An agonist to the A 3 adenosine receptor inhibits colon carcinoma growth in mice via modulation of GSK-3b and NF-jB

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1 (2004) 23, & 2004 Nture Pulishing Group All rights reserved /04 $ An gonist to the A 3 denosine receptor inhiits colon crcinom growth in mice vi modultion of GSK-3 nd NF-jB Pnin Fishmn*,1,2, Sr Br-Yehud 1,2, Gil Ohn 1,2, Fin Brer 1, Avivit Ochion 1, Aigil Erlnger 1 nd Le Mdi 1 1 Cn-Fite Biophrm Ltd, Kiryt-Mtlon, Petch-Tikv 49170, Isrel; 2 Lortory of Clinicl nd Tumor Immunology, The Felsenstein Medicl Reserch Center, Tel-Aviv University Sckler Fculty of Medicine, Rin Medicl Center, Petch-Tikv, Isrel A 3 denosine receptor (A 3 AR)ctivtion with the specific gonist hs een shown to inhiit the development of colon crcinom growth in syngeneic nd xenogrft murine models. In the present study, we looked into the effect of on the moleculr mechnisms involved in the inhiition of HCT-116 colon crcinom in mice. In tumor lesions derived from -treted mice, decrese in the expression level of protein kinse A (PKA)nd n increse in glycogen synthse kinse-3 (GSK-3)ws oserved. This gve rise to downregultion of -ctenin nd its trnscriptionl gene products cyclin D1 nd c-myc. Further mechnistic studies in vitro reveled tht these responses were countercted y the selective A 3 AR ntgonist MRS 1523 nd y the GSK-3 inhiitors lithium nd SB216763, confirming tht the oserved effects were A 3 AR nd GSK-3 medited. downregulted PKB/Akt expression level, resulting in decrese in the level nd DNA-inding cpcity of NF-jB, oth in vivo nd in vitro. Furthermore, the PKA nd PKB/Akt inhiitors H89 nd Worthmnnin mimicked the effect of, supporting their involvement in mediting the response to the gonist. This is the first demonstrtion tht A 3 AR ctivtion induces colon crcinom growth inhiition vi the modultion of the key proteins GSK-3 nd NF-jB. (2004) 23, doi: /sj.onc Pulished online 15 Mrch 2004 Keywords: A 3 denosine receptor; ; colon crcinom; -ctenin; GSK-3 Introduction The A 3 AR is G i -protein-coupled receptor contining seven helicl spnning memrne domins. A 3 AR ws found to e expressed in different tumor cell lines, including Jurkt T, pinel glnd, strocytom, melnom s well s colon nd prostte crcinom (Gessi et l., 2001; Merighi et l., 2001; Suh et l., 2001; Trincvelli *Correspondence: P Fishmn, Cn-Fite BioPhrm Ltd, Kiryt Mtlon, Petch-Tikv, 49170, Isrel; E-mil: pnin@cnfite.co.il Received 31 July 2003; revised 5 Novemer 2003; ccepted 12 Novemer 2003 et l., 2002; Fishmn et l., 2003; Mdi et l., 2003; Ohn et l., 2003). A 3 AR ctivtion leds to inhiition of denylyl cyclse ctivity, camp formtion nd PKA expression, resulting in the initition of vrious signling pthwys which my include the MAPK nd the PI3K (Poulsen nd Quinn, 1998; Olh nd Stiles, 2000; Trincvelli et l., 2002). Our erlier studies demonstrted tht melnom cells highly express A 3 AR, nd suggested tht it my serve s trget for tumor growth inhiition. A 3 AR ctivtion y the synthetic gonist 1-deoxy-1-[6-[[(3-iodophenyl)- methyl]mino]-9h-purine-9-yl]-n-methyl--d-riofurnuronmide (IB-MECA) inhiited the growth of melnom oth in vitro nd in vivo (Fishmn et l., 2001, 2002,, 2003; Ohn et l., 2001; Mdi et l., 2003). The mechnistic pthwy involved downregultion of the Wnt signling pthwy. It ws found tht IB-MECA inhiited the expression of PKAc nd PKB/Akt, therey preventing the phosphoryltion nd inctivtion of GSK-3. Consequently, GSK-3 ws shown to phosphorylte -ctenin nd prevent its trnsloction to the nucleus, resulting in downregultion of cyclin D1 nd c- Myc (Fishmn et l., 2002; Mdi et l., 2003). PKB/ Akt is lso known to control NF-kB level y phosphorylting downstrem proteins, which in turn relese NFkB from its complex (Mdrid et l., 2001). Similr to - ctenin, NF-kB trnsloctes to the nucleus, where, mong other genes, it induces the trnscription of c-myc nd cyclin D1 (Joyce et l., 2001). Our previous studies showed tht is efficcious in suppressing the growth of primry nd liver metstsis of CT-26 colon crcinom cells in syngeneic experimentl tumor models in mice (Ohn et l., 2003). In ddition, inhiited the growth of sucutneous HCT-116 humn colon crcinom cells in xenogrft model in mice. Aerrnt ctivtion of Wnt signling, cused y muttions in -ctenin or APC, is criticl event in the development of colorectl tumors. In these cses, GSK-3 fils to phosphorylte -ctenin, which ccumultes in the cytoplsm. -ctenin then trnsloctes to the nucleus where, in ssocition with Lef/Tcf, it induces the trnscription of cyclin D1 nd c-myc (Morin, 1999). The present study is focused on the moleculr mechnism involved in the inhiition of colon crcinom growth y. We explored the signling

2 2466 modultion of GSK-3 nd NF-kB, oth of which re ffected y PKB/Akt (which is downstrem to PI3K) nd re known to regulte the level of the importnt oncogenes cyclin D1 nd c-myc. A mjor role for GSK- 3 in mediting these responses is discussed. Results inhiits colon crcinom growth in vivo nd modultes the expression level of A 3 AR nd downstrem cell growth-regultory proteins in tumor lesions HCT-116 colon crcinom cells were engrfted sucutneously into nude mice. When tumor reched the size of mm 3, the mice were treted dily orlly with. Tumor growth ws suppressed in the -treted group in comprison to the vehicletreted group (Figure 1). On the dy of study termintion, 52 þ 6.1% (Po0.001) tumor growth inhiition ws oserved. To evlute the effect of chronic tretment on A 3 AR expression nd downstrem cell growth-regultory proteins, extrcts were prepred from tumor lesions nd sujected to Western lot (WB) nlysis. In the group of mice killed 2 h fter the lst tretment, the expression level of A 3 AR, PKAc, - ctenin, NF-kB, c-myc nd cyclin-d1 ws downregulted, wheres GSK-3 ws upregulted. In the group of mice killed 16 h fter the lst tretment, A 3 AR expression ws similr to tht of the vehicle-treted group. Interestingly, in this group, most of the cell growthregultory proteins were decresed in comprison to the control group, indicting tht continuous downregultion is chieved upon chronic tretment. Tken together, these dt show tht receptor downregultion occurs shortly (2 h) fter tretment, leding to modultion of downstrem proteins, nd tht A 3 AR ws not desensitized despite chronic ctivtion (over 20-dy period). The expression of the receptor returned to norml levels 16 h fter dministrtion, demonstrting tht, even fter chronic ctivtion, the receptor is fully expressed (Figure 1). Tumor size (mm 3 ) A 3 AR PKAc β-ctenin NF-κB Cyclin D1 c-myc Vehicle Dys fter tumor incultion 2h Time fter lst tretment 16h modultes the expression level of A 3 AR nd downstrem cell growth-regultory proteins in vitro To further study the ssocition etween A 3 AR ctivtion nd the expression of downstrem cell growthregultory proteins, HCT-116 colon crcinom cells were incuted in the presence of (10 nm) for 15 min. Proteins were extrcted nd nlysed y WB. Similr effects of to those seen in vivo were recorded. The expression level of the two kinses PKAc nd PKB/Akt ws downregulted, while the expression of their downstrem sustrte GSK-3 ws upregulted. The levels of the coctivtor -ctenin nd the downstrem trget genes cyclin D1 nd c-myc were decresed (Figure 2). To confirm tht these responses re medited vi the A 3 AR, the ntgonist MRS 1523 ws introduced to the culture system. The ntgonist countercted the effect of, therey retining the Figure 1 Inhiition of colon crcinom cell growth in nude mice nd modultion of cell growth-regultory proteins in tumor lesions. HCT-116 cells were sucutneously engrfted to nude mice. (10 mg/kg) tretment ws initited when tumor reched size of 150 mm 3, nd ws given twice dily for 21 consecutive dys. On dy 21, the mice were killed 2 or 16 h fter tretment. Tumor lesions were removed nd protein extrcts were prepred. () Tumor size ws mesured every 4 dys. The curve represents comprison etween the vehicle nd - treted groups. () Immunolots showing the effect of on cell growth-regultory proteins derived from the colon crcinom tumor lesions. A 3 AR ws downregulted 2 h fter tretment nd fully expressed fter 16 h. Downstrem cell growth-regultory proteins were modulted upon tretment control levels of PKAc, GSK-3 nd cyclin D1, demonstrting the specificity of the response (Figure 2). To further elucidte the role of PKA nd

3 PKB in mediting cell response to, their ctivity ws mimicked y H89 nd Worthmnnin (PKA nd PKB/Akt inhiitors, respectively). Figure 2c depicts n PKAc P-PKB/AKT β-ctenin Cyclin D1 c-myc CONTROL increse in GSK-3 level upon tretment with the two inhiitors. deregultes GSK-3 nd downstrem key signling proteins The next set of experiments ws crried out to ssure tht decresed cyclin D1 nd c-myc levels vi modultion of GSK-3. We therefore compred the ctive nonphosphorylted GSK-3 level to its nonctive phosphorylted form. Consistent with the former dt, we found tht, upon tretment, the nonphosphorylted form ws upregulted, wheres the phosphorylted one ws decresed (Figure 3). SB216763, n inhiitor to GSK-3, countercted the ility of to downregulte c-myc, confirming tht this response ws GSK-3 medited (Figure 3). Furthermore, mrked increse in the ctivity of GSK-3 ws lso noted fter 15 nd 30 min (Figure 3c). To ssess whether the decrese in -ctenin is medited vi its phosphoryltion y GSK-3, HCT-116 cells were treted with lithium chloride tht inhiits the serine/threonine phosphoryltion ctivity of GSK-3. Indeed, lithium tretment reversed the decrese in -ctenin expression level (36 þ 3.4%, Po0.002), confirming tht this response is GSK-3 medited (Figure 3d). In ddition, the nucler level of LEF-1 in the -treted cells ws downregulted (Figure 3e), supporting the notion tht less -ctenin ws ssocited with LEF-1 nd susequently trnslocted to the nucleus Effect of on the level nd trnscription ctivity of NF-kB Activted PKB/Akt cn phosphorylte IkB kinse, leding to further phosphoryltion events nd the relese of NF-kB from its complex with IkB. Accordingly, we exmined whether the downregultion of PKB/Akt will ffect the protein expression nd DNAinding cpcity of NF-kB, lso known to induce cyclin D1 nd c-myc trnscription. Indeed, decresed NF-kB level ws seen in protein extrcts derived from - treted HCT-116 cells (Figure 4). This decrese ws locked when the ntgonist MRS 1523 ws present in the culture medium together with, demonstrting the specificity of this response. Moreover, electrophoretic moility shift ssy (EMSA) conducted with cell nuclei extrcts reveled mrked reduction in NF-kB PKAc Cyclin D1 +MRS1523 MRS1523 c Worthmnnin +H89 H89 Worthmnnin 5 Figure 2 Modultion of cell growth-regultory proteins in HCT- 116 colon crcinom cells upon tretment in vitro. () Immunolots showing the effect of 10 nm on the expression levels of PKAc, PKB/Akt, GSK-3, -ctenin, cyclin D1 nd c-myc in HCT-116 cells. Serum-strved cells (for 18 h) were treted for 15 min with in the presence of 1% FBS. () To test the specificity of this response, the ntgonist MRS 1523 (100 nm) ws introduced to the culture system. Immunolots showing the effect of on the cell growth-regultory proteins in the presence nd sence of MRS 1523 re depicted. (c) Immunolots showing the effect of H89 (10 mm) nd Worthmnnin (100 nm) on the expression level of GSK-3

4 2468 DNA-inding cpcity t 15, 30 nd 60 min, suggesting reduction in the NF-kB trnscription ctivity t these time points (Figure 4). -P 0.01µM µM Discussion In the present study, we followed the downstrem signling events tking plce susequent to A 3 AR ctivtion, resulting in tumor growth inhiition. These studies were conducted in xenogrft nude mice model, nd were confirmed in vitro. In mice treted chroniclly for 20 dys with, receptor protein downregultion ws noted shortly fter dministrtion. Lter on, prominent A 3 AR expression ws noted, demonstrting tht A 3 AR ws fully expressed in the tumor cells fter chronic tretment with. These fluctutions my e ttriuted to receptor internliztion, degrdtion nd re-synthesis, which occurs susequent to receptor ctivtion. These dt re the first to show A 3 AR expression in vivo, nd c-myc SB SB MRS1523 MRS1523 NF-kB c Activity 15' 30' Actin Competition 15' 30' 60' 15' 30' 60' d +LiCl β-ctenin e Aritry units LiCl 15 min. 60 min. LEF Figure 4 NF-kB expression level in cell lystes nd EMSA in nucler extrcts. HCT-116 colon crcinom cells were incuted for 15, 30 nd 60 min t 371C with 10 nm. () WB nlysis of whole-cell protein extrcts conducted t 15 min of incution in the sence nd presence of the ntgonist MRS 1523 (100 nm) nd () EMSA of HCT-116 nucler extrcts t different time points Figure 3 Increse in GSK-3 expression level nd ctivity upon tretment of HCT-116 cells with leds to decresed - ctenin expression level. Cells were depleted from serum for 18 h nd treted with vehicle (control) or with (10 nm or 10 mm) in the presence of 1% FBS for the times nd concentrtions indicted. () The expression of nonphosphorylted GSK-3 nd phosphorylted GSK-3 (GSK-3-P) ws determined in cell protein extrcts y WB nlysis. () The ility of to inhiit the expression level of c-myc ws countercted y SB216763, n inhiitor of GSK-3 (c). GSK-3 ctivity in HCT- 116 colon crcinom cells ws incuted for 15 nd 30 min t 371C with 10 mm. (d) HCT-116 cells were treted with (10 nm) for 15 nd 30 min in the presence nd sence of lithium chloride. The ltter countercted the decrese in -ctenin expression level, indicting tht the response is GSK-3 medited. (e) LEF-1 nlysis in the nucler extrcts of HCT-116 cells treted with, s detiled ove, for 30 nd 60 min

5 support the notion tht colon crcinom cells do not develop resistnce or tolernce to chronic tretment with synthetic A 3 AR gonist. Supporting the ove is our recent puliction demonstrting tht, upon ctivtion of B16-F10 melnom cells with IB-MECA, A 3 AR ws internlized nd sorted to the lysosome for degrdtion. Lter on, the receptor ws resynthesized nd recycled to the cell surfce (Mdi et l., 2003). In the present study, receptor functionlity ws demonstrted y the modultion in the expression level of key signling cell growth-regultory proteins downstrem to receptor ctivtion. This included downregultion of PKAc nd PKB/Akt nd upregultion of GSK-3. Additionlly, the protein expression levels of -ctenin, LEF-1 nd the two oncogenes cyclin D1 nd c-myc were found to e decresed. These results re in ccordnce with our previous studies, which showed decresed PKAc nd PKB/Akt levels upon tretment of B16-F10 melnom cells with IB-MECA (Fishmn et l., 2002; Mdi et l., 2003). PKAc is the ctlytic suunit of PKA, known to e ctivted susequent to increse in camp level. Activtion of A 3 AR is known to decrese denylyl cyclse ctivity nd camp formtion, resulting in decline in PKAc level. PKB/Akt hs recently een shown to e phosphorylted nd therey ctivted y PKAc (Fng et l., 2000). The PI3K rm ws reported to e upregulted upon A 3 AR ctivtion vi the g-suunit (Schutle nd Fredholm, 2002), leding to n increse in the phosphorylted form of PKB/Akt. Here, we show tht, in colon crcinom cells, downregultion of PKB/ Akt tkes plce upon receptor ctivtion, suggesting tht in tumor cells modultion of the PKA rm is the dominnt event, leding to the downregultion of PKB/ Akt. PKAc nd PKB/Akt utilize GSK-3 s sustrte nd, upon phosphoryltion, GSK-3 ctivity is inhiited. The ltter hs een widely implicted in cell homeostsis, y its ility to phosphorylte rod rnge of sustrtes including -ctenin, key component of the Wnt pthwy (Ferkey nd Kimelmn, 2000). In norml cells, GSK-3 phosphoryltes -ctenin, therey inducing its uiquitintion nd degrdtion y the proteosome system (Morin, 1999). However, in tumor cells, GSK-3 fils to phosphorylte -ctenin, leding to its ccumultion in the cytoplsm. It then trnsloctes to the nucleus, where it cts in concert with LEF-1 to induce the trnscription of the cell cycle progression genes such s cyclin D1 nd c-myc (Kolligs et l., 2002). In previous studies, we showed tht A 3 AR ctivtion induced downregultion of cyclin D1 nd c-myc in melnom nd prostte crcinom cells, vi deregultion of some Wnt signling proteins (Fishmn et l., 2002, 2003; Mdi et l., 2003). We thus ssume tht the decresed expression level of -ctenin is responsile for the diminished level of cyclin D1 nd c-myc. In the present study, we exmined the effect of on HCT-116 colon crcinom cells, known to e mutted in the -ctenin gene (CTNNB1) (Lovig et l., 2002). Muttions of CTNNB1 were found t the GSK- 3 consensus phosphoryltion site of -ctenin, tht is, deletion of serine 45 tht occurs t puttive phosphoryltion trget of GSK-3 (Ilys et l., 1997). Surprisingly, we found tht downregultion of -ctenin expression, which occurred upon tretment, ws susequent to n increse in the level of GSK-3, notwithstnding the previously descried, forementioned muttion. Moreover, tretment of the cells with lithium, which directly inhiits the ctivity of GSK-3, reversed the -ctenin level to tht of the control. It thus seems tht circumvents the inility of GSK-3 to phosphorylte -ctenin, leding to its susceptiility to degrdtion. Support for the involvement of - ctenin in the downregultion of cyclin D1 nd c-myc my e found in the dt showing tht nucler level of LEF-1 ws downregulted upon tretment. Furthermore, the GSK-3 inhiitor SB countercted the ility of to downregulte c-myc, thus confirming tht the events downstrem to -ctenin re lso medited vi GSK-3. An dditionl mechnism which my ccount for the downregultion of c-myc nd cyclin D1 is the direct phosphoryltion of the two oncogenes y GSK-3. It ws recently shown tht GSK-3 phosphoryltes c-myc t Thr-58 nd cyclin D1 t Thr-286, therey triggering their degrdtion (Alt et l., 2000; Sers et l., 2000). The decresed level of PKB/Akt prompted us to exmine the involvement of n dditionl importnt signling protein, NF-kB, known to e phosphorylted nd ctivted y PKB/Akt nd dditionl downstrem kinses. Since NF-kB is lso involved in the trnscription of cyclin D1 nd c-myc (Krin et l., 2002), its decresed level my lso ttriute to the diminished expression of the two cell cycle genes. The Wnt nd the NF-kB signling pthwys re interconnected t the level of cyclin D1 nd c-myc. Both -ctenin nd NF-kB control the trnscription of these genes, therey cting s sensor for growth signls. Tken together, we propose here model in which ctivtion of the A 3 AR induces modultion of PKAc nd PKB, which on one hnd upregultes GSK-3, leding to phosphoryltion nd uiquitintion of - ctenin. On the other hnd, remrkly, the similrity etween the in vitro nd in vivo dt supports the notion tht signling proteins involved with the Wnt nd NFkB pthwys re responsile for the oserved modultion of cell growth-regultory proteins. The finding tht cyclin D1 nd c-myc were downregulted upon A 3 AR ctivtion oth in vitro nd in vivo is highly importnt in light of the ulk literture showing tht most humn cncers re chrcterized y overexpression of the two oncogenes (Hosokw nd Arnold, 1998; Prrell, 2001; Msud et l., 2002). In some mlignncies, overexpression of these proteins my serve s mrker of poor prognosis (Chn et l., 2002; Nguyen et l., 2003). The importnce of these two oncogenes in modulting the tumorigenic response ws evidenced y the introduction of n ntisense cyclin D1 or c-myc sequence to mlignnt cells. This led to the inhiition of growth, the induction of poptosis nd the enhncement of sensitivity to chemotherpeutic gents (Vn Wrdenurg et l., 1997). Additionlly, Jin et l. 2469

6 2470 (2002) showed tht rief MYC inctivtion induced sustined loss of neoplstic phenotype. Tken together, the moleculr model tht trnspires upon ctivtion of A 3 AR with includes downregultion of PKAc with susequent decrese in PKB/ Akt expression level. This my led on one hnd to upregultion of the unphosphorylted form of GSK-3 nd the phosphoryltion nd uiquitintion of - ctenin, resulting in the inhiition of trnsltion of cyclin D1 nd c-myc. Additionl events tking plce downstrem to PKB/Akt include decresed expression nd DNA-inding cpility of NF-kB, leding lso to downregultion of cyclin D1 expression level. The cpility of, smll orlly ioville molecule, to downregulte cyclin D1 nd c-myc levels oth in vitro nd in vivo suggest tht the compound is n ttrctive cndidte to e developed s n nticncer gent. Mterils nd methods Regents is GMP grde of the A 3 AR gonist 1-deoxy-1- mino]-9h-purine-9-yl]-n-methyl-(-d-riofurnuronmide) (IB-MECA), nd ws synthesized for Cn-Fite BioPhrm y Alny Moleculr Reserch Inc., Alny, NY, USA. MRS 1523, highly selective A 3 AR ntgonist, ws purchsed from RBI/Sigm (Ntick, MA, USA). For oth regents, stock solution of 10 mm ws prepred in DMSO nd further dilutions in RPMI medium were performed. Lithium chloride nd H89 were purchsed from Sigm Isrel, nd SB ws purchsed from Biomol Reserch Lortories Inc. (Plymouth, USA). RPMI, fetl ovine serum (FBS) nd ntiiotics for cell cultures were otined from Beit Hemek, Hif, Isrel. Rit polyclonl ntiodies ginst murine nd humn A3AR nd the cell growth-regultory proteins PKAc, PKB/ Akt, c-myc, GSK-3, phosphor-specific GSK-3 (S9), - ctenin, cyclin D1 nd LEF-1 nd -ctin were purchsed from Snt Cruz Biotechnology Inc., CA, USA. Effect of on the growth of HCT-116 colon crcinom in nude mice nd ssessment of A 3 AR expression nd cell growthregultory proteins in tumor lesions Mice were mintined on stndrdized pelleted diet nd supplied with tp wter. Experiments were performed in ccordnce with the guidelines estlished y the Institutionl Animl Cre nd Use Committee t Cn-Fite BioPhrm, Peth Tikv, Isrel. Nude mle Bl/c mice, ged 2 months, weighing n verge of 20 g, were otined from Hrln Lortories, Jeruslem, Isrel. HCT-116 colon crcinom cells ( ) were sucutneously injected into the flnk of the mice. When tumor reched 150 mm 3 in size, (10 mg/kg ody weight) ws dministered orlly twice dily for 20 dys. The control group ws treted orlly twice dily with the vehicle only. Tumor size (width (W) nd length (L)) ws mesured twice weekly with clier, nd clculted ccording to the following formul: tumor size ¼ (W) 2 L/2. After 20 dys of tretment nd prior to terminting the study, the -treted mice were divided into two groups. (A) mice treted for 20 dys with nd killed 16 h fter lst tretment; (B) mice treted for 20 dys with, received dditionl tretment on dy 21 nd killed 2 h lter. Tumor lesions from the two groups nd the control were then excised, homogenized (Polytron, Kinemtic) nd protein ws extrcted. WB nlysis ws crried out to determine the A 3 AR expression level nd dditionl cell growth-regultory proteins. Ech group contined 15 mice nd the study ws repeted three times. The results depicted re representtive experiment. WB nlysis WB nlysis of the following smples ws crried out: (A) tumor lesions derived from nd vehicle-treted nude mice inoculted with HCT-116 colon crcinom cells (detiled ove). (B) HCT-116 humn colon crcinom cells were serum strved overnight nd then incuted with (10 nm or 10 mm) in the presence nd sence of MRS 1523 (100 nm), H89 (10 mm), Worthmnin (100 nm), nd/or SB (1 mm) for time periods, s specified elow, t 371C. Smples were rinsed with ice-cold PBS nd trnsferred to ice-cold lysis uffer (TNN uffer, 50 mm Tris uffer ph ¼ 7.5, 150 mm NCl, 0.5% NP-40). Cell deris were removed y centrifugtion for 10 min, t 7500 g. Protein concentrtions were determined using the Bio-Rd protein ssy dye regent. Equl mounts of the smple (50 mg) were seprted y SDS PAGE, using 12% polycrylmide gels. The resolved proteins were then electrolotted onto nitrocellulose memrnes (Schleicher & Schuell, Keene, NH, USA). Memrnes were locked with 1% BSA nd incuted with the desired primry ntiody (dilution 1:1000) for 24 h t 41C. Blots were then wshed nd incuted with secondry ntiody for 1 h t room temperture. Bnds were recorded using BCIP/NBT color development kit (Promeg, Mdison, WI, USA). Dt presented in the different figures re representtive of t lest four different experiments. Preprtion of nucler extrcts Nucler extrct proteins from -treted nd control HCT-116 cells were prepred y incuting the cells for 15 min on ice in uffer contining 10 mm HEPES (ph 7.9), 10 mm KCl, 0.1 mm EDTA, 1 mm DTT nd 0.5 mm PMSF. Following incution, Nonident P-40 (10%) ws dded, cells were vortexed for 10 s nd centrifuged. The pellet ws resuspended in uffer contining 20 mm HEPES (ph ¼ 7.9), 400 mm NCl, 1 mm EDTA, 1 mm DTT nd 1 mm PMSF, rocked on shker for 15 min t 41C nd centrifuged. Protein ws quntified utilizing Bio-Rd protein ssy dye regent. GSK-3 immunoprecipittion HCT-116 humn colon crcinom cells were serum strved overnight nd then incuted with (10 mm) for 30 min t 371C. After isolting protein, 300 mg from ech smple ws removed for immunoprecipittion. The smples were clered y incuting for 2 h with 1 mg/smple of rit IgG nd 10 ml/ smple of GmmBind Sephrose (Phrmci, Pisctwy, NJ, USA). After centrifuging, the superntnts were trnsferred to tue contining 3 mg/smple of A ginst GSK-3 ound to GmmBind Sephrose, nd then rotted t 41C overnight. The eds were susequently wshed three times with high slt uffer (1 M Tris-HCl ph 7.4, 0.50 M NCl, nd 1% Nonidet P-40) nd three times with lysis uffer without protese inhiitors. The immunoprecipitted complexes were used in kinse ctivity ssy.

7 GSK-3 ctivity ssy After immunoprecipitting GSK-3 from HCT-116 cells, the protein-contining pellet ws wshed twice with kinse uffer (20 mm MgCl 2, 25mM HEPES, 20 mm glycerophosphte, 20 mm p-nitrophenylphosphte, 20 mm sodium orthovndte nd 2 mm DTT). The pellet ws then suspended in 20 ml kinse uffer nd the following ingredients were dded: 20 mm ATP, 5 mci ATP (BLU 002Z; DuPont-NEN, Boston, MA, USA) nd 10 mg myelin sic protein (MBP; Sigm). The totl volume of smple plus dditions t this point ws 25 ml. The rection ws continued for 30 min t 251C nd then stopped y the ddition of 25 ml/smple of 2 smple uffer. The smples were oiled for 5 min, then run on 12% SDS PAGE gel. The gel ws dried, nd utordiogrphy performed to visulize the 32 P-leled MBP. EMSA of NF-kB To crry out the gel shift ssy, doule-strnded oligonucleotides for the consensus sequence of NF-kB (5 0 -AGTT- References Alt JR, Clevelnd JL, Hnnink M nd Diehl JA. (2000). Genes Dev., 14, Chn JS, Grover R, Tulley P, Lohrer H, Snders R, Groelr AO nd Wilson GD. (2002). Br. J. Plst. Surg., 55, Fng X, Yu SX, Lu Y, Bst RC, Woodgett JR nd Mills GB. (2000). Proc. Ntl. Acd. Sci. USA, 97, Ferkey DM nd Kimelmn D. (2000). Dev. Biol., 225, Fishmn P, Br-Yehud S, Mdi Lnd Cohn I. (2002). Anticncer Drugs, 13, 1 8. Fishmn P, Br-Yehud S, Ohn G, Pthk S, Wssermn L, Brer F nd Multni AS. (2001). Exp. Cell Res., 269, Fishmn P, Mdi L, Br-Yehud S, Brer F, Del Vlle L nd Khlili K. (2002)., 21, Fishmn P, Br-Yehud S, Rth-Wolfson L, Ardon E, Brrer F, Ochion A nd Mdi L. (2003). Anticncer Res., 23, Gessi S, Vrni K, Merighi S, Morelli A, Ferrri D, Leung E, Brldi PG, Splluto G nd Bore PA. (2001). Br. J. Phrmcol., 134, Hosokw Y nd Arnold A. (1998). Genes Chromosomes Cncer, 22, Ilys M, Tomlinson A, Rown M, Pigntelli WF nd Bodmer E. (1997). Proc. Ntl. Acd. Sci. USA, 94, Jin M, Arvnitis C, Chu K, Dewey W, Leonhrdt E, Trinh M, Sunderg CD, Bishop JM nd Felsher DW. (2002). Science, 297, Joyce D, Alnese C, Steer J, Fu M, Bouzhzh B nd Pestell RG. (2001). Cytokine Growth Fctor Rev., 12, Krin M, Co Y, Greten FR nd Li ZW. (2002). Nt. Rev. Cncer, 2, Kolligs FT, Bommer G nd Goke B. (2002). Digestion, 66, Lovig T, Meling GI, Diep CB, Thorstensen L, Norheim Andersen S, Lothe RA nd Rognum TO. (2002). Scnd. J. Gstroenterol., 37, GAGGGGACTTTCCCAGGC-3 0 ) were end-leled with 32 ATP (Amershm) using polynucleotide kinse (Promeg). Nucler protein extrcts (3 mg) were incuted for 30 min t room temperture with the end-leled DNA (1 mg) in inding uffer contining 5 mm MgCl 2, 250 mm NCl, 2.5 mm DTT, 25 mm EDTA, 20% glycerol, 50 mm Tris-HCl ph 7.5 nd 2 mg/smple of poly (di-dc), in finl volume of 25 ml. Competition with unleled oligonucleotide of NF-kB inding sequence t 100-fold molr excess ws used to nlyse specific nds. The rection product ws nlysed y 6% nondenturting polycrylmide gel electrophoresis. The specific nds were visulized y X-ry utordiogrphy. Sttisticl nlysis The results were evluted using the Student s t-test, with sttisticl significnce set t Po0.05. Comprison etween the men vlues of different experiments ws crried out. Mdi L, Br-Yehud S, Brer F, Ardon E, Ochion A nd Fishmn P. (2003). J. Biol. Chem., 278, Mdrid LV, Myo MW, Reuther JY nd Bldwin AS. (2001). J. Biol. Chem., 276, Msud M, Suzui M, Ysumtu R, Nkshim T, Kurtomi Y, Azum K, Tomit K, Komiym S nd Weinstein I. (2002). Cncer Res., 62, Merighi S, Vrni K, Gessi S, Cttrig E, Innott V, Ulouglu C, Leung E nd Bore PA. (2001). Br. J. Phrmcol., 134, Morin JP. (1999). BioEssys, 21, Nguyen DC, Prs B, Close A, Mgnusson B, Crowe DLnd Sinh UK. (2003). Int. J. Oncol., 22, Ohn G, Br-Yehud S, Brer F nd Fishmn P. (2001). J. Cell. Physiol., 186, Ohn G, Br-Yehud S, Arich A, Volfsson-Rt L, Mdi L, Dreznick Z, Silermn D, Slosmn G nd Fishmn P. (2003). Br. J. Cncer, 89, Olh ME nd Stiles GL. (2000). Phrmcol. Ther., 85, Prrell P, Cllero OL, Sidrnsky D nd Mers SL. (2001). Invest. Ophthlmol. Vis. Sci., 42, Poulsen SA nd Quinn RJ. (1998). Bioorg. Med. Chem., 6, Schutle G nd Fredholm BB. (2002). Mol. Phrmcol., 62, Sers R, Nuckolls F, Hur E, Ty Y, Tmi K nd Nevins JR. (2000). Genes Dev., 14, Suh BC, Kim TD, Lee JU, Seong JK nd Kim KT. (2001). Br. J. Phrmcol., 134, Trincvelli ML, Tuscno D, Mrroni M, Flleni A, Gremigni V, Ceruti S, Arcchio MP, Jcoson KA, Ctteni F nd Mrtini C. (2002). Mol. Phrmcol., 62, Trincvelli ML, Tuscno D, Mrroni M, Klotz KN, Luccchini A nd Mrtini C. (2002). Biochim. Biophys. Act, 1591, Vn Wrdenurg RC, Meijer C, Burger H, Nooter K, De Vries EG, Mulder NH nd De Jong S. (1997). Int. J. Cncer, 73,