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Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0037

Cancer
eventionesearch
arch Article

colchicoside Exhibits Anticancer Effects throughnregulation of NF-κB Pathway and Its Regulated Gene

PrR

ducts Linked to Inflammation and Cancer

e Reuter, Sahdeo Prasad, Kanokkarn Phromnoi, Jayaraj Ravindran, Bokyung Sung, Vivek R. Yadav,
swamy Kannappan, Madan M. Chaturvedi, and Bharat B. Aggarwal
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discovery of new uses for older, clinically approved drugs is one way to expedite drug developmentncer. Thiocolchicoside, a semisynthetic colchicoside from the plant Gloriosa superba, is a muscle re-and used to treat rheumatologic and orthopedic disorders because of its analgesic and anti-matory mechanisms. Given that activation of the transcription factor NF-κB plays a major roleammation and tumorigenesis, we postulated that thiocolchicoside would inhibit NF-κB and exhibitncer effects through the modulation of NF-κB–regulated proteins. We show that thiocolchicosideted proliferation of leukemia, myeloma, squamous cell carcinoma, breast, colon, and kidney cancerFormation of tumor colonies was also suppressed by thiocolchicoside. The colchicoside inducedosis, as indicated by caspase-3 and poly(ADP-ribose) polymerase cleavage, and suppressed the ex-on of cell survival [e.g., Bcl-2, X-linked inhibitor of apoptosis (XIAP), MCL-1, bcl-xL, cIAP-1, cIAP-2,LIP] proteins. Cell proliferation biomarkers such as c-MYC and phosphorylation of phosphoinosi--kinase and glycogen synthase kinase 3β were also blocked by thiocolchicoside. Because most cellal and proliferation gene products are regulated by NF-κB, we studied the effect of thiocolchicosideis transcription factor and found that thiocolchicoside inhibited NF-κB activation, degradation oftory κBα (IκBα), IκBα ubiquitination, and phosphorylation, abolished the activation of IκBα ki-and suppressed p65 nuclear translocation. This effect of thiocolchicoside on the NF-κB pathway ledibition of NF-κB reporter activity and cyclooxygenase-2 promoter activity. Our results indicate that
to inh
thiocolchicoside exhibits anticancer activity through inhibition of NF-κB and NF-κB–regulated gene pro-ducts, which provides novel insight into a half-century old drug. Cancer Prev Res; 3(11); 1462–72. ©2010 AACR.

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ocolchicoside is a semisynthetic drug derived fromicoside, a natural glucoside present in the plantsa superba. Thiocolchicoside has been used clinically5 years as a muscle relaxant as well as an anti-matory and analgesic drug (1). Given these proper-iocolchicoside has long been used to treat a numberhopedic, traumatic, and rheumatologic conditions.

linical trials showed that thiocolchicosidend safe treatment for patients with acute

ducesmyoreeffectOn

may ethe Nflammfactorand thmediaious itokintumotants,

n: Cytokine Research Laboratory, Department ofapeutics, University of Texas M.D. Anderson Cancerexas

ress for M.M. Chaturvedi: Department of Zoology,Delhi, India.

thor: Bharat B. Aggarwal, Cytokine Research Labora-f Experimental Therapeutics, University of Texas M.D.enter, 1515 Holcombe Boulevard, Box 143, Houston,13-794-1817; Fax: 713-745-6339; E-mail: aggarwal@

6207.CAPR-10-0037

ssociation for Cancer Research.

; 3(11) November 2010

Cancer Research. on Mcancerpreventionresearch.aacrjournals.org d from

ack pain accompanied by muscle spasm (2) and isst as effective as tizanidine, the standard drug usedt low back pain (3, 4). Biochemical studies indicatediocolchicoside can interact with γ-aminobutyric acidors, given that it was able to inhibit the binding of[3H]γ-aminobutyric acid and [3H]strychnine to ratocortical and spinal cord membranes, respectively,o and in vivo (5–7). Whereas these findings suggesthiocolchicoside, a γ-aminobutyric acid agonist, in-depression of the central nervous system and, in turn,laxation (8), themechanism of its anti-inflammatoryis still unknown.e possible mechanism by which thiocolchicosidexert its anti-inflammatory effect is by modulatingF-κB pathway, which is commonly involved in in-ation and tumorigenesis. NF-κB is a transcriptionthat resides in the cytoplasm in its resting stageen, when activated, translocates to the nucleus andtes the transcription of >400 different genes (9). Var-nflammatory agents can activate NF-κB, including cy-es [e.g., tumor necrosis factor (TNF)], carcinogens,
r promoters, cigarette smoke, environmental pollu-ionizing radiation, and stress (9). NF-κB activation
ay 14, 2018. © 2010 American Association for

http://cancerpreventionresearch.aacrjournals.org/

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en shown to control the expression of genes linked tomation, apoptosis, survival, proliferation, invasion,genesis, metastasis, chemoresistance, tumor cellrmation, and radioresistance (9).ause of the critical role of NF-κB in inflammationumorigenesis, we postulated that thiocolchicosidetes its anti-inflammatory effect through modulationNF-κB pathway. Indeed, our results show that thio-coside inhibits NF-κB activated by inflammatory cy-s (TNF), okadaic acid (OA), tumor promoter [phorbolristate 13-acetate (PMA)], and lipopolysaccharidethrough inhibition of phosphorylation, ubiquitina-nd degradation of inhibitory κBα (IκBα), the inhibitor-κB. Thiocolchicoside also inhibited the phosphoryla-nd nuclear translocation of p65, the major isoform of. Thiocolchicoside inhibition of NF-κB leads to sup-n of NF-κB–regulated proteins, which are responsibleanticancer effect of thiocolchicoside on various canceres, characterized by induction of apoptosis and inhi-of cell proliferation as well as colony formation. To-, our results provide a new role for thiocolchicosideanticancer agent.

rials and Methods

nts00 mmol/L solution of thiocolchicoside, kindly pro-by Sarv Bio Labs, was prepared in water, stored atand then diluted as needed in cell culture medium.ia-derived recombinant human TNF-α was kindlyed by Genentech.icillin, streptomycin, Iscove's modified Dulbecco'sm, DMEM, RPMI 1640, and fetal bovine serumwere purchased from Invitrogen. The proteasomeitor N-acetyl-leucylleucyl-norleucinal (ALLN) wasased from Calbiochem.A, OA, LPS, and anti-β-actin antibody were pur-from Sigma-Aldrich. Antibodies against p65, poly

-ribose) polymerase (PARP), inhibitor of apoptosisn 1, BCL-2, BCL-xL, c-MYC, and caspase-3 were pur-d from Santa Cruz Biotechnology. The antibodyst the inhibitory subunit of NF-κB (IκBα) was pur-d from Imgenex. Anti-XIAP antibodies wereased from BD Biosciences. Anti-phosphorylated IκBα/36) and anti-phosphorylated p65 (Ser536) wereased from Cell Signaling Technology. Anti-IκBα ki-(IKKα), anti-IKKβ, and anticellular caspase-8–like

tory protein (c-FLIP) antibodies were kindly providedgenex.

inescell lines used in this study were purchased fromcan Type Culture Collection. KBM5 cells were cul-in Iscove's modified Dulbecco's medium supplemen-th 15% FBS. U266, RPMI-8226, Jurkat, MM.1S, Caco-2,T-29 cells were cultured in RPMI 1640, and A293, HCT-
CF-7, and MCF-10A cells were cultured in DMEM
emented with 10% FBS. SCC4 cells were culturedcell ex(1.5 ×

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Cancer Research. on Mcancerpreventionresearch.aacrjournals.org wnloaded from

EM containing 10% FBS, nonessential amino acids,ate, glutamine, and vitamins. All media were alsomented with 100 units/mL penicillin and 100 μg/mLomycin.

oxicity assayl proliferation and cell viability experiments were as-by the modified tetrazolium salt MTT assay as de-d previously (10). For the cell proliferation assay,cells were incubated with various concentrationsocolchicoside, in triplicate, for 1, 3, and 5 days inll plates at 37°C. For the cell viability assay, 5,000ere incubated with various concentrations of thio-coside, in triplicate, for 24 hours in 96-well platesC. Thereafter, an MTT solution was added to eachAfter 2 hours of incubation at 37°C, lysis bufferSDS and 50% dimethylformamide) was added,lls were incubated overnight at 37°C, and then ab-nce was measured at 570 nm using a 96-well multi-er (MRX Revelation, Dynex Technologies).

genic assay for HCT-116clonogenic assay or colony formation assay is ano cell survival assay based on the ability of a singlegrow into a colony (11). HCT-116 cells have beenor the clonogenic assay, because these are adherentnd give a good response for this assay. To test theof thiocolchicoside to inhibit single cells to growolonies, 500 cells were seeded in six-well platescubated overnight to allow attachment. The follow-y, the cells were treated with various concentrationsocolchicoside, in triplicate, for 24 hours. The nextemediumwas changed, and the cells were incubateddays to form colonies. Medium was replaced after. At the end of the ninth day, medium was removed,.3 mL of clonogenic acid reagent was added. Cellsncubated for 30 minutes and washed twice, and blueies were counted (12).

ophoretic mobility shift assayassess NF-κB activation, we did electrophoretic mo-shift assay (EMSA) as described previously (13).rief, nuclear extracts prepared from TNF-treated cells106/mL) were incubated with 32P end-labeled 45-mere-stranded NF-κB oligonucleotide (15 μg of protein6 fmol of DNA) from the HIV long terminal repeat,TTACAAGGGACTTTCCGCTGGGGAC-TTTCCAGG-CGTGG-3′ (boldface indicates NF-κB–binding sites)minutes at 37°C, and the DNA-protein complex

dwas separated from free oligonucleotide on 6.6%na-lyacrylamide gels. The dried gels were visualized withm 820 PhosphorImager, and radioactive bands wereitated using ImageQuant software (GE Healthcare).

rn blot analysisdetermine the levels of protein expression in whole-
tracts or in the cytoplasm or nucleus of treated cells106 cells in 1 mL of medium), we prepared extracts,
Cancer Prev Res; 3(11) November 2010 1463



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0 μg of proteins were fractionated by SDS-PAGE.electrophoresis, the proteins were electrotransferredrocellulose membranes, blotted with the relevantdy, and detected with an electrogenerated chemilu-cence reagent (GE Healthcare).

ssaydetermine the effect of thiocolchicoside on TNF-ed IKK activation, IKK assay was done by a methodscribed previously (14). In brief, the IKK complexwhole-cell extracts was precipitated with antibodyt IKKβ and then treated with protein A/G-agarose(Pierce). After 2 hours, the beads were washed withuffer and then resuspended in a kinase assay mixtureining 50 mmol/L HEPES (pH 7.4), 20 mmol/L, 2 mmol/L DTT, 20 μCi of [γ-32P]ATP, 10 μmol/L un-d ATP, and 2 μg of substrate glutathione transferase-(amino acids 1-54). After incubation at 30°C for 30es, the reaction was terminated by boiling with SDSe buffer for 7 minutes. Finally, the protein was re-on 10% SDS-PAGE, the gel was dried, and the radio-bands were visualized with a Storm820. To determinetal amounts of IKKα and IKKβ in each sample, 30 μgole-cell protein were resolved on 7.5% $SDS-PAGE,transferred to a nitrocellulose membrane, and thend with either anti-IKKα or anti-IKKβ antibody.

–dependent reporter gene expression assayeffect of thiocolchicoside on NF-κB–dependent re-

r gene transcription induced by TNF and variouswas analyzed by secretory alkaline phosphatase) assay, with the following modification. In brief,cells (5 × 105 per well) were plated in six-well platesransiently transfected by the calcium phosphated with pNF-κB-SEAP (0.5 μg). To examine TNF-ed reporter gene expression, we transfected the cells.5 μg of the SEAP expression plasmid and 2 μg ofntrol plasmid pCMV-FLAG1 DNA for 24 hours.then treated the cells for 24 hours with thiocolchico-nd then stimulated them with 1 nmol/L TNF. Thelture medium was harvested after 24 hours of TNFent. To examine reporter gene expression inducedrious genes, we transfected A293 cells with 0.5 μgF-κBSEAP plasmid with 1 μg of an expressing plas-nd 0.5 μg of the control plasmid pCMV-FLAG1 forurs, treated them with 100 μmol/L thiocolchicoside,en harvested them from culture medium after an

ional 24 hours of incubation. Culture mediumnalyzed for SEAP activity according to the protocolially as described by the manufacturer (Clontech)a Victor 3 microplate reader (Perkin-Elmer Lifenalytical Sciences).

nocytochemical analysis of NF-κBocalizationeffect of thiocolchicoside on the nuclear transloca-
f p65 was examined by immunocytochemistry as de-d previously (10). In brief, treated cells were plated
on thmetho

r Prev Res; 3(11) November 2010


poly(L-lysine)–coated glass slide by centrifugationdon Cytospin 4, ThermoFisher Scientific), air-dried,xed with 4% paraformaldehyde after permeabiliza-ith 0.2% Triton X-100. After washing in PBS, thewere blocked with 5% normal goat serum for 1 hourhen incubated with rabbit polyclonal anti-humanntibody at a 1:200 dilution. After overnight incuba-t 4°C, the slides were washed, incubated with goatbbit IgG-Alexa Fluor 594 (Invitrogen) at a 1:200 di-for 1 hour, and counterstained for nuclei withst 33342 (50 ng/mL) for 5 minutes. Stained slidesmounted with mounting medium purchased from-Aldrich and analyzed under a fluorescence micro-(Labophot-2). Pictures were captured using a Photo-s Coolsnap CF color camera (Nikon) and Meta-Morphn 4.6.5 software (Molecular Devices).

rase assayeffect of thiocolchicoside on cyclooxygenase-2

-2) promoter activity induced by TNF was analyzeda luciferase assay. A293 cells (2.5 × 105 per well)seeded in six-well plates. After overnight culture,lls in each well were transfected by the calcium phos-method with 0.5 μg of DNA consisting of COX-2 pro-luciferase reporter. The COX-2 promoter (−375 ± 59)rovided by Dr. Xiao-Chun Xu (University of TexasAnderson Cancer Center). After 24 hours of transfec-the cells were incubated with thiocolchicoside forurs, then exposed to 1 nmol/L TNF for 20 hours,arvested. Luciferase activity was measured using therase assay system (Promega) and detected usingctor3 microplate reader (Perkin-Elmer Life and Ana-Sciences).

tical analysisults from at least three independent experimentsnalyzed for statistical significant differences using thent's t test. They are expressed as the mean ± SD.es below0.05were considered as statistically significant.

lts

present studies were designed to investigate theof thiocolchicoside on the NF-κB cell signalingay and on NF-κB–mediated cellular responses.n myeloid KBM5 cells were used for most studies,se TNF induces robust activation of NF-κB in theseand also these cells have extensively been used inboratory to study the mechanism of inhibition ofby various phytochemicals. In addition to this,

cell types were also used to determine the specificityeffect.

olchicoside inhibits cell proliferation of variousr cell linesinvestigated the effect of thiocolchicoside (Fig. 1A)
e proliferation of various cancer cells using the MTTd, which detects the mitochondrial activity of the
Cancer Prevention Research



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thiococell li

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or this, we treated different cell lines (KBM5, Jurkat,, HCT-116, Caco-2, HT-29, RPMI-8226, MM.1S,

ter 1 d, mediumwas changed, and cells were incubated for 9 d for colony fors was counted (bottom). Data are presented as mean (±SD), and * indicates

7, SCC4, and A293) with 25, 50, and 100 μmol/Llchicoside for 5 days (Fig. 1B). Results showed that

U266three

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lchicoside inhibited the proliferation of all cancernes investigated. Proliferation of KBM5, Jurkat, and

(top). After 9 d, cells were stained with crystal violet, and number of.05, when compared with control.

hiocolchicoside suppresses cell proliferation and colony formation of various cancer cell lines. A, the structure of thiocolchicoside. B, thiocolchicosidecell proliferation of various cancer cell lines. Leukemia (KBM5, Jurkat), colon (HCT-116, Caco-2, HT-29), myeloma (U266, RPMI-8266, MM.1S), breast), squamous (SCC4), and kidney (A293) cells were treatedwith 25, 50, and 100 μmol/L thiocolchicoside for 1, 3, and 5 d, and cell proliferationwas assessedTT method. C, thiocolchicoside has no effect on normal cells. MCF-10A (nontransformed breast epithelial cells) cells and MCF-7 (transformed breastl cells) cells were treated with 25, 50, and 100 μmol/L thiocolchicoside for 24 h, and cell viability was assessed by the MTT method. D, thiocolchicosidecolony formation of HCT-116 cells. HCT-116 cells were plated in six-well plates and treated with 25, 50, and 100 μmol/L of thiocolchicoside for

was completely inhibited after 3 and 5 days at allconcentrations tested. HCT-116 cells showed no




effectited aat 25inhibithiococentraa coma lessthe otinhibiTo

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at 25 μmol/L, but proliferation was strongly inhib-t 50 and 100 μmol/L. Caco-2 cells showed no effectμmol/L after 3 days, but proliferation was stronglyted at 5 days, as it was with 50 and 100 μmol/L oflchicoside. RPMI-8226 and SCC4 cells showed con-tion-dependent inhibition with 100 μmol/L havingplete inhibitory effect. HT-29 and A293 cells showedstrong response to 100 μmol/L thiocolchicoside thanher cells. MM.1S and MCF-7 cells also showed antory effect at all concentrations tested.investigate whether thiocolchicoside is selectively ac-gainst cancer cells and not against normal cells, weared its effect on cell viability of MCF-10A (non-ormed breast epithelial cells) and MCF-7 (trans-d breast epithelial cells) using the MTT methodC). Cells were treated with 25, 50, and 100 μmol/Llchicoside for 24 hours, and cell viability was mea-Results show that thiocolchicoside reduced cell via-of MCF-7 cells in a dose-dependent manner. But,lchicoside had no significant effect on cell viabilitytransformed MCF-10A cells. This result indicates thatlchicoside is only active against cancer cells and nott normal cells.also examined whether thiocolchicoside inhibitedy formation of HCT-116 cells in a long-term assay
D). We found that thiocolchicoside had minimal ef-n colony formation of HCT-116 cells at 25 μmol/L
Becprolif

sphorylated PI3K/p85, and phosphorylated GSK3β proteins. Unphosphorylatedding control. Numbers below each panel indicate fold differences after normaliz



0 and 100 μmol/L treatment completely suppressedlony-forming ability of these tumor cells.

olchicoside inhibits antiapoptotic proteins,es PARP and caspase-3 cleavage, and inhibitsssion of proteins involved in cell proliferationetermine themechanismresponsible for thiocolchicoside-ed inhibition of cancer cell proliferation, we analyzedpression of various proteins linked with cell prolif-n and apoptosis by Western blot. Thiocolchicosidedependently inhibited the antiapoptotic proteinsXIAP, Mcl-1, Bcl-xL, cIAP-1, cIAP-2, and cFlip (Fig. 2A).ntrast, thiocolchicoside induced PARP and caspase-3ge, confirming the proapoptotic effect of thiocol-ide (Fig. 2B).thermore, thiocolchicoside inhibited c-myc as well ashorylation of the p85 subunit of phosphoinositidease (PI3K) and GSK3β without having any effectnonphosphorylated formof these twoproteins, whicholved in cell proliferation (Fig. 2C). Together, these re-how that thiocolchicoside inhibits cancer cell growthucing apoptosis and inhibiting cell proliferation.

olchicoside inhibits TNF-inducedactivation

ause the transcription factor NF-κB is involved in celleration and apoptosis, we investigated the effect of

Thiocolchicoside induces apoptosis and inhibits proteins involved in cell proliferation. A, thiocolchicoside downregulates the expression ofptotic proteins. KBM5 cells were treated with 25, 50, 75, and 100 μmol/L of thiocolchicoside for 24 h; whole-cell extracts were prepared andd by Western blot using antibodies against Bcl-2, XIAP, Mcl-1, Bcl-xL, cIAP1, cIAP2, and cFlip. B, thiocolchicoside induces apoptosis. KBM5 cellsated with 25, 50, 75, and 100 μmol/L of thiocolchicoside for 24 h. Whole-cell extracts were prepared and analyzed by Western blot using antibodiesPARP and caspase-3. C, thiocolchicoside downregulates the expression of proteins involved in cell proliferation. KBM5 cells were treated with75, and 100 μmol/L of thiocolchicoside for 24 h. Whole-cell extracts were prepared and analyzed by Western blot using antibodies against c-Myc,

proteins of PI3K/p85 and GSK3β as well as β-actin were usedation to β-actin.




thiocoκB actwhichshowinducand tialoneWe

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flammatory agents has been shown to activate NF-κB, in-cludin

Fig. 3.A, thiocdependconcenTNF foactivatia time-dthiocolcfor 30 mdirect eNuclea0.1 nmoof thiocEMSA. Numbers below panels indicate fold differences normalizedto the c

Fig. 4.cell typby diffethiocol10 μg/mThe cethiocolCaco-2thiocol30 min.


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lchicoside on NF-κB activation. TNF is the main NF-ivator, so we used this cytokine to stimulate NF-κB,is retained in the cytoplasm when inactive. EMSAed that thiocolchicos ide suppressed TNF-ed NF-κB activation in a dose-dependent (Fig. 3A)me-dependent (Fig. 3B) manner. Thiocolchicosidedid not activate NF-κB.next sought to determine whether thiocolchico-irectly modified the binding of NF-κB complex

ontrol.

DNA. EMSA showed that thiocolchicoside didodify the DNA-binding ability of the NF-κB com-

activatinormal

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Fig. 3C). Therefore, we concluded that thiocolchi-inhibits NF-κB activation indirectly rather than

ly.

ression of NF-κB activation by thiocolchicosideot unique to TNFide variety of carcinogens, tumor promoters, and in-

Thiocolchicoside inhibits TNF-dependent NF-κB activation.olchicoside inhibits TNF-dependent NF-κB activation in a dose-ent manner. KBM5 cells were preincubated with indicatedtrations of thiocolchicoside for 24 h, treated with 0.1 nmol/Lr 30 min, and then subjected to EMSA to test for NF-κBon. B, thiocolchicoside inhibits TNF-dependent NF-κB activation inependent manner. KBM5 cells were preincubated with 100 μmol/Lhicoside for the indicated times, treated with 0.1 nmol/L TNFin, and then subjected to EMSA to test for NF-κB activation. C, theffect of thiocolchicoside on NF-κB complex was investigated.r extracts were prepared from untreated cells or cells treated withl/L TNF and incubated for 30minwith the indicated concentrationsolchicoside. They were then assayed for NF-κB activation by

g LPS, PMA, and OA, through mechanisms that may

Thiocolchicoside-induced NF-κB inhibition is neither inducer nore specific. A, thiocolchicoside suppresses NF-κB activationrent stimuli. KBM5 cells were preincubated with 100 μmol/Lchicoside for 24 h and then treated with 0.1 nmol/L TNF orL LPS for 30 min, 500 nmol/L OA for 4 h or 25 μg/mL PMA for 2 h.

lls were then analyzed for NF-κB activation by EMSA. B-D,chicoside induced NF-κB inhibition is not cell type specific., HT-29, and HCT-116 cells were incubated with 100 μmol/Lchicoside for 24 h and then incubated with 0.1 nmol/L TNF forNuclear extracts were then prepared and assayed for NF-κBon by EMSA. Numbers below panels indicate fold differences
ized to the control.



differ.NF-κBof thesuppr

Inhibnot c

Fig. 5. Tinhibitsindicateand p6Cytoplablot anaof IκBα0.1 nmoAn anti-thiocolcand thethiocolckinaseand examined by Western blot analysis using anti-IKKα and anti-IKKβ antibodies. D, immunocytochemical analysis of p65 localization. Cells were incubatedwith 10 in. Ce

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We investigated whether thiocolchicoside abrogatesactivation by all these agents. EMSA showed that all

0 μmol/L thiocolchicoside for 24 h and then treated with 1 nm TNF for 15 m

se agents activated NF-κB and that thiocolchicosideessed activation (Fig. 4A).

BecNF-κB



ition of NF-κB activation by thiocolchicoside wasell type specific

lls were subjected to immunocytochemical analysis.

hiocolchicoside inhibits TNF-dependent IκBα phosphorylation, IκBαdegradation, p65 phosphorylation, and p65 nuclear translocation. A, thiocolchicosideTNF-induced activation of NF-κB. KBM-5 cells were incubated with 100 μmol/L thiocolchicoside for 24 h, treated with 0.1 nmol/L TNF for thed times, and then analyzed for NF-κB activation by EMSA (left). A, right, effect of thiocolchicoside on TNF-induced IκBα degradation, p65 phosphorylation,5 nuclear translocation. Cells were incubated with 100 μmol/L thiocolchicoside for 24 h and treated with 0.1 nmol/L TNF for the indicated times.smic extracts (CE) and nuclear extracts (NE) were prepared, fractionated on SDS-PAGE, and electrotransferred to nitrocellulose membrane. Westernlysis was done using the indicated antibody. An anti-β-actin antibody was the loading control. B, effect of thiocolchicoside on the phosphorylationby TNF. Cells were preincubated with 100 μmol/L thiocolchicoside for 24 h, incubated with 50 μg/mL ALLN for 30 min, and then treated withl/L TNF for 10 min. Cytoplasmic extracts were fractionated and then subjected to Western blot analysis using a phospho-specific anti-IκBα antibody.β-actin antibody was used as loading control (left). B, right, thiocolchicoside inhibits ubiquitination of IκBα. Cells were preincubated with 100 μmol/Lhicoside for 24 h, incubated with 50 μg/mL ALLN for 30 min, and then treated with 0.1 nmol/L TNF for 10 min. Cytoplasmic extracts were fractionatedn subjected to Western blot analysis using a phospho-specific anti-IκBα antibody. An anti-β-actin antibody used as the loading control. C, direct effect ofhicoside on IKK activation induced by TNF. Whole-cell extracts were immunoprecipitated with antibody against IKKβ and analyzed by an immune complexassay. To examine the effect of thiocolchicoside on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE

ause distinct signal transduction pathways can mediateinduction in different cell types (15), we examined




the efftion inHT-29coside(Fig. 4

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ect of thiocolchicoside on TNF-induced NF-κB activa-three different human colon cancer cells: Caco-2,

, and HCT-116 cells. EMSA showed that thiocolchi-inhibited TNF-activated NF-κB in all three cell typesB-D).

olchicoside inhibited TNF-dependent IκBαhorylation, ubiquitination, and degradationtranslocation of NF-κB to the nucleus is preceded byroteolytic degradation of IκBα (16), so we nextt to determine whether thiocolchicoside-inducedinhibitory activity was due to inhibition of IκBαation. EMSA showed that NF-κB was activated withsing TNF incubation times and that thiocolchicosideatment strongly decreased this activation (Fig. 5A, left).rn blot analysis showed that TNF induced IκBα degra-in control cells after 10 minutes, and thiocolchico-hibited this degradation (Fig. 5A, right).ocolchicoside also inhibited IκBα phosphorylation,occurred in control cells after 5 minutes and disap-

d simultaneously with IκBα degradation (Fig. 5A,. These results indicate that thiocolchicoside inhib-oth TNF-induced NF-κB activation and IκBα degra-, as well as phosphorylation.further confirm that inhibition of TNF-induced IκBαation was due to inhibition of IκBα phosphoryla-nd ubiquitination, we used the proteasome inhibitorto block the degradation of IκBα (17). Western blotsis using an antibody that recognizes the serine-horylated form of IκBα showed that TNF inducedhosphorylation and that thiocolchicoside suppressedhis phosphorylation (Fig. 5B, left) and IκBα ubiqui-n (Fig. 5B, right).

olchicoside inhibitedTNF-induced activationof IKKause IKK is required for TNF-induced phosphoryla-f IκBα and because thiocolchicoside inhibited thehorylation of IκBα, we determined the effect oflchicoside on TNF-induced IKK activation. Resultsimmune complex kinase assay showed that TNF

ed the activation of IKK in a time-dependent man-d that thiocolchicoside down-modulated the TNFtion of IKK (Fig. 5C). Neither TNF nor thiocolchico-ffected the expression of IKKα or IKKβ proteinsC).

olchicoside inhibited nuclear translocation of p65is a subunit of NF-κB that has nuclear localizations, and p65 is retained in the cytoplasm by IκBα. Wexamined whether the degradation of IκBα leads tor translocation of p65. We found that TNF inducedclear translocation of p65 in as little as 10 minutesncubation (Fig. 5A, right) and that thiocolchicosideessed p65 translocation. Phosphorylation of p65 atwas also suppressed by thiocolchicoside (Fig. 5A,. Using an immunocytochemical assay, we further
med that thiocolchicoside suppressed the transloca-f p65 from the cytoplasm to the nucleus (Fig. 5D).
Cells wsubject* indica

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Thiocolchicoside represses NF-κB–dependent reporter geneion induced by TNF and by overexpression of various signalingdiates. A, thiocolchicoside inhibits the NF-κB–dependent reporterpression induced by TNF. A293 cells were transiently transfectedF-κB–containing plasmid for 24 h. After transfection, the cellscubated with the indicated concentrations of thiocolchicoside ford then treated with 1 nmol/L TNF for an additional 24 h. Theatants of the culture media were assayed for SEAP activity. Datasented as mean (±SD). B, thiocolchicoside inhibits the NF-κB–ent reporter gene expression induced by TNF, TNFR1, TRADD,, NIK, IKK, and p65. Cells were transiently transfected with acontaining plasmid alone or with the indicated plasmids. Afterction, cells were incubated with 100 μmol/L thiocolchicoside ford then incubated with the relevant plasmid for an additional 24 h.ated cells were incubated with 100 μmol/L thiocolchicoside ford then treated with 1 nmol/L TNF for an additional 24 h. Theatants of the culture media were assayed for SEAP activity. Datasented as mean (±SD). C, thiocolchicoside inhibits the COX-2er activity induced by TNF. Cells were transiently transfected with2 promoter linked to the luciferase reporter gene plasmid for 24 hated with the indicated concentrations of thiocolchicoside for 24 h.ere then treated with 1 nmol/L TNF for an additional 24 h, lysed, and
ed to a luciferase assay. Data are presented as mean (±SD), andtes P < 0.05 when compared with their respective control.



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olchicoside repressed TNF-induced NF-κB–dent reporter gene expressionough EMSA showed that thiocolchicoside blockedactivation, DNA binding alone does not always cor-with NF-κB–dependent gene transcription, suggest-at there are additional regulatory steps. Therefore,vestigated by a NF-κB–dependent reporter genesion assay whether thiocolchicoside could suppressF-induced NF-κB reporter activity. TNF induced–regulated SEAP reporter gene expression, andlchicoside suppressed the expression in a dose-dent manner (Fig. 6A).

olchicoside repressed NF-κB–dependentter gene expression induced by TNF receptor-1,1-associated death domain, TNFR-associated2, NF-κB–inducing kinase, IKKβ, and p65

F has been shown to activate NF-κB activationgh sequential interaction with the TNF receptor 11), TNFR1-associated death domain (TRADD),

-associated factor 2 (TRAF2), NF-κB–inducing kinase, and IKKβ, resulting in phosphorylation of IκBα9). To determine the effect of thiocolchicoside on–dependent reporter gene expression, cells wereently transfected with TNFR1-, TRADD-, TRAF2-,IKKβ-, and p65-expressing plasmids and then mon-for NF-κB–dependent SEAP expression. We found

ells transfected with any of these plasmids expressedF-κB–regulated reporter gene and that the reportersion induced by each of them was suppressed bylchicoside (Fig. 6B).

olchicoside inhibited TNF-induced COX-2oter activityinduces COX-2, which has NF-κB binding sites inmoter (20). Because we found that downregulation-κB by thiocolchicoside suppressed the expression of–regulated gene products, we also examined the ef-f thiocolchicoside on TNF-induced COX-2 promotery using a COX-2 promoter-luciferase reporter plas-e found that TNF induced COX-2 promoter activity

hat thiocolchicoside suppressed this activity in aependent manner (Fig. 6C). This result suggests thatlchicoside inhibits NF-κB–regulated gene expressionpressing NF-κB binding to the COX-2 promoter.

ssion

ur investigation of the effect of thiocolchicoside, aonly used muscle relaxant drug, on cell proliferationious cancer cells and on the NF-κB signaling path-e found that thiocolchicoside inhibited cell prolif-

n of various cancer cell lines and not of normal cells,ted the colony-forming ability of tumor cells, anded apoptosis by inhibiting antiapoptotic proteinsinducing cleavage of caspase-3 andPARP. In addition,
lchicoside suppressed NF-κB activation stimulated byogens and inflammatory stimuli in a dose- and time-
Bcl-2,protei



dentmanner. Thiocolchicoside-inducedNF-κB inhibi-as not cell type specific, and the mechanism involvedund to be through inhibition of IKK activation, IκBαhorylation, ubiquitination, and degradation, aswell asgh p65 phosphorylation and translocation. Finally, wethat thiocolchicoside inhibited NF-κB–dependentter assay and suppressed NF-κB binding to the2 promoter.our knowledge, this is the first report on the effect oflchicoside on cancer cell proliferation as well as onactivation. Our results show that thiocolchicosidets cell proliferation of a wide variety of cancer cells,ot of normal cells, including leukemia, colon, mye-breast, squamous, and kidney cancer cells, indicat-at the inhibitory effect of thiocolchicoside on canceris not cell type specific. Moreover, we found thatlchicoside inhibits the colony-forming ability ofcancer cells.also found that thiocolchicoside inhibits NF-κB ac-n. NF-κB is a sequence-specific transcription factorinvolved in the inflammatory and innate immune

nses and controls >400 genes (21). The molecularfication of its p50 subunit as a member of the reti-dotheliosis (REL) family provided the first evidenceF-κB is linked with cancer, because v-REL is an on-tein of the REL retrovirus (REV-T; ref. 22). Sincenumerous points of evidence have confirmed the in-ent of activated NF-κB in multiple types of cancer

Moreover, NF-κB is one of the major figures in thematory network, and this inflammation is now re-as a “secret killer” in diseases such as atherosclero-eumatoid arthritis, multiple sclerosis, asthma,imer disease, depression, fatigue, neuropathic pain,f appetite, and cancer (23).ause thiocolchicoside has been shown to have anti-matory properties, we postulated that thiocolchico-hibits the activation of theNF-κB transcription factor.sults show that, indeed, thiocolchicoside suppressedactivation in different cancer cells, indicating that theession is not cell type specific. Moreover, thiocolchi-inhibited NF-κB activation induced by a variety of

li, suggesting that thiocolchicoside must act at a stepon to all these activators. In addition, our resultsed that thiocolchicoside blocked NF-κB activationut directly interfering with the DNA binding of. NF-κB activation in response to different stimuli re-IKK activation, which phosphorylates IκBα at Ser32

er36, leading to degradation of IκBα (16). We foundhis inhibition was mediated through the inhibitionby thiocolchicoside, which led to the suppression

osphorylation and ubiquitination of IκBα and thendegradation. Thiocolchicoside also inhibited p65horylation and translocation.investigate whether thiocolchicoside also modulates–regulated genes, we studied the effect of thiocolchi-on several proteins regulated by NF-κB, including

XIAP,Mcl-1, Bcl-xL, cIAP1, cIAP2, and cFlip. All of thesens are involved in apoptosis, and their downregulation




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iocolchicoside may explain the thiocolchicoside-ed apoptosis as observed by PARP and caspase-3ge. We also found that the expression of gene pro-involved in proliferation (c-Myc, phosphorylatedp85, and phosphorylated GSK3β) were stronglyregulated by thiocolchicoside, which may explainhibited proliferation of various cancer cells byrug.lly, we investigated the effect of thiocolchicoside on-κB–dependent reporter assay. Thiocolchicoside in-d the NF-κB–dependent reporter assay induced byn a dose-dependent manner. Furthermore, over-ssion of proteins in the NF-κB signaling pathway1, TRADD, TRAF2, NIK, IKKβ, p65, and IκBα) stim-NF-κB promoter activity, whereas the inhibitor of, IκBα, had no effect. The canonical NF-κB signalingay is triggered in response to microbial and viral in-s as well as by proinflammatory cytokines, such as16). TRADD is an adaptor protein that interacts with1 and with another adaptor protein, TRAF2, to acti-F-κB signaling (18, 24). The IKK complex, which isosed of the two catalytic subunits (IKKα and IKKβ)e scaffolding protein IKKγ/NEMO (25–27), is re-

d to the TNFR1 adaptor proteins, where it is activatedbsequently phosphorylates the specific inhibitors of, the IκB proteins. Moreover, thiocolchicoside dose-dently inhibits the binding of NF-κB proteins to thepromoter, which contains NF-κB binding sites in

uence (20).il now, thiocolchicoside has been described as ae relaxant with anti-inflammatory properties (1),r results show that thiocolchicoside is also effectivet cancer cells and thus may have potential as an an-er agent, which gives a new use to an existing drug.olchicoside (Muscoril) has been used for >35 yearsuscle relaxant, anti-inflammatory, and analgesic1), and clinical trials (2–4, 28) proved its efficacy

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fety. Peak plasma concentration of thiocolchicoside Rece

tex. Eur J Pharmacol 1999;376:149–57.lduini W, De Angelis V, Mazzoni E, Depoortere H, Cattabeni F,ino M. Autoradiographic localization of [3H]thiocolchicoside

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ding on the mode and route of administration0). These concentrations aremuch lower than the con-tion used to study NF-κB inhibitory effects of thiocol-ide in vitro. The mechanism of thiocolchicoside oninhibition in vivo remains to be worked out. Consti-activation of NF-κB has been associated with a widey of tumors and has also been linked with chemother-nd radiotherapy-induced resistance in many cancersTherefore, inhibition of NF-κB has been a widelyt target for cancer prevention. In in vitro and in vivol models, inhibition of constitutive and induciblehas been shown to enhance therapeutic potentials

ny drugs (21).results of our study show that thiocolchicoside is at inhibitor of NF-κB activation, which may explaintiproliferative, proapoptotic, and anti-inflammatorys. Further studies are needed to explore its in vivotial against cancer and other diseases.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

hank Virginia M. Mohlere for carefully editing this article.

Support

M.D. Anderson's Cancer Center support grant NIH CA-16 672,rogram project grant NIH CA-124787-01A2, and Center ford Therapy at University of Texas M.D. Anderson Cancer Center,Dr. Aggarwal is the Ransom Horne, Jr., Professor of Cancerh. Simone Reuter was supported by Fonds National de la Re-Luxembourg grant PDR-08-017.costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedsement in accordance with 18 U.S.C. Section 1734 solely tothis fact.

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2010;3:1462-1472. Published OnlineFirst October 26, 2010.Cancer Prev Res Simone Reuter, Sahdeo Prasad, Kanokkarn Phromnoi, et al. Products Linked to Inflammation and Cancer

B Pathway and Its Regulated GeneκDownregulation of NF-Thiocolchicoside Exhibits Anticancer Effects through

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