumors collected from
ICAM-1, which play important roles in metastasis. Western
these mice revealed downregulation of NF-kB activity in
blot analysis revealed significant downregulation of the
minocycline-treated animals. This effect was also observed in
endogenous levels of these proteins in the tumor samples
samples from our previous animal experiment where min-
excised from mice treated with a single dose of minocycline
ocycline was administered chronically. In line with our data,
(Fig. 6A). The negative modulation of the tumoral expres-
very recently Kobayashi and colleagues and Naura and
sion of these NF-kB target gene products was also observed
colleagues have reported the inhibition of NF-kB by min-
in mice chronically treated with minocycline in their drink-
ocycline in animal models of amyotrophic lateral sclerosis
ing water (Fig. 6B). This completely agrees with the reported
(29) and asthma (30), respectively. Although encouraging,
antitumor effect of minocycline in that animal experiment
these reports have not described the mechanism through
which minocycline interacts with the NF-kB system to
These in vivo observations fully support our in vitro results
render it inactive.
described above, suggesting effective inhibition of NF-kB
Our results show that minocycline blocks activation of
signaling by minocycline leading to its reported biologic
IKK, thereby inhibiting the phosphorylation as well as
effects in cancer.
degradation of IkBa. Suppression of IKK activity strongly
Mol Cancer Res; 11(10) October 2013
Molecular Cancer Research
Regulation of NF-kB Pathway By Minocycline
agent in advanced ovarian cancer (37). On the basis of thesedata, it has been suggested that in the context of ovariancancer, mutations and/or alterations of the key nodesalong canonical TGF-b pathway shift TGF-b to nonca-nonical pathways such as activation of NF-kB whichdescribes the pro-oncogenic role of TGF-b in advancedovarian cancer (38, 39). In fact, activation of TGF-b/NF-
kB pathway has been defined as the underlying mech-anism in the phenomenon of conversion of TGF-b froma tumor suppressor to a tumor promoter during tumor-igenesis (28, 40, 41). In our study, it was found thatminocycline downregulates the endogenous levels ofTGF-b1. In line with this, minocycline could attenuatethe NF-kB activity surge in cells harboring ectopic TGF-
b1 expression. Besides, minocycline treatment of TGF-
b1–silenced cells with decreased NF-kB activity did notresult in any significant effect on this transcription factor.
Taken together, these results confirm that the effect ofminocycline on NF-kB pathway is rendered throughtargeting TGF-b1. In view of the data presented here,we propose a working model for minocycline (Fig. 7)where inhibition of TGF-b1 by minocycline leads tointerruption of TGF-b/TAB1/TAK1/IKK/NF-kB axis.
The functionality of the minocycline effect on the NF-kB
pathway was firmly shown in the tumoral expression of NF-
kB–responsive gene products. In both acute and chronicallytreated mice, tumoral levels of the antiapoptotic gene pro-
Figure 7. Proposed model for attenuation of NF-kB activities byminocycline. TGF-b1 activates NF-kB signaling through regulation of
ducts, Bcl-2 and Bcl-xL, proteins linked with tumor cell
TAB1-mediated TAK1 activation and the subsequent IKK kinase
proliferation cyclin D1 and c-Myc as well as protein linked
activation leading to phosphorylation and degradation of IkBa,
with adhesion (ICAM-1) and invasion (MMP-9) were
phosphorylation and nuclear translocation of NF-kB subunit p65 and
suppressed. Collective suppression of these gene products
activation of NF-kB downstream targets. Minocycline suppressesTGF-
could be attributed to the inhibition of the transcriptional
b1 expression and activities which results in the blockade of the
downstream TAK1/IKK signaling transduction.
activity of NF-kB by minocycline.
In conclusion, the in vitro and in vivo data presented here
suggests that the upstream kinases might be involved.
confirm the inhibitory effect of minocycline on NF-kB in
Numerous kinases have been linked with the activation of
ovarian cancer. Our results also show the mechanism by
IKK. Accumulating evidence suggests that the well-charac-
which minocycline inhibits NF-kB. Development of NF-
terized MAP3K family member TAK1 is the central IKK
kB inhibitors as potential anticancer agents is being actively
kinase induced by several stimuli (31–33). Here, we found
pursued by many research groups and pharmaceutical com-
that minocycline inhibits TAK1 phosphorylation. Further-
panies. Considering the proven clinical safety of minocy-
more, association of the pseudophosphotase TAB1 to
cline, we suggest pilot clinical trials to evaluate this agent for
TAK1, which is indispensable for phosphorylation-depen-
the management of ovarian cancer.
dent TAK1 activation (34), was also inhibited by minocy-
Disclosure of Potential Conflicts of Interest
cline. This indicates that the target of minocycline might lie
No potential conflicts of interest were disclosed.
above TAK1 in the NF-kB activation pathway.
TAB1 is an important signaling intermediate between
Authors' Contributions
TGF-b1 receptors and TAK1 in noncanonical TGF-b1
Conception and design: P. Ataie-Kachoie, M.H. PourgholamiDevelopment of methodology: P. Ataie-Kachoie, S. Badar, M.H. Pourgholami
pathway, which leads to NF-kB activation (28, 35). TGF-
Acquisition of data (provided animals, acquired and managed patients, provided
b1 stimulation triggers dissociation of TAK1 from TGF-b1
facilities, etc.): P. Ataie-Kachoie, D.L. MorrisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-
receptor, and subsequently TAK1 is phosphorylated through
tational analysis): P. Ataie-Kachoie, M.H. Pourgholami
TAB1-mediated autophosphorylation (35, 36). The so-far
Writing, review, and/or revision of the manuscript: P. Ataie-Kachoie
reported interactions between TGF-b and NF-kB in tumor
Administrative, technical, or material support (i.e., reporting or organizing data,constructing databases): P. Ataie-Kachoie
cells provide a confusing view, with TGF-b playing the role
Study supervision: D.L. Morris, M.H. Pourgholami
of either an inhibitor or activator of NF-kB signaling in
The costs of publication of this article were defrayed in part by the payment of page
different cell types. Besides, although TGF-b acts as a potent
charges. This article must therefore be hereby marked advertisement in accordance with
tumor suppressor in normal ovarian surface epithelial cells,
18 U.S.C. Section 1734 solely to indicate this fact.
the loss of growth-inhibitory responses is an early event in
Received May 9, 2013; revised July 1, 2013; accepted July 2, 2013;
ovarian cancer and yet TGF-b acts as a tumor-promoting
published OnlineFirst July 15, 2013.
Mol Cancer Res; 11(10) October 2013
Ataie-Kachoie et al.
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Source: http://mcr.aacrjournals.org/content/11/10/1279.full-text.pdf
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