Upregulation of tissue inhibitor of matrix metalloproteinases-1 confers the anti-invasive action of cisplatin on human cancer cells

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1 (27) 26, & 27 Nture Pulishing Group All rights reserved /7 $3. SHORT COMMUNICATION Upregultion of tissue inhiitor of mtrix metlloproteinses-1 onfers the nti-invve tion of ispltin on humn ner ells R Rmer, K Eihele nd B Hinz Deprtment of Experimentl nd Clinil Phrmology nd Toxiology, Friedrih Alexnder Univerty Erlngen-Nürnerg, Erlngen, Germny Cner ell invon is one of the ruil events in lol spreding, growth nd metsts of tumors. The present study investigtes the mehnism underlying the ntiinvve tion of the hemotherpeuti ispltin. In humn ervil rinom ells (HeL), ispltin used time- nd onentrtion-dependent suppreson of ell invon through Mtrigel. Inhiition of invon ws ompnied y upregultion of tissue inhiitor of mtrix metlloproteinses-1 (TIMP-1), wheres levels of mtrix metlloproteinse-2 (MMP-2), MMP-9 nd TIMP-2 remined unhnged. pltin s effets on TIMP-1 expreson nd invon were ssoited with phosphoryltions of p38 nd p42/44 mitogen-tivted protein kinses nd were rogted y speifi inhiitors of oth pthwys. The impt of TIMP-1 in the nti-invve tion of ispltin ws proven y trnsfeting ells with smll interfering RNA trgeting TIMP-1, whih ompletely reversed suppreson of invon y ispltin. A funtionl relevne of TIMP-1 upregultion ws sustntited y findings showing onentrtion-dependent inhiition of Mtrigel invon y reominnt TIMP-1. The essentil role of TIMP-1 in the nti-invve tion of ispltin ws onfirmed ung nother humn ervil rinom ell line (C33A) nd humn lung rinom ells (A549). Altogether, our dt demonstrte hitherto unknown mehnism y whih ispltin exerts its ntimetstti properties on highly invve ner ells. (27) 26, ; doi:1.138/sj.on ; pulished online 19 Mrh 27 Keywords: ispltin; mtrigel ell invon; tissue inhiitor of mtrix metlloproteinses-1; mtrix metlloproteinse-2; humn ervil ner ells; humn lung rinom ells pltin (is-dimine-dihloropltinum(ii)) is one of the most importnt hemotherpeuti drugs used for tretment of different ner types inluding testiulr, ovrin, esophgl, hed/nek nd lung ner (for Correspondene: Dr B Hinz, Deprtment of Experimentl nd Clinil Phrmology nd Toxiology, Friedrih Alexnder Univerty Erlngen-Nürnerg, Fhrstrsse 17, D-9154 Erlngen, Germny. E-mil: hinz@phrmkologie.uni-erlngen.de Reeived 11 July 26; revised 15 Jnury 27; epted 26 Jnury 27; pulished online 19 Mrh 27 review, see Loehrer nd Einhorn, 1984). As the mjor ntineoplsti mehnism of this drug, the formtion of intr- nd interstrnd DNA rosslinks hs een reported (Jmieson nd Lipprd, 1999). In ddition, rod rry of tions independent of DNA ddut prodution hs een desried, inluding tivtion of mitohondril (Biroio et l., 24) nd Fs-ssoited poptoti pthwys (Rzzque et l., 1999) s well s indution of mitogen-tivted protein kinses (MAPKs) (Zhng et l., 25; Villedieu et l., 26). Moreover, nti-invve properties of ispltin hve een reported (Morikw et l., 1995; Muhi et l., 24) with the underlying mehnisms still eing poorly understood. Mtrix metlloproteinses (MMPs) hve emerged s group of enzymes exerting n importnt funtion during tumor invon, metsts nd ngiogenes through degrdtion of extrellulr mtrix omponents suh s ollgens nd proteoglyns (Currn nd Murry, 2; Stmenkovi, 2). Furthermore, tissue inhiitors of MMPs (TIMPs) hve een shown to ply key role in determining the proteolyti tivity of tumor tissues y regulting the tivity of MMPs. Aordingly, severl studies demonstrted orreltion etween high invveness nd deresed TIMP-1- (Khokh et l., 1989; Chn et l., 25) or inresed MMP-2 expreson (Mori et l., 24; Choi et l., 25). Conversely, the ntiinvve tion of severl ntirinogeni sustnes hs een ssoited with elevted TIMP-1 (Khokh et l., 1992; Prk et l., 25) or diminished MMP-2 levels (Prk et l., 25; Hong et l., 26). The present study therefore foussed on ispltin s effets on TIMP-1 nd MMP-2 expreson nd its impt on ner ell invon. In view of reent studies demonstrting p38 nd p42/44 MAPK tivtion y ispltin (Zhng et l., 25; Villedieu et l., 26) nd findings implying TIMP-1 s trget of MAPK gnling (Tong et l., 24; Hn nd Smith, 25), we further investigted posle role of oth MAPKs in this respet. To ddress these issues, the highly invve ervil ner ell line HeL ws used for Mtrigel invon ssys. HeL ells showed derese of invon fter 24-h tretment with 3 mm ispltin tht eme even more pronouned over the studied inution period (Figure 1). Inhiition of HeL ell invon y ispltin ourred in onentrtion-dependent mnner (Figure 1).

2 Inhiition of ner ell invon y ispltin pltin (3 µm) Inution time (hours) % Control Invon index Viility [pltin] (µm) d 12 Migrtion (% Control) pltin (3 µm) Viility (% Control) pltin (3 µm) Inution time (hours) Inution time (hours) e 14 Invon index Viility 12 % Control FU Pli CHX C 2 -er Figure 1 Influene of ispltin on HeL ell invon, migrtion nd viility. Time-dependeny of nti-invve tion of 3 mm ispltin (). Conentrtion-dependent effet of ispltin (3 3 mm) on invon nd ellulr viility following 72-h inution (). Time-dependeny of migrtion () nd viility (d) fter tretment of ells with 3 mm ispltin or vehile (.1% v/v dimethyl sulphoxide (DMSO)). Effet of 5-fluorouril (5-FU, 1 mm), plitxel (Pli,.3 mm), yloheximide (CHX, 1 mm) nd C 2 -ermide (C 2 -er, 1 mm) or vehile (.1% v/v DMSO) on ellulr invon nd viility (e). For determintion of viility, ells were seeded into 48-well pltes in volume of 5 ml serum-free Duleo s modified Egle s medium (DMEM) per insert nd treted with test sustnes or vehiles for the indited times. Cell viility ws mesured ung the WST-1 test (Rohe Dignostis, Mnnheim, Germny). For determintion of invveness, upper des of trnswell inserts (8-mm pore ze; BD Biosienes, Oxford, UK) were oted with 28.4 mg Mtrigel per insert in 24-well pltes. Trypnized nd pelleted ells were suspended to finl onentrtion of ells in volume of 5 ml serum-free DMEM per insert nd treted with test sustnes or vehiles for the indited times. DMEM ontining 1% fetl lf serum (FCS) ws used s hemottrtnt in the ompnion plte. Following inution for the indited times, the non-invded ells on the upper surfe of the inserts were removed with otton sw, nd viility of invded ells on the lower surfe ws determined. Quntifition of migrtion ws performed ung the sme experimentl proedure without Mtrigel oting. Invon index ws lulted s the sorne t 49 nm of ells hving invded through Mtrigel-oted Boyden hmers divided y sorne of ells hving migrted through unoted ontrol inserts with equl tretment ((invon/ migrtion) %). Perent ontrol represents omprison with vehile-treted ells (%) in the sene of test sustne. Vlues re mens7s.e.m. of n ¼ 4 experiments. Po.5, Po.1, Po.1, vs orresponding vehile ontrol (, nd d: Student s t-test; ( nd e): one-wy ANOVA plus Bonferroni test). To rule out the posility tht deresed invon y ispltin ws n unspeifi ytotoxiity-relted phenomenon, the time-dependent derese of ellulr viility fter ispltin exposure ws nlysed. Aording to our results, ispltin eliited moderte toxiity tht ws fr elow orresponding inhiitions of invon (Figure 1,

3 5824 TIMP-1 mrna (% Control) Inhiition of ner ell invon y ispltin 1 15 pltin (3 µm) 5 12 h 24 h 48 h 12 h 24 h 48 h MMP-2 mrna (% Control) TIMP-1 protein (% Control) 5 25 pltin (3 µm) 12 h 24 h 48 h -LC [pltin] (3 µm) 12 h 24 h 48 h TIMP-1 protein (% Control) [pltin] (µm) [pltin] (µm) - TIMP-2 - MMP-9 Figure 2 Time- nd onentrtion-dependent effet of ispltin on TIMP-1, MMP-2, TIMP-2 nd MMP-9 expreson in HeL ells. Time ourse of TIMP-1 nd MMP-2 mrna () nd protein expreson () fter tretment of ells with 3 mm ispltin or vehile (.1% v/v DMSO) for the indited times. Conentrtion-dependent effet of ispltin (3 3 mm) or vehile (.1% v/v DMSO) on TIMP-1, MMP-2, TIMP-2 nd MMP-9 protein levels following 48-h inution of ells (). TIMP-1 nd MMP-2 mrna levels were determined y quntittive rel-time reverse trnsription polymerse hin retion (RT PCR) s desried reently (Hinz et l., 25). For determintion of protein levels, ell ulture superntnts were entrifuged nd onentrted ung Miroon YM-1 entrifugl filter units (Millipore GmH, Shwlh, Germny) with 1 kd utoff. Western lot nlys ws performed s desried (Hinz et l., 25) ung speifi ntiodies rised to TIMP-1, MMP-2, TIMP-2 nd MMP-9 ( Reserh Produts, Sn Diego, CA, USA). To ontrol for loding of equl protein mounts, trnsferred proteins on Western lot memrnes were stined with the fluoresent dye Roti-Green (Crl Roth, Krlsruhe, Germny). As orresponding stndrd nd with ze of out 65 kd whih ppered unregulted ws hosen s loding ontrol (LC). Dentometri nlys of TIMP-1 nd intenties (normlized to loding ontrol) ws hieved y n optil snner nd the Multi-Anlyst progrm, veron 1.1 (Bio-Rd Lortories, Herules, CA, USA). In RT PCR experiments () nd dentometri nlyses ( nd ), perent ontrol represents omprison with vehile-treted ells (%) in the sene of test sustne. Vlues re mens7s.e.m. of n ¼ 4 8 (RT PCR) or n ¼ 5 (dentometry) experiments. Po.5 vs orresponding vehile ontrol (Student s t-test). -LC ompre Figure 1 nd d). Aordingly, there ws no gnifint hnge in invon inhiition when Mtrigel ell invon ws normlized to the orresponding ell viility insted of ell migrtion (i.e., invon index): 12 h: 1777%, n ¼ 4; 24 h: 2272%, n ¼ 4, Po.1; 48 h: 1373%, n ¼ 4, Po.1; 72 h: 673%, n ¼ 4, Po.1 (ll vlues vs respetive vehile (%), Student s t-test). Moreover, ispltin lso eliited onderle nti-invve tion t non-toxi onentrtion of 3 mm (Figure 1). We further tested dditionl ntineoplsti or ytotoxi sustnes t equitoxi onentrtions s ispltin (Figure 1e). As mtter of ft, none of these ompounds (i.e., 5-fluorouril, plitxel, yloheximide nd C 2 -ermide) mimiked the ntiinvve tion of ispltin. Finlly, speifi invon-ssoited proess ws sustntited y the finding tht redued invon ws not ssoited with deresed ell motility (Figure 1).

4 Anlys of mrna in ell lystes reveled timedependent indution of TIMP-1 following inution with ispltin (Figure 2). The stimultory tion of ispltin on TIMP-1 mrna expreson eme first evident fter 24-h inution period (Figure 2). In line with these dt, ispltin lso exhiited time- nd onentrtion-dependent indution of TIMP-1 protein, whih ws determined in ell ulture superntnts (Figure 2 nd ). Although n inhiitory tion of ispltin on MMP-2 hs een desried previously in smll-ell lung ner (Morikw et l., 1995) nd gliolstom ells (Chintl et l., 1997), ispltin did not exhiit gnifint suppreson of MMP-2 mrna (Figure 2) nd MMP-2 protein levels (Figure 2 nd ) in HeL ells inditing tumor-type speifi modultion of this enzyme. Aprt from MMP-2, ispltin did lso not influene MMP-9 nd TIMP-2 expreson (Figure 2). The oserved timedependent inrese of TIMP-1 nd MMP-2 in vehile ontrols of the respetive time points (Figure 2) is likely due to n umultion of TIMP-1 nd MMP-2 in ell ulture superntnts. Experiments ddresng the role of p38 nd p42/44 MAPKs in ispltin-eliited suppreson of invon reveled n tivtion of oth kinses y ispltin tht eme first evident fter 15 min, nd ws still detetle fter 24 nd 48 h (Figure 3). To onfirm usl link etween MAPK tivtions, TIMP-1 upregultion nd redution of invveness, ells were pretreted with inhiitors of oth MAPK pthwys. In ft, inhiitors of p38 MAPK tivity (SB2358) nd of p42/44 MAPK tivtion (PD9859) led to omplete reversl of ispltin s nti-invve nd TIMP-1-induing tion (Figure 3). SB2358 nd PD9859 given lone did not gnifintly influene invveness of nd TIMP-1 expreson in HeL ells (Figure 3). Further inhiitor experiments imply n involvement of protein kinse C in the oserved nti-invve nd TIMP-1-upregulting tion of ispltin, ut exlude ontriution of the phosphtidylinotol 3-kinse (PI3K) pthwy in this respet (Figure 3). In n ttempt to link ispltin-indued TIMP-1 expreson to the oserved derese of invon, HeL ells were trnsfeted with TIMP-1 smll-interfering RNA (RNA). Monitoring of TIMP-1 seretion into the ulture medium of the upper Boyden hmer onfirmed profound inhiition of TIMP-1 expreson in ells trnsfeted with TIMP-1 RNA s ompred to ontrol smples (Figure 4). As further shown in Figure 4, knokdown of TIMP-1 expreson led to n lmost omplete rogtion of the ispltin-medited derese of invon. By ontrst, ultures treted with non-lening sequene exhiited the sme invon pttern s ontrol ells treted with trnsfetion gent only (Figure 4). These findings were lso onfirmed y stining invded ells t the lower de of the trns-well inserts (Figure 4). The funtionl relevne of TIMP-1 upregultion ws sustntited y demonstrting tht reominnt TIMP-1 gnifintly inhiited Mtrigel invon (Figure 4). Speifiity of RNA trgeting TIMP-1 ws further proven y nlyng the expreson of TIMP-2 (Figure 4). Inhiition of ner ell invon y ispltin h ## + SB 24 h 48 h + PD To exlude tht the oserved effets re restrited to HeL ells, we performed key experiments in nother humn ervil rinom ell line (C33A) s well s in humn lung rinom ells (A549). Applition of ispltin resulted in gnifint inhiition of Mtrigel - pp38 - p38 - pp42/44 - p42/44 - ß-tin [pltin] (3 µm) Bis Bis + LY SB PD LY -LC Figure 3 Anlys of gnl trnsdution pthwys involved in ispltin-modulted HeL ell invon. Time-ourse of p38 nd p42/44 MAPK tivtion y 3 mm ispltin (). Effet of 1-h pretretment with () 1mM SB2358 (SB; inhiitor of p38 MAPK tivity) nd 1 mm PD9859 (PD; inhiitor of p42/44 MAPK tivtion) or () 1mM indolylmleimide II (Bis, protein kinse C inhiitor) nd 1 mm LY2942 (LY, PI3K inhiitor) on the ntiinvve tion of ispltin (3 mm) or vehile (.2% v/v DMSO) fter 72-h inution nd on TIMP-1 nd MMP-2 protein levels fter 48-h inution period. Western lot nlys ws performed s desried reently (Hinz et l., 25) ung ntiodies rised ginst p38, phospho-p38, p42/44 nd phospho-p42/44 (New Englnd BioLs GmH, Frnkfurt, Germny) nd -tin (Cliohem, Bd Soden, Germny). In invon experiments, perent ontrol represents omprison with vehile-treted ells (%) in the sene of test sustne. Vlues re mens7s.e.m. of n ¼ 4 experiments. Po.1, Po.1, vs orresponding ## vehile ontrol; Po.1, Po.1, vs ispltin (one-wy ANOVA plus Bonferroni test). 5825

5 Inhiition of ner ell invon y ispltin HeL TIMP- 1 - TIMP C33A TIMP A TIMP-2 Invon index (% Control) HeL C33A A [TIMP-1] (ng/ml) [TIMP-1] (ng/ml) [TIMP-1] (ng/ml) Figure 4 Role of TIMP-1 in the nti-invve tion of HeL, C33A nd A549 ells. Effet of TIMP-1 knokdown on the ntiinvve tion of ispltin ( nd ). Effet of reominnt TIMP-1 protein on tumor ell invon (). HeL () C33A nd A549 ells () were trnsfeted with TIMP-1 RNA (;.25 mg/ml; trget sequene: 5 -tttttttgg-3 ; Qigen GmH, Hilden, Germny) or non-lening RNA (;.25 mg/ml; Eurogente, Sering, Belgium; Ct. No. OR-3-neg) with equl rtios (w/v) RNAiFet (Qigen GmH) for 24 h in DMEM supplemented with 1% FCS. Susequently, trypnized nd entrifuged ells were resuspended to finl denty of /5 ml in serum-free DMEM ontining the sme mounts of TIMP-1 RNA or non-lening RNA to provide onstnt trnsfetion onditions, pled into invon hmers, nd stimulted with 3 mm ispltin or vehile (.1% v/v DMSO). Red-out of invon nd Western lot nlys of TIMP-1, TIMP-2 nd MMP-2 protein levels in superntnts olleted from the upper Boyden hmers were performed fter 72-h tretment of trnsfeted ells. Following 72-h inution, HeL ells invded through Mtrigel-oted memrnes were stined with Diff-Quik (Medion Dignostis GmH, Bu dingen, CH, Germny) nd visully doumented under mirosope t 2 mgnifition. Reominnt humn TIMP-1 protein (Cliohem) ws dded to the ells for 72-h inution in Mtrigel-oted Boyden hmers. In invon experiments, perent ontrol represents omprison with vehile-treted ells (%) in the sene of test sustne. Vlues re mens7s.e.m. of n ¼ 4 experiments. Po.5, Po.1, Po.1, vs vehile ontrol; Po.1, vs ispltin (one-wy ANOVA plus Bonferroni test).

6 invon ompnied y inresed TIMP-1 seretion with oth events eing olished in the presene of TIMP-1 RNA (Figure 4). Likewise, 72-h tretment with ispltin eliited no hnge in ell motility (C33A: 9672%, n ¼ 4 vs vehile (%); A549: 1177%, n ¼ 4 vs vehile (%)) nd only minor loss of viility (C33A: 77%, n ¼ 4, Po.5 vs vehile (%), Student s t-test; A549: 73%, n ¼ 4, Po.1 vs vehile (%), Student s t-test) s ompred to its pronouned nti-invve tion. Agin, ispltin left expreson of MMP-2 nd TIMP-2 virtully unltered (Figure 4). Colletively, these results exlude ell line-speifi effet nd suggest tht seletive upregultion of TIMP-1 y ispltin my e more generl nti-invve mehnism. However, given tht the leding pltinum gents in ner hemotherpy my provoke different Referenes Biroio A, Bens B, Fiorentino F, Zupi G. (24). Glutthione depletion indued y -My downregultion triggers poptos on tretment with lkylting gents. Neopl 6: Chn VY, Chn MW, Leung WK, Leung PS, Sung JJ, Chn FK. (25). Intestinl trefoil ftor promotes invon in nontumorigeni Rt-2 firolst ell. Regul Pept 127: Chintl SK, Ali-Osmn F, Mohnm S, Ryford A, Go Y, Goksln ZL et l. (1997). Effet of ispltin nd BCNU on MMP-2 levels in humn gliolstom ell lines in vitro. Clin Exp Metsts 15: Choi J, Choi K, Benveniste EN, Rho SB, Hong YS, Lee JH et l. (25). Bl-2 promotes invon nd lung metsts y induing mtrix metlloproteinse-2. Cner Res 65: Currn S, Murry GI. (2). Mtrix metlloproteinses: moleulr spets of their roles in tumour invon nd metsts. Eur J Cner 36: Hn R, Smith TJ. (25). Indution y IL-1 of tissue inhiitor of metlloproteinse-1 in humn oritl firolsts: modultion of gene promoter tivity y IL-4 nd IFN-g. J Immunol 174: Hrtmnn JT, Lipp HP. (23). Toxiity of pltinum ompounds. Expert Opin Phrmother 4: Hinz B, Rosh S, Rmer R, Tmm ER, Brune K. (25). Ltnoprost indues mtrix metlloproteinse-1 expreson in humn nonpigmented iliry epithelil ells through ylooxygense-2-dependent mehnism. FASEB J 19: Hong JH, Ahn KS, Be E, Jeon SS, Choi HY. (26). The effets of urumin on the invveness of prostte ner in vitro nd in vivo. Prostte Cner Prostti Dis 9: Jmieson ER, Lipprd SJ. (1999). Struture, reognition, nd proesng of ispltin-dna dduts. Chem Rev 99: Khokh R, Wterhouse P, Ygel S, Ll PK, Overll CM, Norton G et l. (1989). Antisense RNA-indued redution in murine TIMP levels onfers onogeniity on Swiss 3T3 ells. Siene 243: Khokh R, Zimmer MJ, Grhm CH, Ll PK, Wterhouse P. (1992). Suppreson of invon y induile expreson of Inhiition of ner ell invon y ispltin ellulr stress responses (Hrtmnn nd Lipp, 23), the nti-invve tion of ropltin nd oxlipltin should lso e ddressed in future studies. To the est of our knowledge, this is the first study tht estlished deive role of TIMP-1 in the ntiinvve tion of ispltin. This hitherto unknown mehnism is expeted to ply n importnt role in the ntineoplsti tions of this outstnding hemotherpeuti drug. Aknowledgements This work ws supported y grnts from the Deutshe Kreshilfe e.v. (Bonn, Germny), Deutshe Forshungsgemeinshft (SFB 539, TP BI.6) nd Johnnes und Fried Mrohn Stiftung (Erlngen, Germny). tissue inhiitor of metlloproteinse-1 (TIMP-1) in B16-F1 melnom ells. J Ntl Cner Inst 84: Loehrer PJ, Einhorn LH. (1984). Drugs five yers lter. pltin. Ann Intern Med : Muhi S, Ohmihi M, Nishio Y, Hysk T, Kimur A, Oht T et l. (24). Inhiition of NF-kB inreses the effiy of ispltin in vitro nd in vivo ovrin ner models. J Biol Chem 279: Mori K, Shinum M, Nose K. (24). Invve potentil indued under long-term oxidtive stress in mmmry epithelil ells. Cner Res 64: Morikw T, Shiuy M, Ski S, Kudo S. (1995). Effet of ntiner drugs on invve pity of humn smll-ell lung ner ells in vitro. Nippon Ik Digku Zsshi 62: Prk MJ, Lee JY, Kwk HJ, Prk CM, Lee HC, Woo SH et l. (25). Arseni trioxide (As 2 O 3 ) inhiits invon of HT18 humn firosrom ells: role of nuler ftor-kb nd retive oxygen speies. J Cell Biohem 95: Rzzque MS, Koji T, Kumtori A, Tguhi T. (1999). pltin-indued poptos in humn proximl tuulr epithelil ells is ssoited with the tivtion of the Fs/Fs lignd system. Histohem Cell Biol 111: Stmenkovi I. (2). Mtrix metlloproteinses in tumor invon nd metsts. Sem Cner Biol 1: Tong L, Smyth D, Kerr C, Ctterll J, Rihrds CD. (24). Mitogen-tivted protein kinses Erk1/2 nd p38 re required for mximl regultion of TIMP-1 y onosttin M in murine firolsts. Cell Signl 16: Villedieu M, Deslndes E, Duvl M, Heron JF, Guduhon P, Poulin L. (26). Aquition of hemorestne following disontinuous exposures to ispltin is ssoited in ovrin rinom ells with progresve ltertion of FAK, ERK nd p38 tivtion in response to tretment. Gyneol Onol 11: Zhng QX, Feng R, Zhng W, Ding Y, Yng JY, Liu GH. (25). Role of stress-tivted MAP kinse p38 in ispltin- nd DTT-indued poptos of the esophgel rinom ell line E 19. World J Gstroenterol 11: