Inorganic sulfur reduces the motility and invasion of MDA-MB-231 human breast cancer cells

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1 Nutrition Reserch nd Prctice (Nutr Res Prct) 211;5(5): Inorgnic sulfur reduces the motility nd invsion of MDA-MB-231 humn rest cncer cells Jin Joo Kim 1, Ae Wh H 1, Hee Sun Kim 2 nd Woo Kyoung Kim 1 1 Deprtment of Food Science nd Nutrition, Dnkook University, 126 Jukjeon-dong, Suji-gu, Yongin-si, Gyunggi , Kore 2 Deprtment of Food nd Nutrition, Bewh Women s University, Seoul , Kore Astrct This study investigted the effects of inorgnic sulfur on metstsis in MDA-MB-231 humn rest cncer cells. MDA-MB-231 cells were cultured in the sence or presence of vrious concentrtions (12.5, 25, or 5 μmol/l) of inorgnic sulfur. Cell motility, invsion, nd the ctivity nd mrna expression of mtrix metlloproteses (MMPs) were exmined. Numers of vile MDA-MB-231 cells did not differ y inorgnic sulfur tretment from to 5 μmol/l within 48 h. Inorgnic sulfur significntly decresed cell motility nd invsion in the MDA-MB-231 cells in dose-dependent mnner (P <.5), s determined using Boyden chmer ssy nd Mtrigel chmer. The ctivities of MMP-2 nd MMP-9 were significntly reduced y inorgnic sulfur in dose-dependent mnner (P <.5). The inorgnic sulfur lso significntly inhiited MMP-2 nd MMP-9 expression in the cells (P <.5). These dt suggest tht inorgnic sulfur cn suppress cncer cell motility nd invsion y inhiiting MMP-2 nd MMP-9 ctivity nd gene expression in MDA-MB-231 cells. Key Words: Inorgnic sulfur, invsion, motility, mtrix metlloprotese, MDA-MB-231 cells Introduction 1) The World Helth Orgniztion (WHO) hs estimted tht from 27 to 23, the numer of cncer ptients will increse from 11.3 million to 15.5 million, nd deth cused y cncer will increse y 45% from 7.9 million to 11.5 million, indicting tht cncer is one of the mjor cuses of deth worldwide [1]. The numer of cncer ptients is incresing in Kore s well; it is reported tht -12 thousnd individuls ecme cncer ptients in 28, with > 55 thousnd lredy eing estlished ptients [2]. In the cse of rest cncer, Kore hd 11,439 new ptients in 28 (oth mle nd femle) nd it ws rnked in 6th in the occurrence rte of new ptients [3]. For femle ptients only, it ws rnked 2nd in occurrence rte, nd 1st in the numer of treted ptients (Ntionl Helth Insurnce, ll rights reserved) [2]. Brest cncer is divided into invsive nd non-invsive ctegories ccording to its metsttic ehvior, nd > 75% of rest cncer is invsive. Thus, lthough it is importnt to prevent the occurrence of rest cncer per se, preventing metstsis fter rest cncer hs occurred cn e crucil wy to reduce the resulting deth rte. In generl, the mechnism of tumor metstsis is s follows. Tumor cells migrte from primry tumors nd ttch to sement memrnes, whose extrcellulr-mtrix (ECM) mde of collgen, is degrded y mtrix metlloproteses (MMPs), cusing invsion into proximl tissues [4-6]. The tumor cells then enter the circultion system including lood / lymphtic vessels, nd circulte through nd penetrte vessels of certin tissues to cuse metsttic cncer [7-9]. More thn 9% of rest cncer occurs t Lctiferous ducts, which hve widely distriuted lymphtic vessels through which cncer cn esily metstsize [1]. Sulfur is chemicl element with n tomic numer of 16 (MW 32.64) nd is denoted y S. It is insolule in wter, ut solule in lcohol, enzene, nd ether [11]. Chemicls contining sulfur re usully involved in redox (oxidtion-reduction) rections in the ody [12]. The humn ody cn ingest sulfur from wter nd food, minly s orgnic sulfur from sulfur-contining mino cids (SAAs) in foods [13]. However, it is reported tht lthough sufficient mount of sulfur-contining mino cids re ingested, sulfte deficiency symptoms cn occur if the mount of dietry sulfte is insufficient, nd thus supply of dietry sulfte is essentil for mice [14]. It is lso reported tht dietry sulfte for nimls cn supply the sulfur needed to form cysteine, nd thus ffect the function of ioctive mterils tht hve cysteine s their precursor [15]. Although inorgnic sulfur is thought to This work ws supported y Koren Reserch Foundtion Grnt funded y the Koren Government (MOEHRD, Bsic Reserch Promotion Fund) (KRF F2). Corresponding Author: Kim, Woo Kyoung, Tel , Fx , Emil. wkkim@dnkook.c.kr Received: Decemer 15, 21, Revised: June 16, 211, Accepted: June 29, 211 c211 The Koren Nutrition Society nd the Koren Society of Community Nutrition This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution Non-Commercil License ( which permits unrestricted non-commercil use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 376 Effect of inorgnic sulfur on metstsis ply n independent physiologicl role, little reserch hs een done on this topic. Toxicity y inorgnic sulfur is uncommon nd is usully limited to the skin [4]. No toxicity ws reported from orl dministrtion of.17 mg/kg to humns, or in other niml tests with rits, rts, guine pigs, nd dogs [5]. The nti-cncer effects of orgnic sulfur contined in foods hve een reported [16-18], ut those of inorgnic sulfur hve rrely een exmined. Thus, in this study, we explored whether inorgnic sulfur cn ffect the motility nd invsion of humn MDA-MB-231 rest cncer cells, s well s MMP-2 nd MMP-9 ctivity nd mrna expression to ffect tumor metstsis. Mterils nd Methods Regents nd chemicls Inorgnic sulfur powder of 99% purity ws otined from Sulfon PS Inc. (Seoul, Kore). The inorgnic sulfur powder ws dissolved in methyl lcohol (Aldrich, 3239, USA) t 5 mm concentrtion nd stored t -2. Dulecco's Modified Egle's medium nd Hm s F12 Nutrient Mixture (DMEM/F12), s well s streptomycin nd penicillin were otined from Gico/BRL (Grnd-Islnd, NY, USA). RIA-grde ovine serum lumin (BSA), trnsferrin, nd other regents were purchsed from Sigm (St. Louis, MO, USA). Cell culture MDA-MB-231 humn rest cncer cells were purchsed from the Americn Type Culture Collection (Rockville, MD, USA). The cells were mintined in DMEM/F12 contining 1 ml/l of fetl ovine serum (FBS) with 1, U/L of penicillin nd 1 mg/l of streptomycin. The medium ws replced every 2-3 dys. To exmine the effects of inorgnic sulfur on rest cncer cell prolifertion, MDA-MB-231 cells were plted in 24-well pltes t density of cells/ml in DMEM/F12 supplemented with 1% FBS. After 48 h of incution, the monolyers were serum-strved with DMEM/F12 supplemented with 5 μg/ml trnsferrin, 5 ng/ml selenium, nd 1 mg/ml ovine serum lumin for 24 h. After serum strvtion, the monolyers were incuted in serum free medium (SFM) with, 12.5, 25, or 5 μmol/l inorgnic sulfur. Vile cell numers were estimted t nd 48 h fter the cells were exposed to inorgnic sulfur y using the 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrzolium romide (MTT) ssy, s previously descried [19]. The experiments were performed independently 3 times. coted with.1% geltin solution for 16 h t room temperture. MDA-MB-231 cells resuspended in SFM in the sence or presence of vrious concentrtions (12.5, 25, or 5 μmol/l) of inorgnic sulfur were crefully trnsferred into the upper chmer. The lower chmer ws filled with 1% FBS medium to ttrct the cells. The Boyden chmer ws incuted t 37 with 5% CO 2 for 8 h. After gently removing the filter from the chmer, the cells on the upper chmer side of the filter were removed y wiping the filter with pper. The filter ws stined with Diff-Quick stin solution (Dde Behring, Network, NJ, USA) nd the cells on the lower surfce of the filter were fixed onto glss slide. The cells in 5 rndomly selected microscopic fields ( 4) of the lower slide were then counted. The experiments were performed independently 3 times. Invsion ssy The invsion ssy ws performed s previously descried [21]. Wells of Mtrigel chmer (BD Bioscience, MA, USA) were filled with SFM nd dpted t room temperture. MDA- MB-231 cells resuspended in SFM with the sence or presence of vrious concentrtions (12.5, 25, or 5 μmol/l) of inorgnic sulfur were crefully trnsferred into the upper chmer. The lower chmer ws filled with 1% FBS medium to ttrct the cells. The Mtrigel chmers were incuted for 12 h t 37 with 5% CO 2. Then, the cells on the upper surfces of the filters were removed y wiping the filter with pper. The filters were stined with Diff-Quick stin solution nd the cells on the lower surfce of the filter were fixed onto glss slide. Cells in 5 rndomly selected microscopic fields ( 4) of the lower slide were counted. The experiments were performed independently 3 times. Mtrix metlloproteses ctivity (geltin zymogrphy) MMP ctivity ws investigted s previously descried [22]. The cells were seeded into 6-well pltes t cells/ml nd incuted in medium contining 1% FBS for 48 h. The monolyers were incuted in the presence of vrious concentrtions of inorgnic sulfur (, 12.5, 25, 5 μmol/l) for 24 h. The superntnts were collected nd concentrted 1-fold in Centricon centrifugl filter devices (Milipore, Bedford, MA, USA), fter which the MMP ctivity in the superntnts ws investigted using geltin zymogrphy. Ech superntnt ws mixed with 2x smple uffer (lnvitrogen), nd zymogrphy ws performed using gels (1% polycrylmide, 1% geltin). The MMP ctivities were visulized y stining with Coomssie lue. The experiments were performed independently 3 times. Boyden chmer motility ssy The Boyden chmer motility ssy ws performed s previously descried [2]. PVPF filters (8 μm dimeter pore size) were Reverse trnscriptse polymerse chin rection MMP mrna levels in the culture medium were determined s previously descried [22]. Totl RNA ws isolted using

3 Jin Joo Kim et l. 377 TRI-regent (Sigm), nd cdna ws synthesized using 2 μg of totl RNA with SuperScript Ⅱ reverse trnscriptse (lnvitrogen). For mplifiction of the cdna, primers for MMP-2 (upstrem primer, 5'-CAGGCTCTTCTCCTTTCSCAAC-3'; downstrem primer, 5'-AAGCCACGGCTTGGTTTTCCTC-3') nd MMP-9 (upstrem primer, 5'-TGGGCTACGTGACCTATGACCAT-3'; downstrem primer, 5'-GCCCAGCCCACCTCCACTCCTC-3'; nneling t 55 for 1 min with 35 cycles) were used. The polymerse chin rection (PCR) products were seprted on 1% grose gel nd stined with ethidium romide. The nds corresponding to ech specific PCR product were quntified y densitometric scnning of the exposed film using the Bio-profile Bio-IL ppliction (Viler-Lourmt). of vile MDA-MB-231 cells did not differ y inorgnic sulfur tretment from to 5 μmol/l within 48 h (Fig. 1). To show tht the nti-metsttic effect of inorgnic sulfur ws independent of decrese in cell prolifertion, the inorgnic sulfur tretment time did not exceed 48 h in this study. Inorgnic sulfur decreses tumor motility nd invsion of MDA- MB-231 cells Inorgnic sulfur significntly decresed tumor motility using Boyden chmer ssy (Fig. 2) (P <.5), nd invsion through Mtrigel chmer (Fig. 3) (P <.5). Sttisticl nlysis Sttisticl nlyses were performed using the Sttisticl Anlysis System softwre (SAS Institute, Cry, NC, USA). The dt were expressed s mens with stndrd errors nd nlyzed vi nlysis of vrince (ANOVA). Sttisticlly significnt differences mong the mens of groups were tested t α =.5 using Duncn's multiple rnge test. Results Inorgnic sulfur did not influence cell prolifertion in MDA- MB-231 cells until 24 h of incution To exmine the effects of inorgnic sulfur on MDA-MB-231 cell prolifertion, cells in monolyer culture were incuted in SFM with, 12.5, 25, or 5 μmol/l inorgnic sulfur. Numers Fig. 2. Effect of inorgnic sulfur on cell motility in MDA-MB-231 cells. Cells were cultured in the presence of vrious concentrtions of inorgnic sulfur (, 12.5, 25, or 5 μmol/l) for 8 h with Boyden chmer. A) Microphotogrphy of cells treted with inorgnic sulfur in cell migrtion ssy. B) Quntittive nlysis of cell motility ssy. The numer of migrted cells ws expressed s percentge of the control (without inorgnic sulfur). Ech r represents the men ± SE from 3 independent experiments. Significnt differences (P <.5) mong groups re indicted y different letters ove ech r % Control ( h) μmol/l 12.5 μmol/l 25 μmol/l 5 μmol/l NS NS (h) Fig. 1. Effect of inorgnic sulfur on cell prolifertion in MDA-MB-231 cells. MDA-MB-231 cells were plted t density of cells/ml in 24 well pltes with DMEM/F12 supplemented with 1% FBS for 48 h. The monolyers were serum-strved with DMEM/F12 supplemented with 5 μg/ml trnsferrin, 5 ng/ml selenium, nd 1 mg/ml ovine serum lumin for 24 h. After serum strvtion, the monolyer were incuted in serum free medium with, 12.5, 25, or 5 μmol/l inorgnic sulfur for 24 h. Vile cell numers were estimted y the MTT ssy. Ech r represents the men ± SE from 3 independent experiments. Significnt differences (P <.5) mong groups re indicted y different letters ove ech r. Fig. 3. Effects of inorgnic sulfur on invsion in MDA-MB-231 cells. Cells were cultured in the presence of vrious concentrtions of inorgnic sulfur (, 12.5, 25, 5 μmol/l) for 12 h in n invsion chmer. A) Microphotogrphy of cells treted with inorgnic sulfur in cell migrtion ssy. B) Quntittive nlysis of cell motility ssy. The numer of migrted cells ws expressed s percentge of the control (without inorgnic sulfur). Ech r represents the men ± SE from 3 independent experiments. Significnt differences (P <.5) mong groups re indicted y different letters ove ech r.

4 378 Effect of inorgnic sulfur on metstsis A () MMP-2 (72KD) B () MMP-9 (92KD) () (%) 15 () (%) c Fig. 4. Effects of inorgnic sulfur on MMP-2 nd MMP-9 ctivity in MDA-MB-231 cells. Cells were plted in 6 well pltes t density cell/ml in DMEM/F12 supplemented with 1% FBS. After 48 h of incution, the monolyers were incuted in the presence of vrious concentrtions of inorgnic sulfur (, 12.5, 25, 5 μmol/l) for 24 h. The medium ws collected nd concentrted for zymogrphy. A-) Photogrphs of MMP-2, A-) Quntittive nlysis of the nds. B-) Photogrphs of MMP-9 nds, B-) Quntittive nlysis of the nds. The enzyme ctivity ws expressed s percentge of the control (without inorgnic sulfur). Ech r represents the men ± SE of 3 independent experiments. Significnt differences (P <.5) mong groups re indicted y different letters ove ech r. A () MMP-2 (32p) A () MMP-9 (31p) β-ctin (318p) () (%) 15 β-ctin (318p) () (%) c Fig. 5. Effect of inorgnic sulfur on MMP-2 nd MMP-9 mrna expression in MDA-MB-231 cells. Cells were cultured in the presence of vrious concentrtions of inorgnic sulfur (, 12.5, 25, or 5 μmol/l) for 24 h. Totl RNA ws isolted nd RT-PCR ws performed. Photogrphs of the ethidium romide-stined gels, which re representtive of 3 independent experiments, re shown. A-) Photogrphs of the MMP-2 nds, A-) Quntittive nlysis of the nds. B-) Photogrphs of the MMP-9 nds, B-) Quntittive nlysis of the nds. The mrna expression of the enzymes ws expressed s percentge of the control (without inorgnic sulfur). Ech r represents the men ± SE, nd significnt differences (P <.5) mong groups re indicted y different letters ove ech r. Inorgnic sulfur reduces MMP-2 nd MMP-9 ctivity in MDA- MB-231 cells To determine the effects of inorgnic sulfur on MMP-2 nd MMP-9, which degrde ECM llowing the metstsis of mlignnt tumors, their ctivities were tested. Here, the induced ctivity of MMP-2 nd MMP-9 ws significntly reduced y inorgnic sulfur in dose-dependent mnner (Fig. 4) (P <.5). Inorgnic sulfur reduces MMP-2 nd MMP-9 mrna expression in MDA-MB-231 cells We performed RT-PCR nlysis to determine whether inorgnic sulfur regultes MMP-2 nd MMP-9 mrna expression. As shown in Fig. 5, inorgnic sulfur significntly inhiited MMP-2 nd MMP-9 mrna expression in the cells (P <.5). Discussion Becuse > 9% of rest cncer occurs t Lctiferous ducts which hve widely distriuted lymphtic vessels through which the cncer cn esily metstsize [1], reserch on dietry components tht cn prevent metstsis hs gret significnce. Sulfur is nutrient whose composition in the ody is the seventh highest [13], nd reserch on orgnic sulfur, such s tht contined in cruciferous vegetles, hs een performed with regrd to its relevnce to cncer [16-18]. On the other hnd,

5 Jin Joo Kim et l. 379 few studies hve focused on inorgnic sulfur. Thus, in the present study, we ttempted to explore the nutritionl nd physiologicl functions of inorgnic sulfur y studying its effects on rest cncer metstsis. When MDA-MB-231 cells were treted with inorgnic sulfur, no effects were oserved until 48 h of incution. However, fter 72 h of incution, it ws oserved from previous tests tht cell prolifertion ws suppressed s the concentrtion of inorgnic sulfur incresed (dt now shown). In this study, we did not exceed 48 h of inorgnic sulfur tretment in order to show tht the effects of inorgnic sulfur on the motility or invsion of tumor cells were independent from the suppression of cell prolifertion. As consequence, tretment with inorgnic sulfur significntly suppressed tumor motility nd invsion in Boyden chmer nd invsion ssys t concentrtion > 25 μmol/l. Wu et l. treted L9981 lung cncer cells with sulfur-contining enzyl isothiocynte (BITC) nd phenethyl isothiocynte (PEITC), nd reported tht the motility of BITC nd PEITC were decresed to 11.1% nd 19.4% (compred to control) fter 24 h, of incution, nd to 8.1% nd 16.5% fter 3 h of incution, respectively [23]. Ho et l. [24] treted AGS gstric cncer cells with BITC (,.25, or.5 μmol/l) in Boyden chmer motility ssy, nd reported tht tumor cell motility decresed y 42% nd 43% for.25 nd.5 μmol/l fter 24 h of incution, respectively, nd 37% nd 44% fter 48 h of incution, respectively. They concluded tht the decrese in tumor motility y orgnic sulfur ws due to decrese in the ERK signl pthwy. Furthermore, Hwng nd Lee [25] treted SK-Hep1 humn heptom cells with llyl isothiocyntes (AITC) nd N-cetylcysteine conjugte isothiocyntes (NAC-AITC) t -5 μmol/l to explore their effects on tumor cell invsion. As result, they reported tht AITC nd NAC-AITC significntly decresed tumor cell invsion t the concentrtion of 1 μmol/l, nd t 5 μmol/l, tumor invsion ws decresed y 6% nd 53% for AITC nd NAC- AITC, respectively. According to the ove dt, sulfur-contining orgnics hve een reported to decrese the motility nd invsion of tumor cells, nd it ws found in this study tht inorgnic sulfur hs the sme effect. Tumor metstsis is the mjor cuse of deth for cncer ptients nd involves the ctivtion nd migrtion of ECM-degrding enzymes [26]. Consisting of collgen, ECM is degrded y MMPs, nd tumor cells will then metstsize to proximl lood vessels [5,6]. Enzymes tht cn degrde cell memrnes nd cuse the invsion nd metstsis of tumor cells include MMPs, serine proteses, cysteine proteses, nd sprtyl protenses; mong these, MMPs re Zn-dependent peptides tht cn degrde ECM nd ply role in wound heling, tumor invsion, metstsis, nd ngiogenesis [5,27,28]. MMPs re clssified into geltinses, collgenses, stromelysins, nd memrne type MMPs ccording to enzyme chrcteristics [29]. High expressions of MMP-2 nd MMP-9 tht hve chrcteristics of geltinses re closely relted to the metstsis of rest cncer [6,3-33]. Dvies et l. [34] reported tht the ctivtion of MMP-2 nd MMP-9 incresed in rest cncer. In this study, tretment of MDA-MB-231 cells with inorgnic sulfur significntly decresed the ctivtion of MMP-2 nd MMP-9 nd their mrna expression. Hwng nd Lee [25] reported tht when SK-Hep1 heptom cells were treted with AITC nd NAC-AITC, oth decresed ctivtion of MMP-2 nd MMP-9, ut AITC hd greter effect. They lso reported tht mrna expression of MMP-2 ws significntly decresed y oth AITC nd NAC-AITC, ut tht of MMP-9 ws decresed y only NAC-AITC. In conclusion, tretment with inorgnic sulfur cn decrese the motility nd invsion of tumor cells without ffecting their prolifertion, which is considered to e due to MMP-2 nd MMP-9 ctivtion nd the suppression of their mrna expression. In the future, more reserch on the mechnism of inorgnic sulfur s ffect on metstsis is necessry. References 1. World Helth Orgniztion [Internet]. Are the numer of cncer cses incresing or decresing in the world? [cited 21 June 3]. Aville from: 2. Ntionl Helth Insurnce [Internet]. Anlysis of cncer ptients in 28. [cited 21 June 5]. Aville from: kr/cms/ord/ord/bord.jsp?ct=view&communitykey=b7 &ordid= Ntionl Cncer Center [Internet]. Types of rest cncer. [cited 21 June 1]. Aville from: /cncer_is/2/ _1751.html. 4. Yip D, Ahmd A, Krpetis CS, Hwkins CA, Hrper PG. Mtrix metlloproteinse inhiitors: pplictions in oncology. Invest New Drugs 1999;17: Duffy MJ. The role of proteolytic enzymes in cncer invsion nd metstsis. 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