Randomized Placebo-Controlled Phase IITrial of Autologous Mesenchymal StemCells in Multiple Sclerosis Sara Llufriu1., Marı´a Sepu´lveda1., Yolanda Blanco1, Pedro Marı´n2,Beatriz Moreno1, Joan Berenguer3, In˜igo Gabilondo1, Eloy Martı´nez-Heras1,Nuria Sola-Valls1, Joan-Albert Arnaiz4, Enrique J. Andreu5, Begon˜a Ferna´ndez1,Santi Bullich1, Bernardo Sa´nchez-Dalmau1,6, Francesc Graus1, Pablo Villoslada1,Albert Saiz1*
Mcr-13-0239 1279.129Signal 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.
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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M&A: Using Uncertainty to Your AdvantageA Survey of European Companies' Merger and Acquisition Plans for 2012 André Kronimus, Peter Nowotnik, Alexander Roos, and Sebastian Stange This report was prepared by The Boston Consulting Group on the basis of a survey of corporate executives in Europe conducted jointly with UBS Investment Bank.December 2011