The effect of thalidomide on non-small cell lung cancer (NSCLC) cell lines: possible involvement in the PPARg pathway

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1 Crcinogenesis vol.25 no.10 pp , 2004 doi: /crcin/gh210 The effect of thlidomide on non-smll cell lung cncer (NSCLC) cell lines: possile involvement in the PPARg pthwy Kthleen L.DeCicco, Tkemi Tnk, Fusto Andreol nd Luigi M.De Luc 1 Lortory of Cellulr Crcinogenesis nd Tumor Promotion, Center for Cncer Reserch, Ntionl Cncer Institute, NIH, Bethesd, MD , USA 1 To whom correspondence should e ddressed Emil: delucl@dc37.nci.nih.gov Lung cncer is the leding cuse of cncer-relted deth in developed countries. Non-smll cell lung cncer (NSCLC) represents 80% of the totl lung cncer cses nd is comprised of denocrcinom, denosqumous crcinom, squmous cell crcinom nd lrge cell crcinom (LCC) sutypes. The ility of LCC to metstsize erlier thn the other forms of lung cncer suggests nti-ngiogenic drugs s effective gents to comt this cncer. Thlidomide is n nti-ngiogenic drug tht hs shown promise in multiple hemtologicl diseses, nd myelom nd other cncers. However, the moleculr mechnism y which thlidomide exerts its effects is poorly understood. Therefore, we evluted the effectiveness of thlidomide on NSCLC cell growth, nd found tht LCC cells were growth inhiited y %. This effect seemed specific to LCC cncer cells, since other forms of NSCLC were only mildly ffected y thlidomide. At the moleculr level, thlidomide incresed peroxisome prolifertor-ctivted receptor gmm (PPARg) protein dose-dependently, nd peroxisome prolifertor response element ctivity. Further, thlidomide tretment of LCC cells decresed nucler fctor kpp B ctivity in dose-dependent fshion, incresed poptosis nd decresed the expression of ngiogenic proteins. In our mouse xenogrft model of lung cncer, we found tht intrtumorl thlidomide cused 64% decrese in tumor growth; moreover, tumors from the thlidomide-treted mice expressed higher PPARg, thn tumors from control mice. This study shows the ntitumor ctivity of thlidomide ginst LCC tumors nd suggests model in which thlidomide exerts its ntitumor effects on LCC cells through the induction of PPARg nd susequent downstrem signling. To our knowledge, this is the first study to show link etween thlidomide nd PPARg. Introduction Lung cncer mortlity ccounts for over deths per yer, mking it the leding cuse of cncer-relted deth in developed countries (1). The most common form of lung cncer, non-smll cell lung cncer (NSCLC), represents Arevitions: COX-2, cyclooxygense-2; IL, interleukin; NFkB, nucler fctor kpp B; NHBE, norml humn ronchil epithelil; NSCLC, nonsmll cell lung cncer; LCC, lrge cell crcinom; PPAR, peroxisome prolifertor-ctivted receptor; PPRE, peroxisome prolifertor response element. ~80% of the totl cses nd comprises four sutypes, nmely denocrcinom, denosqumous crcinom, squmous cell crcinom nd lrge cell crcinom (LCC) (2). Of these sutypes, LCC tumors, clssified y complete lck of ny differentition mrkers, generlly grow nd metstsize t n erlier stge thn other forms of NSCLC (2). Considering the high metsttic rte of this type of lung cncer, prognosis is poor, with % 5-yer survivl rte. Since tumor growth nd metstsis re ngiogenesis-dependent, ttempts hve een mde to trget tumor vsculture through the use of nti-ngiogenic drugs, some of which re now in phse I--III clinicl trils (3,4). Thlidomide is n nti-ngiogenic (5) nd immunomodultory (6) drug tht ws first mrketed in Europe in the lte 1950s for the tretment of pregnncy-ssocited morning sickness. It susequently ws withdrwn in the 1960s when it ws discovered tht women who took this drug in their first trimester gve irth to children with severe mlformtions in the lims nd internl orgns (7). The return of thlidomide s therpy in cncers nd infections stems from its rod rry of nti-inflmmtory nd nti-ngiogenic effects, s demonstrted in diseses such s HIV, Crohn s disese nd leprosy, nd cncers such s myelom (6,8--10). The cellulr trget nd mechnism of ction of thlidomide re poorly understood, ut it hs een reported in myelom to suppress ngiogenic fctors such s vsculr endothelil growth fctor (VEGF), nd inflmmtory genes such s tumor necrosis fctor lph (TNF-) nd interleukin-6 (IL-6) (6,8). Prolifertor-ctivted receptors (PPARs) re memers of the nucler hormone receptor superfmily of lignd-ctivted trnscription fctors (11,12). PPARs heterodimerize with the retinoid X receptor (RXR), nd, upon inding to peroxisome prolifertor response elements (PPREs) locted in the promoter region of trget genes, regulte the expression of genes ssocited with lipid metolism nd dipocyte differentition. Additionlly, PPARs hve een shown to regulte inflmmtory responses y ntgonizing nucler fctor kpp B (NFkB) nd ctivtor protein 1 (AP-1) signling pthwys (13--15). To dte, three PPAR sutypes hve een isolted nd nmed PPAR, PPAR nd PPARg (12). Ech of these sutypes hs distinct tissue distriution nd is encoded y seprte gene. Of the three sutypes, PPARg expression hs een suggested s potentil mrker for lung cncer (16). It hs een shown tht, upon ddition of PPARg selective gonists to lung cncer cells, growth ws inhiited through induction of differentition nd poptosis (17,18). Additionlly, decresed PPARg expression hs een correlted with poor prognosis in ptients with lung cncer, suggesting tht this gene my e lost s lung cncer progresses (16). Becuse LCC metstsizes erlier thn other forms of NSCLC, metstsis is n ngiogenesis-dependent process, nd thlidomide is n nti-ngiogenic drug, we evluted thlidomide s cndidte drug for this solid tumor. We hypothesized tht thlidomide would e n effective nticncer Crcinogenesis vol.25 no.10 # Oxford University Press 2004; ll rights reserved. 1805

2 K.L.DeCicco et l. drug in LCC nd tht its nticncer properties would e medited through PPARg. We used in vitro nd in vivo pproches to ddress the mechnism of ction. Mterils nd methods Cell growth studies NSCLC cell lines (NCI-H1299, NCI-H460, A-549, NCI-H661, NCI-H520, NCI-H596, NCI-H522) were purchsed from ATCC (Mnsses, VA). Norml ronchil epithelil lung cells were purchsed from Cmrex (Wlkersville, MD) nd grown in BEGM defined medium (Cmrex, Wlkersville, MD). NSCLC cell lines were mintined in RPMI medi þ 5% FBS in 37 C, 5% CO 2 incutor. For the growth studies of the primry norml cells nd ech cell line, 1000 cells were plted in 96-well pltes nd mintined in RPMI medi supplemented with 2 mm L-glutmine, 100 U/ml penicillin, 100 mg/ml streptomycin nd 5% het-inctivted FBS (ll from Invitrogen, Crlsd, CA). Thlidomide 1 mg/ml (Cliochem, Sn Diego, CA) or vehicle ws dded dily nd complete medi chnges were performed every 48 h. At the end of 8 dys, cell growth ws mesured y the WST-1 colorimetric ssy (Roche, Indinpolis, IN). In this ssy, the tetrzolium slt WST-1 is cleved y mitochondril dehydrogenses in vile cells producing formzn, whose drk red color cn e mesured spectrophotometriclly t 440 nm. Three independent experiments were conducted nd verges nd stndrd error clculted sed on these findings. Dt re reported s percent inhiition in cell growth, s compred with vehicle-treted controls. Western nlysis NSCLC cells were plted in 6-well dishes (Corning, Corning, NY), nd fter ttchment treted dily with vrying doses of thlidomide or vehicle control. After 48 h, whole cell lystes were prepred y scrping cells in lysis uffer (1% SDS, 10 mm Tris ph 7.4), sonicting extrcts (level 2, pulse 10 s, rest 5 s; three cycles per smple), nd centrifuging (35 min r.p.m.) t 4 C. Protein concentrtion ws detected y the Bio-Rd Protein Assy dye, using IgG s stndrd (Bio-Rd, Hercules, CA). For ech cell line, 60 mg protein ws electrophoresed on 10% Bis--Tris Novex minigel (Invitrogen). After trnsfer, lots were proed with 1:1000 rit polyclonl PPARg, 1:1000 cyclooxygense 2 or 1:1500 mouse ctin (Snt Cruz, Snt Cruz, CA). Secondry ntiody (got nti-rit IgG-HRP or got nti-mouse IgG-HRP) ws used t 1:2000. Detection ws performed using enhnced chemiluminescence ccording to the mnufcturer s instructions (ECL; Pierce, Rockford, IL). Densitometry ws performed using the lph imger 3.24 progrm. Results re reported s mens of three experiments, normlized to ctin. Cell trnsfections/reporter ssys A549, NCI-H1299 nd NCI-H460 cells were seeded in RPMI medi þ 10% FBS in 6-well dishes nd llowed to grow to 50% confluence. Following removl of growth medium, A549 nd NCI-H460 cells were trnsfected with 1.2 ml of serum-free medi contining 1 mg of the luciferse reporter construct PPRE 3 -TK-LUC ( gift from Dr R.M.Evns), TK-LUC (gift from Dr R.M.Evns), ptal-nfkb-luc (Clontech, Plo Alto, CA), or ptal-luc (Clontech) expression vector nd 1 ml of Superfect (Qigen, Vlenci, CA). NCI-H1299 cells were treted similrly, except cells were trnsfected with medi contining serum, since removl of ll serum drsticlly chnged the phenotype of the cells. After 5 h, the trnsfection mix ws replced with medium (contining 10% FBS) with or without incresing doses of thlidomide. After 24 h, cell lystes were prepred nd luminescence ws detected using the protocol from the Luciferse Assy System (Promeg, Mdison, WI). Briefly, ttched cells were rinsed in PBS, hrvested in 1 lysis uffer, pelleted through rief centrifugtion, nd superntnts trnsferred to microfuge tue. A totl of 20 ml of cell lyste nd 100 ml of Luciferse Assy regents were mixed nd trnsferred to 96-well plte. Luminescence ws mesured using Tropix microplte luminometer. Some wells were treted with 10 mm troglitzone (Biomol, Plymouth Meeting, PA) s positive control for PPRE ctivity. Dt re reported s the verge of three independent experiments. Apoptosis ssy NCI-H1299 nd NCI-H460 cells were treted with 1 mg/ml thlidomide for 24 h nd poptosis ws ssessed using Annexin V-FITC stining (BD Biosciences, Plo Alto, CA) per mnufcturer s instructions. Briefly, cells were wshed twice with cold PBS, nd resuspended in 1 inding uffer t concentrtion of cells/ml. One hundred microliters of cells were trnsferred to 5 ml culture tue, 5 ml of Annexin V-FITC nd 5 ml of propidium iodide were dded, smples were vortexed nd incuted for 15 min t room temperture in the drk. A totl of 400 ml of1 inding uffer ws dded to ech tue nd smples were immeditely nlyzed y flow cytometry FACS nlyses were performed on the FACS Cliur (BD Biosciences, Sn Jose, CA) with CellQuest softwre (BD Biosciences) for cquisition nd nlysis of dt. In vivo experimenttion Twenty-four 4--6-week-old Femle Bl/c nude mice were injected with NCI-H1299 cells (suspended in 0.1 ml PBS) in the rer left flnk. Ten dys fter dministrtion, mm 3 tumors were pprent on ll mice. At this time, nimls were divided into four groups (n ¼ 6) nd intrtumorlly received either vehicle [dimethyl formmide (DMF)], or thlidomide (2, 20 or 200 mg/kg) dissolved in DMF. Mice were treted twice per week for 3 weeks with thlidomide (or vehicle) nd tumor growth ws mesured t ech time of tretment with clipers. Tumor volume ws clculted using the formul V (mm 3 ) ¼ [ 2 ]/2, where is the length nd is the width of the tumor. Immunohistochemicl nlysis of PPARg protein At the conclusion of the in vivo experiment, tumors were excised from mice nd stored in 10% formlin. Tissues were prffin-emedded (Moleculr Histology, Montgomery Villge, MD) nd PPARg immunoleling ws performed s follows. After deprffiniztion nd locking of endogenous peroxidse in hydrogen peroxide/methnol, 5 mm tissue sections were microwved in 10 mm citrte uffer ( ph 6.0) for 12 min for ntigen retrievl. The sections were then locked for 1 h in 1.5% horse serum nd incuted overnight with 1:200 mouse polyclonl PPARg (E8) ntiody (Snt Cruz, CA). Antiody inding ws visulized y the vidin--iotin complex technique (Vector Lortories, Burlingme, CA). Protein rry NCI-H460 or NCI-H1299 cells were plted in 6-well dishes, nd fter ttchment treted with 1 mg/ml thlidomide or vehicle control. After 48 h, 1 ml of superntnt ws collected from cells nd humn cytokine protein rry (Ryiotech, Norcross, GA) ws ssyed ccording to mnufcturer s instructions. Briefly, cytokine memrnes were locked in 1 locking uffer for 30 min. After locking, 1 ml of smple ws dded to memrnes nd llowed to incute for 1.5 h. Memrnes were wshed nd 1 ml of iotinconjugted nti-cytokine ntiody mix ws dded to memrnes, nd llowed to incute for 2 h. After memrne wshing, 2 ml of diluted HRP-conjugted streptvidin ws dded. After 60-min incution, followed y wshing, detection ws performed using enhnced chemiluminescence. Results Growth inhiition of NSCLC cells y thlidomide ws sutype specific To study the effect of thlidomide on NSCLC cell growth, cells were treted with 1 mg/ml thlidomide or vehicle control for 8 dys. As shown in Figure 1, fter 8 dys of tretment, cell prolifertion ws reduced % in the three LCC cells tht were tested. This is in contrst to the deno- nd squmous-cell crcinom cell lines, in which the reduction in cell prolifertion fter 8 dys of tretment ws much less (Figure 1). Interestingly, no significnt reduction in growth inhiition ws oserved in the primry norml humn ronchil epithelil (NHBE) cells. Thlidomide significntly incresed PPARg protein expression in ll LCC cell lines To ssess the effect of thlidomide on PPARg protein expression, western nlysis ws performed on cell lystes from thlidomide-treted LCC cells. In ll LCC cells, PPARg protein expression ws lower thn in norml primry ronchil epithelil cells. This reduction ws specific to the PPARg1 isoform since PPARg2 is expressed in norml lung cells. After 48 h of incution of LCC cells with thlidomide, the PPARg1 isoform ws incresed in ll LCC cells (Figure 2), with the highest dose eing comprle with those found in the NHBE cells. Densitometry nlysis of these cells showed this effect to e dose-dependent (Figure 2). Thlidomide lso induced the PPARg2 isoform in dose-dependent fshion. This increse in PPARg expression ws specific to the LCC cell lines, nd not

3 Effect of thlidomide on NSCLC cell lines Fig. 1. Thlidomide inhiits the growth of LCC cells, ut hs miniml effect on deno- nd squmous cell crcinom cell growth LCC cells (NCI-H460, NCI-H661, NCI-H1299) were plted in 96-well pltes nd mintined in RPMI medi supplemented with 2 mm L-glutmine, 100 U/ml penicillin, 100 mg/ml streptomycin nd 5% het-inctivted FBS in 37 C, 5% CO 2 incutor. Thlidomide 1 mg/ml or vehicle ws dded dily nd complete medi chnges were performed every 48 h. At the end of 8 dys, cell growth ws mesured y the WST-1 colorimetric ssy () deno- or squmous cells were treted in similr fshion to the LCC cells, nd growth inhiition mesured y WST-1 regent (). Primry NHBE cells were lso treted to ensure tht thlidomide did not ffect the growth of norml lung cells. oserved in the deno- nd squmous cell crcinom cells (dt not shown). Thlidomide significntly incresed PPRE reporter ctivity in LCC cell lines, ut not in A549 denocrcinom cells To ssess whether the PPARg induction y thlidomide hd functionl consequences, PPRE reporter ssy ws performed. We found tht in oth LCC cell lines tested, thlidomide incresed PPRE reporter ctivity in dose-dependent mnner, with the highest dose (100 mg/ml) yielding times the ctivity of TK controls (Figure 3 nd ). This highest dose ws comprle with the PPARg selective gonist, troglitzone, which served s positive control. In shrp contrst, no differences were found in PPRE reporter ctivity in A549 denocrcinom cells trnsfected in similr mnner (Figure 3c). In this cell line, troglitzone (selective PPARg gonist) still incresed PPRE ctivity, suggesting tht the lck of effect in the thlidomide-treted cells ws not due to ssy conditions. Thlidomide decresed NFkB reporter ctivity in LCC cells treted with thlidomide, ut not in A549 denocrcinom cells Since PPARg hs een shown to interfere with the ntipoptotic NFkB signling pthwy, we mesured NFkB ctivity in cells treted with vrying doses of thlidomide. For the two LCC cell lines tested (NCI-H460 nd NCI-H1299), control cells tht received vector (ptal-nfkb-luc) ut no thlidomide tretment hd very high levels of NFkB. When cells were treted with incresing doses of thlidomide for Fig. 2. PPARg expression is significntly induced in LCC cells treted with thlidomide. NSCLC cells were treted with vrying doses of thlidomide for 48 h, fter which time cell lystes were prepred. Sixty microgrms of protein were loded on 10% Bis--Tris Novex gel, electrophoresed, nd trnsferred to PVDF memrne. Memrnes were proed with 1:1000 rit PPARg, 1:2000 got nti-rit IgG HRP nd nds detected y enhnced chemiluminescence (). Cells were normlized to -ctin nd densitometry nlyzed using n lph imger (). Primry NHBE cells were used s positive control. 1807

4 K.L.DeCicco et l. c c Fig. 3. Thlidomide significntly increses PPRE reporter ctivity in LCC cell lines, ut not in A549 denocrcinom cells. NCI-H460 (), NCI-H1299 () nd A549 (c) cells were trnsfected with PPRE 3 -TK-LUC expression vector nd 1 ml of Superfect for 5 h. After trnsfection, medi thlidomide ws dded. After 24 h, luminescence ws detected in cell lystes using the Luciferse Assy System. Some wells were treted with 10 mm troglitzone s positive control for PPRE ctivity. 24 h, dose-dependent decrese in NFkB expression ws seen, with the highest dose of thlidomide hving times lower NFkB ctivity (Figure 4 nd ). This decrese in NFkB ctivity ws not found in the A549 denocrcinom cells (Figure 4c), even though A549 cells treted with 10 mm troglitzone did hve reduction in the NFkB reporter ctivity. Thlidomide incresed poptosis in LCC cell lines, ut not in A549 denocrcinom cells NFkB is pro-survivl fctor in cncer cells, nd ecuse we found tht thlidomide locks NFkB ctivity, we tested whether thlidomide could induce poptosis in LCC cells. Annexin V stining nd FACS nlysis ws performed on cells treted with 1 mg/ml thlidomide (or vehicle) for 24 h. As demonstrted in Figure 5, NCI-H460 cells underwent 4-fold increse in poptosis (8.07 versus 1.74; sum of qudrnts B þ D) nd NCI-H1299 cells hd twice s mny poptotic cells in thlidomide treted (10.15) versus control treted (5.67) cells (the sum of qudrnts B þ D), when treted with 1808 Fig. 4. NFkB reporter ctivity is reduced in LCC cells ut not in A549 denocrcinom cells. NCI-H460 (), NCI-H1299 () nd A549 (c) cells were trnsfected with ptal-nfkb-luc expression vector nd 1 ml of Superfect for 5 h. After trnsfection, medi thlidomide ws dded. After 24 h, luminescence ws detected from cell lystes using the Luciferse Assy System. Some wells were treted with 10 mm troglitzone s positive control for PPRE ctivity. thlidomide (Figure 5). There ws no significnt difference (P ) in poptosis in A549 cells (dt not shown). Thlidomide tretment of LCC cells decresed inflmmtory nd ngiogenic proteins To ssess whether thlidomide ws n nti-ngiogenic fctor for lung cncer, humn cytokine protein rry ws conducted in thlidomide treted versus control cells. NCI-H460 nd NCI-H1299 LCC cells were treted with 1 mg/ml thlidomide (or vehicle control) nd superntnt collected fter 48 h. As seen in Figure 6, NCI-H460 cells treted with thlidomide hd lower growth-relted oncogene (GRO), epithelil cell derived-neutrophil ctivting peptide-78 (ENA-78) nd ngiotensin levels. In NCI-H1299 cells treted with thlidomide, the ngiogenic cytokine IL-8 ws significntly lower thn in

5 Effect of thlidomide on NSCLC cell lines Fig. 5. Thlidomide induces poptosis in LCC cells NCI-H460 () nd NCI-H1299 () cells were treted with 1 mg/ml thlidomide for 24 h nd poptosis ws performed using Annexin V-FITC stining per mnufcturer s instructions. FACS nlyses were performed on the FACS Cliur with CellQuest softwre for cquisition nd nlysis of dt. See online Supplementry Mteril for color version of this figure. control cells. No differences were found in other cytokines including GM-CSF, GCSF, I-309, IL-1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 13, 15, IFN-g, MCP, MCSF, MDCV, MIG, MIP, RANTES, SCF, SDF-1, TARC, TGF-B, TNF-/, EDG, IGF-1, OSM, VEGF, PDGF, Tpo nd leptin. In oth LCC cell lines, cyclooxygense 2 (COX-2) protein levels were high compred with norml lung cells tht did not express COX-2) (dt not shown). Thlidomide tretment of LCC cells decresed COX-2 protein levels significntly in dosedependent mnner (Figure 6), restoring the levels to the undetected levels found in the NHBE cells. Intrtumorl injection of thlidomide reduced NCI-H1299 tumor growth To ssess whether in vivo thlidomide could decrese lung cncer growth, 24 femle nude mice were injected with NCI-H1299 cells ccording to the timeline shown in Figure 7. Ten dys fter injection, tumor formtion ws seen in ll mice nd tretments were initited. As displyed in Figure 8, there ws no difference etween vehicle-treted mice nd those treted with the lowest dose of thlidomide (2 mg/kg). Mice treted with 20 mg/kg thlidomide hd 20% reduction in tumor growth, nd mice treted with the highest dose (200 mg/kg) hd growth inhiition of 64% (Figure 8). No toxicity ws found in mice treted with ny of the doses of thlidomide (dt not shown). Intrtumorlly injected thlidomide incresed PPARg protein in mouse tumors To exmine whether mouse tumors hd higher immunoleling of PPARg protein, immunohistochemistry on excised tumors ws performed. At the time of deth, mouse tumors were excised, stored in 10% formlin, prffin-emedded nd sectioned (5 mm sections) for immunoleling of PPARg protein. As shown in Figure 9, there is lrge increse in PPARg protein expression in the 200 mg/kg thlidomidetreted mice compred with control tumors tht hd lmost no PPARg expression. Two sections from two different nimls re displyed. Discussion Despite recent dvnces in novel chemotherpeutic gents for the tretment of NSCLC, s well s in the understnding of the moleculr sis of this disese, the numer of deths due to lung cncer is still greter thn for ny other form of cncer (1). The very poor (10--15%) 5-yer survivl rte is primrily due to high rte of metstsis. Of the four sutypes of NSCLC, LCC hs een shown to follow n extremely ggressive clinicl course, growing nd metstsizing erlier thn other NSCLC histologicl sutypes (2). Since the growth nd metstsis of neoplsm is dependent on dequte vsculr support to sustin prolifertion, survivl nd spred of the mlignnt cells, tumor growth nd metstsis re considered to e ngiogenesis-dependent. Therefore, therpeutic strtegies imed t inhiiting ngiogenesis re theoreticlly ttrctive. Thlidomide is n nti-ngiogenic nd nti-inflmmtory drug tht hs proven effective in vrious inflmmtory diseses nd cncers, nd is eginning to e evluted s n gent ginst the growth of solid tumors. However, to dte, the mechnism of ction y which thlidomide exerts its effects is poorly understood. Some hve suggested tht thlidomide exerts its effects through the down-regultion of 1809

6 K.L.DeCicco et l. Fig. 6. Thlidomide inhiits the expression of inflmmtory cytokines, ngiogenic fctors nd COX-2 in LCC cells NCI-H460 or NCI-H1299 cells were plted in 6-well dishes, nd fter ttchment treted with 1 mg/ml thlidomide or vehicle control. After 48 h, 1 ml of superntnts ws collected from cells nd incuted with humn protein rry memrnes s descried in the Mterils nd methods. Differences were detected using enhnced chemiluminescence (). Additionlly, whole cell lystes prepred from cells treted with incresing doses of thlidomide for 48 h were electrophoresed nd western lotted for Cox-2 expression (). Fig. 8. Thlidomide tretment decreses lung tumor growth in xenogrft mice. Twenty-four femle Bl/c mice were injected with NCI-H1299 cells nd fter tumor formtion, treted with thlidomide s descried in Figure 7. () A representtive niml from ech dose tested, s well s photogrph of the excised tumors. () The growth inhiition þ SD for ech group of mice. Fig. 7. Timeline for in vivo experiment. Twenty-four femle Bl/c mice were injected with NCI-H1299 cells. After tumor formtion, mice were treted with 2, 20 or 200 mg/kg thlidomide (n ¼ 6/group) or DMF vehicle control twice weekly for 3 weeks. Tumors were mesured y clipers t ech time of tretment. Tumors were excised t dy 31, nd stored in 10% formlin for immunohistochemicl nlysis of PPARg. tumor-relesed signling molecules tht stimulte ngiogenesis. These molecules include IL-6, FGF, VEGF nd TNF- (5,19). Others elieve thlidomide increses cell dhesion molecules (20), suppresses COX-2 ctivity (21) or lters cytokine expression (22). In this mnuscript, we report tht thlidomide inhiition of growth does not ffect ll NSCLC cells 1810 ut is rther specific to LCC cells. Indeed, wheres LCC cells were lrgely inhiited fter 8 dys of tretment, deno-, denosqumous nd squmous cell crcinoms were only mildly ffected. At the moleculr level, we focused on PPARg, memer of the nucler hormone receptor superfmily whose expression is undnt in norml lung tissue, ut often lcking in lung cncer tissues (16). It hs een documented previously in lung cncer cells tht PPARg-selective gonists such s ciglitzone nd PGJ2 cn inhiit growth of NSCLC cells through the induction of poptosis, promotion of differentition nd the down-regultion of cell cycle proteins such s Cyclin D1 (17,18). Becuse we hve seen tht the PPARg-selective gonist troglitzone is lso effective t inhiiting growth of NSCLC cells, nd morphologiclly seems to differentite the cells, we used this s

7 Effect of thlidomide on NSCLC cell lines Tumor from control mouse Tumor from TD treted mouse Tumor from control mouse Tumor from TD treted mouse Fig. 9. PPARg expression is sustntilly incresed in xenogrft tumors tken from mice treted with 200 mg/kg thlidomide compred with controls Tumors were excised from mice nd stored in 10% formlin. Sections were mde nd incuted with mouse polyclonl PPARg (s descried in the Mterils nd methods) nd visulized using the ABC method. Two representtive sections from two different mice re shown (20 mgnifiction). positive control in our studies. PPARg is present in two isoforms, PPARg1 nd PPARg2, resulting from lternte promoter usge. Structurlly, PPARg2 contins n dditionl 30 t the N-terminl end reltive to PPARg1. Ech of these two isoforms hs specific function nd tissue distriution. PPARg1 is expressed in mny tissues, ut the expression of PPARg2 is limited exclusively to dipose tissue where it hs een shown to ply key role in dipogenesis (23). In our study we found tht PPARg1 expression ws lower in LCC cells thn in primry NHBE cells. Moreover, thlidomide incresed the level of this isoform in dose-dependent mnner in LCC cells, with the highest dose incresing the level of expression to tht of the NHBE cells (Figure 2). This PPARg ws functionl, s demonstrted through PPRE reporter ssy (Figure 3). As expected, the primry NHBE cells do not express PPARg2, ut the expression of this isoform cn e forced upon thlidomide tretment. Others hve suggested tht forced expression of either PPARg1 or PPARg2 promotes differentition long the dipocyte linege (23) nd this differentition seems to e found in responding tumor cells (24). Although we did not mesure dipocyte-specific genes nd the ccumultion of lipid, it is possile tht our tumor cells re eing differentited towrd this phenotype. Unlike the LCC cells tht lose their expression of PPARg compred with NHBE cells, the denocrcinom cells normlly contin mesurle mounts of oth PPARg1 nd PPARg2 (dt not shown); this, possily explins why these cells re not s responsive to thlidomide tretment. Consistent with the fct tht PPARg locks inflmmtory responses y ntgonizing NFkB nd AP-1 signling pthwys (13--15), we found tht the induction of PPARg expression due to thlidomide tretment ws correlted with dose-dependent inhiition in NFkB ctivity (Figure 4). In contrst, untreted LCC cell lines hd very high NFkB ctivity due to the inflmmtory response tht ccompnies this cncer (Figure 4), greeing with previously reported literture (25). Since it is well known tht NFkB ctivtion is pro-survivl phenomenon used y cncer cells to void poptosis, we studied whether the ddition of thlidomide increses poptosis in our LCC cells. We found tht in oth cell lines, thlidomide tretment could induce poptosis (Figure 5), lthough the level of poptosis induction is proly not gret enough to ccount for the totl growth inhiition tht we hve found in the LCC cells. Thlidomide hs een shown in hemtologic diseses, such s myelom, to decrese neoplstic cell growth y ltering some of the pro-inflmmtory cytokines such s TNF nd IL-6. Wheres we filed to detect differences in pro-inflmmtory cytokines upon thlidomide tretment (Figure 6), we did find tht one ngiogenic cytokine (IL-8) in the NCI-H1299 LCC cell line, nd three ngiogenic cytokines in the NCI-H460 cells (Ang, GRO nd ENA-78) were down-regulted in thlidomide treted cells. This is importnt ecuse ENA-78 nd IL-8 hve oth een found to e elevted in NSCLC, consistent with the high vsculrity of this cncer, nd neutrlizing ENA-78 nd IL-8 ntiodies hve een shown to decrese tumor growth, vsculrity nd spontneous metstsis (26,27). Additionlly, we did find tht thlidomide down-regulted the strong COX-2 expression tht ccompnies NSCLC (Figure 6). This is noteworthy ecuse COX-2 is elieved to e involved in the invsiveness of NSCLC, s well s in resistnce to poptosis (28). Moreover, over-expression of COX-2 in NSCLC hs een linked to poor prognosis for this disese (28). It hs een documented tht COX-2 modultes production of ngiogenic fctors in colon cncer cells (29), nd therefore locking this inflmmtory gent my prevent ngiogenesis nd stimulte immune surveillnce nd poptosis. In mcrophges (30), thlidomide cn decrese the expression of COX-2. Our dt support tht this is lso true for LCC cells. In our in vivo experimenttion, we found tht intrtumorl injection of thlidomide (using the highest concentrtion tested) resulted in 64% reduction in tumor growth (Figure 8), ccompnied y decresed vsculrity (oservtion). To confirm tht some of this growth inhiition my e correlted with n elevted level of PPARg, immunohistochemicl nlysis of PPARg ws performed on excised tumors. In control tumors, we rely found ny detectle levels of PPARg (Figure 9). In contst, thlidomide-treted tumors showed significntly more stining thn control tumors, which correltes well with our in vitro western dt (Figure 2). As control, we hve tested norml ronchil tissue nd found significnt numer of PPARg-positive cells, minly in luminl position (dt not shown), suggesting tht the induction of PPARg y thlidomide my e restoring the norml phenotype. This work implictes thlidomide s eing involved in the PPARg pthwy, nd suggests tht thlidomide medites some of the effects of PPARg in LCC cells. To our knowledge, this is the first report of link etween thlidomide nd PPARg. Whether thlidomide cts s direct lignd for PPARg or indirectly through other proteins hs yet to e elucidted. Thus, this work my further help to identify one possile mechnism y which thlidomide exerts its nticncer effects, s well s define new moleculr trgets in the tretment of lung cncer. 1811

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